Gene Replacement Techniques For Escherichia Coli Genome Modification Biology Essay

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Breakthrough in genome sequencing projects has revealed large quantity of potentially new genes. Since then microbial genome sequences became accessible to most research laboratories, reverse genetic analysis has become a standard experimental approach to study bacterial gene function. Mutations can be introduced into bacterial chromosomes which is important for progress in further studies in functional genomics. Analysis of these genes for their functions needs gene manipulation methods which are simple and efficient allowing targeted modification of particular sequences in their chromosomal position. Gene replacement is a technique of modifying endogenous gene at its original locus in the chromosome. The strategies which have been developed for replacing segments of the chromosomal genes are based on recombination between the target locus and a cloned DNA fragment containing the desired modification. Gene replacement in E. coli chromosome can be done by variety of techniques. In this work we present recent methods by which mutations can be introduced into E. coli chromosome by gene replacement.

Gene replacement is an effective technique that takes advantage of the natural ability of an organism to recombine homologous regions of DNA. It enables the specific replacement of targeted genome sequences with copies of those carrying defined mutations. Therefore gene replacement can promote the assignment of function to cloned genes. Obviously there are many potential uses of this method in research on microorganisms. Exploitation of large quantity of potentially new genes from recent microorganism genome sequence projects will, in many cases, require the ability to verify in an efficient manner the gene assignments based on sequence homologies and has opened many new experimental avenues.

The ability to make precise genetic modifications to the bacterial chromosome and then to study the resulting phenotypic behavior is very important for functional studies. Construction of mutant alleles can be done in vitro by recombinant DNA techniques. Gene replacement strategies basically involve three stages. First, a cloned region of the genome is disrupted; for example, by the insertion of an antibiotic-resistance marker. Second, the mutated sequence carried on a suitable vector is introduced into the host organism by transformation. Finally, transformants are screened to identify those in which the desired gene replacement events have occurred. Recombination events between the homologous sequences present on the chromosome and the DNA vector results in the insertion of the transforming DNA into the host chromosome. A double crossover, that is two homologous recombination (HR) events, one on either side of the mutation in the cloned sequence, results in replacement of the wild-type gene with the mutated copy. Recombination at only one end of the wild-type sequence results in a single crossover and the insertion of the transforming DNA into the host chromosome. The DNA introduced in the cells is randomly integrated into the genome by recombination (homologous and illegitimate). Gene replacement technique is developed not only to maximize the frequency of homologous integration which is a rare event compared to illegitimate recombination but also improving selection for this event (Morton and Hooykaas, 1995). Techniques exploiting HR to achieve specific gene replacement have been developed for use in many micro-organisms. Gene replacement on the E. coli chromosome can be done by a variety of techniques. In this review we discuss recent approaches and progress for obtaining gene replacement in E. coli.

Recombination as a genetic tool

Genetic recombination is a process by which a molecule of nucleic acid is broken and then joined to a different DNA molecule. Recombination in E. coli and other bacteria is mediated by RecA whose activity is ATP depended and requires a minimal length of DNA homology between the donor sequence and a double strand recipient DNA. HR requires sequence similarity between the incoming donor DNA and DNA sequence of the recipient. In E. coli a minimum of approximately 20 bp is acquired for successful recombination with circular DNA (Watt et al. 1985).

In most cases, DNA is integrated into the recipient genome through HR. HR is an invaluable aid to the molecular geneticist in both prokaryote and eukaryote systems, providing the means by which DNA can be accurately excised, replaced or inserted into the chromosome. Targeted disruption of genes is commonly achieved using HR in a variety of bacteria (Winans et al. 1985; Miller and Mekalanos 1988; Stibitz et al. 1989). In principle, a simple approach utilizes a plasmid vector carrying a single homologous region of DNA which is complementary to part of a specific gene and entirely internal of the open reading frame. Introduction of this into the cell and resultant Campbell-type recombination (Campbell 1962) would integrate the vector into the open reading frame of the target gene on the chromosome thus creating two truncated genes. A more reliable approach involves using a vector with two regions of homologous DNA flanking an antibiotic marker. Recombination between both homologous vector sequences and the chromosome would result in excision of any chromosomal sequence between the homologous regions and integration of the antibiotic marker: allelic exchange. A chromosomal fragment is cloned into a plasmid not replicating in laboratory strain, and the construction is transferred into laboratory strain. The homologous fragments in the plasmid and in the chromosome recombine, result in merging of the plasmid into the chromosome (Fig. 1), and the integrants can be found with selection. Depending on the arrangement of the homologous fragments, single crossover or double crossover resulting in insertions or deletions in the chromosome will occur (Biswas et al. 1993; Mills 2001).

