BACKGROUND
the λ-red recombinase system can be used to introduce mutations, deletions, or insertions into the e. coli chromosome by recombining regions of homology carried on short single-stranded oligonucleotides or large double-stranded dna molecules  <cit> .
the λ-red system consists of three proteins, the gam, exo and bet gene products.
when expressed in the cell the gam protein protects linear double stranded dna from degradation by the host recbcd complex.
the exo protein generates single stranded dna overhangs, which are substrates for recombination, catalyzed by the bet protein, with homologous regions of the chromosome  <cit> .
several λ-red recombineering techniques have been developed: two in particular are of note, which differ in the way that the target dna is delivered into the cell.
the first technique, and arguably the most widely used, was first described by murphy  <cit>  and later refined by datsenko and wanner  <cit> .
in this method a plasmid is used to express the λ-red genes from an arabinose inducible promoter.
strains expressing λ-red are transformed, by electroporation, with a dsdna pcr product carrying an antibiotic cassette flanked by short regions of homology to the target gene.
λ-red mediated recombination occurs, resulting in replacement of the targeted gene by an antibiotic resistance cassette.
this cassette can then be excised by flp recombinase leaving a ~  <dig> bp dna scar in place of the target gene.
the second technique, "gene gorging", designed by herring and co-workers  <cit> , is a two plasmid method that also utilizes the λ-red system to generate recombinants.
gene gorging eliminates the need to electroporate a dsdna fragment into cells, by supplying the regions of homology to the target gene on a donor plasmid that also contains a dna recognition site for the saccharomyces cerevisiae i-scei endonuclease.
the donor plasmid and the recombineering plasmid, pacbsr , are transformed into the recipient strain.
upon arabinose induction, i-scei cleaves the donor plasmid, providing a linear dsdna target for the λ-red system.
the obvious advantage of this system is that multiple copies of the homologous dna are present in the bacterial cell, which increases the number of potential recombination events.
the frequency of recombination for gene gorging is reported to be 1-15%, eliminating the absolute requirement for an antibiotic resistance marker to select for recombinants.
we have used both systems for making gene knockouts and gene fusions in laboratory e. coli strains.
however, we have had less success with these methods in pathogenic strains such as the o157:h <dig> sakai strain  <cit> , and virtually no success in the cft <dig> upec  <cit> , the o <dig> eaec  <cit>  and the h <dig> etec  <cit>  strains.
since techniques such as transduction by p <dig> phage are incompatible with many pathogenic strains, due to extensive surface antigens that block access to the phage receptor  <cit> , gene deletions have to be made directly in the strain.
our aim in this study was to establish a high-throughput recombineering system, with particular emphasis on the ability to couple epitope tags to genes, which is compatible, without modification, for use in a wide range of laboratory and wild-type e. coli strains.
we have achieved this by enhancing the two-plasmid system of herring and co-workers, making three key modifications.
first, a set of donor plasmids have been generated that readily facilitate the deletion of genes or the c-terminal coupling of genes to epitope tags.
second, the inclusion of the sacb gene on the donor plasmid allows for the counter-selection of all but true recombinants.
third, the inclusion of an i-scei recognition site on a derivative of the recombineering plasmid, called pacbsce means that the plasmid is effectively 'self-cleaving' upon induction of i-scei and λ red genes.
hence cells receive a burst of λ-red before pacbsce is lost, which is sufficient to induce recombination but limits the exposure of cells to λ-red function.
prolonged exposure to λ-red activity is undesirable as it can cause unwanted secondary chromosomal modifications in both k- <dig> and pathogenic e. coli strains  <cit> .
we have termed this method gene doctoring, abbreviated to g-doc , and we have demonstrated its versatility by deleting and coupling genes to epitope tags in pathogenic and laboratory e. coli strains.
RESULTS
current techniques for recombineering in laboratory and pathogenic escherichia coli strains
a. electroporation of linear dna fragments
the method first described by murphy  <cit> , later refined by datsenko and wanner  <cit> , of electroporating linear double stranded dna fragments into cells that are then targets for homologous recombination by the λ-red system, is reported to promote a very low recombination efficiency in e. coli k- <dig> strains: approximately  <dig> in every  <dig>  ×  <dig> e. coli k- <dig> mg <dig> cells that survive electroporation  <cit> .
despite this low frequency, we routinely identify between 10- <dig> mg <dig> recombinants per experiment, however, since we use approximately  <dig> ×  <dig> mg <dig> cells per electroporation  <cit> , the identification of only 10- <dig> recombinants indicates that in our hands the recombination efficiency is approximately  <dig> in every  <dig>  ×  <dig> cells,  <dig> times less than reported.
despite consistently attaining recombinants in mg <dig> using this system we have had virtually no success in pathogenic strains.
since the low recombination frequency of the system has been attributed to the inefficient uptake of linear dsdna fragments during electroporation  <cit> , we determined whether the inefficiency of this system for recombination in pathogenic strains was due to a reduced capacity to uptake dna by electroporation.
thus, we compared the transformation frequencies of mg <dig>  o <dig>  cft <dig> and o157:h <dig> sakai cells when transformed by electroporation with different plasmids.
cells in the exponential phase of growth were transformed by electroporation as previously described  <cit>  with either: puc <dig>  <cit> ,  <dig>  bp , conferring ampicillin resistance; pkd <dig>  <cit> ,  <dig>  bp , conferring ampicillin resistance; pacbsr  <cit> ,  <dig>  bp , conferring chloramphenicol resistance; prw <dig>  <cit> ,  <dig>  bp , conferring tetracycline resistance.
