U.S. patent application number 16/778653 was filed with the patent office on 2020-12-24 for compositions and methods for pesticide degradation.
This patent application is currently assigned to UNIVERSITY OF VIRGINIA PATENT FOUNDATION. The applicant listed for this patent is UNIVERSITY OF VIRGINIA PATENT FOUNDATION. Invention is credited to ZACHERY GEORGE DAVIS, JOSEPH FRANK, MARK KESTER, ANDREI KHOKHLACHEV, SHAUN RAFIE MOSHASHA, Payam POURTAHERI, AMEER HAMZA SHAKEEL, SEPEHR ZOMORODI.
Application Number | 20200399618 16/778653 |
Document ID | / |
Family ID | 1000005062358 |
Filed Date | 2020-12-24 |
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United States Patent
Application |
20200399618 |
Kind Code |
A1 |
POURTAHERI; Payam ; et
al. |
December 24, 2020 |
Compositions and Methods for Pesticide Degradation
Abstract
Compositions and methods related to anucelate cells (e.g.,
bacterial minicells) for pesticide degradation applications
including related cells, polypeptides, and vectors.
Inventors: |
POURTAHERI; Payam;
(STERLING, VA) ; ZOMORODI; SEPEHR; (HERNDON,
VA) ; DAVIS; ZACHERY GEORGE; (RICE, VA) ;
SHAKEEL; AMEER HAMZA; (ASHBURN, VA) ; FRANK;
JOSEPH; (CHARLOTTESVILLE, VA) ; MOSHASHA; SHAUN
RAFIE; (CHARLOTTESVILLE, VA) ; KHOKHLACHEV;
ANDREI; (CHARLOTTESVILLE, VA) ; KESTER; MARK;
(AFTON, VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF VIRGINIA PATENT FOUNDATION |
CHARLOTTESVILLE |
VA |
US |
|
|
Assignee: |
UNIVERSITY OF VIRGINIA PATENT
FOUNDATION
CHARLOTTESVILLE
VA
|
Family ID: |
1000005062358 |
Appl. No.: |
16/778653 |
Filed: |
January 31, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16092697 |
Oct 10, 2018 |
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PCT/US2017/027048 |
Apr 11, 2017 |
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16778653 |
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62320790 |
Apr 11, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/16 20130101; A62D
3/02 20130101; C12Y 301/08002 20130101; A62D 2101/26 20130101; C07K
2319/00 20130101; A62D 2101/04 20130101; C07K 14/195 20130101 |
International
Class: |
C12N 9/16 20060101
C12N009/16; C07K 14/195 20060101 C07K014/195; A62D 3/02 20060101
A62D003/02 |
Claims
1. A composition comprising a plurality of intact,
bacterially-derived minicells, wherein each minicell of said
plurality comprises a biologically active polypeptide displayed on
the surface of the bacterial minicell, wherein said biologically
active polypeptide has pesticide degrading activity.
2. The composition of claim 1, wherein the bacterially-derived
minicells are produced from a strain of Escherichia coli, Bacillus
spp., Salmonella spp., Listeria spp., Mycobacterium spp., Shigella
spp., or Yersinia spp.
3. The composition of claim 1, wherein the pesticide is selected
from the group consisting of organophosphates, glyphosates,
neonicotinoids, carbamates, organochlorines, pyrethroids,
Stribilurins, anilenes, thiophthlamadies (Captan), aromatic
hydrocarbons, nitriles, cyanoimidazoles, demethylation inhibitors,
phenylpyrroles, carboximides, phosphonates, dicarboximides,
phenylamides, polyoxins, benzimidazoles, aryloxyphenoxy
propionates, cyclohexanediones, phenylpyrazolin, imidazolinones,
sulfonylamino-carbonyltriazolinones, amides, sulfonylureas,
pyraxoles, triazolpyramidines, triazolones, dinitroanilines,
benzoic acids, carboxylic acids, piconolinic acids, phenoxys,
phenyl-carbamates, triazines, triazinones, uracils,
benzthiadiazoles, ureas, thiocarbamates, triazoles, aryl triazones,
chloroacetamides, pyrazoles, benzofuranyl alkylsulfonates,
semicarbazones, bipyridyliums, and benzopyrazoles.
4. The composition of claim 1, wherein the pesticide is an
organophosphate selected from the group consisting of paraoxon,
malathion, parathion, diazinon, naled, fenthion, dichlorvos,
chlorpyrifos, phosmet, acephate, ethion, soman, sarin, tabun, VX,
echothiophate, isoflurophate, trichlorfon, tetrachlorvinphos,
azamethiphos, and azinphos-methyl.
5. The composition of claim 4, wherein the pesticide comprises
malathion.
6. The composition of any one of claims 1-5, wherein the
biologically active polypeptide comprises a fusion protein.
7. The composition of claim 6, wherein the fusion protein comprises
at least one surface-expressing moiety, and at least one enzyme
moiety.
8. The composition of claim 7, wherein the at least one
surface-expressing moiety comprises an ice nucleation protein
(INPNC) or an exported bacterial protein, including wild type or
mutant versions thereof.
9. The composition of claim 8, wherein the exported bacterial
protein is selected from the group consisting of LamB (.lamda.
receptor), OprF, OmpA (3a, II), Lpp (lipoprotein), MalE
(maltose-binding protein), PhoA (alkaline phosphatase), Bla (TEM-1
.beta.-lactamse, f1 or M13 major coat, f1 or M13 minor coat, and
any wild type or mutant versions thereof.
10. The composition of claim 9, wherein said at least one enzyme
moiety is selected from the group consisting of hydrolases,
lipases, phospholipases, transacylases, transaminases, pectinase,
proteases, amylases, cellulases, cutinases, esterases, acylases,
invertases, isomerases, lyases, glucosidases, oxidoreductases,
transferases, ligases, amidases, manganese peroxidase, laccase,
lignin peroxidase, horseradish peroxidase or phosphonate
dehydrogenase, cytosolic aldehyde oxidase, carbaryl hydrolases, and
amidases, HCH dehydrochlorinase, haloalkane dehalogenase,
pyrethroid hydrolases (PytH, EspA) and permethrinase.
11. The composition of claim 9, wherein said at least one enzyme
moiety comprises an organophosphate hydrolase.
12. The composition of claim 11, wherein the organophosphate
hydrolase is selected from the group consisting of
phosphotriesterases, methyl parathion hydrolases, and
organophosphorus acid anhydrolases.
13. The composition of anyone of claims 1-12, further comprising a
second polypeptide displayed on the surface of the bacterial
minicell, to increase adhesion to plants.
14. The composition of claim 13, wherein the second polypeptide
comprises a fusion protein.
