(C) SDS-PAGE analysis of the affinity-isolation of LacI::6 CA4P solubility dmso × His. Proteins were stained with Coomassie blue. Lane 1 shows protein standards, lane 2, whole cell extract, lane 3, LacI::6 × His affinity-isolate. Conclusion 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 PI3K inhibitor 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 CHIR-99021 concentration 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, 3-mercaptopyruvate sulfurtransferase 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. Methods Strains The E. coli strains used in this study were MG1655 K-12 strain [21], O157:H7 Sakai EHEC strain (derivative in which the stx1 and stx2 genes were deleted by M. D. Goldberg, University of Birmingham, UK) [8], CFT073 UPEC strain [9] O42 EAEC [10] and H10407 ETEC strains [11] (from Ian R. Henderson, University of Birmingham, UK; Sequenced by the Sanger Institute: unpublished). Primers The primers used in this study are listed in table 4.