U.S. patent application number 11/211039 was filed with the patent office on 2006-03-09 for bioluminescent novelty items.
Invention is credited to Bruce Bryan.
Application Number | 20060053505 11/211039 |
Document ID | / |
Family ID | 27082782 |
Filed Date | 2006-03-09 |
United States Patent
Application |
20060053505 |
Kind Code |
A1 |
Bryan; Bruce |
March 9, 2006 |
Bioluminescent novelty items
Abstract
Novelty items that are combinations of articles of manufacture
with fluorescent proteins are provided. These novelty items,
include combinations of transgenic plants that express a luciferase
or a luciferin with plant food that contains a luciferase and a
luciferin.
Inventors: |
Bryan; Bruce; (Pinetop,
AZ) |
Correspondence
Address: |
Lara A. Northrop;Pietragallo, Bosick & Gordon LLP
One Oxford Centre, 38th Floor
301 Grant Street
Pittsburgh
PA
15219
US
|
Family ID: |
27082782 |
Appl. No.: |
11/211039 |
Filed: |
August 24, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09803211 |
Mar 8, 2001 |
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11211039 |
Aug 24, 2005 |
|
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09444762 |
Nov 22, 1999 |
|
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09803211 |
Mar 8, 2001 |
|
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09135988 |
Aug 17, 1998 |
6152358 |
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09444762 |
Nov 22, 1999 |
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08757046 |
Nov 25, 1996 |
5876995 |
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09444762 |
Nov 22, 1999 |
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08597274 |
Feb 6, 1996 |
6247995 |
|
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09444762 |
Nov 22, 1999 |
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Current U.S.
Class: |
800/20 ;
800/288 |
Current CPC
Class: |
C07K 14/43595 20130101;
A23L 17/00 20160801; C12N 9/0069 20130101; A23G 9/363 20130101;
A23G 9/44 20130101; A61K 8/64 20130101; A61K 2800/434 20130101;
F21K 2/06 20130101; C12N 9/0071 20130101; A61Q 19/00 20130101; A23K
50/80 20160501; B05B 7/2416 20130101; C12C 12/004 20130101; A23L
2/38 20130101; C12C 12/006 20130101; A23L 2/58 20130101; F21W
2121/02 20130101; A45D 34/00 20130101; B05B 7/0408 20130101; F21S
8/00 20130101; A23K 20/00 20160501; A61Q 11/00 20130101; B05C
17/00553 20130101; A23G 3/50 20130101; F21K 2/00 20130101; Y02A
40/818 20180101; F21V 33/0028 20130101; A23L 17/30 20160801; A24D
3/14 20130101; C12G 1/06 20130101; C12N 15/8212 20130101; F41B
9/004 20130101; A23G 3/366 20130101; A23V 2002/00 20130101; F21W
2131/401 20130101; Y10S 493/955 20130101; A23L 29/065 20160801;
A23P 10/30 20160801; B05B 9/0833 20130101; A23K 20/189 20160501;
A23L 29/06 20160801; B05B 7/2472 20130101; A23V 2002/00 20130101;
A23V 2300/21 20130101 |
Class at
Publication: |
800/020 ;
800/288 |
International
Class: |
A01K 67/027 20060101
A01K067/027; A01H 1/00 20060101 A01H001/00 |
Claims
1. A combination, comprising: a transgenic fish that expresses one
or more components of a bioluminescent system; and a composition
that includes one or more components of a bioluminescent system,
wherein introducing the composition to the transgenic fish causes
the transgenic fish to illuminate.
2. The combination of claim 1, wherein the transgenic fish is an
ornamental fish.
3. The combination of claim 1, wherein the transgenic fish is a
fishing lure or bait.
4. The combination of claim 1, wherein the transgenic fish is
substantially translucent.
5. The combination of claim 1, wherein the transgenic fish is
substantially transparent.
6. The combination of claim 1, wherein the transgenic fish has
cells and expresses one or more components of a bioluminescent
system on the surfaces of the cells.
7. The combination of claim 1, wherein the transgenic fish has
mouth and a digestive system and expresses one or more components
of a bioluminescent system on at least one of: the mouth or
digestive system.
8. The combination of claim 1, wherein the components of a
bioluminescent system comprise at least one of the following:
luciferase, luciferin, and fluorescent protein.
9. The combination of claim 8, wherein the luciferase or luciferin
is a Renilla luciferase or luciferin.
10. The combination of claim 8, wherein the luciferase or luciferin
is a Vargula luciferase or luciferin.
11. The combination of claim 8, wherein the fluorescent protein is
a green fluorescent protein.
12. The combination of claim 11, wherein the green fluorescent
protein is derived from the Aequorea fluorescent protein.
13. The combination of claim 11, wherein the green fluorescent
protein is derived from the Renilla fluorescent protein.
14. The combination of claim 8, wherein the fluorescent protein is
a blue fluorescent protein.
15. The combination of claim 14, wherein the blue fluorescent
protein is derived from Vibrio species fluorescent protein.
16. The combination of claim 1, wherein the components of the
bioluminescent system are derived from bacterial, fungal, insect,
invertebrate or vertebrate bioluminescent systems.
17. The combination of claim 1, wherein the transgenic fish emits
sufficient illumination to be viewed by the unassisted eye.
18. The combination of claim 1, wherein the transgenic fish emits
illumination of a plurality of colors.
19. The combination of claim 1, wherein the composition is a fish
food.
20. The combination of claim 1, wherein ingestion of the fish food
by the fish causes the fish to illuminate.
21. The combination of claim 1, wherein the transgenic fish is
contained in water and the composition can be added directly to the
water.
22. A transgenic fish that expresses at least one component of a
bioluminescent system.
23. The transgenic fish of claim 22, wherein the transgenic fish is
an ornamental fish.
24. The transgenic fish of claim 22, wherein the transgenic fish is
a fishing lure or bait.
25. The transgenic fish of clam 22, wherein the transgenic fish is
substantially translucent.
26. The transgenic fish of claim 22, wherein the transgenic fish is
substantially transparent.
27. The transgenic fish of claim 22, wherein the transgenic fish
has cells and expresses one or more components of a bioluminescent
system on the surfaces of the cells.
28. The transgenic fish of claim 22, wherein the transgenic fish
has a mouth and a digestive system and expresses one or more
components of a bioluminescent system on at least one of the mouth
or digestive system.
29. The transgenic fish of claim 22, wherein the components of a
bioluminescent system comprise at least one of the following:
luciferase, luciferin, and fluorescent protein.
30. The transgenic fish of claim 22, wherein the components of the
bioluminescent system are derived from bacterial, fungal, insect,
invertebrate of vertebrate bioluminescent systems.
31. The transgenic fish of claim 22, wherein the transgenic fish
emits sufficient illumination to be viewed by the unassisted
eye.
32. The transgenic fish of claim 22, wherein the transgenic fish
emits illumination of a plurality of colors.
33. A fish food that includes one or more components of a
bioluminescent generating system.
34. The fish food of claim 33, wherein the components of a
bioluminescent generating system comprise at least one of:
luciferase, luciferin, or fluorescent protein.
35. The fish food of claim 33, wherein the luciferase or luciferin
comprises from about 1 part per million to about 1 part per 10
weight/weight.
36. The fish food of claim 33, wherein the luciferase or luciferin
is in lyophilized form.
37. A transgenic fish egg that expresses one or more components of
a bioluminescent system.
38. A method of making a transgenic fish emit illuminate,
comprising: providing a transgenic fish that expresses one or more
components of a bioluminescent system; and contacting the
transgenic fish with a composition that comprises one or more
components of a bioluminescent system, wherein contacting the
transgenic fish with the composition causes the transgenic fish to
illuminate.
39. The method of claim 38, wherein the composition is a fish
food.
40. The method of claim 38, wherein the contacting the transgenic
fish with a composition is consumption by the fish of the
composition.
41. A method of producing a transgenic fish, comprising: providing
fish eggs; and transfecting the eggs with at least one luciferase
or luciferin.
42. The method of claim 41, wherein the eggs further comprise DNA
endoding a membrane-spanning protein, and transfecting the eggs
further includes incorporating one or more components of a
bioluminescent system into the DNA encoding a membrane-spanning
protein.
43. A method of producing a transgenic fish food, comprising:
providing a fish food; and including in the fish food one or more
components of a bioluminescent system.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/803,211 to Bruce Bryan, filed Mar. 8, 2001, entitled
"BIOLUMINESCENT NOVELTY ITEMS", which is a continuation of U.S.
application Ser. No. 09/444,762 to Bruce Bryan, filed Nov. 22,
1999, now U.S. Pat. No. 6,113,886, entitled "BIOLUMINESCENT NOVELTY
ITEMS", and is a continuation of U.S. application Ser. No.
09/135,988 to Bruce Bryan, filed Aug. 17, 1998, now U.S. Pat. No.
6,152,358, entitled "BIOLUMINESCENT NOVELTY ITEMS", and is a
continuation-in-part of U.S. application Ser. No. 08/757,046 to
Bruce Bryan, filed Nov. 25, 1996, now U.S. Pat. No. 5,876,995,
entitled "BIOLUMINESCENT NOVELTY ITEMS", and is also a
continuation-in-part of U.S. application Ser. No. 08/597,274 to
Bruce Bryan, filed Feb. 6, 1996, now U.S. Pat. No. 6,247,995,
entitled "BIOLUMINESCENT NOVELTY ITEMS".
[0002] U.S. application Ser. No. 09/803,211 is a continuation of
U.S. application Ser. No. 09/444,762 which is a continuation of
U.S. application Ser. No. 09/135,988, which is a
continuation-in-part of U.S. application Ser. No. 08/757,046, which
is a continuation-in-part of U.S. application Ser. No. 08/597,274.
The subject matter of each of U.S. application Ser. Nos.
09/444,762, 09/135,988, 08/597,274 and 08/757,046 is herein
incorporated in its entirety by reference thereto. This application
is also related to provisional application Ser. Nos. 60/079,624 and
60/089,367. The disclosures of each of the above noted patents,
applications and provisional applications is incorporated herein by
reference thereto.
FIELD OF INVENTION
[0003] The present invention relates to systems for producing
bioluminescent light, and to combinations of the systems with
articles of manufacture including toys, textiles, food and
beverages, to produce novelty items. By virtue of the combination,
the novelty items glow or produce or expel a bioluminescent
composition. Also, provided are compositions, encapsulated
bioluminescence generating reagents, and methods for producing the
bioluminescence.
BACKGROUND OF THE INVENTION
[0004] Luminescence is a phenomenon in which energy is specifically
channeled to a molecule to produce an excited state. Return to a
lower energy state is accompanied by release of a photon (hy).
Luminescence includes fluorescence, phosphorescence,
chemiluminescence and bioluminescence. Bioluminescence is the
process by which living organisms emit light that is visible to
other organisms. Luminescence may be represented as follows:
A+B.fwdarw.X*+Y X*.fwdarw.X+hv, [0005] where X* is an
electronically excited molecule and hy represents light emission
upon return of X* to a lower energy state. Where the luminescence
is bioluminescence, creation of the excited state derives from an
enzyme catalyzed reaction. The color of the emitted light in a
bioluminescent (or chemiluminescent or other luminescent) reaction
is characteristic of the excited molecule, and is independent from
its source of excitation and temperature.
[0006] An essential condition for bioluminescence is the use of
molecular oxygen, either bound or free in the presence of a
luciferase. Luciferases, are oxygenases, that act on a substrate,
luciferin, in the presence of molecular oxygen and transform the
substrate to an excited state. Upon return to a lower energy level,
energy is released in the form of light [for reviews see, e.g.,
McElroy et al. (1966) in Molecular Architecture in Cell Physiology,
Hayashi et al., eds., Prentice-Hall, Inc., Englewood Cliffs, N.J.,
pp. 63-80; Ward et al., Chapter 7 in Chemi-and Bioluminescence,
Burr, ed., Marcel Dekker, Inc. NY, pp. 321-358; Hastings, J. W. in
(1995) Cell Physiology:Source Book, N. Sperelakis (ed.), Academic
Press, pp 665-681; Luminescence, Narcosis and Life in the Deep Sea,
Johnson, Vantage Press, NY, see, esp. pp. 50-56].
[0007] Though rare overall, bioluminescence is more common in
marine organisms than in terrestrial organisms. Bioluminescence has
developed from as many as thirty evolutionarily distinct origins
and, thus, is manifested in a variety of ways so that the
biochemical and physiological mechanisms responsible for
bioluminescence in different organisms are distinct. Bioluminescent
species span many genera and include microscopic organisms, such as
bacteria [primarily marine bacteria including Vibrio species],
fungi, algae and dinoflagellates, to marine organisms, including
arthropods, mollusks, echinoderms, and chordates, and terrestrial
organism including annelid worms and insects.
[0008] Bioluminescence, as well as other types of
chemiluminescence, is used for quantitative determinations of
specific substances in biology and medicine. For example,
luciferase genes have been cloned and exploited as reporter genes
in numerous assays, for many purposes. Since the different
luciferase systems have different specific requirements, they may
be used to detect and quantify a variety of substances. The
majority of commercial bioluminescence applications are based on
firefly [Photinus pyralis] luciferase. One of the first and still
widely used assays involves the use of firefly luciferase to detect
the presence of ATP. It is also used to detect and quantify other
substrates or co-factors in the reaction. Any reaction that
produces or utilizes NAD(H), NADP(H) or long chain aldehyde, either
directly or indirectly, can be coupled to the light-emitting
reaction of bacterial luciferase.
[0009] Another luciferase system that has been used commercially
for analytical purposes is the Aequorin system. The purified
jellyfish photoprotein, aequorin, is used to detect and quantify
intracellular Ca.sup.2+ and its changes under various experimental
conditions. The Aequorin photoprotein is relatively small
[.about.20 kDa], nontoxic, and can be injected into cells in
quantities adequate to detect calcium over a large concentration
range [3.times.10.sup.-7 to 10.sup.-4 M].
[0010] Because of their analytical utility, many luciferases and
substrates have been studied and well-characterized and are
commercially available [e.g., firefly luciferase is available from
Sigma, St. Louis, Mo., and Boehringer Mannheim Biochemicals,
Indianapolis, Ind.; recombinantly produced firefly luciferase and
other reagents based on this gene or for use with this protein are
available from Promega Corporation, Madison, Wis.; the aequorin
photoprotein luciferase from jellyfish and luciferase from Renilla
are commercially available from Sealite Sciences, Bogart, Ga.;
coelenterazine, the naturally-occurring substrate for these
luciferases, is available from Molecular Probes, Eugene, Oreg.].
These luciferases and related reagents are used as reagents for
diagnostics, quality control, environmental testing and other such
analyses. These reagents have not been used in connection with
entertainment and recreation for the glow, illumination and color
produced upon generation of bioluminescence.
[0011] Thus, it is an object herein to exploit bioluminescence for
use as a recreational product in combination with articles of
manufacture to produce novelty items, including toys, personal
items, foods, fountains, beverages, coating compositions, such as
paints and inks, textiles, including clothing, toy cigarettes, fish
food, particularly for feeding transgenic fish that express a
luciferase, jewelry and other such items. It is also an object
herein to provide such combinations and to provide means for
producing and using such combinations.
SUMMARY OF THE INVENTION
[0012] Systems and apparatus for generating bioluminescence, and
combinations of these systems and apparatus with inanimate articles
of manufacture to produce novelty items are provided. These novelty
items, which are articles of manufacture, are designed for
entertainment, recreation and amusement, and include, but are not
limited to: toys, particularly squirt guns, toy cigarettes, toy
"Halloween" eggs, footbags and board/card games; finger paints and
other paints, slimy play material; textiles, particularly clothing,
such as shirts, hats and sports gear suits, threads and yarns;
bubbles in bubble making toys and other toys that produce bubbles;
balloons; figurines; personal items, such as bath powders, body
lotions, gels, powders and creams, nail polishes, make-up,
toothpastes and other dentifrices, soaps, body paints, and bubble
bath; items such as inks, paper; foods, such as gelatins, popcorn,
icings and frostings; fish food containing luciferins and
transgenic fish, particularly transgenic fish that express a
luciferase; plant food containing a luciferin or luciferase,
preferably a luciferin for use with transgenic plants that express
luciferase; and beverages, such as beer, wine, champagne, soft
drinks, and ice cubes and ice in other configurations; fountains,
including liquid "fireworks" and other such jets or sprays or
aerosols of compositions that are solutions, mixtures, suspensions,
powders, pastes, particles or other suitable form.
[0013] Thus, the novelty items provided herein include but are not
limited to: textiles that glow, ink that glows, paints,
particularly fingerpaints, that glow, paper products that glow,
toys, particularly reloadable squirt guns that eject a
bioluminescent fluid, dolls and dummies with internal organs or
parts that glow, figurines and novelty items that glow; toy
"cigarettes" that produce glowing "smoke" upon exhalation, toy eggs
with glowing yolks and/or whites, toy footbags that glow and toy
board and card games with glowing parts, such as glowing cards,
dice, game boards, etc.; foods and beverages that glow, soapy
compositions for blowing bubbles that produce bubbles that glow,
bubble bath compositions that produce bubbles that glow, fountains
that expel glowing fluid, bioluminescent "fireworks", sparklers,
magic-wand toys, and numerous other such items. Food containing a
luciferin for use with plants and animals that express luciferase,
such as transgenic fish, then when fed a food containing an
appropriate substrate glow, is also contemplated herein.
[0014] Bioluminescence is advantageously used in combination with
such novelty items because it can be generated using reagents that
are nontoxic, noncorrosive and nonstaining. Bioluminescence is also
advantageously used because it can be sustained to provide a glow
that lasts, if desired, from minutes up to hours.
[0015] Any article of manufacture that can be combined with a
bioluminescence-generating system as provided herein and thereby
provide entertainment, recreation and/or amusement, including use
of the items for recreation or to attract attention, such as for
advertising goods and/or services that are associated with a logo
or trademark is contemplated herein. Such uses may be in addition
to or in conjunction with or in place of the ordinary or normal use
of such items. As a result of the combination, the items glow or
produce, such as in the case of squirt guns and fountains, a
glowing fluid or spray of liquid or particles. The novelty in the
novelty item derives from its bioluminescence.
[0016] The preferred bioluminescence-generating reactions are
performed by adding oxygen (or water containing oxygen) or calcium
ions or other appropriate metal ion to luciferin and luciferase
mixtures using apparatus and systems as described herein.
Apparatus, systems and substrates for generating the
bioluminescence are provided. The systems include matrix materials
that are coated with bioluminescence generating reagents, capsular
vehicles containing the reagents and single chamber and multiple
chamber apparatus containing the reagents. The matrix materials are
used, for example, in the fabrication of clothing items and also in
the loading cartridges described herein.
[0017] Methods and compositions for producing bioluminescence in
combination with the novelty items are also provided. Micro- and
macro-capsular vehicles containing bioluminescence generating
reagents are provided. The capsular vehicles are capsules, such as
liposomes, isolated endosomes, isolated vacuoles, gelatin capsules,
and other such delivery vehicles, and the apparatus include
vessels, and single chamber, dual chamber and three chamber or more
apparatus. These vehicles encapsulate bioluminescence generating
system reagents, and typically contain less than all of the
reagents necessary to generate a bioluminescent reaction. The
capsular vehicles include vehicles often used for drug delivery,
such as liposomes, and time release capsules; and also capsules
made of glass, plastic and other such materials.
[0018] For example, the bioluminescence generating reagents (or
components) may be coated on the inside of a glass container, such
as a glass capillary tube [see, eg., U.S. Pat. No. 5,387,526]. Upon
addition of a composition containing the necessary activating
agents, such as molecular oxygen, ATP, a reductase, Ca.sup.2+ [or
other suitable metal ion], the coating will be contacted with the
activator and will produce a glow. The capsular vehicles are
intended for use in combination with the articles of
manufacture.
[0019] Thus, the micro- or macro-capsular vehicles, when crushed,
opened, dissolved or otherwise placed under conditions that cause
delivery of the contents, release material that glows upon contact
with air and/or moisture and/or other activator(s). These vehicles
vary in size [in the largest dimension] from as small as less than
0.1 .mu.m up to 0.1 cm or more.
[0020] Matrix materials, such as glass, plastics, cotton and other
textile material, that contain linked bioluminescence-generating
reagents are also provided. For example, one or more components of
the bioluminescence generating system is (are) linked by
adsorption, absorption or other means, directly or indirectly (such
as via a linker) to a matrix material. Matrix materials, such as
textiles, glass, plastic or ceramic surfaces or particles adapted
for linking molecules, for example such as luciferases or
luciferins, are combined with at least one component of the
bioluminescence generating system, particularly the luciferin,
luciferase, or, where the components are amenable, the luciferin
and luciferase. The components) such as the luciferase are linked
to the matrix, such as cotton, using methods known to those of
skill in the art of protein synthesis for linking peptides or
proteins to solid substrates [see, e.g., Eichler et al. (1993)
Biochemistry 32:11035-11041; Merrifield (1964) Biochemistry
3:1385-1390.] Linkage is effected either covalently or
non-covalently and can be direct or via linkers. Such methods and
linkers are well known to those of skill in the chemical arts. The
matrix materials with linked bioluminescence generating system
components are contacted with an article of manufacture resulting
in a novelty item that, when appropriately treated, such as by
spraying on a composition that contains the remaining components of
the reactions, glows or produces bioluminescence. The matrix
materials are advantageously used in the loading cartridges
provided herein.
[0021] Also provided are single and multi-chamber, particularly
dual chamber, apparatus for producing bioluminescence, and
combinations of these apparatus with bioluminescence generating
reagents are also provided. Such apparatus include at least one
chamber that contains all but at least one reagent or component
required to produce bioluminescence. Upon addition of the component
either to the chamber or after ejection of some or all of the
contents of the chamber a bioluminescent glow or glowing fluid,
spray or jet is produced. Recharging or charging cartridges adapted
for loading these apparatus are also provided.
[0022] The charging, or recharging, cartridges are designed to be
used to load components of a bioluminescence generating system into
or onto an article of manufacture to produce the novelty items, and
also to permit reuse after the bioluminescence generating system is
spent. The cartridge, which contains one or more chambers, is in an
exemplary embodiment fabricated with two-chambers. In a preferred
embodiment, the cartridge includes a matrix material, such as a
porous membrane or a cotton ball to which a bioluminescence
generating agent, such as a luciferase or luciferin, is adsorbed or
absorbed such that when flushed with an appropriate composition
will be released from the matrix. The first chamber contains one or
more components of a bioluminescence generating system used in the
bioluminescent process, and the second chamber contains a
composition that will flush or otherwise desorb a quantity of the
component from the matrix material. Typically, the composition is
contained in an easily puncturable or compressible vial and
positioned adjacent to the matrix material. In operation, a
plunger, a dual pronged plunger where there are two or more
chambers, is aligned so that one prong of the plunger is positioned
in each chamber, or the plunger may be movably attached to the
cartridge, and the output nozzles of the cartridge are aligned
against the filler ports of a novelty item, such as a squirt gun.
The plunger is then forced into the cartridge, thereby dispensing
the components out the nozzle of the first chamber and into the
first chamber in the novelty item, and compressing the vial of
fluid to flush the remaining components of the bioluminescence
generating system from the nozzle of the second chamber and into
the second chamber of the novelty item. In this manner, the novelty
items contemplated herein may be initially charged, or recharged
again and again, by replenishing any or all of the components
necessary for generating bioluminescence.
[0023] Articles of manufacture containing one or more components of
a bioluminescence generating system or a composition, such as a
composition containing ATP or Ca.sup.2+ or other activator, within
the packaging material, and a label that indicates that the
contents is used for generating bioluminescence are also
provided.
[0024] Kits containing an article of manufacture and appropriate
reagents for generating bioluminescence are also provided.
DESCRIPTION OF THE DRAWINGS
[0025] In the accompanying drawings:
[0026] FIG. 1 is a side elevation, with portions cut away, of a
squirt gun incorporating the dual chamber structure;
[0027] FIG. 2 is a sectional view taken on line 2-2 of FIG. 1;
[0028] FIG. 3 is a sectional view taken on line 3-3 of FIG. 1;
[0029] FIG. 4 is a side elevation view, with portions cut away, of
a gas powered toy gun with dual chamber detachable fluid
reservoir;
[0030] FIG. 5 is a top plan view of the toy gun of FIG. 4, with
portions cut away;
[0031] FIG. 6 is a side elevation view, partially cut away of a
gas-charged fluid dispensing apparatus incorporating the dual
chamber system;
[0032] FIG. 7 is a sectional view taken on line 7-7 of FIG. 6;
[0033] FIG. 8 is a top plan view of the structure of FIG. 6,
partially cut away;
[0034] FIG. 9 is a side elevation view of a fountain type
configuration of the gas-charged dual chamber fluid dispensing
apparatus, with portions cut away;
[0035] FIG. 10 is a sectional view taken on line 10-10 of FIG.
9;
[0036] FIG. 11 is a side elevation view, partially cut away, of a
dual chamber compressible dispensing container;
[0037] FIG. 12 is a side elevation view, partially cut away of a
bottle/bladder apparatus designed for use with bubble-blowing
compositions;
[0038] FIG. 13 is a view similar to FIG. 12, with the components
mixed and the bubble blowing wand detached for use; and
[0039] FIG. 14 is a side elevation view, partially cut away, of
beverage container with a bladder apparatus actuated by opening of
the beverage container.
[0040] FIG. 15 is a side elevation view, partially cut away of a
single use, dual chamber fluid packaging apparatus adapted for use
with bubble-blowing compositions.
[0041] FIG. 16 is a side elevation view, partially cut away of a
cap apparatus operated by depression of the plunger assembly to
rupture the capsule contained within the cork cap.
[0042] FIG. 17 is a side elevation view, partially cut away of a
cap apparatus operated by screwing the plunger assembly into the
cork cap to rupture the capsule contained therein.
[0043] FIG. 18 is a side elevation view, partially cut away of a
cap apparatus operated by screwing the screw-cap onto the top of
the bottle forcing the plunger assembly against the capsule
contained within the neck of the bottle, thereby rupturing the
capsule membranes.
[0044] FIG. 19 is a view similar to the view of FIG. 18, with the
cap apparatus tightly secured against the top of the bottle and the
capsule membranes ruptured.
[0045] FIG. 20 is a side elevation view, with portions cut away, of
a spray container or can in which the bottom portion of the
apparatus is not engaged.
[0046] FIG. 21 is a side elevation view, with portions cut away, of
the spray container in which the bottom portion of the container is
engaged.
[0047] FIG. 22 is a side elevation view of an exemplary pellet that
contains bioluminescence-generating reagents and that is adapted
for use with the spray container.
[0048] FIG. 23 is a side elevation, with portions cut away, of
another embodiment of a squirt gun incorporating the dual chamber
structure;
[0049] FIG. 24 is a top view, with portions cut away, of the nozzle
end of the squirt gun of FIG. 23;
[0050] FIG. 25 is a sectional view taken on line 25-25 of FIG. 23;
and
[0051] FIG. 26 is a sectional view taken on line 26-26 of FIG.
23.
[0052] FIG. 27 is a side elevation view of a compressible tube
configuration with a portion cut away.
[0053] FIG. 28 is a pictorial view of a charging, or recharging,
cartridge;
[0054] FIG. 29 is a sectional view taken on line 29-29 of FIG. 28,
with the plunger in the starting position;
[0055] FIG. 30 is a sectional view similar to FIG. 29, showing the
cartridge contents ejected into receiving chambers of a typical
unit as shown in FIG. 2;
[0056] FIG. 31 is a sectional view similar to FIG. 29, showing a
plunger locking device;
[0057] FIG. 32 is a sectional view similar to FIG. 30, showing the
locking device released to allow compression of the plunger;
[0058] FIG. 33 is a sectional view taken along line 33-33 of FIG.
31 and showing the positioning of the locking device; and
[0059] FIG. 34 is a sectional view of an alternative embodiment
dual chamber refill cartridge
DETAILED DESCRIPTION OF THE INVENTION
Table of Contents
[0060] A. Definitions
[0061] B. Bioluminescence generating systems [0062] 1. General
description [0063] a. Luciferases [0064] b. Luciferins [0065] c.
Activators [0066] d. Reactions [0067] 2. Ctenophore and
coelenterate systems [0068] a. The aequorin system [0069] (1)
Aequorin photoprotein [0070] (2) Luciferin [0071] b. The Renilla
system [0072] 3. Crustacean, particular Cyrpidina [Vargula],
systems [0073] a. Vargula luciferase [0074] (1) Purification from
Cypridina [0075] (2) Preparation by Recombinant Methods [0076] b.
Vargula luciferin [0077] c. Reaction [0078] 4. Insect
bioluminescence generating systems including fireflies, click
beetles, and other insect systems [0079] a. Luciferase [0080] b.
Luciferin [0081] c. Reaction [0082] 5. Bacterial systems [0083] a.
Luciferases [0084] b. Luciferins [0085] c. Reactions [0086] 6.
Other systems [0087] a. Dinoflagellate bioluminescence generating
systems [0088] b. Systems from molluscs, such as Latia and Pholas
[0089] c. Earthworms and other annelids [0090] d. Glow worms [0091]
e. Marine polycheate worm systems [0092] f. South American railway
beetle [0093] g. Fish [0094] 7. Fluorescent proteins [0095] a.
Green and blue fluorescent proteins [0096] b. Phycobiliproteins
[0097] C. Practice of the reactions in combination with articles of
manufacture [0098] D. Packaging of Bioluminescence Systems [0099]
1. Dispensing and Packaging Apparatus for Combination with the
Bioluminescence Generating System Components [0100] 2. Capsules,
pellets, liposomes, micronized particles [0101] a. Encapsulating
vehicles-in general [0102] b. Encapsulating vehicles-liposomes
[0103] c. Encapsulating vehicles-gelatin and polymeric vehicles
[0104] d. Endosomes and vacuoles [0105] e. Micronized particles
[0106] 3. Apparatus and substrates [0107] a. Matrix materials
[0108] b. Immobilization and activation [0109] 4. Apparatus
containing a single chamber, housing or a vessel [0110] 5. Dual and
multiple chamber fluid dispensing apparatus [0111] a. Mechanical
pump dispensing apparatus [0112] b. Gas-charged dispensing
apparatus [0113] c. Compressible dispensing apparatus [0114] 6.
Other fluid dispensing and packaging apparatus particularly
designed for single or multiple uses [0115] a. Bottle-type single
chamber container/bladder apparatus [0116] b. Dual chambered bottle
type container/bladder apparatus for use with foods and beverages
[0117] c. Can type container/bladder apparatus for use with foods
and beverages [0118] d. Spray containers for use to produce a
glowing spray [0119] 7. Cap Apparatus for use a single chamber
vessel
[0120] E. Combinations of articles of manufacture and
bioluminescence [0121] 1. Personal care products, including bath
powders, bubble baths, products for use on the nails, hair, skin,
lips and elsewhere [0122] a. Bath powders [0123] b. Glowing dust or
powder [0124] c. Lotions, gels and other topical application
formulations [0125] (1) Lotions [0126] (2) Creams [0127] (3)
Solutions and suspensions for topical application [0128] (4) Gels
[0129] (5) Solids [0130] 2. Glowing toys and other items [0131] a.
Single chamber toy guns and other toy weapons that shoot pellets or
liquid [0132] b. Bubble-making toys [0133] c. Board/Card games
[0134] d. Toy Eggs [0135] e. Footbags, bean bags and balls [0136]
f. Figurines [0137] 3. Glowing textiles and paper products [0138]
4. Foods and beverages, including ice cubes [0139] a. Beverages
[0140] b. Ice [0141] c. Popcorn [0142] 5. Jewelry, Clothing and
Other Items of Manufacture [0143] 6. Fountains [0144] 7.
Non-Tobacco Toy Cigarettes [0145] 8. Fish, Fish Bait and Fish Food
[0146] 9. Plant Food and Animal Food
[0147] F. Cartridges for loading (charging or filling) or reloading
(recharging) the novelty items
[0148] A. Definitions
[0149] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which this invention belongs. All patents
and publications of referred to herein are incorporated by
reference in their entirety.
[0150] As used herein, novelty items refer to inanimate articles of
manufacture that are intended to provide, even for only a few
moments, amusement, entertainment, decoration or recreation. The
use for recreation or entertainment may be the items only use or
may be in addition to other uses or benefits of the items, such as
clothing that is modified, as described herein, by combination with
bioluminescence.
[0151] Novelty items are understood by those of skill in
manufacture of such items as well as by the purchasing public and
are intended herein to include items, such as, toys, including toy
guns, dolls, dummies, figurines, balloons, bubbles, "fairy dust",
such as micronized lyophilized particles, puzzles, and inks and
paints, particularly fingerpaints; theatrical vapors when mixed,
for example with dry ice or a fog; souvenirs; textiles,
particularly clothing, including T-shirts, hats, swimsuits, bathing
suit, wet suits, scuba diving suits, surfing suits, and other water
sport or sports attire; foods and beverages, including gelatins,
ice cubes and ice in other shapes, beer, wine, champagne, soft
drinks, ice creams, sorbets, ices, frostings, and candy; jewelry,
medallions, decorative articles, artificial flowers, articles for
displaying names, business trade names, slogans, trademarks on
promotional or other such items, such as T-shirts, hats, paints,
wrapping paper, gifts intended to promote business goodwill;
personal items, such as body paints, body sprays, bubble baths,
make-up, body lotions, dentifrices; fountains; jets or sprays of
particles or fluids, including "fireworks", sparklers, and
magic-wand toys, and many other such novelty items [see, e.g., U.S.
Pat. Nos. 5,435,010, 5,460,022, 5,458,931, 5,435,787, 5,435,010,
5,432,623, 5,421,583, 5,419,558, 5,416,927, 5,413,454, 5,413,332,
5,411,427, 5,410,962, 5,407,691, 5,407,391, 5,405,958, 5,405,206,
5,400,698, 5,399,122, 5,398,972, 5,397,609, 5,396,408, 5,393,580,
5,390,086, 5,389,033, 5,383,684, 5,374,805, 5,368,518, 5,363,984,
5,360,010, 5,353,378, 5,351,931, 5,346,455, 5,341,538, 5,323,492,
5,283,911, 5,222,797, 5,177,812, 5,158,349, 4,924,358, 3,597,877
and many others, which describe types of items are considered
novelty items]. Any such inanimate item that is combined with
bioluminescence is intended to be encompassed herein.
[0152] Thus, for purposes herein, a novelty item refers to any
inanimate article of manufacture that, upon combination with
bioluminescence, provides amusement, entertainment, recreation or
enjoyment, if only for even a few moments. Addition of the
bioluminescence to the article of manufacture does not add to the
function of the item, but adds entertainment, amusement or
recreational aspects to the item so that the resulting combination
is a novelty item. Therefore, the combinations provided herein are
novelty items by virtue of the combination of an inanimate article
of manufacture with bioluminescence.
[0153] As used herein, inanimate means that the articles of
manufacture are not alive nor formerly living [i.e., dead] items.
Thus, the novelty items herein, do not encompass living organisms,
such as genetically modified fireflies or genetically engineered
plants that express luciferase or other such organisms that produce
bioluminescence. Animal food and plant food containing luciferin
(or luciferase) and/or other activators for use with a transgenic
animal or plant that expresses the corresponding luciferase (or
luciferin) are provided. These are intended to result in an
illuminated animal or plant upon ingestion or consumption or
absorption of the food. Transgenic fish and food therefor are also
provided herein.
[0154] As used herein, personal items include items that are used
on the body, such as toothpastes, dentifrices, make-up, nail
polishes, body lotions, body creams, body paints and body
powders.
[0155] As used herein, chemiluminescence refers to a chemical
reaction in which energy is specifically channeled to a molecule
causing it to become electronically excited and subsequently to
release a photon thereby emitting visible light. Temperature does
not contribute to this channeled energy. Thus, chemiluminescence
involves the direct conversion of chemical energy to light energy.
Bioluminescence refers to the subset of chemiluminescence reactions
that involve luciferins and luciferases (or the photoproteins).
Bioluminescence does not herein include phosphorescence.
[0156] As used herein, "fairy dust" refers to particles, such as
light sensitive liposomes or micronized powdered particles, that
glow upon contact with the air, such as "dust" that a child would
use when pretending to be Tinker Bell or other such character.
[0157] As used herein, reference to ice cubes include ice in any
shape or form, including, but not limited to: cubes; ice formations
made from precast molds, such as figurines, icicles, ice sculptures
and other such novelty items formed from ice.
[0158] As used herein, luminescence refers to the detectable EM
radiation, generally, UV, IR or visible EM radiation that is
produced when the excited product of an exergic chemical process
reverts to its ground state with the emission of light.
Chemiluminescence is luminescence that results from a chemical
reaction. Bioluminescence is chemiluminescence that results from a
chemical reaction using biological molecules [or synthetic versions
or analogs thereof] as substrates and/or enzymes.
[0159] As used herein, bioluminescence, which is a type of
chemiluminescence, refers to the emission of light by biological
molecules, particularly proteins. The essential condition for
bioluminescence is molecular oxygen, either bound or free in the
presence of an oxygenase, a luciferase, which acts on a substrate,
a luciferin. Bioluminescence is generated by an enzyme or other
protein [luciferase] that is an oxygenase that acts on a substrate
luciferin [a bioluminescence substrate] in the presence of
molecular oxygen and transforms the substrate to an excited state,
which upon return to a lower energy level releases the energy in
the form of light.
[0160] As used herein, the substrates and enzymes for producing
bioluminescence are generically referred to as luciferin and
luciferase, respectively. When reference is made to a particular
species thereof, for clarity, each generic term is used with the
name of the organism from which it derives, for example, bacterial
luciferin or firefly luciferase.
[0161] As used herein, luciferase refers to oxygenases that
catalyze a light emitting reaction. For instance, bacterial
luciferases catalyze the oxidation of flavin mononucleotide [FM N]
and aliphatic aldehydes, which reaction produces light. Another
class of luciferases, found among marine arthropods, catalyzes the
oxidation of Cypridina [Vargula] luciferin, and another class of
luciferases catalyzes the oxidation of Coleoptera luciferin.
[0162] Thus, luciferase refers to an enzyme or photoprotein that
catalyzes a bioluminescent reaction [a reaction that produces
bioluminescence]. The luciferases, such as firefly and Renilla
luciferases, that are enzymes which act catalytically and are
unchanged during the bioluminescence generating reaction. The
luciferase photoproteins, such as the aequorin and obelin
photoproteins to which luciferin is non-covalently bound, are
changed, such as by release of the luciferin, during
bioluminescence generating reaction. The luciferase is a protein
that occurs naturally in an organism or a variant or mutant
thereof, such as a variant produced by mutagenesis that has one or
more properties, such as thermal or pH stability, that differ from
the naturally-occurring protein. Luciferases and modified mutant or
variant forms thereof are well known.
[0163] Thus, reference, for example, to "Renilla luciferase" means
an enzyme isolated from member of the genus Renilla or an
equivalent molecule obtained from any other source, such as from
another Anthozoa, or that has been prepared synthetically.
[0164] The luciferases and luciferin and activators thereof are
referred to as bioluminescence generating reagents or components.
Typically, a subset of these reagents will be provided or combined
with an article of manufacture. Bioluminescence will be produced
upon contacting the combination with the remaining reagents. Thus,
as used herein, the component luciferases, luciferins, and other
factors, such as O.sub.2, Mg.sup.2+, Ca.sup.2+ are also referred to
as bioluminescence generating reagents [or agents or
components].
[0165] As used herein, "not strictly catalytically" means that the
photoprotein acts as a catalyst to promote the oxidation of the
substrate, but it is changed in the reaction, since the bound
substrate is oxidized and bound molecular oxygen is used in the
reaction. Such photoproteins are regenerated by addition of the
substrate and molecular oxygen under appropriate conditions known
to those of skill in this art.
[0166] As used herein, bioluminescence substrate refers to the
compound that is oxidized in the presence of a luciferase, and any
necessary activators, and generates light. These substrates are
referred to as luciferins, which are substrates that undergo
oxidation in a bioluminescence reaction. These bioluminescence
substrates include any luciferin or analog thereof or any synthetic
compound with which a luciferase interacts to generate light.
Preferred substrates are those that are oxidized in the presence of
a luciferase or protein in a light-generating reaction.
Bioluminescence substrates, thus, include those compounds that
those of skill in the art recognize as luciferins. Luciferins, for
example, include firefly luciferin, Cypridina [also known as
Vargula] luciferin [coelenterazine], bacterial luciferin, as well
as synthetic analogs of these substrates or other compounds that
are oxidized in the presence of a luciferase in a reaction the
produces bioluminescence.
[0167] As used herein, capable of conversion into a bioluminescence
substrate means susceptible to chemical reaction, such as oxidation
or reduction, that yields a bioluminescence substrate. For example,
the luminescence producing reaction of bioluminescent bacteria
involves the reduction of a flavin mononucleotide group (FMN) to
reduced flavin mononucleotide (FMNH.sub.2) by a flavin reductase
enzyme. The reduced flavin mononucleotide [substrate] then reacts
with oxygen [an activator] and bacterial luciferase to form an
intermediate peroxy flavin that undergoes further reaction, in the
presence of a long-chain aldehyde, to generate light. With respect
to this reaction, the reduced flavin and the long chain aldehyde
are substrates.
[0168] As used herein, bioluminescence system [or bioluminescence
generating system] refers to the set of reagents required for a
bioluminescence-producing reaction. Thus, the particular
luciferase, luciferin and other substrates, solvents and other
reagents that may be required to complete a bioluminescent reaction
form a bioluminescence system. Therefore, a bioluminescence system
(or equivalently a bioluminescence generating system) refers to any
set of reagents that, under appropriate reaction conditions, yield
bioluminescence. Appropriate reaction conditions refers to the
conditions necessary for a bioluminescence reaction to occur, such
as pH, salt concentrations and temperature. In general,
bioluminescence systems include a bioluminescence substrate (a
luciferin), a luciferase, which includes enzymes luciferases and
photoproteins, and one or more activators. A particular
bioluminescence system may be identified by reference to the
specific organism from which the luciferase derives; for example,
the Vargula [also called Cypridina] bioluminescence system (or
Vargula system) includes a Vargula luciferase, such as a luciferase
isolated from the ostracod, Vargula or produced using recombinant
means or modifications of these luciferases. This system would also
include the particular activators necessary to complete the
bioluminescence reaction, such as oxygen and a substrate with which
the luciferase reacts in the presence of the oxygen to produce
light.
[0169] As used herein, recharging or reloading the item refers to
the means by which spent bioluminescence generating components are
added to an item. Recharging generally refers to a process in which
one component, such as a luciferase is added to an item, such as a
textile; reloading refers to the process in which all components
are added to an item, such as a refillable squirt gun.
[0170] As used herein, ATP, AMP, NAD+ and NADH refer to adenosine
triphosphate, adenosine monophosphate, nicotinamide adenine
dinucleotide (oxidized form) and nicotinamide adenine dinucleotide
(reduced form), respectively.
[0171] As used herein, production by recombinant means by using
recombinant DNA methods means the use of the well known methods of
molecular biology for expressing proteins encoded by cloned
DNA.
[0172] As used herein, substantially identical to a product means
sufficiently similar so that the property of interest is
sufficiently unchanged so that the substantially identical product
can be used in place of the product.
[0173] As used herein, substantially pure means sufficiently
homogeneous to appear free of readily detectable impurities as
determined by standard methods of analysis, such as thin layer
chromatography (TLC), gel electrophoresis and high performance
liquid chromatography (HPLC), used by those of skill in the art to
assess such purity, or sufficiently pure such that further
purification would not detectably alter the physical and chemical
properties, such as enzymatic and biological activities, of the
substance. Methods for purification of the compounds to produce
substantially chemically pure compounds are known to those of skill
in the art. A substantially chemically pure compound may, however,
be a mixture of stereoisomers. In such instances, further
purification might increase the specific activity of the
compound.
[0174] As used herein equivalent, when referring to two sequences
of nucleic acids means that the two sequences in question encode
the same sequence of amino acids or equivalent proteins. When
"equivalent" is used in referring to two proteins or peptides, it
means that the two proteins or peptides have substantially the same
amino acid sequence with only conservative amino acid substitutions
[see, e.g., Table 2, below] that do not substantially alter the
activity or function of the protein or peptide. When "equivalent"
refers to a property, the property does not need to be present to
the same extent [eg., two peptides can exhibit different rates of
the same type of enzymatic activity], but the activities are
preferably substantially the same. "Complementary," when referring
to two nucleotide sequences, means that the two sequences of
nucleotides are capable of hybridizing, preferably with less than
25%, more preferably with less than 15%, even more preferably with
less than 5%, most preferably with no mismatches between opposed
nucleotides. Preferably the two molecules will hybridize under
conditions of high stringency.
[0175] As used herein: stringency of hybridization in determining
percentage mismatch is as follows: [0176] 1) high stringency:
0.1.times.SSPE, 0.1% SDS, 65.degree. C. [0177] 2) medium
stringency: 0.2.times.SSPE, 0.1% SDS, 50.degree. C. [0178] 3) low
stringency: 1.0.times.SSPE, 0.1% SDS, 50.degree. C.
[0179] It is understood that equivalent stringencies may be
achieved using alternative buffers, salts and temperatures.
[0180] The term "substantially" varies with the context as
understood by those skilled in the relevant art and generally means
at least 70%, preferably means at least 80%, more preferably at
least 90%, and most preferably at least 95%.
[0181] As used herein, biological activity refers to the in vivo
activities of a compound or physiological responses that result
upon administration of a compound, composition or other mixture.
Biological activities may be observed in vitro systems designed to
test or use such activities. Thus, for purposes herein the
biological activity of a luciferase is its oxygenase activity
whereby, upon oxidation of a substrate, light is produced.
[0182] As used herein, a composition refers to a any mixture. It
may be a solution, a suspension, liquid, powder, a paste, aqueous,
non-aqueous or any combination thereof.
[0183] As used herein, a combination refers to any association
between two or among more items.
[0184] As used herein, fluid refers to any composition that can
flow. Fluids thus encompass compositions that are in the form of
semi-solids, pastes, solutions, aqueous mixtures, gels, lotions,
creams and other such compositions.