Illegitimate recombination (nonhomologous) is a type of recombination that occurs between DNA molecules sharing no homology or with very short regions of homology, typically 4 to 10 nucleotides. It occurs widely in both prokaryotes and eukaryotes and is responsible for major genome rearrangements including deletions, duplications, translocations and insertions. Heterologous recombination events requiring between 150-200 bp DNA similarity, have been named homology-facilitated illegitimate recombination (De Vries and Wackernagel 2002). Illegitimate recombination events are associated with very small sequences ('anchors') that join DNA molecules at sites with no or a few identical nucleotide sequences. These short sequences can be as small as 3 or 8 base pair and do not require RecA protein to be initiated. Such events are rare and happen in low frequency among bacteria (De Vries and Wackernagel 2002).

Gene replacement methods

Recent technical advances have produced several methods to introduce a mutant sequence synthesized in vitro into the E. coli chromosome. The two main previously established methods are the 'in-out' method and the 'linear fragment' method.

'In-out' method

In the 'in-out' method (Hamilton et al. 1989; Martinez-Morales et al. 1999; Posfai et al. 1999) a mutant sequence is introduced into the cell on a multicopy circular plasmid. A Rec mediated single homologous crossover results in cointegration of the whole circle into the genome at the target site with the plasmid vector between a wild type and mutant copy of the target sequence ('in'). The cointegrate is resolved by a second single crossover ('out'). When in and out crossovers span the mutant site, the desired mutation is transferred to the genome. The mutant allele can be delivered on a suicide plasmid into the cell. For insertion of the circular molecule into the chromosome a single crossover between the mutant and the wild type allele is required. The generated cointegrate can be resolved by spontaneous recombination of the allele pair resulting in cells with either a wild type or a mutant allele in the chromosome (Fig 1, Blomfield et al. 1991; Link et al. 1997). The key to this procedure is the use of appropriate vectors that are not capable to replicate under the situation used for selection of the cointegrate. The best used vectors include ColE1-drived plasmids that do not replicate in polA mutants, (Gutterson and Koshland 1983; Saarilhti and Pavla 1985) a temperature-sensitive pSC101 replication (Hamilton et al. 1989; Kato et al. 1989; Link et al. 1997; Le Borgne et al. 2001) and a phage-based vector (Slater and Maurer 1993). Although such a cointegration scheme has been successfully and widely used, a problem is that resolution of the cointegrate occurs at a relatively low frequency and may not always give the replacement desired. For this reason, a counterselectable gene can be introduced into vector and cells which retain the cointegrate structure are then efficiently eliminated at counterselection conditions (Dean 1981; Gay et al. 1985; Russell and Dahlquist 1989). The 'in-out' method uses a positive selection such as a drug resistance marker to select for the 'in' step and a negatively selectable marker such as sucrose resistance to drive the 'out' step. The most commonly used method is the sacB/sucrose counterselection system (Gay et al. 1985, Dozois et al. 2000), but the use of the method is limited by its strain-medium and temperature-dependence (Blomfield et al. 1991; Link et al. 1997). However, if there is a positive-selection device for monitoring resolution of the excised vector, this problem would be overcome. Recently, Link et al. (1997) reported such an integration vector that carries a positive-selection marker, the sacB gene encoding levansucrase.

