Rop+ pUC19 from pBR322 Mutant lacking the Tetracycline Resistance Gene

Abstract

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 Acc I and EcoR I. 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.