[0185] As used herein, plant food refers to any liquids,
water-soluble or water-insoluble solids, such as fertilizers
containing any ratio of nitrogen, potassium and/or phosphorous,
formulations, combinations, polymers or plant growth promoters,
such as auxins and hormones, that is applied to a plant to promote
or maintain growth [e.g., see U.S. Pat. Nos. 4,016,880, 4,711,659,
4,804,403,5,547,486, 5,553,853, RE 35,320, and RE 31,801]. The
plant food may be applied directly to the soil, sprayed on the
foliage of the plant or a combination thereof. The plant food may
be slow releasing or available immediately for consumption by the
plant. The plant food may be applied to any plant that can be
genetically engineered to contain a heterologous gene encoding a
component of a bioluminescence generating system, preferably a
luciferase. Examples of such plants, but not meant to be limiting
to, are grasses, agricultural plants and ornamental plants.
[0186] B. Bioluminescence Generating Systems
[0187] A bioluminescence generating system refers to the components
that are necessary and sufficient to generate bioluminescence.
These include a luciferase, luciferin and any necessary co-factors
or conditions. Virtually any bioluminescence generating system
known to those of skill in the art will be amenable to use in the
apparatus, systems, combinations and methods provided herein.
Factors for consideration in selecting a bioluminescence generating
system, include, but are not limited to: the item used in
combination with the bioluminescence; the medium in which the
reaction is run; stability of the components, such as temperature
or pH sensitivity; shelf life of the components; sustainablity of
the light emission, whether constant or intermittent; availability
of components; desired light intensity; and other such factors.
[0188] 1. General Description
[0189] In general, bioluminescence refers to an energy-yielding
chemical reaction in which a specific chemical substrate, a
luciferin, undergoes oxidation, catalyzed by an enzyme, a
luciferase. Bioluminescent reactions are easily maintained,
requiring only replenishment of exhausted luciferin or other
substrate or cofactor or other protein, in order to continue or
revive the reaction. Bioluminescence generating reactions are well
known to those of skill in this art and any such reaction may be
adapted for use in combination with articles of manufacture as
described herein.
[0190] There are numerous organisms and sources of bioluminescence
generating systems, and some representative genera and species that
exhibit bioluminescence are set forth in the following table
[reproduced in part from Hastings in (1995) Cell Physiology:Source
Book, N. Sperelakis (ed.), Academic Press, pp 665-681]:
TABLE-US-00001 TABLE 1 Representative Luminous Organism Type Of
Organism Representative genera Bacteria Photobacterium Vibrio
Xenorhabdus Mushrooms Panus, Armillaria Pleurotus Dinoflagellates
Gonyaulax Pyrocystis Noctiluca Cnidaria (coelenterates) Jellyfish
Aequorea Hydroid Obelia Sea Pansy Renilla Ctenophores Mnemiopsis
Beroe Annelids Earthworms Diplocardia Marine polychaetes
Chaetopterus, Phyxotrix Syllid fireworm Odontosyllis Molluscs
Limpet Latia Clam Pholas Squid Heteroteuthis Heterocarpus Crustacea
Ostracod Vargula (Cypridina) Shrimp (euphausids) Meganyctiphanes
Acanthophyra Oplophorus Gnathophausia Decapod Sergestes Copepods
Insects Coleopterids (beetles) Photinus, Photuris Firefly
Pyrophorus Click beetles Phengodes, Phrixothrix Railroad worm
Arachnocampa Diptera (flies) Echinoderms Brittle stars Ophiopsila
Sea cucumbers Laetmogone Chordates Tunicates Pyrosoma Fish
Cartilaginous Squalus Bony Ponyfish Leiognathus Flashlight fish
Photoblepharon Angler fish Cryptopsaras Midshipman Porichthys
Lantern fish Benia Shiny loosejaw Aristostomias Hatchet fish
Agyropelecus and other fish Pachystomias Malacosteus Midwater fish
Cyclothone Neoscopelus Tarletonbeania
[0191] Other bioluminescent organisms contemplated for use herein
are Gonadostomias, Gaussia, Halisturia, Vampire squid, Glyphus,
Mycotophids (fish), Vinciguerria, Howella, Florenciella,
Chaudiodus, Melanocostus and Sea Pens.
[0192] It is understood that a bioluminescence generating system
may be isolated from natural sources, such as those in the above
Table, or may be produced synthetically. In addition, for uses
herein, the components need only be sufficiently pure so that
mixture thereof, under appropriate reaction conditions, produces a
glow. Thus it has been found, in some embodiments, a crude extract
or merely grinding up the organism may be adequate. Generally,
however, substantially pure components are used, but, where
necessary, the precise purity can be determined empirically. Also,
components may be synthetic components that are not isolated from
natural sources. DNA encoding luciferases is available [see, e.g.,
SEQ ID NOs. 1-13] and has been modified [see, e.g., SEQ ID NOs. 3
and 10-13] and synthetic and alternative substrates have been
devised. The DNA listed herein is only representative of the DNA
encoding luciferases that is available.
[0193] Any bioluminescence generating system, whether synthetic or
isolated form natural sources, such as those set forth in Table 1,
elsewhere herein or known to those of skill in the art, is intended
for use in the combinations, systems and methods provided herein.
Chemiluminescence systems per se, which do not rely on oxygenases
[luciferases] are not encompassed herein.
[0194] a. Luciferases
[0195] Luciferases refer to any compound that, in the presence of
any necessary activators, catalyze the oxidation of a
bioluminescence substrate [luciferin] in the presence of molecular
oxygen, whether free or bound, from a lower energy state to a
higher energy state such that the substrate, upon return to the
lower energy state, emits light. For purposes herein, luciferase is
broadly used to encompass enzymes that act catalytically to
generate light by oxidation of a substrate and also photoproteins,
such as aequorin, that act, though not strictly catalytically
[since such proteins are exhausted in the reaction], in conjunction
with a substrate in the presence of oxygen to generate light. These
luciferases, including photoproteins, such as aequorin, are herein
also included among the luciferases. These reagents include the
naturally-occurring luciferases [including photoproteins], proteins
produced by recombinant DNA, and mutated or modified variants
thereof that retain the ability to generate light in the presence
of an appropriate substrate, co-factors and activators or any other
such protein that acts as a catalyst to oxidize a substrate,
whereby light is produced.
[0196] Generically, the protein that catalyzes or initiates the
bioluminescent reaction is referred to as a luciferase, and the
oxidizable substrate is referred to as a luciferin. The oxidized
reaction product is termed oxyluciferin, and certain luciferin
precursors are termed etioluciferin. Thus, for purposes herein
bioluminescence encompasses light produced by reactions that are
catalyzed by [in the case of luciferases that act enzymatically] or
initiated by [in the case of the photoproteins, such as aequorin,
that are not regenerated in the reaction] a biological protein or
analog, derivative or mutant thereof.
[0197] For clarity herein, these catalytic proteins are referred to
as luciferases and include enzymes such as the luciferases that
catalyze the oxidation of luciferin, emitting light and releasing
oxyluciferin. Also included among luciferases are photoproteins,
which catalyze the oxidation of luciferin to emit light but are
changed in the reaction and must be reconstituted to be used again.
The luciferases may be naturally occurring or may be modified, such
as by genetic engineering to improve or alter certain properties.
As long as the resulting molecule retains the ability to catalyze
the bioluminescent reaction, it is encompassed herein.
[0198] Any protein that has luciferase activity [a protein that
catalyzes oxidation of a substrate in the presence of molecular
oxygen to produce light as defined herein] may be used herein. The
preferred luciferases are those that are described herein or that
have minor sequence variations. Such minor sequence variations
include, but are not limited to, minor allelic or species
variations and insertions or deletions of residues, particularly
cysteine residues. Suitable conservative substitutions of amino
acids are known to those of skill in this art and may be made
generally without altering the biological activity of the resulting
molecule. Those of skill in this art recognize that, in general,
single amino acid substitutions in non-essential regions of a
polypeptide do not substantially alter biological activity (see,
e.g., Watson et al. Molecular Biology of the Gene, 4th Edition,
1987, The Benjamin/Cummings Pub. co., p. 224). Such substitutions
are preferably made in accordance with those set forth in TABLE 2
as follows: TABLE-US-00002 TABLE 2 Original residue Conservative
substitution Ala (A) Gly; Ser Arg (R) Lys Asn (N) Gln; His Cys (C)
Ser; neutral amino acid Gln (Q) Asn Glu (E) Asp Gly (G) Ala; Pro
His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg;
Gln; Glu Met (M) Leu; Tyr; Ile Phe (F) Met; Leu; Tyr Ser (S) Thr
Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; Leu
[0199] Other substitutions are also permissible and may be
determined empirically or in accord with known conservative
substitutions. Any such modification of the polypeptide may be
effected by any means known to those of skill in this art.
[0200] The luciferases may be obtained commercially, isolated from
natural sources, expressed in host cells using DNA encoding the
luciferase, or obtained in any manner known to those of skill in
the art. For purposes herein, crude extracts obtained by grinding
up selected source organisms may suffice. Since large quantities of
the luciferase may be desired, isolation of the luciferase from
host cells is preferred. DNA for such purposes is widely available
as are modified forms thereof.
[0201] Examples of luciferases include, but are not limited to,
those isolated from the ctenophores Mnemiopsis (mnemiopsin) and
Beroe ovata (berovin), those isolated from the coelenterates
Aequorea (aequorin), Obelia (obelin), Pelagia, the Renilla
luciferase, the luciferases isolated from the mollusca Pholas
(pholasin), the luciferases isolated from the Aristostomias and
Porichthys fish and from the ostracods, such as Cypridina (also
referred to as Vargula). Preferred luciferases for use herein are
the Aequorin protein, Renilla luciferase and Cypridina [also called
Vargula] luciferase [see, e.g., SEQ ID NOs. 1, 2, and 4-13]. Also,
preferred are luciferases which react to produce red and/or near
infrared light. These include luciferases found in species of
Aristostomias, such as A. scintillans, Pachystomias, Malacosteus,
such as M. niger.
[0202] b. Luciferins
[0203] The substrates for the reaction include any molecule(s) with
which the luciferase reacts to produce light. Such molecules
include the naturally-occurring substrates, modified forms thereof,
and synthetic substrates [see, e.g., U.S. Pat. Nos. 5,374,534 and
5,098,828]. Exemplary luciferins include those described herein, as
well as derivatives thereof, analogs thereof, synthetic substrates,
such as dioxetanes [see, e.g., U.S. Pat. Nos. 5,004,565 and
5,455,357], and other compounds that are oxidized by a luciferase
in a light-producing reaction [see, e.g., U.S. Pat. Nos. 5,374,534,
5,098,828 and 4,950,588]. Such substrates also may be identified
empirically by selecting compounds that are oxidized in
bioluminescent reactions.
[0204] c. Activators
[0205] The bioluminescence generating systems also require
additional components discussed herein and known to those of skill
in the art. All bioluminescent reactions require molecular oxygen
in the form of dissolved or bound oxygen. Thus, molecular oxygen,
dissolved in water or in air or bound to a photoprotein, is the
activator for bioluminescence reactions. Depending upon the form of
the components, other activators include, but are not limited to,
ATP [for firefly luciferase], flavin reductase [bacterial systems]
for regenerating FMNH.sub.2 from FMN, and Ca.sup.2+ or other
suitable metal ion [aequorin].
[0206] Most of the systems provided herein will generate light when
the luciferase and luciferin are mixed and exposed to air or water.
The systems that use photoproteins that have bound oxygen, such as
aequorin, however, will require exposure to Ca.sup.2+ [or other
suitable metal ion], which can be provided in the form of an
aqueous composition of a calcium salt. In these instances, addition
of a Ca.sup.2+ [or other suitable metal ion]to a mixture of
luciferase [aequorin] and luciferin [such as coelenterazine] will
result in generation of light. The Renilla system and other
Anthozoa systems also require Ca.sup.2+ [or other suitable metal
ion].
[0207] If crude preparations are used, such as ground up Cypridina
[shrimp] or ground fireflies, it may be necessary to add only
water. In instances in which fireflies [or a firefly or beetle
luciferase] are used the reaction may only require addition ATP.
The precise components will be apparent, in light of the disclosure
herein, to those of skill in this art or may be readily determined
empirically.
[0208] It is also understood that these mixtures will also contain
any additional salts or buffers or ions that are necessary for each
reaction to proceed. Since these reactions are well-characterized,
those of skill in the art will be able to determine precise
proportions and requisite components. Selection of components will
depend upon the apparatus, article of manufacture and luciferase.
Various embodiments are described and exemplified herein; in view
of such description, other embodiments will be apparent.
[0209] d. Reactions
[0210] In all embodiments, up to all but one component of a
bioluminescence generating system will be mixed with or packaged
with or otherwise combined with a selected article of manufacture
to produce the novelty item. When bioluminescence is desired, the
remaining component(s) will be added and light will be
produced.
[0211] In general, since the result to be achieved is the
production of light visible to the naked eye for entertainment,
amusement or recreation, for the purposes herein, the precise
proportions and amounts of components of the bioluminescence
reaction need not be stringently determined or met. They must be
sufficient to produce light. Generally, an amount of luciferin and
luciferase sufficient to generate a visible glow is used; this
amount can be readily determined empirically and is dependent upon
the selected system and selected application.
[0212] For purposes herein, such amount is preferably at least the
concentrations and proportions used for analytical purposes by
those of skill in the such arts. Higher concentrations may be used
if the glow is not sufficiently bright. Also because the conditions
in which the reactions are used are not laboratory conditions and
the components are subject to storage, higher concentration may be
used to overcome any loss of activity. Typically, the amounts are 1
mg, preferably 10 mg and more preferably 100 mg, of a luciferase
per liter of reaction mixture or 1 mg, preferably 10 mg, more
preferably 100 mg, coated on a portion of a T-shirt or other
textile or paper. Such coating may be produced by drying a
composition containing at least about 0.01 mg/l, and typically 0.1
mg/l, 1 mg/l, 10 mg/l or more of each component on the item. The
amount of luciferin is also between about 0.01 and 100 mg/l,
preferably between 0.1 and 10 mg/l, additional luciferin can be
added to many of the reactions to continue the reaction. In
embodiments in which the luciferase acts catalytically and does not
need to be regenerated, lower amounts of luciferase can be used. In
those in which it is changed during the reaction, it also can be
replenished; typically higher concentrations will be selected.
Ranges of concentration per liter [or the amount of coating on
substrate the results from contacting with such composition] of
each component on the order of 0.1 to 20 mg, preferably 0.1 to 10
mg, more preferably between about 1 and 10 mg of each component
will be sufficient. When preparing coated substrates, as described
herein, greater amounts of coating compositions containing higher
concentrations of the luciferase or luciferin may be used.
[0213] Thus, for example, in presence of calcium, 5 mg of
luciferin, such as coelenterazine, in one liter of water will glow
brightly for at least about 10 to 20 minutes, depending on the
temperature of the water, when about 10 mgs of luciferase, such as
aequorin photoprotein luciferase or luciferase from Renilla, is
added thereto. Increasing the concentration of luciferase, for
example, to 100 mg/l, provides a particularly brilliant display of
light.
[0214] If desired, the onset of the bioluminescent reaction can be
delayed by adding an, an inhibitor, for example magnesium, of the
bioluminescence generating reaction. Also, where inhibition is not
desired, the concentration of free magnesium may be reduced by
addition of a sufficient amount of chelating agent, such as
ethylenediamine-tetraacetic acid [EDTA]. The amount of EDTA and
also calcium can be empirically determined to appropriately chelate
magnesium, without inhibiting or preventing the desired
bioluminescence.
[0215] It is understood, that concentrations and amounts to be used
depend upon the selected article of manufacture and they may be
readily determined empirically. Proportions, particularly those
used when commencing an empirical determination, are generally
those used for analytical purposes, and amounts or concentrations
are at least those used for analytical purposes, but the amounts
can be increased, particularly if a sustained and brighter glow is
desired.
[0216] 2. Ctenophore and Coelenterate Systems
[0217] Ctenophores, such as Mnemiopsis (mnemiopsin) and Beroe ovata
(berovin), and coelenterates, such as Aequorea (aequorin), Obelia
(obelin) and Pelagia, produce bioluminescent light using similar
chemistries [see, e.g., Stephenson et al. (1981) Biochimica et
Biophysica Acta 678:65-75; Hart et al. (1979) Biochemistry
18:2204-2210; International PCT Application No. WO94/18342, which
is based on U.S. application Ser. No. 08/017,116, U.S. Pat. No.
5,486,455 and other references and patents cited herein]. The
Aequorin and Renilla systems are representative and are described
in detail herein as exemplary and as among the presently preferred
systems. The Aequorin and Renilla systems can use the same
luciferin and produce light using the same chemistry, but each
luciferase is different. The Aequorin luciferase aequorin, as well
as, for example, the luciferases mnemiopsin and berovin, is a
photoprotein that includes bound oxygen and bound luciferin,
requires Ca.sup.2+[or other suitable metal ion] to trigger the
reaction, and must be regenerated for repeated use; whereas, the
Renilla luciferase acts as a true enzyme because it is unchanged
during the reaction and it requires dissolved molecular oxygen.
[0218] a. The Aequorin System
[0219] The aequorin system is well known [see, e.g., Tsuji et al.
(1986) "Site-specific mutagenesis of the calcium-binding
photoprotein aequorin," Proc. Natl. Acad. Sci. USA 83:8107-8111;
Prasher et al. (1985) "Cloning and Expression of the cDNA Coding
for Aequorin, a Bioluminescent Calcium-Binding Protein,"
Biochemical and Biophysical Research Communications 126:1259-1268;
Prasher et al. (1986) Methods in Enzymology 133:288-297; Prasher,
et al. (1987) "Sequence Comparisons of cDNAs Encoding for Aequorin
Isotypes," Biochemistry 26:1326-1332; Charbonneau et al. (1985)
"Amino Acid Sequence of the Calcium-Dependent Photoprotein
Aequorin," Biochemistry 24:6762-6771; Shimomura et al. (1981)
"Resistivity to denaturation of the apoprotein of aequorin and
reconstitution of the luminescent photoprotein from the partially
denatured apoprotein," Biochem. J. 199:825-828; Inouye et al.
(1989) J. Biochem. 105:473-477; Inouye et al. (1986) "Expression of
Apoaequorin Complementary DNA in Escherichia coli," Biochemistry
25:8425-8429; Inouye et al. (1985) "Cloning and sequence analysis
of cDNA for the luminescent protein aequorin," Proc. Natl. Acad.
Sci. USA 82:3154-3158; Prendergast, et al. (1978) "Chemical and
Physical Properties of Aequorin and the Green Fluorescent Protein
Isolated from Aequorea forskalea" J. Am. Chem. Soc. 17:3448-3453;
European Patent Application 0 540 064 A1; European Patent
Application 0 226 979 A2, European Patent Application 0 245 093 A1
and European Patent Specification 0 245 093 B1; U.S. Pat. Nos.
5,093,240; 5,360,728; 5,139,937; 5,422,266; 5,023,181; 5,162,227;
and SEQ ID NOs. 5-13, which set forth DNA encoding the apoprotein;
and a form, described in U.S. Pat. No. 5,162,227, European Patent
Application 0 540 064 A1 and Sealite Sciences Technical Report No.
3 (1994), is commercially available from Sealite, Sciences, Bogart,
Ga. as AQUALITE.RTM.].
[0220] This system is among the preferred systems for use herein.
As will be evident, since the aequorin photoprotein includes
noncovalently bound luciferin and molecular oxygen, it is suitable
for storage in this form as a lyophilized powder or encapsulated
into a selected delivery vehicle. The system can be encapsulated
into pellets, such as liposomes or other delivery vehicles, or
stored in single chamber dual or other multiple chamber apparatus.
When used, the vehicles are contacted with a composition, even tap
water, that contains Ca.sup.2+[or other suitable metal ion], to
produce a mixture that glows. This system is preferred for use in
numerous embodiments herein, such as in any embodiment that uses
pellets. These embodiments include, squirt guns, fairy dust, bubble
toys, bubble baths, soaps, linked to textiles, for addition to
beverages and foods.
[0221] (1) Aequorin and Related Photoproteins
[0222] The photoprotein, aequorin, isolated from the jellyfish,
Aequorea, emits light upon the addition of Ca.sup.2+ [or other
suitable metal ion]. The aequorin photoprotein, which includes
bound luciferin and bound oxygen that is released by Ca.sup.2+,
does not require dissolved oxygen. Luminescence is triggered by
calcium, which releases oxygen and the luciferin substrate
producing apoaqueorin.
[0223] The bioluminescence photoprotein aequorin is isolated from a
number of species of the jellyfish Aequorea. It is a 22 kilodalton
[kD] molecular weight peptide complex [see, e.g., Shimomura et al.
(1962) J. Cellular and Comp. Physiol. 59:233-238; Shimomura et al.
(1969) Biochemistry 8:3991-3997; Kohama et al. (1971) Biochemistry
10:4149-4152; and Shimomura et al. (1972) Biochemistry
11:1602-1608]. The native protein contains oxygen and a
heterocyclic compound coelenterazine, a luciferin, [see, below]
noncovalently bound thereto. The protein contains three calcium
binding sites. Upon addition of trace amounts Ca.sup.2+[or other
suitable metal ion, such as strontium] to the photoprotein, it
undergoes a conformational change the catalyzes the oxidation of
the bound coelenterazine using the protein-bound oxygen. Energy
from this oxidation is released as a flash of blue light, centered
at 469 nm. Concentrations of calcium ions as low as 10.sup.-6 M are
sufficient to trigger the oxidation reaction.
[0224] Naturally-occurring apoaequorin is not a single compound but
rather is a mixture of microheterogeneous molecular species.
Aequoria jellyfish extracts contain as many as twelve distinct
variants of the protein [see, eg., Prasher et al. (187)
Biochemistry 26:1326-1332; Blinks et al. (1975) Fed. Proc. 34:474].
DNA encoding numerous forms has been isolated [see, eg., SEQ ID
NOs. 5-9 and 13].
[0225] The photoprotein can be reconstituted [see, eq., U.S. Pat.
No. 5,023,1811 by combining the apoprotein, such as a protein
recombinantly produced in E. coli, with a coelenterazine, such as a
synthetic coelenterazine, in the presence of oxygen and a reducing
agent [see, e.g., Shimomura et al. (1975) Nature 256:236-238;
Shimomura et al. (1981) Biochemistry J. 199:825-828], such as
2-mercaptoenthanol, and also EDTA or EGTA [concentrations between
about 5 to about 100 mM or higher for applications herein] tie up
any Ca.sup.2+ to prevent triggering the oxidation reaction until
desired. DNA encoding a modified form of the apoprotein that does
not require 2-mercaptoethanol for reconstitution is also available
[see, eg., U.S. Pat. No. 5,093,240]. The reconstituted photoprotein
is also commercially available [sold, e.g., under the trademark
AQUALITE@, which is described in U.S. Pat. No. 5,162,227].
[0226] The light reaction is triggered by adding Ca.sup.2+ at a
concentration sufficient to overcome the effects of the chelator
and achieve the 10.sup.-6 M concentration. Because such low
concentrations of Ca.sup.2+ can trigger the reaction, for use in
the methods and apparatus herein, higher concentrations of chelator
may be included in the compositions of photoprotein. Accordingly,
higher concentrations of added Ca.sup.2+ in the form of a calcium
salt will be required. Precise amounts may be empirically
determined. For use herein, it may be sufficient to merely add
water to the photoprotein, which is provided in the form of a
concentrated composition or in lyophilized or powdered form. Thus,
for purposes herein, addition of small quantities of Ca.sup.2+,
such as those present in most tap water or in phosphate buffered
saline (PBS) or other suitable buffers or possible in the moisture
on the skin, should trigger the bioluminescence reaction.
[0227] Numerous isoforms of the aequorin apoprotein been identified
isolated. DNA encoding these proteins has been cloned, and the
proteins and modified forms thereof have been produced using
suitable host cells [see, e.g., U.S. Pat. Nos. 5,162,227,
5,360,728, 5,093,240; see, also, Prasher et al. (1985) Biophys.
Biochem. Res. Commun. 126:1259-1268; Inouye et al. (1986)
Biochemistry 25: 8425-8429]. U.S. Pat. Nos. 5,093,240; 5,360,728;
5,139,937; 5,288,623; 5,422,266, 5,162,227 and SEQ ID NOs. 5-13,
which set forth DNA encoding the apoprotein; and a form is
commercially available form Sealite, Sciences, Bogart, Ga. as
AQUALITE.RTM.]. DNA encoding apoaequorin or variants thereof is
useful for recombinant production of high quantities of the
apoprotein. The photoprotein is reconstituted upon addition of the
luciferin, coelenterazine, preferably a sulfated derivative
thereof, or an analog thereof, and molecular oxygen [see, e.g.,
U.S. Pat. No. 5,023,181]. The apoprotein and other constituents of
the photoprotein and bioluminescence generating reaction can be
mixed under appropriate conditions to regenerate the photoprotein
and concomitantly have the photoprotein produce light.
Reconstitution requires the presence of a reducing agent, such as
mercaptoethanol, except for modified forms, discussed below, that
are designed so that a reducing agent is not required [see, e.g.,
U.S. Pat. No. 5,093,240].
[0228] For use herein, it is preferred aequorin is produced using
DNA, such as that set forth in SEQ ID NOs. 5-13 and known to those
of skill in the art or modified forms thereof. The DNA encoding
aequorin is expressed in a host cell, such as E. coli, isolated and
reconstituted to produce the photoprotein [see, e.g., U.S. Pat.
Nos. 5,418,155, 5,292,658, 5,360,728, 5,422,266, 5,162,227].
[0229] Of interest herein, are forms of the apoprotein that have
been modified so that the bioluminescent activity is greater than
unmodified apoaequorin [see, e.g., U.S. Pat. No. 5,360,728, SEQ ID
NOs. 10-12]. Modified forms that exhibit greater bioluminescent
activity than unmodified apoaequorin include proteins having
sequences set forth in SEQ ID NOs. 10-12, in which aspartate 124 is
changed to serine, glutamate 135 is changed to serine, and glycine
129 is changed to alanine, respectively. Other modified forms with
increased bioluminescence are also available.
[0230] For use in certain embodiments herein, the apoprotein and
other components of the aequorin bioluminescence generating system
are packaged or provided as a mixture, which, when desired is
subjected to conditions under which the photoprotein reconstitutes
from the apoprotein, luciferin and oxygen [see, e.g., U.S. Pat. No.
5,023,181; and U.S. Pat. No. 5,093,240]. Particularly preferred are
forms of the apoprotein that do not require a reducing agent, such
as 2-mercapto-ethanol, for reconstitution. These forms, described,
for example in U.S. Pat. No. 5,093,240 [see, also Tsuji et al.
(1986) Proc. Natl. Acad. Sci. U.S.A. 83:8107-8111], are modified by
replacement of one or more, preferably all three cysteine residues
with, for example serine. Replacement may be effected by
modification of the DNA encoding the aequorin apoprotein, such as
that set forth in SEQ ID No. 5, and replacing the cysteine codons
with serine.
[0231] The photoproteins and luciferases from related species, such
as Obelia are also contemplated for use herein. DNA encoding the
Ca.sup.2+-activated photoprotein obelin from the hydroid polyp
Obelia longissima is known and available [see, e.g., Illarionov et
al. (1995) Gene 153:273-274; and Bondar et al. (1995) Biochim.
Biophys. Acta 1231:29-32]. This photoprotein can also be activated
by Mn.sup.2+[see, e.g., Vysotski et al. (1995) Arch. Bioch.
Biophys. 316:92-93, Vysotski et al. (1993) J. Biolumin. Chemilumin.
8:301-305].
[0232] In general for use herein, the components of the
bioluminescence are packaged or provided so that there is
insufficient metal ions to trigger the reaction. When used, the
trace amounts of triggering metal ion, particularly Ca.sup.2+ is
contacted with the other components. For a more sustained glow,
aequorin can be continuously reconstituted or can be added or can
be provided in high excess.
[0233] (2) Luciferin
[0234] The aequorin luciferin is coelenterazine and analogs
therein, which include molecules having the structure [formula
(I)]: ##STR1## [0235] in which R.sub.1 is CH.sub.2C.sub.6H.sub.5 or
CH.sub.3; R.sub.2 is C.sub.6H.sub.5, and R.sub.3 is
p-C.sub.6H.sub.4OH or CH.sub.3 or other such analogs that have
activity. Preferred coelenterazine has the structure in which
R.sup.1 is p-CH.sub.2C.sub.6H.sub.4OH, R.sub.2 is C.sub.6H.sub.5,
and R.sub.3 is p-C.sub.6H.sub.4OH, which can be prepared by known
methods [see, e.g., Inouye et al. (1975) Jap. Chem. Soc., Chemistry
Lttrs. pp 141-144; and Hart et al. (1979) Biochemistry
18:2204-2210]. The preferred coelenterazine has the structure
(formula (II)): ##STR2## [0236] and sulfated derivatives
thereof.
[0237] The reaction of coelenterazine when bound to the aequorin
photoprotein with bound oxygen and in the presence of Ca.sup.2+ can
represented as follows: ##STR3##
[0238] The photoprotein aequorin [which contains apoaequorin bound
to a coelenterate luciferin molecule] and Renilla luciferase,
discussed below, can use the same coelenterate luciferin. The
aequorin photoprotein catalyses the oxidation of coelenterate
luciferin [coelenterazine] to oxyluciferin [coelenteramide] with
the concomitant production of blue light [.lamda..sub.max=469
nm].
[0239] Importantly, the sulfate derivative of the coelenterate
luciferin [lauryl-luciferin] is particularly stable in water, and
thus may be used in a coelenterate-like bioluminescence generating
system. In this system, adenosine diphosphate (ADP) and a
sulpha-kinase are used to convert the coelenterazine to the
sulphated form. Sulfatase is then used to reconvert the
lauryl-luciferin to the native coelenterazine. Thus, the more
stable lauryl-luciferin is used in the item to be illuminated and
the luciferase combined with the sulfatase are added to the
luciferin mixture when illumination is desired.
[0240] Thus, the bioluminescence generating system of Aequorea is
particularly suitable for use in the methods and apparatus herein.
The particular amounts and the manner in which the components are
provided depends upon the selected combination of article of
manufacture. This system can be provided in lyophilized form, that
will glow upon addition of Ca.sup.2+. It can be encapsulated,
linked to matrices, such as porous glass, or in as a compositions,
such as a solution or suspension, preferably in the presence of
sufficient chelating agent to prevent triggering the reaction. The
concentration of the aequorin photoprotein will vary and can be
determined empirically. Typically concentrations of at least 0.1
mg/[, more preferably at least 1 mg/l and higher, will be selected.
In certain embodiments, 1-10 mg luciferin/100 mg of luciferase will
be used in selected volumes and at the desired concentrations will
be used.
[0241] b. The Renilla System
[0242] Representative of coelenterate systems is the Renilla
system. Renilla, also known as sea pansies, are members of the
class of coelenterates Anthozoa, which includes other
bioluminescent genera, such as Cavarnularia, Ptilosarcus,
Stylatula, Acanthoptilum, and Parazoanthus. Bioluminescent members
of the Anthozoa genera contain luciferases and luciferins that are
similar in structure [see, e.g., Cormier et al. (1973) J. Cell.
Physiol. 81:291-298; see, also Ward et al. (1975) Proc. Natl. Acad.
Sci. U.S.A. 72:2530-2534]. The luciferases and luciferins from each
of these anthozoans crossreact and produce a characteristic blue
luminescence.
[0243] Renilla luciferase and the other coelenterate and ctenophore
luciferases, such as the aequorin photoprotein, use imidazopyrazine
substrates, particularly the substrates generically called
coelenterazine [see, formulae (I) and (II), above]. Other genera
that have luciferases that use a coelenterazine include: squid,
such as Chiroteuthis, Eucleoteuthis, Onychoteuthis, Watasenia;
cuttlefish, Sepiolina; shrimp, such as Oplophorus, Sergestes, and
Gnathophausia; deep-sea fish, such as Argyropelecus, Yarella,
Diaphus, and Neoscopelus.
[0244] Renilla luciferase does not, however, have bound oxygen, and
thus requires dissolved oxygen in order to produce light in the
presence of a suitable luciferin substrate. Since Renilla
luciferase acts as a true enzyme [i.e., it does not have to be
reconstituted for further use] the resulting luminescence can be
long-lasting in the presence of saturating levels of luciferin.
Also, Renilla luciferase is relatively stable to heat.
[0245] Renilla luciferase, DNA encoding Renilla luciferase, and use
of the DNA to produce recombinant luciferase, as well as DNA
encoding luciferase from other coelenterates, are well known and
available [see, e.g., SEQ ID NO. 1, U.S. Pat. Nos. 5,418,155 and
5,292,658; see, also, Prasher et al. (1985) Biochem. Biophys. Res.
Commun. 126:1259-1268; Cormier (1981) "Renilla and Aequorea
bioluminescence" in Bioluminescence and Chemiluminescence, pp.
225-233; Charbonneau et al. (1979) J. Biol. Chem. 254:769-780; Ward
et al. (1979) J. Biol. Chem. 254:781-788; Lorenz et al. (1981)
Proc. Natl. Acad. Sci. U.S.A. 88: 4438-4442; Hori et al. (1977)
Proc. Natl. Acad. Sci. U.S.A. 74:4285-4287; Hori et al. (1975)
Biochemistry 14:2371-2376; Hori et al. (1977) Proc. Natl. Acad.
Sci. U.S.A. 74:4285-4287; Inouye et al. (1975) Jap. Soc. Chem.
Lett. 141-144; and Matthews et al. (1979) Biochemistry 16:85-91].
The DNA encoding Renilla luciferase and host cells containing such
DNA provide a convenient means for producing large quantities of
the enzyme [see, eg., U.S. Pat. Nos. 5,418,155 and 5,292,658, which
describe recombinant production of Renilla luciferase and the use
of the DNA to isolate DNA encoding other luciferases, particularly
those from related organisms]. A modified version of a method [U.S.
Pat. Nos. 5,418,155 and 5,292,658] for the recombinant production
of Renilla luciferase that results in a higher level of expression
of the recombinant enzyme is presented in the EXAMPLES herein.
[0246] When used herein, the Renilla luciferase can be packaged,
such as in an toy, in lyophilized form, encapsulated in a vehicle,
either by itself or in combination with the luciferin substrate.
Prior to use the mixture is contacted with an aqueous composition,
preferably a phosphate buffered saline or other suitable buffer,
such a Tris-based buffer [such as 0.1 mm Tris, 0.1 mm EDTA] pH 7-8,
preferably about pH 8; dissolved O.sub.2 will activate the
reaction. Addition of glycerol [about 1%] increases light
intensity. Final concentrations of luciferase in the glowing
mixture will be on the order of 0.01 to 1 mg/l or more.
Concentrations of luciferin will be at least about 10.sup.-8 M, but
1 to 100 or more orders of magnitude higher to produce a long
lasting bioluminescence.
[0247] In certain embodiments herein, about 1 to 10 mg, or
preferably 2-5 mg, more preferably about 3 mg of coelenterazine
will be used with about 100 mg of Renilla luciferase. The precise
amounts, of course can be determined empirically, and, also will
depend to some extent on the ultimate concentration and
application. In particular, about addition of about 0.25 ml of a
crude extract from the bacteria that express Renilla to 100 ml of a
suitable assay buffer and about 0.005 .mu.g was sufficient to
produce a visible and lasting glow [see, U.S. Pat. Nos. 5,418,155
and 5,292,658, which describe recombinant production of Renilla
luciferase].
[0248] Lyophilized mixtures, and compositions containing the
Renilla luciferase are also provided. The luciferase or mixtures of
the luciferase and luciferin may also be encapsulated into a
suitable delivery vehicle, such as a liposome, glass particle,
capillary tube, drug delivery vehicle, gelatin, time release
coating or other such vehicle. Kits containing these mixtures,
compositions, or vehicles and also a selected article of
manufacture, such as a toy gun, bubble composition, balloon, item
of clothing, personal item, are also provided. The luciferase may
also be linked to a substrate, such as cotton, polyester,
polyester-cotton blends, polypropylene, polyvinyltoluene, polyvinyl
propylene, glass, ceramic, or plastics are provided in combination
with or as part of an article of manufacture.
[0249] 3. Crustacean, Particularly Cyrpidina Systems
[0250] The ostracods, such as Vargula serratta, hilgendorfii and
noctiluca are small marine crustaceans, sometimes called sea
fireflies. These sea fireflies are found in the waters off the
coast of Japan and emit light by squirting luciferin and luciferase
into the water, where the reaction, which produces a bright blue
luminous cloud, occurs. The reaction involves only luciferin,
luciferase and molecular oxygen, and, thus, is very suitable for
application herein.
[0251] The systems, such as the Vargula bioluminescence generating
systems, are particularly preferred herein because the components
are stable at room temperature if dried and powdered and will
continue to react even if contaminated. Further, the bioluminescent
reaction requires only the luciferin/luciferase components in
concentrations as low as 1:40 parts per billion to 1:100 parts per
billion, water and molecular oxygen to proceed. An exhausted system
can renewed by addition of luciferin.
[0252] a. Vargula Luciferase
[0253] Vargula luciferase is a 555-amino acid polypeptide that has
been produced by isolation from Vargula and also using recombinant
technology by expressing the DNA in suitable bacterial and
mammalian host cells [see, e.g., Thompson et al. (1989) Proc. Natl.
Acad. Sci. U.S.A. 86:6567-6571; Inouye et al. (1992) Proc. Natl.
Acad. Sci. U.S.A. 89:9584-9587; Johnson et al. (1978) Methods in
Enzymology LVII:331-349; Tsuji et al. (1978) Methods Enzymol.
57:364-72; Tsuji (19740 Biochemistry 13:5204-5209; Japanese Patent
Application No. JP 3-30678 Osaka; and European Patent Application
No. EP 0 387 355 A1].
[0254] (1) Purification from Cypridina
[0255] Methods for purification of Vargula [Cypridina] luciferase
are well known. For example, crude extracts containing the active
can be readily prepared by grinding up or crushing the Vargula
shrimp. In other embodiments, a preparation of Cypridina
hilgendorfi luciferase can be prepared by immersing stored frozen
C. hilgendorfi in distilled water containing, 0.5-5.0 M salt,
preferably 0.5-2.0 M sodium or potassium chloride, ammonium
sulfate, at 0-30.degree. C., preferably 0-10.degree. C., for 1-48
hr, preferably 10-24 hr, for extraction followed by hydrophobic
chromatography and then ion exchange or affinity chromatography
[TORAY IND INC, Japanese patent application JP 4258288, published
Sep. 14, 1993; see, also, Tsuji et al. (1978) Methods Enzymol.
57:364-72 for other methods].
[0256] The luciferin can be isolated from ground dried Vargula by
heating the extract, which destroys the luciferase but leaves the
luciferin intact [see, eq., U.S. Pat. No. 4,853,327].
[0257] (2) Preparation by Recombinant Methods
[0258] The luciferase is preferably produced by expression of
cloned DNA encoding the luciferase [European Patent Application NO.
0 387 355 A1; International PCT Application No. WO90/01542; see,
also SEQ ID NO. 5, which sets forth the sequence from Japanese
Patent Application No. JP 3-30678 and Thompson et al. (1989) Proc.
Natl. Acad. Sci. U.S.A. 86:6567-6571] DNA encoding the luciferase
or variants thereof is introduced into E. coli using appropriate
vectors and isolated using standard methods.
[0259] b. Vargula Luciferin
[0260] The natural luciferin in a substituted imidazopyrazine
nucleus, such a compound of formula (III): ##STR4##
[0261] Analogs thereof and other compounds that react with the
luciferase in a light producing reaction also may be used.
[0262] Other bioluminescent organisms that have luciferases that
can react with the Vargula luciferin include, the genera Apogon,
Parapriacanthus and Porichthys.
[0263] c. Reaction
[0264] The luciferin upon reaction with oxygen forms a dioxetanone
intermediate [which includes a cyclic peroxide similar to the
firefly cyclic peroxide molecule intermediate]. In the final step
of the bioluminescent reaction, the peroxide breaks down to form
CO.sub.2 and an excited carbonyl. The excited molecule then emits a
blue to blue-green light.
[0265] The optimum pH for the reaction is about 7. For purposes
herein, any pH at which the reaction occurs may be used. The
concentrations of reagents are those normally used for analytical
reactions or higher [see, e.g., Thompson et al. (1990) Gene
96:257-262]. Typically concentrations of the luciferase between 0.1
and 10 mg/l, preferably 0.5 to 2.5 mg/l will be used. Similar
concentrations or higher concentrations of the luciferin may be
used.
[0266] 4. Insect Bioluminescence Generating Systems including
Firefly, Click Beetle, and Other Insect Systems
[0267] The biochemistry of firefly bioluminescence was the first
bioluminescence generating system to be characterized [see, e.g.,
Wienhausen et al. (1985) Photochemistry and Photobiology 42:609-61
1; McElroy et al. (1966) in Molecular Architecture in Cell
Physiology, Hayashi et al., eds. Prentice Hall, Inc., Englewood
Cliffs, N.J., pp. 63-80] and it is commercially available [e.g.,
from Promega Corporation, Madison, Wis., see, e.g., Leach et al.
(1986) Methods in Enzymology 133:51-70, esp. Table 1]. Luciferases
from different species of fireflies are antigenically similar.
These species include members of the genera Photinus, Photurins and
Luciola. Further, the bioluminescent reaction produces more light
at 30.degree. C. than at 20.degree. C., the luciferase is
stabilized by small quantities of bovine albumin serum, and the
reaction can be buffered by tricine.
[0268] a. Luciferase
[0269] DNA clones encoding luciferases from various insects and the
use to produce the encoded luciferase is well known. For example,
DNA clones that encode luciferase from Photinus pyralis, Luciola
cruciata [see, e.g., de Wet et al. (1985) Proc. Natl. Acad. Sci.
U.S.A. 82:7870-7873; de We et al. (1986) Methods in Enzymology
133:3; U.S. Pat. No. 4,968,613, see, also SEQ ID No. 3] are
available. The DNA has also been expressed in Saccharomyces [see,
e.g., Japanese Application No. JP 63317079, published Dec. 26,
1988, KIKKOMAN CORP] and in tobacco.
[0270] In addition to the wild-type luciferase modified insect
luciferases have been prepared. For example, heat stable luciferase
mutants, DNA-encoding the mutants, vectors and transformed cells
for producing the luciferases are available. A protein with 60%
amino acid sequence homology with luciferases from Photinus
pyralis, Luciola mingrelica, L. cruciata or L. lateralis and having
luciferase activity is available [see, e.g., International PCT
Application No. WO95/25798]. It is more stable above 30.degree. C.
than naturally-occurring insect luciferases and may also be
produced at 37.degree. C. or above, with higher yield.
[0271] Modified luciferases that generate light at different
wavelengths [compared with native luciferase], and thus, may be
selected for their color-producing characteristics. For example,
synthetic mutant beetle luciferase(s) and DNA encoding such
luciferases that produce bioluminescence at a wavelength different
from wild-type luciferase are known [Promega Corp, International
PCT Application No. WO95/18853, which is based on U.S. application
Ser. No. 08/177,081 Jan. 3, 1994]. The mutant beetle luciferase has
an amino acid sequence differing from that of the corresponding
wild-type Luciola cruciata [see, e.g., U.S. Pat. Nos. 5,182,202,
5,219,737, 5,352,598, see, also SEQ ID No.3] by a substitution(s)
at one or two positions. The mutant luciferase produces a
bioluminescence with a wavelength of peak intensity that differs by
at least 1 nm from that produced by wild-type luciferases.
[0272] Other mutant luciferase have also been produced. Mutant
luciferases with the amino acid sequence of wild-type luciferase,
but with at least one mutation in which valine is replaced by
isoleucine at the amino acid number 233, valine by isoleucine at
239, serine by asparagine at 286, glycine by serine at 326,
histidine by tyrosine at 433 or proline by serine at 452 are known
[see, e.g., U.S. Pat. Nos. 5,219,737, and 5,330,906]. The
luciferases are produced by expressing DNA-encoding each mutant
luciferase in E. coli and isolating the protein. These luciferases
produce light with colors that differ from wild-type. The mutant
luciferases catalyze luciferin to produce red [A 609 nm and 612
nm], orange[A 595 and 607 nm] or green [A 558 nm] light. The other
physical and chemical properties of mutant luciferase are
substantially identical to native wild type-luciferase. The mutant
luciferase has the amino acid sequence of Luciola cruciata
luciferase with an alteration selected from Ser 286 replaced by
Asn, Gly 326 replaced by Ser, His 433 replaced by Tyr or Pro 452
replaced by Ser. Thermostable luciferases are also available [see,
e.g., U.S. Pat. No. 5,229,285; see, also International PCT
Application No. WO 95/25798, which provides Photinus luciferase in
which the glutamate at position 354 is replaced lysine and Luciola
luciferase in which the glutamate at 356 is replaced with
lysine].
[0273] These mutant luciferases as well as the wild type
luciferases are among those preferred herein, particularly in
instances when a variety of colors are desired or when stability at
higher temperatures is desired. It is also noteworthy that firefly
luciferases have alkaline pH optima [7.5-9.5], and, thus, are
suitable for use in combination with articles of manufacture, such
as the bubble compositions that have alkaline pH.
[0274] b. Luciferin
[0275] The firefly luciferin is a benzothiazole: ##STR5##
[0276] Analogs of this luciferin and synthetic firefly luciferins
are also known to those of skill in art [see, e.g., U.S. Pat. Nos.