cells were then plated onto lennox broth  agar plates supplemented with appropriate antibiotics, incubated for  <dig> hours at 37°c and the number of colonies counted.
table  <dig> shows the transformation frequencies of the pathogenic strains by each plasmid, expressed as a percentage of the transformation frequency of mg <dig>  it is clear that the transformation frequencies of the pathogenic strains are dramatically lower than for mg <dig>  particularly for strains cft <dig> and o <dig>  considering that we expect approximately 10- <dig> recombinants in mg <dig>  such low electroporation efficiencies could explain why using this technique in pathogenic strains results in minimal success.
since the datsenko and wanner system relies upon the introduction of pcr generated dna into cells and not plasmids that have been isolated from an e. coli k- <dig> strain, we re-examined the dna uptake efficiencies of the strains when transformed with a pcr generated version of the plasmid, puc <dig>  we reasoned that plasmids isolated from a k- <dig> strain may be subject to host restriction-modification systems in pathogenic strains, hence, using a pcr-generated puc <dig> derivative would not only more closely resemble the conditions used by datsenko and wanner, but also allow us to monitor the transformation efficiencies by means of the acquired ampicillin resistance due to puc <dig> plasmid uptake.
thus, we amplified puc <dig> by pcr and then incubated the reaction with dpni, which specifically digested the methylated template plasmid and not the pcr generated product.
the pcr generated puc <dig> plasmid  was then transformed into mg <dig>  cft <dig>  o157:h <dig> sakai and o <dig> by electroporation.
the results  show that the transformation frequency of the pathogenic strains by puc18pcr was slightly improved when compared with mg <dig>  although the overall transformation frequency remains far lower than mg <dig>  the overall number of mg <dig> colonies identified after transformation with puc <dig> or puc18pcr was comparable.
thus, the electroporation step is likely to be the primary reason for the poor efficiency of this system in pathogenic e. coli strains.
this shortcoming was alleviated somewhat by murphy and campellone  <cit>  who developed an improved electroporation based protocol for recombineering in e. coli ehec and epec strains.
however, we have had mixed success using this protocol, particularly when recombineering in eaec and upec strains, where no increase in recombination frequency was observed.
b. two-plasmid recombineering
the two plasmid gene-gorging method described by herring and co-workers  <cit>  has an immediate advantage for recombineering in pathogenic strains since the method does not rely upon efficient electroporation as a means of introducing target dna into the cell.
instead, the target dna is flanked by recognition sites for the meganuclease i-scei on a donor plasmid that is transformed into cells along with the recombineering plasmid, pacbsr, which carries i-scei and the λ-red genes whose expression is controlled by an arabinose inducible promoter.
induction of i-scei results in donor plasmid cleavage, generating the linear dsdna target, which is a substrate for λ-red gene products.
herring and co-workers disrupted chromosomal genes by introducing amber mutations, using long regions of homology to the chromosome and reported that the recombination frequency for gene gorging was between 1-15%.
to introduce epitope tags at the carboxyl terminus of chromosomal genes and, crucially, to maintain a high-throughput system, we chose to amplify the epitope tag by pcr using oligonucleotide primers that contained  <dig> bp of homology to the chromosomal gene of interest, rather than sequentially cloning regions of homology to either side of the epitope tag.
we reasoned that since short homologous sequences had already been successfully utilised for recombineering by datsenko and wanner,  <cit>  this strategy could be adapted for epitope tagging.
the amplified dna product was cloned into pbr <dig>  modified so that the pcr product would be flanked by two recognition sites for i-scei.
the resulting construct was co-transformed, along with pacbsr, into mg <dig> cells and gene gorging experiments performed as described by herring and co-workers  <cit> .
the results of the experiments  indicated that the recombination efficiency using short regions of homology was very poor; several hundred colonies recovered after gene gorging were screened by pcr and the frequency of recombination was found to be  <dig> - <dig> %, far less than the 1-15% reported by herring and co-workers.
to improve the identification rate of recombinants we modified the technique by including a kanamycin cassette adjacent to the epitope tag on the pbr <dig> based donor plasmid.
we reasoned that after in vivo digestion of the donor plasmid, the ampicillin cassette carried on pbr <dig> would be lost and kanamycin resistance would only be maintained if a successful recombination event had occurred.
hence after gene gorging, cells were plated onto lb agar plates containing kanamycin, and the next day colonies were replica plated onto lb plates containing either ampicillin or kanamycin.
these colonies were screened for candidates which were kanamycin resistant and ampicillin sensitive, indicative of donor plasmid loss and kanamycin cassette retention as a result of recombination with the chromosome.
however, this approach proved to be problematic, since unless the in vivo cleavage rate of the donor plasmid by i-scei approaches 100% efficiency, the ampicillin and kanamycin cassettes are still present on the donor plasmid in the cell, since the plasmid is present in multi-copy, rendering positive selection ineffective.
typically we screened up to  <dig>  colonies by replica plating, identifying no more than  <dig> colonies with the correct phenotype.
taken together these results demonstrate that a more effective technique, that is both rapid and reliable, is required to introduce epitope tags onto the chromosome of pathogenic e. coli strains.
gene doctoring
to address this requirement we have developed an enhanced version of the two-plasmid gene gorging system.
our method, termed gene doctoring , facilitates the coupling of genes to epitope tags or the deletion of chromosomal genes and increases the rate of identifying recombinants.
we have generated a suite of pdoc plasmids which allow for the deletion of chromosomal genes, or the coupling of chromosomal genes to a  <dig> × his, a  <dig> × flag, a  <dig> × proteina or a gfp tag.
