Initial Stages in Creating a lacI Knockout in Escherichia coli C29 Using the Lambda Red Recombinase System

Abstract

An attempt was made in this study to knockout the lac I gene in Escherichia coli C29 cells. This multi-step process, involving PCR and electroporation, was successful in many areas. The initial transformation of the C29 cells with the plasmid pKD46 using electroporation successfully conferred ampicillin resistance. The pKD46 plasmid introduced at this stage contains the lamda-red recombinase genes as well as an ampicillin resistance cassette. Primer design and subsequent amplification of the promoter-less kanamycin resistance gene from the pACYC177 plasmid, preceded the second electroporation of the C29 cells harboring pKD46. The PCR product was thought to be designed such that the flanking ends were homologous to the termini of the lac I gene. However, it was discovered that an upstream DNA binding domain remained and was fused to the kanamycin resistance cassette. Introduction of the linear DNA PCR product into the C29 cells resulted in kanamycin resistance. This result indicates successful integration of the PCR product into the genome of the C29 cells, for only under an endogenous promoter would expression of the kanamycin gene be observed. Supporting this observation is the activation of β -galactosidase ( β -gal) in the absence of IPTG, for the cells expressed a blue phenotype when grown in LB/X-gal solid media. The blue phenotype was less intense than expected, suggesting only partial repression of the lac operon as a result of the DNA binding domain present in the fusion protein. When transformants were tested using an ONPG assay, higher basal levels of β -gal activity was observed when compared to control cells, yet both samples were equally sensitive to IPTG.