5,374,534 and 5,098,828]. These include compounds of formula (IV)
[see, U.S. Pat. No. 5,098,828]: ##STR6## [0277] in which: [0278]
R.sup.1 is hydroxy, amino, linear or branched C.sub.1-C.sub.20
alkoxy, C.sub.2-C.sub.20 alkyenyloxy, an L-amino acid radical bond
via the .alpha.-amino group, an oligopeptide radical with up to ten
L-amino acid units linked via the .alpha.-amino group of the
terminal unit; [0279] R.sup.2 is hydrogen, H.sub.2PO.sub.3,
HSO.sub.3, unsubstituted or phenyl substituted linear or branched
C.sub.1-C.sub.20 alkyl or C.sub.2-C.sub.20 alkenyl, aryl containing
6 to 18 carbon atoms, or R.sup.3--C(O)--; and [0280] R.sup.3 is an
unsubstituted or phenyl substituted linear or branched
C.sub.1-C.sub.20 alkyl or C.sub.2-C.sub.20 alkenyl, aryl containing
6 to 18 carbon atoms, a nucleotide radical with 1 to 3 phosphate
groups, or a glycosidically attached mono- or disaccharide, except
when formula (IV) is a D-luciferin or D-luciferin methyl ester.
[0281] c. Reaction
[0282] The reaction catalyzed by firefly luciferases and related
insect luciferases requires ATP, Mg.sup.2+ as well as molecular
oxygen. Luciferin must be added exogenously. Firefly luciferase
catalyzes the firefly luciferin activation and the subsequent steps
leading to the excited product. The luciferin reacts with ATP to
form a luciferyl adenylate intermediate. This intermediate then
reacts with oxygen to form a cyclic luciferyl peroxy species,
similar to that of the coelenterate intermediate cyclic peroxide,
which breaks down to yield CO.sub.2 and an excited state of the
carbonyl product. The excited molecule then emits a yellow light;
the color, however, is a function of pH. As the pH is lowered the
color of the bioluminescence changes from yellow-green to red.
[0283] Different species of fireflies emit different colors of
bioluminescence so that the color of the reaction will be dependent
upon the species from which the luciferase is obtained.
Additionally, the reaction is optimized at pH 7.8.
[0284] Addition of ATP and luciferin to a reaction that is
exhausted produces additional light emission. Thus, the system,
once established, is relatively easily maintained. Therefore, it is
highly suitable for use herein in embodiments in which a sustained
glow is desired or reuse of the item is contemplated. Thus, the
components of a firefly system can be packaged with the item of
manufacture, such as a toy gun, and then combined with the article
before use. For example, the luciferin and ATP can be added to a
mild bubble or a protein composition that contains luciferase each
time the bubbles are used.
[0285] 5. Bacterial Systems
[0286] Luminous bacteria typically emit a continuous light, usually
blue-green. When strongly expressed, a single bacterium may emit
10.sup.4 to 10.sup.5 photons per second. Bacterial bioluminescence
systems include, among others, those systems found in the
bioluminescent species of the genera Photobacterium, Vibrio and
Xenorhabdus. These systems are well known and well characterized
[see, e.g., Baldwin et al. (1984) Biochemistry 23:3663-3667; Nicoli
et al. (1974) J. Biol. Chem. 249:2393-2396; Welches et al. (1981)
Biochemistry 20:512-517; Engebrecht et al. (1986) Methods in
Enzymology 133:83-99; Frackman et al. (1990) J. of Bacteriology
172:5767-5773; Miyamoto et al. (1986) Methods in Enzymology 133:70;
U.S. Pat. No. 4,581,335].
[0287] a. Luciferases
[0288] Bacterial luciferase, as exemplified by luciferase derived
from Vibrio harveyi [EC 1.14.14.3, alkanol reduced-FMN-oxygen
oxidoreductase 1-hydroxylating, luminescing], is a mixed function
oxidase, formed by the association of two different protein
subunits .alpha. and .beta.. The .alpha.-subunit has an apparent
molecular weight of approximately 42,000 kD and the .beta.-subunit
has an apparent molecular weight of approximately 37,000 kD [see,
e.g., Cohn et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80:102-123].
These subunits associate to form a 2-chain complex luciferase
enzyme, which catalyzes the light emitting reaction of
bioluminescent bacteria, such as Vibrio harveyi [U.S. Pat. No.
4,581,335; Belas et al. (1982) Science 218:791-793], Vibrio
fischeri [Engebrecht et al. (1983) Cell 32:773-781; Engebrecht et
al. (1984) Proc. Natl. Acad. Sci. U.S.A. 81:4154-4158] and other
marine bacteria.
[0289] Bacterial luciferase genes have been cloned [see, e.g., U.S.
Pat. Nos. 5,221,623; 4,581,335; European Patent Application No. EP
386 691 A]. Plasmids for expression of bacterial luciferase, such
as Vibrio harveyi, include pFIT001 (NRRL B-18080), pPALE001 (NRRL
B-18082) and pMR19 (NRRL B-18081)] are known. For example the
sequence of the entire lux regulon from Vibiro fisheri has been
determined [Baldwin et al. (1984), Biochemistry 23:3663-3667;
Baldwin et al. (1981) Biochem. 20: 512-517; Baldwin et al. (1984)
Biochem. 233663-3667; see, also, e.g., U.S. Pat. Nos. 5,196,318,
5,221,623, and 4,581,335]. This regulon includes luxI gene, which
encodes a protein required for autoinducer synthesis [see, e.g.,
Engebrecht et al. (1984) Proc. Natl. Acad. Sci. U.S.A.
81:4154-4158], the luxC, luxD, and luxE genes, which encode enzymes
that provide the luciferase with an aldehyde substrate, and the
luxA and luxB genes, which encode the alpha and beta subunits of
the luciferase.
[0290] Lux genes from other bacteria have also been cloned and are
available [see, e.g., Cohn et al. (1985) J. Biol. Chem.
260:6139-6146; U.S. Pat. No. 5,196,524, which provides a fusion of
the luxA and luxB genes from Vibrio harveyi]. Thus, luciferase
alpha and beta subunit-encoding DNA is provided and can be used to
produce the luciferase. DNA encoding the .alpha. [1065 bp] and
.beta. [984 bp] subunits, DNA encoding a luciferase gene of 2124
bp, encoding the alpha and beta subunits, a recombinant vector
containing DNA encoding both subunits and a transformed E. coli and
other bacterial hosts for expression and production of the encoded
luciferase are available. In addition, bacterial luciferases are
commercially available.
[0291] b. Luciferins
[0292] Bacterial Luciferins Include: ##STR7## [0293] in which the
tetradecanal with reduced flavin mononucleotide are considered
luciferin since both are oxidized during the light emitting
reaction.
[0294] C. Reactions
[0295] The bacterial systems require, in addition to reduced
flavin, five polypeptides to complete the bioluminescent reaction:
two subunits, .alpha. and .beta., of bacterial luciferin and three
units of a fatty acid reductase system complex, which supplies the
tetradecanal aldehyde. Examples of bacterial bioluminescence
generating systems useful in the apparatus and methods provided
herein include those derived from Vibrio fisheri and Vibrio
harveyi. One advantage to this system is its ability to operate at
cold temperatures. It will thus be particularly amenable to use in
ice cubes. All components of a bacterial system can be frozen into
ice cubes. As it the ice cubes melt into a warmer beverage, which
has dissolved O.sub.2 the reaction will proceed, thereby providing
a sustained glow.
[0296] Bacterial luciferase catalyzes the flavin-mediated
hydroxylation of a long-chain aldehyde to yield carboxylic acid and
an excited flavin; the flavin decays to ground state with the
concomitant emission of blue green light [.lamda..sub.max 490 nm;
see, e.g., Legocki et al. (1986) Proc. Natl. Acad. Sci. USA
81:9080; see U.S. Pat. No. 5,196,524]: ##STR8##
[0297] The reaction can be initiated by contacting reduced flavin
mononucleotide [FMNH.sub.2] with a mixture of the bacterial
luciferase, oxygen, and a long-chain aldehyde, usually n-decyl
aldehyde.
[0298] DNA encoding luciferase from the fluorescent bacterium
Alteromonas hanedai is known [CHISSO CORP; see, also, Japanese
application JP 7222590, published Aug. 22, 1995]. The reduced
flavin mononucleotide [FMNH.sub.2; luciferin] reacts with oxygen in
the presence of bacterial luciferase to produce an intermediate
peroxy flavin. This intermediate reacts with a long-chain aldehyde
[tetradecanal] to form the acid and the luciferase-bound hydroxy
flavin in its excited state. The excited luciferase-bound hydroxy
flavin then emits light and dissociates from the luciferase as the
oxidized flavin mononucleotide [FMN] and water. In vivo FMN is
reduced again and recycled, and the aldehyde is regenerated from
the acid.
[0299] Flavin reductases have been cloned [see, e.g., U.S. Pat. No.
5,484,723; see, SEQ ID No. 14 for a representative sequence from
this patent]. These as well as NAD(P)H can be included in the
reaction to regenerate FMNH.sub.2 for reaction with the bacterial
luciferase and long chain aldehyde. The flavin reductase catalyzes
the reaction of FMN, which is the luciferase reaction, into
FMNH.sub.2; thus, if luciferase and the reductase are included in
the reaction system, it is possible to maintain the bioluminescent
reaction. Namely, since the bacterial luciferase turns over many
times, bioluminescence continues as long as a long chain aldehyde
is present in the reaction system.
[0300] The color of light produced by bioluminescent bacteria also
results from the participation of a protein blue-florescent protein
[BFP] in the bioluminescence reaction. This protein, which is well
known [see, e.g., Lee et al. (1978) Methods in Enzymology
LVII:226-234], and may also be added to bacterial bioluminescence
reactions in order to cause a shift in the color.
[0301] 6. Other Systems
[0302] a. Dinoflagellate Bioluminescence Generating Systems
[0303] In dinoflagellates, bioluminescence occurs in organelles
termed scintillons. These organelles are outpocketings of the
cytoplasm into the cell vacuole. The scintillons contain only
dinoflagellate luciferase and luciferin [with its binding protein],
other cytoplasmic components being somehow excluded. The
dinoflagellate luciferin is a tetrapyrrole related to chlorophyll:
##STR9## [0304] or an analog thereof.
[0305] The luciferase is a 135 kD single chain protein that is
active at pH 6.5, but inactive at pH 8 [see, e.g., Hastings (1981)
Bioluminescence and Chemiluminescence, DeLuca et al., eds. Academic
Press, N.Y., pp. 343-360]. Luminescent activity can be obtained in
extracts made at pH 8 by shifting the pH from 8 to 6. This occurs
in soluble and particulate fractions. Within the intact scintillon,
the luminescent flash occurs for .about.100 msec, which is the
duration of the flash in vivo. In solution, the kinetics are
dependent on dilution, as in any enzymatic reaction. At pH 8, the
luciferin is bound to a protein [luciferin binding protein] that
prevents reaction of the luciferin with the luciferase. At pH 6,
however, the luciferin is released and free to react with the
enzyme.
[0306] b. Systems from Molluscs, such as Latia and Pholas
[0307] Molluscs Latia neritoides and species of Phol as are
bioluminescent animals. The luciferin has the structure: ##STR10##
[0308] and has been synthesized [see, e.g., Shimomura et al. (1968)
Biochemistry 7:1734-1738; Shimomura et al. (1972) Proc. Natl. Acad.
Sci. U.S.A. 69:2086-2089]. In addition to a luciferase and
luciferin the reaction has a third component, a "purple protein".
The reaction, which can be initiated by an exogenous reducing agent
is represented by the following scheme: ##STR11##
[0309] XH.sub.2 is a reducing agent.
[0310] Thus for practice herein, the reaction will require the
purple protein as well as a reducing agent.
[0311] c. Earthworms and Other Annelids
[0312] Earthworm species, such as Diplocardia longa, Chaetopterus
and Harmothoe, exhibit bioluminescence. The luciferin has the
structure: ##STR12##
[0313] The reaction requires hydrogen peroxide in addition to
luciferin and luciferase. The luciferase is a photoprotein.
[0314] d. Glow Worms
[0315] The luciferase/luciferin system from the glow worms that are
found in New Zealand caves, Australia and those found in Great
Britain are also intended for use herein.
[0316] e. Marine Polycheate Worm Systems
[0317] Marine polycheate worm bioluminescence generating systems,
such as Phyxotrix and Chaetopterus, are also contemplated for use
herein.
[0318] f. South American Railway Beetle
[0319] The bioluminescence generating system from the South
American railway beetle is also intended for use herein.
[0320] g. Fish
[0321] Of interest herein, are luciferases and bioluminescence
generating systems that generate red light. These include
luciferases found in species of Aristostomias, such as A.
scintillans [see, e.g., O'Day et al. (1974) Vision Res.
14:545-550], Pachystomias, Malacosteus, such as M. niger.
[0322] 7. Fluorescent Proteins
[0323] Fluorescent proteins (FPs), particularly green fluorescent
proteins (GFPs), such as those from Aquorea and Renilla, and other
related proteins can be used in combination with any of the novelty
items provided herein, including toys, beverages, foods, cosmetics,
paper products and others. The FPs may be used alone with these
items or may be added to bioluminescence generating systems or
items with such systems as a means of altering the color of the
items. Mutein GFPs from Aquorea are also known (see, e.g., U.S.
Pat. No. 5,625,048).
[0324] a. Green and Blue Fluorescent Proteins
[0325] Blue light is produced using the Renilla luciferase or the
Aequorea photoprotein in the presence of Ca.sup.2+ and the
coelenterazine luciferin or analog thereof. This light can be
converted into a green light if a green fluorescent protein (GFP)
is added to the reaction. Green fluorescent proteins, which have
been purified [see, e.g., Prasher et al. (1992) Gene 111:229-233]
and also cloned [see, e.g., International PCT Application No. WO
95/07463, which is based on U.S. application Ser. No. 08/119,678
and U.S. application Ser. No. 08/192,274, which are herein
incorporated by reference], are energy-transfer acceptors. GFPs
fluoresce in vivo upon receiving energy from a
luciferase-oxyluciferin excited-state complex or a
Ca.sup.2+-activated photoprotein. The chromophore is formed from
modified amino acid residues within the polypeptide.
[0326] The best characterized GFPs are those of Aequorea and
Renilla [see, e.g., Prasher et al. (1992) Gene 111:229-233; Hart,
et al. (1979) Biochemistry 18:2204-2210]. For example, a green
fluorescent protein [GFP] from Aequorea Victoria contains 238 amino
acids, absorbs blue light and emits green light.
[0327] Thus, for example, inclusion of this protein in a
composition containing the aequorin photoprotein charged with
coelenterazine and oxygen, can, in the presence of calcium, result
in the production of green light. It is contemplated that GFPs may
be included in the bioluminescence generating reactions that employ
the aequorin or Renilla luciferases or other suitable luciferase in
order to enhance or alter color of the resulting
bioluminescence.
[0328] GFPs are activated by blue light to emit green light and
thus may be used in the absence of luciferase and in conjunction
with an external light source with novelty items, as described
herein. Similarly, blue fluorescent proteins (BFPs), such as from
Vibrio fischeri, Vibrio harveyi or Photobacterium phosphoreum, may
be used in conjunction with an external light source of appropriate
wavelength to generate blue light. (See for example, Karatani, et
al., "A blue fluorescent protein from a yellow-emitting luminous
bacterium," Photochem. Photobiol. 55(2):293-299 (1992); Lee, et
al., "Purification of a blue-fluorescent protein from the
bioluminescent bacterium Photobacterium phosphoreum" Methods
Enzymol. (Biolumin. Chemilumin.) 57:226-234 (1978); and Gast, et
al. "Separation of a blue fluorescence protein from bacterial
luciferase" Biochem. Biophys. Res. Commun. 80(1):14-21 (1978),
each, as all references cited herein, incorporated in its entirety
by reference herein.) In particular, GFPs, and/or BFPs or other
such fluorescent proteins may be used in the beverage and/or food
combinations provided herein and served in rooms illuminated with
light of an appropriate wavelength to cause the fluorescent
proteins to fluoresce.
[0329] GFPs and/or BFPs or other such fluorescent proteins may be
used in any of the novelty items and combinations provided herein,
such as the beverages and toys, including bubble making toys,
particularly bubble-making compositions or mixtures and cosmetics.
Such systems are particularly of interest because no luciferase is
needed to activate the photoprotein and because the proteins are
readily digested. These fluorescent proteins may also be used in
addition to bioluminescence generating systems to enhance or create
an array of different colors.
[0330] These proteins may be used alone or in combination with
bioluminescence generating systems to produce an array of colors.
They may be used in combinations such that the color of, for
example, a beverage changes over time, or includes layers of
different colors.
[0331] b. Phycobiliproteins
[0332] Phycobiliproteins are water soluble fluorescent proteins
derived from cyanobacteria and eukaryotic algae [see, e.g., Apt et
al. (1995) J. Mol. Biol. 238:79-96; Glazer (1982) Ann. Rev.
Microbiol. 36:173-198; and Fairchild et al. (1994) J. of Biol.
Chem. 269:8686-8694]. These proteins have been used as fluorescent
labels in immunoassay [see, Kronick (1986) J. of Immunolog. Meth.
92:1-13], the proteins have been isolated and DNA encoding them is
also available [see, e.g., Pilot et al. (1984) Proc. Natl. Acad.
Sci. U.S.A. 81:6983-6987; Lui et al. (1993) Plant Physiol
103:293-294; and Houmard et al. (1988) J. Bacteriol. 170:5512-5521;
the proteins are commercially available from, for example, ProZyme,
Inc., San Leandro, Calif.].
[0333] In these organisms, the phycobiliproteins are arranged in
subcellular structures termed phycobilisomes and function as
accessory pigments that participate in photosynthetic reactions by
absorbing visible light and transferring the derived energy to
chlorophyll via a direct fluorescence energy transfer
mechanism.
[0334] Two classes of phycobiliproteins are known based on their
color: phycoerythrins (red) and phycocyanins (blue), which have
reported absorption maxima between 490 and 570 nm and between 610
and 665 nm, respectively. Phycoerythrins and phycocyanins are
heterogeneous complexes composed of different ratios of alpha and
beta monomers to which one or more class of linear tetrapyrrole
chromophores are covalently bound. Particular phycobiliproteins may
also contain a third y-subunit which often associate with
(.alpha..beta.).sub.6 aggregate proteins.
[0335] All phycobiliproteins contain either phycothrombilin or
phycoerythobilin chromophores, and may also contain other bilins,
such as phycourobilin, cryptoviolin or a 697 nm bilin. The
.gamma.-subunit is covalently bound with phycourobilin, which
results in the 495-500 nm absorbance peak of B- and
R-phycoerythrins. Thus, the spectral characteristics of
phycobiliproteins may be influenced by the combination of the
different chromophores, the subunit composition of the
apo-phycobiliproteins and/or the local environment that affects the
tertiary and quaternary structure of the phycobiliproteins.
[0336] As described above for GFPs & BFPs, phycobiliproteins
are also activated by visible light of the appropriate wavelength
and thus may be used in the absence of luciferase and in
conjunction with an external light source to illuminate novelty
items, particularly, as described herein. In particular,
phycobiliproteins may be used in the novelty items, such as
beverage and/or food combinations provided herein and served in
rooms illuminated with light of an appropriate wavelength to cause
the fluorescent proteins to fluoresce. Cosmetics containing these
proteins are also contemplated.
[0337] As noted above, these proteins may be used in combination
with other fluorescent proteins and/or bioluminescence generating
systems to produce an array of colors or to provide different
colors over time.
[0338] Attachment of phycobiliproteins to solid support matrices is
known (e.g., see U.S. Pat. Nos. 4,714,682; 4,767,206; 4,774,189 and
4,867,908). Therefore, phycobiliproteins may be coupled to
microcarriers coupled to one or more components of the
bioluminescent reaction, preferably a luciferase, to convert the
wavelength of the light generated from the bioluminescent reaction.
Microcarriers coupled to one or more phycobiliproteins may be used
in any of the novelty items and combinations provided herein, such
as the multicolor beverages and toys, including bubble making toys,
particularly bubble-making compositions or mixtures.
[0339] C. Practice of the Reactions in Combination with Articles of
Manufacture
[0340] The particular manner in which each bioluminescence system
will be combined with a selected article of manufacture will be a
function of the article and the desired effect. In general,
however, less than all of the components of the reaction will be
provided with the article and then contact with the remaining
component(s) to produce a glow. There are a multitude of
alternative means for achieving this result; some are described
herein, and others will be apparent by virtue of the disclosure
herein.
[0341] In the simplest embodiments, the organisms can be ground up
and dried. For example, light will be emitted by ground up
fireflies when mixed with water and ATP. Light will also be emitted
merely be combining ground up Vargula shrimp and adding water,
preferably cool water [room temperature or lower]. The only caveat
is that the water must not be too hot; high temperatures destroy
activity of the luciferases.
[0342] In other embodiments, the substantially pure reagents are
combined with the article of manufacture and the article will glow
or spew a glowing spray or jet. The reagents may be provided in
compositions, such as suspensions, as powders, as pastes or any in
other suitable form. They may be provided as sprays, aerosols, or
in any suitable form. The reagents may be linked to a matrix and
combined with the article of manufacture or formed into the article
of manufacture. Typically all but one or more, though preferably
all but one, of the components necessary for the reaction will be
mixed and provided together; reaction will be triggered contacting
the mixed component(s) with the remaining component(s), such as by
adding Ca.sup.2+, FMN with reductase, FMNH.sub.2, ATP, air or
oxygen. The resulting matrix materials are advantageously used in
connection numerous novelty items, such as clothing. They are also
used in the cartridges provided herein.
[0343] In preferred embodiments the luciferase or
luciferase/luciferin, such as the aequorin photoprotein, will be
provided in combination with the article of manufacture or added
before use. The article will then be contacted with the remaining
components. As will become apparent herein, there are a multitude
of ways in which each system may be combined with a selected
article of manufacture.
[0344] D. Packaging of Bioluminescence Systems
[0345] Packaging for bioluminescence generating reagents provided
herein must be chosen according to the article of manufacture with
which the reagents are to be combined. In general, the packaging is
non-reactive with the compositions contained therein and must
exclude water and or air to the degree those substances are
required for the luminescent reaction to proceed. It will be
appreciated, however, that specific uses for the bioluminescence
generating systems may require specific packaging. Following are
some examples of the special packaging requirements of various end
uses of the bioluminescence generating systems. These are offered
as examples only and are in no way intended as limiting.
[0346] The bioluminescence generating reagents may be provided in
pellets, encapsulated as micro or macro-capsules, linked to
matrices and included in or on articles of manufacture, or as
mixtures in chambers within an article of manufacture or in some
other configuration. With respect to other articles of manufacture
that include chambers or vessels, such as certain toys, primary
considerations are that the bioluminescence generating system be
amenable to activation by the user at will and that the container
be non-reactive and, if desired, translucent to the bioluminescent
glow. Examples of vessels include beverage holders, plates or other
dishes, vases, jars, bottles, spray cans and other containers. In
general, vessels for use in practicing the methods herein have an
enclosed, defined space, that contains most of the components of
the bioluminescence generating system, and a separate enclosed,
defined space containing the remaining necessary ingredients; such
that, the two spaces are separated by a readily removable membrane
which, upon removal, permits the components to mix and thereby
react, resulting in illumination. Alternatively, the vessel can
have a single compartment containing all but the final ingredients
of the bioluminescence generating system and being amenable to
addition of the final ingredients by the user; for example through
an opening in the compartment.
[0347] Any toy, vessel or other article of manufacture that is
amenable to having a generally translucent covering defining a
space for containment of the bioluminescence generating system
components and that is amenable to simple manipulation to permit
addition of the final components necessary for the illumination
reaction is contemplated.
[0348] Thus, whether the item that will glow or produce a glowing
fluid, jet or spray, is a toy, vessel or other article of
manufacture, its general design is the same. At least one of the
bioluminescence generating system components is separated from the
remaining components. The remaining components are added prior to
use. They can be included in the article of manufacture and
physically separated from the other components. For example, the
physical separation means are those that are readily removed by the
user, to permit mixing, resulting in illumination of the
components. For example, an article of manufacture may contain a
luciferase and substrate in one compartment and a bioluminescence
activator in an adjacent compartment; or alternatively, one
compartment may contain the luciferase, and the other the substrate
luciferin and dissolved oxygen or other requisite activator(s). The
compartments are separated by a dividing member, such as a
membrane, that, upon compression of the article of manufacture,
ruptures permitting separated components to mix and to thereby
glow. For suitable embodiments, see EXAMPLES, below [see, also,
e.g., containers described in U.S. Pat. Nos. 3,539,794 and
5,171,081].
[0349] Other embodiments contemplated herein, include those in
which a fluid is ejected as a spray or jet and is rendered
bioluminescent prior to ejection from the device, such as a toy or
fountain. In general, the methods will involve addition of the
bioluminescence generating system components to the water just
prior to ejection thereby causing the ejected spray or jet or
stream to glow. Various apparatus for accomplishing this are
provided herein. In light of the disclosure herein other apparatus
can be adapted for such use. Examples include chambers within a toy
that inject the components into a water chamber just prior to
ejection of the water, or a clip-on device housing the components,
perhaps in pre-measured amounts, which is attached to the toy and
manually or automatically engaged to inject the ingredients into a
water chamber. Similarly, the water can be introduced into a
chamber containing the components and then ejected.
[0350] In other embodiments, the components may be packaged as
separate compositions, that, upon mixing, glow. For example, a
composition containing luciferase may be provided separately from,
and for use with, an a separate composition containing a
bioluminescence substrate and bioluminescence activator. In another
instance, luciferase and luciferin compositions may be separately
provided and the bioluminescence activator may be added after, or
simultaneously with, mixing of the other two compositions.
[0351] Similarly, the luciferase and bioluminescence substrate may
be provided in a single packaging apparatus, an composition that is
a mixture, suspension, solution, powder, paste or other suitable
composition, that is designed to exclude the necessary
bioluminescence activator. Upon addition of the bioluminescence
activator to the remaining components or upon addition of the
components to the bioluminescence activator, the reaction commences
and the mixture glows. One example of such a system is "fairy
dust". In this embodiment the luciferase and bioluminescence
substrate, for example, are packaged to exclude water and/or air,
the bioluminescence activator. Release of the components from the
packaging into the air and/or moisture in the air activates the
components thereby generating luminescence. Another example is
packaging the luciferase and substrate in the cap apparatus of a
vessel, such that operation of the cap apparatus releases the
components into the composition contained in the vessel, causing it
to glow.
[0352] 1. Dispensing and Packaging Apparatus for Combination with
the Bioluminescence generating system Components
[0353] In one aspect, the bioluminescent apparatus systems provided
herein are bioluminescence [or bioluminescent] systems in
combination with dispensing or packaging apparatus. The
bioluminescence systems, described in detail elsewhere herein,
include three components: a bioluminescence substrate [e.g., a
luciferin], a luciferase [e.g., a luciferase or photoprotein], and
a bioluminescence activator or activators [e.g., molecular oxygen
or Ca.sup.2+]. The dispensing and packaging apparatus are
configured to keep at least one of the three components separate
from the other two components, until generation of bioluminescence
is desired.
[0354] In general, the dispensing and packaging apparatus are
non-reactive with the bioluminescence generating system components
contained therein and can exclude moisture, air or other
activators, such as O.sub.2 or Ca.sup.2+, or in some manner keep
all necessary components that are required for the bioluminescent
reaction to come into contact until desired.
[0355] It will be appreciated, however, that specific applications
and configurations of the bioluminescence systems may require
specific apparatus. Following are exemplary descriptions of various
dispensers and packages contemplated for use herein. These are
offered as examples only and are in no way intended as limiting. It
is understood that in light of the description herein, other
apparatus may be modified or devised, that would be suitable for
use to produce bioluminescence in combination with novelty
items.
[0356] 2. Capsules, Pellets, Liposomes, Endosomes, Vacuoles,
Micronized Particles
[0357] Certain embodiments of the novelty item combinations
provided herein require sequestering of the components from the
environment prior to use or require the components to be provided
in particulate form. Examples of such embodiments include
beverages, foods and particles, such as for use as fairy dust or in
toy guns, fountains of particles and other such applications. In
particular, embodiments in which the bioluminescence generating
system is manufactured as part of food or beverage producing
glowing beverages or foods require specific packaging
considerations. To be amenable to use as an additive to beverages
for human consumption, the packaging must be non-toxic, and should
be easy to open to provide for contact of the bioluminescence
generating system components with the beverage. Examples of
suitable packaging for such use include encapsulating the
bioluminescence generating system components in one or micro-[up to
about 100.mu.m in size] or macroparticles [larger than 100 .mu.M]
of material that permits release of the contents, such as by
diffusion or by dissolution of the encapsulating material.
Liposomes and other encapsulating vehicles [see, e.g., U.S. Pat.
No. 4,525,306, which describes encapsulation of compounds in
gelatin; U.S. Pat. Nos. 4,021,364, 4,225,581, 4,269,821, 4,322,311,
4,324,683, 4,329,332, 4,525,306, 4,963,368 describe encapsulation
of biologically active materials in various polymers] known to
those of skill in the art, including those discussed herein and
known to those of skill in the art [such as soluble paper, see U.S.
Pat. No. 3,859,125]. Likewise, packaging of the system components
for addition to food products must address the same considerations.
The components may be added to the food substance directly, e.g.,
by sprinkling the dried and powdered ingredients onto the food, or
indirectly, e.g., via addition, to the food, of a capsule
containing the ingredients.
[0358] a. Encapsulating Vehicles in General
[0359] All components of the bioluminescence generating system,
except for the oxygen or water or Ca.sup.2+, depending upon the
selected system can be incorporated into encapsulating material,
such as liposomes, that protect the contents from the environment
until placed into conditions that cause release of the contents
into the environment. Encapsulating material contemplated for use
herein includes liposomes and other such materials used for
encapsulating chemicals, such as drug delivery vehicles.
[0360] b. Encapsulating Vehicles-Liposomes
[0361] For example, liposomes that dissolve and slowly release the
components into the selected beverage, which contains dissolved
oxygen or Ca.sup.2+ or even ATP for the luciferase system are
contemplated herein. They can be formulated in compositions, such
as solutions, suspensions, gels, lotions, creams, and ointments.
Liposomes and other slow release encapsulating compositions are
well known and can be adapted for use in for slow release delivery
of bioluminescence generating components. Typically the luciferin
and luciferase will be encapsulated in the absence of oxygen or
Ca.sup.2+ or ATP or other activating component. Upon release into
the environment or medium containing this component at a suitable
concentration, the reaction will proceed and a glow will be
produced. Generally the concentrations of encapsulated components
should be relatively high, perhaps 0.1-1 mg/ml or more, to ensure
high enough local concentrations upon release to be visible.
[0362] Liposomes or other sustained release delivery system that
are formulated in an ointment or sustained release topical vehicle,
for example, would be suitable for use in a body paint, lotion.
Those formulated as a suspension would be useful as a spray.
Numerous ointments and suitable liposome formulations are known
[see, e.g., Liposome Technology, Targeted Drug Delivery and
Biological Interaction, vol. III, G. Gregoriadis ed., CRC Press,
Inc., 1984; U.S. Pat. Nos. 5,470,881; 5,366,881; 5,296,231;
5,272,079; 5,225,212; 5,190,762; 5,188,837; 5,188,837; 4,921,757;
4,522,811]. For example, an appropriate ointment vehicle would
contain petrolatum, mineral oil and/or anhydrous liquid lanolin.
Sustained release vehicles such as liposomes, membrane or contact
lens delivery systems, or gel-forming plastic polymers would also
be suitable delivery vehicles. Liposomes for topical delivery are
well known [see, e.g., U.S. Pat. No. 5,296,231; Mezei et al. (1980)
"Liposomes-A selective drug delivery system for the topical route
of administration, I. lotion dosage form" Life Sciences
26:1473-1477; Mezei et al. (1981) "Liposomes-A selective drug
delivery system for the topical route of administration: gel dosage
form" Journal of Pharmacy and Pharmacology 34:473-474; Gesztes et
al. (1988) "Topical anaesthesia of the skin by
liposome-encapsulated tetracaine" Anesthesia and Analgesia
67:1079-1081; Patel (1985) "Liposomes as a controlled-release
system", Biochemical Soc. Trans. 13:513-516; Wohlrab et al. (1987)
"Penetration kinetics of liposomal hydrocortisone in human skin"
Dermatologica 174:18-22].
[0363] Liposomes are microcapsules [diameters typically on the
order of less than 0.1 to 20 .mu.m] that contain selected mixtures
and can slowly release their contents in a sustained release
fashion. Liposomes or other capsule, particularly a time release
coating, that dissolve upon exposure to oxygen, air, moisture,
visible or ultraviolet [UV] light or a particular pH or temperature
[see, e.g., U.S. Pat. No. 4,882,165; Kusumi et al. (1989) Chem.
Lett. no. 3 433-436; Koch Troels et al. (1990) Bioconjugate Chem.
4:296-304; U.S. Pat. Nos. 5,482,719; 5,411,730; 4,891,043;
Straubinger et al. (1983) Cell 32:1069-1079; and Straubinger et al.
(1985) FEBS Lttrs. 179:148-154; and Duzgunes et al. in Chapter 11
of the book CELL FUSION, edited by A. E. Sowers; Ellens et al.
(1984) Biochemistry 23:1532-1538; Yatvin et al. (1987) Methods in
Enzymology 149:77-87] may be used for example in the squirt guns or
toy machine guns or fairy dust or toy cigarettes. Liposome
formulations for use in baking [see, e.g., U.S. Pat. No. 4,999,208]
are available. They release their contents when eaten or heated.
Such liposomes may be suitable for incorporation into food products
herein or in embodiments in which release of the components by
heating is desired.
[0364] Liposomes be prepared by methods known to those of skill in
the art [see, e.g., Kim et al. (1983) Bioch. Bioph. Acta
728:339-348; Assil et al. (1987) Arch Ophthalmol. 105:400; and U.S.
Pat. No. 4,522,811, and other citations herein and known to those
of skill in the art].
[0365] Liposomes that are sensitive to low pH [see, e.g., U.S. Pat.
Nos. 5,352,448, 5,296,231; 5,283,122; 5,277,913, 4,789,633] are
particularly suitable for addition to bath powders or to bubble
compositions, just prior to use. Upon contact with the low pH
detergent or soap composition or a high pH composition, the
contents of the liposome will be released. Other components,
particularly Ca.sup.+ or the presence of dissolved O.sub.2 in the
water will cause the components to glow as they are released.
Temperature sensitive liposomes are also suitable for use in bath
powders for release into the warm bath water. [0366] c.
Encapsulating Vehicles-Gelatin and Polymeric Vehicles
[0367] Macro or microcapsules made of gelatin or other such polymer
that dissolve or release their contents in a beverage or food or on
contact with air or light or changes in temperature may also be
used to encapsulate components of the bioluminescence generating
systems.
[0368] Such microcapsules or macrocapsules may also be incorporated
into solid soaps, such that as the soap dissolves the incorporated
capsules or pellets release their contents, which glow upon contact
with the water in which the soap is placed.
[0369] The aequorin system is particularly suitable for this
application. It can be encapsulated in suspension or solution or as
a paste, or other suitable form, of buffer with sufficient
chelating agent, such as EDTA, to prevent discharge of the
bioluminescence. Upon exposure of the capsule [microcapsule or
macrocapsule]to moisture that contains Ca.sup.2+, such as in a food
or beverage, a two chamber apparatus or single chamber apparatus,
such as described herein, or even in a moist environment containing
Ca.sup.2+, the slowly released components will glow.
[0370] Thus, encapsulated bioluminescence generating components can
be used in combination with foods, beverages, ice and ice cubes
(and other geometries of ice), as bullets or pellets, such as
"fairy dust" [pellets that dissolve upon exposure to light and
thereby release the luciferase/luciferin, such as the Renilla
system, which will light upon exposure to air], and other such
items.
[0371] Other encapsulating containers or vehicles for use with the
bioluminescence systems are those that dissolve sufficiently in
water to release their contents, or that are readily opened when
squeezed in the hand or from which the contents diffuse when mixed
with a aqueous mixture. These containers can be made to exclude
water, so that the bioluminescence generating system components may
be desiccated and placed therein. Upon exposure to water, such as
in an aqueous composition or in the atmosphere, the vehicle
dissolves or otherwise releases the contents, and the components
react and glow. Similarly, some portion including less than all of
the bioluminescence generating reagents may be provided in pellet
form or as a concentrated paste. For example, the component(s) may
be mixed with gelatin or similar hardening agent, poured into a
mold, if necessary and dried to produce a water soluble pellet.
[0372] The capsules, encapsulating containers or vehicles may be
formed from gelatin or similar water soluble material. If the
packaging is to be added to food or beverage, then it should be
chosen to be non-toxic, non-reactive and flavorless. To be readily
opened by hand, the packaging may be constructed of thin plastic or
may be configured in two halves which form an airtight seal when
joined but which are readily separated when release of the
components is desired.
[0373] In one aspect, these capsular embodiments of the packaging
apparatus is contemplated for use as an additive to beverages,
creams, sauces, gelatins or other liquids or semi-solids. In
another aspect, it is contemplated that the contents of the
packaging apparatus is released into the air whereby it glows upon
contact with the moisture of the atmosphere and/or with molecular
oxygen.
[0374] d. Endosomes and Vacuoles
[0375] Vehicles may be produced using endosomes or vacuoles from
recombinant host cells in which the luciferase is expressed using
method known to those of skill in the art [see, e.g., U.S. Pat.
Nos. 5,284,646, 5,342,607, 5,352,432, 5,484,589, 5,192,679,
5,206,161, and 5,360,726]. For example, aequorin that is produced
by expression in a host, such as E. coli, can be isolated within
vesicles, such as endosomes or vacuoles, after protein synthesis.
Using routine methods the cells are lysed and the vesicles are
released with their contents intact. The vesicles will serve as
delivery vehicles. When used they will be charged with a luciferin,
such as a coelenterazine, and dissolved oxygen, such as by
diffusion, under pressure, or other appropriate means.
[0376] e. Micronized Particles
[0377] The bioluminescence generating system components that are
suitable for lyophilization, such as the aequorin photoprotein, the
Renilla system, and the Vargula systems, can be micronized to form
fine powder and stored under desiccating conditions, such as with a
desiccant. When used the fine powder can be combined with the
selected article of manufacture, such as a personal item, a chamber
in a gun or fountain, or used as fairy dust. Contact with dissolved
oxygen or Ca.sup.2+ in the air or in a mist that can be supplied or
in added will cause the particles to release their contents and
glow.
[0378] 3. Apparatus and Substrates
[0379] The combinations herein are produced by combining a selected
novelty item and combining it with a system and apparatus for
producing bioluminescence. Selection of the system depends upon
factors such as the desired color and duration of the
bioluminescence desired as well as the particular item. Selection
of the apparatus primarily depends upon the item with which it is
combined.
[0380] Among the simplest embodiments herein, are those in which
the apparatus contains a single chamber [vessel] or matrix material
and, if needed, ejection means. Components, generally all but at
least one necessary component, typically the activator as defined
herein, of the bioluminescence reaction are introduced into the
housing or vessel or onto the substrate as a mixture in liquid
phase or as a powder or other paste or other convenient
composition. Prior to use the final component(s) is added or the
other components are contacted with the final component(s).
[0381] a. Matrix Materials
[0382] For preparation of combinations of articles of manufacture
such as clothing, paper, items fabricated from a textile, plastic,
glass, ceramic or other such material, such as a figurine, and for
use in the cartridges, at least one component of the
bioluminescence generating system is linked to the matrix
substrate. When desired, a mixture or mixtures(s) containing the
remaining component(s), typically a liquid mixture is applied, as
by pouring or spraying onto the matrix substrate, to produce a
glow. For example, the aequorin photoprotein, including
coelenterazine and oxygen, is linked to the substrate. When desired
a liquid containing Ca.sup.2+, such as tap water or, preferably, a
liquid mixture containing the Ca.sup.2+ in an appropriate buffer,
is contacted, such as by spraying, with the matrix with linked
luciferase. Upon contacting the material glows.
[0383] In other embodiments, the luciferase, such as a Vargula
luciferase, is linked to the substrate material, and contacted with
a liquid mixture containing the luciferin in an appropriate buffer.
Contacting can be effected by spraying or pouring or other suitable
manner. The matrix material is incorporated into, onto or is formed
into an article of manufacture, such as clothing or a ceramic,
glass, plastic figurine, toy, balloon, flocking agent, such as a
Christmas tree flocking agent, or other item. The resulting novelty
item can be sold as a kit with a container of the mixture
containing the non-linked components, such as in a canister, spray
bottle or can, or other suitable format.
[0384] The kits may also include containers containing compositions
of the linked components which can be provided in a form, such as
sprayed on as a liquid and air dried, that can be applied to the
substrate so that the item can be made to glow again. Thus, kits
containing a substrate, such as clothing or a plastic, ceramic or
glass item, and a first composition containing a luciferase or a
luciferin or both and luciferin, and a second composition
containing the remaining components. The item as provided in the
kit can be charged with the first composition, such as having the
composition applied and dried, or may require charging prior to the
first use. Alternatively, the item may be sprayed with both
compositions when desired to produce a glow.
[0385] It is understood that the precise components and optimal
means for application or storage are a function of the selected
bioluminescence system. The concentrations of the components, which
can be determined empirically, are not critical, but must be
sufficient to produce a visible glow when combined. Typical
concentrations are as low as nanomoles/l, preferably on the order
of mg/l or higher. The concentration on the substrate is that
produced when a composition containing such typical concentration
is applied to the material. Again, such ideal concentrations can be
readily determined empirically by applying the first composition,
letting it dry, spraying the second composition, and observing the
result.
[0386] The matrix material substrates contemplated herein are
generally insoluble materials used to immobilize ligands and other
molecules, and are those that used in many chemical syntheses and
separations. Such substrates, also called matrices, are used, for
example, in affinity chromatography, in the immobilization of
biologically active materials, and during chemical syntheses of
biomolecules, including proteins, amino acids and other organic
molecules and polymers. The preparation of and use of matrices is
well known to those of skill in this art; there are many such
materials and preparations thereof known. For example,
naturally-occurring matrix materials, such as agarose and
cellulose, may be isolated from their respective sources, and
processed according to known protocols, and synthetic materials may
be prepared in accord with known protocols.
[0387] The substrate matrices are typically insoluble materials
that are solid, porous, deformable, or hard, and have any required
structure and geometry, including, but not limited to: beads,
pellets, disks, capillaries, hollow fibers, needles, solid fibers,
random shapes, thin films and membranes. Thus, the item may be
fabricated from the matrix material or combined with it, such by
coating all or part of the surface or impregnating particles.
[0388] Typically, when the matrix is particulate, the particles are
at least about 10-2000 .mu.M, but may be smaller or larger,
depending upon the selected application. Selection of the matrices
will be governed, at least in part, by their physical and chemical
properties, such as solubility, functional groups, mechanical
stability, surface area swelling propensity, hydrophobic or
hydrophilic properties and intended use.
[0389] If necessary the support matrix material can be treated to
contain an appropriate reactive moiety or in some cases the may be
obtained commercially already containing the reactive moiety, and
may thereby serve as the matrix support upon which molecules are
linked. Materials containing reactive surface moieties such as
amino silane linkages, hydroxyl linkages or carboxysilane linkages
may be produced by well established surface chemistry techniques
involving silanization reactions, or the like. Examples of these
materials are those having surface silicon oxide moieties,
covalently linked to gamma-aminopropylsilane, and other organic
moieties; N-[3-(triethyoxysilyl)propyl]phthelamic acid; and
bis-(2-hydroxyethyl)ami-nopropyltriethoxysilane. Exemplary of
readily available materials containing amino group reactive
functionalities, include, but are not limited to,
para-aminophenyltriethyoxysilane. Also derivatized polystyrenes and
other such polymers are well known and readily available to those
of skill in this art [e.g., the Tentagel.RTM. Resins are available
with a multitude of functional groups, and are sold by Rapp
Polymere, Tubingen, Germany; see, U.S. Pat. Nos. 4,908,405 and
5,292,814; see, also Butz et al. (1994) Peptide Res. 7:20-23;
Kleine et al. (1994) Immunobiol. 190:53-66].
[0390] These matrix materials include any material that can act as
a support matrix for attachment of the molecules of interest. Such
materials are known to those of skill in this art, and include
those that are used as a support matrix. These materials include,
but are not limited to, inorganics, natural polymers, and synthetic
polymers, including, but are not limited to: cellulose, cellulose
derivatives, acrylic resins, glass, silica gels, polystyrene,
gelatin, polyvinyl pyrrolidone, co-polymers of vinyl and
acrylamide, polystyrene cross-linked with divinylbenzene or the
like [see, Merrifield (1964) Biochemistry 3:1385-1390],
polyacrylamides, latex gels, polystyrene, dextran, polyacrylamides,
rubber, silicon, plastics, nitrocellulose, celluloses, natural
sponges. Of particular interest herein, are highly porous glasses
[see, e.g., U.S. Pat. No. 4,244,721] and others prepared by mixing
a borosilicate, alcohol and water.
[0391] Synthetic matrices include, but are not limited to:
acrylamides, dextran-derivatives and dextran co-polymers,
agarose-polyacrylamide blends, other polymers and co-polymers with
various functional groups, methacrylate derivatives and
co-polymers, polystyrene and polystyrene copolymers [see, e.g.,
Merrifield (1964) Biochemistry 3:1385-1390; Berg et al. (1990) in
Innovation Perspect. Solid Phase Synth. Collect. Pap., Int. Symp.,
1st, Epton, Roger (Ed), pp. 453-459; Berg et al. (1989) in Pept.,
Proc. Eur. Pept. Symp., 20th, Jung, G. et al. (Eds), pp. 196-198;
Berg et al. (1989) J. Am. Chem. Soc. 111:8024-8026; Kent et al.
(1979) Isr. J. Chem. 17:243-247; Mitchell et al. (1978) J. Org.
Chem. 43:2845-2852; Mitchell et al. (1976) Tetrahedron Lett.
42:3795-3798; U.S. Pat. No. 4,507,230; U.S. Pat. No. 4,006,117; and
U.S. Pat. No. 5,389,449]. Methods for preparation of such matrices
are well-known to those of skill in this art.
