Exclusion of pBR322 after co-transformation with pUC19 into Escherichia coli is mediated by the rop gene

09/05/2015

Angela Mo, Thomas So, Wendy Zhang, Wouter-Jan van Roosmalen

Department of Microbiology and Immunology, University of British Columbia

Volume 19
Fall 2014 / Winter 2015

Co-transformation of plasmids is often necessary and useful in molecular biology for purposes such as introducing multiple desirable characteristics into cells. It has been observed that co-transformation of pBR322 and pUC19, two commonly used vectors, into Escherichia coli results in the exclusion of pBR322, where cells that have taken up both plasmids lose pBR322 while retaining pUC19. The three characteristics of pUC19 that may contribute to its selective maintenance in the cell relative to pBR322 are (i) its smaller size, (ii) a G to A mutation in RNAII, and (iii) lack of a rop gene. We hypothesized that the presence of the rop gene on pBR322 results in its exclusion when co-transformed with pUC19, and the inactivation of rop would diminish this effect. The influence of the rop gene on plasmid exclusion was tested by using a derivative of pBR322, pCAWK, which contains a 34 bp sequence disrupting the rop gene. To measure the plasmid ratio in co-transformed cells, we developed a β-galactosidase white/blue colony ratio screening assay. When plasmids pCAWK and pUC19 were transformed into E. coli DH5ɑ we observed a pCAWK:pUC19 ratio of 6.25. By comparison, when pBR322 and pUC19 were co-transformed into E. coli DH5α we observed only pUC19. This data suggests that pCAWK, which lacks the rop gene, can be maintained in the presence of pUC19 whereas pBR322 cannot. Taken together these data support the hypothesis that the rop gene on pBR322 contributes to its exclusion upon co-transformation with pUC19.