U.S. patent application number 12/948887 was filed with the patent office on 2011-05-19 for methods and compositions for genetically manipulating clostridia and related bacteria with homologous recombination associated proteins.
Invention is credited to Eleftherios Papoutsakis, Bryan Tracy.
Application Number | 20110117655 12/948887 |
Document ID | / |
Family ID | 44011565 |
Filed Date | 2011-05-19 |
United States Patent
Application |
20110117655 |
Kind Code |
A1 |
Tracy; Bryan ; et
al. |
May 19, 2011 |
METHODS AND COMPOSITIONS FOR GENETICALLY MANIPULATING CLOSTRIDIA
AND RELATED BACTERIA WITH HOMOLOGOUS RECOMBINATION ASSOCIATED
PROTEINS
Abstract
Methods for effecting homologous recombination in a bacterium of
the Clostridia family are described. These methods provide enhanced
capability to genetically modify clostridia.
Inventors: |
Tracy; Bryan; (Wilmington,
DE) ; Papoutsakis; Eleftherios; (Newark, DE) |
Family ID: |
44011565 |
Appl. No.: |
12/948887 |
Filed: |
November 18, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61262288 |
Nov 18, 2009 |
|
|
|
Current U.S.
Class: |
435/471 ;
435/252.3; 435/440 |
Current CPC
Class: |
C07K 14/33 20130101;
C12N 15/1082 20130101; C12N 15/74 20130101 |
Class at
Publication: |
435/471 ;
435/440; 435/252.3 |
International
Class: |
C12N 15/74 20060101
C12N015/74; C12N 15/00 20060101 C12N015/00; C12N 1/21 20060101
C12N001/21 |
Claims
1. A method for effecting an efficient single-crossover or
double-crossover homologous recombination in a population of
bacteria comprising singly or combinatorially expressing a
recombination protein selected from the group consisting of RecG,
RecO and RecA, wherein recombinant bacteria are produced.
2. The method of claim 1, wherein the homologous recombination
proteins are derived from any RecG, RecO, or RecA gene or variant
capable of functioning in the recombinant bacteria.
3. The method of claim 1, wherein functional RecG, RecO, RecA and
combinations thereof are expressed by the recombinant bacteria.
4. A recombinant bacterium produced by the method of claim 1.
5. The method of claim 1, wherein the genetic heterogeneity of the
population of recombinant bacteria is increased.
6. The method of claim 1 further comprising screening the
recombinant bacteria for desired phenotypic traits.
7. A method for integrating a recombinant gene into a host
chromosome for recombinant protein expression comprising the method
of claim 1.
8. The method of claim 7, wherein the integrated recombinant gene
is a synthetic operon.
9. The method of claim 7, wherein expression of a gene is
suppressed.
10. The method of claim 7, wherein expression of a gene is
stimulated.
11. The method of claim 7, wherein the timing of expression of a
gene is altered.
12. The method of claim 7, wherein expression of a first gene is
repressed by the expression of a second gene.
13. The method of claim 7, wherein expression of a first gene is
induced by the expression of a second gene.
14. The bacterium of claim 4, wherein the bacterium is a
Clostridial bacterium.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Patent Application No. 61/262,288, filed Nov. 18, 2009, which is
incorporated herein by reference in entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to methods and compositions
for genetically manipulating bacterial cells, particularly a cell
of the class Clostridia, but also related bacteria which are
difficult to genetically manipulate due to lack of an effective
recombination system. In particular, embodiments of the present
invention relate to the expression of recombinant homologous
recombination proteins in Clostridia and in other bacterial species
as demonstrated in the following provisional patent.
BACKGROUND OF THE INVENTION
Clostridia and its Potential Use in Industry
[0003] Rising and unstable prices for petroleum based chemicals and
fuels have resulted in renewed interest in their production via
alternative approaches (e.g. biochemical approaches). Coupled with
concerns of global climate change and securing a domestic source of
transportation fuels, efforts are being focused towards the
fermentative conversion of inexpensive renewable feedstocks
(biomass) to fuel alcohols and chemicals. Such processes have been
employed for over a century at very large scale, but advanced
genetic and metabolic engineering approaches for generating
second-generation chemical and fuel producing microbes are required
for making these current ventures commercially viable.
