U.S. patent application number 15/254317 was filed with the patent office on 2017-01-05 for method of attaching a cell-of-interest to a microtube.
The applicant listed for this patent is TECHNION RESEARCH & DEVELOPMENT FOUNDATION LTD.. Invention is credited to Michal GREEN, Shiri KLEIN, Jonathan Charles KUHN, Eyal ZUSSMAN.
Application Number | 20170002343 15/254317 |
Document ID | / |
Family ID | 40751233 |
Filed Date | 2017-01-05 |
United States Patent
Application |
20170002343 |
Kind Code |
A1 |
KUHN; Jonathan Charles ; et
al. |
January 5, 2017 |
METHOD OF ATTACHING A CELL-OF-INTEREST TO A MICROTUBE
Abstract
A method of attaching a cell or a membrane-coated
particle-of-interest to a microtube is provided. The method
comprising: co-electrospinning two polymeric solutions through
co-axial capillaries, wherein a first polymeric solution of the two
polymeric solutions is for forming a shell of the microtube and a
second polymeric solution of the two polymeric solutions is for
forming a coat over an internal surface of the shell, the first
polymeric solution is selected solidifying faster than the second
polymeric solution and a solvent of the second polymeric solution
is selected incapable of dissolving the first polymeric solution
and wherein the second polymeric solution comprises the cell or the
membrane-coated particle-of-interest, thereby attaching the cell or
the membrane-coated particle-of-interest to the microtube. Also
provided are microtubes with attached, entrapped or encapsulated
cells or membrane-coated particles and methods of using same
Inventors: |
KUHN; Jonathan Charles;
(Haifa, IL) ; ZUSSMAN; Eyal; (Haifa, IL) ;
GREEN; Michal; (Haifa, IL) ; KLEIN; Shiri;
(Tel-Aviv, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TECHNION RESEARCH & DEVELOPMENT FOUNDATION LTD. |
Haifa |
|
IL |
|
|
Family ID: |
40751233 |
Appl. No.: |
15/254317 |
Filed: |
September 1, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12918365 |
Nov 4, 2010 |
9469919 |
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PCT/IL2009/000169 |
Feb 12, 2009 |
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15254317 |
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61064204 |
Feb 21, 2008 |
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61064206 |
Feb 21, 2008 |
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61064210 |
Feb 21, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/54393 20130101;
C02F 2101/36 20130101; C12Q 1/66 20130101; C12N 11/04 20130101;
C12M 21/18 20130101; Y02W 10/10 20150501; B82Y 30/00 20130101; D01D
5/247 20130101; Y10T 428/1393 20150115; C02F 2101/18 20130101; C02F
2101/306 20130101; A61K 9/0092 20130101; A61K 38/44 20130101; C12M
23/16 20130101; C12Y 114/16001 20130101; C02F 2101/20 20130101;
Y02W 10/15 20150501; D01F 8/14 20130101; C02F 3/102 20130101; A61P
43/00 20180101; C09D 171/02 20130101; G01N 2333/90241 20130101;
C09D 167/04 20130101; C12Y 113/12007 20130101; C02F 2305/08
20130101; C12M 23/06 20130101; D01D 5/003 20130101; D01F 1/10
20130101; C02F 3/342 20130101; C02F 3/34 20130101 |
International
Class: |
C12N 11/04 20060101
C12N011/04; C02F 3/34 20060101 C02F003/34; D01D 5/00 20060101
D01D005/00; C02F 3/10 20060101 C02F003/10 |
Claims
1. A microtube comprising: an electrospun shell, an electrospun
coat polymer over an internal surface of said shell and a cell or
membrane-coated particle-of-interest attached to the microtube,
wherein said electrospun shell is formed of a first polymeric
solution comprising a first solvent and said electrospun coat is
formed of a second polymeric solution comprising a second solvent,
wherein said second solvent of said second polymeric solution is
incapable of dissolving a polymer of said first polymeric solution,
wherein said first polymeric solution solidifies faster than said
second polymeric solution, wherein said second polymeric solution
is capable of wetting said internal surface of said shell during or
following solidification of said first polymeric solution.
2. The microtube of claim 1, wherein said polymer of said first
polymeric solution and a polymer of said second polymeric solution
are different.
3. The microtube of claim 1, wherein said electrospun shell
comprises pores.
4. The microtube of claim 1, wherein said electrospun shell
comprises a polymer selected from the group consisting of poly
(e-caprolactone) (PCL), polyamide, poly(siloxane), poly(silicone),
poly(ethylene), poly(vinyl pyrrolidone), poly(2-hydroxy
ethylmethacrylate), poly(N-vinyl pyrrolidone), poly(methyl
methacrylate), poly(vinyl alcohol), poly(acrylic acid), poly(vinyl
acetate), polyacrylamide, poly(ethylene-co-vinyl acetate),
poly(ethylene glycol), poly(methacrylic acid), polylactide,
polyglycolide, poly(lactide-coglycolide), polyanhydride,
polyorthoester, poly(carbonate), poly(acrylo nitrile),
poly(ethylene oxide), polyaniline, polyvinyl carbazole,
polystyrene, poly(vinyl phenol), polyhydroxyacid,
poly(caprolactone), polyanhydride, polyhydroxyalkanoate,
polyurethane, collagen, albumin, alginate, chitosan, starch, and
hyaluronic acid.
5. The microtube of claim 1, wherein said electrospun coat
comprises a polymer selected from the group consisting of
poly(acrylic acid), poly(vinyl acetate), polyacrylamide,
poly(ethylene-co-vinyl acetate), poly(ethylene glycol),
poly(methacrylic acid), polylactide polyglycolide,
poly(lactide-coglycolide), polyanhydride, polyorthoester,
poly(carbonate), poly(ethylene oxide), polyaniline, polyvinyl
carbazole, polystyrene, poly(vinyl phenol), polyhydroxyacid,
alginate, starch, hyaluronic acid.
6. The microtube of claim 1, wherein at least one of electrospun
shell and electrospun coat comprises a polymer selected from the
group consisting of: collagen, albumin, alginate, chitosan, starch,
and hyaluronic acid. elastin, tropoelastin, thrombin, fibronectin,
poly(amino acids), poly(propylene fumarate), gelatin, pectin,
fibrin, cellulose, oxidized cellulose, chitin, polyethylene,
polyethylene terephthalate, poly(tetrafluoroethylene),
polycarbonate, and polypropylene, or derivatives thereof.
7. The microtube of claim 1, wherein said first solvent of said
first polymeric solution evaporates faster than said second solvent
of said second polymeric solution, and wherein said second solvent
of said second polymeric solution is capable of evaporating through
said internal surface of said shell.
8. The microtube of claim 1, wherein a thickness of said shell is
from about 100 nm to about 20 micrometer.
9. The microtube of claim 1, wherein an internal diameter of the
microtube is from about 50 nm to about 20 micrometer.
10. The microtube of claim 1, wherein said microtube is filled with
a liquid.
11. The microtube of claim 1, wherein said cell or membrane-coated
particle-of-interest is attached to said coat over said internal
surface of said shell.
12. The microtube of claim 1, wherein said cell or said
membrane-coated particle-of-interest is attached to said shell of
the microtube.
13. The microtube of claim 1, wherein said first polymeric solution
further comprises polyethylene glycol (PEG).
14. The microtube of claim 1, wherein said shell prevents diffusion
of the cell or said membrane-coated particle-of-interest
therethrough.
15. A microfluidic device comprising a plurality of the microtubes
of claim 1.
16. The microtube of claim 1, wherein at least one of said first
polymeric solution and second polymeric solution comprises a
co-polymer.
17. The microtube of claim 1, wherein at least one of said first
polymeric solution and second polymeric solution comprises a blend
of polymers.
18. A method of attaching a cell or said membrane-coated
particle-of-interest to a microtube, the method comprising:
co-electrospinning two polymeric solutions through co-axial
capillaries, wherein a first polymeric solution of said two
polymeric solutions is for forming a shell of the microtube and a
second polymeric solution of said two polymeric solutions is for
forming a coat over an internal surface of said shell, said first
polymeric solution is selected solidifying faster than said second
polymeric solution and a solvent of said second polymeric solution
is selected incapable of dissolving said first polymeric solution
and wherein said second polymeric solution comprises the cell or
said membrane-coated particle-of-interest, thereby attaching the
cell or said membrane-coated particle-of-interest to the
microtube.
19. A method of bioremediation, the method comprising contacting a
solution containing a contaminant with the microtube of claim 1,
wherein said cell, a portion of said cell or said membrane-coated
particle-of-interest is capable of degrading or assimilating said
contaminant.
20. A method of depleting a molecule from a solution, comprising
contacting the solution with the microtube of claim 1, wherein the
molecule is capable of binding to or being processed by said cell
or said membrane-coated particle-of-interest, thereby depleting the
molecule from the solution.
Description
RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Application Nos. 61/064,210, 61/064,206 and 61/064,204 filed on
Feb. 21, 2008.
[0002] The teachings of PCT/IB2007/054001 are incorporated herein
by reference.
[0003] The contents of all of the above documents are incorporated
by reference as if fully set forth herein.
FIELD AND BACKGROUND OF THE INVENTION
[0004] The present invention, in some embodiments thereof, relates
to a method of attaching a cell or a membrane-coated
particle-of-interest to a microtube and, more particularly, but not
exclusively, to electrospun microtubes which include cells or
membrane-coated particles attached, entrapped or encapsulated
therein which can be used in various applications such as water
purification, detoxification, mineral enrichment, tissue grafts and
cell-based therapy.
[0005] Water purification usually entails the removal of toxic
chemicals such as mercury, mercurial compounds, and cadmium or
elements such as toluene, chloroform, benzene, pesticides and
herbicides. Interestingly, although these organic compounds are not
found in nature and result from modern industry and motor vehicles,
certain bacterial strains have evolved mechanisms for degrading
them while utilizing their carbon and nitrogen atoms.
[0006] For example, Pseudomonads can be used to degrade toluene
(Moat and Foster, 1995), benzene, phenol, naphthalene (Doelle,
1969) and certain hydrocarbons from oil (van der Linden et al.,
1965). In addition, atrazine, a commonly used toxic herbicide that
enters the water supply, can be detoxified to ammonia and carbon
di-oxide via a dechlorinization reaction mediated by Pseudomonas
ADP. Thus, bioparticles of Pseudomonas ADP grown on granulated
active carbon were shown capable of degrading atrazine in water
(Herzberg et al., 2006). However, since the granulated active
carbon particles are only effective for a limited time, a carbon
source (citrate) must be added to the water as it enters the
purification column, which may be associated with the growth of
other bacterial species and increases the costs of the purification
process.
[0007] Another serious problem which occurs in some water systems
is the presence of toxic heavy metals such as cadmium and mercury.
Several bacterial strains such as Chromobacterium violaceum,
Pseudomonas maltophila, Pseudomonas aeruginosa, Spirulina
planensis, Staphylococcus aureus, Bacillus cereus, Bacillus
subtilis and Escherichia coli have been found capable of removing
metal contamination, destroying the toxic complex containing the
metallic ions (e.g., cadmium and tellurium) or recovering valuable
metals such as platinum and palladium, gold and silver from the
water. For example, certain bacteria have been used to form
nanoparticles of valuable metals (Brayner et al., 2007).
[0008] Nanofibers and polymeric nanofibers in particular can be
produced by the electrospinning process (Reneker D H., et al.,
2006; Ramakrishna S., et al., 2005; Li D, et al., 2004; PCT WO
2006/106506 to Zussman, E., et al.). Sun and co-workers (Sun Z, et
al., 2003) describe the production of core-shell nanofibers (i.e.,
filled fibers) by co-electrospinning of two polymeric solutions
using a spinneret with two co-axial capillaries. US patent
application No. 20060119015 to Wehrspohn R., et al. describes the
production of hollow fibers by introducing a liquid containing a
polymer to a porous template material, and removal of the template
following polymer solidification. PCT/IB2007/054001 to Zussman, E.,
et al. (which is fully incorporated herein by reference) discloses
methods of producing electrospun microtubes (i.e., hollow fibers)
which can be further filled with liquids and be used as
microfluidics.
[0009] Salalha W., et al., 2006, describe the encapsulation of
whole bacterial cells and complex bacterial viruses in electrospun
single-layer fibers, in which the entrapped cells or viruses
retained both physiological activity and some of their viability,
even after storing the dry electrospun mats for a number of
months.
SUMMARY OF THE INVENTION
[0010] According to an aspect of some embodiments of the present
invention there is provided a method of attaching a cell or a
membrane-coated particle-of-interest to a microtube, the method
comprising: co-electrospinning two polymeric solutions through
co-axial capillaries, wherein a first polymeric solution of the two
polymeric solutions is for forming a shell of the microtube and a
second polymeric solution of the two polymeric solutions is for
forming a coat over an internal surface of the shell, the first
polymeric solution is selected solidifying faster than the second
polymeric solution and a solvent of the second polymeric solution
is selected incapable of dissolving the first polymeric solution
and wherein the second polymeric solution comprises the cell or the
membrane-coated particle-of-interest, thereby attaching the cell or
the membrane-coated particle-of-interest to the microtube.
[0011] According to an aspect of some embodiments of the present
invention there is provided a microtube comprising an electrospun
shell, an electrospun coat over an internal surface of the shell
and a cell or a membrane-coated particle-of-interest attached to
the microtube.
[0012] According to an aspect of some embodiments of the present
invention there is provided a method of bioremediation, the method
comprising contacting a solution containing a contaminant with the
microtube of some embodiments of the invention, wherein the cell, a
portion of the cell or the membrane-coated particle-of-interest is
capable of degrading or assimilating the contaminant.
[0013] According to an aspect of some embodiments of the present
invention there is provided a method of depleting a molecule from a
solution, comprising contacting the solution with the microtube of
some embodiments of the invention, wherein the molecule is capable
of binding to or being processed by the cell or the membrane-coated
particle-of-interest, thereby depleting the molecule from the
solution.
[0014] According to an aspect of some embodiments of the present
invention there is provided a method of isolating a molecule from a
solution, comprising: (a) contacting the solution with the
microtube of some embodiments of the invention under conditions
which allow binding of the molecule to the cell or the
membrane-coated particle-of-interest, and; (b) eluting the molecule
from the microtube; thereby isolating the molecule from the
solution.
[0015] According to an aspect of some embodiments of the present
invention there is provided a method of detecting a presence of a
molecule in a sample, comprising: (a) contacting the sample with
the microtube of some embodiments of the invention, wherein the
cell or the membrane-coated particle-of-interest is capable of
binding to or processing the molecule, and; (b) detecting the
binding or the processing; thereby detecting the presence of the
molecule in the sample.
[0016] According to some embodiments of the invention, the
electrospun shell is formed of a first polymeric solution and the
electrospun coat is formed of a second polymeric solution.
[0017] According to some embodiments of the invention, the first
polymeric solution solidifies faster than the second polymeric
solution.
[0018] According to some embodiments of the invention, a solvent of
the second polymeric solution is incapable of dissolving the first
polymeric solution.