Suicide plasmid-mediated gene replacement

The application of HR to bacterial genetics has been difficult in practice. The first problem encountered is how to deliver the mutated allele to the chromosome. One solution is to use 'suicide' vectors. These vectors are based on an ori R6K, which means they will only replicate in permissive E. coli strains in which the π protein encoded by the pir gene is provided in trans (Kolter et al. 1978). A number of tools have been described for introducing mutations in bacterial chromosomes (Martinez-Morales et al. 1999; Murphy et al. 2000; Yu et al. 2000). Suicide plasmids are convenient vehicles for the delivery of DNA into the E. coli chromosome and are the most useful tools to avoid antibiotic resistance markers or scars in the chromosome which also allow combination of multiple mutations. Gene constructs may also be introduced to the bacterial chromosome at random locations by using Tn elements delivered by suicide vectors (Rossignol et al. 2001). These systems involve a two-step procedure with plasmid integration within the target sequence by HR, followed by its excision via a second cross-over event, resulting in allelic exchange. A large number of suicide plamid vectors are now available to use in gram-negative bacteria (Donnenberg and Kaper 1991; Kaniga et al. 1991). Two types of suicide plasmids have been engineered: temperature-sensitive plasmids like pSC101 (Cornet et al. 1994), pKO3 and their derivatives (Link et al. 1997), and plasmids carrying the replication origin of R6K, such as pCVD441 and its derivatives (Donnenberg and Kaper 1991). The replication of the first type of plasmids is possible only at the permissive temperature, while the second type of plasmids are able to replicate only in strains producing the π protein, the product of the pir gene. A temperature-sensitive pSC101 replicon (rep101TS), originally isolated by Hashimoto and Sekiguchi (1977), forms the backbone for most of the conditionally replicating vectors currently used in E. coli and S. enterica serovar Typhimurium (Favre and Viret 2000, Merlin at al. 2002). Integration of the plasmids into the chromosome is selected by an antibiotic resistance marker, either at the restrictive temperature or in a pir− background. Excision of the integrated plasmid for allelic exchange is selected with counter-selectable markers: if the plasmid is still integrated in the chromosome, the cell will die in the presence of the counter-selective compound (Reyrat et al. 1998). For easy identification of the resolved products, a counterselection marker, such as sacB (sucrose sensitivity) (Dedonder 1966, Gay et al. 1985, Link at al. 1997; Dozois et al. 2000) or rpsL (streptomycin sensitivity) (Dean 1981; Hashimoto et al. 2005, Russel et al. 1989, Wang et al. 1993), is integrated in the suicide plasmids. The resolution of the plasmids is thus screened on media supplemented with sucrose or streptomycin, respectively. The sucrose-sensitivity system is widely used in most common suicide plasmids. Another efficient mode of enhancing the plasmid excision step is the introduction of an 18-base pair (bp) meganuclease I-sceI cleavage site in the suicide plasmid (Posfai et al. 1999). Cleavage of the genome at this unique site creates a double-strand break, which simultaneously stimulates recombination and selects for resolution of the integrated plasmid. However, the effectiveness of the 'suicide' strategy is limited by transformation efficiency, a constraint that does not apply to replicating vectors. With replicating vectors there comes a need for positive selection of the recombination event combined with a means of eliminating the plasmid. Although methods that employ suicide plasmids can be used to delete genomic segments, for each deletion experiment, these procedures require the creation of specific targeting vectors before recombining them into the chromosome.

