U.S. patent application number 13/353052 was filed with the patent office on 2012-06-21 for ready-to-use electroporation cuvette including frozen electrocompetent cells.
This patent application is currently assigned to MOLECULAR TRANSFER, INC.. Invention is credited to Robert L. Bebee.
Application Number | 20120156786 13/353052 |
Document ID | / |
Family ID | 34990494 |
Filed Date | 2012-06-21 |
United States Patent
Application |
20120156786 |
Kind Code |
A1 |
Bebee; Robert L. |
June 21, 2012 |
READY-TO-USE ELECTROPORATION CUVETTE INCLUDING FROZEN
ELECTROCOMPETENT CELLS
Abstract
A ready-to-use electroporation cuvette is provided that includes
a cuvette, first and second electrodes positioned within the
cuvette and electroporation competent cells frozen in a suspension
solution within the cuvette, wherein the electroporation cuvette is
configured to permit electroporation of the cells when the cells
are thawed. The electroporation cuvette may be sealed with a cap
that may be color coded to aid the user.
Inventors: |
Bebee; Robert L.;
(Rockville, MD) |
Assignee: |
MOLECULAR TRANSFER, INC.
GAITHERSBURG
MD
|
Family ID: |
34990494 |
Appl. No.: |
13/353052 |
Filed: |
January 18, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11488179 |
Jul 18, 2006 |
8105818 |
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13353052 |
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11072715 |
Mar 7, 2005 |
7078227 |
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11488179 |
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60556380 |
Mar 26, 2004 |
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Current U.S.
Class: |
435/471 ;
435/285.2 |
Current CPC
Class: |
C12M 35/02 20130101 |
Class at
Publication: |
435/471 ;
435/285.2 |
International
Class: |
C12N 15/63 20060101
C12N015/63; C12M 1/42 20060101 C12M001/42 |
Claims
1-43. (canceled)
44. An electroporation cuvette with an internal structure
comprising: first and second electrodes positioned at a first fixed
distance apart within the electroporation cuvette, wherein each of
first and second electrodes is in the form of an I-beam, each
I-beam comprising (i) inner, and (ii) outer plates that are
connected by at least one perpendicular interconnecting plate, the
length of which determines, between the inner and outer plates, a
second fixed distance; a first space formed between the first and
second electrodes; wherein the length of at least one of the
interconnecting plates of the first and second electrodes may be
varied to enable a change in the first fixed distance between the
first and second electrodes.
45. The electroporation cuvette of claim 44 wherein the first and
second electrodes positioned within the electroporation cuvette are
held in position by ledges or walls within the electroporation
cuvette.
46. The electroporation cuvette of claim 44 wherein at least one of
the first and second electrodes is placed against and fixed to one
of the inside walls of the electroporation cuvette.
47. The electroporation cuvette of claim 44 wherein the first and
second electrodes are positioned parallel to each other.
48. The electroporation cuvette of claim 45 wherein the first and
second electrodes are positioned parallel to each other.
49. The electroporation cuvette of claim 46 wherein the first and
second electrodes are positioned parallel to each other.
50. The electroporation cuvette of claim 44 further comprising a
second space formed in the upper portion of the electroporation
cuvette above the first space, wherein the structure defining the
boundaries of the second space comprises a wedge-shaped blend
between the upper walls of the electroporation cuvette and the
first and second electrodes, wherein the wedge-shaped blend
provides a transition from the second space into the first space to
facilitate the flow of liquids into the first space.
51. The electroporation cuvette of claim 50 wherein the structure
defining the boundaries of the second space comprises one or more
ledges to hold first and second electrodes in a fixed position
during electroporation.
52. The electroporation cuvette of claim 44 wherein at least one of
the first and second electrodes is placed against a one of the
lower inside-walls of the electroporation cuvette.
53. The electroporation cuvette of claim 52, wherein the first and
second electrodes are positioned parallel to each other.
54. The electroporation cuvette of claim 44 wherein the inner and
outer plates of the I-beam electrodes have sufficient structural
rigidity to retain their shape, meaning that the fixed distance
between the electrodes is maintained during freezing and thawing
processes.
55. The electroporation cuvette of claim 44 wherein at least one of
the first and second electrodes comprises a metal.
56. The electroporation cuvette of claim 44 wherein at least one of
the first and second electrodes comprises aluminum.