[0004] Clostridia are strictly anaerobic, endospore forming
prokaryotes of major importance to cellulose degradation, human and
animal health and physiology, anaerobic degradation of simple and
complex carbohydrates, acidogenesis, and bioremediation of complex
organics [10]. Solventogenic, butyric-acid clostridia (e.g.,
Clostridium acetobutylicum, C. beijerinckii and C. butyricum) [11]
played a major industrial role in the production of acetone and
butanol in the past (and likely now and in the future) by the
Acetone-Butanol-Ethanol fermentation (ABE) (Jones and Woods 1986;
Rogers 1986; Lesnik, Sampath et al. 2001). Significantly, metabolic
engineering (ME) of solventogenic clostridia, as recently reviewed
[9], may lead to industrial processes for production of additional
chemicals such as butyric acid, butanediol, propanol, and acetoin
(Jones and Woods 1986; Rogers 1986; Lesnik, Sampath et al. 2001),
production of hydrogen [12] or for biotransformations [13]. Some of
these chemicals (butanol, ethanol) can serve as biofuels directly,
while others can be used for chemical conversion to biofuels (e.g.,
butyric acid [14]) or the generation of electricity [12]. Related
clostridia can produce additional chemicals such as propionic and
acrylic acids [15] [16]. Finally, clostridia, as might be expected
from these ancient anaerobic soil organisms, have a great potential
for applications in bioremediation [17].
[0005] Based on the fundamental and applied importance of this
genus, the DOE has completed the genome sequence [18] of C.
acetobutylicum ATCC 824 (referred to as Cac from now on). A number
of ME tools have been developed for this genus of bacteria such as
recombinant DNA expression plasmids [19], antisense RNA approaches
[20], and gene expression libraries [21]. However, the full
potential of any industrially relevant species will not be fully
realized until an efficient chromosomal integration system is
developed that allows for more elaborate and stable genetic
manipulations of the host. Such a system would ideally be suitable
for all clostridia species, be able to disrupt desired genes, be
able to integrate large pieces of recombinant DNA into the host
chromosome, and be easily and rapidly implemented in any research
and R&D setting (academic or industrial). Methods for gene
inactivation in clostridia have been inefficient, first based on
non-replicating vectors [22], and later using a replicating vector
(Harris, Welker et al. 2002). These methods however are tedious,
slow and require substantial time and effort commitments. Moreover,
they are rarely successful in inactivating genes. Most recently,
the TargeTron.TM. system (group II intron principle) was adapted to
clostridia by two different groups (Heap, Pennington et al. 2007;
Shao, Hu et al. 2007). There are also two other more recently
reported methods, the first method was developed by the research
group of Dr. P. Soucaille at INSA of Toulouse, in collaboration
with the company Metabolic Explorer. They developed a novel
endonuclease expression technique to digest replicating plasmid DNA
into linear disruption cassettes within the clostridia host that
can then recombine via homologous recombination into the target
chromosomal region. A patent has been filed based on their work
[23] and a paper has been published that employed this method [24].
The other was a suicide plasmid approach developed by the inventors
(Bryan Tracy and Eleftherios Papoutsakis) and was used to knockout
acid-formation genes in the Cac asporogenous mutant M5 [25].
However, all approaches are severely limited in multiple
regards.
[0006] First, the group II intron and endonuclease based methods
have not demonstrated the ability to incorporate more than
.about.1.5 kB of DNA into the chromosome, and the majority of 1.5
kB is already consumed by a selection marker. Secondly, the suicide
approach has only been demonstrated in one specific strain of C.
acetobutylicum, the M5 strain, and has yet to be successful in any
other clostridia strain.
[0007] Homologous Recombination
[0008] Homologous recombination is a housekeeping process involved
in the maintenance of chromosome integrity and generation of
genetic variability that is nearly ubiquitous to all microorganisms
[26-28]. The cellular machinery involved is not necessarily
conserved, but the general series of events is common to all
microorganisms studied to date. The typical series of events for
homologous recombination are initiation, strand-invasion,
strand-exchange, and Holliday junction resolution [28-30], see FIG.
1. Within specific classes of bacteria, the proteins involved in
homologous recombination are fairly well conserved and are given
for Clostridia in FIG. 1. The specific C. acetobutylicum genes
involved are given in Table 1 and FIG. 1, which were determined by
a best-best blast search to Bacillus subtilis ATCC23857. B.
subtilis serves as the model Gram-positive organism.
[0009] Absense, Importance and Utility of Resolvase Expression
[0010] The most essential protein in the later stages of homologous
recombination is the resolvase. Resolvases are a well-known class
of proteins that perform a key role in Holliday-junction
resolution. There are a number of distinct resolvase enzymes, and
resolvase activity is ubiquitous to nearly all bacteria [28, 31].