[0392] Synthetic matrices include those made from polymers and
co-polymers such as polyvinylalcohols, acrylates and acrylic acids
such as poly-ethylene-co-acrylic acid, polyethylene-co-methacrylic
acid, polyethylene-co-ethylacrylate, polyethylene-co-methyl
acrylate, polypropylene-co-acrylic acid,
polypropylene-co-methyl-acrylic acid,
polypropylene-co-ethylacrylate, polypropylene-co-methyl acrylate,
polyethylene-co-vinyl acetate, polypropylene-co-vinyl acetate, and
those containing acid anhydride groups such as
polyethylene-co-maleic anhydride, polypropylene-comaleic anhydride
and the like. Liposomes have also been used as solid supports for
affinity purifications [Powers et al. (1989) Biotechnol. Bioeng.
33:173].
[0393] For example, U.S. Pat. No. 5,403,750, describes the
preparation of polyurethane-based polymers. U.S. Pat. No. 4,241,537
describes a plant growth medium containing a hydrophilic
polyurethane gel composition prepared from chain-extended polyols;
random copolymerization is preferred with up to 50% propylene oxide
units so that the prepolymer will be a liquid at room temperature.
U.S. Pat. No. 3,939,123 describes lightly crosslinked polyurethane
polymers of isocyanate terminated prepolymers containing
poly(ethyleneoxy) glycols with up to 35% of a poly(propyleneoxy)
glycol or a poly(butyleneoxy) glycol. In producing these polymers,
an organic polyamine is used as a crosslinking agent. Other
matrices and preparation thereof are described in U.S. Pat. Nos.
4,177,038, 4,175,183, 4,439,585, 4,485,227, 4,569,981, 5,092,992,
5,334,640, 5,328,603
[0394] U.S. Pat. No. 4,162,355 describes a polymer suitable for use
in affinity chromatography, which is a polymer of an aminimide and
a vinyl compound having at least one pendant halo-methyl group. An
amine ligand, which affords sites for binding in affinity
chromatography is coupled to the polymer by reaction with a portion
of the pendant halo-methyl groups and the remainder of the pendant
halo-methyl groups are reacted with an amine containing a pendant
hydrophilic group. A method of coating a substrate with this
polymer is also described. An exemplary aminimide is
1,1-dimethyl-1-(2-hydroxyoctyl)amine methacrylimide and vinyl
compound is a chloromethyl styrene.
[0395] U.S. Pat. No. 4,171,412 describes specific matrices based on
hydrophilic polymeric gels, preferably of a macroporous character,
which carry covalently bonded D-amino acids or peptides that
contain D-amino acid units. The basic support is prepared by
copolymerization of hydroxyalkyl esters or hydroxyalkylamides of
acrylic and methacrylic acid with crosslinking acrylate or
methacrylate comonomers are modified by the reaction with diamines,
aminoacids or dicarboxylic acids and the resulting carboxyterminal
or aminoterminal groups are condensed with D-analogs of aminoacids
or peptides. The peptide containing D-amino-acids also can be
synthesized stepwise on the surface of the carrier.
[0396] U.S. Pat. No. 4,178,439 describes a cationic ion exchanger
and a method for preparation thereof. U.S. Pat. No. 4,180,524
describes chemical syntheses on a silica support.
[0397] Immobilized Artificial Membranes [IAMs; see, e.g., U.S. Pat.
Nos. 4,931,498 and 4,927,879] may also be used. IAMs mimic cell
membrane environments and may be used to bind molecules that
preferentially associate with cell membranes [see, e.g., Pidgeon et
al. (1990) Enzyme Microb. Technol. 12:149].
[0398] These materials are also used for preparing articles of
manufacture, such as toys, balloons, figurines, sponges,
knick-knacks, key chains, clothing, translucent or transparent
soaps, preferably mild soaps, and other items, and thus are
amenable to linkage of molecules, either the luciferase, luciferin,
mixtures thereof.
[0399] For example, matrix particles may be impregnated into items
that will then be contacted with an activator. For example, matrix
particles with linked luciferin, preferably a luciferin/luciferase
complex, such as the aequorin photoprotein is incorporated into a
transparent or translucent soaps [see, e.g., U.S. Pat. Nos.
4,081,394, 5,183,429, and 5,141,664, and United Kingdom Patent No.
GB 2,235,931 A], preferably a mild soap. Upon contacting the soap
with water matrix particles near the surface will glow.
[0400] Kits containing the item including the matrix material with
or without the coating of the bioluminescence generating
components, and compositions containing the remaining components
are provided.
[0401] b. Immobilization and Activation
[0402] Numerous methods have been developed for the immobilization
of proteins and other biomolecules onto solid or liquid supports
[see, e.g., Mosbach (1976) Methods in Enzymology 44; Weetall (1975)
Immobilized Enzymes, Antigens, Antibodies, and Peptides; and
Kennedy et al. (1983) Solid Phase Biochemistry, Analytical and
Synthetic Aspects, Scouten, ed., pp. 253-391; see, generally,
Affinity Techniques. Enzyme Purification: Part B. Methods in
Enzymology, Vol. 34, ed. W. B. Jakoby, M. Wilchek, Acad. Press,
N.Y. (1974); Immobilized Biochemicals and Affinity Chromatography,
Advances in Experimental Medicine and Biology, vol. 42, ed. R.
Dunlap, Plenum Press, N.Y. (1974)].
[0403] Among the most commonly used methods are absorption and
adsorption or covalent binding to the support, either directly or
via a linker, such as the numerous disulfide linkages, thioether
bonds, hindered disulfide bonds, and covalent bonds between free
reactive groups, such as amine and thiol groups, known to those of
skill in art [see, e.g., the PIERCE CATALOG, Immuno Technology
Catalog & Handbook, 1992-1993, which describes the preparation
of and use of such reagents and provides a commercial source for
such reagents; and Wong (1993) Chemistry of Protein Conjugation and
Cross Linking, CRC Press; see, also DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Zuckermann et al. (1992) J. Am.
Chem. Soc. 114:10646; Chen et al. (1994) J. Am. Chem. Soc.
116:2661; Bunnin et al. (1994) Proc. Natl. Acad. Sci. U.S.A.
91:4708; Sucholeiki (1994) Tetrahedron Lttrs. 35:7307; and Su-Sun
Wang (1976) J. Org. Chem. 41:3258; Padwa et al. (1971) J. Org.
Chem. 41:3550 and Vedejs et al. (1984) J. Org. Chem. 49:575, which
describe photosensitive linkers]
[0404] To effect immobilization, a composition containing the
protein or other biomolecule is contacted with a support material
such as alumina, carbon, an ion-exchange resin, cellulose, glass or
a ceramic. Fluorocarbon polymers have been used as supports to
which biomolecules have been attached by adsorption [see, U.S. Pat.
No. 3,843,443; Published International PCT Application WO/86
03840].
[0405] A large variety of methods are known for attaching
biological molecules, including proteins and nucleic acids,
molecules to solid supports [see e.g., U.S. Pat. No. 5,451,683].
For example, U.S. Pat. No. 4,681,870 describes a method for
introducing free amino or carboxyl groups onto a silica matrix.
These groups may subsequently be covalently linked to other groups,
such as a protein or other anti-ligand, in the presence of a
carbodiimide. Alternatively, a silica matrix may be activated by
treatment with a cyanogen halide under alkaline conditions. The
anti-ligand is covalently attached to the surface upon addition to
the activated surface. Another method involves modification of a
polymer surface through the successive application of multiple
layers of biotin, avidin and extenders [see, e.g., U.S. Pat. No.
4,282,287]; other methods involve photoactivation in which a
polypeptide chain is attached to a solid substrate by incorporating
a light-sensitive unnatural amino acid group into the polypeptide
chain and exposing the product to low-energy ultraviolet light
[see, e.g., U.S. Pat. No. 4,762,881]. Oligonucleotides have also
been attached using a photochemically active reagents, such as a
psoralen compound, and a coupling agent, which attaches the
photoreagent to the substrate [see, e.g., U.S. Pat. Nos. 4,542,102
and 4,562,157]. Photoactivation of the photoreagent binds a nucleic
acid molecule to the substrate to give a surface-bound probe.
[0406] Covalent binding of the protein or other biomolecule or
organic molecule or biological particle to chemically activated
solid matrix supports such as glass, synthetic polymers, and
cross-linked polysaccharides is a more frequently used
immobilization technique. The molecule or biological particle may
be directly linked to the matrix support or linked via linker, such
as a metal [see, e.g., U.S. Pat. No. 4,179,402; and Smith et al.
(1992) Methods: A Companion to Methods in Enz. 4:73-78]. An example
of this method is the cyanogen bromide activation of polysaccharide
supports, such as agarose. The use of perfluorocarbon polymer-based
supports for enzyme immobilization and affinity chromatography is
described in U.S. Pat. No. 4,885,2501. In this method the
biomolecule is first modified by reaction with a
perfluoroalkylating agent such as perfluorooctylpropylisocyanate
described in U.S. Pat. No. 4,954,444. Then, the modified protein is
adsorbed onto the fluorocarbon support to effect
immobilization.
[0407] The activation and use of matrices are well known and may be
effected by any such known methods [see, e.g., Hermanson et al.
(1992) Immobilized Affinity Ligand Techniques, Academic Press,
Inc., San Diego]. For example, the coupling of the amino acids may
be accomplished by techniques familiar to those in the art and
provided, for example, in Stewart and Young, 1984, Solid Phase
Synthesis, Second Edition, Pierce Chemical Co., Rockford.
[0408] Other suitable methods for linking molecules to solid
supports are well known to those of skill in this art [see, e.g.,
U.S. Pat. No. 5,416,193]. These include linkers that are suitable
for chemically linking molecules, such as proteins, to supports and
include, but are not limited to, disulfide bonds, thioether bonds,
hindered disulfide bonds, and covalent bonds between free reactive
groups, such as amine and thiol groups. These bonds can be produced
using heterobifunctional reagents to produce reactive thiol groups
on one or both of the moieties and then reacting the thiol groups
on one moiety with reactive thiol groups or amine groups to which
reactive maleimido groups or thiol groups can be attached on the
other. Other linkers include, acid cleavable linkers, such as
bismaleimideothoxy propane, acid labile-transferrin conjugates and
adipic acid diihydrazide, that would be cleaved in more acidic
intracellular compartments; cross linkers that are cleaved upon
exposure to UV or visible light and linkers, such as the various
domains, such as C.sub.H1, C.sub.H2, and C.sub.H3, from the
constant region of human IgG, (see, Batra et al. (1993) Molecular
Immunol. 30:379-386). Presently preferred linkages are direct
linkages effected by adsorbing the molecule to the surface of the
matrix. Other linkages are photocleavable linkages that can be
activated by exposure to light [see, e.g., Goldmacher et al. (1992)
Bioconj. Chem. 3:104-107, which linkers are herein incorporated by
reference]. The photocleavable linker is selected such that the
cleaving wavelength that does not damage linked moieties.
Photocleavable linkers are linkers that are cleaved upon exposure
to light [see, e.g., Hazum et al. (1981) in Pept., Proc. Eur. Pept.
Symp., 16th, Brunfeldt, K (Ed), pp. 105-110, which describes the
use of a nitrobenzyl group as a photocleavable protective group for
cysteine; Yen et al. (1989) Makromol. Chem 190:69-82, which
describes water soluble photocleavable copolymers, including
hydroxypropylmethacrylamide copolymer, glycine copolymer,
fluorescein copolymer and methylrhodamine copolymer; Goldmacher et
al. (1992) Bioconj. Chem. 3:104-107, which describes a cross-linker
and reagent that undergoes photolytic degradation upon exposure to
near UV light (350 nm); and Senter et al. (1985) Photochem.
Photobiol 42:231-237, which describes nitrobenzyloxycarbonyl
chloride cross linking reagents that produce photocleavable
linkages]. The selected linker will depend upon the particular
application and, if needed, may be empirically selected.
[0409] Aequorin that is designed for conjugation and conjugates
containing such aequorin have been produced [see, e.g.,
International PCT application No. WO 94/18342; see, also Smith et
al. (1995) in American Biotechnology Laboratory]. Vargula
luciferase has also been linked to other molecules [see, e.g.,
Japanese application No. JP 5064583, Mar. 19, 1993]. Such methods
may be adapted for use herein to produce aequorin coupled to
protein or other such molecules, which are linked to the selected
matrix. Finally, as an alternative, a component of the
bioluminescence generating system may be modified for linkage, such
as by addition of amino acid residues that are particularly
suitable for linkage to the selected substrate. This can be readily
effected by modifying the DNA and expressing such modified DNA to
produce luciferase with additional residues at the N- or
C-terminus.
[0410] 4. Apparatus Containing a Single Chamber, Housing or a
Vessel
[0411] Examples of vessels include beverage containers, plates or
other dishes, vases, jars, balloons, bottles and other
containers.
[0412] Single chamber housings or vessels will include single
chamber water guns, inks, paints and other such items, in which one
or more components of the bioluminescence system up to all of the
components except for one of the components required for
bioluminescence is included in the vessel as a mixture, powder or
suspension of particles. The remaining component(s) is(are)
introduced just prior to use. Thus, for example, for a squirt gun
or a balloon or other such item, the items can be packaged with a
powder in the chamber or inside the item, or a powder or other
composition can be added, and then water is added. Alternatively,
the luciferase, such as Renilla, Vargula, and firefly luciferase,
can be linked to the surface of the item and water added. Depending
upon the bioluminescence generating system selected the water can
be tap water or water that contains the additional component, such
as dissolved oxygen, or Ca.sup.2+ or ATP, or other suitable
composition, and/or appropriate luciferin/bioluminescence
substrate. Similarly, the luciferase/luciferase can be linked to
the surface of the item in association with the appropriate
luciferin/bioluminescence substrate, such that addition of
activator alone generates luminescence.
[0413] For inks or paints the components are suspended in the ink
or paint, and then the final component(s) is(are) added.
Alternatively, pellets containing components of the bioluminescence
generating system, such as the Renilla or Aequorin system can be
added to an ink or paint or other such liquid item, and as the
pellet dissolves or the contents diffuse out, the item will
glow.
[0414] Kits containing the item and the bioluminescence generating
systems are also provided herein. The kits typical its typically
contain a beverage container, balloon or bottle and, may also
contain, the buffer compositions and other ingredients required for
the bioluminescence reaction, as well as instructions for use. The
kits may also include the cartridges for recharging or reloading
the item.
[0415] 5. Dual and Multiple Chamber Fluid Dispensing Apparatus
[0416] An example of a dispensing apparatus contemplated for use
herein is a dual chamber fluid dispensing apparatus. In general,
this apparatus has two chambers thereby maintaining at least one of
the bioluminescence generating system components separate from the
remaining components until illumination is desired. This apparatus
may include a mixing chamber to permit mixing of the components
prior to dispensing from the apparatus. Further, the apparatus may
be used with fluid or semi-fluid bioluminescence systems; for
example, water based compositions or cream/lotion systems. [0417]
a. Mechanical Pump Dispensing Apparatus
[0418] Another embodiment of a dual chamber fluid dispensing
apparatus employs a mechanical pump mechanism in its operation. In
this embodiment, the dispensing apparatus maintains at least one of
the components of the bioluminescence reaction, such as the
substrate, luciferase or activator, in separate chambers. A pump
mechanism operates to withdraw the contents from each chamber and
into a mixing chamber. Within the mixing chamber and upon ejection,
the mixed composition is activated, for example by the oxygen in
the air or by reaction of the components that were in one chamber,
and glows. The pump mechanism may be manually operated, for example
by pulling the trigger of a toy squirt gun, or it may be
mechanically operated, for example by a motor which operates the
pumping mechanism. [0419] b. Gas-Charged Dispensing Apparatus
[0420] Another example of a dual chamber fluid dispensing apparatus
is one that uses CO.sub.2 or, preferably a mixture gases containing
O.sub.2 or other gas, to propel the components of the
bioluminescence system, such as the bioluminescence substrate and
luciferase into a mixing chamber where they combine before being
ejected through a dispensing nozzle. In such a dispensing
apparatus, upon mixing of the contents in the mixing chamber the
contents will glow.
[0421] These apparatus may be configured as, for example, a toy
gun, toy cannon or other toy weapon, a can for shaving cream or
other glowing foam, a decorative fountain or volcano or almost any
fluid squirting or spouting device. A volcano shaped dispensing
apparatus may be used, for example, as a substitute for
conventional, similarly shaped fireworks displays.
[0422] Almost any bioluminescence generating system may be selected
for use with the dual chamber fluid dispensing apparatus. If air is
the bioluminescence activator, then the contents glow after mixing
and upon ejection from the dispensing apparatus. Alternatively, the
bioluminescent activator may be contained in one of the two
chambers along with either the luciferase or bioluminescence
substrate, or it may be located in a third chamber that is also
connected to the mixing chamber. Thus, as with all the combinations
described herein, the critical aspect of these dispensing apparatus
is that at least one of the bioluminescence generating system
components be maintained separate from the other components until
reaction is desired.
[0423] c. Compressible Dispensing Apparatus
[0424] Another embodiment of a dual chamber fluid dispensing
apparatus contemplated for use herein takes the form of a
compressible bottle or tube. The bottle has two compartments within
it that keep at least two of the bioluminescence generating system
components separated. The cap of the bottle can serve as a mixing
chamber or a mixing chamber may be positioned between the two
chambers and the cap. The bioluminescence generating system
components are forced by compression from the bottle into the
mixing chamber. They are then dispensed from the mixing chamber.
For example, the mixed contents may be removed from the bottle by
attaching a plunger/syringe apparatus to the dispensing end and
withdrawing the contents therethrough.
[0425] Such compressible bottle or tube is particularly useful for
dispensing bioluminescent body creams, gels or lotions, finger
paints, dentifrices, shampoos, hair gels, cosmetics and other
viscous fluids and semi-solids. The bottle or tube is preferably
constructed of plastic, plastic/metal laminate or similar
collapsible composite to avoid formation of a vacuum within the
container as its contents are expelled. See, for example, U.S. Pat.
No. 4,687,663, which describes a dual chambered tube for use with
dentifrices and which, as all cited patents and publications
herein, is incorporated herein in its entirety. This tube may be
adapted for use in combination with the bioluminescence generating
systems provided herein. Other tubes and vessels that have dual
chambers, such as those used to keep components of the final
product separate until use, may be used herein [see, e.g., U.S.
Pat. Nos. 5,405,056, 4,676,406, 4,438,869, 5,059,417, 4,528,180,
4,849,213, 4,895,721, 5,085,853, see, esp. 5,038,963]
[0426] 6. Other Fluid Dispensing and Packaging Apparatus
Particularly Designed for Single Use
[0427] Additional embodiments of the dispensing and packaging
apparatus contemplated for use herein include fluid packaging
apparatus, designed for use with bioluminescent fluids. These
apparatus maintain at least one of the bioluminescence generating
system components separate from the remaining components until
illumination is desired. Unlike the dual chamber fluid dispensing
apparatus, however, these apparatus result in illumination of the
entire contents of the package and therefore are typically intended
for a single use applications. They can, however, be recharged by
adding additional substrate, luciferase or other exhausted
component.
[0428] a. Bottle-Type Single Chamber Container/Bladder
Apparatus
[0429] One example of a fluid packaging apparatus, contemplated for
use herein, is a bottle shaped device having a bladder within it
that contains at least one of the bioluminescence generating system
components. A piercing pin or other means for rupturing the bladder
is also located within the bottle. When the bladder is ruptured,
within the bottle, its contents mix with the contents of the bottle
and the resulting mixture becomes illuminated or glows upon contact
with an activator, such as air.
[0430] Because the bioluminescence generating system components are
mixed within the entire bottle, those contents must be used shortly
after mixing. Thus, this type of packaging is particularly suitable
for use with bioluminescence systems that are consumed in a single
use or activity such as bubble-blowing. [0431] b. Dual Chambered
Bottle Type Container/Bladder Apparatus for Use with Foods and
Beverages
[0432] Another example of a fluid packaging apparatus provided
herein is a single use, dual chambered bottle. This apparatus is
configured with a membrane between the two chambers. One chamber is
designed to readily collapse against the other chamber thereby
rupturing the membrane which divides the chambers. The contents of
the two chambers then mix, resulting in illumination of the fluids.
Alternatively, instead of a membrane separation means, a one-way
valve may be situated between the two chambers. Such a single use,
dual chamber apparatus is particularly suitable for use with
bubble-making compositions, beverages, single use amounts of
shampoos, soaps, creams or lotions, or similar substances. [0433]
c. Can Type Container/Bladder Apparatus for Use with Foods and
Beverages
[0434] Another example of a fluid packaging apparatus, which is
amenable to use with bioluminescent food or beverage, is a
container/bladder combination. In one embodiment, the container is
configured like a pop-top can, such as a soda can. A bladder,
containing at least one of the bioluminescence generating system
components, is positioned under the top of the can. Within the can
is a beverage that contains the remaining bioluminescence
generating system components. Upon opening the can, the bladder is
punctured and its contents mixed with the rest of the contents of
the can; thereby illuminating the beverage. Preferably, the
container is clear, so that the illumination will be almost
immediately visible. Other pop top cans that can be modified for
use herein are known [see, e.g., U.S. Pat. No. 5,397,014].
[0435] Alternative configurations of the container/bladder
apparatus are likewise contemplated. For example, the container may
be in any shape and configured with a removable cap to which the
bladder is attached. To cause the beverage to glow, the bladder is
punctured or otherwise compromised and its contents added to the
container; thereby causing illumination of the food or beverage.
The contents of the container need not be a food or beverage, any
fluid or semi-solid may be used and is herein contemplated.
[0436] d. Spray Containers that Produce a Glowing Spray
[0437] Spray containers or cans that are adapted to produce a
glowing spray are provided herein. These containers are also
intended for use in any application in which two components,
particularly solutions or liquid components, are intended to be
mixed just prior to use. These containers include a housing portion
for the first component and a second portion designed to inject or
introduce the second component.
[0438] A preferred embodiment of these containers, which is
illustrated in FIGS. 20-22 [see, also EXAMPLE 10], includes two
portions, a top housing portion and a bottom plunger portion. For
use in generating bioluminescence, the top housing portion includes
all, except one or more, of the components of a bioluminescence
generating system. The remaining components of the bioluminescence
generating system are contained in a pellet or are encapsulated, as
described above.
[0439] The top housing portion is adapted at its bottom end with an
indentation within which the pellet fits. At least one wall of the
indentation includes a rupturable membrane or material. The top
housing portion is further adapted to attach securely to and within
the bottom plunger portion. A plunger is situated within the bottom
plunger portion such that the plunger rests in the indentation of
the top housing portion when the bottom plunger portion is tightly
secured thereto. In operation, the pellet or encapsulated vehicle
is placed within the indentation of the top housing portion and the
bottom plunger portion secured tightly thereto. The plunger within
the bottom plunger portion presses against the pellet forcing it
through the rupturable membrane or material, thereby permitting the
pellet to dissolve in and mix with the contents of the top housing
portion. Alternatively the pellet will include a sharpened portion
that will puncture the rupturable wall of the housing. An angular
seal may be used, situated within the bottom plunger portion, to
set against the bottom of the top housing portion forming a seal to
prevent leakage of the mixed contents of the spray can apparatus.
The top housing portion additionally contains a conduit or other
suitable means for ejecting the contents.
[0440] The top housing portion of the spray container may be
adapted to receive the bottom plunger portion by threading the two
spray can portions so that they may be screwed together. [See,
e.g., FIG. 21, illustrating the spray container apparatus with the
bottom plunger portion fully screwed into place]. Alternatively,
the two portions may be adapted to snap together, such as by
insertion of a tongue from one portion into a groove of the other
portion.
[0441] As stated, the indentation or pocket located in the bottom
end of the top housing portion includes at least one wall formed by
a rupturable membrane. Preferably that wall is the top wall and is
readily ruptured by pressure, for example, from the pellet or
plunger or plunger forcing the pellet, against it. The pellet is
fabricated from material that will release the contents into
aqueous medium. The pellet may also include a sharp tip designed to
puncture the spray container.
[0442] The spray container is fabricated from suitable materials,
such as plastic, aluminum, metal alloys, tin, and other materials
from which spray cans and containers, such as hair spray cans and
other containers designed for delivery of aerosols and sprays, are
fabricated. The size of the spray can apparatus may vary depending
upon the intended use and demands of the market place, but will
typically have a usable volume of from about 100 mls to about a
liter.
[0443] The bottom plunger portion is typically fabricated from a
metal, such as aluminum, and the plunger is shaped and situated
such that it fits into the pocket of the top housing portion when
the bottom plunger portion is screwed tightly in place. It can also
be made from compressible plastic or other such material and
designed to compress and deliver the inserted pellet, which is
designed to fit into the indentation, slot or pocket and be
retained by virtue of the tight fit.
[0444] 7. Cap Apparatus for use a Single Chamber Vessel
[0445] Another example of a packaging apparatus contemplated herein
is a cap apparatus for use with a vessel. In this embodiment, one
or more of the bioluminescence generating system components, up to
all but one component, is [are] within the cap of the vessel and
the remaining components are contained in the vessel. Upon
operation of the cap apparatus, the bioluminescence generating
system components are added to the composition in the vessel and
the composition glows. Preferably the vessel is translucent to the
bioluminescence; however, the glowing composition may be dispensed
from the vessel.
[0446] Generally, the cap is configured with a pocket within it
which opens to the bottom of the cap. For example, the bottom of
the cap can be U-shaped, curving into the cap and thereby forming
the pocket. The cap apparatus contains a capsule or similar
package, containing one or more, up to all but one, of the
bioluminescence generating system components, within the pocket in
the cap. Means for deploying the bioluminescence generating system
components into the vessel are attached to the cap. Such deployment
means can be, for example, a plunger assembly. The cap apparatus is
operated by depressing the plunger, thereby forcing the packaged
components into the composition within the vessel or breaking the
packaging, releasing its contents into the composition within the
vessel. The package should be dissolvable in the composition or
amenable to diffusion of the components contained therein or
readily rupturable upon contact with the plunger assembly.
[0447] Alternatively, the packaging within the cap apparatus can be
a membrane or series of membranes separating the bioluminescence
generating system components from the composition within the vessel
or from the composition within the vessel and from each other. In
this alternative, the plunger can rupture the membrane(s) thereby
permitting the bioluminescence generating system components
contained therein to be released into the composition contained in
the vessel. Again, upon mixture of the components with the
composition, illumination ensues.
[0448] The bioluminescence generating system components contained
within the cap apparatus may be in a composition, such as a
solution, a powder or a suspension of particles or other form
amenable to packaging within the cap apparatus that can be mixed
with the composition contained within the vessel. The cap apparatus
also may be adapted with a screen or filter attached to the bottom
of the cap to prevent membrane fragments from entering the
vessel.
[0449] The cap apparatus, as all the apparatus described herein
that are in contact with a bioluminescence generating system
component, should be non-reactive with the components and is
preferably non-toxic, particularly if used with a composition
intended for human consumption. The cap can be constructed of cork,
for example, and situated in a wine or champagne bottle.
Alternatively, the cap can be a screw-top type cap, having a
plunger integral thereto, such that tightening of the screw-cap
onto the top of the vessel forces the plunger against the packaged
bioluminescence generating system components either rupturing the
packaging or pushing it into the vessel.
[0450] E. Combinations of Articles of Manufacture and
Bioluminescence
[0451] Combinations of articles of manufacture and bioluminescence
are provided herein. By virtue of the bioluminescence the
combinations are novelty items because the bioluminescence provides
entertainment, amusement or recreation. Any such combination of an
article of manufacture with bioluminescence that produces a novelty
item [i.e., provides entertainment, amusement, or recreation] is
intended herein. The combination is formed by contacting the
article of manufacture or materials in the manufacture with a
bioluminescence generating system or an apparatus therefore. The
components of the bioluminescence generating system are
manufactured as part of the item, coated thereon, impregnated
therein, or added after manufacture. Alternatively, the article of
manufacture is combined with an apparatus that contains or to which
components of the bioluminescence generating system are added, and
that produces the bioluminescence.
[0452] The bioluminescence generating systems provided herein are
contemplated for use with various substances to glow the substance.
For example, as discussed below, the bioluminescence generating
system components may be used to produce glowing aqueous mixtures
housed in transparent portions of articles of manufacture, thereby
illuminating that portion of the article of manufacture.
Additionally, the bioluminescence generating system components may
be used to produce glowing food or beverage products, textiles,
creams, lotions, gels, soaps, bubbles, papers, powders or water.
Following are brief examples of combinations of bioluminescence
systems with articles of manufacture and the resulting novelty
items contemplated herein.
[0453] 1. Personal Care Products, Including Bath Powders, Bubble
Baths, Products for Use on the Nails, Hair, Skin, Lips and
Elsewhere
[0454] Personal care products can be in the form of powders,
pressed powders, sprays, foams, aerosols, lotions, gels, ointments
and other suitable formulations. The common element will be the
combination of such items with bioluminescence generating reagents
or fluorescent proteins, so that before use or upon application to
the body or when used the product will glow. These items include,
body powders, lotions, gels, aqueous compositions and solutions,
nail polishes, make-up, body paints, shaving cream and dentifrices.
As described herein, the items are combined with one or more
components of a bioluminescence generating system, and, when a glow
is desired, the remaining components are added or combined with the
other components.
[0455] a. Bath Powders
[0456] Numerous bath powders exemplified herein, are suitable for
use in combination with the bioluminescence generating systems
herein. Such bath powders are preferably non-detergent with a pH
close to neutral. The selected bioluminescence generating system
must be selected to be active at the resulting pH. In addition,
capsular delivery vehicles, such as liposomes or time release
delivery vehicles, preferably microcapsules, that contain a
luciferase and luciferin, such as the Renilla, Vargula, or Aequorin
system, and that are pH, temperature sensitive, or that dissolve in
water or that are otherwise released are preferred for use herein.
In certain embodiments, there will be two types of capsules, one
type containing up to all but one of the components required for
the bioluminescence reaction, and the other containing the
remaining components [except, if desired, for those components that
will be present in the bath water, such as Ca.sup.2+]. Such
capsules may be components of the bath powder or may be added to a
bath to give it a glow. Upon contact with the warm water or with
water of a particular pH the contents of the capsule or pellet will
be released, preferably over time, and will glow.
[0457] In other embodiments, there will be one type of capsule that
contains the luciferase and other components. The luciferin may be
included in the bath powder or added separately. Other ways in
which the components may be combined will, in light of the
disclosure herein, be apparent to those of skill in the art. The
bath powders and bioluminescence generating reactions will be
provided as a combination or in a kit.
[0458] Suitable bath powders and bubble baths and other bubble
compositions for use in these combinations are well known to those
of skill in the art [see, e.g., U.S. Pat. Nos. 5,478,501 4,565,647;
5,478,490; 5,412,118; 5,401,773; and many other examples]. These
may be modified by adding the bioluminescence generating system
components.
[0459] b. Glowing Dust or Powder
[0460] Another embodiment of the combination described herein is as
a glowing dust or powder substance, or a vapor, such as for use in
the theatrical productions. In this embodiment, lyophilized or
desiccated forms, micronized powdered forms, or, a suitable
composition, of up to all but one of the bioluminescence generating
system components are encapsulated in readily rupturable or time
release or temperature or pH or light sensitive microspheres or
capsules, as described above. Preferable encapsulating agents are
light or temperature sensitive so that upon exposure to the
environment, the contents are released from the capsules. Moisture
or oxygen in the air or a spray of water on the skin with dissolved
oxygen in the vicinity of the "dust" will produce a glow.
[0461] The dust can be added to another powder, such as body
powder, provided it is stored in an airtight container. Once the
powder contacts the moisture in the air and on the wearer's skin,
it glows.
[0462] Alternatively, micronized particles of lyophilized powders
are packaged such in manner so that the powder remains dry. Upon
exposure to moist air or to air with water droplets [such as a
fog], the micronized powders will glow. [0463] c. Lotions, Gels and
Other Topical Application Formulations
[0464] For application to the skin, the macro or microparticles or
the luciferase, luciferin or mixture thereof, may be added to
cosmetic compositions. The compositions may be provided in the form
of gels, creams, lotions, solids, and other compositions, such as
solutions and suspensions, aerosols or solid base or vehicle known
in the art to be non-toxic and dermatologically acceptable to which
sufficient number of such particles are added under conditions in
which the contents are released into the gels, creams, lotions,
solids, solutions or suspensions, or aerosols, which contain either
molecular oxygen and/or Ca.sup.2+ to react with the contents of
particles. Upon application to the skin the gels, creams, lotions,
solids, solutions or suspensions, or aerosols glow.
(1) Lotions
[0465] The lotions contain an effective concentration of less than
all reagents for one or more bioluminescence generating systems.
Preferably, the reagents are encapsulated in a vehicle that
releases its contents upon exposure to light or temperature, such
that as the contents of the vehicle are released they react with
oxygen or Ca.sup.2+ in the lotion and/or on the skin. Prior to use
the skin can be sprayed with a mist of water, buffer or other
composition containing the requisite ions. The effective
concentration is that sufficient to produce a visible glow when
contacting the skin. Any emollients, as long as they do not
inactivate the bioluminescent reaction, known to those of skill in
the art as suitable for application to human skin may be used.
These include, but are not limited to, the following:
[0466] (a) Hydrocarbon oils and waxes, including mineral oil,
petrolatum, paraffin, ceresin, ozokerite, microcrystalline wax,
polyethylene, and perhydrosqualene.
[0467] (b) Silicone oils, including dimethylpolysiloxanes,
methylphenylpolysiloxanes, water-soluble and alcohol-soluble
silicone-glycol copolymers.
[0468] (c) Triglyceride fats and oils, including those derived from
vegetable, animal and marine sources. Examples include, but are not
limited to, castor oil, safflower oil, cotton seed oil, corn oil,
olive oil, cod liver oil, almond oil, avocado oil, palm oil, sesame
oil, and soybean oil.
[0469] (d) Acetoglyceride esters, such as acetylated
monoglycerides.
[0470] (e) Ethoxylated glycerides, such as ethoxylated glyceryl
monstearate.
[0471] (f) Alkyl esters of fatty acids having 10 to 20 carbon
atoms. Methyl, isopropyl and butyl esters of fatty acids are useful
herein. Examples include, but are not limited to, hexyl laurate,
isohexyl laurate, isohexyl palmitate, isopropyl palmitate,
isopropyl myristate, decyl oleate, isodecyl oleate, hexadecyl
stearate, decyl stearate, isopropyl isostearate, diisopropyl
adipate, diisohexyl adipate, dihexyldecyl adipate, diisopropyl
sebacate, lauryl lactate, myristyl lactate, and cetyl lactate.
[0472] (g) Alkenyl esters of fatty acids having 10 to 20 carbon
atoms. Examples thereof include, but are not limited to, oleyl
myristate, oleyl stearate, and oleyl oleate.
[0473] (h) Fatty acids having 9 to 22 carbon atoms. Suitable
examples include, but are not limited to, pelargonic, lauric,
myristic, palmitic, stearic, isostearic, hydroxystearic, oleic,
linoleic, ricinoleic, arachidonic, behenic, and erucic acids.
[0474] (i) Fatty alcohols having 10 to 22 carbon atoms, such as,
but not limited to, lauryl, myristyl, cetyl, hexadecyl, stearyl,
isostearyl, hydroxystearyl, oleyl, ricinoleyl, behenyl, erucyl, and
2-octyl dodecyl alcohols.
[0475] (j) Fatty alcohol ethers, including, but not limited to
ethoxylated fatty alcohols of 10 to 20 carbon atoms, such as, but
are not limited to, the lauryl, cetyl, stearyl, isostearyl, oleyl,
and cholesterol alcohols having attached thereto from 1 to 50
ethylene oxide groups or 1 to 50 propylene oxide groups or mixtures
thereof.
[0476] (k) Ether-esters, such as fatty acid esters of ethoxylated
fatty alcohols.
[0477] (l) Lanolin and derivatives, including, but not limited to,
lanolin, lanolin oil, lanolin wax, lanolin alcohols, lanolin fatty
acids, isopropyl lanolate, ethoxylated lanolin, ethoxylated lanolin
alcohols, ethoxylated cholesterol, propoxylated lanolin alcohols,
acetylated lanolin, acetylated lanolin alcohols, lanolin alcohols
linoleate, lanolin alcohols ricinoleate, acetate of lanolin
alcohols ricinoleate, acetate of ethoxylated alcohols-esters,
hydrogenolysis of lanolin, ethoxylated hydrogenated lanolin,
ethoxylated sorbitol lanolin, and liquid and semisolid lanolin
absorption bases.
[0478] (m) Polyhydric alcohols and polyether derivatives,
including, but not limited to, propylene glycol, dipropylene
glycol, polypropylene glycol [M.W. 2000-4000], polyoxyethylene
polyoxypropylene glycols, polyoxypropylene polyoxyethylene glycols,
glycerol, ethoxylated glycerol, propoxylated glycerol, sorbitol,
ethoxylated sorbitol, hydroxypropyl sorbitol, polyethylene glycol
[M.W. 200-6000], methoxy polyethylene glycols 350, 550, 750, 2000,
5000, poly(ethylene oxide) homopolymers [M.W. 100,000-5,000,000],
polyalkylene glycols and derivatives, hexylene glycol
(2-methyl-2,4-pentanediol), 1,3-butylene glycol,
1,2,6,-hexanetriol, ethohexadiol USP (2-ethyl-1,3-hexanediol),
C.sub.15-C.sub.18 vicinal glycol and polyoxypropylene derivatives
of trimethylolpropane.
[0479] (n) Polyhydric alcohol esters, including, but not limited
to, ethylene glycol mono- and di-fatty acid esters, diethylene
glycol mono- and di-fatty acid esters, polyethylene glycol [M.W.
200-6000], mono- and di-fatty esters, propylene glycol mono- and
di-fatty acid esters, polypropylene glycol 2000 monooleate,
polypropylene glycol 2000 monostearate, ethoxylated propylene
glycol monostearate, glyceryl mono- and di-fatty acid esters,
polyglycerol poly-fatty acid esters, ethoxylated glyceryl
monostearate, 1,3-butylene glycol monostearate, 1,3-butylene glycol
distearate, polyoxyethylene polyol fatty acid ester, sorbitan fatty
acid esters, and polyoxyethylene sorbitan fatty acid esters.
[0480] (o) Wax esters, including, but not limited to, beeswax,
spermaceti, myristyl myristate, and stearyl stearate and beeswax
derivatives, including, but not limited to, polyoxyethylene
sorbitol beeswax, which are reaction products of beeswax with
ethoxylated sorbitol of varying ethylene oxide content that form a
mixture of ether-esters.
[0481] (p) Vegetable waxes, including, but not limited to, carnauba
and candelilla waxes.
[0482] (q) Phospholipids, such as lecithin and derivatives.
[0483] (r) Sterols, including, but not limited to, cholesterol and
cholesterol fatty acid esters.
[0484] (s) Amides, such as fatty acid amides, ceramides,
ethoxylated fatty acid amides, and solid fatty acid
alkanolamides.
[0485] The lotions further preferably contain [by weight] from 1%
to 10%, more preferably from 2% to 5%, of an emulsifier. The
emulsifiers can be nonionic, anionic or cationic. Examples of
satisfactory nonionic emulsifiers include, but are not limited to,
fatty alcohols having 10 to 20 carbon atoms, fatty alcohols having
10 to 20 carbon atoms condensed with 2 to 20 moles of ethylene
oxide or propylene oxide, alkyl phenols with 6 to 12 carbon atoms
in the alkyl chain condensed with 2 to 20 moles of ethylene oxide,
mono- and di-fatty acid esters of ethylene oxide, mono- and
di-fatty acid esters of ethylene glycol where the fatty acid moiety
contains from 10 to 20 carbon atoms, diethylene glycol,
polyethylene glycols of molecular weight 200 to 6000, propylene
glycols of molecular weight 200 to 3000, glycerol, sorbitol,
sorbitan, polyoxyethylene sorbitol, polyoxyethylene sorbitan and
hydrophilic wax esters. Suitable anionic emulsifiers include, but
are not limited to, the fatty acid soaps, e.g. sodium, potassium
and triethanolamine soaps, where the fatty acid moiety contains
from 10 to 20 carbon atoms. Other suitable anionic emulsifiers
include, but are not limited to, the alkali metal, ammonium or
substituted ammonium alkyl sulfates, alkyl arylsulfonates, and
alkyl ethoxy ether sulfonates having 10 to 30 carbon atoms in the
alkyl moiety. The alkyl ethoxy ether sulfonates contain from 1 to
50 ethylene oxide units. Among satisfactory cationic emulsifiers
are quaternary ammonium, morpholinium and pyridinium compounds.
Certain of the emollients described in preceding paragraphs also
have emulsifying properties. When a lotion is formulated containing
such an emollient, an additional emulsifier is not needed, though
it can be included in the composition.
[0486] Other conventional components of such lotions may be
included. One such additive is a thickening agent at a level from
1% to 10% by weight of the composition. Examples of suitable
thickening agents include, but are not limited to: cross-linked
carboxypolymethylene polymers, ethyl cellulose, polyethylene
glycols, gum tragacanth, gum kharaya, xanthan gums and bentonite,
hydroxyethyl cellulose, and hydroxypropyl cellulose.
[0487] The balance of the lotion is water or a C.sub.2 or C.sub.3
alcohol, or a mixture of water and the alcohol. The lotions are
formulated by admixing all of the components together. Preferably
bioluminescence generating system reagents are suspended or
otherwise uniformly dispersed in the mixture.
[0488] In certain embodiments the components may be mixed just
prior to use. Devices for effecting such mixture are known to those
of skill in the art or are exemplified herein.
[0489] Kits containing the lotion and powders, capsular vehicles
and, optionally, buffer compositions containing ATP, Ca.sup.2+ and
other ingredients required for the bioluminescence reaction are
also provided.
[0490] (2) Creams
[0491] The creams are similarly formulated to contain an effective
concentration typically at between about 0.1%, preferably at
greater than 1% up to and greater than 50%, preferably between
about 3% and 50%, more preferably between about 5% and 15% [by
weight] of one ore more the bioluminescence generating systems
provided herein. The creams also contain from 5% to 50%, preferably
from 10% to 25%, of an emollient and the remainder is water or
other suitable non-toxic carrier, such as an isotonic buffer. The
emollients, as described above for the lotions, can also be used in
the cream compositions. The cream may also contain a suitable
emulsifier, as described above. The emulsifier is included is in
the composition at a level from 3% to 50%, preferably from 5% to
20%.
[0492] (3) Solutions and Suspensions for Topical Application
[0493] These compositions are formulated to contain an amount
sufficient to produce a visible glow, typically at a concentration
of between about 0.1-10 mg/l preferably between 1 and 5 mg/l of the
luciferase. The amount of luciferin is similarly between about 0.1
and 10 mg/l, although the amount can be selected based on the
desired duration of the glow. The balance is water, a suitable
organic solvent or other suitable solvent or buffer. Suitable
organic materials useful as the solvent or a part of a solvent
system are as follows: propylene glycol, polyethylene glycol [M.W.
200-600], polypropylene glycol [M.W. 425-2025], glycerine, sorbitol
esters, 1,2,6-hexanetriol, ethanol, isopropanol, diethyl tartrate,
butanediol, and mixtures thereof. Such solvent systems can also
contain water.
[0494] Solutions or suspensions used for topical application can
include any of the following components: a diluent, such as water
saline solution, fixed oil, polyethylene glycol, glycerine,
propylene glycol or other synthetic solvent; antimicrobial agents,
such as benzyl alcohol and methyl parabens; antioxidants, such as
ascorbic acid and sodium bisulfite; chelating agents, such as EDTA;
buffers, such as acetates, citrates and phosphates; and agents for
the adjustment of tonicity such as sodium chloride or dextrose.
Liquid preparations can be enclosed in ampules, disposable syringes
or multiple dose vials made of glass, plastic or other suitable
material. Suitable carriers may include physiological saline or
phosphate buffered saline [PBS], and the suspensions and solutions
may contain thickening and solubilizing agents, such as glucose,
polyethylene glycol, and polypropylene glycol and mixtures thereof.
Liposomal suspensions, may also be suitable as pharmaceutically
acceptable carriers. These may be prepared according to methods
known to those skilled in the art.
[0495] These compositions that are formulated as solutions or
suspensions may be applied to the skin, or, may be formulated as an
aerosol or foam and applied to the skin as a spray-on. The aerosol
compositions typically contain [by weight] from 25% to 80%,
preferably from 30% to 50%, of a suitable propellant. Examples of
such propellants are the chlorinated, fluorinated and
chlorofluorinated lower molecular weight hydrocarbons. Nitrous
oxide, carbon dioxide, butane, and propane are also used as
propellant gases. These propellants are used as understood in the
art in a quantity and under a pressure suitable to expel the
contents of the container.
[0496] Solutions, may be formulated as 0.01%-10% isotonic
solutions, pH about 5-8, with appropriate salts, and preferably
containing one or more of the compounds herein at a concentration
of about 0.1%, preferably greater than 1%, up to 50% or more.
Suitable mild solutions are known [see, e.g., U.S. Pat. No.
5,116,868, which describes typical compositions of ophthalmic
irrigation solutions and solutions for topical application]. Such
solutions, which have a pH adjusted to about 7.4, contain, for
example, 90-100 mM sodium chloride, 4-6 mM dibasic potassium
phosphate, 4-6 mM dibasic sodium phosphate, 8-12 mM sodium citrate,
0.5-1.5 mM magnesium chloride, 1.5-2.5 mM calcium chloride, 15-25
mM sodium acetate, 10-20 mM D. L.-sodium .beta.-hydroxybutyrate and
5-5.5 mM glucose.
[0497] The active materials can also be mixed with other active
materials, that do not impair the desired action, or with materials
that supplement the desired action.
[0498] (4) Gels
[0499] Gel compositions can be formulated by admixing a suitable
thickening agent to the previously described [(3)] solution or
suspension compositions. Examples of suitable thickening agents
have been previously described with respect to the lotions.
[0500] The gelled compositions contain an effective amount of one
or more an anti-hyperalgesic amount, typically at a concentration
of between about 0.1 mg/1-10 mg/l or more of one or more of systems
provided herein, from 0% to 75%, from 0.5% to 20%, preferably from
1% to 10% of the thickening agent; the balance being water or other
aqueous carrier.