the pdoc plasmids carry the sacb gene, which allows for the counter-selection of cells still harbouring donor plasmid, thus eliminating the need for screening post recombination.
using this system we routinely identify more than  <dig> recombinants per experiment in both laboratory and pathogenic e. coli strains, using short regions of homology to the chromosome, thus maintaining both a high-throughput and broad-range compatibility system.
g-doc plasmids
the pdoc plasmids are derived from pex100t, a medium copy number plasmid which carries ampicillin resistance and the b. subtilis sacb gene  <cit> .
we have introduced different dna sequences into the pex100t i-scei restriction sites to create a suite of plasmids, schematic diagrams of which are shown in figure  <dig>  the cloning plasmid, pdoc-c, has a large cloning region  flanked by two i-scei recognition sites.
the dna sequence of pdoc-c, from  <dig> bp upstream of the left-hand i-scei site to  <dig> bp downstream of the right-hand i-scei site is shown in figure  <dig>  panel a. the template plasmid, pdoc-k, carries a kanamycin resistance cassette flanked by flp recombinase recognition sites .
on either side of this region are  <dig> cloning regions .
the other template plasmids, pdoc-h, pdoc-f, pdoc-p and pdoc-g are derivatives of pdoc-k that have the coding sequence for a  <dig> × his,  <dig> × flag,  <dig> × protein a and gfp tag respectively, immediately downstream of cr <dig>  figure 2; panel b, shows the dna sequence common to all of the pdoc template plasmids, from  <dig> bp upstream of the left-hand i-scei site to  <dig> bp downstream of the right-hand i-scei site.
the template plasmids differ between the cr <dig> and flp <dig> sequences: this region is outlined by an open box in the figure.
the dna sequence proceeds through cr <dig>  along the respective dna sequence for each plasmid within the open box, and into the flp <dig> sequence below.
the plasmid pdoc-k has  <dig> bp of dna sequence prior to flp <dig>  the plasmid pdoc-h has the coding sequence for the  <dig> × his tag and a stop codon followed by a short dna sequence leading into the flp <dig> site.
the first  <dig> codons of the  <dig> × flag, proteina and gfp tags are shown, followed by the stop codon and short dna sequences leading into flp <dig> site.
other features indicated on the dna sequences of the pdoc plasmids in figure  <dig> are described in the g-doc recombineering protocol below.
the full dna sequence of each pdoc plasmid is provided in additional file  <dig> and is also available from genbank, accession numbers gq88494-gq <dig>
g-doc recombineering protocol
for generating gene:epitope tag fusions, the epitope tag and kanamycin cassette are amplified by pcr, using the relevant pdoc plasmid as a template.
a schematic outline of the cloning strategy for generating gene:epitope tag fusions is shown in figure  <dig>  panel a. the clockwise primer used for the pcr amplification is designed so that it contains between 25- <dig> bp of homology to the 3' end of the target gene , not including the stop codon, followed by  <dig> bp of sequence which anneals to the epitope tag sequence on the pdoc plasmid.
this should be designed so that, after recombination with the target gene on the chromosome, the gene will be in frame with the coding sequence of the epitope tag.
the downstream primer is designed so that it contains 25- <dig> bps of homology to the dna sequence immediately downstream of the target gene  and the primer sequence p-rev.
the two primers are also designed with a restriction site at the 5' end, so that, after amplification by pcr, the dna product can be cloned into the cloning region of pdoc-c, between the two i-scei sites.
for generating gene deletions, the kanamycin cassette from pdoc-k, is amplified by pcr.
a schematic outline of the cloning strategy for generating gene deletions is shown in figure  <dig>  panel b. the clockwise primer used for the pcr amplification is designed so that it contains between 25- <dig> bp of homology to the dna immediately upstream of the start of the gene , followed by  <dig> bp of sequence which anneals to the k-fwd sequence on pdoc-k. the anti-clockwise primer is designed so that it contains 25- <dig> bps of homology to the 3' end of the gene  and the primer sequence p-rev.
again, the two primers are designed with 5' restriction sites for cloning the dna product into pdoc-c.
alternatively, when longer regions of homology to the chromosome are required, sequential cloning steps can be performed, utilising the multiple cloning sites to introduce long regions of chromosomal homology upstream and downstream of the kanamycin cassette and epitope tag.
in this case we recommend sequencing the cloned homology regions, post cloning and before recombineering, using priming sequences s <dig> and s <dig> .
the next step is to transform the pdoc donor plasmid into the recipient strain with the recombineering plasmid, which expresses i-scei and the λ-red gene products.
a schematic protocol, outlining the key steps in generating recombinants is shown in figure  <dig>  we have modified the recombineering plasmid, pacbsr, used by herring and co-workers  <cit>  by introducing an i-scei recognition site adjacent to the replication origin of the plasmid: we have called this plasmid pacbsce.
upon arabinose induction, a burst of i-scei and λ-red expression occurs; i-scei cleaves the donor plasmid resulting in generation of the substrate for λ-red mediated recombination.
in addition, i-scei also cleaves the pacbsce recombineering plasmid, resulting in loss of plasmid and loss of λ-red expression, thus avoiding prolonged λ-red activity, which can result in unwanted chromosomal modification  <cit> .
recombination occurs between homologous regions on the linear dna substrate and the chromosome, transferring the kanamycin cassette, and in the case of gene:coupling, the epitope tag, onto the chromosome .
recombinant clones are selected for by growing cells on lb agar plates containing kanamycin and sucrose: only true recombinants, which have lost the sacb gene due to donor plasmid loss and have retained the kanamycin cassette due to recombination, are able to survive and grow on this medium.
examination of recombinants, to ensure that the correct chromosomal modification has been generated, is achieved by amplifying the target region by pcr, using primers that anneal adjacent to the homology regions  and chromosomal check priming sequences cc <dig> and cc <dig> .
once recombination has been confirmed, the kanamycin cassette can be excised from the chromosome using the flp recombinase sites, as described previously.