Double strand break (DSB) - stimulated gene replacement

Recently, new approach for targeted insertion and deletion mutagenesis at specific loci was developed. Plasmids have been constructed incorporating an 18 bp recognition site for the ultrarare cutting meganuclease I-SceI, encoded by the mobile group I intron of the mitochondrial 21S rRNA gene from Saccharomyces cerevisiae (Colleaux et al. 1986). Expression of I-SceI in hosts that contain a plasmid carrying the recognition site yields linearized DNA that is highly recombinogenic. This strategy has been used to delete genomic segments (both large and small) of the E. coli chromosome by triggering DSB repair recombination (Kolisnychenko et al. 2002). By using a combination of techniques, including Red-mediated linear DNA recombination and I-SceI-induced DSB recombination, an E. coli strain with a significantly reduced genome size has been engineered (Kolisnychenko et al. 2002). DSB stimulated gene replacement offers an efficient and simple way to manipulate chromosomal sequences used in recombination-proficient wild type E. coli as it produces replacements at high efficiency and does not require specific growth conditions. This method permits the targeted construction of markerless insertions, point mutations, as well as large deletions in the E. coli genome, and is potentially applicable in other microorganisms as well. In this method the integration of the mutant gene which is carried on a circular plasmid (suicide plasmid) is inserted at a homologous locus into the genome by HR between the mutant and the wild-type alleles resulting in a direct duplication. The cointegrate formed via intramolecular recombination and the resolution of the allele pair results in either a mutant or a wild type chromosome which can be distinguished by allele-specific PCR screening. The cointegrate is resolved by introducing a unique DSB by the meganuclease I-SceI into the chromosome (Fig. 2). The enzyme recognizes an 18 bp sequence and generates a DSB with a 4-base 3' hydroxyl overhang (Montelheit et al. 1990). Cleavage by the nuclease not only enhances the frequency of resolution by two to three orders of magnitude, but also selects for the resolved products. Use of the method was demonstrated by Pósfai et al. (1999) where they constructed a 17 bp and a 62 kb deletion in the MG1655 chromosome. Cleavage of the chromosome induced the SOS response but did not lead to an increased mutation rate. It can be assumed that modified versions of this system can be applied in several E. coli strains, and various microorganisms including pathogens to introduce mutations into the genome since DSBs are known to stimulate HR in a wide range of systems. Wong (2004) described a new gene replacement scheme termed "SCE jumping" in P. aeruginosa by using plasmids incorporating I-SceI sites to perform allelic exchange at a high frequency. The "SCE jumping" technique involved engineering an R6K γori suicide vector so that I-SceI recognition sites flank an insert of P. aeruginosa chromosomal DNA that had been randomly mutagenized with a mariner Tn element (Wong 2004). Use of 'SCE jumping' for generating transposon insertion mutants is anticipated to be widely applicable to other bacterial organisms. Yu at al. 2008 described an improved method for the rapid markerless deletion with linear DNA where a single helper plasmid carrying genes encoding Red proteins and I-sceI nuclease under the control of arabinose and rhamnose promoters, respectively. Genomic deletions are performed by first growing the bacteria in a medium that contains arabinose as the carbon source (to stimulate Red proteins synthesis) and then changing the carbon source in the growth medium from arabinose to rhamnose (to stimulate production of I-SceI nuclease, which introduces a DSB that stimulates intramolecular recombination).

Gene replacement by Xer recombination

Gene replacement by Xer recombination is an efficient method for unlabelled and stable insertion of gene into bacterial chromosomes and for selectable marker gene excision. This technique makes use of native Xer recombinases that normally function to restore the plasmid as well as chromosomal dimers produced by RecA back to monomers. Xer recombinases are present in bacteria naturally to excise gene for antibiotic resistance followed by chromosomal integration of a gene, by that means eliminate the requirement for an exogenous site-specific recombinase system (SSR). Xer recombinases present in E. coli are XerC and XerD (Leslie and Sherratt 1995), and have homologues in Bacillus subtilis such as RipX and CodV (Sciochetti et al. 2001) and are present in majority of bacterial species and have been used for gene insertions and deletions successfully. Bloor and Cranenburgh (2006) proposed the term 'Xer-cise' to describe this technique. They constructed cassette consisting of gene for antibiotic resistance flanked by dif sites and regions of homology to the chromosomal target. This gene was integrated into the chromosome after amplified or cloned into a plasmid. Cells that have undergone intramolecular Xer recombination at dif sites during further culture were identified by antibiotic sensitivity and verified by PCR. The inclusion of a counter-selectable gene is not necessary as the Xer recombination frequency is high enough for recombinant clone detection without the antibiotic selection. This is beneficial, since certain counter-selectable genes can be mildly toxic such as sacB even when counter-selection is not present, leading to an accumulation of cells carrying mutations in sacB and for that reason generating false-positive results during clone selection (Bloor and Cranenburgh 2006). Xer-cise technique will simplify and enhance the production of unlabeled E. coli mutants and other bacteria for which native dif site has been elucidated (Chalker et al. 2000). Xer-cise technology can be applied in gene deletion which is helpful in accurate excision of target alleles preventing reversion in mutant strains and in gene insertion by inserting new genes for protein expression or for altering the phenotype. The benefits of this method are; chromosomal insertion was followed by excision of the antibiotic resistance gene by native Xer recombinases, no requirement of exogenous recombinases such as Cre, Flp; Xer-cise technology works in a wide species range of bacteria and it makes able multiple gene integration events in the identical strain. This approach has some disadvantages such as: expression of surrounding genes altered by insertion of foreign gene permanently and the presence of antibiotic resistance gene on the chromosome can affect the use of the same gene for plasmid maintenance. To address this, genes for antibiotic resistance have been flanked with sites for site-specific recombinase enzyme supplied in trans on a plasmid. When the new DNA is inserted into the host cell chromosome, the site-specific recombinase cuts its two target sites and joins them together to create a single site, thereby excising the intervening antibiotic resistance gene (Recchia and Sherratt 1999).

