Determination of Exclusion Effects of Potentially rop Deficient Mutant pBR322 during Co-transformation with the Wild-type Plasmid

Abstract

It has been shown that when pBR322 is co-transformed with pUC19 into the Escherichia coli DH5 α host bacterial cell, pBR322 is selectively excluded. Furthermore, pBR322 encodes a functional rop gene that regulates copy number. The Rop protein is involved in stabilizing the interaction between RNA I and RNA II, which in turn prevents the replication of pBR322. Since pUC19 lacks rop, it is of particular interest to investigate whether Rop is responsible for a relatively lower plasmid copy number in pBR322 and the observed selective exclusion. Previously, a mutant pBR322 was created by site-directed mutagenesis such that a mutation was introduced in the ribosome binding site along with a new Alu I restriction site. The focus of this study was to determine whether the pBR322 mutant, which should produce less of the Rop protein, will result in a higher copy number and exclude the wild-type plasmid during co-transformation of both plasmids into DH5 α. Through extensive gel electrophoresis analysis, it was observed that when mutant and wildtype pBR322 plasmids were co-transformed, the mutant plasmid was selectively excluded from the cell. The observed effect was contrary to expected observation. However, due to time constraints, the results could not be replicated to confirm the probable exclusion effect of mutant pBR322 by the wild-type plasmid.