Construction of a Mutant pBR322 Using Site-Directed Mutagenesis to Investigate the Exclusion Effects of pBR322 During Co-transformation with pUC19

It has been observed that when pBR322 and pUC19 plasmids (derivatives of pColE1) are co-transformed the pBR322 plasmid is selectively excluded from the cell. There are several potential factors that may explain this observation. One particular factor may be that pBR322 encodes the gene for the Rop protein, not seen in pUC19. This protein is involved in stabilizing the interaction between RNA I and RNAII, which in turn prevents the replication of pBR322. The focus of this study was to create an altered pBR322 plasmid such that it contains a mutation in the ribosome binding site. In theory, a mutation in the ribosome binding site would lead to a decreased production of the Rop protein, which may result in a higher copy number pBR322 and a reduction in exclusion effect during cotransformation with pUC19. Site-directed mutagenesis was used to introduce a mutation in the ribosome binding site along with a new Alu I restriction site not present in wild-type pBR322. Following mutagenic PCR, the mixture was transformed into Escherichia coli DH5α cells and five colonies were chosen for restriction digests. All five colonies showed a consistent restriction pattern, which was different from the wild-type pBR322 plasmid. Future work will confirm the identities of these colonies, and co-transformation tests will assess the role of Rop. _______________________________________________________________