<cit>
g-doc validation
to test our system we designed oligonucleotides to couple the rsd and yacl genes to the  <dig> × flag epitope tag.
one primer contained homology to the first  <dig> codons of the  <dig> × flag sequence,  <dig> bp of homology to the 5' end of the target gene, excluding the stop codon, and an ecori restriction site .
the second primer contained homology to the p-rev sequence,  <dig> bp of homology to the chromosome, immediately downstream of the target gene primer and a kpni restriction site .
dna fragments generated by pcr using pdoc-f as a template were cloned into pdoc-c, which was subsequently co-transformed with pacbsce into k- <dig> mg <dig>  ehec o157:h <dig> sakai and upec cft <dig> cells.
the gene doctoring protocol was followed and the results are reported in table  <dig>  for both genes, in all three strains, a large number of colonies were identified with a kanamycin resistant, sucrose insensitive phenotype.
after pcr analysis of the relevant chromosomal region  and d <dig>  and d <dig>  and d <dig> for rsd and d <dig> and d <dig>  and d <dig> and d <dig> for yacl) the vast majority of candidates were found to be true recombinants and in each case, more than 90% were sensitive to both ampicillin and chloramphenicol, indicating loss of both pdoc donor and pacbsce plasmids.
where a candidate was found to have the wild-type form of the gene after pcr verification, we assumed that the kanamycin cassette had inserted into a different part of the chromosome, since we were unable to isolate any donor plasmid dna from cells using standard plasmid isolation techniques.
hence, for each gene, in each strain, more than  <dig> recombinants were identified that had the correct chromosomal modification and were free of the recombineering plasmid pacbsce.
to test the effectiveness of recombination using our recombineering plasmid pacbsce, compared with the recombineering plasmid pacbsr, used by herring and co-workers  <cit>  we repeated the gene coupling analysis of the rsd gene.
the results in table  <dig> show that more kanamycin resistant, sucrose insensitive recombinants were identified in each strain when pacbsr was used as the recombineering plasmid, with a comparable percentage being free of pdoc donor plasmid, when compared to using pacbsce as the recombineering plasmid.
however, very few candidates had lost the recombineering plasmid, and in strain cft <dig>  all of the recombinant candidates still carried pacbsr, thus exposing cells to the potential effects of excess of λ-red expression and requiring additional steps to cure cells of the plasmid  <cit> .
we have further validated the system by making several gene deletions in both laboratory and pathogenic strains.
dna fragments, generated by pcr amplification, using pdoc-k as a template were cloned into pdoc-c, and the resulting donor plasmids used for gene doctoring.
to date we have made deletions of the rpos, fur, flhdc and soxs genes in mg <dig>  o157:h <dig> sakai, cft <dig> and h <dig> strains .
functionality of the epitope tags
to examine the functionality of the epitope tags we coupled each to the lac repressor protein in mg <dig>  the experimental details and primer design for each recombination experiment are given in the methods section.
for each epitope tag we identified more than  <dig> candidates that were kanamycin resistant, sucrose insensitive.
after verification by pcr amplification and dna sequencing of the chromosomal region , we tested the functionality of the epitope tags.
the laci:: <dig> × flag, laci:: <dig> × proteina and laci::gfp fusion proteins were analyzed by western blotting.
whole cell extracts were separated by sds-page and proteins transferred to nitrocellulose membranes, which were then probed with primary antibodies specific to the tag.
the membranes were then washed and probed with secondary antibodies conjugated to horse-radish peroxidase.
figure 5; panel b, shows an image of the membranes after exposure to x-ray film; the fusion proteins are indicated.
in a recent study we validated the functionality of the laci:: <dig> × flag fusion protein by isolating dna fragments carrying laci binding sites from cells  <cit> .
we also confirmed the fluorescence of the laci::gfp fusion protein, in whole cells using fluorescent microscopy .
finally, we tested the integrity of the  <dig> × his fusion proteins by isolating the protein fusion by affinity purification using nickel agarose affinity media .
purified proteins were analysed by sds-page.
figure 5; panel c, shows a scanned image of the sds-page gel on which the fusion protein is highlighted.
CONCLUSIONS
we have developed a version of the two-plasmid recombineering system for generating chromosomal modifications in e. coli strains which, we have termed gene doctoring.
this method relies on homologous recombination, mediated by the λ-red genes, of a linear dna fragment that is supplied in vivo by restriction of a pdoc donor plasmid by i-scei endonuclease.
the identification of recombinants is highly efficient and reproducible, since counter-selection using the sacb gene identifies true recombinants.
this eliminates the requirement for screening large numbers of candidates by pcr, which is both costly and time consuming.
in addition, we have made a modified recombineering plasmid, pacbsce, which carries a dna recognition site for i-scei in the origin of replication, meaning that recombinants are not over-exposed to the potential mutagenic side-effects of the λ-red gene products.
the gene doctoring system is principally effective for recombineering in different pathogenic e. coli strains, which as we have demonstrated, are not particularly amenable to chromosomal modification using existing systems.
this system is designed to facilitate the coupling of genes to epitope tags, though the deletion of genes can also be readily achieved.
we have demonstrated the versatility of gene doctoring by deleting genes in both laboratory and pathogenic e. coli strains, in addition to coupling several genes to epitope tags, and we have confirmed the functionality of epitope tagged fusion proteins using biochemical methods.
we believe that the gene doctoring system can be transferable to other bacteria, in which the pdoc and pacbsce plasmids are stable and will replicate.