[0501] (5) Solids
[0502] Compositions of solid forms may be formulated as stick-type
compositions intended for application to the lips or other parts of
the body. Such compositions contain an effective amount of one or
more of the compounds provided herein. The amount is typically an
amount effective to glow when contacted with moist skin, such as
lips, typically at a concentration of between about 0.1 mg/1-10
mg/l or more of one or more of the systems provided herein. The
solids also contain from about 40% to 98%, preferably from about
50% to 90%, of the previously described emollients. This
composition can further contain from 1% to 20%, preferably from 5%
to 15%, of a suitable thickening agent, and, if desired or needed,
emulsifiers and water or buffers. Thickening agents previously
described with respect to lotions are suitably employed in the
compositions in solid form.
[0503] Other ingredients, such as preservatives, including
methyl-paraben or ethyl-paraben, perfumes, dyes or the like, that
are known in the art to provide desirable stability, fragrance or
color, or other desirable properties, such as shielding from
actinic rays from the sun, to compositions for application to the
skin may also be employed in a composition for such topical
application.
[0504] 2. Glowing Toys and Other Items
[0505] Examples of uses of the bioluminescence generating systems
in toys include illumination of dolls, toy vehicles, hoolahoops,
yo-yos, balloons, immersible bubble generating toys, such as a toy
submarine that blows glowing bubbles, and any other toy amenable to
having a generally translucent covering defining a space for
containment of the bioluminescence generating system and addition
of the final ingredients necessary for the illumination reaction.
Also contemplated herein are toys that eject or spew a fluid. For
example, toy or game projectiles are contemplated that contain a
luciferase and bioluminescence substrate in an oxygen-free
environment. The projectiles rupture upon impact with a hard
surface thereby exposing the contents to moisture in the air that
contains dissolved oxygen, the bioluminescence activator, and
causing reaction.
[0506] Dolls and dummies containing one or two of the
bioluminescence generating system components within a transparent
or translucent portion of their bodies are also contemplated
herein. Addition of the remaining bioluminescence generating system
component(s) results in illumination of that body part or area. For
example, a doll can have a visible, translucent digestive system
containing a luciferase and substrate in a water-free environment.
Upon "ingestion" of water by the doll, that is addition of water
through its mouth, for example, the digestive system glows or is
illuminated.
[0507] Other examples of uses of the bioluminescence generating
systems in toys include, but are not limited to illuminated
hoolahoops, yo-yos, slimy play materials, such as those based on
sodium alginate and glycerine [U.S. Pat. No. 5,310,421], such as
those sold by MATTEL.RTM. as FLOAM.RTM., GAK.RTM., and SMUD.RTM.
and moldable play materials, such as those described in U.S. Pat.
Nos. 2,541,851, 3,384,498, 3,565,815, 3,634,280, 3,661,790,
3,804,654, 3,873,485, 4,076,547, 4,172,054, 4,229,790, 4,624,976
and 4,735,660, all of which are incorporated herein in their
entirety. With respect to the slimy and moldable play materials,
the bioluminescence generating components can be incorporated into
the play material during manufacture, as liposomes, or linked to
the material.
[0508] In one embodiment, the slimy play materials are fabricated
from self cross-linking sodium alginate, a glycerin solution
[concentration over 90%], water and preservatives.
[0509] In an alternative embodiment, the slimy play materials are
fabricated from polyvinyl alcohol and tetraborate. In another
embodiment, discussed further below and in the Examples, the slimy
play material is packaged in a compressible dispensing apparatus,
for example, as illustrated in FIG. 27. In such an apparatus, all
but one of the bioluminescence generating reagents may be provided
in a compartment situated within the dispensing apparatus. A second
compartment within the apparatus may contain less than all the
components required to complete the slimy play material
composition, and the main body of the apparatus may contain the
remaining bioluminescence generating reagents and/or remaining
slimy play material components.
[0510] Alternatively, three compartments within the compressible
dispensing apparatus may be provided where, the third compartment
contains either or both of the remaining bioluminescence generating
reagents or the remaining slimy play material components. The main
body of the apparatus would then contain an aqueous composition
within which to mix the contents of the three compartments or the
bioluminescence generating reagents or slimy play material
components not contained within the third compartment.
[0511] In still other embodiments, the slime material is provided
without bioluminescence generating reagents and the bioluminescence
generating reagents are provided as separate compositions, in time
release vehicles or other delivery vehicles, and are mixed into the
material prior to use.
[0512] Another slimy material provided herein is prepared from 24%
sodium tetraborate 2-3 ml and 2-8% polyvinyl alcohol mixed with 10
ml add 100 .mu.gs charged aequorin or other suitable luciferase.
When used with aequorin, addition of a little water [tap water or
other calcium-containing aqueous medium] results in slime material
that lights up. As mentioned above, one embodiment of an apparatus
designed for containing and delivering the slimy play material is
shown in FIG. 27. The apparatus is a compressible apparatus, for
example, like a toothpaste tube, having one, two or three,
preferably two, compartments inside the compressible apparatus. The
compartments are formed, at least in part, of a readily rupturable
material, such as plastic, such that upon squeezing the
compressible apparatus, the contents of the compartments are
released into the main body of the apparatus and are thereby
mixed.
[0513] One compartment of the compressible apparatus may contain
slime material with a luciferase and the other compartment contain
the remaining bioluminescence generating components or the
remaining components in slime. Alternatively, one compartment
contains sodium tetraborate and luciferase and the other
compartment contains the polyvinyl alcohol. In a three compartment
system, one compartment may contain luciferin and luciferase
packaged in the absence of oxygen. The second compartment may
contain the polyvinyl alcohol and the third compartment contain the
sodium tetraborate. The main body of the compressible apparatus
would then contain the remaining slime material ingredients and the
remaining bioluminescence generating reagents, such as calcium ion.
If oxygen is the final bioluminescence generating reagent required,
it may be present in the aqueous slime material composition present
in the main body of the apparatus, or it may be provided by the
atmosphere when the slime material is expelled. Other variations in
which the components are separated are also contemplated
herein.
[0514] Other alternative embodiments of the moldable play materials
include those fabricated from dimethyl silicone treated with a
compound of boron preferably followed by further treatment using
heat and/or a catalyst, as described in U.S. Pat. No. 2,541,851;
those fabricated from manogalactan gum, alkali metal borate, boric
acid, high molecular weight polysaccharide, bacteriostat,
fungistat, filler, colorant and perfume, as described in U.S. Pat.
No. 3,384,498; those fabricated from material fillers, such as clay
and talc, together with hydrocarbon petroleum distillate oil, waxy
paraffinic hydrocarbon oil, a liquid silicone compound, an
astringent, a humectant, glue and water, such as described in U.S.
Pat. No. 3,804,654; those fabricated from synthetic resin and a
wooden powder together with an oil formulation, where the synthetic
resin is a rubber reinforced styrene resin and the oil used is a
hydrocarbon oil utilizing an aromatic ring forming carbon, such as
described in U.S. Pat. No. 4,624,976; or those fabricated from wood
flower combined with a water-based gel using cross-linkable guar
gum as a gellant, such as described in U.S. Pat. No. 4,735,660.
[0515] The glycerin based slimy play materials, such as those
described in U.S. Pat. No. 5,310,4211 contains 2.5-4.0 by weight
3.33 weight %, of a self-crosslinking sodium alginate; 1.0-3.5
weight % of a glycerin and water composition in excess of 90%
glycerin; a preservative; 4.0 weight % NaCl; and water, and can
include 0.04-0.08 weight % of a colorant. As modified herein, it
will also include up to all but one component of a bioluminescence
generating system, such as a luciferase, such as Renilla or Vargula
or a firefly luciferase, or a luciferin and luciferase, such as the
Aequorin photoprotein and EDTA. A second mixture of the slime
material will contain the remaining components.
[0516] A preferred slimy material contains 2.5-4.0% by weight,
preferably 3.33% by weight, of a self-crosslinking sodium alginate;
1.0-3.5% by weight of a glycerin and water solution in excess of
90% glycerin; one or more preservatives; 2.0-7.0%, preferably about
4%, by weight NaCl; and water, and can include 0.04-0.08% by weight
of one or more colorants. The material will also include up to all
but one component of a bioluminescence generating system, such as a
luciferase, such as Renilla or Vargula or a firefly luciferase, or
a luciferin and luciferase, such as the Aequorin photoprotein and
EDTA.
[0517] The slimy play material may be made to glow by contacting it
with a second mixture of the slime material containing the
remaining components of the bioluminescence generating system or by
contacting it with the air or an aqueous composition, where
molecular oxygen or calcium ion is required to complete the
reaction. The second mixture can also contain a different colorant,
so that upon mixing not only will the material glow, it will change
color.
[0518] The concentrations of bioluminescence system components,
such as luciferase, will be those sufficient to generate a visible
glow. The concentrations of luciferase can be empirically
determined, but generally will be between about 0.1 and 1 mg per
liter of material. The amount of luciferin generally will be in
excess. The luciferases and luciferin and other components can also
be provided as time release vehicles in the material or provided
separately for subsequent addition.
[0519] This slime material can be packaged as a kit or article of
manufacture containing a first slime composition containing all but
at least one bioluminescence generating reagent, and a second slime
composition containing the remaining components. The kit will
include instructions for mixing the two compositions to produce a
glowing composition. The kit can also contain additional
compositions or vehicles or dried powders of bioluminescence
generating reagents so that they can be added prior to use so that
the material can be reused.
[0520] In another embodiment, discussed further below and in the
Examples, the slimy play material is packaged in a compressible
dispensing apparatus, for example, as illustrated in FIG. 27. In
such an apparatus, up to all except for one of the bioluminescence
generating reagents may be provided in a compartment situated
within the dispensing apparatus. A second compartment within the
apparatus may contain less than all the components required to
complete the slimy play material composition, and the main body of
the apparatus may contain the remaining bioluminescence generating
reagents and/or remaining slimy play material components.
[0521] Alternatively, for example, three compartments within the
compressible dispensing apparatus may be provided such that the
third compartment contains one or all of the remaining
bioluminescence generating reagents or the remaining slimy play
material components. The main body of the apparatus would then
contain a composition, typically an aqueous solution within, which
to mix the contents of the three compartments or the
bioluminescence generating reagents or slimy play material
components not contained within the third compartment.
[0522] In still other embodiments, the slime material is provided
without bioluminescence generating reagents and the bioluminescence
generating reagents are provided as separate compositions, in time
release vehicles or other delivery vehicles, and are mixed into the
material prior to use.
[0523] Another slimy material provided herein is prepared from 24%
sodium tetraborate 2-3 ml and 2-8% polyvinyl alcohol mixed with 10
ml add 100 .mu.gs charged aequorin or other suitable luciferase.
When used with aequorin, addition of a little water [tap water or
other calcium-containing composition] results in slime material
that lights up. As mentioned above, one embodiment of an apparatus
designed for containing and delivering the slimy play material is
shown in FIG. 27. The apparatus is a compressible apparatus, for
example, like a toothpaste tube, having one, two or three,
preferably two, compartments inside the compressible apparatus. The
compartments are formed, at least in part, of a readily rupturable
material, such as plastic, such that upon squeezing the
compressible apparatus, the contents of the compartments are
released into the main body of the apparatus and are thereby
mixed.
[0524] One compartment of the compressible apparatus may contain
slime material with a luciferase and the other compartment contain
the remaining bioluminescence generating components or the
remaining components in slime. Alternatively, one compartment
contains sodium tetraborate and luciferase and the other
compartment contains the polyvinyl alcohol. In a three compartment
system, one compartment may contain luciferin and luciferase
packaged in the absence of oxygen. The second compartment may
contain the polyvinyl alcohol and the third compartment contain the
sodium tetraborate. The main body of the compressible apparatus
would then contain the remaining slime material ingredients and the
remaining bioluminescence generating reagents, such as calcium ion.
If oxygen is the final bioluminescence generating reagent required,
it may be present in the aqueous slime material composition present
in the main body of the apparatus, or it may be provided by the
atmosphere when the slime material is expelled. Other variations in
which the components are separated are also contemplated
herein.
[0525] Other toys, games, novelty items, clothes, accessories,
foods, beverages, fountains, water dispensing apparatus, soaps,
creams, cosmetics and sporting equipment amenable to
bioluminescence are further embodiments of the presently disclosed
combination. Thus, any article of manufacture or substance capable
of modification to allow bioluminescence thereof is contemplated
herein.
[0526] Articles of manufacture that are amenable to use with the
bioluminescence generating systems provided herein are well known
[see, e.g., U.S. Pat. Nos. 5,415,151, 5,018,449, 3,539,794,
5,171,081, 4,687,663, 5,038,963, 4,765,510, 4,282,678, 5,366,108,
5,398,827, 5,397,014, 5,219,096, 5,305,919, 5,184,755, 5,029,732,
4,214,674,4,750,641, 4,676,406], which describe devices useful as
toy water guns or vessels for beverages or creams and lotions. To
be amenable to use in the embodiments described herein, each may
require some modification, such as, for example, addition of a
mixing chamber.
[0527] In light of the disclosure herein, such modification will be
apparent. Some of the patents describe other toy devices, training
mock weapon devices, dolls, and beverage containers and dentifrice
containers [i.e., toothpaste tubes]. In the simplest modification,
powdered or capsular vehicles containing bioluminescence generating
systems may be added to the water-holding chambers of the toy gun
or other water spewing toy. As the powder dissolves or the vehicle
releases its contents, typically luciferin and luciferase, contact
with the water in the gun will cause the bioluminescence reaction
to occur.
[0528] As is apparent from the above, toy guns are well known items
and materials and specifications for manufacture thereof are also
well known (see, the above list and see, also, U.S. Pat. Nos.
5,029,732, and 5,415,151]. Any single chamber squirt gun may used
in combination with bioluminescence generating systems herein by
mixing the components in the gun chamber. Of course the selected
system should be one that has sustained illumination.
Alternatively, pellets of encapsulated bioluminescent components,
such as the aequorin photoprotein or the Renilla luciferase and
luciferin, may be added to water in the gun chamber. In the case of
the aequorin photoprotein and Renilla luciferase, added tap water
may be sufficient. For the Renilla system the pellets may contain
the luciferase and luciferin or either. The remaining component
will be added to the gun chamber. If pellets are used, the pellets
will slowly release their contents thereby providing for a
continuous glow.
[0529] Similar apparatus and designs are also used for any fountain
or water propelling device. Any such device [see, e.g., U.S. Pat.
No. 5,360,142] may be modified to include a bioluminescence system
to produce a glowing stream.
[0530] In all of these devices, the water, for example, can be tap
water or a selected buffer, particularly phosphate buffered saline.
The items may packaged as kits with the packaged luciferin,
luciferase, and including the water.
[0531] a. Single Chamber Toy Guns and Other Toy Weapons that Shoot
Pellets or Liquid
[0532] Numerous toy guns and other toy weapons that shoot pellets
or liquid, in addition to those exemplified herein, are suitable
for use in combination with the bioluminescence generating systems
herein. The toy weapons may be loaded with a composition containing
microspheres of luciferin and/or luciferase, or with lyophilized
luciferin/luciferin, or other mixtures as described herein.
Suitable toy weapons and devices that shoot jets or sprays of water
are described in the following sampling of U.S. Pat. No. 5,462,469
[toy gun that shoots bubbles]; U.S. Pat. No. 5,448,984 [toy gun
that shoots balls and water and can be modified to shoot light or
temperature sensitive pellets, which should be stored under
appropriate conditions or appropriately packaged, that release
luciferin/luciferase when exposed to light]; U.S. Pat. Nos.
5,439,139; 5,427,320; 5,419,458; 5,381,928; 5,377,656; 5,373,975;
5,373,833 and 5,373,832 [which describe toy guns that rely upon a
pressurizable bladder for release of air-pressure to shoot a
projectile, which can be modified to shoot projectiles of
encapsulated luciferin/luciferase]; U.S. Pat. Nos. 5,370,278 [which
describes liquid from a port mounted to a headband]; U.S. Pat. Nos.
5,366,108; 5,360,142 [which describes a supply and delivery
assembly for use in combination with a pump type water gun or other
water propelling device]; U.S. Pat. Nos. 5,346,418; 5,343,850
[which describes a projectile launcher for use in combination with
the pellets provided herein]; U.S. Pat. Nos. 5,343,849; 5,339,987
[which describes water guns that have at least one pressurizable
air/water storage tank, a pressurizing mechanism, a channel of
release for shooting water and a release mechanism]; U.S. Pat. Nos.
5,326,303; 5,322,191; 5,305,919; 5,303,847 [which describes a
device worn on a user's hand with sheaths for the tips of the
fingers that includes a housing for a water reservoir, a water pump
and electrical motor and a battery pack to be secured to the user's
body]; U.S. Pat. Nos. 5,292,032; 5,284,274 [which describes an
action to system including a capsule for containing water, which
will herein contain components of a bioluminescence generating
system, having an orifice and a plunger and a spring loaded
mechanism for driving the water from the orifice. The action toy
may be configured as a shotgun accepting a plurality of prefilled
shell capsules into its breechblock for firing through its barrel,
as a missile launcher in which the capsules are mounted to the
front of the launcher and the water is ejected directly from the
capsule against the target, or as a crossbow with the bow loading
the spring-loaded mechanism and a water stream obtained on release
of the bow]; U.S. Pat. No. 5,284,272 [which describes a bottle and
cap combination for spewing liquid]; U.S. Pat. Nos. 5,256,099;
5,244,153; 5,241,944; 5,238,149; 5,234,129; 5,224,625; 5,213,335;
4,854,480; 5,213,089; 5,184,755; 5,174,477; 5,150,819; 5,141,467;
5,141,462; 5,088,950; 5,071,387 [which describes a figurine-shaped
water squirting toy]; U.S. Pat. No. 5,064,095 [which describes a
water cannon apparatus]; U.S. Pat. Nos. 5,029,732; 5,004,444;
4,892,228; 4,867,208 [which describes an apparatus for storing and
dispensing fluid under pressure]; U.S. Pat. Nos. 4,808,143;
4,784,293, 4,768,681; 4,733,799; 4,615,488 and many others. U.S.
Pat. No. 5,415,151 describes a toy gun that launches projectiles
that can be adapted for shooting the pellets, such as light
sensitive pellets that are degraded upon exposure to light,
provided herein.
[0533] b. Bubble-Making Toys
[0534] Soap bubbles are blown from water solutions or other aqueous
composition containing soap or another surfactant. A great variety
of bubble formulations are available, including those that feature
special effects in bubble making. There are solutions for making
large bubbles, "long lasting" bubbles, split bubbles, self-healing
bubbles, multiple bubbles, vanishing bubbles, flaking bubbles,
bursting bubbles, high and/or far-flying bubbles, sinking bubbles
etc. In general, many anionic, non-ionic or amphoteric aqueous
solutions with low surface tension are suitable for bubble or
foam-making when air or other gases are blown into such
compositions.
[0535] Such compositions, preferably those that have near neutral
pH, can be combined with the components of the bioluminescence
generating systems provided herein. In particular, a mixture of
luciferase and luciferin, such as the Renilla system or firefly
system or Cypridina system, preferably in the form of pellets or
microspheres, such as liposomes or other time release capsule, can
be added to the bubble mixture. When used, the air added to the
mixture will cause a glow, or a glow will be produced as the
contents of the pellets are released into the composition.
Alternatively, one or more component of the bioluminescence
generating system may be added to the bubble making composition,
such as, for example, a luciferase and any necessary activators,
and the remaining component(s), e.g., a luciferin, may be directly
applied to bubbles using a fine spray from an atomizer or other
suitable spray or misting means.
[0536] In addition, a fluorescent protein, such as GFP, BFP or a
phycobiliprotein, may be added to the bubble-making composition and
then illuminated using an external light source. For example,
bubbles containing a fluorescent protein may be produced in a room
illuminated with light of an appropriate wavelength to cause the
fluorescent protein to fluoresce.
[0537] Alternatively, the fluorescent protein may be added to the
bubble-making composition containing all the components of the
bioluminescence generating system to effect a change of the color
of the bubbles. For example, the fluorescent proteins may be added
to the bubble-making composition directly or may be added in
time-released or slowly-dissolving microspheres or liposomes, such
that release of a fluorescent protein in the bubble composition,
such as, for example, GFP or a phycobiliprotein, introduces a
change in the color of the bubbles. It is particularly advantageous
to have the fluorescent protein released into the composition after
the container has been opened and used. A single bottle of
bubble-making solution will be amenable to the production of more
than one color of bubbles. For example, microparticles or liposomes
suspectible to breakdown by exposure to air or by agitation by the
wand or stick used for blowing bubbles are of particular
interest.
[0538] Kits containing such soap compositions, with preferably a
moderate pH [between 5 and 8] and bioluminescence generating
reagents, including luciferase and luciferin and the fluorescent
protein are provided herein. These kits, for example, can be used
with a bubble-blowing or producing toy. These kits can also include
a reloading or charging cartridge, such as the cartridges provided
herein.
[0539] Toys that produce bubbles include bubbles with wand for
blowing, bicycles, flying toys, dolls, swords, toy musical
instruments, bubble beards, and numerous other toys are well known
[see, e.g., U.S. Pat. No. RE 32,973, which describes a toy
bubble-blowing lawn mower; U.S. Pat. No. 4,511,497, which describes
a non-toxic non-irritating bubble composition for toys, U.S. Pat.
Nos. 2,579,714; 5,480,334; 5,041,042; 5,478,267; 5,462,469;
5,419,728; 5,393,256; 5,366,402; 5,348,507; 5,322,464; 5,304,085;
5,269,715; 5,224,893; 5,183,428; 5,181,875; 5,156,564; 5,135,422;
5,080,623; 5,078,636; 4,957,464; 4,955,840; 4,943,255; 4,923,426,
4,867,724; 4,861,303; 4,840,597; 4,808,138; 4,804,346; 4,764,141;
4,700,965; 4,556,392 4,334,383; 4,292,754; 4,246,717; and many
others].
[0540] c. Board/Card Games
[0541] Board games, card games and similar entertainment items may
be used in combination with the bioluminescence generating systems
described herein. The boards or cards may be constructed of paper
or fabric, as described herein, or may be constructed of plastic or
other polymer amenable to covalent or non-covalent attachment of
bioluminescence generating compontents.
[0542] A particular portion of the game board or a card piece is
covered or impregnated one or more up to all but one of the
bioluminescence components. A developing wand or sponge or similar
apparatus is impregnated or coated or dispenses the remaining
bioluminescence component(s) [developing reagents]. Contacting,
such as by wiping, the card piece or game board with the developing
wand or sponge or contents of the dispensing apparatus will produce
a glow.
[0543] The developing reagents can be applied to the developing
wand or sponge in various forms. For example, the developing
reagents may be in solution or suspension and the sponge or wand
soaked in the solution then sealed in an air-tight packaging to be
opened immediately before use.
[0544] Alternatively, the developing reagents may be lyophilized or
dessicated and applied in powder form to the wand or sponge.
Immediately before use, water is added to the wand or sponge and
then wiped on the game board or card piece.
[0545] Alternatively, the board and pieces may include adsorbed or
absorbed lyophilized bioluminescence-generating reagents.
Contacting these items with water, containing the appropriate salts
and buffers, such as calcium, if for example, the aqueorin system
is used, or ATP if the firefly system is used.
[0546] The bioluminescence components applied to the game board or
card piece can be applied in a particular pattern, for example to
spell a word or illustrate an instruction. Preferably, the
bioluminescence system chosen will be capable of repeated use. For
example, the Renilla system, is among the preferred systems. The
luciferase can be linked to the pieces, and the luciferin can be
applied to the board or card and a new developing wand or sponge
used each time the game is played.
[0547] Alternative embodiments will be appreciated, for example,
the game can be an educational one in which the player uses the
developing wand or sponge to reveal the correct answer to a
question. Similarly, the game board may be a puzzle where a
"hidden" illustration or message is revealed by wiping the
completed puzzle with the developing wand or sponge.
[0548] d. Toy "Eggs" or Other Encapsulated Items
[0549] Egg-shaped (or any other desired shape) toys containing a
liquid or paste that glows upon exposure to ambient air are a
further example of a combination contemplated herein. The
ingredients of the egg composition include a luciferin and
luciferase, such as the Cypridina or Vargula luciferin and
luciferase, which requires oxygen for activation. The liquid or
paste is introduced into the "eggs" the eggs are sealed under
nitrogen or other suitable gas, other than oxygen or air. Upon
exposure to air, by opening or cracking the egg, the egg
composition glows. This principle can be adapted to other uses,
such as sphere shaped macrocapsules that may be shot from a toy gun
and burst upon impact, in a manner similar to paint ball guns
currently used to shoot paint balls at targets for marking. In
practice, water is de-oxygenated, for example by bubbling argon or
nitrogen gas through it. The de-oxygenated water is then used to
mix the bioluminescence generating components, other than molecular
oxygen. The mixing should take place under strictly conditions in
which air or oxygen is excluded, such as in a hood under nitrogen,
in order to prevent exhaustion of the bioluminescence-generating
components.
[0550] In one embodiment, to produce a realistic egg-like mixture,
approximately 1 to 2 mg of a luciferin/luciferase composition per
30 ml of egg volume is combined with a suitable thickener, such as
hydroxymethyl cellulose, to provide the consistency of a real egg.
The "shell" of the egg is formed of a suitable material which
excludes oxygen (air) and is readily opened by the consumer before
use. For example, the egg mixture can be packed into paper mache
and covered with wax to provide an airtight seal. Similarly, the
"shell" may be formed from a polymer, such as a plastic, that is
airtight but readily broken when desired.
[0551] e. Footbags, Bean Bags and Balls
[0552] Glowing footbags, bean bags and balls are also provided
herein. Footbags, such as the HACKY SACK, which is a registered
Trademark of Wham-O Corporation, described in U.S. Pat. No.
4,151,994, are generally constructed of an outer leather casing
having a diameter of about three inches, which is filled with small
granules, such as beans or other granular material [see, also, U.S.
Pat. Nos. 5,429,351, 4,963,117, 4,717,158, and 4,002,839]. The sack
is used to play a game in which players kick the sack between one
another, trying to keep the sack in motion and off the ground,
without using their hands.
[0553] Contemplated herein are footbags and balls that glow as they
are kicked about by the players. The bags are fabricated from an
inflatable translucent material, such as a plastic, Similar to the
egg mixture described above, the granules in the footbag are made
in an oxygen free environment and packaged such that air/oxygen is
excluded until the sack is in use. For example, the granules are
made of a gelatinized mixture of bioluminescence generating system
components excluding molecular oxygen and are packaged in an oxygen
free package, such as dry nitrogen packaging, commonly used in
marine electronics, or in rupturable liposomal pellets.
[0554] The granules can be covered in a flexible plastic of varying
thicknesses to allow for the timed ingress of oxygen across the
plastic membrane. As the footbag is repeatedly kicked by the
players, the mechanical stress on the granules allows more oxygen
to react with the bioluminescence generating components contained
therein, creating more light.
[0555] An alternative embodiment contemplated herein involves
partitioning the granules within the footbag using, for example, a
semi-permeable membrane material that permits slow permeation of
the compositions contained in the two compartments thereby formed.
One compartment is then filled with all but one or more
bioluminescence components and the other compartment is filled with
the remaining components. As the footbag is kicked about, the
mechanical stresses on the separating membraned force the contents
of the two compartments to mix, thereby providing flashes of light
or periods of illumination followed by non-illumination, For
example, in one compartment, a calcium containing composition can
be added to the beads, and in the other compartment, a
coelenterazine-charged aequorin is added. When the footbag is
kicked, flashes of light are produced.
[0556] The covering of the footbag must be translucent, transparent
or some combination thereof to allow the light generated to be
visible. Thus, the "sack" can be formed from clear nylon webbing,
translucent or transparent pliable plastic, translucent or
transparent cloth or similar material.
[0557] f. Figurines
[0558] Glowing figurines are also provided herein. Figurines may be
of any size or shape and preferably contain at least one chamber
that holds liquid. The figurine may be cast, molded or manufactured
from any suitable material. Preferably a portion of or the entire
figurine is translucent to the wavelength of light produced in the
bioluminescence generating reaction. The figurine may be in any
design or theme, such as characterizations of entertainment and
sport celebrities, memorabilia, slogans and logos, trademarks or
other promotional items, animals, Christmas ornaments or other
inanimate objects. For example, small figurines may be placed in
areas of dim lighting, e.g., on tables in restaurants, that contain
one or more component of the bioluminescence generating system,
such as a luciferase. The remaining components of the
bioluminescent reaction, i.e., a luciferin and any necessary
activators, are added at a the desired time and the figurine
glows.
[0559] In another embodiment, one or more components of the
bioluminescence generating system may incorporated into or linked
to the material from which the figurine is fabricated. The
remaining components of the bioluminescence generating reaction may
be sprayed or applied to the surface of the figurine to initiate
the bioluminescent reaction.
[0560] 3. Glowing Textiles and Paper Products
[0561] The bioluminescence generating systems described herein are
also contemplated for use with textiles and paper. One or two of
the bioluminescence generating system reagents are applied to the
textile or paper and the remaining components are added when
illumination is desired. For example, the luciferase in association
with the bioluminescence substrate may be applied to the textile or
paper, through covalent or non-covalent interaction. When water, or
other appropriate activator, is applied to the material,
illumination ensues. Examples of uses for the textile include the
fabric portion of an umbrella, clothing, towels, the fabric portion
of artificial plants or flowers, toys having a fabric component or
any item susceptible to manufacture from textile material.
[0562] With respect to paper, the luciferase may be applied to the
paper in association with the bioluminescence substrate. The paper
glows upon addition of the bioluminescence activator to the paper.
Thus, if the bioluminescence activator is water, addition of water
to the paper, for example as an aerosol, produces a glow on the
paper. The paper may also be illuminated by "writing" upon it with
one or two of the bioluminescence generating system components then
"writing" or spraying over those components with the remaining
component(s). As with the other systems disclosed herein, the
critical aspect to operation is maintaining at least one of the
bioluminescence generating system components separate from the
other components until illumination is desired. The paper may be in
almost any form or of almost any type, such as writing paper,
wrapping paper, boxes, poster paper, books, paper jewelry, paper
towels, napkins or other paper products.
[0563] 4. Foods and Beverages, Including Ice Cubes
[0564] Examples of beverages and foodstuffs amenable to combination
with bioluminescence systems include, but are not limited to,
alcoholic beverages, as well as sodas and juices, and such foods as
applesauce and mashed potatoes. Further, bioluminescence generating
systems can be chosen and adapted for use in such foodstuffs as
cakes and ice creams or almost any other edible item.
Considerations in combining bioluminescence systems with food
and/or beverages are primarily the stability of the system
throughout processing of the food or beverage, unless the system is
added subsequent to any such processing; the ability to contact the
system with its finally required ingredients to produce
bioluminescence; and taste of the components of the system with the
foodstuffs to which they are added.
[0565] Bioluminescent food products are also contemplated herein.
Such products, amenable to combination with the bioluminescence
generating systems described herein, include those that may be
stored between about 0.degree. C. and 35.degree. C. Generally, once
the luciferase or bioluminescence substrate is added to the food
product, it cannot be heated above about 100.degree. C. Thus, food
products requiring cooking prior to consumption also can be cooked
prior to addition of either the luciferase or bioluminescence
substrate.
[0566] Examples of food products amenable for use in combination
with the bioluminescence generating systems described herein
include, but are not limited to, icings and other toppings or
sauces, cookies, biscuits, and similar prepared foods.
Bioluminescent icings, for example, may be prepared by including
the luciferase and bioluminescence substrate in a dehydrated icing
mixture. Addition of water, just prior to use causes the mixture to
glow. Alternatively, the bioluminescence activator and either the
luciferase or bioluminescence substrate may be included in the
prepared icing mixture and the absent bioluminescence generating
system component stirred into the icing just prior to use.
[0567] Alternatively, food products may be produced to include a
fluorescent protein, such as a phycobiliprotein or a green or blue
fluorescent protein, and then illuminated using an external light
source. For example, icing containing fluorescent protein may be
served in a room illuminated with light of an appropriate
wavelength to cause the fluorescent protein to fluoresce.
Similarly, a fluorescent protein may be included in an ice cream
mixture, in an ice cream topping sauce, in a salad dressing, in
cakes, puddings or similar food product and the food then subjected
to an external light source of appropriate wavelength to initiate
the fluorescence.
[0568] a. Beverages
[0569] Beverage products are likewise contemplated for use herein
in combination with the bioluminescence generating systems
described herein. As with other embodiments, at least one of the
bioluminescence generating system components is excluded from the
beverage until bioluminescence is desired. For example, a
container/bladder apparatus, as described generally above and in
detail below, maintains the luciferase and bioluminescence
substrate separate from the beverage. Upon opening of the
container, the luciferase and substrate are added to the beverage
causing it to glow.
[0570] Alternatively, the beverage may be produced and packaged
already containing one or two of the bioluminescence generating
system components, such that addition of the remaining components
causes a glow. An example of such a beverage is bioluminescent
beer, wine, champagne or a soft drink. In this embodiment, the
yeast used to produce the alcohol component of the beer or other
beverage, are genetically transformed to contain, for example, a
gene encoding a luciferase and the complementary genes necessary to
direct the yeast to manufacture and secrete the luciferase.
Assuming O.sub.2 or air is the bioluminescence activator, then when
a glow is desired, the bioluminescence substrate is added to the
beer.
[0571] Another example of a bioluminescent beverage contemplated
herein is a soft drink containing two of the three bioluminescence
generating system components. When bioluminescence is desired, a
third bioluminescence generating system component is added. If the
bioluminescence generating system is, for example, the Aequorin
system or the Renilla system, then the Aequorin luciferase with
bound luciferin or the Renilla luciferase and the luciferin may be
included in the soft drink and the bioluminescence activator,
Ca.sup.2+ [for the aequorin system] or dissolved O.sub.2, added to
the beverage to cause a glow. Suitable vessels for such beverages
are provided herein [see, EXAMPLES] and also are known to those of
skill in the art [see, e.g., U.S. Pat. No. 5,398,827].
[0572] Similarly, a soft drink beverage can be produced containing
all the bioluminescence generating system components except, for
example, dissolved oxygen where the bioluminescence generating
selected requires oxygen to complete the bioluminescent reaction.
In lieu of carbon dioxide, the beverage may have another gas or
gasses dissolved therein, for example nitrogen, helium, nitrous
oxides or helium oxygen (heliox). The soft drink is packaged under
oxygen free conditions and, upon opening of the soft drink
container and exposure of its contents to the air, the oxygen in
the air activates the bioluminescent reaction causing the soft
drink to glow.
[0573] In each of the above embodiments, it is also contemplated
that slowly-dissolving or time releasing microparticies, such as,
but not limited to liposome or isolated endosomes, may be included
in the beverage that contains additional bioluminescent components.
Microparticles may contain, for example, one or more luciferases, a
phycobiliprotein, a green or blue fluorescent protein, a luciferin
or any mixture or combination thereof. Upon dissolution of the
microparticle or release of the contents by other means, the
contents of the microparticle are released into the beverage or
other liquid, resulting, for example, in a change in the color of
the emitted light the beverage, an change in the color of the
bioluminescent light and/or an increase in the intensity of the
emitted light of the entire beverage or just a portion thereof. By
selecting the appropriate microparticle(s), the release of one or
more component of the reaction may be effected sequentially or
concurrently. Thus, drinks in which several glowing colors are
produced are contemplated herein. Multiple color changes are
effected by the appropriate selection of bioluminescence generating
agents and/or fluorescent proteins.
[0574] For example, an appropriate time-released or
slowly-dissolving microparticle containing a GFP or a
phycobiliprotein may be added to a beverage containing the Renilla
or aequorin bioluminescence generating system. Upon dissolution or
release of the fluorescent protein into the medium, the initial
blue color of the glowing beverage is converted to another color,
e.g., converted to a green color by the GFP. The inclusion of an
additional microparticle containing a phycobiliprotein with an
absorption maxima in the green spectra, in which the microparticle
has been selectively designed to dissolve or release into the
beverage after release of the GFP, would result in the beverage
once again changing color to, for example, red. The color of the
beverage may be changed sequentially and repeated as many times as
desired. The number of possible color changes will depend on the
type of beverage, the desired colors and the duration of each
color. Any beverage is contemplated for the color changes as
described herein, such as soft drinks, alcoholic beverages, juices
and the like.
[0575] Alternatively, the color change may be designed to be
effected in only a portion of the beverage. For example,
microparticles that contain a fluorescent protein in combination
with a composition that has a higher or lower specific density than
the beverage [e.g., a saturated sucrose solution or any suitable
non-toxic, highly viscous solution having a higher specific
density]. Dissolution or release of the contents of the
microparticle results in the formation of a biphasic solution in
which, for example, the top portion of the beverage glows blue
whereas the bottom portion of the beverage containing the released
fluorescent protein [e.g., GFP or a phycobiliprotein] glows green,
red or another color. The concentration of the fluorescent proteins
and the selection of a higher or lower density liquid and
percentages to be used herein may be determined empirically by one
of skill in the art.
[0576] The color of each layer may be changed sequentially or the
color change may be effectively repeated in any order depending on
the microparticle or macroparticle employed [e.g., inclusion by
direct addition, time releasing particles or thermal or pH
sensitive microparticles].
[0577] b. Ice
[0578] Ice containing bioluminescent components, such as
lyophilized components or encapsulated components is contemplated
herein. Upon addition to a liquid containing any remaining
components or exposure to air, the contents of the ice will be
released as they melt to produce a glow. The ice may be in any
shape or form. Examples of ice formations, include but are not
limited to, geometric shapes, such as spheres and cubes; ice
formations made from precast molds, such as figurines, icicles,
popsicles; shaved ice, such as snow cones or imitation snow for
recreational activity like skiing, sledding or snow-mobiling; ice
sculptures, where the ice glows and/or in combination an inanimate
object frozen within the ice that glows. In addition, ice used as a
surface for recreational ice skating or hockey is also contemplated
herein.
[0579] The ice may contain one or more of the bioluminescence
generating components. For example, the ingredients of ice may
include a luciferin and/or luciferase, such as the Cypridina or
Vargula luciferin and luciferase, which requires oxygen for
activation. Luciferases isolated from different specie that result
in the production of light other than green or blue, e.g.,
Aristostomias or Pachystomias which emit red light, or additional
components which alter the wavelength of the emitted light, e.g., a
green fluorescent protein or a phycobiliprotein, used in
conjunction with the luciferase are also contemplated herein.
[0580] In practice, water is de-oxygenated, for example, by
bubbling argon or nitrogen gas through it. The de-oxygenated water
is used to mix all of the bioluminescence generating components
besides molecular oxygen. The mixing should take place under strict
conditions in which air or oxygen is excluded, such as in a fume
hood under nitrogen, in order to prevent exhaustion of the
bioluminescence-generating components.
[0581] The water is placed in a tray, a vessel, a precast form of a
particular shape or design, stored or maintained under an inert
atmosphere and snap frozen using liquid nitrogen. The resulting ice
is packaged in a sealed container under an inert atmosphere lacking
molecular oxygen (e.g., argon or nitrogen). Upon exposure to air or
a liquid containing dissolved oxygen, the ice glows.
[0582] Alternatively, one or more component of the bioluminescence
generating system may be applied to the surface of the ice to
initiate or re-generate the bioluminescent reaction. This method is
particularly suitable for production of a glowing ice surface, such
as an ice skating rink. The components of the reaction may be added
to the water contained within the Zamboni ice cleaning machine. The
water from the machine is overlayed over the existing ice, which
contains (or is first coated on the surface) at least one component
of the bioluminescence generating system, as a thin coating of a
composition that contains the other one or more component(s) of the
bioluminescence generating system. As the two layers meet, the
bioluminescence generating system is produced or restored and the
ice glows.
[0583] Furthermore, microparticles containing additional
bioluminescence generating components may be added to water prior
to snap freezing. For example, microparticles containing or coupled
to a phycobiliprotein or a green/and or blue fluorescent protein
(GFP) can be produced. The additional components may also be added
to the surface of the ice after freezing. As with the beverages,
described above, as the microparticles dissolve in the ice or as
the ice melts, the fluorescent protein or other components are
released. The presence of the fluorescent protein converts the
wavelength of the light emitted from the surface or interior of the
ice, which can include the components of a bioluminescence
generating system, thereby changing the color of the ice or liquid,
for example, from blue to green or red. The addition of GFP also
increases the intensity of the green light emitted about 2-5-fold.
Thus, a beverage containing such ice would not only change color as
time proceeds but also glow more brilliantly. The light insenity of
the liquid could also be enhanced by the addition of microparticles
containing an appropriate luciferin or activator that upon
dissolving would provide additional substrate to promote the
bioluminescent reaction.
[0584] The components may also be combined with dry ice, which as
it sublimes, will release the components that contact with moisture
condensing in the air. This will produce a glowing fog for use, for
example, in theatrical productions.
[0585] c. Other Foods
[0586] Other foods contemplated herein include a transgenic corn
that expresses luciferase. The corn is served with butter
containing luciferin, particularly coelentrazine, and as result
glows.
[0587] Alternatively, the popcorn is engineered to express a
fluorescent protein. When exposed to the appropriate light, the
corn will glow.
[0588] 5. Jewelry, Clothing and Other Items of Manufacture
[0589] The bioluminescence generating systems and fluorescent
proteins can be used in combination with articles of manufacture
that include jewelry, clothing, figurines and other such items. In
particular, these items may be manufactured from matrix materials
or from mixtures of the matrix material and other materials.
Alternatively, the matrix material may be coated on or impregnated
in such articles. Bioluminescence generating reagents,
particularly, luciferases can be linked to the matrix material.
When a glow is desired the article can be contacted with
composition containing the remaining components.
[0590] In addition, articles, such as clothing, particularly,
T-shirts and sports gear, and paper items may be sprayed with two
compositions, the first containing less than all of the necessary
reagents and the second containing the remaining reagents.
[0591] In other embodiments, the article may be made of two vessels
separated by a removable separating means, so that when desired
components contained therein communicate and react resulting in
bioluminescence.
[0592] 6. Fountains
[0593] Numerous fountains and other water spraying apparatus and
devices for use in such apparatus, in addition to those exemplified
herein, are suitable for use in combination with the
bioluminescence generating systems herein [see, e.g., U.S. Pat.
Nos. 5,480,094; 5,472,140; 5,439,170; 5,402,836; 5,388,285;
5,381,956; 5,337,956; 5,288,018; 5,167,368; 4,852,801; 3,894,689;
3,889,880; 3,838,816; 3,820,715; 3,773,258; 3,749,311]. For use
herein, the fountains will be modified or adapted [see, e.g.,
EXAMPLES] so that jets of liquid containing bioluminescent will
spew.
[0594] Fountains can be recharged, for example, by adding
additional substrate and other activators. Spent substrate should
be removed, such as by passing the water through an affinity matrix
specific for the oxidized substrate.
[0595] 7. Non-Tobacco Cigarettes
[0596] Also contemplated herein is a novelty item that is shaped
like a cigarette and that includes a bioluminescence generating
system, which produces glowing "smoke" upon exhalation by the user.
The user contemplated herein is an adult former smoker who derived
pleasure from blowing smoke rings. The toy cigarette can be made,
for example, by placing, under oxygen free conditions, a
lyophilized, micropulverized mixture of the bioluminescence
generating system components into liposomes, as described above, or
other packaging material, such as porous plastic microspheres, made
from TYGON or other biocompatible non-toxic material. The liposomes
(or other packaging) are selected to be of a suitable size to
facilitate or permit passage into the bronchioles of the user. The
liposomes are preferably on the order of 5-10 .mu.M in diameter and
are situated in a tubular delivery vehicle [the "cigarette"].
[0597] An example of an appropriate delivery vehicle is a thin
glass vial surrounded by plastic, similar to vials known to those
of skill in the art that are used for storing amyl nitrate,
betadine and benzoin solutions. The delivery vehicle is preferably
shaped and sized like a standard cigarette. The plastic covering is
preferably cylindrical with each end open to allow for the passage
of air upon inspiration. The plastic covering is surrounded by a
filter material that allows passage of the liposomes from the
device, but prevents the accidental inhalation of particulates,
such as glass, if the vial is broken. Additional filters, having
pore sizes of about 10 .mu.M, are placed at either end of the
"cigarette" as a further barrier to inhalation of any material
larger than the liposomes. Solid plastic or similar material caps
may be situated over each end of the "cigarette" to prevent the
liposomes contained therein from falling out. These caps would be
removed just prior to use of the "cigarette", to permit the free
flow of air through the device. The liposomes are preferably held
within the delivery vehicle by friction.
[0598] In operation, the user will inhale the liposomes or similar
encapsulating vehicles, which release their contents upon
contacting the lungs. The humid environment of the bronchial tree
then provides the water and oxygen necessary to complete the
bioluminescence reaction. Upon exhalation, the air leaving the
users lungs is illuminated, providing glowing "smoke". If the
packaging apparatus chosen for the bioluminescence generating
components is a porous plastic microsphere, such as TYGON, then the
bronchiolar-ciliary transport mechanism of the body will transport
the spent microspheres out of the bronchia and into the digestive
system. Because plastic is biologically non-reactive, the
microspheres will be passed from the body through normal excretory
pathways without eliciting an immune or toxic reaction.
[0599] 8. Fish and Fish Food and Fish Bait
[0600] Also contemplated herein are genetically engineered fish
that express luciferin or, preferably luciferase, and food
therefor. Such fish may be produced may any method known to those
of skill in the art for preparation of transgenic fish. For
example, to produce the fish, fish eggs are transfected with a gene
encoding a particular luciferase and any other genes or regulatory
sequences necessary to direct the fish to manufacture and express
the luciferase, using methods known to those of skill in the art.
Methods for generating transgenic fish are known [see, e.g., U.S.
Pat. Nos. 5,512,421, 5,510,099, 5,489,742, 5,476,779, 5,416,017 and
5,166,065; see, also, Ozato et al. (1986) Cell Differ. Devel.