Holliday-junctions are four-way DNA intermediate complexes formed
during homologous recombination [32]. There are two major
resolvases found on the genomes of Gram-negative and Gram-positive
bacteria. These are ruvC and recU, respectively [28, 33]. The
significance of resolvases, and more specifically of recU in
Gram-positive organisms was studied via deletion mutants and tested
by the deficiency in DNA repair and intramolecular recombination
[33-35]. These studies strongly support the essential role of RecU
in Holliday-junction resolution for Gram-positive organisms, such
as clostridia and bacilli. Subsequent studies determined
high-resolution structures of RecU from Bacillus subtilis and B.
stearothermophilus and proposed detailed models for how the RecU
protein physically interacts with the Holliday-junction [36, 37]. A
recent comparative genomic analysis suggests that clostridia do not
contain genes for any recognizable resolvase protein [28]. Thus, we
hypothesized that the lack of resolvase activity is responsible for
the experimental difficulty in generating homologous recombination
events for gene disruptions in all clostridia [38].
[0011] The analysis by Rocha et al. [28] demonstrated the absence
of resolvase activity in Clostridium perfringens, C. tetani and
Cac, which were all the clostridia genomes they analyzed.
Significantly only 10 genomes out of the 110 analyzed appeared to
be resolvase deficient. Of those 10, four were void of any sort of
recombination system. To further extend their analysis, we
performed our own homology searches of the B. subtilis recU gene
against six annotated or draft clostridia genome sequences, based
upon the first round of orthology assignment performed in the Rocha
et al. paper. The final analysis included Cac, C. perfringens, C.
tetani, C. difficile 630, C. novyi NT, C. thermocellum, C.
beijerincki, and C. cellulolyticum. All genomes were void of any
discernable RecU resolvase, which is the conserved resolvase for
Gram-positive organisms.
[0012] We thus tested the hypothesis that RecU expression in
clostridia will result in efficient recombination. Specifically, we
over-expressed the heterologous resolvase from B. subtilis, RecU
(coded by recU), in Cac under the control of the strong Cac
thiolase promoter. For this, we used a replicating plasmid that
contained two contiguous regions of homology for a gene on the Cac
chromosome. Initial investigations targeted the sigma factor sigE,
which is a known transcriptional regulator in clostridia
sporulation and possibly also solvent formation [10]. The final
plasmid, targeted against a specific gene is referred to as a
site-specific chromosomal integration plasmid (SSCI plasmid). For
sigE we refer to this as the sigE SSCI plasmid. The two regions of
homology were disrupted by a thiamphenicol (TH) antibiotic
resistance gene. There was also an erythromycin (EM) antibiotic
resistance gene on the plasmid, outside of the regions of homology.
Therefore if a double crossover event between the host chromosome
and plasmid were to occur, the TH marker would be incorporated into
the chromosome and the EM marker would be lost upon plasmid curing,
which describes the loss of the plasmid. If a single crossover
occurred, both TH and EM markers would be incorporated in the
chromosome, but EM resistance would not be as strong upon plasmid
curing, because cells require multiple copies of the EM resistance
gene to be very EM resistant. A single copy of the TH resistance
gene is sufficient for strong TH resistance.
[0013] Resolvase Expression is not Sufficient for Efficient
Double-Crossover Recombination
[0014] Previous analyses, for which a utility patent was filed,
showed that resolvase expression improved single-crossover
efficiency very well, but double-crossover homologous recombination
was still only witnessed in one example out of many. Therefore, we
hypothesized that homologous recombination proteins are not
well-expressed and/or down-regulated during exponential growth, the
period of culture during which we believe homologous recombination
is occurring.
[0015] Current State of the Technology for Generating Targeted Gene
Disruptions in Clostridia
[0016] In regards to gene disruption via homologous recombination,
the current state of the art is to employ the Clostridia host's
homologous recombination machinery for double crossover
recombination. Recombination occurs between parent chromosome and
plasmid-borne homologous regions that flank a selectable marker,
see FIGS. 1-3.
[0017] For C. acetobutylicum there are only five published reports
of site-specific integration; three via non-replicating (suicide)
plasmids [22, 25, 39], one via a replicating plasmid [40] and a
third via linear DNA created within the host cell [24]. The first
attempt utilized a suicide plasmid with an integration cassette
composed of .about.225 bp nucleotide sequences of contiguous
homology flanking a macrolide-lincosamide-streptogramin B
resistance (MLSr) gene. The MLSr gene confers erythromycin (EM)
resistance. The plasmid was introduced into C. acetobutylicum via
electroporation, and knockout mutants were selected for EM
resistance. Only mutants that had undergone a recombination event
could maintain EM resistance. This technique was successful three
times for the generation of pta, bk and aad mutants [22, 39].