 the λ-red recombinase system can be used to introduce mutations, deletions, or insertions into the e. coli chromosome by recombining regions of homology carried on short single-stranded oligonucleotides or large double-stranded dna molecules. the λ-red system consists of three proteins, the gam, exo and bet gene products. when expressed in the cell the gam protein protects linear double stranded dna from degradation by the host recbcd complex. the exo protein generates single stranded dna overhangs, which are substrates for recombination, catalyzed by the bet protein, with homologous regions of the chromosome. several λ-red recombineering techniques have been developed: two in particular are of note, which differ in the way that the target dna is delivered into the cell. the first technique, and arguably the most widely used, was first described by murphy and later refined by datsenko and wanner. in this method a plasmid is used to express the λ-red genes from an arabinose inducible promoter. strains expressing λ-red are transformed, by electroporation, with a dsdna pcr product carrying an antibiotic cassette flanked by short regions of homology to the target gene. λ-red mediated recombination occurs, resulting in replacement of the targeted gene by an antibiotic resistance cassette. this cassette can then be excised by flp recombinase leaving a ~ bp dna scar in place of the target gene. the second technique, "gene gorging", designed by herring and co-workers, is a two plasmid method that also utilizes the λ-red system to generate recombinants. gene gorging eliminates the need to electroporate a dsdna fragment into cells, by supplying the regions of homology to the target gene on a donor plasmid that also contains a dna recognition site for the saccharomyces cerevisiae i-scei endonuclease. the donor plasmid and the recombineering plasmid, pacbsr, are transformed into the recipient strain. upon arabinose induction, i-scei cleaves the donor plasmid, providing a linear dsdna target for the λ-red system. the obvious advantage of this system is that multiple copies of the homologous dna are present in the bacterial cell, which increases the number of potential recombination events. the frequency of recombination for gene gorging is reported to be 1-15%, eliminating the absolute requirement for an antibiotic resistance marker to select for recombinants. we have used both systems for making gene knockouts and gene fusions in laboratory e. coli strains. however, we have had less success with these methods in pathogenic strains such as the o157:h sakai strain, and virtually no success in the cft upec, the o eaec and the h etec strains. since techniques such as transduction by p phage are incompatible with many pathogenic strains, due to extensive surface antigens that block access to the phage receptor, gene deletions have to be made directly in the strain. our aim in this study was to establish a high-throughput recombineering system, with particular emphasis on the ability to couple epitope tags to genes, which is compatible, without modification, for use in a wide range of laboratory and wild-type e. coli strains. we have achieved this by enhancing the two-plasmid system of herring and co-workers, making three key modifications. first, a set of donor plasmids have been generated that readily facilitate the deletion of genes or the c-terminal coupling of genes to epitope tags. second, the inclusion of the sacb gene on the donor plasmid allows for the counter-selection of all but true recombinants. third, the inclusion of an i-scei recognition site on a derivative of the recombineering plasmid, called pacbsce means that the plasmid is effectively'self-cleaving' upon induction of i-scei and λ red genes. hence cells receive a burst of λ-red before pacbsce is lost, which is sufficient to induce recombination but limits the exposure of cells to λ-red function. prolonged exposure to λ-red activity is undesirable as it can cause unwanted secondary chromosomal modifications in both k- and pathogenic e. coli strains. we have termed this method gene doctoring, abbreviated to g-doc, and we have demonstrated its versatility by deleting and coupling genes to epitope tags in pathogenic and laboratory e. coli strains.  current techniques for recombineering in laboratory and pathogenic escherichia coli strains a. electroporation of linear dna fragments the method first described by murphy, later refined by datsenko and wanner, of electroporating linear double stranded dna fragments into cells that are then targets for homologous recombination by the λ-red system, is reported to promote a very low recombination efficiency in e. coli k- strains: approximately in every × e. coli k- mg cells that survive electroporation. despite this low frequency, we routinely identify between 10- mg recombinants per experiment, however, since we use approximately × mg cells per electroporation, the identification of only 10- recombinants indicates that in our hands the recombination efficiency is approximately in every × cells, times less than reported. despite consistently attaining recombinants in mg using this system we have had virtually no success in pathogenic strains. since the low recombination frequency of the system has been attributed to the inefficient uptake of linear dsdna fragments during electroporation, we determined whether the inefficiency of this system for recombination in pathogenic strains was due to a reduced capacity to uptake dna by electroporation. thus, we compared the transformation frequencies of mg o cft and o157:h sakai cells when transformed by electroporation with different plasmids. cells in the exponential phase of growth were transformed by electroporation as previously described with either: puc, bp, conferring ampicillin resistance; pkd, bp, conferring ampicillin resistance; pacbsr, bp, conferring chloramphenicol resistance; prw, bp, conferring tetracycline resistance. cells were then plated onto lennox broth agar plates supplemented with appropriate antibiotics, incubated for hours at 37°c and the number of colonies counted. table shows the transformation frequencies of the pathogenic strains by each plasmid, expressed as a percentage of the transformation frequency of mg it is clear that the transformation frequencies of the pathogenic strains are dramatically lower than for mg particularly for strains cft and o considering that we expect approximately 10- recombinants in mg such low electroporation efficiencies could explain why using this technique in pathogenic strains results in minimal success. since the datsenko and wanner system relies upon the introduction of pcr generated dna into cells and not plasmids that have been isolated from an e. coli k- strain, we re-examined the dna uptake efficiencies of the strains when transformed with a pcr generated version of the plasmid, puc we reasoned that plasmids isolated