19:237-244, Inoue et al. (1990) Cell Differ. Devel. 29:123-128,
Rokkones et al. (1989) J. Comp. Phyiol.B 158::751-758, and Guyomard
et al. (1989) Biochimie 71:857-863, which describe preparation of
transgenic medaka, medaka, salmon and trout, respectively].
Transgenic fish of numerous species have been prepared, providing
the skilled artisan with a variety of procedures for developing
transgenic fish. Thus, using a transfection methods known to those
of skill in the art and methods for introduction and expression of
luciferase, transgenic fish that express a luciferase are prepared.
Desirably, the fish express the luciferase on cell surfaces, such
as by incorporating the luciferase into DNA encoding a
membrane-spanning protein, or express the luciferase so that it is
secreted into the digestive systems or mouths of the fish.
[0601] The resulting fish are fed food containing an appropriate
luciferin or luciferins [or luciferase] and any additional
bioluminescence generating reagents required. Typically, the
luciferin will be present in the fish food at concentrations
ranging from about 1 part per million (ppm) to about 1 part per 10,
weight/weight. As the luciferin, bioluminescent activators and
other system components come in contact with the luciferase
expressed by the transgenic fish, the fish or selected organs or
tissues will glow. For example, if the luciferase is expressed on
the tissues lining the transgenic fish's mouth, then its mouth will
light up as it eats the fish food. Similarly, if the fish
transfected with the luciferase gene is translucent, then the
digestive organs, particularly the stomach, will glow as the
bioluminescence generating components come into contact and
complete the bioluminescent reaction. The selected
luciferase/luciferin systems should be one that is resistant to
conditions, such as the acidic pH of the digestive system, in the
fish.
[0602] Thus, for purposes herein, fish food that includes
luciferin, preferably in lyophilized form, particularly, Renilla
coelenterazine and Vargula luciferin, is provided. The transgenic
fish that express luciferase or luciferin are also provided.
[0603] Also contemplated herein, are glowing fish lures for use as
fish bait.
[0604] 9. Plant Food
[0605] Plant food, containing a luciferase or luciferin, for use
with transgenic plants that express a luciferin or luciferase. For
example, transgenic plants that express a luciferase are known
[see, e.g., U.S. Pat. Nos. 5,464,758, 5,436,392, 5,432,081,
5,412,085, 5,362,865, 5,268,463, and 5,015,580]. When treated with
[i.e., fed] plant food containing a luciferase and other needed
components of the bioluminescence generating system, these plants
glow.
[0606] Plant food containing one or more components of the
bioluminescence generating system, preferably a luciferin, is
provided herein for administration to transgenic plants that
express a luciferase. The plant food containing a luciferin and any
necessary activators may be in the form of any composition that is
typically applied to a plant to promote or maintain growth [e, see
U.S. Pat. Nos. 4,016,880, 4,711,659, 4,804,403, 5,547,486,
5,553,853, RE 35,320, and RE 31,801]. The luciferin and any
activators may be added directly to the plant food mixture or
housed in a separate compartment and added to the plant food
immediately prior to use. The plant food may be applied to the
soil, sprayed on the foliage of the plant or any combination
thereof.
[0607] F. Cartridges for Loading or Reloading the Novelty Items
[0608] In order to effectively charge, recharge or refill the
bioluminescence generating systems that are part of the novelty
items, a variety of cartridges are contemplated herein. It is to be
appreciated that any charging device discussed herein is capable of
either initially charging a novelty item, such as a squirt gun, or
recharging such a novelty item once one or more component(s) of the
bioluminescence generating system is depleted. Exemplary
embodiments are set forth in FIGS. 28-34 and described in EXAMPLE
14 below.
EXAMPLES
[0609] The following examples are included for illustrative
purposes only and are not intended to limit the scope of the
invention.
Example 1
[0610] Dual Chamber Fluid Dispensing Apparatus--Toy Water Gun
[0611] An exemplary embodiment of the dual chamber fluid dispensing
apparatus is a toy water gun as illustrated in FIGS. 1 through 3.
The following description of that preferred embodiment is made with
reference to those figures. The toy water gun includes two housings
[or chambers] 10, 12 that conveniently may be constructed of
injection-molded plastic or other suitable material. The two
housings 10, 12 are affixed, such as glued, heat sealed or by other
such means, along a median seam 46 to form the body of the water
gun. See especially FIGS. 2 and 3.
[0612] In operation, one housing 10 contains a mixture having less
than all the components necessary for generating bioluminescence
and the other housing 12 contains a mixture having the remaining
components or the remaining components, save the bioluminescent
activator. Depression of the trigger 14 pushes the pistons 26, 36
into their respective cylinders 38, 48 compressing the trigger
springs 28, 43 and pushing the contents of the cylinder through the
second check-valve 34, into the mixing chamber 20 and out the
nozzle orifice 22. As the trigger 14 is released, the trigger
springs 28, 43 return to their relaxed state pushing the pistons
26, 36 out of the cylinders 38, 48 creating a vacuum therein which
pulls the contents of the housings 10, 12 past the first
check-valves 33, 32, respectively and into the cylinders 38, 48
respectively. Pumping the trigger, that is repeatedly depressing
and releasing it, moves the mixtures contained in the housings
through the gun and out the nozzle orifice 22.
[0613] As the mixtures leave the cylinders 38, 48, they enter the
mixing chamber 20, via the conduit means 44 and second check-valve
34. Luminescence begins either upon mixing of the components or as
the mixed composition contacts the air upon expulsion from the toy
gun. The mixtures may be powdered, such as those produced by
lyophilization, or they may be liquid. If powdered, water can be
added prior to use.
[0614] The housings 10, 12 may be filled and refilled through the
filling caps 17, 19, respectively, located at the top of each
housing. A trigger 14 is attached to a trigger guide 13 which
serves to guide the trigger 14 towards two piston assemblies 25.
Depression of the trigger 14 activates the two piston assemblies
25. This causes a portion of the composition located in each
housing 10,12 to move through the water gun into a mixing chamber
20 and out a nozzle orifice 22. The preferred embodiment
illustrated has a trigger guard 15 which helps prevent accidental
discharge of the gun and makes the gun appear more realistic. The
sighting aids 21, 23 aid in aiming the toy gun and also serve to
make the gun appear realistic.
[0615] Only one of the two piston assemblies 25 is completely
illustrated, and it is visible in FIG. 1. The other piston assembly
is adjacent to and, in this preferred embodiment, identical to the
one illustrated. These assemblies operate by substantially
identical means and are activated by depression of the single
trigger 14. The piston assembly 25 includes a piston 26 which
passes through a sealing o-ring 30, is connected to a trigger
spring 28 and moves within a cylinder 38. The piston assembly also
includes a spring retainer 40 that secures one end of the trigger
spring 28 to the end wall of the cylinder. The cylinder 38 is in
communication with one end of a pick-up tube 18 and lies about
perpendicular to the pick-up tube 18. The cylinder 38 also
communicates with the mixing chamber 20 via conduit means 44.
[0616] In the sectional views of the water gun, illustrated in
FIGS. 2 and 3, portions of the second, adjacent piston assembly are
visible. Namely, the second trigger spring retainer 42 and trigger
spring 43 are visible in FIG. 2, and the second piston 36 is
visible in FIG. 3.
[0617] Referring to the piston assembly 25 illustrated in FIG. 1,
the piston 26 passes into the water gun through the sealing o-ring
30 and into the cylinder 38. The trigger spring 28 is attached by
one end to the piston and by its other end to the spring retainer
40 located at the opposite end of the cylinder from the piston. As
the trigger 14 is depressed, the piston 26 moves into the cylinder
38 and through the sealing o-ring 30. This compresses the trigger
spring 28 within the cylinder 38. As the trigger 14 is released,
the trigger spring 28 expands, returning it and the piston 26 to a
resting position.
[0618] Because the piston 26 is sealed within the cylinder 38 by
the sealing o-ring 30, its repeated movement causes the air within
the cylinder to be displaced thereby creating a vacuum within the
pick-up tube 18 of the water gun. The composition located in the
housing 12 is then drawn into the pick-up tube 18, past a first
check valve 32, past the trigger spring 28, past a second check
valve 34, into the mixing chamber 20 and out the nozzle orifice 22
via an outlet tube 24. The second check valve 34 is illustrated
with a spring mechanism 35 which serves to maintain the check valve
34 in a closed position isolating the piston assembly cylinders 28
and conduit means 44 from the mixing chamber 20, allowing a vacuum
to form within the gun during operation.
[0619] The same mechanism operates to simultaneously withdraw
composition from the complementary housing 10 into the mixing
chamber 20 via a pick-up tube 16. Thus, referring to FIGS. 2 and 3,
the action of the piston 36 within its cylinder compresses the
trigger spring 43 against the spring retainer 42 creating a vacuum
within the pick-up tube 16 and moving some of the composition
located in the housing 10 through the pick-up tube 16 into the
mixing chamber 20 and out-the nozzle orifice 22.
[0620] As illustrated in FIG. 2, the two pick-up tubes 16 and 18
originate in the housings 10 and 12, respectively. Each pick-up
tube 16, 18 includes a check valve 32 and 33, respectively. The
first check valves 32, 33 serve to prevent fluid flow from the
piston assembly cylinders 38, 48 back into the housings 10, 12. The
single second check valve 34 prevents the mixed compositions from
flowing out of the mixing chamber 20 back into the piston assembly
cylinders 38, 48.
[0621] Thus, repeated depression of the trigger 14 increases the
pressure within the gun, thereby filling the mixing chamber 20 with
a combination of the compositions located in the two housings 10,
12, then forcing the mixed compositions through the outlet-tube 24
and out the nozzle orifice 22.
Example 2
[0622] Dual Chamber Fluid Dispensing Apparatus--Gas-Charged Toy
Water Gun
[0623] In contrast to the above-described toy water gun, the
gas-charged toy water gun operates using pressurized gas, rather
than the piston assembly, to move the bioluminescent mixtures
through the system. A preferred embodiment of this device is
illustrated in FIGS. 4 and 5. In this embodiment the butt of the
water gun 86 houses the two chambers 64, 74 that contain the
bioluminescence generating system components. Further, the butt 86
is detachable and thus readily replaced.
[0624] To pressurize the gun for operation, a CO.sub.2 or air [or
other suitable gas or mixtures thereof] canister 50 is inserted
into a gas chamber 56 as shown. A screw cap 52, located at the base
of the gas chamber, secures the canister 50 into the chamber 56. As
the screw cap 52 is tightened, the CO.sub.2 or air canister is
forced against a piercing pin 54, thereby releasing CO.sub.2 or air
into the gas chamber 56 and charging the water gun for use.
[0625] Depression of a trigger 58 aligns a gas cock 60 with each of
two gas conduits 62 and 72 and the gas chamber 56. With the gas
cock 60 so-aligned, CO.sub.2 gas or air enters the gas conduits 62
and 72 and passes into the two chambers 64 and 74. The pressure of
the gas forces some of each mixture out of the chambers 64, 74, via
composition pick-up tubes 66, 76. The composition pick-up tubes 66,
76 are connected to outlet conduits 78 and 80 through which the
mixtures pass into a mixing chamber 68, and are combined. The
continued pressure of the CO.sub.2 gas or air forces the combined
mixture from the mixing chamber 68 and out a nozzle orifice 70.
[0626] The gas conduits 62, 72 and outlet conduits 78, 80 are
housed within the main body of the water gun and extend beyond it
in the region where the butt 86 of the gun is attached to the main
body. The composition pick-up tubes 66, 76 are completely within
the butt of the water gun 86. In order to obtain a leak-free
assembly of the butt of the gun to the main body, the gas conduits
62, 72 and outlet conduits 78, 80 each pass through a leak seal 88
located within the butt of the gun 86. The leak seals 88 may be
constructed of rubber or similar soft sealing material and should
be covered, either with a removable cap or with a material
susceptible to piercing, to prevent spillage of the compositions
contained therein.
[0627] In attaching the butt of the gun 86 to the main body, the
gas conduits 62, 72 and outlet conduits 78, 80 pass through the
leak seals 88 forming a tight seal between the tubes and the butt
of the gun. Also, as can be seen in FIG. 4, the delivery tubes 78,
80 set within the composition pick-up tubes 66, 76 at the point
where they enter the butt of the gun. This permits fluid
communication between the composition pick-up tubes 66, 76 and the
outlet conduits 78, 80.
[0628] Additional features of the preferred embodiment, as
illustrated in FIGS. 4 and 5 include retaining hooks or latches 90,
92 and 94 positioned on the main body of the water gun and used to
secure the butt of the gun to the main body. Additionally, the two
chambers 64 and 74 can be configured with filler caps 82 and 84, as
illustrated, thereby allowing them to be refilled as an alternative
to replacement.
[0629] It will be appreciated that the gas used to operate the
gas-charged fluid dispensing apparatus described herein may be
other than carbon dioxide. Any gas or mixture of gases, such as air
or mixtures of O.sub.2 and CO.sub.2, that operates in the same
manner may be used.
Example 3
[0630] Dual Chamber Fluid Dispensing Apparatus--Gas-Charged
[0631] FIGS. 6, 7 and 8 illustrate a preferred embodiment of a
gas-charged fluid dispensing apparatus as provided herein. This
embodiment may be adapted for particular uses; for example, it may
be housed within a decorative sculpture, thereby functioning as a
decorative water fountain. Alternative embodiments incorporating
this embodiment are illustrated in FIGS. 4 and 5 [EXAMPLE 2] and
FIGS. 9 and 10 [EXAMPLE 4].
[0632] Referring to FIGS. 6 and 7, the gas-charged dual chamber
dispensing apparatus has two chambers 100 and 102. In a preferred
embodiment as illustrated, the two chambers 100 and 102 are
refillable via filler caps 104 and 106 located on the upper end of
the chambers. A gas chamber 108 is situated about equidistant from
the two chambers and communicates with each of them via gas
conduits 117. The gas conduits 117 end at gas inlets 118 that
communicate with the two chambers 100, 102. The gas inlets 118 are
positioned near the upper end of the chambers 100 and 102. While
one gas inlet 118 is depicted, it is understood that each chamber
100, 102 has such an inlet.
[0633] A gas canister 112 fits into the gas chamber 108, being
secured therein by a screw cap 110. Screwing the screw cap 110
tightly into place forces the top of the gas canister 112 against a
piercing needle 114, thereby releasing gas into the gas chamber
108. A gas control valve 116 is used to control the flow of the gas
from the gas chamber 108 into the gas conduits 118.
[0634] A mixing chamber 124 is also situated about equidistant from
the two chambers 100 and 102 and communicates with them via outlet
conduit means 122, such as fluid ports. The outlet conduits [fluid
ports] 122 are located sufficiently near the bottom of the chambers
100 and 102 to permit the chamber contents to empty. Near the lower
end of the two chambers 100, 102 are fluid outlets that connect to
the fluid ports 122. Blow-out plugs 120 prevent the compositions
contained therein from leaving the chambers and entering the fluid
ports before activation of the device. One-way valves or similar
devices can be substituted for the blow-out plugs 120. The mixing
chamber 124, having bottom inlets and a top outlet, is associated
with a nozzle 126, which may be attached or integral to the mixing
chamber. Optionally, the nozzle 126 has a closure cap 132 distal to
the mixing chamber 124.
[0635] In a preferred embodiment, illustrated in FIGS. 6, 7 and 8,
an upper support 130 is shown. This upper support 130 spans the
upper ends of both chambers 100 and 102 and over the top end of the
gas chamber 108. The gas conduits 118 and inlets 117 are within the
upper support 130. The nozzle 126 passes through the upper support
130 and is supported thereby.
[0636] Also illustrated in this preferred embodiment, is a base
support 123 that spans across the lower ends of the chambers 100
and 102 and that is integral to the mixing chamber 124. The fluid
ports 122 connecting the chambers 100 and 102 with the mixing
chamber 124 are contained within the base support 123 [see, FIGS. 6
and 7].
[0637] To operate the basic dual chamber gas-charged fluid
dispensing apparatus, a gas canister 112 containing gas under
pressure, for example pressurized CO.sub.2, is inserted into the
gas chamber 108. The screw cap 110 is tightened, forcing the gas
canister against the piercing needle 114. As gas escapes from the
canister, it fills the gas chamber. The gas control valve 116 is
opened, permitting the gas to enter the gas conduits 117 and pass
into the chambers 100 and 102 through the gas inlets 118.
[0638] The pressure of the gas in the chambers pushes the mixtures
therein against the blow-out plugs 120, or through the one-way
valves, out the fluid outlets, into the fluid ports 122 or other
fluid conduit means, and into the mixing chamber 124 via the bottom
inlets. In the mixing chamber 124, the mixtures combine, while the
continued pressure from the gas propels the combined mixtures
through the nozzle 126 and out the nozzle orifice 128.
Example 4
[0639] Dual Chamber Fluid Dispensing Apparatus and Volcano-Shaped
Gas-Charged Apparatus
[0640] FIGS. 9 and 10 illustrate a preferred embodiment of the
gas-charged fluid dispensing apparatus illustrated in FIGS. 6, 7
and 8 and described above. In this embodiment, each chamber has a
generally half-conical shape, or other suitable shape [depending
upon the intended use], such that, when attached they form, in this
embodiment, a volcano-shaped apparatus. The gas chamber 160 and gas
conduit 162 are defined by the inner walls 176, 178 of the chambers
150, 152, respectively. Similarly, the mixing chamber 170 and
nozzle 172 are defined by the inner walls 176, 178 of the chambers
150, 152, respectively.
[0641] As in the apparatus, FIGS. 6, 7 and 8, a gas canister 154 is
housed in the gas chamber 160 and is activated by tightening a gas
screw-cap 156 which forces the gas canister 154 against a piercing
needle 158 thereby releasing the gas into the gas chamber 160. The
gas enters the gas conduits 162, forces out the blow-out plugs 164
and passes into the chambers 150, 152 via the gas inlets 166.
Alternatively, a control valve, or other suitable control means, is
situated between the gas chamber and gas conduits or within the gas
conduit means and used to control the flow of gas into the gas
chambers.
[0642] Within the two chambers 150, 152, one containing, for
example, up to all except one component necessary for the
bioluminescence generating reaction and the other the remaining
components, the gas forces the bioluminescence generating mixtures
into the mixing chamber 170. Blow-out plugs 168, situated between
the chambers 150, 152 and mixing chamber 170, prevent the
bioluminescence mixtures from entering the mixing chamber 170 until
the apparatus is activated. The continued pressure of the gas
forces the combined mixtures from the mixing chamber 170 through
the nozzle 172 and out the nozzle orifice 174.
[0643] This apparatus is particularly designed for use as
"fireworks" configured in the shape of a volcano. As the combined
bioluminescent mixtures are forced from the apparatus into the air,
they glow in a similar manner to traditional fireworks.
[0644] Alternatives to the specific embodiment described herein are
likewise contemplated. For example, blow-out plugs may be replaced
by one-way or control valves. Manually operated valves may be
replaced by electronically or mechanically controlled valves. The
apparatus does not have to be in the shape of a volcano, but may be
formed into any shape, such as animals, humans, plants or abstract
forms.
[0645] In another alternative embodiment, not illustrated, the
nozzle 172, through which the mixed bioluminescent composition
exits from the apparatus, is shortened, moving the mixing chamber
170 closer to the nozzle orifice 174. This is particularly
appropriate where the bioluminescence generating system used in the
apparatus produces short bursts of light or is quickly exhausted
once activated, such that the bioluminescent components are
preferably kept separated until just before expulsion from the
apparatus. In such an alternative embodiment, outlet tubes (or
conduits) may be provided that maintain the bioluminescence
generating components separate until just before expulsion from the
apparatus. The outlet tubes illustrated in FIGS. 23, 24 and 26 and
described in EXAMPLE 11, could likewise be employed in this
alternative embodiment.
Example 5
[0646] Compressible Dispensing Apparatus--Lotion/Cream
Container
[0647] FIG. 11 illustrates a preferred embodiment of a compressible
dispensing apparatus particularly useful for dispensing waxy, pasty
or semi-solid compositions such as body lotions or finger paints.
In this embodiment, the container, preferably a tube, has two
chambers 200, 202. In certain embodiments, within one chamber are
all, except for one or more, components of the bioluminescence
generating system, and in the other chamber are the remaining
components. The composition, such as body lotion or cream is in one
or, preferably, both chambers. The container is preferably
constructed of a pliable collapsible or compressible material, such
as plastic, plastic/metal laminate or similar collapsible
composite, which can be squeezed by hand. Numerous such tubes are
known to those of skill in this art are used to dispense products
such as finger paints, toothpaste, gels, lotions and other such
items.
[0648] A membrane seal 204 at the top end [dispensing end] of the
container prevents the contents of the chambers from mixing. The
cap apparatus 206 of the container has a dispensing cap at the top
210 and is configured such that a space 208 exists between the
membrane seal 204 and the dispensing cap 210, which space acts as a
mixing chamber 208.
[0649] Thus, to operate the lotion/cream container, the membrane
seal 204 is punctured, or otherwise opened, and a portion of the
contents of the two chambers 200, 202 are simultaneously squeezed
into the mixing chamber 208 by applying pressure to the container.
The dispensing cap 210 is removed and the contents of the mixing
chamber 208 are squeezed out the dispensing orifice 212. The mixed
composition may be dispensed by squeezing the container or by
squeezing the cap apparatus 206. Alternatively, a plunger/syringe
device [not illustrated] may be attached to the dispensing orifice
and the mixed cream composition thereby withdrawn from the mixing
chamber 208.
[0650] The membrane seal, 204 situated between the chambers 200,
202 and the mixing chamber 208, functions to prevent the contents
of the mixing chamber 208 from returning into either of the
chambers 200, 202. It may be constructed, for example, of a thin
layer of rubber, plastic, or other suitable porous material, having
a small hole or holes through which the contents pass. As the sides
of the container are compressed, portions of the contents of the
chambers are forced through the holes in the membrane and into the
mixing chamber, with the membrane returning to its "sealed" state
once the pressure is relieved. A one-way valve or similar device
may be substituted for the membrane seal 204, provided it too
prevents the contents of the mixing chamber 208 from flowing back
into either of the chambers 200, 202.
Example 6
[0651] Bottle/Bladder Apparatus--Bubble Composition Bottle
[0652] FIGS. 12 and 13 illustrate a preferred embodiment of the
bottle/bladder apparatus adapted for use with bioluminescent bubble
compositions. This bubble composition bottle has a bladder 300
positioned within it and held in place, in the neck 302 of the
bottle, by friction. A collar 304 is positioned on the neck of the
bottle 302, preventing the cap 306 from being screwed completely
onto the top of the bottle. The cap 306 contains a plunger 308
which operates to push the bladder 300 into the body of the bottle
when the collar 304 is removed and the cap 306 is screwed down
tightly. Upon entering the body of the bottle, the bladder is
pierced by a piercing pin 310 located on the bottom of the bottle;
thereby releasing the contents of the bladder into the bottle. FIG.
13 shows the bottle with the collar 304 removed, the cap 306
screwed on tightly, and the bladder 300 collapsed within it.
[0653] Component(s) [less than all] of the bioluminescence
generating reaction are contained in the bladder. The components
may be in the form of a solution, suspension, suspended particles,
or particles. Prior to use the bottle may be gently agitated. The
particles may be time release capsules that release their contents
upon exposure to the bubble composition or from which the contents
diffuse upon mixing of the contents of the bladder with the bubble
composition. The remaining component(s), such as Ca.sup.2+ or ATP,
are contained in the bubble composition 314, which is preferably a
mild bubble forming composition. Selection of the bioluminescence
generating composition depends upon the selected bubble composition
and also the desired action. In other embodiments, remaining
components, such as ATP, FMN, a flavin reductase or other component
that may be somewhat sensitive to the bubble composition, of the
bioluminescence generating system may be added to the bubble
composition just prior to use.
[0654] The collar 304 of the bottle is adapted with a bubble
blowing ring 312, with arm, integral thereto. Thus, the collar 304
is removed, the bladder 300 pierced within the bottle as described
and the bubble blowing ring 312 dipped into the mixed composition,
withdrawn and bioluminescent bubbles blown. A standard bubble
blowing wand [arm with ring] may be used and/or provided in place
of that depicted in FIG. 12.
[0655] The bladder 300 should be constructed of a material that can
be pierced by a piercing means, such as a needle or pin, made for
example of thin plastic or other polymeric film. Preferably the
distance from the base of the neck of the bottle to the tip of the
piercing needle is less than the length of the bladder, so that the
bladder will be pierced by the needle before its top edge clears
the base of the neck of the bottle.
[0656] The bottle 316 may be fabricated of any material ordinarily
used for dispensing bubbles. It may be transparent or translucent
to the bioluminescent light so that any glow in the bottle can be
seen.
Example 7
[0657] Container/Bladder Apparatus--Beverage Can
[0658] An exemplary of the container/bladder apparatus, illustrated
in FIG. 14, is suitable for use as a beverage can or bottle. It is
configured similarly to a pop-top aluminum drink can but has a
bladder 400 under the top which is pierced by the pop-top 402 when
the can is opened. The bladder may be centered under the top of the
can, as illustrated, may be off-center or may be attached to the
top and side of the can. Positioning of the bladder is chosen such
that it may be readily pierced and its contents mixed with the
contents of the container 404. Thus, the bladder should be
sufficiently thin that the pop-top 402 is able to pierce it
allowing its contents to mix with the contents of the beverage can.
The can is preferably fabricated of translucent or transparent
material such that the glowing beverage can be observed.
[0659] An alternative embodiment includes a beverage container with
two pop-tops, in which one is designed, such as including by having
a point at the end, to puncture the bladder and the other can be a
typical pop-top that is used for emptying the contents of the can,
such as by pouring into a glass or into a person's mouth. Since the
novelty of these items resides in the resulting glow in the
beverage, the beverage should be poured into a glass, or the
container should be transparent or translucent to the
bioluminescent light.
[0660] Another alternative contemplated herein includes a mesh
filter surrounding the bladder and functioning to prevent small
pieces of the ruptured bladder from mixing with the contents of the
can. The contents of the bladder are in aqueous composition; thus,
the density of the mesh of the filter that is permeable to the
luciferase and other bioluminescence generating components.
[0661] Similarly, embodiments employing other opening types are
contemplated herein. For example, the bladder and corresponding
container opening may be pierced with a point-ended straw, or other
sharp device. Likewise, the dispensing opening [which may be the
same as the bladder-associated opening] may be covered with a thin
aluminum pull tab. Critical to the operation of the can/bladder
combination is that the bladder preclude mixing of the contents of
the bladder and the can until the consumer takes action to rupture
the bladder.
[0662] The bladder may be constructed of any material which is
amenable to being pierced as described and is preferably
constructed of a material which will rarely if ever break into
small pieces when pierced. For example, aluminum foil with a thin
plastic coating, when pierced with a point-ended straw in
particular, will rarely break into small pieces. The body of the
can may be constructed of aluminum, plastic or similar material and
is preferably constructed of a translucent material such as
plastic.
[0663] The bladder includes up to all except for one component of
the bioluminescence generating system, and the beverage includes
the remaining component(s). For example, the bladder includes the
aequorin photoprotein [typically 0.1 to 1 mg or more] in a
composition containing a chelator to prevent activation of the
photoprotein, and the beverage contains Ca.sup.2+.
Example 8
[0664] Single Use, Dual Chamber Fluid Packaging Apparatus
[0665] FIG. 15 illustrates an exemplary embodiment of the single
use, dual chamber fluid packaging apparatus or bottle described
generally above, and the following description is with reference to
that FIGURE. The bottle has a first chamber 500 which contains a
composition including one or more, up to all but one, of the
bioluminescence generating system components. Below the first
chamber and operatively attached thereto, is a second chamber 502,
containing the remaining bioluminescence generating system
components in composition. In the embodiment illustrated, the first
chamber 500 is seated in the second chamber 502 along a side seam
506 and a separation membrane 504.
[0666] The second chamber 502 is constructed of pliable material,
such as plastic, that is convoluted 508 such that it can be readily
collapsed against the bottom of the first chamber in the direction
of the illustrated arrow. When collapsed in this way, the force of
the composition contained within the second chamber ruptures the
separation membrane 504A, permitting the compositions to mix. Once
mixed, the compositions begin to illuminate.
[0667] This apparatus, as illustrated, is adapted for use with
bubble-blowing compositions in that the cap of the bottle 510 has a
bubble-blowing wand 512 attached to it. Alternatively, the
apparatus may be used with a beverage and, if so used, would not
have the illustrated bubble-blowing wand 512.
[0668] Another embodiment of this apparatus, not illustrated, but
contemplated herein, is a bottle in which the second chamber may be
secured to the first chamber or to itself in a collapsed position.
For example, the second chamber can be adapted with a hooking
mechanism on its exterior such that it can be hooked to itself when
collapsed.
Example 9
[0669] Cap Apparatus for Use with Composition Vessels
[0670] FIGS. 16, 17 and 18 & 19 illustrate three exemplary
embodiments of the cap apparatus for use with composition
vessels.
[0671] A. Cork Cap Apparatus
[0672] Referring to FIG. 16, a cork 600, situated within the neck
602 of a bottle and having a rupturable capsule 604 housed within
it, is illustrated. In this embodiment, the bottom edge of the cork
600 is substantially U-shaped such that a pocket is formed.
Contained within the pocket is the capsule which is in
communication with the screen 608 which is permanently attached to
the bottom of the cork. The capsule contains one or more, up to all
but one, of the bioluminescence generating system components. A
plunger assembly 606 is positioned, partially within the cork, such
that depression of the plunger assembly 606 results in rupture of
the capsule and release of its contents into the composition within
the bottle. The screen 608 or other filtering device prevents
fragments of the ruptured capsule from entering the vessel.
[0673] The plunger assembly 606, illustrated in FIG. 16, has a top
portion 610 integral to the stem portion 612. Pressing on the top
portion 610 forces the stem 612 to move within the cork 600 and
against the capsule 604, thereby rupturing the capsule and
releasing its contents into the vessel.
[0674] FIG. 17 illustrates an alternative embodiment of the cork
cap apparatus. In this embodiment, the cork 700 is illustrated as
being about flush with the top of the neck 702 of the bottle. The
plunger apparatus 704 is adapted with a finger ring 706 for ease in
handling. The stem 708, which may be pointed or blunt or any
combination thereof, is threaded 710. In operation, the plunger
assembly 704 is screwed into the cork 700 where it contacts a
capsule 712, rupturing it and releasing its contents against the
screen 714 or filter. The capsule will preferably contain powdered
or otherwise condensed bioluminescence generating components.
[0675] It will be appreciated that the cork cap alone, with
encapsulated compositions encased within and screen or filter
attached thereto, is an alternative embodiment of the two
illustrated cork cap apparatus. In this embodiment a corkscrew may
be employed to rupture the capsule and to remove the cork cap.
[0676] B. Screw-Top Cap Apparatus
[0677] FIGS. 18 and 19 illustrate another exemplary embodiment of
the cap apparatus for use with composition vessels. FIG. 18 shows
the cap apparatus before activation or engagement. This is
particularly adapted for use with a wine or champagne bottle, and
includes encapsulated bioluminescence generating system
components.
[0678] This embodiment generally includes a bottle-shaped vessel
with a collar 802 situated about the neck 804 of the bottle and a
cap 800 attached to the top of the bottle just above the collar
802. The neck of the bottle 804 is threaded to receive the screw-on
cap 800. The collar 802 is situated such that a lower portion of
the threads on the neck of the bottle 804 are covered thereby
preventing the screw-on cap 800 from being completely attached to
the bottle. Enough threads remain exposed on the top of the bottle
such that the screw-on cap 800 is securely, though not completely,
attached to the top of the bottle.
[0679] The screw-on cap 800 has a plunger 806 integral thereto
which extends into the bottle neck 804. A screen or filter assembly
812 is attached to the interior of the bottle within the bottle
neck 804. A membrane system 808, 810 or capsule or similar
composition packaging is situated between the plunger 806 of the
screw-on cap 800 and the screen/filter assembly 812. In operation,
the collar 802 is removed, for example by removing the screw-cap
800 and lifting off or screwing off the collar 802 or by tearing
off the collar 802, and the screw-on cap 800 is tightened against
the top of the bottle. This forces the plunger 806 through the
membranes 808, 810, rupturing them and releasing the compositions)
contained therein. The composition(s) pass through the screen
assembly 812 and are mixed with the contents of the bottle. FIG. 19
illustrates the cap apparatus fully engaged with the membrane
system ruptured.
[0680] In the embodiment illustrated, the screen assembly 812 is
attached along the interior of the neck of the bottle 804 as well
as across the interior of the neck, thereby forming a basket within
which the membrane system 808, 810 sits. Alternatively, the screen
assembly can be attached around the circumference of the bottle
neck only and not along its sides to the top of the bottle, as
illustrated.
[0681] The precise height of the collar 802 will be determined by
the length of the plunger 806 and location of the membrane system
808, 810. The height will be sufficient to prevent the plunger 806
from being engaged through the membrane system 808, 810 prior to
activation by the user, while permitting the screw-on cap 800 to be
secured to the top of the bottle.
[0682] The membrane system 808, 812 contains one or more, up to all
but one, of the bioluminescence generating system components.
Typically the components will include the luciferase and luciferin
in lyophilized form.
[0683] The illustrated embodiment is shown and described as
attached to a bottle. It will be appreciated, however, that the
vessel to which the cap apparatus is attached may be a can, tube or
any other container. Additionally, the embodiment is exemplified
and illustrated with reference to the neck of the bottle. It is not
necessary that the vessel have a "neck" for the cap apparatus to
function. For example, if the vessel does not have a neck, other
means may be employed to hold the collar in place below the
screw-on cap, such as, a lip formed on the container, below the
threads, to stop the collar at an appropriate point.
[0684] With respect to these three embodiments of the cap apparatus
adapted for use with composition vessels, the stem of the plunger
assembly is short enough not to pierce the screen or filter device,
yet long enough to effectively rupture the capsule, membrane or
other packaging once engaged. The bioluminescence generating system
component(s) contained within the cap apparatus may be powdered or
in composition or in any form amenable to addition to the
composition contained within the vessel. Additionally, the
components may be contained in more than one capsule, membrane or
other packaging. In this case, the component packages are
adjacently positioned, such that each is ruptured by engagement of
the plunger. Preferably, the remaining components required for
completion of the bioluminescent reaction are contained within the
vessel within any composition. These embodiments are particularly
adapted to use with wine or champagne or other beverage.
Example 10
[0685] Spray Container Apparatus
[0686] FIGS. 20, 21 and 22 illustrate an exemplary embodiment of a
spray container provided herein. This container is typically a can
apparatus intended for use in combination with the bioluminescence
generating systems as described herein. The following description
of that exemplary embodiment is made with reference to those
figures.
[0687] The spray container apparatus includes two portions, a top
housing portion 902 and a bottom plunger portion 904. The contents
of the top housing portion 902 include all, except one or more, of
the components of a bioluminescence generating system. The top
housing portion 902 also contains a conduit 912 operatively
attached to a spray nozzle 920.
[0688] The top housing portion 902 of the spray container apparatus
is adapted to receive the bottom plunger portion 904. In this
embodiment, the top housing portion 902 and bottom plunger portion
are threaded 903 and 910, respectively, such that the bottom
plunger portion 904 can be screwed onto the top housing portion
902. [See FIG. 21, illustrating the spray container apparatus with
the bottom plunger portion fully screwed into place.]
[0689] The top housing portion 902 additionally has a pocket 926
defined by a conical side wall 922 and a top wall/rupture membrane
916. The pocket 926 is adapted to receive a pellet 906, that
contains the remaining component(s) necessary for generating
bioluminescence.
[0690] The bottom plunger portion 904 of the spray container
apparatus has a plunger 914 shaped and situated such that it fits
into the pocket 926 of the top housing portion 902 when the bottom
plunger portion 904 is screwed tightly in place. The bottom plunger
portion 904 is adapted with an angular seal 918 that serves to seal
the bottom plunger portion 904 against the top housing portion 902
thereby preventing leakage of the contents of the spray container
apparatus.
[0691] In operation, the pellet 906 is placed into the pocket 926
of the top housing portion 902 where it contacts the top
wall/rupture membrane 916 of the pocket 926. The bottom plunger
portion 904 is then screwed onto the top housing portion 902,
thereby forcing the plunger 914 against the pellet 906, which
presses against the top wall/rupture membrane 916 of the pocket
926, rupturing the same. The pellet dissolves or is suspended in
the composition contained in the top housing portion 902 and the
composition glows. Depression of the spray nozzle 920 releases the
contents of the spray container apparatus.
[0692] Alternative embodiments of this spray container apparatus
will be appreciated. For example, the pellet 906 may be a vessel
containing the necessary bioluminescence generating components that
is fabricated from material that can dissolve or that will be
suspended in the composition contained in the top housing portion
902 of the spray container apparatus 900 or that will release its
contents upon contacting the composition, such as by passive
diffusion. Examples of such material include, but are not limited
to liposomes, gelatin, soluble paper and other such materials that
will dissolve or release contents into aqueous compositions.
Further, the spray container apparatus 900 can be adapted such that
the bottom plunger portion 904 snaps onto the top housing portion
902, rather than screwing into place.
Example 11
[0693] Alternative Embodiment of Dual Chamber Fluid Dispensing
Apparatus--Toy Water Gun
[0694] Another embodiment of the dual chamber fluid dispensing
apparatus is a toy water gun, such as that illustrated in FIGS. 23
through 26. This toy water gun includes two housings [or chambers]
406, 408 that may be constructed of injection-molded plastic or
other suitable material. The two housings 406, 408 are affixed,
such as glued, heat sealed or by other such means, along a median
seam 462 to form the body of the water-gun. See especially FIGS. 25
and 26.
[0695] In operation, one housing 406 contains a mixture having less
than all the components necessary for generating bioluminescence
and the other housing 408 contains a mixture having the remaining
components or the remaining components except for air. Depressing
the trigger 410 pushes the pistons 428, 430 into their respective
cylinders 450, 452 compressing the trigger springs 432, 434 and
pushing the contents of the cylinder through the respective conduit
means 458, 460, past the second check-valves 442, 444, out the
outlet tubes 424, 426, into the mixing chamber 420 and out the
nozzle orifice 422. As the trigger 410 is released, the trigger
springs 434, 432 return to their relaxed state pushing the pistons
430, 428 out of the cylinders 452, 450 creating a vacuum therein
that pulls the contents of the housings 406, 408 through the
pick-up tubes 412, 414, past the first check-valves 438, 440 and
into the cylinders 450, 452. Pumping the trigger, such as by
repeatedly depressing and releasing it, moves the mixtures
contained in the housings through the gun into the mixing chamber
420 and out the nozzle orifice 422.
[0696] As the mixtures leave the outlet tubes 424, 426, just prior
to expulsion from the toy gun via the nozzle orifice 422, they
enter the mixing chamber 420. Bioluminescence begins either upon
mixing of the components or as the mixed composition contacts the
air as it exits the toy gun. The mixtures may be powdered, such as
those produced by lyophilization, or they may be condensed into a
paste, or they may be liquid. If powdered or condensed, water or a
suitable composition, such as a suitable buffer can be added prior
to use.
[0697] The housings 406, 408 may be filled and refilled through the
filling caps 464, 466 located at the top of each housing. The
trigger 410 is attached to a trigger guide 416 which serves to
guide the trigger 410 towards the two piston assemblies 472. Only
one of the two piston assemblies 472 is completely illustrated, and
it is visible in FIG. 23. The other piston assembly is adjacent to
and, in this embodiment, identical to the one illustrated.
Depression of the trigger 410 activates the two piston assemblies,
e.g., 472. This causes a portion of the composition located in each
housing 406, 408 to move through the toy gun into a mixing chamber
420 and out a nozzle orifice 422, as detailed above.
[0698] The piston assemblies e.g., 472 each include a piston 430,
428 which passes through a sealing o-ring 436, 429 is connected to
a trigger spring 434, 432 and moves within a cylinder 452, 450. The
piston assemblies each also include a spring retainer 456, 454 that
secures one end of the trigger spring 434, 432 to the end wall of
the cylinder. Each cylinder 452, 450 is in communication with one
end of a pick-up tube 414, 412 and is about perpendicular to the
pick-up tubes 414, 412. Each cylinder 452, 450 also communicates
with the conduit means 458, 460.
[0699] Because the pistons 428, 430 are sealed within their
cylinders 450, 452 by a sealing o-ring 429, 436, repeated movement
of the pistons within the cylinders causes the air within the
cylinders to be displaced thereby creating a vacuum within the
pick-up tubes 412, 414 of the toy gun. This initiates the operation
of the toy gun as described in detail above.
[0700] The illustrated embodiment has a trigger guard 411 that acts
to prevent accidental discharge of the gun and makes the gun appear
more realistic. The sighting aids 468, 470 aid in aiming the toy
gun and also serve to make the gun appear realistic.
[0701] As illustrated in FIG. 25, the two pick-up tubes 412 and 414
originate in the housings 406 and 408, respectively. Each pick-up
tube 412, 414 includes a check-valve 440, 438, respectively. The
first check-valves 440, 438 serve to prevent fluid flow from the
piston assembly cylinders 450, 452 back into the housings 406, 408.
The second check-valves 442, 444, similarly prevent the fluids from
flowing out of the outlet tubes 424, 426 and back into the piston
assembly cylinders 452, 450.
[0702] Thus, in operation, repeated depression of the trigger 410
increases the pressure within the gun, thereby filling the mixing
chamber 420 with a combination of the compositions located in the
two housings 406, 408, then forcing the mixed compositions out of
the toy gun through the nozzle orifice 422.
Example 12
[0703] Compressible Dispensing Apparatus
[0704] FIG. 27 illustrates an alternative exemplary embodiment of a
compressible dispensing apparatus. This embodiment is particularly
adapted for containing and dispensing bioluminescent slimy play
material as described herein, but may be used to dispense other
ingredients. The primary difference between the embodiment
illustrated in FIG. 11 and that illustrated in FIG. 27 is that the
latter has one or more small compartments 942, 944 located within
the apparatus. These compartments are located such that compression
of the apparatus expels the contents of the compartments into the
main body 940 of the apparatus where those contents and any
contents contained within the main body 940 mix.
[0705] The embodiment illustrated in FIG. 27 has a first
compartment 942 and a second compartment 944 contained within the
main body 940 of the compressible dispensing apparatus. The
compartments 942, 944 are preferably formed, along at least one
edge 950, 952, by rupturable membranes, such as plastic membranes,
or other readily punctured dividing means. At least one other edge
of each compartment 946, 948 is permanently affixed to the interior
of the main body 940 of the apparatus. Thus, upon compression of
the apparatus, the contents of the two compartments 942, 944 press
against and rupture the rupturable membranes 950, 952, resulting in
expulsion of the contents of the two compartments 942, 944 into the
main body 940 of the apparatus. Because at least one edge of each
compartment 946, 948 is permanently affixed to the interior of the
apparatus, the compartments remain in position and readily rupture
during compression.
[0706] Preferably the two compartments 942, 944 are large enough to
contact one another along one contact edge 954 within the
apparatus. As the sides of the apparatus are compressed, the
contents of the two compartments are pressed against this contact
edge 954 and against the rupturable membranes 950, 952, which
membranes then rupture. Preferably, the cap 956 to the apparatus
remains in place until the two compartments have been ruptured and
the contents mixed within the apparatus.
[0707] The compressible dispensing apparatus is illustrated in FIG.
27 with two compartments 942, 944; however, it will be appreciated
that one, three or more compartments may be included as
appropriate. Factors to be considered in determining the
appropriate number of compartments are the bioluminescence
generating system to be used, the ingredients, particularly slimy
play material ingredients to be used, the desired timing and
duration of illumination, and the ultimate use for resulting
composition, such as the slimy play material.
[0708] By way of example only, where two compartments are included
in the apparatus, as illustrated in FIG. 27, one compartment may
contain the charged luciferin/luciferase mixture, such as aequorin
photoprotein with coelenterazine and oxygen and the second
compartment may contain a polyvinyl alcohol mixture. The main body
of the apparatus contains the remaining ingredients, such calcium
ions, necessary to complete the bioluminescence generating
reaction, and also contains the other ingredients of the slimy play
material, such as sodium tetraborate.
[0709] Alternatively, where the apparatus is configured with three
compartments within the main body, one or more of the ingredients
contained within the main body of the two compartment embodiment
may instead be contained within the third compartment. For example,
the sodium tetraborate may be included in the third compartment and
the calcium ions, in an aqueous medium, may be in the main body of
the apparatus. It will further be appreciated that the contents of
each compartment and/or the main body may be in powder, liquid or
semi-solid form. The liquid or semi-solid form are preferred.
Example 13
[0710] Recombinant Production Renilla Reniformis Luciferase
[0711] The phagemid pTZ18R (Pharmacia) is a multi-purpose DNA
vector designed for in vitro transcriptions and useful for
expression of recombinant proteins in bacterial hosts. The vector
contains the .beta.-lactamase gene, which allows for the selection
of transformants by resistance to ampicillin, and a polylinker site
adjacent to the lacZ' gene. The heterologous gene of interest is
inserted in the polylinker and transcribed from the lac promoter by
induction, for example, with
isopropyl-.beta.-D-thiogalactopyranoside (IPTG).
[0712] The DNA encoding the Renilla reniformis luciferase has been
cloned (e.g., see U.S. Pat. Nos. 5,292,658 and 5,418,155). The
plasmid pTZRLuc-1 encodes the Renilla luciferase on a 2.2 Kbp EcoRI
to SstI DNA fragment inserted in EcoRI and SstI sites of pTZ18R
(plasmid construction is described U.S. Pat. Nos. 5,292,658 and
5,418,155; see also Lorenz et al. (1991) Isolation and Expression
of a cDNA encoding Renilla reniformis Luciferase, Proc. Natl. Acad.
Sci. U.S.A. 88:4438-4442). The initiation of transcription of the
Renilla luciferase cDNA is under the control of the lacZ' promoter.