15. The composition of claim 14, wherein the fusion protein
comprises at least one surface-expressing moiety and at least one
plant cell adhesion moiety.
16. The composition of claim 15, wherein said at least one
surface-expressing moiety comprises an exported bacterial protein
selected from the group consisting of LamB (.lamda. receptor),
OprF, OmpA (3a, II), Lpp (lipoprotein), MalE (maltose-binding
protein), PhoA (alkaline phosphatase), Bla (TEM-1 .beta.-lactamse,
f1 or M13 major coat, f1 or M13 minor coat and any wild type or
mutant versions thereof.
17. The composition of claim 16, wherein said at least one plant
cell adhesion moiety comprises a cellulose binding domain.
18. The composition of any one of claims 1-17, wherein the
minicells have at least 90-95% purity.
19. The composition of any one of claims 1-18, wherein the
minicells are about 0.4 .mu.m-0.5 .mu.m in diameter.
20. A method for pesticide degradation, the method comprising
contacting a crop or an agricultural product with the composition
of any one of claims 1-19, wherein the composition mediates
degradation of the pesticide to yield inert by-products.
21. A method for bioremediation, the method comprising contacting a
crop or an agricultural product with the composition of any one of
claims 1-19, wherein the composition mediates degradation of the
pesticide to yield inert by-products.
22. A method for reducing the half-life of a pesticide, the method
comprising contacting a crop or agricultural product with the
composition of any one of claims 1-19.
23. The method of claim 22, wherein the pesticide comprises
malathion.
24. The method of claim 22 or claim 23, wherein the half-life is
about 4 hours.
25. The method of any one of claims 20-24, wherein the crop or
agricultural product is selected from the group consisting of
grapes, peaches, apples, sweet bell peppers, celery, nectarines,
strawberries, cherries, pears, spinach, lettuce, and potatoes
26. The method of any one of claims 20-25, wherein the crop or
agricultural product comprises grapes.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 16/092,697, filed Oct. 10, 2018, which is a 371 national phase
of International Application No. PCT/US2017/027048, filed Apr. 11,
2017 and published as WO 2017/0180650, which claims priority to
U.S. Provisional Patent Application No. 62/320,790 filed on Apr.
11, 2016, each of which are incorporated herein by reference in its
entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA
EFS-WEB
[0002] The entire content of the following electronic submission of
the sequence listing submitted via the USPTO EFS-WEB server is
incorporated herein by reference in its entirety for all purposes.
The sequence listing is within the electronically filed text file
that is identified as follows:
File Name: UVA_SEQL_ST25.txt
Date of Creation: 8 Oct. 2018
[0003] Size (bytes): 1,055 bytes
TECHNICAL FIELD OF THE INVENTION
[0004] The invention relates generally to compositions and methods
concerning pesticide degradation. In particular, the invention
relates to compositions and methods concerning production of
bacterial minicells for use in pesticide degradation. The
aforementioned compositions and methods also concern related cells
(e.g., minicell-producing host cells), polypeptides, and
vectors.
BACKGROUND OF THE INVENTION
[0005] Pesticides (e.g., insecticides, herbicides, and fungicides)
are widely used in agriculture to control weeds, insect infestation
and diseases. While the widespread use of pesticides has had
several benefits for the agricultural industry in terms of
combating the adverse economic impact of pest-related crop loss,
excessive pesticide use poses significant problems to the
environment and health. For example, the appearance and quality of
crops and fruits sprayed with massive amounts of pesticide are
significantly compromised. Further, excessive pesticide exposure
leads to detrimental health effects such as nausea, seizures, and
even death.
[0006] In view of the adverse effects produced by exposure to these
compounds, it is essential to reduce, eliminate, or detoxify
pesticides from crops and the environment. Various bioremediation
approaches have been developed, such as enzyme-mediated
decontamination and genetically-engineered organisms for specific
bioremediation applications. However, biosafety concerns hinder
synthetic biology applications because microorganisms (e.g.,
bacteria) rapidly replicate and may contain one or more antibiotic
resistance genes. Thus, there is a critical need to develop safe,
convenient, and economically-feasible methods for pesticide
detoxification or removal.
[0007] As discussed herein, aspects of the present invention
address the aforementioned challenges and unmet needs by providing
bacterially-derived minicells for pesticide degradation, which are
devoid of chromosomal DNA, and therefore unable to replicate and
cause infection or ecological harm.
[0008] The compositions and methods of the present invention
provide a solution for optimizing and/or reducing pre-harvest
intervals (PHI) for a crop or an agricultural product, such as
grapes, peaches, apples, sweet bell peppers, celery, nectarines,
strawberries, cherries, pears, spinach, lettuce, and potatoes. The
pre-harvest interval (PHI) is established by the Environmental
Protection Agency (EPA) and describes the minimum period of time
that must elapse between the application of pesticides to for a
crop or an agricultural product and their harvest. The pre-harvest
interval is a function of a pesticide's use pattern and of the
amount of pesticide residues allowed on the crop at harvest. PHIs
may range from a week to more than two months depending upon the
pesticide, and limits the frequency at which a crop or an
agricultural product may be harvested. Accordingly, the
compositions and methods of the present invention potentially
provide farmers with much needed solution to achieve premium
harvests and premium marketing opportunities by encouraging
regulatory changes in view of the reduction in the half-life of
pesticides.
BRIEF SUMMARY OF THE INVENTION
[0009] The present invention provides compositions comprising a
plurality of intact, bacterially-derived minicells, wherein each
minicell of said plurality comprises a biologically active
polypeptide displayed on the surface of the bacterial minicell,
wherein said biologically active polypeptide has pesticide
degrading activity. In certain embodiments, the bacterially-derived
minicells are produced from a strain of Escherichia coli, Bacillus
spp., Salmonella spp., Listeria spp., Mycobacterium spp., Shigella
spp., or Yersinia spp. In certain embodiments, the pesticide is
selected from the group consisting of organophosphates,
glyphosates, neonicotinoids, carbamates, organochlorines,
pyrethroids, Stribilurins, anilenes, thiophthlamadies (Captan),
aromatic hydrocarbons, nitriles, cyanoimidazoles, demethylation
inhibitors, phenylpyrroles, carboximides, phosphonates,
dicarboximides, phenylamides, polyoxins, benzimidazoles,
aryloxyphenoxy propionates, cyclohexanediones, phenylpyrazolin,
imidazolinones, sulfonylamino-carbonyltriazolinones, amides,
sulfonylureas, pyraxoles, triazolpyramidines, triazolones,
dinitroanilines, benzoic acids, carboxylic acids, piconolinic
acids, phenoxys, phenyl-carbamates, triazines, triazinones,
uracils, benzthiadiazoles, ureas, thiocarbamates, triazoles, aryl
triazones, chloroacetamides, pyrazoles, benzofuranyl
alkylsulfonates, semicarbazones, bipyridyliums, and benzopyrazoles.