However, integration efficiency was very low (, 0.5 mutants/.mu.g
transformed KO plasmid DNA), the approach was and unsuccessful for
many additional targets, and the nature of integration is still not
fully characterized (i.e. the region of integration has yet to be
sequenced and confirmed). A similar approach was performed to
knockout the bk and ak genes in the degenerate C. acetobutylicum
strain M5 [25]. This approach employed a "stronger" antibiotic
marker (i.e. chloramphenicol under the strong ptb promoter). The
integration was confirmed (i.e. sequenced) to have perfectly
integrated through a single region of homology. Unfortunately this
approach has not been successful in the WT C. acetobutylicum
strain.
[0018] A second approach was later developed that employed a
replicating plasmid. When using the replicating approach, an
additional selection marker is required outside the integration
cassette in order to prove the loss of plasmid after a successful
of double crossover recombination. For this approach a
thiamphenicol (CM/TH) resistance gene was employed. Following
electroporation, transformed cells are selected for EM resistance.
Transformants were then vegetatively transferred six times on
non-antibiotic nutrient plates. The seventh and eighth transfers
were onto EM and TH containing plates, respectively. These two
plates were compared for regions of growth on EM but not on TH,
suggesting double crossover recombination and loss of plasmid. So
far this approach has been successful at generating only a handful
of mutants such as spo0A mutant [40], CAC8241 mutant and ctfAB
mutant [41], and all subsequent attempts at additional targets have
been unsuccessful.
[0019] Among other clostridia species there have been few
successful attempts at generating targeted chromosomal integrations
via suicide and replicating plasmids [42-44]. Most notable is the
use of multimeric, suicide plasmids in C. perfringens, but this
approach is unreliable (i.e. low frequency of integration) the
mechanism is very poorly characterized [45]. Thus a different sort
of gene disruption system was adapted to Clostridia in order to
increase site-specific integration efficiency. The group II intron
system developed by the Lambowitz lab at University of
Texas--Austin, now commercialized by Sigma-Aldrich (TargeTron.TM.),
has been employed on multiple occasions to generate gene
disruptions in C. perfringens and C. acetobutylicum [46-48]. A more
intensive study modified the TargeTron.TM. specifically for
application in clostridia species. The ClosTron system has been
employed to generate gene disruptions in C. acetobutylicum, C.
difficile, C. botulinum and C. sporogenes [38]. Group II introns
are naturally occurring autocatalytic retrotransposable elements
that include a six stem-loop RNA structure complexed with an
intron-encoded protein (IEP). The IEP exhibits four unique
activities: 1) maturase for intron splicing, 2) DNA binding for
target site recognition, 3) endonuclease for nicking host
chromosome and 4) reverse transcriptase for forming intron cDNA.
Group II introns can insert RNA directly into target DNA sequences
and then reverse transcribe themselves. DNA is targeted mainly by
base pairing of the intron RNA, however the IEP also recognizes a
few base pairs. Subsequently, group II introns can theoretically be
engineered to target any desired DNA sequence by modifying the
intron RNA [49].
SUMMARY OF THE INVENTION
[0020] The engineering of microbes for specialty chemical
conversion, biofuel generation, bioremediation and pharmaceutical
production remains an immediate scientific and industrial goal.
Specifically for the class Clostridia among prokaryotes, the
pursuit of industrial scale biofuel production, replacement of
fossil feedstock chemicals with biomass, and the production of
value added chemicals from industrial and municipal waste streams
is motivating a tremendous amount of strain development. Clostridia
are naturally some of the most prolific cellulosic-material
fermenting microbes [1-4], or exhibit great potential to be
metabolically engineered into cellulose utilizing bacteria [5-8].
Additionally, due to the anaerobic and spore forming
characteristics, clostridia are being engineered to target the
necrotic and anaerobic cores of malignant tumors and to essentially
kill tumors, inside out (REFS).
[0021] The tools for genetically manipulating clostridia remain
limiting and insufficient for harboring the total potential of this
class of bacteria [9]. Advances have occurred slowly over the past
thirty years, but need to be dramatically accelerated, especially
given the recent interest in biofuels and white biotechnology.
Three of the more. notable limitations, which can be addressed with
our technology, are engineering gene specific mutants for gene
inactivation, generating genetically diverse mutant populations for
genome scale library screenings, and being able to add/delete large
lengths of chromosomal DNA in any host clostridia strain.