E. coli strains harboring plasmid pTZRLuc-1 express Renilla
luciferase that is functional in bioluminescence assays and retains
the most of the critical properties of the native enzyme (see,
e.g., U.S. Pat. Nos. 5,292,658 and 5,418,155).
[0713] A derivative of pTZRLuc-1, pTZRLuc-3.6, produces
approximately 7-fold higher levels of recombinant Renilla
luciferase than pTZRLuc-1 when transformed into the same E. coli
host. Competent E. coli strain XL-1 was transformed using purified
pTZRLuc-3.6 according to the instructions provided by the
manufacturer (XL-1 Supercompetent cells.TM. and protocol;
Stratagene, Inc., La Jolla, Calif.). Transfectants were selected by
plating on Luria Broth (LB) plates supplemented with 100 .mu.g/ml
ampicillin.
[0714] Single ampicillin resistant colonies were grown in LB medium
supplemented with 100 .mu.g/ml ampicillin at ambient temperature
using continuous shaking until cell growth reached mid-log phase
(i.e., cell culture reaches an O.D..sub.600 nm=0.6-0.8 units).
Transcription from the lac promoter was induced by addition of 1 mM
IPTG and cell culture was shaken at ambient temperature for an
additional 8 hours.
[0715] Cells were harvested by centrifugation at 10,000.times.g and
frozen at -20.degree. C. The cell pellet was thawed and resuspended
at a 1:5 ratio (w/w) in a compositions containing 10 mM EDTA, pH
8.0, containing 4 mg/ml lysozyme (Sigma Chemical Corp.). The cells
were placed in a 25.degree. C. water bath for 30 minutes and then
transferred to ice for 1 hour. The cells were lysed by sonication
at 0.degree. C. using a 1 minute pulse from an Ultrasonics, Inc.
cell disrupter.
[0716] The lysed cellular debris was removed by centrifugation at
30,000.times.g for 3 hours and the supernatant was decanted and
retained. The pellet was resuspended at a 1:5 ratio in the
above-described compositions, and the subsequent incubations, lysis
and centrifugation steps were repeated. The two supernatants were
combined and stored at -70.degree. C.
[0717] The resulting "clarified lysate" was employed as a source of
recombinant luciferase. Alternatively, the lysate may be subjected
to additional purification steps (e.g., ion exchange chromatography
or immunoaffinity chromatography) to further enrich the lysate or
provide a homogeneous source of the purified enzyme (see e.g., U.S.
Pat. Nos. 5,292,658 and 5,418,155).
Example 14
[0718] Cartridges for Loading, Charging, Recharging and/or Filling
Bioluminescent Novelty Items
[0719] An exemplary loading, recharging or charging cartridge is
depicted in FIGS. 28-34. Referring first FIG. 28, a charging
cartridge is shown and generally designated 1000. This charging
cartridge includes a block 1002 having two cylinders, a first
cylinder 1010 and a second cylinder 1012, and a plunger 1004 having
a first piston 1006 and a second piston 1008. Additional chambers
may be included. Also, the device may be adapted for use with the
single chamber apparatus provided herein.
[0720] As shown, the block is formed with two cylinders 1010 and
1012, and the plunger is formed with two cylindrical pistons 1006
and 1008. It is to be appreciated that a triangular, rectangular,
or any other geometry vessel may be substituted for either
cylinder, so long as the shape of the pistons provides for
insertion into the block. Additionally, for example, the plunger
1004 may be formed such that the two pistons 1006 and 1008 are
separate from the other to permit the insertion of pistons 1006 and
1008 into the block 1002 at different times.
[0721] The block 1002 and plunger 1004 may be made of any material
known to one of skill in the art that does not react with the
components of a bioluminescence generating system. In a preferred
embodiment, the block 1002 and plunger 1004 are made of a plastic
material that can be readily injection molded into a selected
particular shape. Suitable plastics include, but are not limited to
polyvinyl chloride (PVC), or any other plastic, TEFLON,
polyethylene, or any other material that is inert to components
stored and dispensed from the block 1002. Alternatively, the block
1002 and plunger 1004 can be made from a metal that is machined,
cast, or otherwise formed into the particular shape.
[0722] Referring now to FIG. 29, the first cylinder 1010 has a plug
1016 which retains, for example, dry ingredients 1018 containing
one or more components of a bioluminescence generating system,
preferably including a luciferase and/or luciferin and any
necessary buffers and activators, e.g., ATP or Ca.sup.2+, and more
preferably a luciferase, buffers and any necessary activators, in
lyophilized or other suitable form, in the cylinder 1010 and
against the seal 1022. Thus, the dry or condensed ingredients 1018
are trapped within the first cylinder 1010 between the plug 1016
and the seal 1022 until the plunger 1004 and piston 1006 are forced
into the first cylinder 1010. At that time, theses ingredients 1018
are forced through the funnel means 1020, thereby breaking the seal
1022, and forcing the ingredients 1018 out of the block 1002
through nozzle 1024 and out aperture 1026. The seal 1022 is
preferably made of a material which is capable of being broken with
only minimal pressure asserted on the plunger 1004. Such a material
includes, for example, a paper, wax-covered paper, plastic sheet,
foil, cellophane or any other material exhibiting the requisite
properties.
[0723] The second cylinder 1012 is formed within a fluid sleeve
1014 that is inserted into the block 1002. In this way, the sleeve
1014 may be a sealed tube made from, for example, plastic, glass,
or any other material that is compressible and/or breakable,
thereby allowing the fluid 1030 to be forced from the sleeve 1014.
The sleeve 1014 may be prefabricated and loaded with the fluid 1030
prior to insertion into the block 1002, or the fluid 1030 may be
added to the sleeve 1014 once it is positioned within the block
1002, and retained therein by plug 1028.
[0724] The piston 1008 slides into the second cylinder 1012 and
strikes plug 1028, advancing it into the block 1002. The advancing
plug 1028 creates a fluid pressure within the sleeve 1014 which
eventually breaks seal 1032 and optionally bathes the matrix
material 1034 in fluid 1030. Like the seal 1022 in the first
cylinder 1010, the seal 1032 in the second cylinder 1014 can be
made of any material that can be broken or torn or ruptured with
only minimal pressure being asserted on the plunger 1008. Such a
material may be a paper, wax-covered paper, plastic sheet, foil,
cellophane or any other material which exhibits the necessary
characteristics.
[0725] The matrix material 1034 may be any porous material to which
the bioluminescence generating component can be adsorbed, absorbed
or otherwise linked, as described herein, that is non-reactive with
the components of the bioluminescence generating system. When
necessary, the matrix material 1034 is included and bathed in the
fluid 1030 such that the component(s) of the bioluminescence
generating system affixed to the matrix material are released into
the fluid 1030. As the piston is continually advanced, the fluid,
containing bioluminescence generating components eluted from the
matrix material, is forced through the filter 1036 and out the
nozzle 1038 and aperture 1040. Filter 1036 is used to prevent the
expulsion of matrix material 1034 from the second cylinder 1014. As
a result, the filter 1036 may be made from a cloth or metallic
weave, or any other material that will not react with the various
components and compositions present within the second cylinder
1014.
[0726] It is to be appreciated, however, that the various
components of the bioluminescent reaction may be distributed in
different combinations between the two cylinders 1010, 1012, and
the matrix material 1034. One cylinder, such as the first cylinder
1010, typically contains the dry or condensed ingredients 1018 and
the second cylinder 1012 typically contains a fluid 1030 and the
matrix material containing the remaining components necessary for
the bioluminescent reaction. The dry or condensed ingredients may
contain any combination of the components of the bioluminescence
generating system, such as a luciferase and/or a luciferin, buffer
salts, ATP, Ca.sup.2+ or any other necessary activator. The fluid
1030 may be water, a buffer, an organic solvent or any other
aqueous medium suitable for solubilizing or suspending one or more
components of a bioluminescence generating system to be dispensed
into the bioluminescent novelty item.
[0727] In a preferred embodiment, the dry ingredients 1018 include
lyophilized luciferase and buffer salts in powder form, and the
fluid includes an alcohol that is used to dissolve or suspend a
quantity of luciferin affixed to the matrix material.
Alternatively, all of the components of a bioluminescence
generating system, such as the Vargula system, may be added and
packaged in the first and/or second cylinders in the absence of
molecular oxygen such that components are activated when combined
and exposed to air.
[0728] Referring now to FIG. 30, the cartridge 1000 is shown as
used in conjunction with a typical bioluminescent novelty item
1042. As shown, the plunger 1004 has been pressed completely
against the block 1002 causing the first piston 1006 and the second
piston 1008 to be inserted completely into the block 1002. As the
piston 1006 is advanced into the block 1002, the dry or condensed
ingredients 1018, for example, are forced out of the first cylinder
1010, through the funnel 1020 thereby breaking the seal 1022, and
out the nozzle 1024 and aperture 1026 into the chamber 1044 in
novelty item 1042. Likewise, as the piston 1008 is advanced into
the block 1002, the seal 1032 on the sleeve 1014 is ruptured
causing the fluid 1030 to be dispensed, optionally bathing matrix
material 1034. As the piston 1008 is advanced further, the fluid
1030 is forced through filter 1036, out nozzle 1038 and aperture
1040, and into chamber 1046 of novelty item 1042. In this manner,
the novelty item is fully recharged with the components of a
bioluminescence generating system necessary for a bioluminescent
reaction, while maintaining the separation of the chemicals as
required for some novelty items.
[0729] The cartridge 1000 is shown inserted into the filler holes
of a typical novelty item 1042, such as those described elsewhere
in this application. For example, the cartridge could be adapted to
fit the numerous of the novelty items, such as the following
novelty items: the filler caps 17, 19 associated with chambers 10,
12 shown in FIGS. 1 and 3; the filler caps 82, 84 shown in FIGS. 4
and 5; the filler caps 104, 106 shown in FIGS. 6, 7, and 8; and the
filler caps 406, 408 on housing 466 in FIGS. 23 through 26. It
should be appreciated that although several novelty items have be
identified as being either chargeable or rechargeable using the
cartridges disclosed herein, such identification is merely
exemplary and is in no way to be intended as limiting the
application of the cartridges to those particular novelty items. On
the contrary, the cartridges described herein may be adaptable to
charge, or recharge, virtually any bioluminescent novelty item.
[0730] Referring now to FIG. 31, a second embodiment of a charging
cartridge is shown and generally designated 1100. The cartridge
1100 is shaped substantially like the cartridge 1000, with the
addition of a safety feature that prevents the accidental or
inadvertent discharge of the cartridge when not inserted properly
within a novelty item. While an accidental discharge would not be
physically harmful to a human or non-human animal, such a discharge
could prematurely release the bioluminescent materials. The
likelihood of such an accidental discharge could, perhaps, be
increased when considering the intended user of many of the novelty
items, such as children.
[0731] In this exemplary embodiment, cartridge 1100 contains a
block 1102 and a plunger 1104 which, like the cartridge 1000, has a
first piston 1106 and a second piston 1108. Unlike the cartridge
1000, however, each of the pistons 1106 and 1108 is equipped with a
piston head 1110 and 1112, respectively. These piston heads, in
conjunction with cap 1118 prevent the removal of the plunger 1104
from the block 1102. As a result, the cartridge 1100 cannot be
disassembled to yield direct access to the contents of the
cylinders 1114 and 1116. In addition to the piston heads 1110,
1112, the cartridge 1100 is also equipped with a stop 1120 and a
slide 1122 to prevent the accidental compression of the plunger
1104 into the block 1102 while the cartridge is not inserted into a
novelty item. More specifically, the stop 1120 is normally
positioned in the path of the first piston 1106 to prevent the
advance of the first piston 1106 into the block 1102. Once the
cartridge 1100 is positioned on an appropriate novelty item, the
slide 1122 is automatically pressed upwards thereby moving the stop
1120 out of the path of the dry piston 1106. Once the stop 1120 is
out of the way, the two pistons 1106, and 1108, may be pressed into
the block 1102, thereby releasing the contents of the first
cylinder 1114 and the second cylinder 1116 in the same manner as
discussed above in conjunction with FIGS. 28 through 30.
[0732] Referring now to FIG. 32, the cartridge 1100 is shown as
used in conjunction with a properly equipped novelty item 1152. As
shown, the novelty item 1152 is equipped with a pin 1162 which
extends upwards from the novelty item 1152. As the cartridge 1100
is placed over the novelty item 1152, the pin 1162 forces the slide
1122 upwards thereby moving the stop 1120 from the path of piston
1106. Once piston 1106 is able to be pressed into the block, the
piston 1106 and piston 1108 are forced into the block 1102. More
specifically, as piston 1106 is forced into the block 1102, the
piston advances plug 1126 which in turn forces the dry or condensed
ingredients 1128 to break seal 1130. Once the seal 1130 is broken,
the dry or condensed ingredients 1128 are further forced through
nozzle 1132 and out aperture 1134, and into the first chamber 1154
of the novelty item 1152. Similarly, as the plunger is depressed,
the wet piston 1108 is forced into the fluid cylinder 1116 and
strikes plug 1138. As the wet piston is advanced, the plug 1138
creates a fluid pressure within the sleeve 1136, thereby rupturing
the seal 1142 causing the fluid 1140 to be forced through the
matrix material 1144, through filter 1146, and through nozzle 1148
and out aperture 1150 and into the second chamber 1156 in novelty
item 1152.
[0733] FIG. 33 provides a cross-sectional view of the cartridge
1100, showing in detail the placement of the stop 1120 and slide
1122 in relation to the dry piston head 1110. As shown, the stop
1120 extends into cylinder 1114 sufficiently to prevent the
advancement of piston 1126 in cylinder 1114. It should be
appreciated that while the stop 1120 is blocking the advance of
only the piston 1110, that piston 1112 could be held in place in
addition to, or instead of, piston 1110. Moreover, the stop 1120
and slide 1122 could be positioned anywhere in the block 1102 such
that the pin 1162 could be positioned on the novelty device in an
alternative location. It should also be appreciated that a spring
(not shown) may be used to hold the stop 1120 in a resting position
such that only with the movement of the slide 1122 can the dry
piston 1106 be advanced into the block. Additionally, a spring (not
shown) may be positioned to naturally urge the slide towards hole
1124 in block 1102, thereby preventing the accidental movement of
the slide without the aid of a pin 1162.
[0734] In addition to the cartridges as shown above, other means
may be employed to minimize the leakage of the contents of the
bioluminescence generating systems in combination with the various
novelty items described herein. More specifically, the novelty item
1152 may be equipped with a removable cap 1164 that is used to seal
the chambers 1154 and 1156 of the novelty item 1152 to minimize the
leakage of any components of the bioluminescence generating system.
Further, a series of seals 1158 and 1160, or one way seal valves,
can be used to prevent the escape of the components once they have
been placed in the chambers of the novelty item 1152. Seal 1160 is
of a type which is normally biased to a closed position to prevent
the passage of material in one direction. In this application, the
seal 1160 is biased closed such that any material within the
chambers 1154 and 1156 is retained within the chamber. Only upon
the insertion of nozzles 1132 and 1148 through the seals 1158 and
1160 is it possible for material to pass through the seal. Thus,
once the nozzles 1132 and 1148 are inserted into the novelty item
1152 through the seals 1158 and 1160, the contents of the cylinders
1128 and 1140 are easily injected. Once the contents are injected,
however, the nozzles are removed, and the seals 1158, 1160 return
to their normal biased closed position to prevent the escape of the
chemicals from the chambers 1154, 1156.
[0735] In yet another alternative embodiment of a cartridge, a
dispensing syringe is shown in FIG. 34 and generally designated
1200. Syringe 1200 has a body 1202 which is equipped with a
circumferential flange 1204 (or a pair of tabs extending from each
side of the body), and a plunger 1206. This construction provides
for a one-handed operation recharging a novelty item. More
specifically, by holding the body adjacent to the circumferential
flange between the index finger and middle finger of a user, and
using the thumb to advance the plunger 1206 into the body 1202, the
entire contents of the dispensing syringe 1200 can be injected into
the novelty item.
[0736] The plunger 1206 has two pistons 1210 and 1208 which are
formed with plugs 1212 and 1220 respectively. These plugs 1212 and
1220 are sized to be snugly received inside the cylinders, e.g.,
cylinders 1213 and 1221. One cylinder, e.g., cylinder 1213, is
filled with dry ingredients 1214 and held in place against the seal
1216. Like the cartridges 1000 and 1100 discussed above, as piston
1212 is advanced into cylinder 1213, the seal 1216 is ruptured
allowing the expulsion of the dry ingredients 1214 out of nozzle
1218 and into chamber 1234 of novelty item 1232.
[0737] Plug 1220 is positioned in the cylinder 1221 to retain, for
example, the fluid 1222 between seal 1224 and plug 1220. As with
the cartridges discussed above, as piston 1208 is advanced into the
body 1202, fluid pressure is created within the cylinder 1221,
thereby rupturing the seal 1224. Once the seal is ruptured, the
fluid is dispensed, and optionally bathes matrix material 1226 to
dissolve the one or more component of the bioluminescence
generating system into the fluid. As the piston 1212 is further
advanced, the fluid 1222 is forced through filter 1228 and out
nozzle 1230 and into chamber 1236 of novelty item 1232.
[0738] As an alternative to the nozzles 1218 and 1230, a mixing
chamber (not shown) can be formed in the body 1202 or attached
thereto. Such a chamber would provide for the thorough mixing of
the dry ingredients 1214 and the fluid 1222, prior to introduction
of the chemicals into the novelty item. Such a mixing would be
advantageous where it is not feasible to keep the components of the
bioluminescence generating system separate until the instant the
reaction is desired, such as in a single-chambered novelty item
having a single chemical input port. It is also to be appreciated
that a mixing chamber can be easily formed within the cartridge
1000 and/or 1100 or attached thereto.
[0739] The charging cartridges 1000, 1100, and 1200 shown and
described herein have substantially cylindrical chambers within
which to store the components of the bioluminescence generating
system, separately or together, in liquid or solid form. It should
be appreciated, however, that any shape chamber is contemplated
herein. Specifically, in cartridge 1000 and 1100 may be formed with
a pair of chambers having a rectangular cross-section, or may be
formed with each chamber having a semi-circular cross-section,
representing one half of a cylindrical block. Virtually any shape
for the block and chambers is contemplated herein, and the
particular embodiments shown in FIGS. 28 through 34 are only
exemplary.
[0740] In yet another alternative embodiment (not shown), the
cylindrical chambers of the cartridges 1000 and 1100 are replaced
by compressible tubing which are positioned within the block and
filled with the necessary chemicals, but are also easily compressed
to expel the chemicals within them. The compressible tubing can be
made from any other material which is sufficiently rigid to contain
the chemicals, such plastic, rubber or other such material, but
pliable enough to allow the expulsion of the chemicals using a
piston. The tubing can be formed in an accordion-shape which has
pre-formed creases in the walls of the tubing, or may be formed in
any manner which simplifies expulsion of the chemicals. Such a
tubing construction would eliminate the need for plugs to retain
the chemicals within the block, and will also simplify the
manufacturing of the cartridge by eliminating the direct handling
of the bioluminescent components.
[0741] As an alternative to a cartridge having a block and plunger,
a cartridge may be constructed having a block made from a pliable
material that allows compression of the chemical tubing or other
suitable material by squeezing the sides of the block. In other
words, instead of requiring a plunger having pistons which compress
the chemical tubing, the block may be sealed with the chemical
tubing contained inside the block, with the chemicals being
expelled by squeezing the sides of the block to create the pressure
necessary to rupture the chemical tubing inside.
[0742] In addition to a charging cartridge for charging and/or
recharging bioluminescent novelty items, the cartridge
incorporating compressible tubing can be formed to allow
replacement of the compressible tubing portions within the block.
More specifically, once a cartridge has been used to charge or
recharge a novelty item, the compressible tubing having a fluid and
at least one component of the bioluminescent reaction, and the
compressible tubing having the dry ingredients, may be removed from
the block, and a new set of chemical tubing may be positioned
within the block. As a result, the cartridge may be repeatedly
used, replacing only the chemical tubing portions. This would
provide for the minimization of the costs associated with the use
and repeated use of the novelty items because only the chemical
tubing portions would have to be replaced, instead of discarding
the entire cartridge following each use.
[0743] Since modifications will be apparent to those of skill in
this art, it is intended that this invention be limited only by the
scope of the appended claims.
[0744] Summary of Sequences of Representative Luciferases and the
Reductase Set Forth in the Sequence Listing [0745] 1. SEQ ID NO. 1
Renilla reinformis Luciferase [U.S. Pat. No. 5,418,155] [0746] 2.
SEQ ID NO. 2 Cypridina hilgendorfii Luciferase [EP 0 387 355]
[0747] 3. SEQ ID NO. 3 Modified Luciola cruciata Luciferase
[firefly; U.S. Pat. No. 4,968,613] [0748] 4. SEQ ID NO. 4 Vargula
(Cypridina) luciferase [Thompson et al. (1989) Proc. Natl. Acad.
Sci. U.S.A. 86:6567-657] and from JP 3-30678 Osaka [0749] 5. SEQ ID
NO. 5 Apoaequorin-encoding gene [U S. Pat. No. 5,093,240, pAQ440]
[0750] 6. SEQ ID NO. 6 Recombinant Aequorin AEQ1 [Prasher et al.
(1987) "Sequence Comparisons of cDNAs Encoding for Aequorin
Isotypes," Biochemistry 26:1326-1332] [0751] 7. SEQ ID NO. 7
Recombinant Aequorin AEQ2 [Prasher et al. (1987)] [0752] 8. SEQ ID
NO. 8 Recombinant Aequorin AEQ3 [Prasher et al. (1987)] [0753] 9.
SEQ ID NO. 9 Aequorin photoprotein [Charbonneau et al. (1985)
"Amino Acid Sequence of the Calcium-Dependent Photoprotein
Aequorin," Biochemistry 24:6762-6771] [0754] 10. SEQ ID NO. 10
Aequorin mutant with increased bioluminescence activity [U.S. Pat.
No. 5,360,728; Asp 124 changed to Ser] [0755] 11. SEQ ID NO. 11
Aequorin mutant with increased bioluminescence activity [U.S. Pat.
No. 5,360,728; Glu 135 changed to Ser] [0756] 12. SEQ ID NO. 12
Aequorin mutant with increased bioluminescence activity [U.S. Pat.
No. 5,360,728 Gly 129 changed to Ala] [0757] 13. SEQ ID NO. 13
Recombinant apoaequorin [sold by Sealite, Sciences, Bogart, Ga. as
AQUALITE.RTM., when reconstituted to form aequorin] [0758] 14. SEQ
ID NO. 14 Vibrio fisheri Flavin reductase [U.S. Pat. No. 5,484,723]
Sequence CWU 0
0
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 14 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 1196 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (ix) FEATURE: (A) NAME/KEY: Coding
Sequence (B) LOCATION: 1...942 (D) OTHER INFORMATION: Renilla
Reinformis Luciferase (x) PUBLICATION INFORMATION: (H) DOCUMENT
NUMBER: 5,418,155 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: AGC TTA
AAG ATG ACT TCG AAA GTT TAT GAT CCA GAA CAA AGG AAA CGG 48 Ser Leu
Lys Met Thr Ser Lys Val Tyr Asp Pro Glu Gln Arg Lys Arg 1 5 10 15
ATG ATA ACT GGT CCG CAG TGG TGG GCC AGA TGT AAA CAA ATG AAT GTT 96
Met Ile Thr Gly Pro Gln Trp Trp Ala Arg Cys Lys Gln Met Asn Val 20
25 30 CTT GAT TCA TTT ATT AAT TAT TAT GAT TCA GAA AAA CAT GCA GAA
AAT 144 Leu Asp Ser Phe Ile Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu
Asn 35 40 45 GCT GTT ATT TTT TTA CAT GGT AAC GCG GCC TCT TCT TAT
TTA TGG CGA 192 Ala Val Ile Phe Leu His Gly Asn Ala Ala Ser Ser Tyr
Leu Trp Arg 50 55 60 CAT GTT GTG CCA CAT ATT GAG CCA GTA GCG CGG
TGT ATT ATA CCA GAT 240 His Val Val Pro His Ile Glu Pro Val Ala Arg
Cys Ile Ile Pro Asp 65 70 75 80 CTT ATT GGT ATG GGC AAA TCA GGC AAA
TCT GGT AAT GGT TCT TAT AGG 288 Leu Ile Gly Met Gly Lys Ser Gly Lys
Ser Gly Asn Gly Ser Tyr Arg 85 90 95 TTA CTT GAT CAT TAC AAA TAT
CTT ACT GCA TGG TTG AAC TTC TTA ATT 336 Leu Leu Asp His Tyr Lys Tyr
Leu Thr Ala Trp Leu Asn Phe Leu Ile 100 105 110 TAC CAA AGA AGA TCA
TTT TTT GTC GGC CAT GAT TGG GGT GCT TGT TTG 384 Tyr Gln Arg Arg Ser
Phe Phe Val Gly His Asp Trp Gly Ala Cys Leu 115 120 125 GCA TTT CAT
TAT AGC TAT GAG CAT CAA GAT AAG ATC AAA GCA ATA GTT 432 Ala Phe His
Tyr Ser Tyr Glu His Gln Asp Lys Ile Lys Ala Ile Val 130 135 140 CAC
GCT GAA AGT GTA GTA GAT GTG ATT GAA TCA TGG GAT GAA TGG CCT 480 His
Ala Glu Ser Val Val Asp Val Ile Glu Ser Trp Asp Glu Trp Pro 145 150
155 160 GAT ATT GAA GAA GAT ATT GCG TTG ATC AAA TCT GAA GAA GGA GAA
AAA 528 Asp Ile Glu Glu Asp Ile Ala Leu Ile Lys Ser Glu Glu Gly Glu
Lys 165 170 175 ATG GTT TTG GAG AAT AAC TTC TTC GTG GAA ACC ATG TTG
CCA TCA AAA 576 Met Val Leu Glu Asn Asn Phe Phe Val Glu Thr Met Leu
Pro Ser Lys 180 185 190 ATC ATG AGA AAG TTA GAA CCA GAA GAA TTT GCA
GCA TAT CTT GAA CCA 624 Ile Met Arg Lys Leu Glu Pro Glu Glu Phe Ala
Ala Tyr Leu Glu Pro 195 200 205 TTC AAA GAG AAA GGT GAA GTT CGT CGT
CCA ACA TTA TCA TGG CCT CGT 672 Phe Lys Glu Lys Gly Glu Val Arg Arg
Pro Thr Leu Ser Trp Pro Arg 210 215 220 GAA ATC CCG TTA GTA AAA GGT
GGT AAA CCT GAC GTT GTA CAA ATT GTT 720 Glu Ile Pro Leu Val Lys Gly
Gly Lys Pro Asp Val Val Gln Ile Val 225 230 235 240 AGG AAT TAT AAT
GCT TAT CTA CGT GCA AGT GAT GAT TTA CCA AAA ATG 768 Arg Asn Tyr Asn
Ala Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met 245 250 255 TTT ATT
GAA TCG GAT CCA GGA TTC TTT TCC AAT GCT ATT GTT GAA GGC 816 Phe Ile
Glu Ser Asp Pro Gly Phe Phe Ser Asn Ala Ile Val Glu Gly 260 265 270
GCC AAG AAG TTT CCT AAT ACT GAA TTT GTC AAA GTA AAA GGT CTT CAT 864
Ala Lys Lys Phe Pro Asn Thr Glu Phe Val Lys Val Lys Gly Leu His 275
280 285 TTT TCG CAA GAA GAT GCA CCT GAT GAA ATG GGA AAA TAT ATC AAA
TCG 912 Phe Ser Gln Glu Asp Ala Pro Asp Glu Met Gly Lys Tyr Ile Lys
Ser 290 295 300 TTC GTT GAG CGA GTT CTC AAA AAT GAA CAA TAA
TTACTTTGGT TTTTTATTTA 965 Phe Val Glu Arg Val Leu Lys Asn Glu Gln
305 310 CATTTTTCCC GGGTTTAATA ATATAAATGT CATTTTCAAC AATTTTATTT
TAACTGAATA 1025 TTTCACAGGG AACATTCATA TATGTTGATT AATTTAGCTC
GAACTTTACT CTGTCATATC 1085 ATTTTGGAAT ATTACCTCTT TCAATGAAAC
TTTATAAACA GTGGTTCAAT TAATTAATAT 1145 ATATTATAAT TACATTTGTT
ATGTAATAAA CTCGGTTTTA TTATAAAAAA A 1196 (2) INFORMATION FOR SEQ ID
NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1822 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: Coding
Sequence (B) LOCATION: 1...1665 (D) OTHER INFORMATION: Cypridina
hilgendorfii luciferase (x) PUBLICATION INFORMATION: (H) DOCUMENT
NUMBER: EP 0 387 355 TORAY (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
ATG AAG CTA ATA ATT CTG TCT ATT ATA TTG GCC TAC TGT GTC ACA GTC 48
Met Lys Leu Ile Ile Leu Ser Ile Ile Leu Ala Tyr Cys Val Thr Val 1 5
10 15 AAC TGC CAG GAT GCA TGT CCT GTA GAA GCT GAA GCA CCG TCA AGT
ACA 96 Asn Cys Gln Asp Ala Cys Pro Val Glu Ala Glu Ala Pro Ser Ser
Thr 20 25 30 CCA ACA GTC CCA ACA TCT TGT GAA GCT AAA GAA GGA GAA
TGT ATC GAT 144 Pro Thr Val Pro Thr Ser Cys Glu Ala Lys Glu Gly Glu
Cys Ile Asp 35 40 45 ACC AGA TGC GCA ACA TGT AAA CGA GAC ATA CTA
TCA GAC GGA CTG TGT 192 Thr Arg Cys Ala Thr Cys Lys Arg Asp Ile Leu
Ser Asp Gly Leu Cys 50 55 60 GAA AAT AAA CCA GGG AAG ACA TGC TGT
AGA ATG TGC CAG TAT GTA ATT 240 Glu Asn Lys Pro Gly Lys Thr Cys Cys
Arg Met Cys Gln Tyr Val Ile 65 70 75 80 GAA TCC AGA GTA GAA GCT GCT
GGA TAT TTT AGA ACG TTT TAC GCC AAA 288 Glu Ser Arg Val Glu Ala Ala
Gly Tyr Phe Arg Thr Phe Tyr Ala Lys 85 90 95 AGA TTT AAT TTT CAG
GAA CCT GGT AAA TAT GTG CTG GCT CGA GGA ACC 336 Arg Phe Asn Phe Gln
Glu Pro Gly Lys Tyr Val Leu Ala Arg Gly Thr 100 105 110 AAG GGT GGC
GAC TGG TCT GTA ACC CTC ACC ATG GAG AAT CTA GAT GGA 384 Lys Gly Gly
Asp Trp Ser Val Thr Leu Thr Met Glu Asn Leu Asp Gly 115 120 125 CAG
AAG GGA GCT GTA CTG ACT AAG ACA ACA CTG GAG GTA GTA GGA GAC 432 Gln
Lys Gly Ala Val Leu Thr Lys Thr Thr Leu Glu Val Val Gly Asp 130 135
140 GTA ATA GAC ATT ACT CAA GCT ACT GCA GAT CCT ATC ACA GTT AAC GGA
480 Val Ile Asp Ile Thr Gln Ala Thr Ala Asp Pro Ile Thr Val Asn Gly
145 150 155 160 GGA GCT GAC CCA GTT ATC GCT AAC CCG TTC ACA ATT GGT
GAG GTG ACC 528 Gly Ala Asp Pro Val Ile Ala Asn Pro Phe Thr Ile Gly
Glu Val Thr 165 170 175 ATT GCT GTT GTC GAA ATA CCC GGC TTC AAT ATT
ACA GTC ATC GAA TTC 576 Ile Ala Val Val Glu Ile Pro Gly Phe Asn Ile
Thr Val Ile Glu Phe 180 185 190 TTT AAA CTA ATC GTG ATA GAT ATT CTG
GGA GGA AGA TCT GTG AGA ATT 624 Phe Lys Leu Ile Val Ile Asp Ile Leu
Gly Gly Arg Ser Val Arg Ile 195 200 205 GCT CCA GAC ACA GCA AAC AAA
GGA CTG ATA TCT GGT ATC TGT GGT AAT 672 Ala Pro Asp Thr Ala Asn Lys
Gly Leu Ile Ser Gly Ile Cys Gly Asn 210 215 220 CTG GAG ATG AAT GAC
GCT GAT GAC TTT ACT ACA GAC GCA GAT CAG CTG 720 Leu Glu Met Asn Asp
Ala Asp Asp Phe Thr Thr Asp Ala Asp Gln Leu 225 230 235 240 GCG ATC
CAA CCC AAC ATA AAC AAA GAG TTC GAC GGC TGC CCA TTC TAC 768 Ala Ile
Gln Pro Asn Ile Asn Lys Glu Phe Asp Gly Cys Pro Phe Tyr 245 250 255
GGG AAT CCT TCT GAT ATC GAA TAC TGC AAA GGT CTC ATG GAG CCA TAC 816
Gly Asn Pro Ser Asp Ile Glu Tyr Cys Lys Gly Leu Met Glu Pro Tyr 260
265 270 AGA GCT GTA TGT CGT AAC AAT ATC AAC TTC TAC TAT TAC ACT CTG
TCC 864 Arg Ala Val Cys Arg Asn Asn Ile Asn Phe Tyr Tyr Tyr Thr Leu
Ser 275 280 285 TGC GCC TTC GCT TAC TGT ATG GGA GGA GAA GAA AGA GCT
AAA CAC GTC 912 Cys Ala Phe Ala Tyr Cys Met Gly Gly Glu Glu Arg Ala
Lys His Val 290 295 300 CTT TTC GAC TAT GTT GAG ACA TGC GCT GCA CCG
GAA ACG AGA GGA ACG 960 Leu Phe Asp Tyr Val Glu Thr Cys Ala Ala Pro
Glu Thr Arg Gly Thr 305 310 315 320 TGT GTT TTA TCA GGA CAT ACT TTC
TAT GAC ACA TTC GAC AAA GCC AGA 1008 Cys Val Leu Ser Gly His Thr
Phe Tyr Asp Thr Phe Asp Lys Ala Arg 325 330 335 TAT CAA TTC CAG GGC
CCA TGC AAA GAG CTT CTG ATG GCC GCA GAC TGT 1056 Tyr Gln Phe Gln
Gly Pro Cys Lys Glu Leu Leu Met Ala Ala Asp Cys 340 345 350 TAC TGG
AAC ACA TGG GAT GTA AAG GTT TCA CAT AGA GAT GTT GAG TCA 1104 Tyr
Trp Asn Thr Trp Asp Val Lys Val Ser His Arg Asp Val Glu Ser 355 360
365 TAC ACT GAG GTA GAG AAA GTA ACA ATC AGG AAA CAG TCA ACT GTA GTA
1152 Tyr Thr Glu Val Glu Lys Val Thr Ile Arg Lys Gln Ser Thr Val
Val 370 375 380 GAT TTG ATT GTG GAT GGC AAG CAG GTC AAG GTT GGA GGA
GTG GAT GTA 1200 Asp Leu Ile Val Asp Gly Lys Gln Val Lys Val Gly
Gly Val Asp Val 385 390 395 400 TCT ATC CCG TAC AGT TCT GAG AAC ACA
TCC ATA TAC TGG CAG GAT GGA 1248 Ser Ile Pro Tyr Ser Ser Glu Asn
Thr Ser Ile Tyr Trp Gln Asp Gly 405 410 415 GAC ATC CTG ACG ACG GCC
ATC CTA CCT GAA GCT CTT GTC GTT AAG TTC 1296 Asp Ile Leu Thr Thr
Ala Ile Leu Pro Glu Ala Leu Val Val Lys Phe 420 425 430
AAC TTT AAG CAG CTC CTT GTA GTT CAT ATC AGA GAT CCA TTC GAT GGA
1344 Asn Phe Lys Gln Leu Leu Val Val His Ile Arg Asp Pro Phe Asp
Gly 435 440 445 AAG ACA TGC GGC ATA TGT GGT AAC TAT AAT CAA GAT TCA
ACT GAT GAT 1392 Lys Thr Cys Gly Ile Cys Gly Asn Tyr Asn Gln Asp
Ser Thr Asp Asp 450 455 460 TTC TTT GAC GCA GAA GGA GCA TGC GCT CTG
ACC CCC AAT CCC CCA GGA 1440 Phe Phe Asp Ala Glu Gly Ala Cys Ala
Leu Thr Pro Asn Pro Pro Gly 465 470 475 480 TGT ACA GAG GAG CAG AAA
CCA GAA GCT GAG CGA CTC TGC AAT AGT CTA 1488 Cys Thr Glu Glu Gln
Lys Pro Glu Ala Glu Arg Leu Cys Asn Ser Leu 485 490 495 TTT GAT AGT
TCT ATC GAC GAG AAA TGT AAT GTC TGC TAC AAG CCT GAC 1536 Phe Asp
Ser Ser Ile Asp Glu Lys Cys Asn Val Cys Tyr Lys Pro Asp 500 505 510
