Construction of an enlarged pUC19 vector with a rop gene designed to study plasmid maintenance in Escherichia coli

09/05/2015

Benson Chang, Arnab Ray, Thomas Tsuei, Rachel Wan

Department of Microbiology and Immunology, University of British Columbia

Volume 19
Fall 2014 / Winter 2015

Experimental evidence has demonstrated that when pUC19 and pBR322 are co-transformed into DH5α Escherichia coli, only pUC19 is maintained after a few generations. Possible selective factors include the difference in size between the two plasmids, the presence of a negative regulator rop gene encoded by pBR322 and a G→A point mutation in the origin of replication of pUC19. The focus of this study was the construction of a novel enlarged pUC19 vector, pBART, which carries the rop and tetracycline resistance genes derived from pBR322. Previous attempts at producing a rop+ pUC19 were unsuccessful and failures were attributed to low DNA insert concentration and poor ligation. To circumnavigate these problems, the pBR322 fragment was amplified using PCR and through the process of primer design, a unique XbaI restriction site was created. The PCR fragment was digested and ligated into pUC19 and subsequently transformed into DH5α cells. Disruption of lacZ by inserting the pBR322 fragment into the multiple cloning site of pUC19 assisted in isolating transformants carrying the pBR322 fragment. The pBART construct was confirmed by restriction mapping and sequencing. The pUC19 derived pBART construct is comparable in size to pBR322 and can be used to explore how the role of plasmid size, presence of rop and the origin of replication G→A point mutation contributes to differences in plasmid copy number during cotransformation of pUC19 and pBR322.