Assessment of Targeting the Lambda Red Recombinase System to the Intended Disruption of the lacI gene in Escherichia coli C29

An attempt was made to knockout the Escherichia coli C29 lacI gene with a kanamycin resistance cassette isolated from the plasmid pACYC177. This process used the lambdared recombinase system to allow for homologous recombination, and subsequent insertion of the kanamycin cassette into the lacI gene. The E. coli C29 cells were initially transformed with pKD46, which contains the lambda-red recombinase genes, along with an ampicillin resistance cassette. The resulting ampicillin transformants were transformed with a linear DNA PCR product, the kanamycin cassette with flanking ends that were homologous to the termini of the lacI gene. If the lacI gene was successfully knocked out, then the β-galactosidase gene should be constitutively expressed. However, when the kanamycin resistant cells were grown on media containing X-gal, the colonies had a faint blue phenotype. The blue phenotype was not the intense blue phenotype that was expected in cells with high levels of β-galactosidase production. When these transformants were tested using an ONPG assay, the cells showed an increase in β-galactosidase activity in the presence of IPTG, at the same levels that were seen the original C29 cells. _____________________________________________________