15 pKD46 100 5 2 0.26 pACBSR 100 2.8 1.5 0 pRW50 100 1.2 1.1 0 pUC18PCR 100 57 15 1 Since the Datsenko and Wanner system

relies upon the introduction of PCR generated DNA into cells and not plasmids that have been isolated from selleck chemical an E. coli K-12 strain, we re-examined the DNA uptake efficiencies of the strains when transformed with a PCR generated version of the plasmid, pUC18. We reasoned that plasmids isolated from a K-12 strain may be subject to host restriction-modification systems in pathogenic strains, hence, using a PCR-generated pUC18 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 pUC18 plasmid uptake. Thus, we amplified pUC18 by PCR and then incubated the reaction with DpnI, which specifically digested the methylated template plasmid and not the PCR generated MK-1775 product. The PCR generated pUC18 plasmid (pUC18PCR) was then transformed into MG1655, CFT073, O157:H7 Sakai and O42 by electroporation. The results (table 1) show that the transformation frequency of the pathogenic strains by pUC18PCR was slightly improved when compared with MG1655, although the overall transformation frequency remains far lower than MG1655. The overall number of MG1655

colonies identified after transformation with pUC18 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

Florfenicol [15] 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 [4] 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%.