Attempts to Use PCR Site-Directed Mutagenesis to Create a Non-functional rop Gene in the Plasmid pBR322

Abstract

The co-transformation of two ColE1-type plasmids, pBR322 and pUC19 results in the exclusion of pBR322 from the cell. Several factors could potentially contribute to this exclusion effect; one potential factor is the Rop protein that is only encoded in the pBR322 plasmid. The Rop protein stabilizes the interaction between the replication primer, RNA II, and its antisense strand, RNA I. By complexing RNA I to RNA II, the replication of the plasmid is inhibited. Therefore, the absence of rop in pUC could directly lead to a higher replication rate and eventually a higher copy number, which then leads to the eventual exclusion of pBR322 during a co-transformation with pUC. This study attempted to create a pBR322 plasmid with a non-functional rop gene, by inserting stop codons into the rop gene using PCR site-directed mutagenesis. Theoretically, by creating a non-functional rop gene, the RNA II transcript should be free to anneal to the plasmid in order to act as a primer for replication. This increased rate of replication for pBR322 could potentially lead to a higher copy number, and perhaps result in the cancellation of the exclusion effect in a co-transformation experiment. However, the incorporation of the mutations into the plasmid was unsuccessful and so the co-transformation step was never completed.