[0022] We have invented a novel approach for genetically altering
clostridia. Our invention in its simplest form is the recombinant
expression of the homologous recombination associated proteins
RecO, RecG, and RecA derived from any heterologous source that is
naturally compatible (i.e. can be transcribed and translated) or
engineered to be compatible (e.g. codon usage of heterologous gene
is varied to be readily transcribed in the bacteria host) any
clostridia species or other difficult to genetically manipulate
species. Expression can be independent or in combinations,
particulary with RecU. We demonstrate that during independent or
combinatorial expression, homologous recombination is stimulated at
higher frequency than in the absence of recombinant homologous
recombination protein expression. Additionally, we demonstrate that
this approach is feasible and delivers similar advantages in other
clostridia species, particulary C. cellulolyticum and C.
cellulovorans, but also including C. butyricum, C. thermocellum, C.
tyrobutyricum, C. beijerinckii, C. perfringens, C. tetani, C.
difficile, C. botulinum, C. sporogenes, and C. novyi.
[0023] The utility of our technology is the enhanced capability to
genetically modify clostridia. We demonstrate this utility through
the proceeding examples in Clostridium acetobutylicum: 1) enhanced
frequency for site-specific homologous recombination by independent
recombinant expression of a homologous recombination protein, 2)
enhanced frequency of site-specific homologous recombination by
combinatorial recombinant expression of homologous recombination
proteins (particularly with RecU), 3) enhanced frequency of
site-specific double crossover homologous recombination by
combinatorial recombinant expression of homologous recombination
proteins (particularly with RecU), and 4) We also demonstrate the
exact same advantages in C. cellulolyticum and C.
cellulovorans.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 is a DNA-microarray analysis of the essential
homologous recombination proteins for Cac. Expression data is shown
as both differential expression (left) and expression intensity
(right). For differential expression, green means down-regulated
compared to the pooled RNA from the entire time course and red
means up-regulated. For the expression intensity, darker blue means
the gene gave higher fluorescence intensity for its specific probes
on the microarray against all other genes and vice versa for the
lighter blue. Grey refers to time points whereby a gene was not
differentially expressed or did not generate a detectable signal on
the microarray.
[0025] FIG. 2 is a schematic of a SSCI plasmid incorporating into
its targeted genomic region through a double crossover integration,
which is commonly believed to occur via a Campbell-like mechanism.
This schematic differs from what is described in Task 1 by the
antibiotic markers being switched. The schematic shows the EM
marker (MLSr) being present in the disruption cassette and the TH
marker being on the plasmid backbone. However, to screen for single
crossovers, the EM marker must be on the vector backbone and TH
marker in the disruption cassette. We are interested in both single
and double crossovers, thus we switched these markers.
[0026] FIG. 3 is a schematic of all possible integration events and
the PCR reactions that we perform to determine the nature of the
integration. PCR primers are as listed: red--Conf-F; blue--Conf-R;
green--CM/TH-F; and yellow--CM/TH-R.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The simplest explanation of our technology is the
recombinant expression of any individual or combination of the
homologous recombination proteins RecG, RecO or RecA. This can be
in any clostridia host or related bacteria species and source of
the recG, recO or recA can be any natural or engineered
heterologous gene. Specific applications include complementing a
clostridia or related species with the aforementioned genes. The
genes can be expressed individually, in combination, from the
site-specific chromosome targeted integration plasmid, from a
separate plasmid, or from chromosomal integration into a host
organism. The expression is used for gene knockins, gene knockouts,
constructing gene knockin/knockout libraries, creating chromosomal
expressed fusion proteins, etc.
Example 1
Analysis of Publicly Provided DNA Microarray Data for Expression of
Homologous Recombination Proteins in Clostridium acetobutylicum
[0028] We analyzed the expression profiles and absolute expression
levels of each homologous recombination proteins from a detailed
time profile of a batch culture of the WT Cac strain (ATCC824)
[50]. Expression profiles refer to the differential expression of
the gene over all growth phases (exponential, transition,
early-stationary, mid-stationary and late-stationary), which were
determined by hybridizing cDNA from a specific period of growth
against a pool of cDNA from all periods of growth. The expression
level was determined by ranking each gene from the full genome
microarray on a percentage scale of 0 to 100. Genes with greater
expression at a specific time point, as determined by greater
fluorescence intensity on the microarray, are ranked closer to 100.
Genes that showed very low intensity were ranked closer to 0. FIG.
