Attempt to Construct a Rop pUC19 from pBR322 Mutant lacking the Tetracycline Resistance Gene

The two commonly used plasmid vectors, pBR322 and pUC19, are both derivatives of the parent plasmid, pColE-1. However, it has been observed that pBR322 is excluded from the host cells following co-transformation of pBR322 and pUC19. One potential explanation for this exclusion effect is the presence of a functional rop gene in pBR322. The rop gene encodes for a protein that stabilizes the interaction between RNA I and RNA II, thus inhibiting the RNA II from carrying out its function as a primer to begin DNA replication. It is hypothesized that the lack of a functional rop gene in pUC19 accounts for its dominance in the co-transformation with pBR322. An attempt was made to isolate a fragment containing the rop gene from the tet pBR322 mutant, and inserting it into pUC19 to create a rop pUC19 construct. The rop gene was successfully excised using restriction endonucleases AccI and EcoRI. However, ligation and subsequent transformation produced no viable clones. Due to time constraints, ligation and transformation conditions could not be optimized, but a number of different options are discussed in this study. The ultimate success of creating a rop pUC19 will allow comparisons of co-transformation to be performed using pBR322 and either the wildtype or mutant rop pUC19. Insights will be gained about whether the rop gene contributes significantly to the exclusion effect. ________________________________________________________________