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Abstract
Sequence alignments done between cloning vectors pBR322 and pUC19 revealed that pUC19 contained residual 32 bp sequence of the pBR322 rop gene, which is responsible for plasmid replication control and thought to play a role in pBR322 exclusion that occurs with co-transformation. Attempts were made to construct a pUC19 plasmid completely without the rop gene, by digesting out a segment containing the residual rop, and then performing a – fill-in reaction and blunt-end ligation to create a rop pUC19. Efforts at creating such a – construct were not successful and rop pUC19 transformants were not obtained, although positive control results suggest that both the fill-in and ligation reactions were functional. The lack of desired construct could be due to the deletion of a sequence/structure required for replication (e.g. essential region of the H strand Y effector site). Further experiments to test this hypothesis could be done by first inserting pBR322 rop gene into pUC19, before removal of the segment containing residual rop. Initial attempts at inserting the pBR322 rop gene into pUC19 were made during the course of this study. The rop gene was amplified from pBR322 template DNA by PCR, and efforts were made to insert the PCR ® ® product into an Invitrogen pCR II-TOPO Vector. However, results on the identity of potential clones obtained are pending sequencing results. Further studies should include the optimization of fill-in, ligation, and transformation reactions to help increase transformation efficiency.