Linear fragment method

Linear DNA fragment approach has been introduced to avoid the inconvenience of constructing the targeting vectors in the in-out method of gene replacement. Transformation with short (<10 kb) linear fragments occurs at a very low or undetectable frequency in wild-type (rec+ sbc+) E. coli but does occur in E. coli mutant expressing the RecF (recBC sbcBC or recD) pathway (Jasin and Schimmel 1984; Winans et al. 1985; Shevell et al. 1988; Russel et al. 1989). The linear fragment method (Murphy 1998; Datsenko and Wanner 2000; Murphy et al. 2000; Yu et al. 2000), utilizes the Red recombination system encoded by bacteriophage lambda gam, bet and exo genes which operates on linear DNA. It is an alternative procedure to deliver the mutant allele on a linear DNA-fragment into the cell (Jasin and Schimmel 1984; Dabert and Smith 1997; Zhang et al. 1998). Electroporation is used to introduce a linear DNA fragment carrying the synthesized mutation directly into the cell where Red favors double crossover events in the progeny since a single crossover would result in a chromosome break. Incorporation of the mutation into the chromosome occurs where the double crossover spans the mutant site. An appropriate linear DNA, containing a deleted or mutated gene flanked by homologous regions of the chromosome, is transferred into recombination-proficient strains, such as recBC, sbcBC, or recD. Double cross-over recombination between the E. coli chromosome and both ends of the linear DNA fragment results in gene replacement in these particular genetic backgrounds at a high frequency. A disadvantage of this method is that it is restricted to specific nuclease-deficient recombination-proficient genetic backgrounds. Variations of linear fragment method depend on requirement of extensive DNA engineering using long PCR primers, introduction of a marker along with the mutant gene into the genome and specifically altered host cells. This introduction of a marker along with the mutant gene can have polar effects or can prevent multiple manipulations of the genome and can be eliminated only in a second round of allele replacement (Zhang et al. 1998). But this step requires the use of a counterselection system with its intrinsic limitations. The linear fragment method is limited by the very low efficiency of electroporation. Even with high DNA concentrations and cells of the highest competency, it is impossible to introduce donor DNA into more than a tiny fraction of target cells. To overcome this limitation, a two step process may be used to first select for the insertion of a cassette with both negative and positive selectable markers at the chromosomal target site and then to perform a second (negative) selection to create the desired mutation. The second step can either employ a second electroporation with Red, site specific recombinases (in which a 'scar' is typically left behind) (Datsenko and Wanner 2000) or Rec mediated reduction of the genetic intermediate with a duplication incorporated in the original fragment (Kolisnychenko et al. 2002). The latter is especially useful in the production of deletions.

