The Study of Exclusion Effect of pBR322 using its rop-inactivated Mutant, During Co-transformation with pBR322 and pUC19: Plasmid Copy Number

Abstract

It has been shown that when Escherichia coli DH5α cells are co-transformed with pBR322 and pUC19, pUC19 is retained in the host cell while pBR322 is excluded. A possible explanation this phenomenon is that the Rop protein produced in pBR322, but not in pUC19, has caused pBR322 to have a lower plasmid copy number, leading to the exclusion of pBR322. The purpose of this study was to investigate the role of Rop protein in the exclusion effect of pBR322 by using pCAWK, a pBR322 mutant that lacked functional rop gene. The inactivation of rop gene in pCAWK was verified using plasmid copy number test. Cotransformation tests of pBR322 with pUC19, pBR322 with pCAWK and pCAWK with pUC19 were done to determine the ability of different plasmids to co-exist in the same host and the ability of each plasmid to be retained in the host when co-existence was not permissible. Based on the results, plasmid copy number did not influence the sustainability and co-existence of plasmids. There were two ways to interpret the result for the co-transformation of pUC19 with pCAWK. The first interpretation suggested that the inability of plasmids to co-exist was due to competition for replication resources after sharing the same origin of replication, whereas the second interpretation suggested that the presence of Rop protein prevented the coexistence of plasmids with functional rop gene and plasmids without functional rop gene. The factor that decides which plasmids are retained in the host cell when plasmids co-exist was not yet determined.