Determining the Importance of a Specific rom Mutation in Plasmid Exclusion of pBR322 During pUC19 Co-Transformation

Abstract

Co-transformation of the plasmids pBR322 and pUC19 has been shown to result in exclusion of pBR322 from transformed cells. The underlying mechanism for this exclusion is believed to be due to differences between the two plasmids including size, genetic mutations, and the presence or absence of specific genes. One such gene of potential importance is the rom gene, which has been shown to stabilize an interaction between RNAI and RNAII, inhibiting DNA replication in pBR322. This study investigates a derivative of the plasmid pBR322 with a specific mutation in the ribosome binding site of rom, pIK054, and compares it to pUC19 and pBR322 in co-transformation studies. It was expected that because of the mutation to the rom ribosome binding sequence, the rom gene product would not be formed and replication would increase in pIK054. It was found that despite this mutation, pIK054 did not outcompete pBR322 directly, and was still excluded during co-transformation with pUC19. The data suggests either that additional changes to the pBR322 plasmid are necessary to account for the improved replication of pUC19, or that the mutation to the ribosome binding site did not inhibit translation of the rom gene product. Future studies can investigate the presence of the rom gene product or the relative affinities of the ribosome to the ribosome binding site of the rom gene. In addition, a future study can systematically investigate the regions of pBR322 (or pIK054) that can improve replication.