λ Red-promoted gene replacement

This method makes use of the fact that the phage lambda Red (gam, bet, exo) function promotes a greatly enhanced rate of recombination when using linear DNA (Datsenko and Wanner 2000, Murphy 1998, Yu at al. 2000). It is known that bacteriophage λ recombination system Red, have the ability to act on linear DNA substrates promoting HR in the absence of RecBCD activity of the E. coli. Given the absence of any hotspot requirements and the break-join mechanism recommended for λ Red-mediated recombination (Stahl et al. 1990), it seems that for the promotion of E. coli gene replacement λ Red would be best suited. Murphy et al. (1998) developed an extremely efficient system that uses the bacteriophage λ recombination functions (exo, bet, and gam) expressed from a multicopy plasmid and transformation carried out with linear DNA substrates to stimulate gene replacement. Red system offers a number of advantages over other recombination systems in the bacteria as it is extremely efficient, there is no noticeable increase of spontaneous mutation rates when Red proteins are transiently expressed, very short homology sequence is required (targeting vector for DNA modification can be easily obtained from PCR) and single strand DNA oligos are better substrates for recombination with Red system. Therefore, generating point mutations can be achieved with DNA oligos. Moreover, when single strand oligos are used, the only known recombination protein required is the beta-protein (Lee et al. 2001). One requirement for using Red system is that one needs to have the genomic sequence of a gene, at least the region that needs to be manipulated (Liu et al. 2003). Hashimoto et al. (2005) developed a deletion method that goes through two serial Red-mediated recombination events. In the first recombination, the targeted genomic region is replaced with the CmR-rpsL-sacB (CRS) cassette flanked by DNA fragments that are homologous to chromosomal target. In the second step, the inserted CRS cassette is replaced with a linear DNA fragment that consists of only the chromosomal sequences producing a markerless deletion. In the first step, the chloramphenicol resistance gene (CmR) is used as a positive selection marker for the deletion mutants and in the second step rpsL and sacB are used as counterselection markers. Red/ET recombination (ET cloning/recombineering) presented by Zhang et al. 1998 is an easy to use modification system for prokaryotic functional genomics. They demonstrated that a pair of phage coded proteins (RecE and RecT) only need 42 bp long homology arms to mediate the HR between a linear DNA molecule (e.g. a PCR product) and circular DNA (plasmid, BAC or E. coli chromosome) (Copeland et al. 2001; Court et al. 2002). Later this system was extended by Muyrers et al. 1999 in replacing recE and recT by their respective functional counterparts of phage lambda redα and redβ. Recombineering takes advantage of the phage derived protein pairs, either RecE/RecT from the Rac phage or Redα/Redβ from the λ phage, to aid cloning of segments of DNA into vectors without the requirement of restriction enzyme sites or ligases (Zhang et al. 1998, 2000). Earlier HR technique had to be studied in strains deficient in RecBCD nuclease as it degrades linear DNA. This was overcome by the discovery that Redα and Redβ were assisted by Redγ, which inhibits RecBCD nuclease activity making it possible to use the technique in E. coli and other bacterial strains (Murphy et al. 1998) increasing the recombination efficiency 10-100 times (Murphy et al. 2000). The union of these three enzymes (α, β and γ, or E, T and γ) in one vector was named Red/ET recombination and the basic principles of the method are that it requires two homology regions of 442 bp in a linear fragment, DSBs in both ends, and another linear or circular plasmid in order for recombination to take place. Since the year 2000 the system, which is protected by several international patents from Gene Bridges, has been used by other academic groups to disrupt several chromosomal genes in E. coli (Datsenko and Wanner 2000; Yu et al. 2000). Rivero-Müller et al. (2007) reported a method, Assisted Large Fragment Insertion by Red/ET-recombination (ALFIRE), where they have overcome both the size limitations and the restrictions of the cloning/subcloning procedures used in the Red/ET recombineering system and this method can be used for the construction of large and complex vectors.