1 presents the results.
[0029] Based upon the microarray analysis, differential expression
suggests that a number of homologous recombination initiation
proteins are upregulated during exponential growth, such as recO,
recN, recJ and recD. However, the most important "strand exchange"
protein, RecA, is down-regulated during exponential growth. Looking
at the expression rankings, it suggests that recO and recG are very
lowly expressed (<23rd percentile ranking for all timepoints for
recO and <31st percentile for recG). Based upon these findings
and recU over-expression results, we believe homologous
recombination can be enhanced by over-expressing recO, recG and/or
recA.
Example 2
Assess the Feasibility of Stimulating Recombination Via Expression
of Homologous Recombination Proteins in Conjunction with RecU
[0030] Base upon our findings from the DNA-microarray
transcriptional analysis, as discussed above, we believe that RecG,
RecO and RecA are ideal targets for over-expression. Additionally
we will test the expression of a heterologous RecA (from B.
subtilis) since this approach was successful with the B. subtilis
resolvase (RecU) expression.
Example 2.A
To Express Homologous Recombination Proteins from a Replicating,
Site-Specific Chromosomal Integration Plasmid (SSCI Plasmid)
[0031] Each homologous recombination protein (RecO, RecG,
endogenous RecA and heterologous RecA) will be PCR amplified from
Cac genomic DNA with an appended thiolase promoter (P.sub.thl) on
the 5'-primer and a rho-independent transcription terminator
sequence on the 3'-primer. The thiolase promoter is a strong,
growth-associated promoter, commonly used in Cac for gene
over-expression, and was used in our previous studies with RecU
expression. The rho-independent terminator is a palindromic
sequence that forms a stem-loop, hairpin structure when
transcribed, causing the RNA polymerase to dissociate from the DNA
thus terminating transcription. Due to the presence of the
P.sub.thl and the rho-independent terminator, each PCR product is a
single transcriptional unit. The resulting PCR products will be
individually cloned into the sigE-targeted, replicating, SSCI
plasmid (sigE-SSCI plasmid). This is the same SSCI plasmid we
previously employed for disrupting the sigE locus via a single
crossover event with RecU over-expression, thus already has RecU
over-expression.
[0032] The 5' region of homology is 253 basepairs (bp) and the 3'
region is 306 bp. The regions of homology are contiguous to the
targeted region of the chromosome, and are disrupted on the plasmid
by a thiamphenicol (TH) antibiotic resistance marker (refer to FIG.
2 for a schematic of such a plasmid and integration event). The
regions of homology and disrupting TH marker (also a
chloramphenicol (CM) resistance marker in E. coli) are collectively
referred to as the disruption cassette. In addition to the
disruption cassette, the SSCI plasmid contains an erythromycin (EM)
antibiotic marker and origins of replication for both Gram-negative
and Gram-positive bacteria, which we collectively refer to as the
plasmid backbone. The plasmid is shuttled through a strain of E.
coli containing methylase activity, which methylates the SSCI
plasmid prior to transforming Cac [51]. Cac is electrotransformed
via a well-established protocol developed in the Papoutsakis' lab
(Mermelstein, Welker et al. 1992; Mermelstein and Papoutsakis 1993)
and transformants are confirmed via TH and EM resistance.
Expression of each homologous recombination protein will be
confirmed by reverse transcription PCR.
Example 2.B
Inducing and Screening for Site-Specific Chromosomal Integration
(SSCI)
[0033] To induce SSCI, we grow cells harboring the SSCI plasmid for
5 days under vegetative growth conditions and under TH selection.
This is done by replica plating cells every 24 hours onto a fresh
nutrient plate with TH selection. Cells grow exponentially to
create a "lawn" of growth within 24 hours and are then replica
plated again with velveteen squares and a replica-plating device.
TH selection is maintained for a period of 5 days in order to
either maintain cells harboring the SSCI plasmid or to maintain
cells that have integrated the SSCI plasmid into the chromosome via
either a single or double crossover event. A single-crossover event
incorporates the entire SSCI plasmid (disruption cassette and
plasmid backbone), and a double-crossover event replaces the
endogenous regions of homology with the disruption cassette, and
excises the SSCI plasmid backbone. Therefore, SSCI plasmid
harboring cells, single-crossover and double-crossover cells will
be maintained during the TH replica plating. Cells that lose the
plasmid and have not undergone a crossover event will be lost from
the population.
