PCR Amplification, and Sequence Comparison of lacI gene in WT E. coli C29 cells and a presumptive lacI Knockout E. coli C29 cells to Determine the Difference in the Basal Expression Level of lacZ in Lac Operon

LacI gene encodes for the repressor of Lac operon. An Esherichia coli (E. coli) strain with a lacI knockout would yield cells that can constitutively express ß-galactosidase. Use of these cells can considerably reduce the use on an inducer isopropyl-ß-D-1-thiogalactoside (IPTG) and reduce the induction time. Jaeger et al (2) prepared a presumptive lacI knockout in E. coli C29 strain using a λ Red recombination system. This knockout would, in theory, delete a large portion of gene that encodes 54348 amino acids from a total of 360 amino acids in a repressor. The presumptive deleted region of the lacI gene was replaced by a kanamycin resistant gene, which would confer the recombinant cells resistant to the antibiotic kanamycin. Interestingly, these cells were found to have low expression of ß-galactosidase, probably, due to partial repression of Lac operon. Also, it was found that when an inducer was added, these cells would show higher expression of ß-galactosidase. This study was carried out to determine why there was still repression in the expression of ß-galactosidase, whereas there should not be any repression at all. The multi-step approach used in this study was to PCR amplify the lacI gene from a WT E. coli C29 and the presumptive lacIE. coli C29 cells, sequence the gene and confirm whether the deletional mutation took place in the presumptive lacIE. coli C29 cells as specified. The sequencing results from this experiment showed that the deletional mutation did not take place as specified. The results suggest that the recombinant cells were kanamycin resistant either because recombination took place at some other location in the genome, or that the cells had acquired kanamycin resistance from some other source. They also indicate that the lacI gene may posses a point mutation that can cause the basal expression of lacI gene to vary between WT E. coli C29 cells and the kanamycin resistant E. coli C29 cells. _______________________________________________________________