Attempt to Construct a Rop+ pUC19 by Using NdeI, AatII, and AfeI Restriction Endonucleases with Separate Blunt and Staggered-end Ligations

Abstract

Cloning vector, pUC19, was originally derived from parent plasmid pBR322. But pUC19 has a higher copy number in host cells compared to pBR322. pBR322 plasmids possess a functional rop gene, expressed by readthrough transcription of the upstream tet gene, which generally controls copy number by facilitating the association of RNA II and RNA I molecules. It is theorized that the lack of a functional rop gene in pUC19 and the presence of a point mutation in the coding region of RNA II is responsible for the increase in copy + number. In this study, it was attempted to create a rop pUC19 plasmid construct to investigate the effect of a functional rop gene on pUC19 copy number compared to the unmodified pUC19 and the parent pBR322 plasmid. The first attempt to ligate the pBR322 fragment encoding the tet and rop genes into the pUC19 plasmid in place of the existing partial rop gene resulted in no clones. Experimental evidence showed that an inability to ligate Nde I cut DNA ends was a likely problem. An alternate method was explored that involved first excising the tet coding region from pBR322 with a different restriction – enzyme and recircularizing the intermediate tet pBR322 plasmid. Clones were successfully obtained that contained the correct intermediate plasmid. Subsequently, the rop gene along with the upstream tet gene P2 promoter and -10 and -35 regions could be excised and ligated into the pUC19 plasmid as before. Due to time constraints, this final step will need to be performed in future experiments which can then determine whether the lack of a rop gene in pUC19 is the sole reason for its high copy number compared to pBR322.