Gene replacement by electrotransformation

Due to exonucleolytic degradation of incoming DNA gene targeting using linear dsDNA fragments in wild-type E. coli transformation is generally inefficient. To overcome transformation inefficiency, strains having high recombination-proficiency in which the exonucleolytic activity of RecBCD is inactivated, have been used as transformation recipients (such as recB recC sbcA, recB recC sbcB sbcCD, and recD mutants or strains expressing bacteriophage recombination functions). Recently, an approach was developed to achieve gene replacement in wild-type cells by transformation with linear DNA containing Chi sequences (5'-GCTGGTGG-3') at both ends (Dabert and Smith 1997). These sequences are known to decrease the activity of RecBCD exonuclease and stimulate its activity of recombination (Dixon and Kowalczykowski 1993; Myers and Stahl 1994; Karoui et al. 1999). The use of electrocompetent cells and electrotransformation technique appears to lessen the exonucleolytic activity of RecBCD in E. coli, in this way allowing gene replacement to occur and gene replacements can be achieved on a plasmid as well as on a chromosomal target with the use of linear DNA without Chi sequences in wild-type E. coli. The exonuclease activity of RecBCD is reduced after electroporation thus reducing degradation of the linear DNA fragment. The linear DNA containing the Chi sites had no effect on gene replacement efficiency although the Chi sites are known to block DNA degradation and stimulate recombination in E. coli. In electroporated cells the RecBCD-mediated exonucleolytic activity was found to be diminished. In this way electrotransformation may constitute a simple, direct and extremely efficient way to perform gene replacement in many E. coli strains and does not necessitate special DNA constructions (Karoui et al. 1999). The frequencies of gene replacement events obtained (with a chromosomal target) are comparable to those obtained in the Chi-stimulated recombination method (Dabert and Smith 1997). It may also be used to make gene disruptions on plasmid-carried targets which can then be transferred to the organism of interest. It has also been reported that electroporation of single-stranded oligonucleotides results in even higher recombination efficiency in Red-expressing cells (Ellis et al. 2001), leading to the feasibility of introducing point mutations, insertions, and deletions in a single step without the need for counterselection markers. Since the frequency of recombination approaches 1 out of 90 to 260 electroporated cells, mutants can be screened directly by PCR (Swaminathan et al. 2001). The potential of this technology to construct additional plasmid tools has yet to be fully realized.

PCR-mediated gene replacement

The hyper-recombinogenic properties of an E. coli strain in which the recBCD genes have been replaced by λ Red recombination functions were exploited in the development of a general PCR-mediated gene replacement scheme for E. coli. An experimental system that allows for PCR-mediated disruption of genes in yeast has greatly aided studies of this organism (Baudin et al. 1993; Wach et al. 1994; Lorenz et al. 1995). In this scheme, PCR- generated DNA fragments containing a selectable marker flanked by ~50 bases of sequences upstream and downstream of the gene of interest are introduced into yeast cells by electroporation (Fig. 4). Given their high rate of HR, nearly 95% of the transformed yeast cells carry the designed gene disruption. E. coli does not recombine as readily as Saccharomyces cerevisiae (Russell et al. 1989). Transformation and/or electroporation of these strains with linear DNA substrates containing a drug resistance-conferring marker near or in place of the gene of interest results in integration of the mutant or deleted gene by HR. However, successful gene replacement with these strains usually requires extensive regions of homology and/or occurs at a low frequency, limitations that have precluded the development of an efficient PCR-mediated gene disruption method for E. coli. The gene replacement technology described by Murphy et al. (2000) allows for the easy one-step replacement of almost any gene in E. coli. The system takes advantage the hyper-recombinogenic environment of an E. coli strain containing the Red system encoded by bacteriophage lambda gam, bet and exo genes. This method of gene replacement offers an advantage over existing gene replacement techniques presently employed for E. coli: that is, the gene of interest does not require prior cloning, and can be used to easily construct precise gene disruptions and moreover plasmid-chromosome co-integrants do not have to be formed and resolved. This technique is well suited for the generation of precise deletions of unknown open reading frames predicted from the genome sequence of E. coli (and possibly those of other bacteria) in an effort to identify essential functions that might serve as suitable targets for drug design (Baba et al. 2006).