[0019] According to some embodiments of the invention, the
electrospun shell comprises a polymer selected from the group
consisting of poly (e-caprolactone) (PCL), polyamide,
poly(siloxane), poly(silicone), poly(ethylene), poly(vinyl
pyrrolidone), poly(2-hydroxy ethylmethacrylate), poly(N-vinyl
pyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol),
poly(acrylic acid), poly(vinyl acetate), polyacrylamide,
poly(ethylene-co-vinyl acetate), poly(ethylene glycol),
poly(methacrylic acid), polylactide, polyglycolide,
poly(lactide-coglycolide), polyanhydride, polyorthoester,
poly(carbonate), poly(acrylo nitrile), poly(ethylene oxide),
polyaniline, polyvinyl carbazole, polystyrene, poly(vinyl phenol),
polyhydroxyacid, poly(caprolactone), polyanhydride,
polyhydroxyalkanoate, polyurethane, collagen, albumin, alginate,
chitosan, starch and hyaluronic acid, and whereas the electrospun
coat comprises a polymer selected from the group consisting of
poly(acrylic acid), poly(vinyl acetate), polyacrylamide,
poly(ethylene-co-vinyl acetate), poly(ethylene glycol),
poly(methacrylic acid), polylactide polyglycolide,
poly(lactide-coglycolide), polyanhydride, polyorthoester,
poly(carbonate), poly(ethylene oxide), polyaniline, polyvinyl
carbazole, polystyrene, poly(vinyl phenol), polyhydroxyacid,
alginate, starch and hyaluronic acid.
[0020] According to some embodiments of the invention, a solvent of
the first polymeric solution evaporates faster than a solvent of
the second polymeric solution.
[0021] According to some embodiments of the invention, the
electrospinning is effected using a rotating collector.
[0022] According to some embodiments of the invention, the solvent
of the second polymeric solution is capable of evaporating through
the internal surface of the shell.
[0023] According to some embodiments of the invention, the second
polymeric solution is capable of wetting the internal surface of
the shell.
[0024] According to some embodiments of the invention, a thickness
of the shell is from about 100 nm to about 20 micrometer.
[0025] According to some embodiments of the invention, an internal
diameter of the microtube is from about 50 nm to about 20
micrometer.
[0026] According to some embodiments of the invention, the first
and the second polymeric solutions are selected from the group
consisting of: 10% poly (e-caprolactone) (PCL) in chloroform
(CHCl.sub.3) and dimethylforamide (DMF) (80:20 by weight) as the
first polymeric solution and 4% poly(ethylene oxide) (PEO) in water
(H.sub.2O) and ethanol (60:40 by weight) as the second polymeric
solution, 10% PCL in CHCl.sub.3 and DMF (80:20 by weight) as the
first polymeric solution and 6% PEO in H.sub.2O and ethanol (60:40
by weight) as the second polymeric solution, 9% PCL in CHCl.sub.3
and DMF (90:10 by weight) as the first polymeric solution and 7%
PEO in H.sub.2O as the second polymeric solution, 10% PCL in
CHCl.sub.3 and DMF (80:20 by weight) as the first polymeric
solution and 9% poly(vinyl alcohol) (PVA) in water and ethanol
(50:50 by weight) as the second polymeric solution, and 10% PCL in
CHCl.sub.3 and DMF (90:10 by weight) as the first polymeric
solution and 4% (w/w) PEO in ethanol:H.sub.2O (26:74 by weight) as
a second polymeric solution.
[0027] According to some embodiments of the invention, the
microtube is filled with a liquid.
[0028] According to some embodiments of the invention, the first
and the second polymeric solutions are biocompatible.
[0029] According to some embodiments of the invention, the cell or
the membrane-coated particle-of-interest is attached to the coat
over the internal surface of the shell.
[0030] According to some embodiments of the invention, the cell or
the membrane-coated particle-of-interest is attached to the shell
of the microtube.
[0031] According to some embodiments of the invention, the first
polymeric solution comprises polyethylene glycol (PEG).
[0032] According to some embodiments of the invention, the shell
comprises pores.
[0033] According to some embodiments of the invention, the shell
prevents diffusion of the cell or the membrane-coated
particle-of-interest therethrough.
[0034] According to some embodiments of the invention, the cell
comprises a prokaryotic cell.
[0035] According to some embodiments of the invention, the cell
comprises a cell wall.
[0036] According to some embodiments of the invention, the
contaminant comprises atrazine.
[0037] According to some embodiments of the invention, the method
further comprising collecting said solution following the
contacting.
[0038] Unless otherwise defined, all technical and/or scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which the invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of
embodiments of the invention, exemplary methods and/or materials
are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be necessarily limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] Some embodiments of the invention are herein described, by
way of example only, with reference to the accompanying drawings.
With specific reference now to the drawings in detail, it is
stressed that the particulars shown are by way of example and for
purposes of illustrative discussion of embodiments of the
invention. In this regard, the description taken with the drawings
makes apparent to those skilled in the art how embodiments of the
invention may be practiced.
[0040] In the drawings:
[0041] FIGS. 1A-B are images depicting high resolution scanning
electron microscope (HRSEM) micrographs of the microtubes according
to an embodiment of the invention which are attached to cells of
microorganisms such that the cells are entrapped therein.
Electrospinning was performed using a first polymeric solution (for
forming the shell) which consisted of 9% [weight/weight (w/w)]
polycaprolactone (PCL) dissolved in chloroform/DMSO [9:1 (w/w)];
and a second polymeric solution (for forming the coat over the
internal surface of the shell) which consisted of 8% poly(ethylene
oxide) (PEO) in water (w/w). FIG. 1A--Magnification of 5000.times.;
FIG. 1B--Magnification of 10,000.times..
[0042] FIG. 2 is a microscopic image of Pseudomonas putida
bacterial cells transformed with the DsRed expression vector
encoding the red fluorescent protein [GenBank Accession No. Q9U6Y8
(SEQ ID NO:7)] and entrapped within a microtube of an embodiment of
the invention. A microtube was formed by co-electrospinning of the
two polymeric solutions: 9% (w/w) polycaprolactone (PCL) dissolved
in chloroform/DMSO [9:1 (w/w)] as a first polymeric solution (for
forming the shell); and 8% poly(ethylene oxide) (PEO) in water
(w/w) as second polymeric solution (for forming the coat over the
internal surface of the shell) which also included 100 .mu.l of
Pseudomonas putida bacterial cells (at a concentration of 10.sup.9
cells/ml). Detection of the bacterial cells within the microtube
was performed using a fluorescence microscope. Red fluorescence of
the red fluorescent protein (RFP) encoded by the DsRed expression
vector was visualized at a wavelength of 359 nm for excitation and
examining emitted light at a wavelength of 361 nm. Magnification:
200.times.; Size bar: 20 .mu.m.
[0043] FIGS. 3A-B are schematic illustrations depicting the
encapsulation of bacteria (FIG. 3A) and the growth of entrapped
bacteria (FIG. 3B) within the microtube of some embodiments of the
invention. FIG. 3A--A microtube is formed from two polymeric
solutions, the first one, for forming the shell is insoluble in
water, and the second one, for forming the coat over the internal
surface of the shell is soluble in water (see for example, the
description of solutions with FIG. 2, hereinabove). Following
microtube formation, the microtube can be filled with an aqueous
solution (e.g., water or phosphate buffer) by simply exposing the
microtube to an aqueous solution (e.g., by immersing the microtube
in the aqueous solution). The aqueous solution dissolves some of
the polymer of the inner layer (the coat over the internal surface
of the shell) which is mixed with the bacterial cells and the
bacteria are released to the internal volume of the microtube. The
bacteria (red circles) reside within the soluble layer. FIG.
3B--The bacteria (red circles) can proliferate within the soluble
layer of the microtube when supplied with appropriate
nutrients.
[0044] FIG. 4 is a schematic illustration of a bioremediation
system generated according to some embodiments of the invention.
System 300 comprises conduit [220, e.g., part of an aqueous system,
can be a plastic or metal (e.g., copper) pipe/tube] having borders
(100) includes microtube (230) with shell borders (120) and
entrapped bacterial cells (210, red). For purification (e.g.,
detoxification of water), a liquid (e.g., drinking water)
containing molecule (150, blue) flows within the conduit. Pores
(180) within the microtube shell enable the diffusion of molecule
(150) through the microtube shell to the microtube lumen (140)
containing bacterial cells (210). Cells (210) interact with
molecule (150) and reaction product (160, green) diffuses out of
the microtube outside of the microtube lumen (140) through the
microtube shell pores (180).
[0045] FIG. 5 depicts the degradation of atrazine by Pseudomonas
ADP bacterial cells which are attached to (entrapped or
encapsulated within) the microtube of the invention. Atrazine is
degraded by the Pseudomonas ADP endogenous enzymes: atrazine
chlorohydrolase, e.g., GenBank Accession No. NP_862474 (SEQ ID NO:
1) encoded by the gene atzA, hydroxyatrazine hydrolase, e.g.,
GenBank Accession No. NP_862481 (SEQ ID NO:2) encoded by atzB,
N-isopropylammelide isopropylamino hydrolase, e.g., GenBank
Accession No. NP_862508 (SEQ ID NO:3) encoded by atzC, cyanuric
acid amidohydrolase, e.g., GenBank Accession No. NP_862537 (SEQ ID
NO:4) encoded by atzD, biuret hydrolase, e.g., GenBank Accession
No. NP_862538 (SEQ ID NO:5) encoded by atzE and allophanate
hydrolase, e.g., GenBank Accession No. AAK50333 (SEQ ID NO:6)
encoded by atzF.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0046] The present invention, in some embodiments thereof, relates
to methods of attaching a cell or a membrane-coated particle to a
microtube and, more particularly, but not exclusively, to
microtubes with cells or membrane-coated particles attached,
entrapped or encapsulated therein and uses thereof in various
purification, bioremediation, isolation, detection, and therapeutic
applications.
[0047] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not
necessarily limited in its application to the details set forth in
the following description or exemplified by the Examples. The
invention is capable of other embodiments or of being practiced or
carried out in various ways.
[0048] While reducing the present invention to practice, the
present inventors have devised a method of attaching a cell or a
membrane-coated particle to a microtube, to thereby obtain
attached, entrapped or encapsulated cells or membrane-coated
particles within the microtube.
[0049] Thus, as described in Example 1 of the Examples section
which follows, the present inventors were capable of attaching
cells (e.g., bacterial cells) to a microtube. In addition as shown
in FIG. 2, cells attached within electrospun microtubes remained
intact and viable. Moreover, as shown in Tables 4 and 5 and
described in Example 2 of the Examples section which follows, the
attached cells preserved their catalytic activity following the
electrospinning process and were capable of degrading atrazine from
a solution. These results suggest the use of the microtubes of some
embodiments of the invention in various applications such a
bioremediation of solutions (flow-through applications) and soils,
purification, detoxification, and synthesis.
[0050] According to one aspect of the invention there is provided a
method of attaching a cell or a membrane-coated
particle-of-interest to a microtube, the method comprising:
co-electrospinning two polymeric solutions through co-axial
capillaries, wherein a first polymeric solution of the two
polymeric solutions is for forming a shell of the microtube and a
second polymeric solution of the two polymeric solutions is for
forming a coat over an internal surface of the shell, the first
polymeric solution is selected solidifying faster than the second
polymeric solution and a solvent of the second polymeric solution
is selected incapable of dissolving the first polymeric solution
and wherein the second polymeric solution comprises the cell or the
membrane-coated particle-of-interest, thereby attaching the cell or
the membrane-coated particle-of-interest to the microtube.
[0051] As used herein the term "microtube" refers to a hollow tube
having an inner diameter from about 50 nm to about 50 .mu.m and an
outer diameter from about 0.5 .mu.m to about 100 .mu.m.
[0052] According to some embodiments of the invention the inner
diameter of the microtube shell of the invention can vary from
about 100 nm to about 20 .mu.m, e.g., from about 200 nm to about 10
.mu.m, e.g., from about 500 nm to about 5 .mu.m, e.g., from about 1
.mu.m to about 5 .mu.m, e.g., about 3 .mu.m.
[0053] According to some embodiments of the invention the thickness
of the microtube shell of the invention can vary from a few
nanometers to several micrometers, such as from about 100 nm to
about 20 .mu.m, e.g., from about 200 nm to about 10 .mu.m, from
about 100 nm to about 5 .mu.m, from about 100 nm to about 1 .mu.m,
e.g., about 500 nm.
[0054] According to some embodiments of the invention, the
microtube may have a length which is from about 0.1 millimeter (mm)
to about 20 centimeter (cm), e.g., from about 1-20 cm, e.g., from
about 5-10 cm.
[0055] As used herein the term "cell" refers to a eukaryotic or
prokaryotic cell.
[0056] According to some embodiments of the invention, the
cell-of-interest comprises a cell wall. Non-limiting examples of
cells which comprise a cell wall and which can be attached to the
microtube of the invention include plant cells, bacteria (e.g.,
Gram positive and Gram negative bacteria), archaea, protozoa,
fungi, and algae.
[0057] According to some embodiments of the invention, the
cell-of-interest comprises a dermis (e.g., insect cells).
[0058] According to some embodiments of the invention, the cell has
a diameter from about 500 nanometers to about 30 microns, e.g.,
from about 1-10 microns.
[0059] According to some embodiments of the invention, the cell has
a diameter from about 1-2 microns.
[0060] The cell-of-interest which is attached to the microtube may
have an activity (e.g., a catalytic activity or a binding activity)
which is beneficial (e.g., expression of specific enzymes, e.g.,
atrazine-degrading enzyme, binding to a specific substrate via a
cell receptor or via interaction with a molecule present in the
cells, e.g., a DNA, RNA or protein). According to some embodiments
of the invention, the cell is genetically modified to express a
gene or a protein-of-interest (e.g., a mutant form which is capable
of degrading a substrate with an improved catalytic activity as
compared to a wild-type form; a specific label, e.g., a green
fluorescent protein). Genetic modification can be done using known
recombinant DNA technology and include, but not limited to, mutant
isolation, know-out or knock-in mutagenesis, site-directed
mutagenesis, gene silencing (e.g., siRNA, Ribozyme, DNAzyme,
antisense) and gene overexpression (e.g., by transfection with an
expression vector).
[0061] As used herein the phrase "membrane-coated particle" refers
to a lipid membrane coated particle. The membrane may enable
passage of molecules (e.g., organic and inorganic molecules,
polymeric molecules) therethrough.
[0062] The membrane may be a naturally occurring membrane (e.g., a
cell membrane), a portion of a naturally occurring membrane (e.g.,
a vesicle), an artificial membrane formed of natural membrane
components (e.g., a liposome with a lipid bilayer), and/or an
artificial membrane formed of non-natural components such as a
polymer, a surfactant [e.g., dioleoylphosphatidylethanolamine
(DOPE)], a ceramic, a glass and/or a metal.