In certain embodiments, the pesticide is an organophosphate
selected from the group consisting of paraoxon, malathion,
parathion, diazinon, naled, fenthion, dichlorvos, chlorpyrifos,
phosmet, acephate, ethion, soman, sarin, tabun, VX, echothiophate,
isoflurophate, trichlorfon, tetrachlorvinphos, azamethiphos, and
azinphos-methyl. In particular embodiments, the pesticide comprises
malathion.
[0010] In some embodiments, the biologically active polypeptide
comprises a fusion protein. In some embodiments, the fusion protein
comprises at least one surface-expressing moiety and at least one
enzyme moiety. In some embodiments, the surface-expressing moiety
comprises an ice nucleation protein (INPNC) or an exported
bacterial protein, including wild type or mutant versions thereof.
Contemplated exported bacterial proteins include LamB (.lamda.
receptor), OprF, OmpA (3a, II), Lpp (lipoprotein), MalE
(maltose-binding protein), PhoA (alkaline phosphatase), Bla (TEM-1
.beta.-lactamse, f1 or M13 major coat, f1 or M13 minor coat, and
any wild type or mutant versions thereof. Contemplated enzyme
moieties hydrolases, lipases, phospholipases, transacylases,
transaminases, pectinase, proteases, amylases, cellulases,
cutinases, esterases, acylases, invertases, isomerases, lyases,
glucosidases, oxidoreductases, transferases, ligases, amidases,
manganese peroxidase, laccase, lignin peroxidase, horseradish
peroxidase or phosphonate dehydrogenase, cytosolic aldehyde
oxidase, carbaryl hydrolases, and amidases, HCH dehydrochlorinase,
haloalkane dehalogenase, pyrethroid hydrolases (PytH, EspA) and
permethrinase.
[0011] In particular embodiments, the enzyme moiety comprises an
organophosphate hydrolase. In some embodiments, a hydrolase is a
phosphotriesterase, methyl parathion hydrolase, or organophosphorus
acid anhydrolases.
[0012] In some embodiments, a second polypeptide is displayed on
the surface of the bacterial minicell to increase adhesion to
plants. In some embodiments, the second polypeptide comprises a
fusion protein. In some embodiments, the fusion protein comprises
at least one surface-expressing moiety and at least one plant cell
adhesion moiety. Contemplated surface-expressing moieties include
exported bacterial proteins such as LamB (.lamda. receptor), OprF,
OmpA (3a, II), Lpp (lipoprotein), MalE (maltose-binding protein),
PhoA (alkaline phosphatase), Bla (TEM-1 .beta.-lactamse, f1 or M13
major coat, f1 or M13 minor coat and any wild type or mutant
versions thereof. Contemplated plant cell adhesion moieties include
fusion proteins comprising a cellulose binding domain.
[0013] In some embodiments, minicells are at least 90-95% purity
(i.e., free of detectable contaminants). In some embodiments,
minicells are about 0.4 .mu.m to about 0.5 .mu.m in diameter.
[0014] The present invention also provides methods for pesticide
degradation, comprising contacting a crop or an agricultural
product with a composition in accordance with the present
invention, wherein the composition mediates degradation of the
pesticide to yield inert by-products.
[0015] The present invention also provides methods for
bioremediation, comprising contacting a crop or an agricultural
product with a composition in accordance with the present
invention, wherein the composition mediates degradation of the
pesticide to yield inert by-products.
[0016] The present invention also provides methods for reducing the
half-life of a pesticide, the method comprising contacting a crop
or agricultural product with the composition in accordance with the
present invention. In some embodiments, the pesticide comprises
malathion. In some embodiments, the half-life of malathion is
reduced to about 1 hour, to about 2 hours, to about 3 hours, or to
about 4 hours.
[0017] The present invention provides compositions and methods that
are useful in degrading pesticides applied to, or otherwise present
on, a crop or agricultural product such as grapes, peaches, apples,
sweet bell peppers, celery, nectarines, strawberries, cherries,
pears, spinach, lettuce, or potatoes.
BRIEF SUMMARY OF THE SEVERAL VIEWS OF THE DRAWINGS
[0018] FIG. 1: Symmetric bacterial cell division by overexpression
of ftsZ protein.
[0019] FIGS. 2A-2B: Construction of an exemplary bacterial
minicell-inducing plasmid.
[0020] FIG. 2A depicts a synthesized K-12 E. coli filamenting
temperature-sensitive mutant Z (ftsZ) gene cloned into pSB1C3
(BBa_K1011000). FIG. 2B depicts a minicell-inducing construct
(BBa_K1011001) which includes IPTG-inducible promoter (BBa_R0011),
ribosomal binding sequence (RBS, BBa_B0034), synthesized K-12 E.
coli ftsZ gene (BBa_K1011000), RBS (BBa_B0034), GFP (BBa_E0040),
and double terminator (BBa_B0015) in pSB1C3.
[0021] FIG. 3: Fluorescence microscopy visualization of
BBa_K1011001-transformed XL1-Blue E. coli cells grown in the
presence of 40 .mu.M IPTG. As shown in the inset and marked by the
red triangles, asymmetric cell division successfully produced
minicells after 14 h of induction. Scale bar is 35 .mu.m and inset
scale bar is 9 .mu.m.
[0022] FIG. 4: Exemplary plasmid constructs for producing green
fluorescent protein (GFP)-expressing minicells.
[0023] FIGS. 5A-5B: Fluorescence microscopy visualization of
induction of minicell formation and purified minicells. FIG. 5A
depicts the appearance of green glowing minicells 90 minutes
following induction of the bacterial cells. The tiny brighter green
spots appear to be minicells. FIG. 5B depicts minicells separated
using centrifugation, antibiotics, and a sequence of filters to
achieve about 95% purity of a minicell sample.
[0024] FIGS. 6A-6C: Fluorescence staining demonstrating that
minicells lack DNA but retain membrane proteins. The mixed solution
of E. coli and minicells were stained with DAPI (FIG. 6A); DAPI and
GFP (FIG. 6B), and the resulting fluorescent photographic images
were overlapped to visualize and identify cells that were green but
lacked the blue staining (FIG. 6C), which correspond to the
minicells.
[0025] FIGS. 7A-7B: Exemplary plasmid construct for surface
expression of organophosphate hydrolase (OPH). FIGS. 7A and 7B show
representations of the same construct. The construct expresses a
truncated ice nucleation protein (INPC)-OPH fusion protein where
the INPC serves as a surface anchor to display OPH on the minicell
surface.
