Preparation and expression of fusion gene clbS, colibactin resistance protein from Escherichia coli bacterium, and lacZ gene

Colibactin is a secondary metabolite/genotoxin produced by extraintestinal pathogenic Escherichia coli strains. Genes coding for colibactin synthesis are placed in a 54 - kilobase long genomic island, pks+ (polyketide synthase). An increase in the prevalence of E.coli strains harboring the pks genomic island has been recorded in developed countries. In addition, pks+ strains are often isolated from human colon cancer biopsies. Colibactin causes DNA double-strand breaks, chromosome irregularities, enescence, and in model organisms, such as the mouse, it causes tumorigenesis. ClbA is the key enzyme for colibactin synthesis while the ClbS protein binds to colibactin and protects the producer from its own toxin. It was previously shown that ClbS is also one of the few DNA-binding proteins that physically protects DNA from damage, including double-strand breaks. There is currently no data on (mechanisms controlling) clbS gene expression nor on its role in a population of pks+ E. coli strains. The results of the presented Master's thesis include the studied clbS gene expression level on the basis of fusion of the clbS gene promoter region with the promoterless lacZ gene. For that purpose, clbS gene promoter region was amplified with the appropriate oligonucleotide primers and PCR reaction, followed by ligation into a low-copy-number plasmid vector, pRW50, into which the lacZ gene was incorporated. The activity of the clbS-lacZ fusion was studied using the β-galactosidase test, under various bacterial growth conditions