[0063] Liposomes include emulsions, foams, micelles, insoluble
monolayers, liquid crystals, phospholipid dispersions, lamellar
layers and the like. The liposomes may be prepared by any of the
known methods in the art [Monkkonen, J. et al., 1994, J. Drug
Target, 2:299-308; Monkkonen, J. et al., 1993, Calcif. Tissue Int.,
53:139-145; Lasic D D., Liposomes Technology Inc., Elsevier, 1993,
63-105. (chapter 3); Winterhalter M, Lasic D D, Chem Phys Lipids,
1993 September; 64(1-3):35-43]. The liposomes may be positively
charged, neutral or negatively charged. The liposomes may have a
single lipid layer or may be multilamellar. If the therapeutic
agent is hydrophilic, its delivery may be further improved using
large unilamellar vesicles because of their greater internal
volume. Conversely, if the therapeutic agent is hydrophobic, its
delivery may be further-improved using multilamellar vesicles.
Alternatively, the therapeutic agent (e.g. oligonucleotide) may not
be able to penetrate the lipid bilayer and consequently would
remain adsorbed to the liposome surface. In this case, increasing
the surface area of the liposome may further improve delivery of
the therapeutic agent. The liposomes can be non-toxic liposomes
such as, for example, those prepared from phosphatidyl-choline
phosphoglycerol, and cholesterol. The diameter of the liposomes
used can range from 0.1-1.0 microns.
[0064] The particle may comprise an atom, an isotope, a molecule
(e.g., a bio-molecule such as an amino acid, a nucleic acid, a
polypeptide, a DNA or an RNA), a drug, a virus, a portion of a cell
(e.g., a cell vesicle, enzymes of a cell), a bead (e.g., a glass
bead, a magnetic bead) or any combination thereof, e.g., a magnetic
bead conjugated to a molecule such as a polypeptide, a DNA and/or
an RNA.
[0065] As used herein the term "attaching" refers to the binding of
the cell or the membrane-coated particle-of-interest to the
polymer(s) comprised in the microtube of the invention via covalent
or non-covalent binding (e.g., via an electrostatic bond, a
hydrogen bond, a van-Der Waals interaction) so as to obtain an
absorbed, embedded or immobilized cell or membrane-coated
particle-of-interest to the microtube of the invention.
[0066] According to some embodiments of the invention, the length
(L) of the microtube can be several orders of magnitude higher
(e.g., 10 times, 100 times, 1000 times, 10,000 times, e.g., 50,000
times) than the microtube's diameter (D). Accordingly, a cell or a
membrane-coated particle-of-interest which is attached to a
microtube is referred to as being entrapped or encapsulated within
the microtube.
[0067] According to some embodiments of the invention, covalent
attachment of the cell or membrane-coated particle can be via
functional groups such as SH groups, amino groups, carboxyl groups
which are added to the polymer(s) forming the microtube.
[0068] As used herein the phrase "co-electrospinning" refers to a
process in which at least two polymeric solutions are electrospun
from co-axial capillaries (i.e., at least two capillary dispensers
wherein one capillary is placed within the other capillary while
sharing a co-axial orientation) forming the spinneret within an
electrostatic field in a direction of a collector. The capillary
can be, for example, a syringe with a metal needle or a bath
provided with one or more capillary apertures from which the
polymeric solution can be extruded, e.g., under the action of
hydrostatic pressure, mechanical pressure, air pressure and/or high
voltage.
[0069] The collector serves for collecting the electrospun element
(e.g., the electrospun microtube) thereupon. Such a collector can
be a rotating collector or a static (non rotating) collector. When
a rotating collector is used, such a collector may have a
cylindrical shape (e.g., a drum), however, the rotating collector
can be also of a planar geometry (e.g., an horizontal disk). The
spinneret is typically connected to a source of high voltage, such
as of positive polarity, while the collector is grounded, thus
forming an electrostatic field between the dispensing capillary
(dispenser) and the collector. Alternatively, the spinneret can be
grounded while the collector is connected to a source of high
voltage, such as with negative polarity. As will be appreciated by
one ordinarily skilled in the art, any of the above configurations
establishes motion of a positively charged jet from the spinneret
to the collector. Reverse polarity for establishing motions of a
negatively charged jet from the spinneret to the collector are also
contemplated.
[0070] For electrospinning, the first polymeric solution is
injected into the outer capillary of the co-axial capillaries while
the second polymeric solution is injected into the inner capillary
of the co-axial capillaries. In order to form a microtube (i.e., a
hollow structure, as mentioned above), the first polymeric solution
(which is for forming the shell of the microtube) solidifies faster
than the second polymeric solution (also referred herein as a core
polymeric solution, and is for forming a coat over the internal
surface of the shell). In addition, the formation of a microtube
also requires that the solvent of the second polymeric solution be
incapable of dissolving the first polymeric solution.
[0071] The solidification rates of the first and second polymeric
solutions are critical for forming the microtube. For example, for
a microtube of about 100 .mu.m, the solidification of the first
polymer (of the first polymeric solution) can be within about 30
milliseconds (ms) while the solidification of the second polymer
(of the second polymeric solution) can be within about 10-20
seconds. The solidification may be a result of polymerization rate
and/or evaporation rate.
[0072] According to some embodiments of the invention, the solvent
of the first polymeric solution evaporates faster than the solvent
of second polymeric solution (e.g., the solvent of the first
polymeric solution exhibits a higher vapor pressure than the
solvent of the second polymeric solution).
[0073] According to some embodiments of the invention, the rate of
evaporation of the solvent of the first polymeric solution is at
least about 10 times faster than that of the solvent of the second
polymeric solution. The evaporation rate of the solvent of the
first polymeric solution can be at least about 100 times faster or
at least about 1000 times faster than the evaporation rate of the
solvent of second polymeric solution. For example, the evaporation
of chloroform is significantly faster than the evaporation of an
aqueous solution (water) due to the high vapor pressure at room
temperature of the chloroform (195 mmHg) vs. that of the aqueous
solution (23.8 mmHg).
[0074] When selecting a solvent of the second polymeric solution
which is incapable of dissolving the first polymeric solution
(i.e., a non-solvent of the first polymeric solution), the polymer
of the first polymeric solution can solidify (e.g., through
precipitation) and form a strong microtube shell which does not
collapse, and which is characterized by an even thickness.
According to some embodiments of the invention, the first polymeric
solution (e.g., the solvent of the first polymer) is substantially
immiscible in the solvent of the second polymeric solution.
[0075] The solvent of the second polymeric solution may evaporate
while the polymer (of the second polymeric solution) forms a thin
layer on the internal surface of the shell.
[0076] According to some embodiments of the invention, the solvent
of the second polymeric solution is capable of evaporating through
the internal surface of the shell.
[0077] The flow rates of the first and second polymeric solutions
can determine the microtube outer and inner diameter and thickness
of shell. Non-limiting examples are shown in Table 1
hereinbelow.
TABLE-US-00001 TABLE 1 Effect of the flow rates of the two
polymeric solutions during electrospinning on microtube diameter
and thickness of shell System: First polymeric R solution/ Outer d
Electro- Second Flow Fiber Shell V static System polymeric rates
radius thickness Voltage field No. solution (ml/hr) (.mu.m) (.mu.m)
(kV) kV/cm M5 First 4 3.0-4.5 0.5 .+-. 0.1 8.5 0.43 polymeric
solution Second 0.5 polymeric solution M10 First 10 2.3-4.0 1.0
.+-. 0.1 8 0.5 polymeric solution Second 0.3 polymeric solution M11
First 10 3-6 1.0 .+-. 0.1 9 0.56 polymeric solution Second 2
polymeric solution Table 1: Electrospinning was performed with the
following solutions: First polymeric solution (for forming the
shell) was 10% PCL in CHCl.sub.3/DMF (8:2 weight/weight); Second
polymeric solution (for forming the coat) was 4% PEO in
H.sub.2O/EtOH (6:4, weight/weight). PCL 80K; PEO 600K. The
temperature during electrospinning was of 22-26.degree. C. The
relative humidity during electrospinning was 58%, 52% and 53% for
systems M5, M10 and M11, respectively. The flow rates were measured
in milliliter per hour (ml/hr); the outer microtube radius and the
shell thickness were measured in microns (.mu.m). The resulting
tubes were hollow (good tubes in systems M5 and M11, and mostly
good in system M10).
[0078] As used herein the phrase "polymeric solution" refers to a
soluble polymer, i.e., a liquid medium containing one or more
polymers, co-polymers or blends of polymers dissolved in a solvent.
The polymer used by the invention can be a natural, synthetic,
biocompatible and/or biodegradable polymer.
[0079] The phrase "synthetic polymer" refers to polymers that are
not found in nature, even if the polymers are made from naturally
occurring biomaterials. Examples include, but are not limited to,
aliphatic polyesters, poly(amino acids), copoly(ether-esters),
polyalkylenes, oxalates, polyamides, tyrosine derived
polycarbonates, poly(iminocarbonates), polyorthoesters,
polyoxaesters, polyamidoesters, polyoxaesters containing amine
groups, poly(anhydrides), polyphosphazenes, and combinations
thereof.
[0080] Suitable synthetic polymers for use by the invention can
also include biosynthetic polymers based on sequences found in
naturally occurring proteins such as those of collagen, elastin,
thrombin, fibronectin, or mutant or synthetic derivatives thereof
or, starches, poly(amino acids), poly(propylene fumarate), gelatin,
alginate, pectin, fibrin, oxidized cellulose, chitin, chitosan,
tropoelastin, hyaluronic acid, polyethylene, polyethylene
terephthalate, poly(tetrafluoroethylene), polycarbonate,
polypropylene and poly(vinyl alcohol), ribonucleic acids,
deoxyribonucleic acids, polypeptides, proteins, polysaccharides,
polynucleotides and combinations thereof.
[0081] The phrase "natural polymer" refers to polymers that are
naturally occurring. Non-limiting examples of such polymers
include, silk, collagen-based materials, chitosan, hyaluronic acid,
albumin, fibrinogen, and alginate.
[0082] As used herein, the phrase "co-polymer" refers to a polymer
of at least two chemically distinct monomers. Non-limiting examples
of co-polymers include, polylactic acid (PLA)-polyethyleneglycol
(PEG), polyethylene glycol terephthalate (PEGT)/polybutylene
terephthalate (PBT), PLA-polyglycolic acid (PGA),
PEG-polycaprolactone (PCL) and PCL-PLA.
[0083] As used herein, the phrase "blends of polymers" refers to
the result of mixing two or more polymers together to create a new
material with different physical properties.
[0084] The phrase "biocompatible polymer" refers to any polymer
(synthetic or natural) which when in contact with cells, tissues or
body fluid of an organism does not induce adverse effects such as
immunological reactions and/or rejections, cellular death, and the
like. A biocompatible polymer can also be a biodegradable
polymer.
[0085] According to some embodiments of the invention, the first
and the second polymeric solutions are biocompatible.
[0086] Non-limiting examples of biocompatible polymers include
polyesters (PE), PCL, calcium sulfate, PLA, PGA, PEG, polyvinyl
alcohol, polyvinyl pyrrolidone, polytetrafluoroethylene (PTFE,
teflon), polypropylene (PP), polyvinylchloride (PVC),
polymethylmethacrylate (PMMA), polyamides, segmented polyurethane,
polycarbonate-urethane and thermoplastic polyether urethane,
silicone-polyether-urethane, silicone-polycarbonate-urethane
collagen, PEG-DMA, alginate, hydroxyapatite and chitosan, blends
and copolymers thereof.
[0087] The phrase "biodegradable polymer" refers to a synthetic or
natural polymer which can be degraded (i.e., broken down) in a
physiological environment such as by proteases or other enzymes
produced by living organisms such as bacteria, fungi, plants and
animals. Biodegradability depends on the availability of
degradation substrates (i.e., biological materials or portion
thereof which are part of the polymer), the presence of
biodegrading materials (e.g., microorganisms, enzymes, proteins)
and the availability of oxygen (for aerobic organisms,
microorganisms or portions thereof), lack of oxygen (for anaerobic
organisms, microorganisms or portions thereof) and/or other
nutrients. Examples of biodegradable polymers/materials include,
but are not limited to, collagen (e.g., collagen I or IV), fibrin,
hyaluronic acid, polylactic acid (PLA), polyglycolic acid (PGA),
polycaprolactone (PCL), polydioxanone (PDO), trimethylene carbonate
(TMC), polyethyleneglycol (PEG), collagen, PEG-DMA, alginate,
chitosan copolymers or mixtures thereof.
[0088] According to some embodiments, the polymeric solution can be
made of one or more polymers, each can be a polymer or a co-polymer
such as described hereinabove.
[0089] According to some embodiments of the invention, the
polymeric solution is a mixture of at least one biocompatible
polymer and a co-polymer (either biodegradable or
non-biodegradable).
[0090] According to some embodiments of the invention, the first
polymeric solution for forming the shell can be made of a polymer
such as poly (e-caprolactone) (PCL), polyamide, poly(siloxane),
poly(silicone), poly(ethylene), poly(vinyl pyrrolidone),
poly(2-hydroxy ethylmethacrylate), poly(N-vinyl pyrrolidone),
poly(methyl methacrylate), poly(vinyl alcohol), poly(acrylic acid),
poly(vinyl acetate), polyacrylamide, poly(ethylene-co-vinyl
acetate), poly(ethylene glycol), poly(methacrylic acid),
polylactide, polyglycolide, poly(lactide-coglycolide),
polyanhydride, polyorthoester, poly(carbonate), poly(acrylo
nitrile), poly(ethylene oxide), polyaniline, polyvinyl carbazole,
polystyrene, poly(vinyl phenol), polyhydroxyacid,
poly(caprolactone), polyanhydride, polyhydroxyalkanoate,
polyurethane, collagen, albumin, alginate, chitosan, starch,
hyaluronic acid, and blends and copolymers thereof.
[0091] According to some embodiments of the invention, the second
polymeric solution for forming the coat over the internal surface
of the shell can be made of a polymer such as poly(acrylic acid),
poly(vinyl acetate), polyacrylamide, poly(ethylene-co-vinyl
acetate), poly(ethylene glycol), poly(methacrylic acid),
polylactide polyglycolide, poly(lactide-coglycolide),
polyanhydride, polyorthoester, poly(carbonate), poly(ethylene
oxide), polyaniline, polyvinyl carbazole, polystyrene, poly(vinyl
phenol), polyhydroxyacid, alginate, starch, hyaluronic acid, and
blends and copolymers thereof.
[0092] During the formation of the microtube shell (e.g., following
the solidification of the first polymeric solution) the second
polymeric solution flows within the internal surface of the
shell.
[0093] According to some embodiments of the invention, the second
polymeric solution is selected capable of wetting the internal
surface of the shell.
[0094] Various polymeric solutions are capable of wetting other
polymeric surfaces (for forming the shell). Following is a
non-limiting list of pairs of polymeric solutions in which the
second polymeric solution is capable of wetting the internal
surface of the shell formed by the first polymeric solution.
