Consistency and Applicability of the λ-Red Recombinase System in Escherichia coli Tested on the Target Genes lacI and phoA

Abstract

The λ -Red recombinase system has been previously used to successfully delete the lacI gene from Escherichia coli strains C29, BW2553 and MG1655, after many earlier failures. In this study, the previous successful method was tested to determine whether the system could be applied to different genes, and whether the method gives consistent results. To that end, the lacI gene in E. coli C29 was targeted to answer the second aim, and serve as a positive control, while the phoA gene was targeted to test the first aim. A kanamycin resistance cassette ( kanR) was used as a template to create lacI-kanR constructs using the previous PCR primers, and a phoA-kanR construct using newly designed primers. Two phoA-kanR products were attempted; the second construct was not successfully created when Luk’s primers were used. The PCR products were electroporated into competent E. coli C29/pKD46 cells, which contain the pKD46 plasmid that encodes ampicillin resistance and the λ -Red recombinase. Screening for the mutants on selective media including antibiotics and colorimetric substrates showed that the target genes were still functioning. These results indicate that further work is necessary to refine the λ -Red recombinase method, as it was not able to knock out the previously successfully targeted lacI, nor the new target, phoA.