Chi sites enhanced gene replacement

This technique uses the characteristic of Chi sites to govern RecBCD exonuclease activity and stimulate recombination. Chi sites are a cis-acting octameric nucleotide sequences in DNA that stimulate the RecBCD pathway of HR in E. coli. Chi stimulates recombination by interaction with RecBCD enzyme, which has multiple enzymatic activities and multiple physiological roles in recombination, repair, and replication. Chi appears to be active throughout the enteric bacteria; other nucleotide sequences may similarly interact with RecBCD-like enzymes in other bacteria. The nucleotide sequence of Chi was shown to be 5´GCTGGTGG3´ (or its complement or the duplex) (Smith et al. 1984, Myers and Stahl 1994). In vivo, Chi stimulates HR 5-10 fold (Cheng and Smith, 1989; Myers at al. 1995). Stimulation is maximal at the Chi site, decreases approximately factor of two for each 2-3 kb to one side, but is insignificant to the other side of Chi. Inclusion of two properly oriented and positioned Chi sites on the linear fragment enhances gene replacement during transformation about 40-fold in wild-type cells (Dabert and Smith 1997). Transformation or transduction in this way offers a useful method of "gene targeting" with cloned DNA fragments in wild-type E. coli. These observations indicate that Chi is an important element in E. coli HR. In its absence, recombination following conjugation or transduction in wild-type cells would presumably occur at very low or undetectable frequency. Testing this proposal is thwarted by the ≈ 1000 Chi sites in the E. coli genome. When wild-type E. coli cells are made competent by treatment with CaCl2, Chi sites occurring near the ends of linear DNA fragments stimulate the frequency of gene replacement events (Dabert and Smith 1997). One disadvantage of this technique is that it needs DNA constructions that add Chi sites at the fragment extremities.

Gene replacement without selection: 'gene gorging' method

The term 'gene gorging' comes from forcibly incorporating the desired allele into the genome by imposing copious quantities of it into the cell. This method greatly simplifies the process of mutagenesis by combining the efficient steps (uses both 'in-out' and linear fragment methods) and eliminating the multiple selections needed for the earlier methods. The key difference in gene gorging is the linearization of the plasmid in vivo. Incorporation of mutant allele into the genome is achieved at least for some extent since practically every cell takes in linear donor, and Red is highly efficient (Herring et al. 2003). Gene gorging enables investigators to introduce precise sequence changes into genome of E. coli in a direct way without leaving undesirable drug markers or other 'scars'. This technique can be used to make variety of mutations which are introduced at extremely high frequency where no selection is required but just by simple screening. This method does not introduce a large number of unintended mutations. Similar studies by Poteete and Fenton (2000) used lambda phage as the vehicle to efficiently introduce recombinogenic fragments into E. coli for Red recombination. Ellis et al. (2001) have reported the introduction of unselected mutations in up to 7% of viable cells using electroporated single stranded DNA. Herring et al. (2003) presented gene gorging. This approach resulted in generally higher levels of replacement for wild type cells and is greatly simplified by using plasmid rather than lambda techniques and meganuclease I-SceI, which does not cut in the genome of E. coli or most other organisms (Fig. 3). The efficiency of gene gorging is achieved by establishing the linear donor fragment in greater numbers and in a higher proportion of cells than could be achieved by electroporation. Gene gorging may prove especially practical in poorly transformable enteric bacteria since supercoiled plasmid DNA is considerably easier to transform than linear DNA. A method using in vivo production of a recombinogenic linear DNA fragment has been used for directed mutation in Drosophila (Rong and Golic, 2000), and may prove useful in making directed mutations in other important organisms as well.

Conclusion

In the emerging post-genome sequencing era, high-throughput evaluation of uncharacterized open reading frames becomes a necessity. Gene replacement techniques would be the ultimate tool to help investigators to systematically assign functions to the vast number of these new open reading frames. With these new techniques and findings in E. coli gene replacement, genomic modifications can be created with enhanced efficiency and speed by reducing the time and work load compared to previous strategies. These approaches provide powerful tools to clone and modify genes precisely for functional analysis and thus perform biochemical or behavioral experiments to clarify functions. Application of these techniques is not problem-free but further expansion in this research area is likely to continue with several excellent and technically demanding protocols. However, we are hopeful that in future we could see new gene replacement strategies making the more traditional strategies obsolete.

Acknowledgments

This work was supported by the grants of Slovak grant agencies APVT-20-017102, APVV-20-054005 and VEGA 1/0344/10.

Fig. 1. Gene replacement strategy

Fig. 2. General procedure of the DSB-stimulated gene replacement

Fig. 3. Gene gorging method

Fig. 4. Schematic of PCR-mediated gene replacement

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