Relevance of Primer Design to the Effective Disruption of the lacI gene in Escherichia coli C29 using the Lambda Red Recombinase System

Abstract

Previous investigators have been unsuccessful in using the Lambda Red Recombinase system to delete the lacI gene from Escherichia coli C29 cells. The last investigator hypothesized that this failure was likely due to the presence of inverted repeat (IR) sequences within the kanamycin resistance cassette, which is intended to replace the lacI gene. Therefore, in order to address this hypothesis, we designed novel primers to amplify the kanamycin resistance cassette without the flanking IR sequences. As done previously, PCR products containing this kanamycin cassette flanked by lacI homologous regions were transformed into cells containing the Lambda Red Recombinase system on plasmid pKD46. Unfortunately, our attempts to knockout the lacI gene were unsuccessful. Like previous groups, experiments performed on putative transformants strongly suggest that sensitivity of these cells to IPTG was retained, and hence lacI was not deleted. Upon investigation into problems with the experimental protocol, it came to our attention that, similar to previous groups, we had in fact failed to use L-arabinose to induce the expression of the Lambda Red Recombinase genes from the pKD46 plasmid. Lack of induction resulted in the absence of an operational Recombinase system, thus preventing successful replacement of lacI with the kanamycin resistance cassette. Hence, future attempts to use the Lambda Red Recombinase system to delete genes in E. coli should make use of the inducer, L-arabinose. Our initial hypothesis regarding the IR sequences therefore remains unaddressed.