Introduction of a Specific Point Mutation into the RNA II Locus on a rop-deficient pBR322 Plasmid

When the cloning vectors pUC19 and pBR322 are co-transformed into E. coli DH5 α cells, pBR322 is excluded from the cells. This exclusion has been attributed to regulatory elements present in pBR322 that slow down plasmid replication compared to pUC19 where the same elements are absent or mutated. One of these elements is an RNA molecule called RNA I that hybridizes and sequesters the RNA II replication primer. Another of these elements is a protein called Rop that stabilizes the interactions between RNA I and RNA II. In pUC19, the Rop protein is not expressed and a point mutation in RNA II hinders its hybridization and sequestration by RNA I. A previous study focused on the role of Rop in the pBR322 exclusion effect by creating a rop -defient pBR322 derivative, but this construct did not show any mitigation of the exclusion effect. The current study created a rop -deficient pBR322 derivative that also contains the RNA II point mutation present on pUC19 so that the importance of this point mutation in the absence of Rop-stabilized RNA interactions can be investigated. The mutant plasmid called pGK13W was created using site-directed PCR mutagenesis, and a diagnostic restriction digest was performed to confirm the introduction of the desired point mutation.