Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Homologous recombination mediated by the lambda-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 lambda-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 lambda-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-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the lambda-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 6xHis, 3xFLAG, 4xProteinA 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 lambda-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:H7 (EHEC), uropathogenic CFT073 (UPEC), enteroaggregative O42 (EAEC) and enterotoxigenic H10407 (ETEC) E. coli strains as well as in K-12 laboratory strains.