TABLE-US-00002 TABLE 2 Pairs of polymeric solutions for producing
the microtube of the invention First polymeric solution forming the
Second polymeric solution capable of shell wetting the internal
surface of the shell 10% poly (e-caprolactone) (PCL); in 4%
poly(ethylene oxide) (PEO); in water chloroform (CHCl.sub.3) and
(H.sub.2O) and ethanol (60:40 by weight) dimethylforamide (DMF)
(80:20 by weight) Nylon 6,6 in formic acid 7 to 12 wt % 4%
poly(ethylene oxide) (PEO); in water (H.sub.2O) and ethanol (60:40
by weight) Poly(L-lactide-co-glycolide) (PLGA 4% poly(ethylene
oxide) (PEO) in water 10:90) in hexafluroisopropanol (HFIP)
(H.sub.2O) and ethanol (60:40 by weight) concentrations ranging
from 2 to 7 weight % solution. Poly(L-lactide-co-glycolide) (PLGA
4% poly(ethylene oxide) (PEO); in water 15:85) hexafluroisopropanol
(HFIP) (H.sub.2O) and ethanol (60:40 by weight) concentrations
ranging from 2 to 7 weight % solution. poly(lactide-co-glycolide)
(PLGA; 4% poly(ethylene oxide) (PEO); in water
1-lactide/glycolide_50/50) (H.sub.2O) and ethanol (60:40 by weight)
1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) concentrations ranging
from 2 to 7 weight % solution. polyglycolide (PGA) in chloroform
3-10 9% poly(vinyl alcohol) (PVA); in water weight % solution. and
ethanol (50:50 by weight) poly(L-lactide) (PLA) in chloroform 3-10
9% poly(vinyl alcohol) (PVA); in water weight % solution. and
ethanol (50:50 by weight) Segmented polyurethane in DMF and 9%
poly(vinyl alcohol) (PVA); in water THF (80:20 by weight) and
ethanol (50:50 by weight) Polyurethane in DMF and 9% poly(vinyl
alcohol) (PVA); in water tetrahydrofuran, THF (80:20 by weight) and
ethanol (50:50 by weight) PLGA (poly lactic-co-glycolic acid); in
9% poly(vinyl alcohol) (PVA); in water chloroform and DMSO
(dimethyl and ethanol (50:50 by weight) sulfoxide) in chloroform
and DMSO (80:20 by weight). 10% PCL in CHCl.sub.3/DMF (80:20 by 6%
PEO in H.sub.2O/EtOH (60:40 by weight) weight) 9% PCL in
CHCl.sub.3/DMSO (90:10 by 7% PEO in H.sub.2O weight) 10% PCL in
CHCl.sub.3/DMF (80:20 by 9% PVA in ethanol/water (50:50 by weight)
weight) 10% PCL 80 K CHCl.sub.3:DMF (90:10 by 4% (w/w) PEO 600 K;
in ethanol:H.sub.2O weight) (26:74 by weight) 10% PCL 80 K + 1% PEG
6 K 4% (w/w) PEO 600 K; in ethanol:H.sub.2O CHCl.sub.3:DMF (90:10
by weight) (26:74 by weight) Table 2. The polymers forming the
solutions and the solvents are provided by weight ratios, i.e., a
weight/weight (w/w) ratio.
[0095] According to some embodiments of the invention, the first
and the second polymeric solutions are selected from the group
consisting of: 10% poly (e-caprolactone) (PCL) in chloroform
(CHCl.sub.3) and dimethylforamide (DMF) (80:20 by weight) as the
first polymeric solution and 4% poly(ethylene oxide) (PEO) in water
(H.sub.2O) and ethanol (60:40 by weight) as the second polymeric
solution, 10% PCL in CHCl.sub.3 and DMF (80:20 by weight) as the
first polymeric solution and 6% PEO in water and ethanol (60:40 by
weight) as the second polymeric solution, 9% PCL in CHCl.sub.3 and
DMF (90:10 by weight) as the first polymeric solution and 7% PEO in
water as the second polymeric solution, 10% PCL in CHCl.sub.3 and
DMF (80:20 by weight) as the first polymeric solution and 9%
poly(vinyl alcohol) (PVA) in water and ethanol (50:50 by weight) as
the second polymeric solution and 10% PCL in CHCl.sub.3 and DMF
(90:10 by weight) as the first polymeric solution and 4% (w/w) PEO
in ethanol:water (26:74 by weight) as a second polymeric
solution.
[0096] According to some embodiments of the invention, the
microtube can be filled with a liquid.
[0097] To enable a flow of a liquid-of-interest within the
microtube, i.e., along the coat polymer covering the internal
surface of the shell (which originates from the second polymer
solution), the surface (thin film) formed by the coat polymer
should be designed such that it can be wetted by the
liquid-of-interest. The ability to-wet (wettability) polymer films
by liquids is known in the art. For example, silicone oil or water
can wet a surface made of a PEO polymer. The wettability of the
coat polymer covering the internal surface of the shell can be
controlled (e.g., improved) for example by attaching (e.g., using
plasma treatment) functional groups such as hydroxyl groups (OH)
which increase the hydrophilicity of the coat [see Thurston R M,
Clay J D, Schulte M D, Effect of atmospheric plasma treatment on
polymer surface energy and adhesion, Journal of Plastic Film &
Sheeting 23 (1): 63-78 Jan. 2007; which is incorporated within by
reference].
[0098] For certain applications the microtube shell may comprise
pores, thus creating a "breathing" tube. Methods of forming
"breathing" microtube (i.e., microtubes with pores in the shell
thereof) are described in PCT/IB2007/054001 to Zussman E., et al.,
which is fully incorporated herein by reference. Briefly,
"breathing" tubes can be formed by the inclusion of a high percent
(e.g., at least 80%) of a volatile component such as
tetrahydrofuran (THF), chloroform, acetone, or trifluoroethanol
(TFE) in the first polymeric solution forming the shell, and/or by
the inclusion of a water-soluble polymer such as polyethylene
glycol (PEG) in the first polymeric solution forming the shell so
that the first polymeric solution comprises a blend of polymers in
which one is water-soluble and the other is water-insoluble (e.g.,
a blend of PEG and PCL). Alternatively, "breathing" microtubes can
be formed by inducing pores in the shell after the completion of
the electrospinning process, essentially as described in PCT WO
2006/106506 to the present inventors, which is fully incorporated
herein by reference, such as by passing an electrical spark or a
heated puncturing element through the electrospun shell, or by
using a pulsed or continuous laser beam through the electrospun
shell.
[0099] According to some embodiments of the invention, the first
polymeric solution comprises PEG for inducing pores in the shell.
For example, to generate pores greater (>) than 150 nm in
diameter, the first polymeric solution may include about 4% PEG MW
35 kDa. Similarly, to generate pores smaller (<) 150 nm in
diameter, the first polymeric solution may include about 2% PEG MW
6 kDa.
[0100] The microtube shell can be designed for selective passage of
certain molecules or particles. The passage through the shell pores
depends on the size and/or the electrical charge of the
molecules/particles with respect to the geometry (length and
radius), surface energy, electrical charge of the shell pores, and
the viscosity and surface tension of the liquid containing the
molecules/particles.
[0101] According to some embodiments of the invention, the porosity
[i.e., the ratio of the volume of the shell pores to the volume of
the shell mass] and pore size can control the release of the cell
or the membrane-coated particle-of-interest from the microtube. For
example, a shell with pores larger than 1 .mu.m in diameter (e.g.,
about 1-2 .mu.m) can enable the release of a cell therethrough. In
addition, increased porosity can result in a greater rate of
release through the shell pores.
[0102] Alternatively, the microtube shell can be made such that it
prevents diffusion or passage of the cell, the membrane-coated
particle-of-interest or any molecule therethrough (e.g.,
substantially devoid of pores, or with pores having a diameter
which is smaller than the cell or the membrane-coated
particle-of-interest, or which exhibit a geometry which prevents
passage of cells or membrane-coated particles therethrough).
[0103] According to some embodiments of the invention, the cell or
the membrane-coated particle-of-interest is attached to the polymer
of the coat over the internal surface of the shell. For example, as
shown in FIG. 2 and described in Example 1 of the Examples section
which follows, Pseudomonas putida cells which express the DsRed
fluorescent protein were attached to the internal surface of the
shell.
[0104] According to some embodiments of the invention, the cell or
the membrane-coated particle is attached to the microtube
shell.
[0105] According to some embodiments of the invention attachment of
the cell (e.g., a eukaryotic cell such as a mammalian cell) or the
membrane-coated particle is performed following microtube
formation. For example, the microtube can be soaked with a solution
containing the cell/membrane-coated particle. The
cell/membrane-coated particle can diffuse through the shell pores
and enter the inner lumen of the microtube.
[0106] In addition, the microtube can be covalently attached to the
cell/membrane-coated particle (e.g., via SH groups).
[0107] Regardless of the method of production, the present
invention provides a microtube which comprises an electrospun
shell, an electrospun coat over an internal surface of the shell
and a cell or a membrane-coated particle-of-interest attached to
the microtube.
[0108] As used herein, the phrase "electrospun shell" refers to a
hollow element of a tubular shape, made of one or more polymers,
produced by the process of electrospinning as detailed above.
[0109] As used herein the phrase "electrospun coat" refers to a
thin layer covering the internal surface of the shell of the
microtube of the invention which is made of one or more polymers by
the process of electrospinning as detailed above.
[0110] One of ordinary skill in the art will know how to
distinguish an electrospun object from objects made by means which
do not comprise electrospinning by the high orientation of the
macromolecules, the skin (e.g., shell) morphology, and the typical
dimensions of the microtube which are unique to
electrospinning.
[0111] The microtube of the invention can form an individual (e.g.,
single or separated) microtube or can form part of a plurality
(e.g., an aligned array) of microtubes which can be either
connected to each other or separated (as single, not-connected
microtubes).
[0112] For the production of a single microtube a fork like clip is
attached to the edge of the rotating disk. The disk is rotated for
1-2 seconds and individual microtubes are formed between the sides
of the clip. In a similar way individual electrospun fibers were
collected [see E. Zussman, M. Burman, A. L. Yarin, R. Khalfin, Y.
Cohen, "Tensile Deformation of Electrospun Nylon 6,6 Nanofibers,"
Journal of Polymer Science Part B: Polymer Physics, 44, 1482-1489,
(2006), herein incorporated by reference in its entirety].
[0113] Alternatively, when using a rotating collector, a plurality
of microtubes can be formed and collected on the edge of the
collector as described elsewhere for electrospun fibers [A. Theron,
E. Zussman, A. L. Yarin, "Electrostatic field-assisted alignment of
electrospun nanofibers", Nanotechnology J., 12, 3: 384-390, (2001);
herein incorporated by reference in its entirety].
[0114] The plurality of microtubes can be arranged on a single
layer, or alternatively, the plurality of microtubes define a
plurality of layers hence form a three dimensional structure. The
microtubes can have a general random orientation, or a preferred
orientation, as desired. For example, when the fibers are collected
on a cylindrical collector such as a drum, the microtubes can be
aligned predominantly axially or predominantly circumferentially.
Different layers of the electrospun microtubes can have different
orientation characteristics. For example, without limiting the
scope of the present invention to any specific ordering or number
of layers, the microtubes of a first layer can have a first
predominant orientation, the microtubes of a second layer can have
a second predominant orientation, and the microtubes of third layer
can have general random orientation.
[0115] The microtube of the invention can be available as a dry
fibrous mat(s) (e.g., as spun dry microtubes) or as a wetted mat(s)
(e.g., following immersing or filling the microtube with a
liquid).
[0116] According to some embodiments of the invention, the
microtube which is attached to the cell or the membrane-coated
particle-of-interest is configured as or in a microfluidics device.
"Lab-on-a-chip" is described in a series of review articles [see
for example, Craighead, H. "Future lab-on-a-chip technologies for
interrogating individual molecules". Nature 442, 387-393 (2006);
deMello, A. J. "Control and detection of chemical reactions in
microfluidic systems". Nature 442, 394-402 (2006); El-Ali, J.,
Sorger, P. K. & Jensen, K. F. "Cells on chips". Nature 442,
403-411 (2006); Janasek, D., Franzke, J. & Manz, A. "Scaling
and the design of miniaturized chemical-analysis systems". Nature
442, 374-380 (2006); Psaltis, D., Quake, S. R. & Yang, C. H.
"Developing optofluidic technology through the fusion of
microfluidics and optics". Nature 442, 381-386 (2006); Whitesides,
G. M. "The origins and the future of microfluidics". Nature 442,
368-373 (2006); Yager, P. et al. "Microfluidic diagnostic
technologies for global public health". Nature 442, 412-418 (2006)]
each of which is fully incorporated herein by reference].
[0117] According to some embodiments of the invention, the liquid
which fills in, flows in or surrounds the microtube enables the
desorption (detachment) of the cell or the membrane-coated particle
from the microtube (e.g., from the polymer included in the coat
over the internal surface of the shell). According to of some
embodiments of the invention the desorption process facilitates the
interaction between the entrapped or encapsulated
cell/membrane-coated particle with a molecule-of-interest (e.g., a
substrate of an enzyme contained within the cell/membrane coated
particle). According to some embodiments of the invention, the
desorption process enables the flow and/or the release of the
cell/membrane-coated particle within and from the microtube.
[0118] The cell or the membrane-coated particle-of-interest which
is attached, entrapped or encapsulated within the microtube of the
invention can be either an intact cell (i.e., having an un-ruptured
membrane/cell wall) or non-intact cell (i.e., with a ruptured
membrane/cell wall).
[0119] According to some embodiments of the invention, the cell or
the membrane-coated particle-of-interest which is attached to,
entrapped or encapsulated within the microtube of the invention
maintains the activity, or at least a portion thereof, which it
possessed prior to the attachment (e.g., of the same cell or the
membrane-coated particle-of-interest prior to the electrospinning
process, or when unattached to the microtube). For example, a
bacterial cell with a ruptured cell wall/membrane may still contain
the enzymatic activity of its proteins.
[0120] The term "activity" as used herein refers to any of a
catalytic activity, kinetics, and/or affinity to a substrate or a
ligand which the cell or the membrane-coated particle may have.
Such an activity can be any biological activity such as catalysis,
binding (with a specific affinity), hybridization, chelation,
degradation, synthesis, catabolism, hydrolysis, polymerization,
transcription, drug activity and the like.
[0121] As used herein the phrase "at least a portion of the
activity" refers to at least about 10%, at least about 20-50%,
e.g., more than about 50%, e.g., more than about 60%, e.g., more
than about 70%, e.g., more than about 75%, e.g., more than about
80%, e.g., more than about 90%, e.g., more than about 95% of the
activity which the cell or the membrane-coated particle possessed
prior to the attachment to the microtube.
[0122] For example, as mentioned before and described in the
Examples section which follows, the bacterial cells entrapped
within the microtube of some embodiments of the invention preserved
the specific activity to their substrates (atrazine).
[0123] The microtube of the invention with the attached, entrapped
or encapsulated active cell or the membrane-coated
particle-of-interest can be used in various applications which
require the attachment of active cells (including portions thereof)
or membrane-coated particles to a support and optionally also the
controlled release therefrom.
[0124] According to some embodiments of the invention, the
microtube of the invention is attached to more than one type of
cells/membrane-coated particles. The microtube can be attached to a
mixture of cells from several species or from a single species. The
combination of cells can be selected according to the intended use.
For example, several cells which are involved in complex reactions
(e.g., processing of a substrate or a mixture of substrates) can be
used.
[0125] According to some embodiments of the invention the microtube
can be used as a micro-reactor (e.g., bioreactor) for chemical
transition reactions [e.g., a multi-step reaction (cascade)]
requiring high concentrations of several enzymes, e.g., enzymes
produced by a certain cell (e.g., a bacterial cell) or by several
cells or several different species (e.g., several types of
bacterial cells).
