We reasoned that since short homologous sequences had already been successfully

utilised for recombineering by Datsenko and Wanner, [2] this strategy NSC 683864 mouse could be adapted for epitope tagging. The amplified DNA product was cloned into pBR322, 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 MG1655 cells and gene gorging experiments performed as described by Herring and co-workers [4]. The results of the experiments (not shown) 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 0.01-0.05%, 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 pBR322 based donor plasmid. We reasoned that after in vivo digestion of the donor plasmid, the ampicillin cassette carried on pBR322 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 Fludarabine order 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 BCKDHA 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 30,000 colonies by replica plating, identifying no more than 5 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 (G-DOC), 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 6 × His, a 3 × FLAG, a 4 × ProteinA or a GFP tag.