Determination of Exclusion Effect in Wild Type and Rop Deficient Mutated pBR322 Co-transformations

Abstract

pBR322 is an expression vector previously reported to be selectively excluded when cotransformed with pUC19 into Escherichia coli DH5 α. Rop is a regulatory protein encoded on pBR322 but not pUC19. It is involved in stabilizing the RNAI/RNAII complex that inhibits replication of the plasmid. The focus of this study was to determine the role of the Rop protein in the selective exclusion of pBR322 when co-transformed with a mutated pBR322 that is deficient in Rop. This mutation was previously created by site-directed mutagenesis such that a mutation that created a new Alu I restriction site was introduced in the presumptive Rop ribosome binding site. When transformed into host cells, mutated pBR322 should produce no Rop protein, therefore resulting in a high mutant copy number. Competent E. coli DH5 α were cotransformed with equal amounts of wild type and mutated pBR322 to test the relative numbers of transformations. Plasmid DNA was then isolated from the selected transformants, run on an agarose gel, and analyzed for the plasmid band intensities for each plasmid. Our findings showed that when wild type and mutated pBR322 successfully co-transformed into host cells, no exclusion was observed and that doubling the wild type plasmid concentration increased the number of co-transformants by 10 folds. Both findings contradict our hypothesis that Rop is the determining cause of the selective exclusion of pBR322.