Investigation of the pBR322 Exclusion Effect using Putative Rop Mutant pBR322 Plasmid pCAWK

Rop, repressor of primer, protein has been implicated in causing or contributing to plasmid pBR322 exclusion from Escherichia coli host cells. A recent study successfully created pCAWK, a putative mutated pBR322 plasmid with a non-functioning Rop protein. Whole cell PCR was performed with DH5α E. coli colonies respectively containing pUC19, pBR322, pCAWK1, and pCAWK2 (plasmids with different orientations of the 34 bp insert disrupting the rop gene) to compare plasmid copy numbers. Unique forward and reverse primers were designed through manual selection of sequences for each plasmid, complemented and optimized by analyses using primer3, REPFIND, and EMBOSS programs. Following PCR, the plasmid copy numbers of each plasmid were assessed by visual and quantitative agarose gel analysis to observe whether pCAWK in either orientation exists at a quantity that is higher, lower, or comparable to the other plasmids. Both pCAWK1 and pCAWK2 persisted poorly in host cells when compared to the parental pBR322 or pUC19, with pUC19 being most abundant. Although superficially these observations suggest that Rop may not be an important factor in the exclusion effect, the semi-quantitative technique used makes definitive conclusions difficult. The results may instead, however, reveal a problem with pCAWK being a viable tool in studying the exclusion effect and that more quantitative investigations should be conducted to conclusively determine pCAWK’s utility before applying it in future Rop studies.