CGT ATT GCA CGA TGT ATG TAC GAG TAT TGC CTG AGG GGA CAG CAA GGA
1584 Arg Ile Ala Arg Cys Met Tyr Glu Tyr Cys Leu Arg Gly Gln Gln
Gly 515 520 525 TTC TGT GAC CAT GCT TGG GAG TTC AAA AAA GAA TGC TAC
ATA AAG CAT 1632 Phe Cys Asp His Ala Trp Glu Phe Lys Lys Glu Cys
Tyr Ile Lys His 530 535 540 GGA GAC ACT CTA GAA GTA CCA CCT GAA TGC
CAA TAA ATGAACAAAG 1678 Gly Asp Thr Leu Glu Val Pro Pro Glu Cys Gln
545 550 555 ATACAGAAGC TAAGACTACT ACAGCAGAAG ATAAAAGAGA AGCTGTAGTT
CTTCAAAAAC 1738 AGTATATTTT GATGTACTCA TTGTTTACTT ACATAAAAAT
AAATTGTTAT TATCATAACG 1798 TAAAGAAAAA AAAAAAAAAA AAAA 1822 (2)
INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 1644 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 1...1644 (D) OTHER
INFORMATION: Luciola Cruciata Luciferase (Firefly) (x) PUBLICATION
INFORMATION: (H) DOCUMENT NUMBER: 4,968,613 (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:3: ATG GAA AAC ATG GAA AAC GAT GAA AAT ATT
GTA GTT GGA CCT AAA CCG 48 Met Glu Asn Met Glu Asn Asp Glu Asn Ile
Val Val Gly Pro Lys Pro 1 5 10 15 TTT TAC CCT ATC GAA GAG GGA TCT
GCT GGA ACA CAA TTA CGC AAA TAC 96 Phe Tyr Pro Ile Glu Glu Gly Ser
Ala Gly Thr Gln Leu Arg Lys Tyr 20 25 30 ATG GAG CGA TAT GCA AAA
CTT GGC GCA ATT GCT TTT ACA AAT GCA GTT 144 Met Glu Arg Tyr Ala Lys
Leu Gly Ala Ile Ala Phe Thr Asn Ala Val 35 40 45 ACT GGT GTT GAT
TAT TCT TAC GCC GAA TAC TTG GAG AAA TCA TGT TGT 192 Thr Gly Val Asp
Tyr Ser Tyr Ala Glu Tyr Leu Glu Lys Ser Cys Cys 50 55 60 CTA GGA
AAA GCT TTG CAA AAT TAT GGT TTG GTT GTT GAT GGC AGA ATT 240 Leu Gly
Lys Ala Leu Gln Asn Tyr Gly Leu Val Val Asp Gly Arg Ile 65 70 75 80
GCG TTA TGC AGT GAA AAC TGT GAA GAA TTT TTT ATT CCT GTA ATA GCC 288
Ala Leu Cys Ser Glu Asn Cys Glu Glu Phe Phe Ile Pro Val Ile Ala 85
90 95 GGA CTG TTT ATA GGT GTA GGT GTT GCA CCC ACT AAT GAG ATT TAC
ACT 336 Gly Leu Phe Ile Gly Val Gly Val Ala Pro Thr Asn Glu Ile Tyr
Thr 100 105 110 TTA CGT GAA CTG GTT CAC AGT TTA GGT ATC TCT AAA CCA
ACA ATT GTA 384 Leu Arg Glu Leu Val His Ser Leu Gly Ile Ser Lys Pro
Thr Ile Val 115 120 125 TTT AGT TCT AAA AAA GGC TTA GAT AAA GTT ATA
ACA GTA CAG AAA ACA 432 Phe Ser Ser Lys Lys Gly Leu Asp Lys Val Ile
Thr Val Gln Lys Thr 130 135 140 GTA ACT ACT ATT AAA ACC ATT GTT ATA
CTA GAT AGC AAA GTT GAT TAT 480 Val Thr Thr Ile Lys Thr Ile Val Ile
Leu Asp Ser Lys Val Asp Tyr 145 150 155 160 CGA GGA TAT CAA TGT CTG
GAC ACC TTT ATA AAA AGA AAC ACT CCA CCA 528 Arg Gly Tyr Gln Cys Leu
Asp Thr Phe Ile Lys Arg Asn Thr Pro Pro 165 170 175 GGT TTT CAA GCA
TCC AGT TTC AAA ACT GTG GAA GTT GAC CGT AAA GAA 576 Gly Phe Gln Ala
Ser Ser Phe Lys Thr Val Glu Val Asp Arg Lys Glu 180 185 190 CAA GTT
GCT CTT ATA ATG AAC TCT TCG GGT TCT ACC GGT TTG CCA AAA 624 Gln Val
Ala Leu Ile Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys 195 200 205
GGC GTA CAA CTT ACT CAC GAA AAT ACA GTC ACT AGA TTT TCT CAT GCT 672
Gly Val Gln Leu Thr His Glu Asn Thr Val Thr Arg Phe Ser His Ala 210
215 220 AGA GAT CCG ATT TAT GGT AAC CAA GTT TCA CCA GGC ACC GCT GTT
TTA 720 Arg Asp Pro Ile Tyr Gly Asn Gln Val Ser Pro Gly Thr Ala Val
Leu 225 230 235 240 ACT GTC GTT CCA TTC CAT CAT GGT TTT GGT ATG TTC
ACT ACT CTA GGG 768 Thr Val Val Pro Phe His His Gly Phe Gly Met Phe
Thr Thr Leu Gly 245 250 255 TAT TTA ATT TGT GGT TTT CGT GTT GTA ATG
TTA ACA AAA TTC GAT GAA 816 Tyr Leu Ile Cys Gly Phe Arg Val Val Met
Leu Thr Lys Phe Asp Glu 260 265 270 GAA ACA TTT TTA AAA ACT CTA CAA
GAT TAT AAA TGT ACA AGT GTT ATT 864 Glu Thr Phe Leu Lys Thr Leu Gln
Asp Tyr Lys Cys Thr Ser Val Ile 275 280 285 CTT GTA CCG ACC TTG TTT
GCA ATT CTC AAC AAA AGT GAA TTA CTC AAT 912 Leu Val Pro Thr Leu Phe
Ala Ile Leu Asn Lys Ser Glu Leu Leu Asn 290 295 300 AAA TAC GAT TTG
TCA AAT TTA GTT GAG ATT GCA TCT GGC GGA GCA CCT 960 Lys Tyr Asp Leu
Ser Asn Leu Val Glu Ile Ala Ser Gly Gly Ala Pro 305 310 315 320 TTA
TCA AAA GAA GTT GGT GAA GCT GTT GCT AGA CGC TTT AAT CTT CCC 1008
Leu Ser Lys Glu Val Gly Glu Ala Val Ala Arg Arg Phe Asn Leu Pro 325
330 335 GGT GTT CGT CAA GGT TAT GGT TTA ACA GAA ACA ACA TCT GCC ATT
ATT 1056 Gly Val Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala
Ile Ile 340 345 350 ATT ACA CCA GAA GGA GAC GAT AAA CCA GGA GCT TCT
GGA AAA GTC GTG 1104 Ile Thr Pro Glu Gly Asp Asp Lys Pro Gly Ala
Ser Gly Lys Val Val 355 360 365 CCG TTG TTT AAA GCA AAA GTT ATT GAT
CTT GAT ACC AAA AAA TCT TTA 1152 Pro Leu Phe Lys Ala Lys Val Ile
Asp Leu Asp Thr Lys Lys Ser Leu 370 375 380 GGT CCT AAC AGA CGT GGA
GAA GTT TGT GTT AAA GGA CCT ATG CTT ATG 1200 Gly Pro Asn Arg Arg
Gly Glu Val Cys Val Lys Gly Pro Met Leu Met 385 390 395 400 AAA GGT
TAT GTA AAT AAT CCA GAA GCA ACA AAA GAA CTT ATT GAC GAA 1248 Lys
Gly Tyr Val Asn Asn Pro Glu Ala Thr Lys Glu Leu Ile Asp Glu 405 410
415 GAA GGT TGG CTG CAC ACC GGA GAT ATT GGA TAT TAT GAT GAA GAA AAA
1296 Glu Gly Trp Leu His Thr Gly Asp Ile Gly Tyr Tyr Asp Glu Glu
Lys 420 425 430 CAT TTC TTT ATT GTC GAT CGT TTG AAG TCT TTA ATC AAA
TAC AAA GGA 1344 His Phe Phe Ile Val Asp Arg Leu Lys Ser Leu Ile
Lys Tyr Lys Gly 435 440 445 TAC CAA GTA CCA CCT GCC GAA TTA GAA TCC
GTT CTT TTG CAA CAT CCA 1392 Tyr Gln Val Pro Pro Ala Glu Leu Glu
Ser Val Leu Leu Gln His Pro 450 455 460 TCT ATC TTT GAT GCT GGT GTT
GCC GGC GTT CCT GAT CCT GTA GCT GGC 1440 Ser Ile Phe Asp Ala Gly
Val Ala Gly Val Pro Asp Pro Val Ala Gly 465 470 475 480 GAG CTT CCA
GGA GCC GTT GTT GTA CTG GAA AGC GGA AAA AAT ATG ACC 1488 Glu Leu
Pro GLy Ala Val Val Val Leu Glu Ser Gly Lys Asn Met Thr 485 490 495
GAA AAA GAA GTA ATG GAT TAT GTT GCA AGT CAA GTT TCA AAT GCA AAA
1536 Glu Lys Glu Val Met Asp Tyr Val Als Ser Gln Val Ser Asn Ala
Lys 500 505 510 CGT TTA CGT GGT GGT GTT CGT TTT GTG GAT GAA GTA CCT
AAA GGT CTT 1584 Arg Leu Arg Gly Gly Val Arg Phe Val Asp Glu Val
Pro Lys Gly Leu 515 520 525 ACT GGA AAA ATT GAC GGC AGA GCA ATT AGA
GAA ATC CTT AAG AAA CCA 1632 Thr Gly Lys Ile Asp Gly Arg Ala Ile
Arg Glu Ile Leu Lys Lys Pro 530 535 540 GTT GCT AAG ATG 1644 Val
Ala Lys Met 545 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 1820 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (ix) FEATURE: (A) NAME/KEY: Coding Sequence (B) LOCATION:
1...1664 (D) OTHER INFORMATION: Vargula (cypridina) luciferase (x)
PUBLICATION INFORMATION: (A) AUTHORS: Thompson et al. (C) JOURNAL:
Proc. Natl. Acad. Sci. U.S.A. (D) VOLUME: 86 (F) PAGES: 1326-1332
(G) DATE: (1989) (H) DOCUMENT NUMBER: JP 3-30678 Osaka (Tsuji) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:4: ATG AAG ATA ATA ATT CTG TCT GTT
ATA TTG GCC TAC TGT GTC ACC GAC 48 Met Lys Ile Ile Ile Leu Ser Val
Ile Leu Ala Tyr Cys Val Thr Asp 1 5 10 15 AAC TGT CAA GAT GCA TGT
CCT GTA GAA GCG GAA CCG CCA TCA AGT ACA 96 Asn Cys Gln Asp Ala Cys
Pro Val Glu Ala Glu Pro Pro Ser Ser Thr 20 25 30 CCA ACA GTT CCA
ACT TCT TGT GAA GCT AAA GAA GGA GAA TGT ATA GAT 144 Pro Thr Val Pro
Thr Ser Cys Glu Ala Lys Glu Gly Glu Cys Ile Asp 35 40 45 ACC AGA
TGC GCA ACA TGT AAA CGA GAT ATA CTA TCA GAT GGA CTG TGT 192 Thr Arg
Cys Ala Thr Cys Lys Arg Asp Ile Leu Ser Asp Gly Leu Cys 50 55 60
GAA AAT AAA CCA GGG AAG ACA TGC TGT AGA ATG TGC CAG TAT GTG ATT 240
Glu Asn Lys Pro Gly Lys Thr Cys Cys Arg Met Cys Gln Tyr Val Ile 65
70 75 80 GAA TGC AGA GTA GAA GCA GCT GGT TAT TTT AGA ACG TTT TAC
GGC AAA 288 Glu Cys Arg Val Glu Ala Ala Gly Tyr Phe Arg Thr Phe Tyr
Gly Lys 85 90 95 AGA TTT AAT TTT CAG GAA CCT GGT AAA TAT GTG CTG
GCT AGG GGA ACC 336 Arg Phe Asn Phe Gln Glu Pro Gly Lys Tyr Val Leu
Ala Arg Gly Thr
100 105 110 AAG GGT GGC GAT TGG TCT GTA ACC CTC ACC ATG GAG AAT CTA
GAT GGA 384 Lys Gly Gly Asp Trp Ser Val Thr Leu Thr Met Glu Asn Leu
Asp Gly 115 120 125 CAG AAG GGA GCT GTG CTG ACT AAG ACA ACA CTG GAG
GTT GCA GGA GAC 432 Gln Lys Gly Ala Val Leu Thr Lys Thr Thr Leu Glu
Val Ala Gly Asp 130 135 140 GTA ATA GAC ATT ACT CAA GCT ACT GCA GAT
CCT ATC ACA GTT AAC GGA 480 Val Ile Asp Ile Thr Gln Ala Thr Ala Asp
Pro Ile Thr Val Asn Gly 145 150 155 160 GGA GCT GAC CCA GTT ATC GCT
AAC CCG TTC ACA ATT GGT GAG GTG ACC 528 Gly Ala Asp Pro Val Ile Ala
Asn Pro Phe Thr Ile Gly Glu Val Thr 165 170 175 ATT GCT GTT GTT GAA
ATA CCG GGC TTC AAT ATC ACA GTC ATC GAA TTC 576 Ile Ala Val Val Glu
Ile Pro Gly Phe Asn Ile Thr Val Ile Glu Phe 180 185 190 TTT AAA CTA
ATC GTG ATT GAT ATT CTG GGA GGA AGA TCT GTC AGA ATT 624 Phe Lys Leu
Ile Val Ile Asp Ile Leu Gly Gly Arg Ser Val Arg Ile 195 200 205 GCT
CCA GAC ACA GCA AAC AAA GGA CTG ATA TCT GGT ATC TGT GGT AAT 672 Ala
Pro Asp Thr Ala Asn Lys Gly Leu Ile Ser Gly Ile Cys Gly Asn 210 215
220 CTG GAG ATG AAT GAC GCT GAT GAC TTT ACT ACA GAT GCA GAT CAG CTG
720 Leu Glu Met Asn Asp Ala Asp Asp Phe Thr Thr Asp Ala Asp Gln Leu
225 230 235 240 GCG ATC CAA CCC AAC ATA AAC AAA GAG TTC GAC GGC TGC
CCA TTC TAT 768 Ala Ile Gln Pro Asn Ile Asn Lys Glu Phe Asp Gly Cys
Pro Phe Tyr 245 250 255 GGC AAT CCT TCT GAT ATC GAA TAC TGC AAA GGT
CTG ATG GAG CCA TAC 816 Gly Asn Pro Ser Asp Ile Glu Tyr Cys Lys Gly
Leu Met Glu Pro Tyr 260 265 270 AGA GCT GTA TGT CGT AAC AAT ATC AAC
TTC TAC TAT TAC ACT CTA TCC 864 Arg Ala Val Cys Arg Asn Asn Ile Asn
Phe Tyr Tyr Tyr Thr Leu Ser 275 280 285 TGT GCC TTC GCT TAC TGT ATG
GGA GGA GAA GAA AGA GCT AAA CAC GTC 912 Cys Ala Phe Ala Tyr Cys Met
Gly Gly Glu Glu Arg Ala Lys His Val 290 295 300 CTT TTC GAC TAT GTT
GAG ACA TGC GCT GCG CCG GAA ACG AGA GGA ACG 960 Leu Phe Asp Tyr Val
Glu Thr Cys Ala Ala Pro Glu Thr Arg Gly Thr 305 310 315 320 TGT GTT
TTA TCA GGA CAT ACT TTC TAT GAC ACA TTC GAC AAA GCA AGA 1008 Cys
Val Leu Ser Gly His Thr Phe Tyr Asp Thr Phe Asp Lys Ala Arg 325 330
335 TAT CAA TTC CAG GGC CCA TGC AAG GAG ATT CTG ATG GCC GCA GAC TGT
1056 Tyr Gln Phe Gln Gly Pro Cys Lys Glu Ile Leu Met Ala Ala Asp
Cys 340 345 350 TAC TGG AAC ACA TGG GAT GTA AAG GTT TCA CAT AGA GAC
GTC GAA TCA 1104 Tyr Trp Asn Thr Trp Asp Val Lys Val Ser His Arg
Asp Val Glu Ser 355 360 365 TAC ACT GAG GTA GAG AAA GTA ACA ATC AGG
AAA CAG TCA ACT GTA GTA 1152 Tyr Thr Glu Val Glu Lys Val Thr Ile
Arg Lys Gln Ser Thr Val Val 370 375 380 GAT CTC ATT GTG GAT GGC AAG
CAG GTC AAG GTT GGA GGA GTG GAT GTA 1200 Asp Leu Ile Val Asp Gly
Lys Gln Val Lys Val Gly Gly Val Asp Val 385 390 395 400 TCT ATC CCG
TAC AGC TCT GAG AAC ACT TCC ATA TAC TGG CAG GAT GGA 1248 Ser Ile
Pro Tyr Ser Ser Glu Asn Thr Ser Ile Tyr Trp Gln Asp Gly 405 410 415
GAC ATC CTG ACG ACG GCC ATC CTA CCT GAA GCT CTT GTC GTT AAG TTC
1296 Asp Ile Leu Thr Thr Ala Ile Leu Pro Glu Ala Leu Val Val Lys
Phe 420 425 430 AAC TTT AAG CAG CTC CTT GTA GTT CAT ATC AGA GAT CCA
TTC GAT GCA 1344 Asn Phe Lys Gln Leu Leu Val Val His Ile Arg Asp
Pro Phe Asp Ala 435 440 445 AAG ACA TGC GGC ATA TGT GGT AAC TAT AAT
CAA GAT TCA ACT GAT GAT 1392 Lys Thr Cys Gly Ile Cys Gly Asn Tyr
Asn Gln Asp Ser Thr Asp Asp 450 455 460 TTC TTT GAC GCA GAA GGA GCA
TGC GCT CTA ACC CCC AAC CCC CCA GGA 1440 Phe Phe Asp Ala Glu Gly
Ala Cys Ala Leu Thr Pro Asn Pro Pro Gly 465 470 475 480 TGT ACA GAG
GAA CAG AAA CCA GAA GCT GAG CGA CTT TGC AAT AAT CTC 1488 Cys Thr
Glu Glu Gln Lys Pro Glu Ala Glu Arg Leu Cys Asn Asn Leu 485 490 495
TTT GAT TCT TCT ATC GAC GAG AAA TGT AAT GTC TGC TAC AAG CCT GAC
1536 Phe Asp Ser Ser Ile Asp Glu Lys Cys Asn Val Cys Tyr Lys Pro
Asp 500 505 510 CGG ATT GCC CGA TGT ATG TAC GAG TAT TGC CTG AGG GGA
CAA CAA GGA 1584 Arg Ile Ala Arg Cys Met Tyr Glu Tyr Cys Leu Arg
Gly Gln Gln Gly 515 520 525 TTT TGT GAC CAT GCT TGG GAG TTC AAG AAA
GAA TGC TAC ATA AAA CAT 1632 Phe Cys Asp His Ala Trp Glu Phe Lys
Lys Glu Cys Tyr Ile Lys His 530 535 540 GGA GAC ACT CTA GAA GTA CCA
CCT GAA TGT CAA TAA ACGTACAAAG 1678 Gly Asp Thr Leu Glu Val Pro Pro
Glu Cys Gln 545 550 555 ATACAGAAGC TAAGGCTACT ACAGCAGAAG ATAAAAAAGA
AACTGTAGTT CCTTCAAAAA 1738 CCGTGTATTT TATGTACTCA TTGTTTAATT
AGAGCAAAAT AAATTGTTAT TATCATAACT 1798 TAAACTAAAA AAAAAAAAAA AA 1820
(2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 958 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: (vi)
ORIGINAL SOURCE: (ix) FEATURE: (A) NAME/KEY: Coding Sequence (B)
LOCATION: 115...702 (D) OTHER INFORMATION: apoaequorin-encoding
gene (x) PUBLICATION INFORMATION: (A) AUTHORS: Inouye et al. (C)
JOURNAL: Proc. Natl. Acad. Sci. U.S.A. (D) VOLUME: 82 (F) PAGES:
3154-3158 (G) DATE: (1985) (H) DOCUMENT NUMBER: 5,093,240 (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:5: GGGGGGGGGG GGGGGGGGGG GGGGGGGGGG
GGGAATGCAA TTCATCTTTG CATCAAAGAA 60 TTACATCAAA TCTCTAGTTG
ATCAACTAAA TTGTCTCGAC AACAACAAGC AAAC ATG 117 Met 1 ACA AGC AAA CAA
TAC TCA GTC AAG CTT ACA TCA GAC TTC GAC AAC CCA 165 Thr Ser Lys Gln
Tyr Ser Val Lys Leu Thr Ser Asp Phe Asp Asn Pro 5 10 15 AGA TGG ATT
GGA CGA CAC AAG CAT ATG TTC AAT TTC CTT GAT GTC AAC 213 Arg Trp Ile
Gly Arg His Lys His Met Phe Asn Phe Leu Asp Val Asn 20 25 30 CAC
AAT GGA AAA ATC TCT CTT GAC GAG ATG GTC TAC AAG GCA TCT GAT 261 His
Asn Gly Lys Ile Ser Leu Asp Glu Met Val Tyr Lys Ala Ser Asp 35 40
45 ATT GTC ATC AAT AAC CTT GGA GCA ACA CCT GAG CAA GCC AAA CGA CAC
309 Ile Val Ile Asn Asn Leu Gly Ala Thr Pro Glu Gln Ala Lys Arg His
50 55 60 65 AAA GAT GCT GTA GAA GCC TTC TTC GGA GGA GCT GGA ATG AAA
TAT GGT 357 Lys Asp Ala Val Glu Ala Phe Phe Gly Gly Ala Gly Met Lys
Tyr Gly 70 75 80 GTG GAA ACT GAT TGG CCT GCA TAT ATT GAA GGA TGG
AAA AAA TTG GCT 405 Val Glu Thr Asp Trp Pro Ala Tyr Ile Glu Gly Trp
Lys Lys Leu Ala 85 90 95 ACT GAT GAA TTG GAG AAA TAC GCC AAA AAC
GAA CCA ACG CTC ATC CGT 453 Thr Asp Glu Leu Glu Lys Tyr Ala Lys Asn
Glu Pro Thr Leu Ile Arg 100 105 110 ATA TGG GGT GAT GCT TTG TTT GAT
ATC GTT GAC AAA GAT CAA AAT GGA 501 Ile Trp Gly Asp Ala Leu Phe Asp
Ile Val Asp Lys Asp Gln Asn Gly 115 120 125 GCC ATT ACA CTG GAT GAA
TGG AAA GCA TAC ACC AAA GCT GCT GGT ATC 549 Ala Ile Thr Leu Asp Glu
Trp Lys Ala Tyr Thr Lys Ala Ala Gly Ile 130 135 140 145 ATC CAA TCA
TCA GAA GAT TGC GAG GAA ACA TTC AGA GTG TGC GAT ATT 597 Ile Gln Ser
Ser Glu Asp Cys Glu Glu Thr Phe Arg Val Cys Asp Ile 150 155 160 GAT
GAA AGT GGA CAA CTC GAT GTT GAT GAG ATG ACA AGA CAA CAT TTA 645 Asp
Glu Ser Gly Gln Leu Asp Val Asp Glu Met Thr Arg Gln His Leu 165 170
175 GGA TTT TGG TAC ACC ATG GAT CCT GCT TGC GAA AAG CTC TAC GGT GGA
693 Gly Phe Trp Tyr Thr Met Asp Pro Ala Cys Glu Lys Leu Tyr Gly Gly
180 185 190 GCT GTC CCC TAAGAAGCTC TACGGTGGTG ATGCACCCTA GGAAGATGAT
GTGATTTTGA 752 Ala Val Pro 195 ATAAAACACT GATGAATTCA ATCAAAATTT
TCCAAATTTT TGAACGATTT CAATCGTTTG 812 TGTTGATTTT TGTAATTAGG
AACAGATTAA ATCGAATGAT TAGTTGTTTT TTTAATCAAC 872 AGAACTTACA
AATCGAAAAA GTAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 932
AAAAAAAAAA AAAAAAAAAA AAAAAA 958 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 591 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v)
FRAGMENT TYPE: (vi) ORIGINAL SOURCE: (ix) FEATURE: (A) NAME/KEY:
Coding Sequence (B) LOCATION: 1...588 (D) OTHER INFORMATION:
Recombinant Aequorin AEQ1 (x) PUBLICATION INFORMATION: (A) AUTHORS:
Prasher et al. (B) TITLE: Sequence Comparisons of Complementary
DNAs Encoding Aequorin Isotypes (C) JOURNAL: Biochemistry (D)
VOLUME: 26 (F) PAGES: 1326-1332
(G) DATE: 1987 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ATG ACC AGC
GAA CAA TAC TCA GTC AAG CTT ACA CCA GAC TTC GAC AAC 48 Met Thr Ser
Glu Gln Tyr Ser Val Lys Leu Thr Pro Asp Phe Asp Asn 1 5 10 15 CCA
AAA TGG ATT GGA CGA CAC AAG CAC ATG TTT AAT TTT CTT GAT GTC 96 Pro
Lys Trp Ile Gly Arg His Lys His Met Phe Asn Phe Leu Asp Val 20 25
30 AAC CAC AAT GGA AGG ATC TCT CTT GAC GAG ATG GTC TAC AAG GCG TCC
144 Asn His Asn Gly Arg Ile Ser Leu Asp Glu Met Val Tyr Lys Ala Ser
35 40 45 GAT ATT GTT ATA AAC AAT CTT GGA GCA ACA CCT GAA CAA GCC
AAA CGT 192 Asp Ile Val Ile Asn Asn Leu Gly Ala Thr Pro Glu Gln Ala
Lys Arg 50 55 60 CAC AAA GAT GCT GTA GAA GCC TTC TTC GGA GGA GCT
GGA ATG AAA TAT 240 His Lys Asp Ala Val Glu Ala Phe Phe Gly Gly Ala
Gly Met Lys Tyr 65 70 75 80 GGT GTA GAA ACT GAA TGG CCT GAA TAC ATC
GAA GGA TGG AAA AGA CTG 288 Gly Val Glu Thr Glu Trp Pro Glu Tyr Ile
Glu Gly Trp Lys Arg Leu 85 90 95 GCT TCC GAG GAA TTG AAA AGG TAT
TCA AAA AAC CAA ATC ACA CTT ATT 336 Ala Ser Glu Glu Leu Lys Arg Tyr
Ser Lys Asn Gln Ile Thr Leu Ile 100 105 110 CGT TTA TGG GGT GAT GCA
TTG TTC GAT ATC ATT GAC AAA GAC CAA AAT 384 Arg Leu Trp Gly Asp Ala
Leu Phe Asp Ile Ile Asp Lys Asp Gln Asn 115 120 125 GGA GCT ATT TCA
CTG GAT GAA TGG AAA GCA TAC ACC AAA TCT GAT GGC 432 Gly Ala Ile Ser
Leu Asp Glu Trp Lys Ala Tyr Thr Lys Ser Asp Gly 130 135 140 ATC ATC
CAA TCG TCA GAA GAT TGC GAG GAA ACA TTC AGA GTG TGC GAT 480 Ile Ile
Gln Ser Ser Glu Asp Cys Glu Glu Thr Phe Arg Val Cys Asp 145 150 155
160 ATT GAT GAA AGT GGA CAG CTC GAT GTT GAT GAG ATG ACA AGA CAA CAT
528 Ile Asp Glu Ser Gly Gln Leu Asp Val Asp Glu Met Thr Arg Gln His
165 170 175 TTA GGA TTT TGG TAC ACC ATG GAT CCT GCT TGC GAA AAG CTC
TAC GGT 576 Leu Gly Phe Trp Tyr Thr Met Asp Pro Ala Cys Glu Lys Leu
Tyr Gly 180 185 190 GGA GCT GTC CCC TAA 591 Gly Ala Val Pro * 195
(2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 591 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: (vi)
ORIGINAL SOURCE: (ix) FEATURE: (A) NAME/KEY: Coding Sequence (B)
LOCATION: 1...588 (D) OTHER INFORMATION: Recombinant Aequorin AEQ2
(x) PUBLICATION INFORMATION: (A) AUTHORS: Prasher et al. (B) TITLE:
Sequence Comparisons of Complementary DNAs Encoding Aequorin
Isotypes (C) JOURNAL: Biochemistry (D) VOLUME: 26 (F) PAGES:
1326-1332 (G) DATE: 1987 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
ATG ACC AGC GAA CAA TAC TCA GTC AAG CTT ACA TCA GAC TTC GAC AAC 48
Met Thr Ser Glu Gln Tyr Ser Val Lys Leu Thr Ser Asp Phe Asp Asn 1 5
10 15 CCA AGA TGG ATT GGA CGA CAC AAG CAT ATG TTC AAT TTC CTT GAT
GTC 96 Pro Arg Trp Ile Gly Arg His Lys His Met Phe Asn Phe Leu Asp
Val 20 25 30 AAC CAC AAT GGA AAA ATC TCT CTT GAC GAG ATG GTC TAC
AAG GCA TCT 144 Asn His Asn Gly Lys Ile Ser Leu Asp Glu Met Val Tyr
Lys Ala Ser 35 40 45 GAT ATT GTC ATC AAT AAC CTT GGA GCA ACA CCT
GAG CAA GCC AAA CGA 192 Asp Ile Val Ile Asn Asn Leu Gly Ala Thr Pro
Glu Gln Ala Lys Arg 50 55 60 CAC AAA GAT GCT GTA GAA GCC TTC TTC
GGA GGA GCT GGA ATG AAA TAT 240 His Lys Asp Ala Val Glu Ala Phe Phe
Gly Gly Ala Gly Met Lys Tyr 65 70 75 80 GGT GTG GAA ACT GAT TGG CCT
GCA TAT ATT GAA GGA TGG AAA AAA TTG 288 Gly Val Glu Thr Asp Trp Pro
Ala Tyr Ile Glu Gly Trp Lys Lys Leu 85 90 95 GCT ACT GAT GAA TTG
GAG AAA TAC GCC AAA AAC GAA CCA ACG CTC ATC 336 Ala Thr Asp Glu Leu
Glu Lys Tyr Ala Lys Asn Glu Pro Thr Leu Ile 100 105 110 CGT ATA TGG
GGT GAT GCT TTG TTC GAT ATC GTT GAC AAA GAT CAA AAT 384 Arg Ile Trp
Gly Asp Ala Leu Phe Asp Ile Val Asp Lys Asp Gln Asn 115 120 125 GGA
GCC ATT ACA CTG GAT GAA TGG AAA GCA TAC ACC AAA GCT GCT GGT 432 Gly
Ala Ile Thr Leu Asp Glu Trp Lys Ala Tyr Thr Lys Ala Ala Gly 130 135
140 ATC ATC CAA TCA TCA GAA GAT TGC GAG GAA ACA TTC AGA GTG TGC GAT
480 Ile Ile Gln Ser Ser Glu Asp Cys Glu Glu Thr Phe Arg Val Cys Asp
145 150 155 160 ATT GAT GAA AGT GGA CAA CTC GAT GTT GAT GAG ATG ACA
AGA CAA CAT 528 Ile Asp Glu Ser Gly Gln Leu Asp Val Asp Glu Met Thr
Arg Gln His 165 170 175 TTA GGA TTT TGG TAC ACC ATG GAT CCT GCT TGC
GAA AAG CTC TAC GGT 576 Leu Gly Phe Trp Tyr Thr Met Asp Pro Ala Cys
Glu Lys Leu Tyr Gly 180 185 190 GGA GCT GTC CCC TAA 591 Gly Ala Val
Pro * 195 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 591 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: (ix) FEATURE: (A) NAME/KEY: Coding Sequence
(B) LOCATION: 1...588 (D) OTHER INFORMATION: Recombinant Aequorin
AEQ3 (x) PUBLICATION INFORMATION: (A) AUTHORS: Prasher et al. (B)
TITLE: Sequence Comparisons of Complementary DNAs Encoding Aequorin
Isotypes (C) JOURNAL: Biochemistry (D) VOLUME: 26 (F) PAGES:
1326-1332 (G) DATE: 1987 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
ATG ACC AGC GAA CAA TAC TCA GTC AAG CTT ACA TCA GAC TTC GAC AAC 48
Met Thr Ser Glu Gln Tyr Ser Val Lys Leu Thr Ser Asp Phe Asp Asn 1 5
10 15 CCA AGA TGG ATT GGA CGA CAC AAG CAT ATG TTC AAT TTC CTT GAT
GTC 96 Pro Arg Trp Ile Gly Arg His Lys His Met Phe Asn Phe Leu Asp
Val 20 25 30 AAC CAC AAT GGA AAA ATC TCT CTT GAC GAG ATG GTC TAC
AAG GCA TCT 144 Asn His Asn Gly Lys Ile Ser Leu Asp Glu Met Val Tyr
Lys Ala Ser 35 40 45 GAT ATT GTC ATC AAT AAC CTT GGA GCA ACA CCT
GAG CAA GCC AAA CGA 192 Asp Ile Val Ile Asn Asn Leu Gly Ala Thr Pro
Glu Gln Ala Lys Arg 50 55 60 CAC AAA GAT GCT GTA GGA GAC TTC TTC
GGA GGA GCT GGA ATG AAA TAT 240 His Lys Asp Ala Val Gly Asp Phe Phe
Gly Gly Ala Gly Met Lys Tyr 65 70 75 80 GGT GTG GAA ACT GAT TGG CCT
GCA TAC ATT GAA GGA TGG AAA AAA TTG 288 Gly Val Glu Thr Asp Trp Pro
Ala Tyr Ile Glu Gly Trp Lys Lys Leu 85 90 95 GCT ACT GAT GAA TTG
GAG AAA TAC GCC AAA AAC GAA CCA ACG CTC ATC 336 Ala Thr Asp Glu Leu
Glu Lys Tyr Ala Lys Asn Glu Pro Thr Leu Ile 100 105 110 CGT ATA TGG
GGT GAT GCT TTG TTC GAT ATC GTT GAC AAA GAT CAA AAT 384 Arg Ile Trp
Gly Asp Ala Leu Phe Asp Ile Val Asp Lys Asp Gln Asn 115 120 125 GGA
GCC ATT ACA CTG GAT GAA TGG AAA GCA TAC ACC AAA GCT GCT GGT 432 Gly
Ala Ile Thr Leu Asp Glu Trp Lys Ala Tyr Thr Lys Ala Ala Gly 130 135
140 ATC ATC CAA TCA TCA GAA GAT TGC GAG GAA ACA TTC AGA GTG TGC GAT
480 Ile Ile Gln Ser Ser Glu Asp Cys Glu Glu Thr Phe Arg Val Cys Asp
145 150 155 160 ATT GAT GAA AAT GGA CAA CTC GAT GTT GAT GAG ATG ACA
AGA CAA CAT 528 Ile Asp Glu Asn Gly Gln Leu Asp Val Asp Glu Met Thr
Arg Gln His 165 170 175 TTA GGA TTT TGG TAC ACC ATG GAT CCT GCT TGC
GAA AAG CTC TAC GGT 576 Leu Gly Phe Trp Tyr Thr Met Asp Pro Ala Cys
Glu Lys Leu Tyr Gly 180 185 190 GGA GCT GTC CCC TAA 591 Gly Ala Val
Pro * 195 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 567 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: (ix) FEATURE: (A) NAME/KEY: Coding
Sequence
(B) LOCATION: 1...567 (D) OTHER INFORMATION: Aequorin photoprotein
(x) PUBLICATION INFORMATION: (A) AUTHORS: Charbonneau et al. (B)
TITLE: Amino acid sequence of the calcium-dependent photoprotein
aequorin (C) JOURNAL: Am. Chem. Soc. (D) VOLUME: 24 (E) ISSUE: 24
(F) PAGES: 6762-6771 (G) DATE: 1985 (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:9: GTC AAG CTT ACA CCA GAC TTC GAC AAC CCA AAA TGG ATT GGA
CGA CAC 48 Val Lys Leu Thr Pro Asp Phe Asp Asn Pro Lys Trp Ile Gly
Arg His 1 5 10 15 AAG CAC ATG TTT AAT TTT CTT GAT GTC AAC CAC AAT
GGA AGG ATC TCT 96 Lys His Met Phe Asn Phe Leu Asp Val Asn His Asn
Gly Arg Ile Ser 20 25 30 CTT GAC GAG ATG GTC TAC AAG GCG TCC GAT
ATT GTT ATA AAC AAT CTT 144 Leu Asp Glu Met Val Tyr Lys Ala Ser Asp
Ile Val Ile Asn Asn Leu 35 40 45 GGA GCA ACA CCT GAA CAA GCC AAA
CGT CAC AAA GAT GCT GTA GAA GCC 192 Gly Ala Thr Pro Glu Gln Ala Lys
Arg His Lys Asp Ala Val Glu Ala 50 55 60 TTC TTC GGA GGA GCT GCA
ATG AAA TAT GGT GTA GAA ACT GAA TGG CCT 240 Phe Phe Gly Gly Ala Ala
Met Lys Tyr Gly Val Glu Thr Glu Trp Pro 65 70 75 80 GAA TAC ATC GAA
GGA TGG AAA AGA CTG GCT TCC GAG GAA TTG AAA AGG 288 Glu Tyr Ile Glu
Gly Trp Lys Arg Leu Ala Ser Glu Glu Leu Lys Arg 85 90 95 TAT TCA
AAA AAC CAA ATC ACA CTT ATT CGT TTA TGG GGT GAT GCA TTG 336 Tyr Ser
Lys Asn Gln Ile Thr Leu Ile Arg Leu Trp Gly Asp Ala Leu 100 105 110
TTC GAT ATC ATT GAC AAA GAC CAA AAT GGA GCT ATT TCA CTG GAT GAA 384
Phe Asp Ile Ile Asp Lys Asp Gln Asn Gly Ala Ile Ser Leu Asp Glu 115
120 125 TGG AAA GCA TAC ACC AAA TCT GCT GGC ATC ATC CAA TCG TCA GAA
GAT 432 Trp Lys Ala Tyr Thr Lys Ser Ala Gly Ile Ile Gln Ser Ser Glu
Asp 130 135 140 TGC GAG GAA ACA TTC AGA GTG TGC GAT ATT GAT GAA AGT
GGA CAG CTC 480 Cys Glu Glu Thr Phe Arg Val Cys Asp Ile Asp Glu Ser
Gly Gln Leu 145 150 155 160 GAT GTT GAT GAG ATG ACA AGA CAA CAT TTA
GGA TTT TGG TAC ACC ATG 528 Asp Val Asp Glu Met Thr Arg Gln His Leu
Gly Phe Trp Tyr Thr Met 165 170 175 GAT CCT GCT TGC GAA AAG CTC TAC
GGT GGA GCT GTC CCC 567 Asp Pro Ala Cys Glu Lys Leu Tyr Gly Gly Ala
Val Pro 180 185 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 588 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: (ix) FEATURE: (A) NAME/KEY: Coding Sequence
(B) LOCATION: 1...588 (D) OTHER INFORMATION: Aequorin mutant
w/increased bioluminescence activity (x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: 5,360,728 (K) RELEVANT RESIDUES IN SEQ ID NO:
10: Asp 124 changed to Ser (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
ATG ACC AGC GAA CAA TAC TCA GTC AAG CTT ACA CCA GAC TTC GAC AAC 48
Met Thr Ser Glu Gln Tyr Ser Val Lys Leu Thr Pro Asp Phe Asp Asn 1 5
10 15 CCA AAA TGG ATT GGA CGA CAC AAG CAC ATG TTT AAT TTT CTT GAT
GTC 96 Pro Lys Trp Ile Gly Arg His Lys His Met Phe Asn Phe Leu Asp
Val 20 25 30 AAC CAC AAT GGA AGG ATC TCT CTT GAC GAG ATG GTC TAC
AAG GCG TCC 144 Asn His Asn Gly Arg Ile Ser Leu Asp Glu Met Val Tyr
Lys Ala Ser 35 40 45 GAT ATT GTT ATA AAC AAT CTT GGA GCA ACA CCT
GAA CAA GCC AAA CGT 192 Asp Ile Val Ile Asn Asn Leu Gly Ala Thr Pro
Glu Gln Ala Lys Arg 50 55 60 CAC AAA GAT GCT GTA GAA GCC TTC TTC
GGA GGA GCT GCA ATG AAA TAT 240 His Lys Asp Ala Val Glu Ala Phe Phe
Gly Gly Ala Ala Met Lys Tyr 65 70 75 80 GGT GTA GAA ACT GAA TGG CCT
GAA TAC ATC GAA GGA TGG AAA AGA CTG 288 Gly Val Glu Thr Glu Trp Pro
Glu Tyr Ile Glu Gly Trp Lys Arg Leu 85 90 95 GCT TCC GAG GAA TTG
AAA AGG TAT TCA AAA AAC CAA ATC ACA CTT ATT 336 Ala Ser Glu Glu Leu
Lys Arg Tyr Ser Lys Asn Gln Ile Thr Leu Ile 100 105 110 CGT TTA TGG
GGT GAT GCA TTG TTC GAT ATC ATT TCC AAA GAC CAA AAT 384 Arg Leu Trp
Gly Asp Ala Leu Phe Asp Ile Ile Ser Lys Asp Gln Asn 115 120 125 GGA
GCT ATT TCA CTG GAT GAA TGG AAA GCA TAC ACC AAA TCT GCT GGC 432 Gly
Ala Ile Ser Leu Asp Glu Trp Lys Ala Tyr Thr Lys Ser Ala Gly 130 135
140 ATC ATC CAA TCG TCA GAA GAT TGC GAG GAA ACA TTC AGA GTG TGC GAT
480 Ile Ile Gln Ser Ser Glu Asp Cys Glu Glu Thr Phe Arg Val Cys Asp
145 150 155 160 ATT GAT GAA AGT GGA CAG CTC GAT GTT GAT GAG ATG ACA
AGA CAA CAT 528 Ile Asp Glu Ser Gly Gln Leu Asp Val Asp Glu Met Thr
Arg Gln His 165 170 175 TTA GGA TTT TGG TAC ACC ATG GAT CCT GCT TGC
GAA AAG CTC TAC GGT 576 Leu Gly Phe Trp Tyr Thr Met Asp Pro Ala Cys
Glu Lys Leu Tyr Gly 180 185 190 GGA GCT GTC CCC 588 Gly Ala Val Pro
195 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 588 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: (vi)
ORIGINAL SOURCE: (ix) FEATURE: (A) NAME/KEY: Coding Sequence (B)
LOCATION: 1...588 (D) OTHER INFORMATION: Recombinant site-directed
Aequorin mutant w/increased biolum. activity (x) PUBLICATION
INFORMATION: (H) DOCUMENT NUMBER: 5,360,728 (K) RELEVANT RESIDUES
IN SEQ ID NO: 11: Glu 135 changed to Ser (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:11: ATG ACC AGC GAA CAA TAC TCA GTC AAG CTT ACA CCA GAC
TTC GAC AAC 48 Met Thr Ser Glu Gln Tyr Ser Val Lys Leu Thr Pro Asp
Phe Asp Asn 1 5 10 15 CCA AAA TGG ATT GGA CGA CAC AAG CAC ATG TTT
AAT TTT CTT GAT GTC 96 Pro Lys Trp Ile Gly Arg His Lys His Met Phe
Asn Phe Leu Asp Val 20 25 30 AAC CAC AAT GGA AGG ATC TCT CTT GAC
GAG ATG GTC TAC AAG GCG TCC 144 Asn His Asn Gly Arg Ile Ser Leu Asp
Glu Met Val Tyr Lys Ala Ser 35 40 45 GAT ATT GTT ATA AAC AAT CTT
GGA GCA ACA CCT GAA CAA GCC AAA CGT 192 Asp Ile Val Ile Asn Asn Leu
Gly Ala Thr Pro Glu Gln Ala Lys Arg 50 55 60 CAC AAA GAT GCT GTA
GAA GCC TTC TTC GGA GGA GCT GCA ATG AAA TAT 240 His Lys Asp Ala Val
Glu Ala Phe Phe Gly Gly Ala Ala Met Lys Tyr 65 70 75 80 GGT GTA GAA
ACT GAA TGG CCT GAA TAC ATC GAA GGA TGG AAA AGA CTG 288 Gly Val Glu
Thr Glu Trp Pro Glu Tyr Ile Glu Gly Trp Lys Arg Leu 85 90 95 GCT
TCC GAG GAA TTG AAA AGG TAT TCA AAA AAC CAA ATC ACA CTT ATT 336 Ala
Ser Glu Glu Leu Lys Arg Tyr Ser Lys Asn Gln Ile Thr Leu Ile 100 105
110 CGT TTA TGG GGT GAT GCA TTG TTC GAT ATC ATT TCC AAA GAC CAA AAT
384 Arg Leu Trp Gly Asp Ala Leu Phe Asp Ile Ile Ser Lys Asp Gln Asn
115 120 125 GGA GCT ATT TCA CTG GAT TCA TGG AAA GCA TAC ACC AAA TCT
GCT GGC 432 Gly Ala Ile Ser Leu Asp Ser Trp Lys Ala Tyr Thr Lys Ser
Ala Gly 130 135 140 ATC ATC CAA TCG TCA GAA GAT TGC GAG GAA ACA TTC
AGA GTG TGC GAT 480 Ile Ile Gln Ser Ser Glu Asp Cys Glu Glu Thr Phe
Arg Val Cys Asp 145 150 155 160 ATT GAT GAA AGT GGA CAG CTC GAT GTT
GAT GAG ATG ACA AGA CAA CAT 528 Ile Asp Glu Ser Gly Gln Leu Asp Val
Asp Glu Met Thr Arg Gln His 165 170 175 TTA GGA TTT TGG TAC ACC ATG
GAT CCT GCT TGC GAA AAG CTC TAC GGT 576 Leu Gly Phe Trp Tyr Thr Met
Asp Pro Ala Cys Glu Lys Leu Tyr Gly 180 185 190 GGA GCT GTC CCC 588
Gly Ala Val Pro 195 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 588 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: (ix) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 1...588 (D) OTHER
INFORMATION: Recombinant site-directed Aequorin mutant w/increased
biolum. activity (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER:
5,360,728 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: ATG ACC AGC GAA
CAA TAC TCA GTC AAG CTT ACA CCA GAC TTC GAC AAC 48 Met Thr Ser Glu
Gln Tyr Ser Val Lys Leu Thr Pro Asp Phe Asp Asn 1 5 10 15 CCA AAA
TGG ATT GGA CGA CAC AAG CAC ATG TTT AAT TTT CTT GAT GTC 96 Pro Lys
Trp Ile Gly Arg His Lys His Met Phe Asn Phe Leu Asp Val 20 25 30
AAC CAC AAT GGA AGG ATC TCT CTT GAC GAG ATG GTC TAC AAG GCG TCC 144
Asn His Asn Gly Arg Ile Ser Leu Asp Glu Met Val Tyr Lys Ala Ser 35
40 45 GAT ATT GTT ATA AAC AAT CTT GGA GCA ACA CCT GAA CAA GCC AAA
CGT 192 Asp Ile Val Ile Asn Asn Leu Gly Ala Thr Pro Glu Gln Ala Lys
Arg 50 55 60 CAC AAA GAT GCT GTA GAA GCC TTC TTC GGA GGA GCT GCA
ATG AAA TAT 240 His Lys Asp Ala Val Glu Ala Phe Phe Gly Gly Ala Ala
Met Lys Tyr 65 70 75 80 GGT GTA GAA ACT GAA TGG CCT GAA TAC ATC GAA
GGA TGG AAA AGA CTG 288 Gly Val Glu Thr Glu Trp Pro Glu Tyr Ile Glu
Gly Trp Lys Arg Leu 85 90 95 GCT TCC GAG GAA TTG AAA AGG TAT TCA
AAA AAC CAA ATC ACA CTT ATT 336 Ala Ser Glu Glu Leu Lys Arg Tyr Ser
Lys Asn Gln Ile Thr Leu Ile 100 105 110 CGT TTA TGG GGT GAT GCA TTG
TTC GAT ATC ATT TCC AAA GAC CAA AAT 384 Arg Leu Trp Gly Asp Ala Leu
Phe Asp Ile Ile Ser Lys Asp Gln Asn 115 120 125 GCA GCT ATT TCA CTG
GAT GAA TGG AAA GCA TAC ACC AAA TCT GCT GGC 432 Ala Ala Ile Ser Leu
Asp Glu Trp Lys Ala Tyr Thr Lys Ser Ala Gly 130 135 140 ATC ATC CAA
TCG TCA GAA GAT TGC GAG GAA ACA TTC AGA GTG TGC GAT 480 Ile Ile Gln
Ser Ser Glu Asp Cys Glu Glu Thr Phe Arg Val Cys Asp 145 150 155 160
ATT GAT GAA AGT GGA CAG CTC GAT GTT GAT GAG ATG ACA AGA CAA CAT 528
Ile Asp Glu Ser Gly Gln Leu Asp Val Asp Glu Met Thr Arg Gln His 165
170 175 TTA GGA TTT TGG TAC ACC ATG GAT CCT GCT TGC GAA AAG CTC TAC
GGT 576 Leu Gly Phe Trp Tyr Thr Met Asp Pro Ala Cys Glu Lys Leu Tyr
Gly 180 185 190 GGA GCT GTC CCC 588 Gly Ala Val Pro 195 (2)
INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 567 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 1...567 (D) OTHER
INFORMATION: Recombinant apoaequorin (AQUALITE ) (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:13: GTC AAG CTT ACA CCA GAC TTC GAC AAC CCA
AAA TGG ATT GGA CGA CAC 48 Val Lys Leu Thr Pro Asp Phe Asp Asn Pro
Lys Trp Ile Gly Arg His 1 5 10 15 AAG CAC ATG TTT AAT TTT CTT GAT
GTC AAC CAC AAT GGA AGG ATC TCT 96 Lys His Met Phe Asn Phe Leu Asp
Val Asn His Asn Gly Arg Ile Ser 20 25 30 CTT GAC GAG ATG GTC TAC
AAG GCG TCC GAT ATT GTT ATA AAC AAT CTT 144 Leu Asp Glu Met Val Tyr
Lys Ala Ser Asp Ile Val Ile Asn Asn Leu 35 40 45 GGA GCA ACA CCT
GAA CAA GCC AAA CGT CAC AAA GAT GCT GTA GAA GCC 192 Gly Ala Thr Pro
Glu Gln Ala Lys Arg His Lys Asp Ala Val Glu Ala 50 55 60 TTC TTC
GGA GGA GCT GGA ATG AAA TAT GGT GTA GAA ACT GAA TGG CCT 240 Phe Phe
Gly Gly Ala Gly Met Lys Tyr Gly Val Glu Thr Glu Trp Pro 65 70 75 80
GAA TAC ATC GAA GGA TGG AAA AAA CTG GCT TCC GAG GAA TTG AAA AGG 288
Glu Tyr Ile Glu Gly Trp Lys Lys Leu Ala Ser Glu Glu Leu Lys Arg 85
90 95 TAT TCA AAA AAC CAA ATC ACA CTT ATT CGT TTA TGG GGT GAT GCA
TTG 336 Tyr Ser Lys Asn Gln Ile Thr Leu Ile Arg Leu Trp Gly Asp Ala
Leu 100 105 110 TTC GAT ATC ATT GAC AAA GAC CAA AAT GGA GCT ATT CTG
TCA GAT GAA 384 Phe Asp Ile Ile Asp Lys Asp Gln Asn Gly Ala Ile Leu
Ser Asp Glu 115 120 125 TGG AAA GCA TAC ACC AAA TCT GAT GGC ATC ATC
CAA TCG TCA GAA GAT 432 Trp Lys Ala Tyr Thr Lys Ser Asp Gly Ile Ile
Gln Ser Ser Glu Asp 130 135 140 TGC GAG GAA ACA TTC AGA GTG TGC GAT
ATT GAT GAA AGT GGA CAG CTC 480 Cys Glu Glu Thr Phe Arg Val Cys Asp
Ile Asp Glu Ser Gly Gln Leu 145 150 155 160 GAT GTT GAT GAG ATG ACA
AGA CAA CAT TTA GGA TTT TGG TAC ACC ATG 528 Asp Val Asp Glu Met Thr
Arg Gln His Leu Gly Phe Trp Tyr Thr Met 165 170 175 GAT CCT GCT TGC
GAA AAG CTC TAC GGT GGA GCT GTC CCC 567 Asp Pro Ala Cys Glu Lys Leu
Tyr Gly Gly Ala Val Pro 180 185 (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 236 amino acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix)
FEATURE: (D) OTHER INFORMATION: Vibrio fisheri Flavin reductase (x)
PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: 5,484,723 (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:14: Met Pro Ile Asn Cys Lys Val Lys
Ser Ile Glu Pro Leu Ala Cys Asn 1 5 10 15 Thr Phe Arg Ile Leu Leu
His Pro Glu Gln Pro Val Ala Phe Lys Ala 20 25 30 Gly Gln Tyr Leu
Thr Val Val Met Gly Glu Lys Asp Lys Arg Pro Phe 35 40 45 Ser Ile
Ala Ser Ser Pro Cys Arg His Glu Gly Glu Ile Glu Leu His 50 55 60
Ile Gly Ala Ala Glu His Asn Ala Tyr Ala Gly Glu Val Val Glu Ser 65
70 75 80 Met Lys Ser Ala Leu Glu Thr Gly Gly Asp Ile Leu Ile Asp
Ala Pro 85 90 95 His Gly Glu Ala Trp Ile Arg Glu Asp Ser Asp Arg
Ser Met Leu Leu 100 105 110 Ile Ala Gly Gly Thr Gly Phe Ser Tyr Val
Arg Ser Ile Leu Asp His 115 120 125 Cys Ile Ser Gln Gln Ile Gln Lys
Pro Ile Tyr Leu Tyr Trp Gly Gly 130 135 140 Arg Asp Glu Cys Gln Leu
Tyr Ala Lys Ala Glu Leu Glu Ser Ile Ala 145 150 155 160 Gln Ala His
Ser His Ile Thr Phe Val Pro Val Val Glu Lys Ser Glu 165 170 175 Gly
Trp Thr Gly Lys Thr Gly Asn Val Leu Glu Ala Val Lys Ala Asp 180 185
190 Phe Asn Ser Leu Ala Asp Met Asp Ile Tyr Ile Ala Gly Arg Phe Glu
195 200 205 Met Ala Gly Ala Ala Arg Glu Gln Phe Thr Thr Glu Lys Gln
Ala Lys 210 215 220 Lys Glu Gln Leu Phe Gly Asp Ala Phe Ala Phe Ile
225 230 235
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