[0034] Prior to screening, we "cure" cells of the SSCI plasmid by
replica plating for 5 days under vegetative growth conditions
without any antibiotic selection. During this time, cells are
likely to lose the replicating plasmid since there is no selection
for its maintenance, but copies of the TH marker that have
integrated into the chromosome are maintained. Additionally, copies
of the EM marker that have integrated into the chromosome via
single-crossover events are also maintained, unless a second
crossover event occurs and excises out the plasmid backbone.
[0035] For screening, plates are replica plated after the 5th day
of no antibiotic pressure onto fresh nutrient plates with TH
selection. Cells that were "cured" of the plasmid and did not
undergo a crossover event will be lost from the population under TH
selection. Cells are allowed to grow for 24 hours under TH
selection and then replica plated onto fresh nutrient plates with
EM selection. Cells that still harbor the SSCI plasmid or have
undergone a single-crossover event will grow on the EM plates in 24
to 48 hours. Cells that have plasmid borne resistance to EM grow
within 24 hours of replica plating. Cells that have
single-crossover, chromosomal borne EM resistance require at least
36 and more often 48 hours to grow because there is only a single
copy of the EM resistance gene compared to 5-15 copies from the
replicating SSCI plasmid (the average copy number of these plasmids
is 7). Cells that do not grow at all on EM plates, but do grow on
TH are indicative of double-crossover events. Table 1 outlines the
selection criteria and likely explanation for each cell type.
TABLE-US-00001 TABLE 1 TH Resistance EM resistance Likely Genotype
+ - Double-cross + + Single-cross + ++ Plasmid - - No cross or
plasmid
[0036] Table 1. The possible phenotypes from SSCI screening and the
likely genotype associated with each phenotype. Cross is in
reference to crossover.
Example 2.C
Confirming Site-Specific Chromosomal Integration and Determining
the Relative Effectiveness of Each Homologous Recombination Protein
Expression in Conjunction with RecU
[0037] The current standard for confirming SSCI is sequencing the
genomic region about which the integration event occurred. For
double-crossover integrations, this is a simple task of PCR
amplifying the region where integration occurred (refer to FIG. 3).
We will use PCR primers (Conf-F and Conf-R) flanking the regions of
homology where the integration is expected to occur. PCR product
should include the chromosomal region and the disrupting TH marker.
This is then sequenced for confirmation.
[0038] In the case of single-crossover integrations, the PCR
amplification of the region of integration is not easy to perform
because the PCR product would typically be greater than 6000 bp and
will be susceptible to a lot of mispriming due to incomplete
product extension. However, by knowing the orientation of the TH
marker in relation to the gene we are attempting to disrupt (i.e.,
whether the TH marker is in the same or opposite coding strand of
the gene of interest), we can perform two PCR reactions to
determine if crossover occurred through the first or second region
of homology. This is depicted in FIG. 3. If in the same coding
strand, a single-crossover through the first region of homology is
confirmed by a .about.1500 bp PCR product, when using the PCR
primer combination of Conf-F, and CM/TH-R. The Conf-F refers to the
5' flanking PCR primer for the region of integration and the
CM/TH-R refers to the 3' PCR primer for amplifying the TH marker.
If crossover occurred through the second region of homology you
will obtain a .about.1500 bp PCR product when using the PCR primer
combination of Conf-R and CM/TH-F. The Conf-R refers to the 3'
flanking PCR primer for the region of integration and the CM/TH-F
refers to the 5' PCR primer for amplifying the TH marker. If the TH
marker and disrupted gene sequence are in opposite coding strands
you would employ different primer sets and expect different results
from the PCR reactions. Table 2 outlines the appropriate primer
sets for confirming which region of homology a single-crossover
occurred for both orientations of TH marker and disrupted gene.
TABLE-US-00002 TABLE 2 Orientation of CM/TH marker Appropriate
Results from in relation to primer sets for colony PCR(+,
Interpretation disrupted gene confirming region PCR product; -, of
PCR sequence of integration no PCR product) results CM/TH and 1.
Conf-F with 1. + Integration disrupted gene CM/TH-R through
1.sup.st sequence are in 2. Conf-R with 2. - region of the same
coding CM/TH-F homology strand 1. Con-F with 1. - Integration
CM/TH-R through 2.sup.nd 2. Conf-R with 2. + region of CM/TH-F
homology CM/TH and 1. Conf-R with 1. + Integration disrupted gene
CM/TH-R through 1.sup.st sequence are in 2. Conf-F with 2. - region
of opposite coding CM/TH-F homology strands 1. Conf-R with 1. -
Integration CM/TH-R through 2.sup.nd 2. Conf-F with 2. + region of
CM/TH-F homology
[0039] Table 2. List of appropriate primer sets to use when
confirming a single integration event through the 1.sup.st and
2.sup.nd region of homology. The table also details possible
results and the most probably explanation of such results.