[0026] FIG. 8: Scanning electron micrograph showing minicell
formation in E. coli.
[0027] FIGS. 9A-9C: Determination of minicell size. FIGS. 9A-9B
show scanning electron microscope images of minicells expressing
OPH expressing on their surface. The average size of a minicell is
around 0.5 micrometers (FIG. 9A). Minicells increase in size after
each separation because the parent bacterial cell elongates, making
the next minicell a little bigger than the first (FIG. 9B). FIG. 9C
shows a graphical representation of the size distribution by
intensity. Two peaks were obtained during sizing. The smaller peak
is believed to be due to minor levels of contamination (e.g.,
dust). The higher peak around 500 nanometers is the minicell
peak.
[0028] FIGS. 10A-10B: Breakdown of organophosphate (paraoxon) by
minicells (produced using the plasmid construct shown in FIGS.
7A-7B) expressing organophosphate hydrolase (OPH) on their surface.
Paraoxon is broken down into p-nitrophenol, a chromogenic product
with absorbance at 412 nm. As illustrated by the graphs, the
surface OPH-expressing minicells showed very efficient degradation
compared to the controls. FIG. 10A corresponds to paraoxon
degradation observed after one hour after induction of the
bacterial cells to produce minicells expressing OPH. FIG. 10B
corresponds to paraoxon degradation observed 24 hours following
induction of bacterial cells to produce surface OPH-expressing
minicells. As shown in FIG. 10B, more efficient degradation is
observed after 24 hours.
[0029] FIG. 11: Field data showing malathion concentration vs. time
after an application of surface OPH-expressing E. coli minicells
(produced using the plasmid construct shown in FIGS. 7A-7B). The
graph depicted in FIG. 11 shows the decrease in concentration of
the organophosphate class pesticide malathion over time after an
application of minicells with a surface expressed organophosphate
hydrolase to plants that had been sprayed with malathion. All data
points were created by collecting leaves in triplicate and
extracting malathion from the leaves on site at the appropriate
time points. The extraction contained no water and was stored at
-80.degree. C. so any further breakdown of malathion was halted.
These three experimental conditions were chosen in order to best
isolate the action of the OPH enzyme on the minicells. The natural
breakdown condition was chosen to demonstrate that this pesticide
does not breakdown quickly on its own. The minicells with surface
expressed OPH (without/(-) cofactor) was chosen as a condition to
demonstrate that the solution containing the minicells without the
enzyme's cofactor (zinc) are not functional and do not contribute
to the reduction in malathion concentration. The minicells with
surface expressed OPH (with/(+) cofactor) was our fully active
solution. Each extraction was diluted into 3 aliquots and analyzed
via LC/MS and the concentrations calculated from an internal
malathion standard curve. The error bars were calculated from the
standard error between data points corresponding to their
respective time points with n=9. A t-test and ANOVA calculation on
the data points revealed that there is no significant difference
between the natural breakdown and minicells with surface expressed
OPH (without/(-) cofactor), while there was a significant
difference between both of those conditions and the minicells with
surface expressed OPH (with/(+) cofactor).
DETAILED DESCRIPTION OF THE INVENTION
[0030] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the disclosure is related. For
example, the Concise Dictionary of Biomedicine and Molecular
Biology, Juo, Pei-Show, 2.sup.nd ed. 2002, CRC Press; The
Dictionary of Cell and Molecular Biology, 3.sup.rd ed., 1999,
Academic Press; and the Oxford Dictionary of Biochemistry and
Molecular Biology, Revised 2000, Oxford University Press, provide
one of skill in the art with a general dictionary of many of the
terms used herein. Additional definitions are set forth throughout
the detailed description.
[0031] The present invention relates generally to compositions and
methods concerning pesticide degradation. In particular, the
invention relates to compositions and methods concerning production
of minicells, including but not limited to eubacterial minicells
for use in pesticide degradation. In certain aspects, the present
invention provides minicells (e.g., bacterial minicells) comprising
an organophosphate degrading enzyme (e.g., an organophosphorous
hydrolase (OPH)) displayed on the minicell surface to biologically
degrade organophosphates. In certain aspects, the present invention
provides minicells comprising an organophosphate degrading enzyme
(e.g., an organophosphorous hydrolase (OPH)) and/or a polypeptide
having plant adhesion properties (e.g., a cellulose binding domain
(CBD)) displayed on the minicell surface to selectively adhere to
target plants and to biologically degrade organophosphates.
[0032] Minicells are the result of aberrant, asymmetric cell
division, and contain membranes, peptidoglycan, ribosomes, RNA,
protein, and often plasmids but no chromosome. (Frazer and Curtiss
III, Production, Properties and Utility of Bacterial Minicells, 69,
Curr. Top. Microbiol. Immunol. 1-3 (1975)). Because minicells lack
chromosomal DNA, minicells cannot divide or grow, but they can
continue other cellular processes, such as ATP synthesis,
replication and transcription of plasmid DNA, and translation of
mRNA. Although chromosomes do not segregate into minicells,
extrachromosomal and/or episomal genetic expression elements will
segregate, or may be introduced into minicells after segregation
from parent cells.
[0033] In certain aspects, the invention provides a composition
comprising a plurality of minicells, wherein each minicell of said
plurality comprises a biologically active polypeptide displayed on
the surface of the minicell, wherein said biologically active
polypeptide has pesticide degrading activity. In certain aspects,
the invention provides a composition comprising a plurality of
intact, bacterially-derived minicells, wherein each minicell of
said plurality comprises a biologically active polypeptide
displayed on the surface of the bacterial minicell, wherein said
biologically active polypeptide has pesticide degrading activity.
In some embodiments, the composition comprises minicells which
further comprise a second polypeptide displayed on the surface of
the bacterial minicell, to increase adhesion to plants. In some
embodiments, the composition comprises a mixture of minicells,
wherein certain minicells within the mixed minicell population
display the biologically active polypeptide or display the plant
adhesion increasing polypeptide or display both. In certain
embodiments, the bacterially-derived minicells are produced from a
strain, including, but not limited to a strain of Escherichia coli,
Bacillus spp., Salmonella spp., Listeria spp., Mycobacterium spp.,
Shigella spp., or Yersinia spp. In some embodiments, the
bacterially-derived minicells are produced from a strain that
naturally produces minicells. Such natural minicell producing
strains produce minicells, for example, at a 2:1 ratio (2 bacterial
cells for every one minicell) In certain embodiments, exemplary
bacterial strains that naturally produce minicells include, but are
not limited to E. coli strain number P678-54 CGSC number: 4928. B.
