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Abstract
The Lamda Red Recombinase system is a site-specific mutagenesis method allowing for the swapping of targeted genes in the bacterial chromosome with an antibiotic selection factor. This study, along with previous studies, strived to employ this method to knockout the lacI gene from the Escherichia coli C29 host strain in order to remove the repressor activities of the lacI gene product allowing for constitutive expression of β -galactosidase. Previous attempts at disrupting lacI in this strain may have failed due to partial or complete curing of the temperature sensitive recombinase plasmid pKD46 with growth at 37°C, as well as sub-optimal and inconsistent Larabinose induction of the recombinase genes. In this study, a PCR product containing kanamycin was produced using primers which allow for genotypic identification of the inserted construct. This product was transformed into C29 cells containing the recombinase plasmid, with selection for kanamycin resistance revealing transformants. Examination of kanamycin resistant colonies using an enzyme plate assay indicated tight regulation of the β -galactosidase enzyme via lacI repression and as such, unsuccessful recombination using the Lambda Red Recombinase method in C29 cells. From these results it was concluded that the influence of growth at the required 30°C and L-arabinose induction, were insufficient to promote disruption of the lacI gene. Additionally, the presence of a short linking region within the PCR produced insertion construct compared to the previous constructs would allow improved identification of the isolates containing the construct.