57. The electroporation cuvette of claim 44 wherein the first
electrode is fixed to the inside wall of the cuvette and the second
electrode is placed against the inside wall opposite the first
electrode of the cuvette, wherein the electrodes are positioned at
a fixed distance apart.
58. The electroporation cuvette any of claim 50 wherein the
structure defining the boundaries of the second space comprises one
or more ledges to hold the second electrode in a position during
electroporation.
59. An electroporation cuvette with a structure comprising; first
and second electrodes (i) positioned at a fixed distance apart, and
(ii) fused to two of the directly opposing, lower walls of the
electroporation cuvette; wherein each of first and second
electrodes is in the form of an I-beam, comprising: parallel (i)
inner, and (ii) outer plates that are connected by at least one
perpendicular and variable-length interconnecting plate, the length
of which determines a second fixed distance between the inner and
outer plates of each of first and second electrodes; wherein the
directly opposing, lower walls of the electroporation cuvette pass
through a second fixed distance between the inner and outer plates
of each of the first and second electrodes; wherein the outer plate
of each of the first and second electrodes forms the lower
exterior, and the inner plate of each of the first and second
electrodes forms the lower interior, of two of the lower walls of
the cuvette-electrode combination; wherein a first space is formed
within the electroporation cuvette between the inner plates of both
the first and second electrodes; wherein a second space is formed
in the electroporation cuvette, above the first space; wherein the
second space is continuous with the first space; and wherein the
structure defining the side boundaries of the second space
facilitates the flow of liquids into the first space.
60. The electroporation cuvette of claim 59 the volume of the first
space may be varied by varying the length of at least one of the
interconnecting plates of the first and second electrodes to alter
a second fixed distance between the inner and outer plates of at
least one of the first and second electrodes.
61. The electroporation cuvette of claim 59 wherein the parallel
inner and outer plates of the I-beam electrodes have sufficient
structural rigidity to retain their shape, meaning that the first
fixed distance between the electrodes is maintained during freezing
and thawing processes.
62. The electroporation cuvette of claim 59 wherein the inner plate
of one of the first or second electrodes is configured to function
as the electrode surface; and the outer plate of the other of the
first or second electrodes is configured to be in contact with one
of the lower inside walls of the cuvette.
63. An electroporation cuvette with an internal structure
comprising: first and second electrodes positioned within the
electroporation cuvette and placed against two of the directly
opposing, lower inside walls of the electroporation cuvette,
wherein the first and second electrodes are positioned at a first
fixed distance apart; wherein each of first and second electrodes
is in the form of an I-beam, each I-beam comprising (i) inner, and
(ii) outer plates that are connected by at least one perpendicular
interconnecting plate, the length of which determines the second
fixed distance between the inner and outer plates of each of the
electrodes; a first space formed between the first and second
electrodes; and a second space formed in the upper portion of the
electroporation cuvette, above the first-space; wherein the length
of at least one of the interconnecting plates of the first and
second electrodes may be varied to enable a change in distance
between the first and second electrodes, resulting in a change in
the volume of the first space; wherein the second space is
continuous with the first space; and wherein the structure defining
the side boundaries of the second space facilitates the flow of
liquids into the first space.
64. A method of using an electroporation cuvette of claim 44 for
transformation of a bacteria or yeast cell, comprising: positioning
at least one the electrodes inside the cuvette; adjusting and
fixing the length of the interconnecting plate; maintaining the
fixed length of the interconnecting plate throughout
electroporation; wherein the electrode maintains sufficient
structural rigidity to control the distance between the electrodes.
Description
[0001] This application claims priority to U.S. Provisional
Application No. 60/556,380 filed Mar. 26, 2004, the contents of
which are incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention provides an apparatus and methods for
the transformation of cells by electroporation. More particularly,
the invention provides a ready-to-use cuvette for electroporation
and methods Of making and using such a cuvette.
[0004] 2. Description of the Related Art
[0005] Introducing nucleic acids into cells is central to many
types of biological experiments and biotechnology development
methods. For example, when searching for a gene of interest in a
cDNA library, the library must be transferred into a host organism.
Among the various methods used for introducing nucleic acids into
host cells, electroporation has gained widespread use. Exemplary
methods and kits for performing electroporation are disclosed in
U.S. Pat. Nos. 4,910,140 and 5,186,800 to Dower, and U.S. Pat. No.
6,338,965 to Greener et al., each of which is incorporated herein
by reference in their entirety.
