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.
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Key words: gene replacement, E. coli, recombination, genome modification
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 available. To study the effect of mutation by introducing the mutant allele into the cell where it will replace the wild-type gene by homologous recombination (HR) then the effect of mutation can be tested by expressing the gene at its native location. 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 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 to maximize the frequency of homologous integration as it is a rare event compared to illegitimate recombination and also the selection of the 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. Mutant phenotypes can be confirmed by reintroduction of the wild type gene on a plasmid. 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, and results 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).
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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 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 (Blomfield et al. 1991; Link et al. 1997). Because the rare resolution event, to eliminate cells which retain the cointegrate structure and carry a counterselectable gene located on the inserted plasmid effective counterselection is needed (Dean 1981; Gay et al. 1985; Russell and Dahlquist 1989). The most commonly used method is the sacB/sucrose counterselection system (Gay et al. 1985), but the use of the method is limited by its strain-medium and temperature-dependence (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) 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. 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.
Double-stranded break - stimulated (DSB) gene replacement
Recently, new approach for targetted 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 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 and also to delete both large and small segments of the E. coli chromosome by triggering DSB repair recombination (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 markerless replacements at high efficiency and does not require specific growth conditions. This method is based on the recombination and repair activities of the cell and permits the targeted construction of markerless insertions, point mutations, as well as large deletions can be created 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, 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 the development of 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. Use of SCE jumping for generating transposon insertion mutants is anticipated to be widely applicable to other bacterial organisms.
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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, 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 by Xer-cise technology. 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; the presence of antibiotic resistance gene on the chromosome can affect the use of the same gene for plasmid maintenance and the difficulty of multiple gene integration events due to the deficiency of suitable genes for antibiotic resistance. To address this, genes for antibiotic resistance have been flanked with sites for site-specific recombinase enzymes supplied in trans on a plasmid. Additional transformation is required as an extra step followed by further culturing to remove the helper plasmid. Additionally, this technique has only been optimized for a small range of bacteria. This technology should be applicable to all prokaryotes with the ubiquitous Xer dimer resolution system (Recchia and Sherratt 1999).
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. Incorporatation of mutant allele into the genome is achieved atleast 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. 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. Previous methods of directed mutagenesis in E. coli rely on the use of positive and negative selections for recombination intermediates because the desired events occur at very low frequency. Herring et al. (2003) presented gene gorging, in which the efficiency of gene replacement is high enough to make selection of recombinants unnecessary. 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. This method does not introduce a large number of unintended mutations and most mutants generated are supressible. 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.
Linear fragment method
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 genes gam, bet and exo 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.
Both the 'in-out' and 'linear fragment' methods include inefficient steps and require strong genetic selections to achieve useful frequencies. 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 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.
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, in which the transformation of linear DNA containing Chi sequences (5'-GCTGGTGG-3') at both ends flanking the homologies (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). If electrocompetent cells are used, gene replacements with the use of linear DNA without Chi sequences can be achieved in wild-type E. coli, on a plasmid as well as a chromosomal target. 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. The exonuclease activity of RecBCD is reduced after electroporation thus reducing degradation of the linear DNA fragment. The Chi sequences present on linear DNA fragments do not affect the frequency of gene replacement due to an inactivation of RecBCD nucleolytic activity during electroporation (Karoui et al. 1999). This system of gene replacement by electrotransformation provides a simple and extremely efficient way to perform gene replacement in many E. coli strains but requires a particular E. coli strain thus limiting its range of use. In contrast, the method described by Karoui et al. (1999) to achieve gene replacement can be used in many different E. coli strains and does not necessitate special DNA constructions. 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). In this way electrotransformation may constitute a direct method to get gene replacements with linear DNA in wild-type E. coli on plasmid and chromosomal targets. 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. Thus elctrotransformation provides a easy way to carry out gene replacements in several E. coli strains (Karoui et al. 1999). It may also be used to make gene disruptions on plasmid-carried targets which can then be transferred to the organism of interest.
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 5Â´GCTGGTGG3Â´ (or its complement or the duplex) by a comparison of the sequences of active and inactive Chi sites and their flanking sequences (Smith et al. 1984, Myers and Stahl 1994). In vivo, Chi stimulates HR 5-10 fold unidirectionally, with maximum stimulation occuring at Chi and decaying downstream relative to the entry site of RecBCD enzyme (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. Transformation with short (<10 kb) linear fragments (gene replacement) 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). 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; dual Chi sites also enhance a specialized transduction in which intracellular EcoRI restriction enzyme cuts the linear Chi-containing fragment out of infecting Î» DNA (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 (as opposed to recBC sbc or recD mutants used previously). 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 â‰ˆ 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.
PCR-mediated gene replacement
The hyper-recombinogenic properties of an E. coli strain in which the recBCD genes have been replaced by Î» 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, requiring researchers to rely on recombination-proficient mutant strains (e.g. recBCsbcBC or recD) to perform gene replacement (Marinus et al. 1983; Jasin and Schimmel 1984; 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 of PCR-promoted recombination to generate marked gene deletions and the hyper-rec environment of an E. coli strain containing the Red for recBCD allele. 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 of the gene, and can be used to easily construct precise gene disruptions and moreover plasmid-chromosome co-integrants do not have to be formed and resolved. The cloning-free gene replacement scheme is suitable for cases where secondary stage products generating marked gene deletions are difficult to clone. 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.
Î» Red-promoted gene replacement
It is known that bacteriophage Î» recombination system Red, have the ability to act on linear DNA substrates promoting HR in the absence of RecBCD activity of the E. coli. The absence of any hotspot requirements for Î» Red-mediated recombination and the break-join mechanism (Stahl et al. 1990), it seems that for the promotion of E. coli gene replacement Î» Red would be best suited. Murphy et al. 1998 developed a new extremely efficient system that uses the bacteriophage Î» recombination functions (exo, bet, and gam) expressed from a multicopy plasmid and transformation carried out with linear DNA substrates to stimulate gene replacement which is not dependent on a cointegrate, and does not require cloning of the gene in advance, producing gene replacement in almost any E. coli strain (also possibly for other bacteria as well) at high frequency. Gene replacement was promoted by lambda recombination functions within the lacZ gene at a higher rate i.e. about 15 to 130 times than recBC sbcBC or recD strains. The reason for this is since HR is raised to the higher level by the addition of functions to the host, rather than induced by alteration of host functions. The value of this method is emphasized by gene replacement with DNA fragments generated by PCR. Red system offers a number of advantages over other recombination systems in the bacteria. First, it is extremely efficient. Secondly, there is no noticeable increase of spontaneous mutation rates when Red proteins are transiently expressed. Thirdly, very short homology sequence is required (45 bp) so a targeting vector for DNA modification can be easily obtained from PCR. Fourthly, 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). 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). Later this system was extended by Muyrers et al. 1999 in replacing recE and recT by their respective functional counterparts of phage lambda redÎ± and redÎ². 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).
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.
This work was supported by the grants of Slovak grant agencies APVT-20-017102, APVV-20-054005 and VEGA 1/0344/10.