from a k- strain may be subject to host restriction-modification systems in pathogenic strains, hence, using a pcr-generated puc derivative would not only more closely resemble the conditions used by datsenko and wanner, but also allow us to monitor the transformation efficiencies by means of the acquired ampicillin resistance due to puc plasmid uptake. thus, we amplified puc by pcr and then incubated the reaction with dpni, which specifically digested the methylated template plasmid and not the pcr generated product. the pcr generated puc plasmid was then transformed into mg cft o157:h sakai and o by electroporation. the results show that the transformation frequency of the pathogenic strains by puc18pcr was slightly improved when compared with mg although the overall transformation frequency remains far lower than mg the overall number of mg colonies identified after transformation with puc or puc18pcr was comparable. thus, the electroporation step is likely to be the primary reason for the poor efficiency of this system in pathogenic e. coli strains. this shortcoming was alleviated somewhat by murphy and campellone who developed an improved electroporation based protocol for recombineering in e. coli ehec and epec strains. however, we have had mixed success using this protocol, particularly when recombineering in eaec and upec strains, where no increase in recombination frequency was observed. b. two-plasmid recombineering the two plasmid gene-gorging method described by herring and co-workers has an immediate advantage for recombineering in pathogenic strains since the method does not rely upon efficient electroporation as a means of introducing target dna into the cell. instead, the target dna is flanked by recognition sites for the meganuclease i-scei on a donor plasmid that is transformed into cells along with the recombineering plasmid, pacbsr, which carries i-scei and the λ-red genes whose expression is controlled by an arabinose inducible promoter. induction of i-scei results in donor plasmid cleavage, generating the linear dsdna target, which is a substrate for λ-red gene products. herring and co-workers disrupted chromosomal genes by introducing amber mutations, using long regions of homology to the chromosome and reported that the recombination frequency for gene gorging was between 1-15%. to introduce epitope tags at the carboxyl terminus of chromosomal genes and, crucially, to maintain a high-throughput system, we chose to amplify the epitope tag by pcr using oligonucleotide primers that contained bp of homology to the chromosomal gene of interest, rather than sequentially cloning regions of homology to either side of the epitope tag. we reasoned that since short homologous sequences had already been successfully utilised for recombineering by datsenko and wanner, this strategy could be adapted for epitope tagging. the amplified dna product was cloned into pbr modified so that the pcr product would be flanked by two recognition sites for i-scei. the resulting construct was co-transformed, along with pacbsr, into mg cells and gene gorging experiments performed as described by herring and co-workers. the results of the experiments indicated that the recombination efficiency using short regions of homology was very poor; several hundred colonies recovered after gene gorging were screened by pcr and the frequency of recombination was found to be - %, far less than the 1-15% reported by herring and co-workers. to improve the identification rate of recombinants we modified the technique by including a kanamycin cassette adjacent to the epitope tag on the pbr based donor plasmid. we reasoned that after in vivo digestion of the donor plasmid, the ampicillin cassette carried on pbr would be lost and kanamycin resistance would only be maintained if a successful recombination event had occurred. hence after gene gorging, cells were plated onto lb agar plates containing kanamycin, and the next day colonies were replica plated onto lb plates containing either ampicillin or kanamycin. these colonies were screened for candidates which were kanamycin resistant and ampicillin sensitive, indicative of donor plasmid loss and kanamycin cassette retention as a result of recombination with the chromosome. however, this approach proved to be problematic, since unless the in vivo cleavage rate of the donor plasmid by i-scei approaches 100% efficiency, the ampicillin and kanamycin cassettes are still present on the donor plasmid in the cell, since the plasmid is present in multi-copy, rendering positive selection ineffective. typically we screened up to colonies by replica plating, identifying no more than colonies with the correct phenotype. taken together these results demonstrate that a more effective technique, that is both rapid and reliable, is required to introduce epitope tags onto the chromosome of pathogenic e. coli strains. gene doctoring to address this requirement we have developed an enhanced version of the two-plasmid gene gorging system. our method, termed gene doctoring, facilitates the coupling of genes to epitope tags or the deletion of chromosomal genes and increases the rate of identifying recombinants. we have generated a suite of pdoc plasmids which allow for the deletion of chromosomal genes, or the coupling of chromosomal genes to a × his, a × flag, a × proteina or a gfp tag. the pdoc plasmids carry the sacb gene, which allows for the counter-selection of cells still harbouring donor plasmid, thus eliminating the need for screening post recombination. using this system we routinely identify more than recombinants per experiment in both laboratory and pathogenic e. coli strains, using short regions of homology to the chromosome, thus maintaining both a high-throughput and broad-range compatibility system. g-doc plasmids the pdoc plasmids are derived from pex100t, a medium copy number plasmid which carries ampicillin resistance and the b. subtilis sacb gene. we have introduced different dna sequences into the pex100t i-scei restriction sites to create a suite of plasmids, schematic diagrams of which are shown in figure the cloning plasmid, pdoc-c, has a large cloning region flanked by two i-scei recognition sites. the dna sequence of pdoc-c, from bp upstream of the left-hand i-scei site to bp downstream of the right-hand i-scei site is shown in figure panel a. the template plasmid, pdoc-k, carries a kanamycin resistance cassette flanked by flp recombinase recognition sites. on either side of this region are cloning regions. the other template plasmids, pdoc-h, pdoc-f, pdoc-p and pdoc-g are derivatives of pdoc-k that have the coding sequence for a × his, × flag, × protein a and gfp tag respectively, immediately downstream of cr figure 2; panel b, shows the dna sequence common to all of the pdoc template plasmids, from bp upstream of the left-hand i-scei site to bp downstream of the right-hand i-scei site. the