[0006] In general, electroporation involves the transfer of nucleic
acids into a host cell by exposure of the cell to a high voltage
electric impulse in the presence of the nucleic acids, such as
genes or gene fragments. Typically, host cells are stored by
freezing them at a temperature that preserves their viability for a
long term. The frozen cells are stored in a separate container and
must be defrosted, mixed with nucleic acid and subsequently
transferred to a cuvette prior to electroporation.
[0007] An example of an electroporation method is disclosed in U.S.
Pat. No. 5,186,800 and involves growing bacteria in enriched media
(of any sort) and concentrating the bacteria by washing in a buffer
containing 10% glycerol. DNA is added to the cells, the DNA and
cells are mixed and the resulting mixture is subjected to an
electrical discharge (pulse), which temporarily disrupts the outer
cell wall of the bacterial cells and permits the DNA to enter the
cells.
[0008] The efficiency of nucleic acid transfer depends on a variety
of factors, including the electrical field strength, the pulse
decay time, the pulse shape, the temperature in which the
electroporation is conducted, the type of cell, the type of
suspension buffer, and the concentration and size of the nucleic
acid to be transferred. Researchers have modified the host cell
suspension materials to aid in freezing the cells before the
electrical treatment. Methods disclosed in U.S. Pat. No. 6,338,965
include the addition of sugars or sugar derivatives, e.g., sugar
alcohols, to host cells suspended in a substantially non-ionic
solution, either prior to initial freezing, or after thawing, but
prior to electrotransformation, which improve electroporation
efficiency.
[0009] Known methods of preparing frozen cells for electroporation
require thawing the cells and mixing with nucleic acid prior to
adding them to a suitable electroporation cuvette. This sequence of
steps has always been deemed essential for at least two reasons:
first, the structures of an electroporation cuvette are precisely
dimensioned in order to provide reproducible electrical field
strengths in the cell solution, and the freezing procedures
necessary to store cells in the cuvette were considered too harsh
to maintain these precise dimensions; second, the size of the
cuvette chamber was considered too small to allow efficient mixing
of cells with nucleic acid. Efficient mixing of cells and nucleic
acid is essential to achieving a desired level of cell
transformation by electroporation. The steps of thawing and mixing
host cells prior to electroporation require experimenter's time and
presents an opportunity for contamination or experimental errors
that may impact results or diminish electrotransformation yields.
Accordingly, there is a need for a method and equipment that will
eliminate the need to separately thaw and prepare cells before
placing them in an electroporation cuvette.
SUMMARY OF THE INVENTION
[0010] According to an embodiment of the present invention, an
electroporation cuvette includes a cuvette, first and second
electrodes positioned within the cuvette, and cells in a suspension
solution frozen within the cuvette, wherein the electroporation
cuvette is configured to permit electroporation of the cells when
the cells are thawed.
[0011] According to another embodiment of the present invention, a
method of making a ready-to-use electroporation cuvette includes
fabricating an electroporation cuvette comprising a cuvette, and
first and second electrodes positioned within the cuvette,
sterilizing the electroporation cuvette, placing an aliquot of
electrocompetent cells in the electroporation cuvette, placing a
sterile cap on the electroporation cuvette, and freezing the
aliquot of electrocompetent cells within the electroporation
cuvette, such as flash freezing, such as by dipping the cuvette in
liquid nitrogen. The method may also include sealing the
electroporation cuvette in a sterile package.
[0012] According to another embodiment of the present invention, a
method of using a ready-to-use electroporation cuvette includes
thawing cells within the electroporation cuvette, adding nucleic
acid to the cells, placing the electroporation cuvette in an
electroporation machine and conducting electroporation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a diagram of an exemplary ready-to-use
electroporation cuvette according to an embodiment of the present
invention.
[0014] FIG. 2A is a plane view of an exemplary electrode suitable
for use in a ready-to-use electroporation cuvette according to an
embodiment of the present invention. FIG. 2b is a cross-sectional
diagram of the electrode illustrated in FIG. 2A.
[0015] FIG. 3 is a diagram of a ready-to-use electroporation
cuvette according to another embodiment of the present
invention.
[0016] FIG. 4 is a plane view of a ready-to-use electroporation
cuvette according to another embodiment of the present
invention.
[0017] FIG. 5 is a process flow diagram for making an
electroporation cuvette according to an embodiment of the present
invention.
[0018] FIG. 6 is a process flow diagram for using an
electroporation cuvette according to an embodiment of the present
invention.
DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS
[0019] Surprisingly it has been found that host cells, such as
electrocompetent host cells, may be frozen in a suitable
electroporation container, such as an electroporation cuvette
according to the present invention, and then thawed, mixed with
nucleic acid, and efficiently transformed by electroporation
directly in the same container. This result is highly surprising
because the conventional wisdom has been that containers such as
electroporation cuvettes cannot withstand the rigors of the
freezing procedures necessary to store competent cells, the
relatively large mass of the cuvette would interfere with the rapid
freezing of the cell suspension believed to be crucial to the
long-term viability of the cells, and that the containers are too
small to allow adequate mixing of the cells with nucleic acid after
thawing. This surprising discovery permits the preparation and
distribution of "ready to use" electroporation containers, such as
cuvettes, that contain host cells suitable for electroporation and
that permit rapid "one pot" transformation of the cells with a
desired nucleic acid source.
[0020] Reference will now be made in detail to exemplary
embodiments of the present invention. Wherever possible, the same
reference numbers will be used throughout the drawings to refer to
the same or like parts.
[0021] Referring to FIG. 1, according to an embodiment of the
present invention, a ready-to-use electroporation cuvette 1
includes a vessel 2, referred to herein as a cuvette 2, containing
first and second electrodes 3, 4 between which is a volume 5 in
which an aliquot of cells in a suspension solution 6 are deposited
and maintained in the frozen state until ready for use. The
electroporation cuvette may also include a cap 7 which can be
securely attached to the top of the cuvette 2.
[0022] The means for attaching the cap 7 to the cuvette 2 may be
chosen from a number of structures that permit a fluid-tight and/or
biologically-proof barrier, such as with a plastic deformation fit,
compression fit, screw or bayonet fitting, detent structure 8 and
groove fit, gasket and sealing surface fit, or similar removable
closure mechanism. Alternatively or additionally, the cap may be
sealed to the cuvette such as by an adhesive, shrink-wrap plastic,
or glass that may be broken to remove the cap in preparation for
using the cuvette.
[0023] The cuvette 2 is preferably made of a material suitable for
exposure to cold temperatures, such as for example liquid nitrogen
temperatures (about 77K, -196.degree. C.), and very high voltages
or electric field strengths. The cuvette material is selected to be
compatible both with rapid codling rates, such as may be
experienced when the cuvette 2 is dipped into a low temperature
bath (such as that used for "flash freezing" cells, for example, a
liquid nitrogen bath, or a dry ice/ethanol bath), and with high
voltage fields generated in the electroporation process. It is also
preferable that the cuvette material be transparent. In an
embodiment, the cuvette is made from a polycarbonate material, for
example the polycarbonate material conventionally used for
commercially available cuvettes, such as those available from, for
example, Bio-Rad Laboratories (Hercules, Calif.). The cuvette 2 may
be manufactured by injection molding or similar mechanism that
produces a seamless structure With dimensional control over
internal and external surfaces.
[0024] The electrodes 3, 4 are made of a conductive) material,
preferably a metal and more preferably aluminum. In an embodiment,
the electrodes 3, 4 are plates that are positioned to be parallel
to each other and at a fixed distance apart. In an embodiment, the
internal structure of the cuvette includes structure, such as
ledges or walls, that hold the electrode plates in a precise
position, parallel to each other and at a fixed distance apart from
top to bottom. In an alternative embodiment, the electrodes are
formed so as to be self-positioning in the cuvette, such as with a
wall structure 9 that can be placed against or glued to a wall of
the cuvette 2, to hold the electrode surface at a precise
position.
[0025] In an embodiment, electrodes 3, 4 are fabricated to be
smooth in order to deliver a consistent electrical field across the
entire surface, and therefore a uniform electric current through
the cells. Such electrodes may be formed from aluminum plates by
cleaning and then etching the surfaces to remove raised points and
contaminants, rendering the electrodes smooth.
[0026] The electrodes 3, 4 may be separated by a dimension that is
set to provide a predetermined electrical field through the
suspension solution and cells 6 that is determined to result in a
high yield of electroporation transformation of the cells. For
example, cuvettes containing bacteria, such as E. coli, may have
electrodes 3, 4 separated by about 0.1 cm to about 0.2 cm. In
another example, cuvettes containing yeast may have electrodes 3, 4
separated by about 0.2 cm. In yet another example, cuvettes
containing mammalian cells may have electrodes 3, 4 separated by
about 0.4 cm.