[0126] According to an aspect of the invention, there is provided a
method of processing a substrate-of-interest. The method is
effected by contacting the substrate-of-interest with the microtube
of the invention, wherein the cell or the membrane-coated particle
which is attached to, entrapped or encapsulated within the
microtube is capable of processing the substrate, thereby
processing the substrate-of-interest.
[0127] As used herein the term "processing" refers to a catalytic
activity performed by the cell or the membrane-coated particle
which is attached to, entrapped or encapsulated within the
microtube on its cognate substrate.
[0128] According to some embodiments of the invention processing
involves enzymatic-dependent conversion (catalysis) of a substrate
from a given chemical form to a distinct one. Examples of such
catalysis reactions include, but are not limited to degradation,
digestion, hydrolysis, nucleic acid cleavage, nucleic acid
ligation, proteolytic cleavage, polymerization, transfer of an atom
or functional group from one molecule to another and addition of a
chemical group to a molecule.
[0129] According to some embodiments of the invention, such a
process simply incorporates (endocytose) the substrate-of-interest
such as for use in a reaction (synthesis) catalyzed by the cell or
the membrane-coated particle.
[0130] According to some embodiments of the invention, the
microtube can be used for the synthesis of rare biochemicals such
as intermediates in biosynthesis pathways which are normally
present at very low intracellular concentrations. For example, for
the synthesis of indole glycerol phosphate the microtube according
to some embodiments of the invention may be attached to a cell
which expresses the enzymes participating in indole glycerol
phosphate synthesis. For example, to accumulate indole glycerol
phosphate the cell can be a mutant cell lacking the enzyme (or
having a non-functional enzyme) converting indole glycerol
phosphate to indole.
[0131] According to some embodiments of the invention, such a
process can be the incorporation of the substrate-of-interest in a
catabolism reaction catalyzed by the cell or membrane-coated
particle which is attached, entrapped or encapsulated within the
microtube.
[0132] For example, the catabolism reaction can be the degradation
(e.g., by hydrolysis) of a toxic molecule for the purpose of
detoxification (e.g., detoxifying water) or decomposition of an
unwanted molecule.
[0133] According to an aspect of the invention there is provided a
method of depleting a molecule from a composition containing the
molecule, the method is effected by contacting the composition with
the microtube of the invention, wherein the molecule is capable of
binding to or being processed by the cell or the membrane-coated
particle-of-interest, thereby depleting the molecule from the
composition.
[0134] The composition containing the molecule may be in a liquid
form (e.g., a solution), a solid form (e.g., soil) or a gel form.
The molecule can be mixed with the composition or bound to the
composition (by covalent or non-covalent bindings).
[0135] According to some embodiments of the invention, the method
further comprising collecting the composition following the
contacting.
[0136] As used herein the term "depleting" refers to removing an
amount e.g., at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, at least about 95%,
e.g., 99%, e.g., 100% of the molecule from the composition.
[0137] According to some embodiments of the invention, the
depletion (removal) of the molecule from the composition is
effected within a short time period, such as within minutes, hours
or several days (e.g., about 1-7 days)
[0138] As used herein the phrase "contacting" refers to enabling
the interaction between the molecule and the cell or the
membrane-coated particle-of-interest which is attached to,
entrapped or encapsulated within the microtube, for a time period
which is sufficient for depleting the molecule from the composition
(e.g., solution, soil). Such a contact can take place, for example,
while the composition (e.g., solution) is passing through (e.g.,
via capillary forces) the end(s) of the hollow structure of the
microtube and/or through the shell pores. Additionally or
alternatively, such a contact between the molecule and the cell or
the membrane-coated particle-of-interest can take place by
incubating the microtube in the composition (e.g., by placing the
microtube in a container including a solution, a soil or a
gel)).
[0139] The solution can be a water-based or an oil-based solution
which includes inorganic or organic molecules, such as a biological
sample or a sample from a non-living source such as stream,
industrial waste or ocean waters. As used herein the phrase
"biological sample" refers to any sample derived from a living
organism such as plant, bacteria or animals, and can include cells
or alternatively be cell-free (i.e., include only a biological
fluid).
[0140] According to some embodiments of the invention, the solution
is an aqueous solution such as drinking water, groundwater and/or
industrial waste water. According to some embodiments of the
invention, the microtube of the invention forms part of an aqueous
system designed for treatment of the aqueous solution (e.g., for
depleting, eliminating or removing toxic moieties therefrom).
[0141] According to some embodiments of the invention, depleting is
effected via bioremediation (i.e., the process of biodegradation
and/or assimilation of a contaminant by microorganisms, fungi,
green plants or their enzymes). Bioremediation can be used to
remove contaminants such as soil contaminants, chlorinated
hydrocarbons, crude oil herbicides, heavy metals, and the like.
[0142] According to some embodiments of the invention, depleting is
effected via biodegradation.
[0143] As used herein the term "biodegradation" refers to
degradation of a substrate using bio-molecules (e.g., enzymes)
which are expressed by (or contained within) the cell or the
membrane-coated particle which is attached to the microtube of the
invention.
[0144] For example, to remove petroleum pollutants [e.g.,
aliphatics (e.g., C5-C36) and aromatics (e.g., C9-C22) such as
benzene, toluene, ethylbenzene and xylenes (BTEX), phenol,
naphthalene or certain hydrocarbons from oil] from water
(biodegradation of petroleum pollutants), the microtube of the
invention may be attached to bacteria utilizing alkanes as a sole
source of carbon and energy or to membrane-coated particles
including alkane-degrading enzymes. Such alkanes can be, for
example, methane, ethane, propane, butane and mixtures
thereof).
[0145] Non-limiting examples of butane-utilizing bacteria include
Gram negative and Gram positive aerobic rods and cocci, facultative
anaerobic Gram negative rods, non-photosynthetic, non-fruiting
gliding bacteria and irregular non-sporing Gram positive rods. Of
the Pseudomonadaceae family comprising Gram-negative aerobic rods
and cocci, species of the following genera may be suitable:
Pseudomonas; Variovorax; Chryseobacterium; Comamonas; Acidovorax;
Stenotrophomonas; Sphingobacterium; Xanthomonas; Frateuria;
Zoogloea; Alcaligenes; Flavobacterium; Derxia; Lampropedia;
Brucella; Xanthobacter; Thermus; Thermomicrobium; Halomonas;
Alteromonas; Serpens; Janthinobacterium; Bordetella; Paracoccus;
Beijerinckia; and Francisella. Of the Nocardioform Actinomycetes
family comprising Gram-positive Eubacteria and Actinomycetes, the
following genera may be suitable: Nocardia; Rhodococcus; Gordona;
Nocardioides; Saccharopolyspora; Micropolyspora; Promicromonospora;
Intrasporangium; Pseudonocardia; and Oerskovia. Of the
Micrococcaceae family comprising Gram-positive cocci, the following
genera may be suitable: Micrococcus; Stomatococcus; Planococcus;
Staphylococcus; Aerococcus; Peptococcus; Peptostreptococcus;
Coprococcus; Gemella; Pediococcus; Leuconostoc; Ruminococcus;
Sarcina; and Streptococcus. Of the Vibrionaceae family comprising
facultative anaerobic Gram-negative rods, the following genera may
be suitable: Aeromonas; Photobacterium; Vibrio; Plesiomonas;
Zymomonas; Chromobacterium; Cardiobacterium; Calymmatobacterium;
Streptobacillus; Eikenella; and Gardnerella. Of the Rhizobiaceae
family comprising Gram-negative aerobic rods and cocci, the
following genera may be suitable: Phyllobacterium; Rhizobium;
Bradyrhizobium; and Agrobacterium. Of the Cytophagaceae family
comprising non-photosynthetic, non-fruiting, gliding bacteria, the
following genera may be suitable: Cytophaga; Flexibacter;
Saprospira; Flexithrix; Herpetosiphon; Capnocytophaga; and
Sporocytophaga. Of the Corynebacterium family comprising irregular,
non-sporing Gram-positive rods, the following genera may be
suitable: Aureobacterium; Agromyces; Arachnia; Rothia;
Acetobacterium; Actinomyces; Arthrobactera; Arcanobacterium;
Lachnospira; Propionibacterium; Eubacterium; Butyrivibria;
Brevibacterium; Bifidobacterium; Microbacterium; Caseobacter; and
Thernoanaerobacter.
[0146] Non-limiting examples of methane-utilizing bacteria include
Methylomonas (e.g., Methylomonas albus such as the BG 8 strain;
Methylomonas methanica such as the PM strain), Methylobacter (e.g.,
Methylobacterium organophilum), Methylococcus [e.g., Methylococcus
capsulatus, such as the Texas strain ATCC 19069 and the Bath strain
National Collection of Industrial Bacteria (NCIB) 11132],
Methylocystis (Methylocystis parvus), and Methylosinus (e.g.,
Methylosinus trichosporium such as the OB 3b strain, e.g., NCIB No.
11131 and The Fermentation Research Institute (FRI), Japan (as
FERM-P4981)].
[0147] For example, for the biodegradation of cyanide [depletion of
cyanide from a solution such as industrial waste water (e.g., of
silver mining)], bacterial cells or membrane-coated particles
capable of degrading free (CN.sup.- or HCN) or complexed (e.g.,
metal-cyanide complex) cyanide can be attached to the microtube of
the invention. For example, degradation of free cyanide can be
performed using the archeae bacterium Sulfolobus which is also used
in gold extraction from low grade minerals [see Knowles, C. J. and
Bunch, A. W., "Microbial cyanide metabolism. Advances in Microbial
Physiology", (1986); 27: 73 111]; Complexed cyanide such as
silver-cyanide [Ag(CN).sub.2] can be degraded using the Citrobacter
sp. MCM B-181, Pseudomonas sp. MCM B-182, Pseudomonas sp. MCM B-183
and Pseudomonas sp. MCM B-184 bacteria [see Patil Y B, Paknikar K
M, Letters in Applied Microbiology, (2000), 30: 33-37] which
utilize metal-cyanide complexes as a nitrogen source and release
ammonia and carbon dioxide as degradation products.
[0148] To remove toxic moieties of herbicides that enters the water
supply, such as the chlorine entity of atrazine (biodegradation of
atrazine), a microtube which includes cells or membrane-coated
particles (e.g., portions of cells, liposomes) capable of degrading
atrazine can be used. For example, bacterial cells or portions of
cells including the atrazine degrading enzymes: atrazine
chlorohydrolase, hydroxyatrazine hydrolase, N-isopropylammelide
isopropylamino hydrolase, cyanuric acid amidohydrolase, biuret
hydrolase, and allophanate hydrolase (FIG. 5) can be used. Such
bacteria can be the Pseudomonas ADP (which endogenously express the
ATZ genes) or any other bacterial strain which exogenously express
the ATZ genes (atzA-atzF genes), such as the Pseudomonas putida S12
described in the Examples section which follows.
[0149] For example, as described in Tables 4 and 5 and Example 2 of
the Examples section which follows, efficient atrazine degradation
was achieved by microtubes which were attached to the Pseudomonas
ADP or Pseudomonas putida S12 that express the atzA-atzF genes.
[0150] According to some embodiments of the invention,
biodegradation of atrazine is effected such that more than about
50%, e.g., more than about 60%, e.g., more than about 70%, e.g.,
more than about 80% of atrazine is removed from a solution
containing about 20 mg atrazine per liter following 1-4 days of
contacting the solution with the microtube. According to exemplary
embodiments of the invention, biodegradation of atrazine is
effected such that more than about 90%, more than about 95%, e.g.,
100% of atrazine is removed from a solution containing about 20 mg
atrazine per liter following 1-2 days of contacting the solution
with the microtube.
[0151] As mentioned hereinabove, the microtube of the invention may
form part of an aqueous system designed for treatment of the
aqueous solution.
[0152] FIG. 4 schematically illustrates a single configuration of a
bioremediation system generated according to some embodiments of
the invention. System 300 comprises conduit [220, e.g., part of an
aqueous system, can be a plastic or metal (e.g., copper) pipe/tube]
having borders (100) includes microtube (230) with shell borders
(120) and entrapped bacterial cells (210). For purification (e.g.,
detoxification of water), a liquid (e.g., drinking water)
containing molecule (150) flows within the conduit. Pores (180)
within the microtube shell enable the diffusion of molecule (150)
through the microtube shell to the microtube lumen (140) containing
bacterial cells (210). Cells (210) interact with molecule (150) and
reaction product (160) diffuses out of the microtube outside of the
microtube lumen (140) through the microtube shell pores (180).
[0153] According to some embodiments of the invention, a microtube
with entrapped bacterial cells expressing the atrazine degrading
enzymes is placed (or packed within) a column [e.g., conduit (220)
as shown in FIG. 4] filled with an aqueous solution (e.g., drinking
water). The small diameter of the microtube (e.g., 3-5 .mu.m) and
the significant length (e.g., 20 cm) provides an enormous surface
area for degrading atrazine. As mentioned above, due to the
dimensions of the microtube (wherein the length is several orders
of magnitude larger than the diameter), the cells are entrapped
within the microtube and are not washed away. Accordingly, there is
no need to add a carbon source to the bacterial cells.
[0154] According to some embodiments of the invention, the cells
entrapped within the microtube can continue to degrade atrazine for
an extended time period without the formation of a biofilm (i.e., a
complex aggregation of microorganisms marked by the excretion of a
matrix). According to some embodiments of the invention, the
microtube shell prevents passage of predators such as
Bdellovibrios, protozoa, and bacteriophage thus protecting the
entrapped bacterial cells from such predators.
[0155] According to some embodiments of the invention, the
bacterial cells may be refreshed by immersing the microtube in
growth medium which contains usable carbon, nitrogen, phosphorus
and sulfur sources and thereby both allowing the cells to
proliferate and/or renew their metabolic potential.
[0156] According an aspect of the invention, there is provided a
method of isolating a molecule from a solution. The method is
effected by (a) contacting the solution with the microtube of the
invention under conditions which allow binding of the molecule to
the cell or the membrane-coated particle-of-interest, and; (b)
eluting the molecule from the microtube; thereby isolating the
molecule from the solution.
[0157] As used herein the term "isolating" refers to physically
separating the molecule from the solution or its other components
by binding the molecule to the cell or membrane-coated particle
which is attached to, entrapped or encapsulated within the
microtube and eluting the bound molecule therefrom. As used herein
the term "eluting" refers to dissociating the bound molecule from
the microtube. Those skilled in the art are capable of adjusting
the conditions required for eluting (e.g., releasing) the molecule
from the microtube and/or separating the molecule from the cell or
membrane-coated particle.
[0158] According to an aspect of the invention, there is provided a
method of detecting a presence of a molecule in a sample. The
method is effected by (a) contacting the sample with the microtube
of the invention, wherein the cell or the membrane-coated
particle-of-interest is capable of binding to or processing the
molecule, and (b) detecting the binding or the processing; thereby
detecting the presence of the molecule in the sample.