template plasmids differ between the cr and flp sequences: this region is outlined by an open box in the figure. the dna sequence proceeds through cr along the respective dna sequence for each plasmid within the open box, and into the flp sequence below. the plasmid pdoc-k has bp of dna sequence prior to flp the plasmid pdoc-h has the coding sequence for the × his tag and a stop codon followed by a short dna sequence leading into the flp site. the first codons of the × flag, proteina and gfp tags are shown, followed by the stop codon and short dna sequences leading into flp site. other features indicated on the dna sequences of the pdoc plasmids in figure are described in the g-doc recombineering protocol below. the full dna sequence of each pdoc plasmid is provided in additional file and is also available from genbank, accession numbers gq88494-gq g-doc recombineering protocol for generating gene:epitope tag fusions, the epitope tag and kanamycin cassette are amplified by pcr, using the relevant pdoc plasmid as a template. a schematic outline of the cloning strategy for generating gene:epitope tag fusions is shown in figure panel a. the clockwise primer used for the pcr amplification is designed so that it contains between 25- bp of homology to the 3' end of the target gene, not including the stop codon, followed by bp of sequence which anneals to the epitope tag sequence on the pdoc plasmid. this should be designed so that, after recombination with the target gene on the chromosome, the gene will be in frame with the coding sequence of the epitope tag. the downstream primer is designed so that it contains 25- bps of homology to the dna sequence immediately downstream of the target gene and the primer sequence p-rev. the two primers are also designed with a restriction site at the 5' end, so that, after amplification by pcr, the dna product can be cloned into the cloning region of pdoc-c, between the two i-scei sites. for generating gene deletions, the kanamycin cassette from pdoc-k, is amplified by pcr. a schematic outline of the cloning strategy for generating gene deletions is shown in figure panel b. the clockwise primer used for the pcr amplification is designed so that it contains between 25- bp of homology to the dna immediately upstream of the start of the gene, followed by bp of sequence which anneals to the k-fwd sequence on pdoc-k. the anti-clockwise primer is designed so that it contains 25- bps of homology to the 3' end of the gene and the primer sequence p-rev. again, the two primers are designed with 5' restriction sites for cloning the dna product into pdoc-c. alternatively, when longer regions of homology to the chromosome are required, sequential cloning steps can be performed, utilising the multiple cloning sites to introduce long regions of chromosomal homology upstream and downstream of the kanamycin cassette and epitope tag. in this case we recommend sequencing the cloned homology regions, post cloning and before recombineering, using priming sequences s and s. the next step is to transform the pdoc donor plasmid into the recipient strain with the recombineering plasmid, which expresses i-scei and the λ-red gene products. a schematic protocol, outlining the key steps in generating recombinants is shown in figure we have modified the recombineering plasmid, pacbsr, used by herring and co-workers by introducing an i-scei recognition site adjacent to the replication origin of the plasmid: we have called this plasmid pacbsce. upon arabinose induction, a burst of i-scei and λ-red expression occurs; i-scei cleaves the donor plasmid resulting in generation of the substrate for λ-red mediated recombination. in addition, i-scei also cleaves the pacbsce recombineering plasmid, resulting in loss of plasmid and loss of λ-red expression, thus avoiding prolonged λ-red activity, which can result in unwanted chromosomal modification. recombination occurs between homologous regions on the linear dna substrate and the chromosome, transferring the kanamycin cassette, and in the case of gene:coupling, the epitope tag, onto the chromosome. recombinant clones are selected for by growing cells on lb agar plates containing kanamycin and sucrose: only true recombinants, which have lost the sacb gene due to donor plasmid loss and have retained the kanamycin cassette due to recombination, are able to survive and grow on this medium. examination of recombinants, to ensure that the correct chromosomal modification has been generated, is achieved by amplifying the target region by pcr, using primers that anneal adjacent to the homology regions and chromosomal check priming sequences cc and cc. once recombination has been confirmed, the kanamycin cassette can be excised from the chromosome using the flp recombinase sites, as described previously. g-doc validation to test our system we designed oligonucleotides to couple the rsd and yacl genes to the × flag epitope tag. one primer contained homology to the first codons of the × flag sequence, bp of homology to the 5' end of the target gene, excluding the stop codon, and an ecori restriction site. the second primer contained homology to the p-rev sequence, bp of homology to the chromosome, immediately downstream of the target gene primer and a kpni restriction site. dna fragments generated by pcr using pdoc-f as a template were cloned into pdoc-c, which was subsequently co-transformed with pacbsce into k- mg ehec o157:h sakai and upec cft cells. the gene doctoring protocol was followed and the results are reported in table for both genes, in all three strains, a large number of colonies were identified with a kanamycin resistant, sucrose insensitive phenotype. after pcr analysis of the relevant chromosomal region and d and d and d for rsd and d and d and d and d for yacl) the vast majority of candidates were found to be true recombinants and in each case, more than 90% were sensitive to both ampicillin and chloramphenicol, indicating loss of both pdoc donor and pacbsce plasmids. where a candidate was found to have the wild-type form of the gene after pcr verification, we assumed that the kanamycin cassette had inserted into a different part of the chromosome, since we were unable to isolate any donor plasmid dna from cells using standard plasmid isolation techniques. hence, for each gene, in each strain, more than recombinants were identified that had the correct chromosomal modification and were free of the recombineering plasmid pacbsce. to test the effectiveness of recombination using our recombineering plasmid pacbsce, compared with the recombineering plasmid pacbsr, used by herring and co-workers we repeated the gene coupling analysis of the rsd gene. the results in table show that more kanamycin resistant, sucrose insensitive recombinants were identified in each strain when pacbsr was used as the recombineering plasmid, with a comparable percentage being free of pdoc donor