[0027] In an embodiment, the electrodes 3 are in the form of an "I"
beam as illustrated in FIGS. 2A and 2B. In this embodiment, an
electrode 3 comprises a first plate 20 and a second plate 22
connected by a perpendicular interconnecting plate 23 or plates to
form the "I" beam shape. As illustrated in the cross-sectional view
of FIG. 2B, the interconnecting plate 23 positions the first and
second plates 20, 22 a fixed distance apart. The interconnecting
plate 23 also maintains the first plate 20 parallel to the second
plate 22. The distance separating plates 20, 22, which forms a gap
24, is determined by the span of interconnecting plate 23. This
configuration has advantages in fabrication because either the
first or second plates (20 or 22) may contact the cell suspension
fluid to function as the electrode surface, permitting the other
surface to contact and/or be attached to a wall of the cuvette 2.
If the cuvette 2 has a rectangular or square cross section, then
the position and orientation of the electrode 3 in the cuvette 2 is
determined by the electrode itself, in particular by the
interconnecting plate 23. In this manner, a standard size cuvette
(e.g., one with a square cross-section) may be used to provide
electroporation cuvettes with different distances between the
electrodes by using "I" beam electrodes having different spans of
interconnecting plate 23. Further, the "I" beam electrodes have
sufficient structural rigidity to retain their shape, and thus the
controlled distance between respective electrodes, during the
freezing and thawing processes.
[0028] Returning to FIG. 1, the cap 7 may be made by injection
molding of a similar or different material as the cuvette. In a
preferred embodiment the cap may be color coded or otherwise marked
to indicate the type of cells contained in the cuvette and/or the
dimension separating the electrodes 3, 4, since such information is
useful to users of the ready-to-use cuvette. The color coding
permits the user to easily select the proper cuvette for a
particular experiment by observing the cap, which is advantageous
when a number of cuvettes are stored together in a freezer.
[0029] Alternative configurations of the cuvette are contemplated.
FIG. 3 illustrates an alternative embodiment featuring structures
to permit positioning within an electroporation machine. For
example, the cuvette 1 may include a seating ledge 30 that may
interface with a complementary structure on the electroporation
machine. Also, the cuvette 1 may include a handling structure or
flange 31 to permit easy handling of the cuvette 1 without
conducting heat into the cells or risking contamination of the
interior. The cuvette 1 may also include electrical connections 32,
33 for ease of connecting the electrodes (not shown in FIG. 3) to
the electroporation machine.
[0030] In another alternative embodiment illustrated in FIG. 4, the
electroporation cuvette 1 may be formed by fusing the cuvette 2
with "I" beam electrodes 3. In this embodiment, two walls 41 of the
cuvette 2 pass through the gap 24 (see FIG. 2B) in the "I" beam so
that one of the electrode plates 20 forms a portion of the exterior
to the cuvette 2, permitting that plate 20 to serve as an
electrical contact surface 40 for interfacing electrically with the
electroporation machine. On the inside, the other electrode plate
22 forms a portion of the interior of the cuvette 2, where it
functions as the electrode surface 44. In this embodiment, the
interconnecting plate 23 (FIG. 2B) conducts electricity between the
exterior facing plate 20 and the interior face plate 22. The
exterior surface of the cuvette 2 may also feature
position-orienting structures, such as a tab 45, that interfaces
with a corresponding structure in the electroporation machine to
ensure the cuvette is inserted in a proper orientation. The cuvette
2 may also feature structures to strengthen the assembly and to
facilitate flow of liquids into the gap 46 between electrode
surfaces 44, such as a wedge-shaped blend 47. between the upper
wall 48 of the cuvette 2 and the electrode surface 44. As described
above with respect to FIG. 2A, 2B, the size of the gap 46 in the
cuvette 2 may be varied by changing the span of the interconnecting
plate 23 (FIG. 2B), varying the gap 24 between plates 20, 21 of
each electrode, without varying the outside dimensions of the
cuvette 2.
[0031] Manufacture and assembly of the ready-to-use cuvette is
summarized in FIG. 5. The cuvette is fabricated by a suitable
method, such as injection molding, and electrodes are positioned in
the cuvette, which may be accomplished by fusing the electrodes
into the cuvette, or gluing or force-fitting the electrodes into
electrode positioning structure. Step 50. The cuvette may thus be
designed and manufactured for a single use. An example of a
suitable cuvette is the Gene Pulser.RTM. Cuvette, Catalog No.