Subtilis designated number 18197, 168 strain CU403. In certain
embodiments, minicells are produced by overexpressing certain
proteins in bacteria, which proteins cause or increase minicell
production (e.g., ftsZ protein).
[0034] As used herein, the term "display" refers to the exposure of
the polypeptide of interest on the outer surface of the minicell.
By way of non-limiting example, the displayed polypeptide may be
protein or a protein domain which is either expressed on the
minicell membrane or is associated with the minicell membrane such
that the extracellular domain or domain of interest is exposed on
the outer surface of the minicell (expressed and displayed on the
surface of the minicell or expressed in the parental cell to be
displayed on the surface of the segregated/budded minicell). In all
instances, the "displayed" protein or protein domain is available
for interaction with extracellular components. A
membrane-associated protein may have more than one extracellular
domain, and a minicell of the invention may display more than one
membrane-associated protein.
[0035] As used herein, the terms "polypeptide", "protein" and
"protein domain" refer to a macromolecule made up of a single chain
of amino acids joined by peptide bonds. Polypeptides of the
invention may comprise naturally occurring amino acids, synthetic
amino acids, genetically encoded amino acids, non-genetically
encoded amino acids, and combinations thereof. Polypeptides may
include both L-form and D-form amino acids.
[0036] As used herein, the term "biologically active polypeptide"
refers to a polypeptide which encodes a biologically functional
protein. The term "biologically active polypeptide" includes fusion
proteins which perform a biological function. Exemplary
biologically active polypeptides, include enzymes/enzyme moiety
(e.g. wild type, variants, or engineered variants) that
specifically bind to certain receptors or chemical substrates to
effect a biological function such as biological signal transduction
or chemical inactivation.
[0037] In certain embodiments, the biologically active polypeptide
comprises a fusion protein. In certain embodiments, the fusion
protein comprises at least one surface-expressing moiety, and at
least one enzyme moiety.
[0038] Biologically active polypeptides that are within the scope
of the present invention include, but are not limited to,
enzymes/enzyme moieties such as hydrolases, lipases,
phospholipases, transacylases, transaminases, pectinase, proteases,
amylases, cellulases, cutinases, esterases, acylases, invertases,
isomerases, lyases, glucosidases, oxidoreductases, transferases,
ligases, amidases, manganese peroxidase, laccase, lignin
peroxidase, horseradish peroxidase or phosphonate dehydrogenase,
cytosolic aldehyde oxidase, carbaryl hydrolases, and amidases, HCH
dehydrochlorinase, haloalkane dehalogenase, pyrethroid hydrolases
(PytH, EspA) and permethrinase. In certain embodiments, the
biologically active polypeptides displayed by the minicells of the
invention comprise an organophosphate hydrolase. Exemplary
organophosphate hydrolases that are within the scope of the
invention include, but are not limited to sequences corresponding
to Genbank Accession Nos. KM076938.1, KC562907.1, M29593.1.
Organophosphate hydrolase targets the phosphorous ester bond of
organophosphate pesticides leading to degradation of the pesticide
into inert byproducts (e.g., two smaller inert by-products). In
certain embodiments, the organophosphate hydrolase is selected from
the group consisting of phosphotriesterases, methyl parathion
hydrolases, and organophosphorus acid anhydrolases. In certain
embodiments, the enzyme is fused to surface-expressing moiety.
Exemplary surface-expressing moieties include but are not limited
to ice nucleation protein (INPNC) or an exported bacterial protein.
"Exported bacterial proteins," generally refers to bacterial
proteins that are transported across the plasma membrane and
function as an anchor for membrane proteins. Exemplary exported
bacterial proteins encompassed by the present invention, include,
but are not limited to LamB, OprF, OmpA, Lpp, MalE, PhoA, Bla, F1
or M13 major coat, and F1 or M13 minor coat.
[0039] As discussed above, in certain embodiments, the invention
provides a composition comprising minicells which further comprise
a second polypeptide displayed on the surface of the bacterial
minicell, to increase adhesion to plants. Exemplary proteins or
protein domains that mediate increased adhesion to plants include,
but are not limited to cellulose binding domain (CBD). CBDs have
been shown to bind tightly and rapidly to cellulose fibers, and
thus enable strong attachment of the minicell compositions provided
by the present invention to plants for efficient degradation of
organophosphates. Exemplary CBD sequences that are within the scope
of the invention include, but are not limited to sequences
corresponding to Genbank Accession No. AB021656.1.
[0040] As used herein, the term "pesticide" refers to any substance
used to kill, repel, or control certain forms of plant or animal
life that are considered to be pests. Pesticides include herbicides
for destroying weeds and other unwanted vegetation, insecticides
for controlling a wide variety of insects, fungicides used to
prevent the growth of molds and mildew, disinfectants for
preventing the spread of bacteria, and compounds used to control
mice and rats."
[0041] Pesticides within the scope of the present invention
include, but are not limited to the classes of organophosphates,
glyphosates (degraded using e.g., ligninolytic enzymes: manganese
peroxidase, laccase, lignin peroxidase, horseradish peroxidase or
phosphonate dehydrogenase), neonicotinoids (degraded using e.g.,
nitroreductases such as cytosolic aldehyde oxidase), carbamates
(degraded using e.g., methyl carbamate-degrading hydrolases,
carbaryl hydrolases, and amidases to degrade them), organochlorines
(degrading using e.g., monooxygenases, HCH dehydrochlorinase,
haloalkane dehalogenase), and pyrethroids (degraded using e.g.,
pyrethroid hydrolases (PytH, EspA) and permethrinase). Additional
classes of pesticides within the scope of the present invention,
include, but are not limited to stribilurins, anilenes,
thiophthlamadies (Captan), aromatic hydrocarbons, nitriles,
cyanoimidazoles, demethylation inhibitors, phenylpyrroles,
carboximides, phosphonates, dicarboximides, phenylamides,
polyoxins, benzimidazoles, aryloxyphenoxy propionates,
cyclohexanediones, phenylpyrazolin, imidazolinones,
sulfonylamino-carbonyltriazolinones, amides, sulfonylureas,
pyraxoles, triazolpyramidines, triazolones, dinitroanilines,
benzoic acids, carboxylic acids, piconolinic acids, phenoxys,
phenyl-carbamates, triazines, triazinones, uracils,
benzthiadiazoles, ureas, thiocarbamates, triazoles, aryl triazones,
chloroacetamides, pyrazoles, benzofuranyl alkylsulfonates,
semicarbazones, bipyridyliums, benzopyrazoles.
