Abstract
BackgroundHomologous 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-12 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.ResultsOur 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-12 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 6 × His, 3 × FLAG, 4 × 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.ConclusionWe 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:H7 (EHEC), uropathogenic CFT073 (UPEC), enteroaggregative O42 (EAEC) and enterotoxigenic H10407 (ETEC) E. coli strains as well as in K-12 laboratory strains.
Highlights
Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli
Current techniques for recombineering in laboratory and pathogenic Escherichia coli strains A. electroporation of linear DNA fragments The method first described by Murphy [5], later refined by Datsenko and Wanner [2], of electroporating linear double stranded DNA fragments into cells that are targets for homologous recombination by the λ-Red system, is reported to promote a very low recombination efficiency in E. coli K-12 strains: approximately 1 in every 3.5 × 106 E. coli K-12 MG1655 cells that survive electroporation [4]
(page number not for citation purposes) http://www.biomedcentral.com/1471-2180/9/252 the system has been attributed to the inefficient uptake of linear dsDNA fragments during electroporation [4], we determined whether the inefficiency of this system for recombination in pathogenic strains was due to a reduced capacity to uptake DNA by electroporation
Summary
Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. 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. The first technique, and arguably the most widely used, was first described by Murphy [5] and later refined by Datsenko and Wanner [2] 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. This cassette can be excised by FLP recombinase leaving a ~ 80 bp DNA scar in place of the target gene
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
