Troubleshooting the Single-step PCR Site-directed Mutagenesis Procedure intended to Create a Non-functional rop Gene in the pBR322 Plasmid

Abstract

The co-transformation of ColE1-type plasmids, specifically pBR322 and pUC19 results in the exclusion of pBR322 in the transformed cells. The mechanism for this effect is not fully known, but it is speculated that this observed exclusion is due to the molecular differences of the two plasmids. The RNA II in pUC19 has a point mutation that is absent in the RNA II of pBR322. This point mutation causes the RNA I to bind less effectively to RNA II and thus RNA II would not be blocked and can initiate the primer formation. Also, pBR322 has a sequence that codes for a protein called Rop (repressor of primer) and pUC19 does not. Rop has negative regulatory functions and contributes to the inhibition of replication by stabilizing the interaction between RNA I and RNA II. The successful creation of a mutant pBR322 with a non-functional rop gene would give insight into the relative impact of the rop gene in the exclusion effect during co-transformation with pUC19. The non-functional rop gene can be created by inserting stop codons into the rop gene via single-step PCR sitedirected mutagenesis. There have been difficulties creating such a mutant. This study investigated and troubleshoots the creation of a non-functional rop gene via single-step PCR site-directed mutagenesis by modifying the previously attempted PCR conditions, varying the primer combinations (3mrop5, 5mrop3, BR299A and BR229B primers) and testing the effect of linear and circular template DNA. It was found that the structural type of template DNA and annealing temperature have relatively small impact on the PCR for site-directed mutagenesis. The addition of the 5mrop3 primer caused the amplification of a product to be inhibited, thus acting as a inhibitor to the PCR. Future experiments include designing a new primer and modify the PCR to include a thermostable DNA ligase.