Characterizing a Lambda Red Recombinase Induced Presumptive Partial Deletion of lacI in Escherichia coli C29 that Affects Regu

The λ Red recombination system was used in this study in an attempt to inactivate the lacI gene in Escherichia coli C29 cells. The proposed model retained the first 41 amino acids of the lacI gene, and replaced the rest of the gene with a linear double-stranded DNA PCR product that confers kanamycin resistance. The PCR products encode a kanamycin cassette with lacI flanking region. The initial step was to transform wild type E.coli C29 with plasmid pKD46 using electroporation. This transformation was highly successful and yielded ampicillin resistance cells conferring pKD46. pKD46 encodes enzymes required for the recombination process. The second step was to introduce the PCR products into cells conferring plasmid pKD46 again using electroporation. Since the PCR products contain lacI flanking regions, recombination should occur at the homologous lacI sequence in the bacterial chromosome. The second transformation produced Kan E.coli C29. βgalactosidase activity of Kan E.coli C29 cells was measured using the β-galactosidase assay. No significant difference in β-galactosidase activity was observed between Kan and wild type E.coli C29 cells. Evidence showed that lacI was still active and the partial deletion (if it occurred) of the lacI gene was not enough to inactivate the protein. Further studies are needed to determine if the λ red recombination system was successful in inserting the kanamycin gene in place of the distal part of lacI. _______________________________________________________