165-2089 manufactured by Bio-Rad Laboratories of Hercules,
Calif.
[0032] Once assembled, the cuvette is sterilized, step 51.
Sterilization may include one or more of chemical cleaning, heat
treatment and exposure to gamma or X-ray radiation, or other
suitable sterilization process.
[0033] The cuvette may then be prechilled, such as by suspending it
in a water ice bath, in preparation for depositing cells into the
cuvette. Step 54.
[0034] Cells for electroporation are added to a suitable suspension
solution, and then an aliquot of cells and solution are added to
the chilled cuvette. Step 54. The cells may be any cells suitable
for electroporation, including for example, bacteria such as E.
coli, yeast, plant or mammalian cells. The cells may be treated to
render them competent for electroporation, which may include using
a suspension solution that renders the cells electroporation
competent. Suitable suspension solutions are well known in the
art.
[0035] Once cells and suspension solution have been added, the
cuvette is sealed with a cap providing a sterile barrier to prevent
contamination. Step 54. Cells and suspension solution are then
rapidly frozen, such as by flash freezing, step 55, dipping or
submerging the cuvette in liquid nitrogen, step 56, dipping or
submerging the cuvette in a bath of ethanol and dry ice, step 57,
or placing the cuvette in a freezer, such as a freezer at about
-85.degree. C. or a rate-controlled freezer. In various embodiments
of the present invention, the cap may be attached to: the cuvette
before or after freezing.
[0036] Once the cells and suspension solution have been frozen, the
cuvette is stored in a freezer or other suitable cold storage means
until ready for use. Step 58. By maintaining the cuvette below
0.degree. C., such as in ultra-cold storage at about -78.degree.
C., the cells may be maintained ready for use for an extended
period of time.
[0037] Use of the ready-to-use cuvette is illustrated in FIG. 6.
Once the appropriate cuvette is selected, such as based upon the
color code on the cap, the cells and suspension solution are gently
thawed, such as by placing the cuvette on wet ice. Step 60. Once
thawed, the cap is removed, step 61, and a suitable amount of a
nucleic acid, such as in a suspension solution, is added by a user.
Step 62. The nucleic acid is mixed with the cells and suspension
solution. Step 63. A suitable method of mixing nucleic acid and
cells/suspension solution within the electroporation cuvette
comprises alternately drawing solution up into a micropipette and
expelling the solution down into the cuvette a number of times,
such as 3 or 4 times, followed by rapping the cuvette on a solid
surface, such as a laboratory bench.
[0038] Once the nucleic acid and cells/suspension have been mixed,
the cuvette may be placed in a suitable electroporation machine,
step 64, and electroporation conducted, step 65. Electroporation
machines and methods for using them to conduct, electroporation
transformation of cells are well known in the art.
[0039] Following electroporation, the cells are placed on or in an
appropriate medium to promote growth of the transformed cells. The
chosen medium should propagate the transformed cells that either
transiently express or have nucleic acids integrated into the host
cells' genome. Further, the medium advantageously should be
selected so as to assist the cells in recovering from the
electrical treatment.
[0040] Suitable cell suspension solutions are substantially
non-ionic solutions in order to ensure a predictable electric
current is produced in the cells. An appropriate non-ionic solution
may be a buffer solution with minimal or no ions present. Non-ionic
solutions may also be non-polar. The concentration of ions in the
buffer is adequately low so that when electricity is discharged
into the host cells, little or no additional current is carried
into the cells. The presence of ions in the buffer may result in
additional current being carried into the cells and can lower the
survival rate of the host cells. In some embodiments of the
invention, the non-ionic solution includes glycerol at about 5% to
about 10% solution or dimethyl sulfoxide from about 2% to about 15%
solution, depending upon the application.
[0041] The solution may also comprise at least one sugar or sugar
derivative, such as D-stereoisomeric or the L-forms (enantiomers)
form. The concentration of the sugar or derivative may be about
2.0% to about 2.5%. In specific embodiments, the added sugar
derivative is sorbitol and its concentration is about 2.5%.