[0159] As used herein the phrase "detecting binding or the
processing" refers to identifying a change in the concentration,
conformation, spectrum or electrical charge of the molecule in the
sample and/or of the cell or membrane-coated particle that is
attached to the microtube following the binding therebetween or
following the processing of the molecule by the cell or the
membrane-coated particle. Identification of the binding or
processing can be performed using methods known in the art such as
following the fluorescence or the color of the sample,
radioactivity of the sample, the electrical conductivity of the
sample and the like. Binding of a molecule to a cell may be via a
specific receptor on the cell.
[0160] According to some embodiments of the invention, the cell or
the membrane-coated particle-of-interest which is attached to the
microtube is labeled or comprises a label [e.g., by genetic
modification as described above, or by conjugation to a dye,
fluorophore, radio-isotope, or an enzyme (e.g., horse radish
peroxidase) capable of producing a colorimetric product], and
detecting the binding or processing of a molecule is performed by
following such a label.
[0161] The microtube according to some embodiments of the invention
can be used as a biosensor, for the detection of molecules in a
sample. Such a biosensor can be advantageous over known open field
biosensors (e.g., sensors in which the cell or membrane-coated
particle is conjugated to a solid support not having a tubular
structure, such as a flat support) especially due to the increased
ratio between the size of the microtube surface (which attaches the
cell/membrane-coated particle) and the volume of the sample being
in contact therewith.
[0162] The microtube according to some embodiments of the invention
can be used to release the attached cell or membrane-coated
particle through pores in the shell or the microtube openings
(i.e., the ends of the tubular structure). According to some
embodiments of the invention, the microtube can be placed in a
supercritical fluid (e.g., liquid nitrogen) for a short period of
time (e.g., 2-10 minutes), following which the microtube is cut
(e.g., with a sharp knife, razor blade) close to its end. The
microtube can be also subject to cross linking using, e.g.,
glutaraldehyde which assists in preserving the openness of the
microtube.
[0163] The microtube according to some embodiments of the invention
can be attached to bacteria that produce and contain nano-magnets.
Such bacteria were discovered and isolated more than 30 years ago
(Blakemore, R 1975 Magnetotactic bacteria. Science 190:377-379;
Bayzlinski, D. A., Frankel, R. B. and Jannasch, H. W. 1989
Anaerobic magnetite production by marine, magnetotactic bacteria.
Nature 334:518-519). Some of these strains contain a string of
magnets whose poles, N and S, are aligned: Without being bound by
any theory, during the electrospinning process and the flow of the
second polymeric solution the bacteria can be oriented such that
their long axis is parallel to that of the microtube (see an
example of rod bacteria aligned within the microtube; FIG. 2). When
using bacteria that contain magnets, the bacteria can be aligned by
applying magnetic force and their tendency to be aligned by flow
such that the attached bacteria are aligned in a polar manner where
all their N's face in the same direction.).
[0164] Microtubes with attached magnet producing bacteria can be
used in industry (e.g., microelectonics) and medical applications
(such as imaging).
[0165] The microtube according to some embodiments of the invention
can be used in medical dialysis to remove materials from blood and
other bodily fluids such as urine.
[0166] The microtube of some embodiments the invention can be used
in various ex vivo and in vivo applications. For example, the
microtube can be attached to cells (e.g., mammalian) capable of
inducing tissue formation [e.g., stem cells such as adult stem
cells (tissue stem cells) or embryonic stem cells]. Briefly, the
microtube can be contacted with a medium (e.g., tissue culture
medium, physiological solution, blood) which is suitable for
proliferation, differentiation and/or migration of the cell so as
to enable tissue formation.
[0167] The microtube of some embodiments of the invention (e.g., a
microtube made of biocompatible polymers) can be implanted in a
subject in need thereof.
[0168] As used herein the phrase a "subject in need thereof" refers
to any animal subject e.g., a mammal, e.g., a human being which
suffers from a pathology (disease, disorder or condition) which can
be treated by the cell/membrane coated particle which is attached,
entrapped or encapsulated within the microtube of the
invention.
[0169] The term "treating" as used herein refers to inhibiting,
preventing or arresting the development of a pathology and/or
causing the reduction, remission, or regression of a pathology.
Those of skill in the art will understand that various
methodologies and assays can be used to assess the development of a
pathology, and similarly, various methodologies and assays may be
used to assess the reduction, remission or regression of a
pathology.
[0170] According to some embodiments of the invention, the
microtube is implanted in a subject to induce in vivo formation of
a tissue.
[0171] According to some embodiments of the invention, the
microtube directs the attached (or entrapped) cell or
membrane-coated particle-of-interest (e.g., a drug in a liposome)
to a target tissue of a subject (e.g., targeted delivery of a
drug).
[0172] Methods of implanting grafts such as the microtube of the
invention into a subject are known in the art. For example, the
microtube can be implanted subcutaneously, intradermally, or into
any body cavity (e.g., abdomen), as well as into the vascular
system (using e.g., a hollow catheter delivery system).
Alternatively, the microtube of the invention can be connected to a
body conduit (e.g., a blood vessel such as a vein or an artery)
such that it enables the flow of a fluid therethrough.
[0173] The invention further envisages the use of the microtube of
the invention, which includes a cell or a membrane-coated
particle-of-interest attached thereto, for guiding cell growth ex
vivo or in vivo. For example, neuronal cells which are attached or
entrapped within the microtube can be contacted with solution
containing growth factors and/or nutrients needed for neuronal
growth. It will be appreciated that once an initial neuronal growth
has occurred ex vivo, such a system (i.e., the microtube with the
neuronal cells) can be implanted in a subject in need thereof
(e.g., a subject with degenerated, damaged or injured neuronal
cells) to thereby enable neuronal growth and guidance.
[0174] The microtube of some embodiments of the invention can be
included in a kit/article of manufacture along with a packaging
material and/or instructions for use in any of the above described
methods or applications.
[0175] The methods described herein may be conducted batchwise.
[0176] It will be appreciated that the microtubes of the some
embodiments of the present invention can find wide use in waste,
commodity, food, agrotec, cosmetic and pharma industries. A
detailed discussion of some embodiments is not meant to be
limiting.
[0177] As used herein the term "about" refers to .+-.10%.
[0178] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to".
[0179] The term "consisting of means "including and limited
to".
[0180] The term "consisting essentially of" means that the
composition, method or structure may include additional
ingredients, steps and/or parts, but only if the additional
ingredients, steps and/or parts do not materially alter the basic
and novel characteristics of the claimed composition, method or
structure.
[0181] As used herein, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a compound" or "at least one
compound" may include a plurality of compounds, including mixtures
thereof.
[0182] Throughout this application, various embodiments of this
invention may be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible sub-ranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed sub ranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5, and 6. This applies regardless of the breadth of the
range.
[0183] Whenever a numerical range is indicated herein, it is meant
to include any cited numeral (fractional or integral) within the
indicated range. The phrases "ranging/ranges between" a first
indicate number and a second indicate number and "ranging/ranges
from" a first indicate number "to" a second indicate number are
used herein interchangeably and are meant to include the first and
second indicated numbers and all the fractional and integral
numerals therebetween.
[0184] As used herein the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including,
but not limited to, those manners, means, techniques and procedures
either known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0185] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental support in the following examples.
EXAMPLES
[0186] Reference is now made to the following examples, which
together with the above descriptions illustrate some embodiments of
the invention in a non limiting fashion.
[0187] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan
J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical
Immunology" (8th Edition), Appleton & Lange, Norwalk, Conn.
(1994); Mishell and Shiigi (eds), "Selected Methods in Cellular
Immunology", W. H. Freeman and Co., New York (1980); available
immunoassays are extensively described in the patent and scientific
literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;
3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;
3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;
5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J.,
ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins
S. J., eds. (1985); "Transcription and Translation" Hames, B. D.,
and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R.
I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986);
"A Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
General Materials and Experimental Methods
[0188] Eletrospinning Solutions:
[0189] The compositions of the shell and core solutions are given
in Table 3, hereinbelow. All polymers and solvents were purchased
from Sigma-Aldrich and were used as is.
[0190] Bacterial Cells and Growth Medium--
[0191] Pseudomonas putida S12 (exogenously expressing atz genes by
transfection with a plasmid including the coding sequences of the
atz genes depicted in FIG. 5) and Pseudomonas ADP (which
endogenously express the atz genes), both of which are capable of
degrading atrazine and using it as a source of nitrogen, were grown
to stationary phase in an ATZ culture medium (modified from
Mandelbaum R. T., Wackett L. P., Allan D. L., 1993. "Mineralization
of the s-triazine ring of atrazine by stable bacterial mixed
cultures". Applied and Environmental Microbiology 59: 1695-1701) in
which atrazine was included as the sole source of nitrogen. The ATZ
culture medium was prepared as follows: For each liter of double
distilled water the following were added: KH.sub.2PO.sub.4 1.6
grams, K.sub.2HPO.sub.4 0.4 grams, MgSO.sub.4.7H.sub.2O 0.2 grams,
NaCl 1 gram, CaCl.sub.2 25 mg and citrate (sodium-citrate) 2 g; 20
mg atrazine; 20 ml of a vitamin and trace element stock solution.
The vitamin and trace element stock solution contains per liter:
Nitrilotriacetic acid 10 grams, KOH 7.3 grams, MgSO.sub.4.7H.sub.2O
14.45 grams, (NH.sub.4).sub.6Mo.sub.7O.sub.24.times.4H.sub.2O 9.25
grams, FeSO.sub.4.times.7H.sub.2O 100 mg, nicotinic acid 50 mg,
biotin 0.5 mg, thiamin HCl 2.5 mg, and 44 metal solution 50 ml. 44
metal solution contains per 100 ml: EDTA 250 mg, ZnSO.sub.4
7H.sub.2O 1.1 grams, FeSO.sub.4 7H.sub.2O 0.5 grams,
MnSO.sub.4H.sub.2O 154 mg, CuSO.sub.4 5H.sub.2O 50 mg,
Co(NO.sub.3).sub.2 6H.sub.2O 75 mg, Na.sub.2B.sub.4O.sub.7
10H.sub.2O 20 mg.
[0192] The bacterial cells (10.sup.9 cells/ml) grown in the above
medium were added to the core solution (e.g., PEO in water or
PEO+40% ethanol) (e.g., at a ratio of 9:1 (volume/volume) core
solution to cells) and electrospining was performed using the shell
and core solutions shown in Table 3, hereinbelow, so as to form a
microtube in which the bacterial cells are attached to the internal
surface (coat) of the shell. The presence and localization of the
bacterial cells following formation of the microtube was determined
using a fluorescence microscope.
TABLE-US-00003 TABLE 3 Two types of core-shell microtubes:
composition of the solutions Type Shell solution Core solution 1
10% PCL 80 K; in 4% (w/w) PEO 600 K in ethanol:H.sub.2O
CHCl.sub.3:DMF (90:10 by (26:74 by weight) and bacterial cells
(10.sup.9 cells/ml) weight) in a 10:1 ratio (900 .mu.l of polymer
solution and 100 .mu.l of bacterial cell) 1 9% PCL 80 K; in 8%
(w/w) PEO 600 K in water + bacterial CHCl.sub.3:DMSO (90:10 by
cells (10.sup.9 cells/ml) in a 10:1 ratio (900 .mu.l weight) of
polymer solution and 100 .mu.l of bacterial cell) 2 10% PCL 80 K +
1% PEG 4% (w/w) PEO 600 K in ethanol:H.sub.2O 6 K; in
CHCl.sub.3:DMF (90:10 (26:74 by weight) + bacterial cells (10.sup.9
cells/ml) by weight) in a 10:1 ratio (900 .mu.l of polymer solution
and 100 .mu.l of bacterial cell) Table 3. Microtubes were formed by
co-electrospinning of the shell solution (a first polymeric
solution for forming the shell) and a core solution (a second
polymeric solution for forming the coat over the internal surface
of the shell). Type 1 microtubes - do not include PEG in the shell
solution; Type 2 microtubes - include PEG in the shell
solution.
[0193] Electrospinning--
[0194] Hollow microtubes (core-shell hollow fibers) were fabricated
by a co-electrospinning process using the set up described by Sun
et al. (2003) and Zussman et al. (2006) with the polymeric
solutions (for forming the shell and coat over the internal surface
of the shell) as described in Table 3 above. All experiments were
conducted at room temperature (about 22.degree. C.) and a relative
humidity of about 35%. The spinning parameters were as follow: the
electrostatic field used was approximately 0.44 kV/cm and the
distance between the spinneret and collector plate was 16 cm. The
flow rates of both the core and shell solutions were controlled by
two syringe pumps and were 3.5 ml/hour for the shell solution and 1
ml/hour for the core solution. The fibers were collected as a strip
on the edge of a vertical rotating wheel (Theron A., et al.,
(2001)) having a velocity of 1.2 m/second. For fluorescence
microscopy, a few fibers were collected directly onto a microscope
slide.
[0195] Imaging--
[0196] Images of the fibers were obtained using a Leo Gemini high
resolution scanning electron microscope (HRSEM) at an acceleration
voltage of 3 kV and a sample to detector distance of 3-5 mm. The
specimens were coated with a thin gold film to increase their
conductivity. Fluorescence microscope Leica DM IRE2 at excitation
and emission wave lengths of 359 and 361 nm, respectively, was used
for the imaging of fibers filled with fluorescent product.
[0197] Measurement of Atrazine Degradation--
[0198] The mat containing the entrapped bacterial cells was
immersed in a medium [ATZ-containing phosphate buffer (20 mg
Atrazine per liter of phosphate buffer)] and following
predetermined time periods (e.g., 1 to 6 days) aliquots from the
medium surrounding the mat(s) were taken and the amount of atrazine
was measured using HPLC. The aliquots were mixed with an equal
volume of methanol and were centrifuged for 10 minutes at
10,000.times.g to remove salts and other insoluble materials.
High-performance liquid chromatography (HPLC) was conducted on
Hewlett Packard HPLC (HP110 series). Separation was done at a flow
rate of 0.6 ml/minute on a LiChrospher 100 RP-18 (5 .mu.m) column
(LiChroCART HPLC Cartridge system 250-4; Merck, Darmstadt,
Germany), using 200 mM NH.sub.4CH.sub.3COO in 70:30% [volume/volume
(v/v)] methanol/water as a mobile phase. Concentration of atrazine
was quantified relative to authentic standard by integrating peak
area at 220 nm.
Example 1
Entrapment of Bacterial Cells within Microtubes
[0199] Experimental Results
[0200] Attachment of Bacterial Cells within an Electrospun
Microtube--
[0201] A microtube with cells entrapped therein was formed by
co-electrospinning of the two polymeric solutions: 9%
[weight/weight (w/w)] polycaprolactome (PCL) dissolved in
chloroform/DMSO (9:1; w/w) as a first polymeric solution (for
forming the shell); and 8% poly(ethylene oxide) (PEO) in water as
second polymeric solution (for forming the coat over the internal
surface of the shell) which also included Pseudomonas putida or
Pseudomonas ADP bacterial cells (at a concentration of 10.sup.9
cells/ml) at a ratio of 9:1 [volume/volume (v/v); 900 .mu.l
polymeric solution and 100 .mu.l of bacterial cells]. The
Pseudomonas putida S12 cells had been previously transformed to
express the DsRed fluorescent protein (GenBank Accession No.