plasmid, when compared to using pacbsce as the recombineering plasmid. however, very few candidates had lost the recombineering plasmid, and in strain cft all of the recombinant candidates still carried pacbsr, thus exposing cells to the potential effects of excess of λ-red expression and requiring additional steps to cure cells of the plasmid. we have further validated the system by making several gene deletions in both laboratory and pathogenic strains. dna fragments, generated by pcr amplification, using pdoc-k as a template were cloned into pdoc-c, and the resulting donor plasmids used for gene doctoring. to date we have made deletions of the rpos, fur, flhdc and soxs genes in mg o157:h sakai, cft and h strains. functionality of the epitope tags to examine the functionality of the epitope tags we coupled each to the lac repressor protein in mg the experimental details and primer design for each recombination experiment are given in the methods section. for each epitope tag we identified more than candidates that were kanamycin resistant, sucrose insensitive. after verification by pcr amplification and dna sequencing of the chromosomal region, we tested the functionality of the epitope tags. the laci:: × flag, laci:: × proteina and laci::gfp fusion proteins were analyzed by western blotting. whole cell extracts were separated by sds-page and proteins transferred to nitrocellulose membranes, which were then probed with primary antibodies specific to the tag. the membranes were then washed and probed with secondary antibodies conjugated to horse-radish peroxidase. figure 5; panel b, shows an image of the membranes after exposure to x-ray film; the fusion proteins are indicated. in a recent study we validated the functionality of the laci:: × flag fusion protein by isolating dna fragments carrying laci binding sites from cells. we also confirmed the fluorescence of the laci::gfp fusion protein, in whole cells using fluorescent microscopy. finally, we tested the integrity of the × his fusion proteins by isolating the protein fusion by affinity purification using nickel agarose affinity media. purified proteins were analysed by sds-page. figure 5; panel c, shows a scanned image of the sds-page gel on which the fusion protein is highlighted.  we have developed a version of the two-plasmid recombineering system for generating chromosomal modifications in e. coli strains which, we have termed gene doctoring. this method relies on homologous recombination, mediated by the λ-red genes, of a linear dna fragment that is supplied in vivo by restriction of a pdoc donor plasmid by i-scei endonuclease. the identification of recombinants is highly efficient and reproducible, since counter-selection using the sacb gene identifies true recombinants. this eliminates the requirement for screening large numbers of candidates by pcr, which is both costly and time consuming. in addition, we have made a modified recombineering plasmid, pacbsce, which carries a dna recognition site for i-scei in the origin of replication, meaning that recombinants are not over-exposed to the potential mutagenic side-effects of the λ-red gene products. the gene doctoring system is principally effective for recombineering in different pathogenic e. coli strains, which as we have demonstrated, are not particularly amenable to chromosomal modification using existing systems. this system is designed to facilitate the coupling of genes to epitope tags, though the deletion of genes can also be readily achieved. we have demonstrated the versatility of gene doctoring by deleting genes in both laboratory and pathogenic e. coli strains, in addition to coupling several genes to epitope tags, and we have confirmed the functionality of epitope tagged fusion proteins using biochemical methods. we believe that the gene doctoring system can be transferable to other bacteria, in which the pdoc and pacbsce plasmids are stable and will replicate. 
a common technique is to electroporate linear dna fragments into cells. alternatively, dna fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. in both cases the λ-red gene products recombine homologous regions carried on the linear dna fragments with the chromosome. homologous recombination mediated by the λ-red genes is a common method for making chromosomal modifications in escherichia coli. we have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacb gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. to that end we have designed a series of donor plasmids for use with the λ-red recombination system, which when cleaved in vivo by the i-scei meganuclease generate a discrete linear dna fragment, allowing for c-terminal tagging of chromosomal genes with a × his, × flag, × proteina or gfp tag or for the deletion of chromosomal regions. our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and k- e. coli strains. however, we have had limited success with these λ-red based recombination techniques in pathogenic e. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.
 homologous recombination mediated by the λ-red genes is a common method for making chromosomal modifications in escherichia coli. several protocols have been developed that differ in the mechanisms by which dna, carrying regions homologous to the chromosome, are delivered into the cell. a common technique is to electroporate linear dna fragments into cells. alternatively, dna fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. in both cases the λ-red gene products recombine homologous regions carried on the linear dna fragments with the chromosome. we have successfully used both techniques to generate chromosomal mutations in e. coli k- strains. however, we have had limited success with these λ-red based recombination techniques in pathogenic e. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.  our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and k- e. coli strains. to that end we have designed a series of donor plasmids for use with the λ-red recombination system, which when cleaved in vivo by the i-scei meganuclease generate a discrete linear dna fragment, allowing for c-terminal tagging of chromosomal genes with a × his, × flag, × proteina or gfp tag or for the deletion of chromosomal regions. we have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacb gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. this method has the added advantage of limiting the exposure of cells to the potential damaging effects of the λ-red system, which can lead to unwanted secondary alterations to the chromosome.  we have developed a counter-selective recombineering technique for epitope tagging or for deleting genes in e. coli. we have demonstrated the versatility of the technique by modifying the chromosome of the enterohaemorrhagic o157:h, uropathogenic cft, enteroaggregative o and enterotoxigenic h e. coli strains as well as in k- laboratory strains. 