Patent Application: US-94888710-A

Abstract:
methods for effecting homologous recombination in a bacterium of the clostridia family are described . these methods provide enhanced capability to genetically modify clostridia .

Description:
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 . analysis of publicly provided dna microarray data for expression of homologous recombination proteins in clostridium acetobutylicum 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 . fig1 presents the results . 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 (& lt ; 23rd percentile ranking for all timepoints for reco and & lt ; 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 . assess the feasibility of stimulating recombination via expression of homologous recombination proteins in conjunction with recu 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 . to express homologous recombination proteins from a replicating , site - specific chromosomal integration plasmid ( ssci plasmid ) 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 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 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 . 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 fig2 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 &# 39 ; 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 . 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 . 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 . 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 . 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 fig3 ). 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 . 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 fig3 . if in the same coding strand , a single - crossover through the first region of homology is confirmed by a ˜ 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 ˜ 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 2 . list of appropriate primer sets to use when confirming a single integration event through the 1 st and 2 nd region of homology . the table also details possible results and the most probably explanation of such results . 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 . 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 . 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 . 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 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 . 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 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 . varying the presentation of dna for homologous recombination : linear dna versus circular dna on a suicide plasmid versus circular dna on a replicating plasmid 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 thl from a separate plasmid than the ssci plasmid . 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 μg to 0 . 1 μg of dna per transformation . we typically use 0 . 5 μg 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 . 1 . doi , r . h . and a . kosugi , cellulosomes : plant - 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