[0042] As used herein, the term "organophosphate pesticide" refers
to a pesticide belonging to class of compounds which are esters of
phosphoric acid, and have the following general chemical
structure:
##STR00001##
[0043] Organophosphates function by inhibiting the
acetylcholinesterase of many living organisms, including human
beings. High-level exposure to organophosphates results in
acetylcholine accumulation, which interferes with muscular
responses and leads to serious damage of vital organs and
eventually death. Organophosphates within the scope of the present
invention include, but are not limited to paraoxon, malathion,
parathion, diazinon, naled, fenthion, dichlorvos, chlorpyrifos,
phosmet, acephate, ethion, soman, sarin, tabun, VX, echothiophate,
isoflurophate, trichlorfon, tetrachlorvinphos, azamethiphos, and
azinphos-methyl.
[0044] In certain aspects, the present invention provides bacterial
host cells that naturally produce minicells comprising one or more
plasmid constructs for expression of one or more proteins within
the same host cells. An expression construct may drive expression
of a single protein, or multiple proteins under the expression of a
single promoter (e.g., to produce a fusion protein), or multiple
promoters. In some embodiments, one or more of the proteins can be
expressed on the surface of the host cells, and the minicells
produced by them. Expression of the protein(s) of interest can be
driven by one or more promoters, including but not limited to
inducible promoters. In certain aspects, the present invention
provides minicell-producing host cells comprising an expression
construct, wherein the expression construct comprises a gene that
causes or enhances the production of minicells operably linked to
expression sequences that are inducible, and wherein induction of
the gene causes or enhances the production of minicells. In some
embodiments, the minicell-producing host cells, include, but are
not limited to a bacterial strain of Escherichia coli, Bacillus
spp., Salmonella spp., Listeria spp., Mycobacterium spp., Shigella
spp., or Yersinia spp. In some embodiments, the gene that causes or
enhances the production of minicells, includes, but is not limited
to, the tubulin-homolog ftsZ, and the inducible expression
sequences can include, but are not limited to isopropyl
.beta.-D-1-thiogalactopyransoide (IPTG) inducible sequences (e.g.,
promoter from the lac operon). Additional exemplary prokaryotic
promoters, which can be regulated (e.g., induced and/or repressed),
and are within the scope of the present invention are shown in
Table 1. In certain aspects, the expression construct further
comprises nucleic acid sequences encoding a reporter polypeptide or
amino acid sequence for monitoring, visualizing, or assaying
protein expression and/or minicell formation. In some embodiments,
the reporter polypeptides or amino acid sequences, includes, by way
of non-limiting example, a green fluorescent protein (GFP), a
luciferase, a beta-galactosidase, a His tag, an epitope, or a
biotin-binding protein such as streptavidin or avidin.
TABLE-US-00001 TABLE 1 Additional Promoter Primarily Used For
Description Expression Considerations T7 In Vitro Promoter
Constitutive, but Can be used for in vitro transcription/general
from T7 requires T7 RNA transcription only if 2 expression
bacteriophage polymerase different phage promoters are present in
opposite orientations to the gene T7lac High levels of gene
Promoter Negligible basal Commonly found in pET expression from T7
expression when not vectors. Very tightly bacteriophage induced.
Requires T7 regulated by the lac plus lac RNA polymerase, which
operators. Good for operators is also controlled by lac modulating
gene operator. Can be induced expression through varied by IPTG
inducer concentrations. Sp6 In vitro Promoter Constitutive, but Can
be used for in vitro transcription/general from Sp6 requires SP6
RNA transcription only if 2 expression bacteriophage polymerase
different phage promoters are present in opposite orientations to
gene Trp High levels of gene Promoter Repressible Gets turned off
with high expression from E. coli levels of cellular tryptophan
tryptophan operon Lac General Expression Promoter Constitutive in
the Leaky promoter with from lac absense of lac repressor somewhat
weak operon (lacI or lacIq). Can be expression. LacIq mutation
induced by IPTG or increases expression of the lactose. repressor
10x, thus tightening regulation of lac promoter. Good for
modulating gene expression through varied inducer concentrations.
Ptac General Expression Hybrid Regulated like the lac Contains -35
region from promoter of promoter trpB and -10 region from lac and
trp lac. Very tight regulation. Good for modulating gene expression
through varied inducer concentrations. Generally better expression
than lac alone. pL High levels of gene Promoter Can be temperature
Often paired with the expression from regulatable temperature
sensitive bacteriophage cl857 repressor lambda araBAD General
Expression Promoter of Inducible by arabinose Weaker. Commonly
found the arabinose and repressed catabolite in pBAD vectors. Good
metabolic repression in the for rapid regulation and operon
presence of glucose or low basal expression; by competitive binding
however, not well-suited of the anti-inducer for modulating gene
fucose expression through varied inducer concentrations
[0045] In certain aspects, the present invention provides a method
for making minicells, the method comprising (a) culturing a
minicell-producing parent cell, wherein the parent cell comprises
an expression construct, wherein the expression construct comprises
a ftsZ gene operably linked to an IPTG-inducible sequence, and
wherein induction of the ftsZ gene causes or enhances the
production of minicells; and (b) separating the minicells from the
parent cell, thereby generating a composition comprising minicells.
In certain embodiments, the method further comprises (c) purifying
the minicells from the composition by centrifugation and/or
filtration. In certain embodiments, one or more additional
expression constructs can be introduced into the minicell-producing
parent cell which comprise ftsZ gene. In such instances, the
expression constructs may be simultaneously or sequentially
introduced into the parent cell prior to induction for minicell
formation, and can comprise one or more selection markers (e.g.,
antibiotic resistance genes) and/or reporter genes to allow for
selection and/or visualization of minicells expressing the
protein(s) of interest. In certain embodiments, the ftsZ expression
construct can express one or more additional proteins, which are
driven by the same or different promoters, including inducible
promoters.
[0046] In certain aspects, the present invention provides a method
for making minicells, the method comprising (a) culturing a
minicell-producing parent cell, wherein the parent cell comprises
at least one expression construct, wherein the at least one
expression construct comprises a gene encoding a biologically
active polypeptide operably linked to an IPTG-inducible sequence,
and wherein induction causes expression of the biologically active
polypeptide; and (b) separating the minicells from the parent cell,
thereby generating a composition comprising minicells. In certain
embodiments, the method further comprises (c) purifying the
minicells from the composition by centrifugation and/or filtration.
In certain embodiments, the parent cell is a bacterial strain that
naturally produces minicells. In certain embodiments, the gene is
INPNC-OPH, which encodes organophosphate hydrolase (OPH) fused to
an ice nucleation protein (INPC). In certain embodiments, the
minicell-producing parent cell can contain more than one expression
construct. In such instances, the parent cell may comprise more
than one expression construct which are introduced either
simultaneously or sequentially into the parent cell, and can
comprise one or more selection markers (e.g., antibiotic resistance
genes) and/or reporter genes to allow for selection and/or
visualization of minicells expressing the protein(s) of
interest.
