Effect of L-arabinose on the specific homologous recombination efficiency using the Lambda Red Recombinase system for gene disruption of lacI in Escherichia coli C29 cells

Abstract

Specific disruption of lacI in Escherichia coli C29 cells was attempted by using the Lambda Red Recombinase plasmid pKD46. Previous attempts to disrupt lacI using this system may have failed due to the absence of L-arabinose in the media required for expression of the recombinase genes. In this experiment, a kanamycin PCR product with flanking regions homologous to lacI was used to disrupt the lacI gene in L-arabinose induced cells harbouring pKD46. Putative transformants were assayed for the production of β -galactosidase, in the presence or absence of the inducer IPTG, using X-gal plates and a discontinuous enzyme assay. The tested kanamycin resistant transformants were all still inducible by IPTG indicating that lacI was not disrupted. This suggested that the addition of L-arabinose alone did not improve efficiency of specific recombination.