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Abstract
Jaeger et al. originally conducted an experiment to deactivate the lactose repressor gene ( lac I) in Escherichia coli C29 using the λ -red recombinase system following the success of Datsenko et al. with the same system. The results obtained by Jaeger et al. showed that while integration of the kanamycin-resistance cassette occurred, it did not lead to deactivation of lac I, suggesting that the system had mistargeted the integration of the cassette. Many of the follow-up studies carried out were not able to account for the lack of success in deactivating the lac I gene. This project extended the previous studies by looking at the effectiveness of the λ -red recombinase system in two variations of the original experiment performed by Jaeger et al. In one experiment, the λ -red recombinase system was used to disable the alkaline phosphatase gene ( pho A) in Escherichia coli C29 to demonstrate that mistargeting was not gene specific. In the second experiment, the same system was used to deactivate lac I in Escherichia coli MG1655 to determine if the system was strain-specific. Similar to the previous studies, the kanamycin-resistance cassette from pACYC177 was amplified and used for site-directed disruption of both lac I and pho A. The transformation steps carried out in this study were successful, yielding kanamycin-resistant colonies. A brief screening test performed on C29 immediately showed that the pho A gene was still functional and not disrupted. A similar screening test performed on MG1655 initially suggested that lac I was disrupted, suggesting constitutive β -galactosidase production. However, a β -galactosidase assay showed that the use of IPTG resulted in elevated levels of β -galactosidase production. These results indicate that the λ -red recombinase is mistargeting the kanamycin-resistance cassette since disruption of the targeted genes was not achieved.