Successful Application of the λ-Red Recombinase System to Inactivate lacI in Escherichia coli C29, BW25993, and MG1655.

Abstract

The Lambda Red Recombinase system ( λ -Red) is a simple and efficient site-specific mutagenesis method. This study details the successful utilization of this method to inactivate lacI in Escherichia coli strains C29, BW25993, and MG1655 in the context of the Microbiology 421 laboratory. Previous studies performed in this setting have attempted to use this method to knockout the lacI gene from E. coli C29, thereby disrupting the repressor activities of the lacI gene product allowing for constitutive expression of β -galactosidase. Potential explanations for the lack of success in the past include mis-annotated primer design, sub-optimal L-arabinose induction of the recombinase genes, and low recombination frequency. In this study a PCR product containing a kanamycin resistance gene was produced using primers that allow for genotypic identification of the inserted construct, flanked with homologous sequence found upstream and downstream of the lacI gene. This product was transformed into all three E.coli strains containing the recombinase plasmid. Selection for kanamycin resistance confirmed the successful transformants. Preliminary evidence shows that deletion of lacI was successful in all three strains, and conclusive enzyme assay data supported this in both E. coli C29 and BW25993 strains. This study provides the first evidence of successful use of the λ -Red recombinase system in the Microbiology 421 laboratory. This work provides a basis for future studies; the λ -Red system is a rapid and specific method for gene interruption and this work is an important first step in its practical application for gene-function analysis.