Q9U6Y8, SEQ ID NO:7), such that following microtube formation, the
red fluorescence originating from the Pseudomonas putida cells can
be observed using a fluorescence microscope. As is shown in FIG. 2,
the Pseudomonas putida cells were entrapped within the electrospun
microtube. In addition, the observed red fluorescence demonstrates
that the cells were intact.
Example 2
A Triazine Degradation Using Bacterial Cells Entrapped within the
Microtube
[0202] Experimental Results
[0203] Degradation of Atrazine Using Bacterial Cells which are
Entrapped within the Microtube of the Invention--
[0204] Pseudomonas putida S12 or Pseudomonas ADP, both of which are
capable of degrading atrazine and using it as a source of nitrogen,
were grown to stationary phase in a medium in which atrazine was
included as the sole source of nitrogen. The cells were added to
the core solution [e.g., 4% PEO in 40% ethanol (w/w); or 8% PEO in
water (w/w)] and electrospun with the shell-forming solution
(without the addition of PEG to the shell solution) to render
microtubes with a core-shell structure. The microtubes (2 types for
each strain) were tested for their ability to degrade atrazine. The
microtubes were immersed in a flask containing phosphate buffer
with atrazine (20 mg atrazine in 1 liter of phosphate buffer) and
degradation was followed by examining the amount of residual
atrazine using HPLC (conducted on Hewlett Packard HPLC, HP110
series), in aliquots taken at predetermined time periods (from one
day to several days). The microtubes were then transferred to a new
flask and the procedure repeated.
[0205] The percentages of atrazine removal following incubation of
the Pseudomonas ADP cells--containing microtubes in ATZ-containing
phosphate buffer are summarized in Table 4, hereinbelow.
TABLE-US-00004 TABLE 4 Degradation of atrazine by electrospun
microtubes containing Pseudomonas ADP % Atrazine % Atrazine Days
from Incubation time in removal using removal using beginning ATZ
culture microtubes with microtubes with of medium (days) solution A
as solution B as experiment Transfer No. before transfer core core
0 1 1 59.0 32.3 1 2 2 51.8 20.8 3 3 3 87.9 94.0 6 4 1 76.5 91.5 7 5
2 91.2 94.3 14 6 6 85.0 85.0 15 7 1 96.0 96.0 16 8 1 94.2 94.5 17 9
3 100.0 100.0 20 10 1 100.0 100.0 21 11 2 97.5 97.5 Table 4: The
degradation of atrazine by electrospun microtubes containing
Pseudomonas ADP bacterial cells. Shown are the percentages of
atrazine removal following predetermined incubation periods of
microtubes with entrapped Pseudomonas ADP bacterial cells in a
phosphate buffer which contains ATZ (20 mg atrazine per liter). The
core solutions used for forming the microtube coat over the
internal surface of the shell were: solution A - 4% PEO in 40%
ethanol; and solution B - 8% PEO in water.
[0206] As shown in Table 4, hereinabove, when a microtube which was
prepared with a core solution containing 4% PEO in 40% ethanol was
used, efficient removal of atrazine (e.g., about 95% removal) was
observed throughout the experiment and the entrapped bacterial
cells within the microtubes continued to remove atrazine even after
11 transfers. When a microtube which was prepared with a core
solution containing 8% PEO in water was used, efficient removal of
atrazine (e.g., about 95% removal) was observed throughout the time
of the experiment and the microtube preparation (with entrapped
cells) remained competent in this regard even after 21 days and 11
transfers.
[0207] Microtubes with attached cells were incubated ATZ growth
medium (for 4 days, 30.degree. C.), and then the microtubes were
transferred to ATZ-containing phosphate buffer for the beginning of
experiment.
[0208] The percentages of atrazine removal following incubation of
the Pseudomonas putida S12 bacterial cells--containing microtubes
in an ATZ-containing phosphate buffer are summarized in Table 5,
hereinbelow.
TABLE-US-00005 TABLE 5 Degradation of atrazine by electrospun
microtubes containing Pseudomonas putida S12 Incubation % Atrazine
time in ATZ removal using culture microtubes Days from medium with
5% PEO beginning of (days) before in water as a experiment Transfer
No. transfer core solution 0 1 4 100 4 2 3 100 7 3 2 100 9 4 2 100
11 5 3 100 16 6 2 98.3 21 7 5 100 Table 5: The degradation of
atrazine by electrospun microtubes containing Pseudomonas putida
S12 bacterial cells. Shown are the percentages of atrazine removal
following predetermined incubation periods of microtubes with
entrapped S12 bacterial cells in a phosphate buffer which contains
ATZ (20 mg atrazine per liter). The core solution used for forming
the microtube coat over the internal surface of the shell was 5%
PEO in water.
[0209] As shown in Table 5, hereinabove, 100% removal of atrazine
from the solution was observed following 4 days incubation of the
S12-containing microtubes of the invention in the ATZ-containing
phosphate buffer. Efficient removal of atrazine continued through
seven transfers and remained complete in microtube material to the
21.sup.st day.
[0210] This is a new and efficient way to remove atrazine from
water in e.g., a continuous flow system composed of columns filled
with electrospun hollowfibers containing suitable bacterial cells
(e.g., which are capable of degrading atrazine). Thus, the influent
water which contains atrazine penetrates (e.g., by diffusion) into
the hollow fibers (the microtubes) and the atrazine is broken down
to non-toxic material, such that the effluent water is free of
atrazine.
[0211] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0212] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention. To the extent that section headings are used,
they should not be construed as necessarily limiting.
REFERENCES
Additional References are Cited in Text
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(2003), 15, 1929-1932.
Sequence CWU 1
1
71474PRTPseudomonas sp. 1Met Gln Thr Leu Ser Ile Gln His Gly Thr
Leu Val Thr Met Asp Gln 1 5 10 15 Tyr Arg Arg Val Leu Gly Asp Ser
Trp Val His Val Gln Asp Gly Arg 20 25 30 Ile Val Ala Leu Gly Val
His Ala Glu Ser Val Pro Pro Pro Ala Asp 35 40 45 Arg Val Ile Asp
Ala Arg Gly Lys Val Val Leu Pro Gly Phe Ile Asn 50 55 60 Ala His
Thr His Val Asn Gln Ile Leu Leu Arg Gly Gly Pro Ser His 65 70 75 80
Gly Arg Gln Phe Tyr Asp Trp Leu Phe Asn Val Val Tyr Pro Gly Gln 85
90 95 Lys Ala Met Arg Pro Glu Asp Val Ala Val Ala Val Arg Leu Tyr
Cys 100 105 110 Ala Glu Ala Val Arg Ser Gly Ile Thr Thr Ile Asn Glu
Asn Ala Asp 115 120 125 Ser Ala Ile Tyr Pro Gly Asn Ile Glu Ala Ala
Met Ala Val Tyr Gly 130 135 140 Glu Val Gly Val Arg Val Val Tyr Ala
Arg Met Phe Phe Asp Arg Met 145 150 155 160 Asp Gly Arg Ile Gln Gly
Tyr Val Asp Ala Leu Lys Ala Arg Ser Pro 165 170 175 Gln Val Glu Leu
Cys Ser Ile Met Glu Glu Thr Ala Val Ala Lys Asp 180 185 190 Arg Ile
Thr Ala Leu Ser Asp Gln Tyr His Gly Thr Ala Gly Gly Arg 195 200 205
Ile Ser Val Trp Pro Ala Pro Ala Thr Thr Thr Ala Val Thr Val Glu 210
215 220 Gly Met Arg Trp Ala Gln Ala Phe Ala Arg Asp Arg Ala Val Met
Trp 225 230 235 240 Thr Leu His Met Ala Glu Ser Asp His Asp Glu Arg
Ile His Gly Met 245 250 255 Ser Pro Ala Glu Tyr Met Glu Cys Tyr Gly
Leu Leu Asp Glu Arg Leu 260 265 270 Gln Val Ala His Cys Val Tyr Phe
Asp Arg Lys Asp Val Arg Leu Leu 275 280 285 His Arg His Asn Val Lys
Val Ala Ser Gln Val Val Ser Asn Ala Tyr 290 295 300 Leu Gly Ser Gly
Val Ala Pro Val Pro Glu Met Val Glu Arg Gly Met 305 310 315 320 Ala
Val Gly Ile Gly Thr Asp Asn Gly Asn Ser Asn Asp Ser Val Asn 325 330
335 Met Ile Gly Asp Met Lys Phe Met Ala His Ile His Arg Ala Val His
340 345 350 Arg Asp Ala Asp Val Leu Thr Pro Glu Lys Ile Leu Glu Met
Ala Thr 355 360 365 Ile Asp Gly Ala Arg Ser Leu Gly Met Asp His Glu
Ile Gly Ser Ile 370 375 380 Glu Thr Gly Lys Arg Ala Asp Leu Ile Leu
Leu Asp Leu Arg His Pro 385 390 395 400 Gln Thr Thr Pro His His His
Leu Ala Ala Thr Ile Val Phe Gln Ala 405 410 415 Tyr Gly Asn Glu Val
Asp Thr Val Leu Ile Asp Gly Asn Val Val Met 420 425 430 Glu Asn Arg
Arg Leu Ser Phe Leu Pro Pro Glu Arg Glu Leu Ala Phe 435 440 445 Leu
Glu Glu Ala Gln Ser Arg Ala Thr Ala Ile Leu Gln Arg Ala Asn 450 455
460 Met Val Ala Asn Pro Ala Trp Arg Ser Leu 465 470
2481PRTPseudomonas sp. 2Met Thr Thr Thr Leu Tyr Thr Gly Phe His Gln
Leu Val Thr Gly Asp 1 5 10 15 Val Ala Gly Thr Val Leu Asn Gly Val
Asp Ile Leu Val Arg Asp Gly 20 25 30 Glu Ile Ile Gly Leu Gly Pro
Asp Leu Pro Arg Thr Leu Ala Pro Ile 35 40 45 Gly Val Gly Gln Glu
Gln Gly Val Glu Val Val Asn Cys Arg Gly Leu 50 55 60 Thr Ala Tyr
Pro Gly Leu Ile Asn Thr His His His Phe Phe Gln Ala 65 70 75 80 Phe
Val Arg Asn Leu Ala Pro Leu Asp Trp Thr Gln Leu Asp Val Leu 85 90
95 Ala Trp Leu Arg Lys Ile Tyr Pro Val Phe Ala Leu Val Asp Glu Asp
100 105 110 Cys Ile Tyr His Ser Thr Val Val Ser Met Ala Glu Leu Ile
Lys His 115 120 125 Gly Cys Thr Thr Ala Phe Asp His Gln Tyr Asn Tyr
Ser Arg Arg Gly 130 135 140 Gly Pro Phe Leu Val Asp Arg Gln Phe Asp
Ala Ala Asn Leu Leu Gly 145 150 155 160 Leu Arg Phe His Ala Gly Arg
Gly Cys Ile Thr Leu Pro Met Ala Glu 165 170 175 Gly Ser Thr Ile Pro
Asp Ala Met Arg Glu Ser Thr Asp Thr Phe Leu 180 185 190 Ala Asp Cys
Glu Arg Leu Val Ser Arg Phe His Asp Pro Arg Pro Phe 195 200 205 Ala
Met Gln Arg Val Val Val Ala Pro Ser Ser Pro Val Ile Ala Tyr 210 215
220 Pro Glu Thr Phe Val Glu Ser Ala Arg Leu Ala Arg His Leu Gly Val
225 230 235 240 Ser Leu His Thr His Leu Gly Glu Gly Glu Thr Pro Ala
Met Val Ala 245 250 255 Arg Phe Gly Glu Arg Ser Leu Asp Trp Cys Glu
Asn Arg Gly Phe Val 260 265 270 Gly Pro Asp Val Trp Leu Ala His Gly
Trp Glu Phe Thr Ala Ala Asp 275 280 285 Ile Ala Arg Leu Ala Ala Thr
Gly Thr Gly Val Ala His Cys Pro Ala 290 295 300 Pro Val Phe Leu Val
Gly Ala Glu Val Thr Asp Ile Pro Ala Met Ala 305 310 315 320 Ala Ala
Gly Val Arg Val Gly Phe Gly Val Asp Gly His Ala Ser Asn 325 330 335
Asp Ser Ser Asn Leu Ala Glu Cys Ile Arg Leu Ala Tyr Leu Leu Gln 340
345 350 Cys Leu Lys Ala Ser Glu Arg Gln His Pro Val Pro Ala Pro Tyr
Asp 355 360 365 Phe Leu Arg Met Ala Thr Gln Gly Gly Ala Asp Cys Leu
Asn Arg Pro 370 375 380 Asp Leu Gly Ala Leu Ala Val Gly Arg Ala Ala
Asp Phe Phe Ala Val 385 390 395 400 Asp Leu Asn Arg Ile Glu Tyr Ile
Gly Ala Asn His Asp Pro Arg Ser 405 410 415 Leu Pro Ala Lys Val Gly