Specific sugars may include, but are not limited to: aldoses, such
as monosaccharides which include trioses (i.e. glyceraldehyde),
tetroses (i.e. erythrose, threose), pentoses (i.e. arabinose,
xylose, ribose, lyxose), hexoses (i.e. glucose, marinose,
galactose, idose, gulose, altrose, allose, talose), heptoses (i.e.
sedoheptulose), octoses (i.e. glycero-D-manno-octulose), pentose
ring sugars (i.e. ribofurandse, ribopyranose); disaccharides (i.e.,
sucrose, lactose, trehalose, maltose, cellobiose, gentiobiose); and
trisaccharides (i.e., raffinose), oligosaccharides (i.e., amylose,
amylopectin, glycogen).
[0042] Sugar derivatives that may be used include, but are not
limited to: alditols or aldose alcohol, which include erythritol,
glucitol, sorbitol, or mannitol; ketoses, e.g., dihydroxyacetone,
erythrulose, ribulose, xylulose, psicose, fructose, sorbose, and
tagatose; aminosugars such as glucosamine, galactosamine,
N-acetylglucosamine, N-acetylgalactosamine, muramic acid, N-acetyl
muramic acid, and N-acetylneuraminic acid (sialic acid);
glycosides, such as glucopyranose and methyl-glucopyranose; and
lactones, such as gluconolactone.
[0043] Nucleic acids that may be added to the ready-to-use
electroporation cuvette may include, but are not limited to, RNA,
DNA, or non-naturally occurring nucleic acid sequences that encode
functional or non-functional proteins, and fragments of those
sequences, polynucleotides, or oligonucleotides. The nucleic acids
of interest may be obtained naturally or synthetically, e.g., using
PCR or mutagenesis. Further, the nucleic acids may be circular,
linear, or supercoiled in their topology. Preferably, the nucleic
acids may range from about 3 kb to about 300 kb.
[0044] i 1
EXAMPLES
[0045] Preparation of Ready-To-Use Electroporation Cuvettes. A
recA-minus derivative of E. coli strain MC1061 was inoculated into
1 liter of SOB (minus magnesium) growth medium and incubated at
37.degree. C. and 275 rpm overnight (approximately 15 hours). The
overnight culture was diluted 1:50 into 1.5 liters of SOB (minus
magnesium) and grown at 39.degree. C., 275 rpm until an OD550 of
1.0 was reached. Cells were harvested by centrifugation and then
washed by resuspending in an equal (to the original) volume of cold
(approx. 4.degree. C.) 10% glycerol. The washing step was repeated
one time. The final cell pellet was resuspended in a minimal amount
of 10% glycerol. The final concentration of the cells was adjusted
to .about.250 OD550 units/ml with cold 10% glycerol. The cell
suspension was dispensed in 20 .mu.l aliquots into pre-chilled
electroporation cuvettes (Gene Pulser.RTM. Cuvette from Bio-Rad
Laboratories), flash-frozen by partial immersion of the cuvettes in
liquid nitrogen, and then stored in an ultra-cold freezer at about
-75.degree. C.
[0046] Use of Ready-To-Use Electroporation Cuvettes in
Transformation: The cuvettes prepared according to the procedure
described above were removed from the freezer and placed on ice for
about 10 minutes to thaw the cells. One microliter of pUC19 (10 pg)
nucleic acid was pipetted directly into the thawed cells and the
material was mixed with the cells in the cuvette by pipetting the
combined solution up and down several times. Further mixing of the
material was achieved by rapping the cuvette sharply on the bench
top several times. The mixture was then subjected to electroshock
using a Bio-Rad Micropulser from Bio-Rad Laboratories on
pre-programmed setting of "Ec1". Immediately after pulsing, the
cells were removed from the cuvette by rinsing out the electrode
gap with 980 .mu.l of SOC, and transferring the resulting liquid to
sterile snap-cap polypropylene tubes (Falcon 2059). The tubes were
shaken at 275 rpm, 37.degree. C. for about 1 hour. The liquid was
then diluted 1:100, and 100 .mu.l of this dilution was plated onto
LB+100 .mu.g/ml ampicillin plates and incubated overnight at
37.degree. C.
[0047] The foregoing description of various embodiments of the
invention has been presented for purposes of illustration and
description. It is not intended to be exhaustive or to limit the
invention to the precise form disclosed, and modifications and
variations are possible in light of the above teachings or may be
acquired from practice of the invention, the embodiments were
chosen and described in order to explain the principles of the
invention and its practical application to enable one skilled in
the art to utilize the invention in various embodiments and with
various modifications as are suited to the particular use
contemplated.
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