[0047] In certain embodiments, the invention provides minicells
having about 90-99% purity, or about 90-95%. Purity refers to the
absence of contaminating parental bacteria in the composition.
Minicells can be separated from their parent cells based on their
much smaller size. The minicells provided by the present invention
are typically about 0.4 .mu.m-0.5 .mu.m in diameter. In certain
embodiments, the composition comprises an aqueous solution of
minicells (e.g., E. coli minicells) at a concentration from about
1.times.10.sup.6 to about 1.times.10.sup.9 cells/mL. In certain
embodiments, the minicell solution is prepared in water containing
0.5% zinc (a co-factor for OPH), and applied to the crop or
agricultural product using a spray bottle.
[0048] In certain aspects, the invention provides a method for
pesticide degradation, the method comprising contacting a crop or
an agricultural product with any of the compositions of the
invention disclosed herein, wherein the composition mediates
degradation of the pesticide to yield inert by-products. In certain
embodiments, pesticide degradation can be evaluated by measuring
the levels of one or more by-products.
[0049] In other embodiments, pesticide degradation can be evaluated
by measuring the amount of residual pesticide. In addition, or
alternatively, the half-life of the pesticide may be used to
measure the efficacy of any of the compositions of the invention
disclosed herein. The half-life is the time it takes for a certain
amount of a pesticide to be reduced by half. In general, a
pesticide will break down to 50% of the original amount after a
single half-life. After two half-lives 25% will remain. In some
embodiments, the half-life of the pesticide may be reduced by a
factor of about 2, about 3, about 4, about 5, about 6, about 7,
about 8, about 9, about 10, about 20, about 30, about 40, or about
50 or more. In certain embodiments, invention provides a method for
reducing the half-life of a pesticide (e.g., organophosphate
pesticide). In certain embodiments, the invention provides a method
for reducing the half-life of malathion. In certain embodiments,
the half-life of malathion is reduced to less than 4 hours (<4
h.).
[0050] In certain aspects, the invention provides a method for
bioremediation, the method comprising contacting a crop or an
agricultural product with any of the compositions of the invention
disclosed herein the composition mediates degradation of the
pesticide to yield inert by-products.
EXAMPLES
[0051] The invention is now described with reference to the
following Examples. These Examples are provided for the purpose of
illustration only, and the invention is not limited to these
Examples, but rather encompasses all variations which are evident
as a result of the teachings provided herein.
Example 1
[0052] Construction of an IPTG-Inducible ftsZ Operon for the
Production of E. coli Minicells
[0053] Despite many attractive qualities for synthetic biology,
Escherichia coli and other engineering-amenable bacteria suffer
from a number of inherent biosafety issues that limit their
practical application. A safer alternative exists in the
little-known nano-chassis called a minicell--the anucleoid product
of an aberrant, asymmetric cell division. To introduce minicells to
synthetic biology, a minicell-inducing operon from standardized
parts was engineered. Through IPTG-tunable overexpression of the
tubulin-homolog FtsZ, this operon enables optimization of E. coli
minicell production. Whereas a spatially oscillating "Z ring"
composed of FtsZ protein marks cleavage at the cell midline in wild
type E. coli, overexpression of FtsZ initiates Z-ring assembly
distal to the midline to form a minicell (FIG. 1). These engineered
minicells are achromosomal bacterial particles that are unable to
replicate and cause infection or ecological harm.
[0054] Synthesis and Cloning of ftsZ: Integrated DNA Technologies
(Coralville, Iowa, USA) synthesized the coding sequence of the ftsZ
gene based on NCBI database sequence 944786 flanked by the BioBrick
prefix (gaattcgcggccgcttctag; SEQ ID NO: 1) and suffix
(tactagtagcggccgctgcag; SEQ ID NO: 2). Prior to synthesis, the
sequence was codon optimized to remove an EcoRI restriction site
112 bp 3' of the start site. The synthesized ftsZ gene was cloned
into the vector pSB1C3 (Registry of Standard Biological Parts, iGEM
Foundation, Cambridge, Mass., USA) using EcoRI and PstI restriction
sites, forming construct BBa_K1011000. Using primers VF2
(tgccacctgacgtctaagaa; SEQ ID NO: 3) and VR (attaccgcctttgagtgagc;
SEQ ID NO: 4), the sequence identity was validated by DNA
sequencing and comparison to a ftsZ reference sequence (NCBI
Accession No. 944786).
[0055] Formation of IPTG-Inducible ftsZ Operon: To construct an
IPTG-inducible operon that contains, in order, ftsZ and GFP, an
EcoRI-SpeI fragment containing BioBrick BBa_S05213 was cloned into
the EcoRI and XbaI restriction sites that are 5' of BioBrick
BBa_S05212 in vector pSB1C3, forming BioBrick BBa_K1011001. To
construct BioBrick BBa_S05212 (875 bp), an EcoRI-SpeI fragment
containing BioBrick BBa_S05211 was cloned into the EcoRI and XbaI
restriction sites that are 5' of a double terminator sequence
(BioBrick BBa_B0015) in vector pSB1AK3. BioBrick BBa_S05212 was
subsequently cloned into pSB1C3 using EcoRI and PstI restriction
sites. To construct BioBrick BBa_S05211 (738 bp), an XbaI-PstI
fragment containing GFP coding sequence (BioBrick BBa_E0040) was
cloned into the SpeI and PstI restriction sites that are 3' of the
RBS sequence (BioBrick BBa_B0034) in vector pSB1A3. To construct
BioBrick BBa_S05213 (1233 bp), an XbaI-PstI fragment containing
ftsZ coding sequence (BioBrick BBa_K1011000) was cloned into the
SpeI and PstI sites 3' of an IPTG-inducible promoter and a RBS
(BioBrick BBaK215000) in vector pSB1A3. Using primers VF2 and VR,
the sequence of each construct was validated before submission to
the Registry of Standard Biological Parts (iGEM Foundation,
Cambridge, Mass.).
[0056] Reagents and Culturing of E. coli: Transformed XL1-Blue E.
coli were cultured at 37.degree. C. in Luria Broth (LB) with
antibiotics as appropriate (chloramphenicol 25 mg/L, ampicillin 100
mg/L, or kanamycin 50 mg/L). Competent cells were obtained from
Sigma (St. Louis, Mo., USA). Restriction and DNA modifying enzymes
were obtained from New England BioLabs, Inc. (Ipswich, Mass.,
USA).