Phe Ser Gly Pro Val Asp Met Thr Val Ile 420 425 430 Asn Gly Lys Val
Val Trp Arg Asn Gly Glu Phe Pro Gly Leu Asp Glu 435 440 445 Met Glu
Leu Ala Arg Ala Ala Asp Gly Val Phe Arg Arg Val Ile Tyr 450 455 460
Gly Asp Pro Leu Val Ala Ala Leu Arg Arg Gly Thr Gly Val Thr Pro 465
470 475 480 Cys 3403PRTPseudomonas sp. 3Met Ser Lys Asp Phe Asp Leu
Ile Ile Arg Asn Ala Tyr Leu Ser Glu 1 5 10 15 Lys Asp Ser Val Tyr
Asp Ile Gly Ile Val Gly Asp Arg Ile Ile Lys 20 25 30 Ile Glu Ala
Lys Ile Glu Gly Thr Val Lys Asp Glu Ile Asp Ala Lys 35 40 45 Gly
Asn Leu Val Ser Pro Gly Phe Val Asp Ala His Thr His Met Asp 50 55
60 Lys Ser Phe Thr Ser Thr Gly Glu Arg Leu Pro Lys Phe Trp Ser Arg
65 70 75 80 Pro Tyr Thr Arg Asp Ala Ala Ile Glu Asp Gly Leu Lys Tyr
Tyr Lys 85 90 95 Asn Ala Thr His Glu Glu Ile Lys Arg His Val Ile
Glu His Ala His 100 105 110 Met Gln Val Leu His Gly Thr Leu Tyr Thr
Arg Thr His Val Asp Val 115 120 125 Asp Ser Val Ala Lys Thr Lys Ala
Val Glu Ala Val Leu Glu Ala Lys 130 135 140 Glu Glu Leu Lys Asp Leu
Ile Asp Ile Gln Val Val Ala Phe Ala Gln 145 150 155 160 Ser Gly Phe
Phe Val Asp Leu Glu Ser Glu Ser Leu Ile Arg Lys Ser 165 170 175 Leu
Asp Met Gly Cys Asp Leu Val Gly Gly Val Asp Pro Ala Thr Arg 180 185
190 Glu Asn Asn Val Glu Gly Ser Leu Asp Leu Cys Phe Lys Leu Ala Lys
195 200 205 Glu Tyr Asp Val Asp Ile Asp Tyr His Ile His Asp Ile Gly
Thr Val 210 215 220 Gly Val Tyr Ser Ile Asn Arg Leu Ala Gln Lys Thr
Ile Glu Asn Gly 225 230 235 240 Tyr Lys Gly Arg Val Thr Thr Ser His
Ala Trp Cys Phe Ala Asp Ala 245 250 255 Pro Ser Glu Trp Leu Asp Glu
Ala Ile Pro Leu Tyr Lys Asp Ser Gly 260 265 270 Met Lys Phe Val Thr
Cys Phe Ser Ser Thr Pro Pro Thr Met Pro Val 275 280 285 Ile Lys Leu
Leu Glu Ala Gly Ile Asn Leu Gly Cys Ala Ser Asp Asn 290 295 300 Ile
Arg Asp Phe Trp Val Pro Phe Gly Asn Gly Asp Met Val Gln Gly 305 310
315 320 Ala Leu Ile Glu Thr Gln Arg Leu Glu Leu Lys Thr Asn Arg Asp
Leu 325 330 335 Gly Leu Ile Trp Lys Met Ile Thr Ser Glu Gly Ala Arg
Val Leu Gly 340 345 350 Ile Glu Lys Asn Tyr Gly Ile Glu Val Gly Lys
Lys Ala Asp Leu Val 355 360 365 Val Leu Asn Ser Leu Ser Pro Gln Trp
Ala Ile Ile Asp Gln Ala Lys 370 375 380 Arg Leu Cys Val Ile Lys Asn
Gly Arg Ile Ile Val Lys Asp Glu Val 385 390 395 400 Ile Val Ala
4363PRTPseudomonas sp. 4Met Tyr His Ile Asp Val Phe Arg Ile Pro Cys
His Ser Pro Gly Asp 1 5 10 15 Thr Ser Gly Leu Glu Asp Leu Ile Glu
Thr Gly Arg Val Ala Pro Ala 20 25 30 Asp Ile Val Ala Val Met Gly
Lys Thr Glu Gly Asn Gly Cys Val Asn 35 40 45 Asp Tyr Thr Arg Glu
Tyr Ala Thr Ala Met Leu Ala Ala Cys Leu Gly 50 55 60 Arg His Leu
Gln Leu Pro Pro His Glu Val Glu Lys Arg Val Ala Phe 65 70 75 80 Val
Met Ser Gly Gly Thr Glu Gly Val Leu Ser Pro His His Thr Val 85 90
95 Phe Ala Arg Arg Pro Ala Ile Asp Ala His Arg Pro Ala Gly Lys Arg
100 105 110 Leu Thr Leu Gly Ile Ala Phe Thr Arg Asp Phe Leu Pro Glu
Glu Ile 115 120 125 Gly Arg His Ala Gln Ile Thr Glu Thr Ala Gly Ala
Val Lys Arg Ala 130 135 140 Met Arg Asp Ala Gly Ile Ala Ser Ile Asp
Asp Leu His Phe Val Gln 145 150 155 160 Val Lys Cys Pro Leu Leu Thr
Pro Ala Lys Ile Ala Ser Ala Arg Ser 165 170 175 Arg Gly Cys Ala Pro
Val Thr Thr Asp Thr Tyr Glu Ser Met Gly Tyr 180 185 190 Ser Arg Gly
Ala Ser Ala Leu Gly Ile Ala Leu Ala Thr Glu Glu Val 195 200 205 Pro
Ser Ser Met Leu Val Asp Glu Ser Val Leu Asn Asp Trp Ser Leu 210 215
220 Ser Ser Ser Leu Ala Ser Ala Ser Ala Gly Ile Glu Leu Glu His Asn
225 230 235 240 Val Val Ile Ala Ile Gly Met Ser Glu Gln Ala Thr Ser
Glu Leu Val 245 250 255 Ile Ala His Gly Val Met Ser Asp Ala Ile Asp
Ala Ala Ser Val Arg 260 265 270 Arg Thr Ile Glu Ser Leu Gly Ile Arg
Ser Asp Asp Glu Met Asp Arg 275 280 285 Ile Val Asn Val Phe Ala Lys
Ala Glu Ala Ser Pro Asp Gly Val Val 290 295 300 Arg Gly Met Arg His
Thr Met Leu Ser Asp Ser Asp Ile Asn Ser Thr 305 310 315 320 Arg His
Ala Arg Ala Val Thr Gly Ala Ala Ile Ala Ser Val Val Gly 325 330 335
His Gly Met Val Tyr Val Ser Gly Gly Ala Glu His Gln Gly Pro Ala 340
345 350 Gly Gly Gly Pro Phe Ala Val Ile Ala Arg Ala 355 360
5457PRTPseudomonas sp. 5Met Lys Thr Val Glu Ile Ile Glu Gly Ile Ala
Ser Gly Arg Thr Ser 1 5 10 15 Ala Arg Asp Val Cys Glu Glu Ala Leu
Ala Thr Ile Gly Ala Thr Asp 20 25 30 Gly Leu Ile Asn Ala Phe Thr
Cys Arg Thr Val Glu Arg Ala Arg Ala 35 40 45 Glu Ala Asp Ala Ile
Asp Val Arg Arg Ala Arg Gly Glu Val Leu Pro 50 55 60 Pro Leu Ala
Gly Leu Pro Tyr Ala Val Lys Asn Leu Phe Asp Ile Glu 65 70 75 80 Gly
Val Thr Thr Leu Ala Gly Ser Lys Ile Asn Arg Thr Leu Pro Pro 85 90
95 Ala Arg Ala Asp Ala Val Leu Val Gln Arg Leu Lys Ala Ala Gly Ala
100 105 110 Val Leu Leu Gly Gly Leu Asn Met Asp Glu Phe Ala Tyr Gly
Phe Thr 115 120 125 Thr Glu Asn Thr His Tyr Gly Pro Thr Arg Asn Pro
His Asp Thr Gly 130 135 140 Arg Ile Ala Gly Gly Ser Ser Gly Gly Ser
Gly Ala Ala Ile Ala Ala 145 150 155 160 Gly Gln Val Pro Leu Ser Leu
Gly Ser Asp Thr Asn Gly Ser Ile Arg 165 170 175 Val Pro Ala Ser Leu
Cys Gly Val Trp Gly Leu Lys Pro Thr Phe Gly 180 185 190 Arg Leu Ser
Arg Arg Gly Thr Tyr Pro Phe Val His Ser Ile Asp His 195 200 205 Leu
Gly Pro Leu Ala Asp Ser Val Glu Gly Leu Ala Leu Ala Tyr Asp 210 215
220 Ala Met Gln Gly Pro Asp Pro Leu Asp Pro Gly Cys Ser Ala Ser Arg
225 230 235 240 Ile Gln Pro Ser Val Pro Val Leu Ser Gln Gly Ile Ala
Gly Leu Arg 245 250 255 Ile Gly Val Leu Gly Gly Trp Phe Arg Asp Asn
Ala Gly Pro Ala Ala 260 265 270 Arg Ala Ala Val Asp Val Ala Ala Leu
Thr Leu Gly Ala Ser Glu Val 275 280 285 Val Met Trp Pro Asp Ala Glu
Ile Gly Arg Ala Ala Ala Phe Val Ile 290 295 300 Thr Ala Ser Glu Gly
Gly Cys Leu His Leu Asp Asp Leu Arg Ile Arg 305 310 315 320 Pro Gln
Asp Phe Glu Pro Leu Ser Val Asp Arg Phe Ile Ser Gly Val 325 330 335
Leu Gln Pro Val Ala Trp Tyr Leu Arg Ala Gln Arg Phe Arg Arg Val 340
345 350 Tyr Arg Asp Lys Val Asn Ala Leu Phe Arg Asp Trp Asp Ile Leu
Ile 355 360 365 Ala Pro Ala Thr Pro Ile Ser Ala Pro Ala Ile Gly Thr
Glu Trp Ile 370 375 380 Glu Val Asn Gly Thr Arg His Pro Cys Arg Pro
Ala Met Gly Leu Leu 385 390 395 400 Thr Gln Pro Val Ser Phe Ala Gly
Cys Pro Val Val Ala Ala Pro Thr 405 410 415 Trp Pro Gly Glu Asn Asp
Gly Met Pro Ile Gly Val Gln Leu Ile Ala 420 425 430 Ala Pro Trp Asn
Glu Ser Leu Cys Leu Arg Ala Gly Lys Val Leu Gln 435 440 445 Asp Thr
Gly Ile Ala Arg Leu Lys Cys 450 455 6605PRTPseudomonas sp. 6Met Asn
Asp Arg Ala Pro His Pro Glu Arg Ser Gly Arg Val Thr Pro 1 5 10 15
Asp His Leu Thr Asp Leu Ala Ser Tyr Gln Ala Ala Tyr Ala Ala Gly 20
25 30 Thr Asp Ala Ala Asp Val Ile Ser Asp Leu Tyr Ala Arg Ile Lys
Glu 35 40 45 Asp Gly Glu Asn Pro Ile Trp Ile Ser Leu Leu Pro Leu
Glu Ser Ala 50 55 60 Leu Ala Met Leu Ala Asp Ala Gln Gln Arg Lys
Asp Lys Gly Glu Ala 65 70 75 80 Leu Pro Leu Phe Gly Ile Pro Phe Gly
Val Lys Asp Asn Ile Asp Val 85 90 95 Ala Gly Leu Pro Thr Thr Ala
Gly Cys Thr Gly Phe Ala Arg Thr Pro 100 105
110 Arg Gln His Ala Phe Val Val Gln Arg Leu Val Asp Ala Gly Ala Ile
115 120 125 Pro Ile Gly Lys Thr Asn Leu Asp Gln Phe Ala Thr Gly Leu
Asn Gly 130 135 140 Thr Arg Thr Pro Phe Gly Ile Pro Arg Cys Val Phe
Asn Glu Asn Tyr 145 150 155 160 Val Ser Gly Gly Ser Ser Ser Gly Ser
Ala Val Ala Val Ala Asn Gly 165 170 175 Thr Val Pro Phe Ser Leu Gly
Thr Asp Thr Ala Gly Ser Gly Arg Ile 180 185 190 Pro Ala Ala Phe Asn
Asn Leu Val Gly Leu Lys Pro Thr Lys Gly Leu 195 200 205 Phe Ser Gly
Ser Gly Leu Val Pro Ala Ala Arg Ser Leu Asp Cys Ile 210 215 220 Ser
Val Leu Ala His Thr Val Asp Asp Ala Leu Ala Val Ala Arg Val 225 230
235 240 Ala Ala Gly Tyr Asp Ala Asp Asp Ala Phe Ser Arg Lys Ala Gly
Ala 245 250 255 Ala Ala Leu Thr Glu Lys Ser Trp Pro Arg Arg Phe Asn
Phe Gly Val 260 265 270 Pro Ala Ala Glu His Arg Gln Phe Phe Gly Asp
Ala Glu Ala Glu Ala 275 280 285 Leu Phe Asn Lys Ala Val Arg Lys Leu
Glu Glu Met Gly Gly Thr Cys 290 295 300 Ile Ser Phe Asp Tyr Thr Pro
Phe Arg Gln Ala Ala Glu Leu Leu Tyr 305 310 315 320 Ala Gly Pro Trp
Val Ala Glu Arg Leu Ala Ala Ile Glu Ser Leu Ala 325 330 335 Asp Glu
His Pro Glu Val Leu His Pro Val Val Arg Asp Ile Ile Leu 340 345 350
Ser Ala Lys Arg Met Ser Ala Val Asp Thr Phe Asn Gly Ile Tyr Arg 355
360 365 Leu Ala Asp Leu Val Arg Ala Ala Glu Ser Thr Trp Glu Lys Ile
Asp 370 375 380 Val Met Leu Leu Pro Thr Ala Pro Thr Ile Tyr Thr Val
Glu Asp Met 385 390 395 400 Leu Ala Asp Pro Val Arg Leu Asn Ser Asn
Leu Gly Phe Tyr Thr Asn 405 410 415 Phe Val Asn Leu Met Asp Leu Ser
Ala Ile Ala Val Pro Ala Gly Phe 420 425 430 Arg Thr Asn Gly Leu Pro
Phe Gly Val Thr Phe Ile Gly Arg Ala Phe 435 440 445 Glu Asp Gly Ala
Ile Ala Ser Leu Gly Lys Ala Phe Val Glu His Asp 450 455 460 Leu Ala
Lys Gly Asn Ala Ala Thr Ala Ala Pro Pro Lys Asp Thr Val 465 470 475
480 Ala Ile Ala Val Val Gly Ala His Leu Ser Asp Gln Pro Leu Asn His
485 490 495 Gln Leu Thr Glu Ser Gly Gly Lys Leu Arg Ala Thr Thr Arg
Thr Ala 500 505 510 Pro Gly Tyr Ala Leu Tyr Ala Leu Arg Asp Ala Thr
Pro Ala Lys Pro 515 520 525 Gly Met Leu Arg Asp Gln Asn Ala Val Gly
Ser Ile Glu Val Glu Ile 530 535 540 Trp Asp Leu Pro Val Ala Gly Phe
Gly Ala Phe Val Ser Glu Ile Pro 545 550 555 560 Ala Pro Leu Gly Ile
Gly Thr Ile Thr Leu Glu Asp Gly Ser His Val 565 570 575 Lys Gly Phe
Leu Cys Glu Pro His Ala Ile Glu Thr Ala Leu Asp Ile 580 585 590 Thr
His Tyr Gly Gly Trp Arg Ala Tyr Leu Ala Ala Gln 595 600 605
7225PRTDiscosoma sp. 7Met Arg Ser Ser Lys Asn Val Ile Lys Glu Phe
Met Arg Phe Lys Val 1 5 10 15 Arg Met Glu Gly Thr Val Asn Gly His
Glu Phe Glu Ile Glu Gly Glu 20 25 30 Gly Glu Gly Arg Pro Tyr Glu
Gly His Asn Thr Val Lys Leu Lys Val 35 40 45 Thr Lys Gly Gly Pro
Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln 50 55 60 Phe Gln Tyr
Gly Ser Lys Val Tyr Val Lys His Pro Ala Asp Ile Pro 65 70 75 80 Asp
Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val 85 90
95 Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser Ser
100 105 110 Leu Gln Asp Gly Cys Phe Ile Tyr Lys Val Lys Phe Ile Gly
Val Asn 115 120 125 Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr
Met Gly Trp Glu 130 135 140 Ala Ser Thr Glu Arg Leu Tyr Pro Arg Asp
Gly Val Leu Lys Gly Glu 145 150 155 160 Ile His Lys Ala Leu Lys Leu
Lys Asp Gly Gly His Tyr Leu Val Glu 165 170 175 Phe Lys Ser Ile Tyr
Met Ala Lys Lys Pro Val Gln Leu Pro Gly Tyr 180 185 190 Tyr Tyr Val
Asp Ser Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr 195 200 205 Thr
Ile Val Glu Gln Tyr Glu Arg Thr Glu Gly Arg His His Leu Phe 210 215
220 Leu 225
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