[0040] Eventually we need to determine the exact sequence of the
entire region of integration. So after confirming a putative
single-crossover clone by the aforementioned PCR method, we perform
XL (extra-long) PCR reactions under an assortment of reaction and
annealing temperature conditions to obtain specific and large
quantities of PCR product that can then be sequenced.
[0041] To determine the relative overall effectiveness of each
homologous recombination protein in conjunction with RecU at
stimulating and enhancing recombination, we first determine whether
single or double-crossovers occur at all. Our comparison control is
the sigE-SSCI plasmid without any homologous recombination protein
expression. Previously, such experiments never generated single of
double-crossover events without the expression of the RecU protein.
Thus the ability to generate either a single or double-crossover
event is a positive outcome. However, there are no established
protocols for quantitatively determining the effectiveness of
stimulating homologous recombination. Therefore we propose the
following semi-quantitative approach, which will likely be
necessary for comparing the results from each homologous protein
expression against each other.
[0042] Semi-quantitative analysis will be performed by first
quantifying the physical area on each TH screening plate that
indicates single or double crossover integration. Subsequently we
will determine the frequency of single and double-crossover events
per colony screened as determined by PCR confirmation. This value,
multiplied by the physical area of single or double integration
from the TH screening plates will represent the relative
effectiveness (RE) for enhancing and stimulating chromosomal
integration.
Example 3
Feasibility Analysis of Further Enhancing Homologous Recombination
Via the Resolvase Only Over-Expression by Varying Length of
Homologous DNA and the Presentation of the Disruption Cassette
[0043] We have already demonstrated the utility of resolvase (RecU)
expression for stimulating homologous recombination. However, we
will continue to investigate various parameters that affect the
frequency of the recombination events, as well as parameters that
affect the frequency of single versus double-crossover events.
Example 3.A
The Impact of the Length of the Homologous Regions for
Recombination
[0044] As mentioned, the majority of our experiments has and will
continue to use regions of homology that are 250-300 bp long.
However, the majority of clostridia literature that has attempted
chromosomal integration via homologous recombination, reports using
regions of homology that are significantly longer. Therefore we
will investigate the significance of the length of the homologous
regions. Specifically we will test 1000, 500, 250 and 100 bp
regions of homology aiming as above to integrate into the sigE
locus. We will construct new disruption cassettes and clone them
into the already made sigE-SSCI plasmid that contains the
RecU-P.sub.thl expression. We will stimulate, screen, confirm and
determine the relative effectiveness of enhancing recombination for
each length of homology by the methods described above.
Example 3.B
Varying the Presentation of DNA for Homologous Recombination:
Linear DNA Versus Circular DNA on a Suicide Plasmid Versus Circular
DNA on a Replicating Plasmid
[0045] Other common approaches for integrating DNA into the
chromosome include linear DNA (i.e. the Longtine approach employed
in yeast [52]) and suicide/non-replicating plasmids, which has been
reported in Cac but cannot be routinely performed. We will attempt
these same approaches by creating the strain 824(pRecU), which
expresses RecU-P.sub.thl from a separate plasmid than the SSCI
plasmid.
[0046] EM resistance provided on the pRecU plasmid will maintain
RecU expression. We will transform 824(pRecU) with either a linear
DNA-disruption cassette or a suicide SSCI plasmid that contains a
disruption cassette but no origin of replication for Gram-positive
organisms, such as pAKKO from a recent publication from the
Papoutsakis group [25]. Transformants that survive TH selection
theoretically must have undergone a chromosomal integration event
because suicide plasmids and linear DNA cannot replicate. In this
approach, RecU is under the expression of the strong,
growth-associated thiolase promoter. Thus, at the time of
transformation, the competent cells should be actively expressing
RecU and RecU will serve the same purpose of promoting
recombination as demonstrated via the replicating SSCI plasmid
approach. RecU expression will again be verified by reverse
transcription PCR. Resulting TH resistant mutants are readily cured
of the pRecU plasmid by vegetatively transferring without EM
selection. We will test a range of DNA amounts for each approach,
from 50 .mu.g to 0.1 .mu.g of DNA per transformation. We typically
use 0.5 .mu.g of DNA for transforming a replicating plasmid. We
will stimulate, screen, confirm and determine the relative success
at enhancing recombination by the methods described previously.
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