[0057] Microscopy: Cells transformed with BioBrick BBa_K1011001 in
pSB1C3 were incubated in LB medium with or without 40 .mu.M IPTG
for 14 h prior to microscopy. Images were acquired at 400.times.
total magnification using an Olympus BX50 microscope with Olympus
DP25 camera attachment, Scion Image software (Scion Corporation,
Frederick, Md., USA), and 40.times. Olympus UPlanFL objective.
[0058] Minicell Purification: Minicells were purified by
differential centrifugation at 2,000 g for 10 min at 4.degree. C.
to pellet the parent bacteria followed by centrifugation of the
supernatant at 10,000 g for 10 min at 4.degree. C. to pellet
minicells. The minicell pellet was then re-suspended in 50 mL LB
and incubated at 37.degree. C., 180 rpm for 90 min with 100 mg/L
ceftriaxone. This dose of ceftriaxone is sufficient to cause cell
lysis without having any detrimental effect on minicell integrity.
The resulting minicell preparation was filtered at room temperature
through a 0.45 .mu.m dead end filter (Millipore SE1M003M00) to
remove any remaining parent cells, followed by additional
filtration with a 0.22 .mu.m cross-flow filter (Millipore
GVWP04700) to remove small cell debris and endotoxins.
[0059] To facilitate the production of minicells two plasmid
constructs were designed containing either (1) the ftsZ gene alone
(FIG. 2A), or (2) an operon consisting of an IPTG-inducible ftsZ
gene and Green Fluorescent Protein (GFP) reporter gene (FIG. 2B).
The first construct (BBa_K1011000) served as an intermediary for
the production of the second construct. Here, this first construct
is made available to the synthetic biology community because this
copy of ftsZ lacks an internal EcoRI restriction site and is thus
compatible with standardized DNA assembly methods. The second
construct (BBa_K1011001) was used for the production of minicells
and contains an operon with two genes, ftsZ, which, when
overexpressed, drives minicell formation, and GFP. GFP expression
serves as a reporter for the transcriptional expression of ftsZ and
enables easy visualization of cells transformed with this
construct.
[0060] To test the functionality of the minicell-producing
construct, XL1-Blue E. coli cells were transformed with
BBa_K1011001. As shown in FIG. 3, fluorescence microscopy revealed
minicell production 14 h post-induction with 40 .mu.M IPTG.
Elongated, rod-shaped cells were also observed as a byproduct of
multiple rounds of asymmetric cell division. As expected, minicell
production occurred post-IPTG induction, and GFP provided a visual
readout of induction and aided minicell detection.
Example 2
[0061] Plasmid Construct for Surface Expression of Organophosphate
Hydrolase (OPH) on Minicells
[0062] An exemplary plasmid construct for surface expression of
organophosphate hydrolase (OPH) on minicells is shown in FIGS.
7A-7B. The Kn.sup.r designation represents the kanamycin resistance
gene used in order to ascertain whether isolated minicells
expressed OPH; any minicell lacking the plasmid would die upon
exposure to sufficient levels of kanamycin. The P.sub.AllacO-1
designation represents the lac promoter that regulates the
expression of our INPNC-OPH protein. In the absence of IPTG,
expression of the INPNC-OPH protein will be actively repressed
until IPTG is in solution. The p15A represents the origin of
replication of the plasmid. This allows for the plasmid to be
copied within the cell in order to ensure that maximal expression
of the desired protein--INPNC-OPH--upon induction with IPTG. The
INPNC-OPH gene codes for organophosphate hydrolase fused to an ice
nucleation protein. The ice nucleation protein allows the OPH to be
expressed on the surface of an E. coli parent cell and thus on the
surface of a resultant E. coli minicell.
Example 3
[0063] Induction and Purification of Minicells
[0064] Bacterial strains that naturally produce minicells (commonly
at a 2:1 ratio (2 bacterial cells for every one minicell) were used
to obtain OPH-expressing minicells (produced using the construct
shown in FIGS. 7A-7B and described in Example 2). Minicells were
purified by differential centrifugation at 2,000 g for 10 min at
4.degree. C. to pellet the parent bacteria followed by
centrifugation of the supernatant at 10,000 g for 10 min at
4.degree. C. to pellet minicells. The minicell pellet was then
re-suspended in 50 mL LB and incubated at 37.degree. C., 180 rpm
for 90 min with 100 mg/L ceftriaxone. This dose of ceftriaxone is
sufficient to cause cell lysis without having any detrimental
effect on minicell integrity. The resulting minicell preparation
was filtered at room temperature through a 0.45 .mu.m dead end
filter (Millipore SE1M003M00) to remove any remaining parent cells,
followed by additional filtration with a 0.22 .mu.m cross-flow
filter (Millipore GVWP04700) to remove small cell debris and
endotoxins.
Example 4
[0065] Degradation of Paraoxon (Organophosphate) by Minicells
Expressing/Displaying Organophosphate Hydrolase (OPH) on their
Surface.
[0066] Degradation of paraoxon by the OPH-expressing minicells
(produced using the construct shown in FIGS. 7A-7B and described in
Example 2) was studied. The results are shown on FIGS. 10A-10B.
Example 5
[0067] Field Studies of Malathion Degradation by Minicells
Expressing/Displaying Organophosphate Hydrolase (OPH) on their
Surface.
[0068] A minicell solution prepared from purified E. coli minicells
(e.g., as described in Example 3 above) The minicell solution
comprises about 1.times.10.sup.6 to about 1.times.10.sup.9
minicells/milliliter of the solution. The minicell solution was
prepared in water containing 0.5% zinc (the cofactor for OPH), and
applied with a spray bottle to grapes grown in several vineyards.
The application was performed once, but could be applied multiple
times if necessary or desirable. In order to assay for pesticide
degradation, samples of leaves, grapes, and runoff were collected
at varying time points up to six hours (t=0 min, t=0.1 min, t=15
min, t=30 min, t=45 min, t=60 min, t=90 min, t=120 min, t=150 min,
t=180 min, t=240 min, t=300 min, t=360 min), and the residual
pesticide was extracted with ethyl acetate, and analyzed with mass
spectrometry. The results of the study are shown in FIG. 11.
Sequence CWU 1
1
4120DNAArtificial SequenceBioBrick prefix 1gaattcgcgg ccgcttctag
20221DNAArtificial SequenceBioBrick suffix 2tactagtagc ggccgctgca g
21320DNAArtificial SequencePrimer VF2 3tgccacctga cgtctaagaa
20420DNAArtificial SequencePrimer VR 4attaccgcct ttgagtgagc 20
* * * * *