Construction of pCAWK, a Novel pBR322-derived Plasmid with Insertional Inactivation of the rop Gene

Abstract

The ColE1-type plasmid pBR322 is commonly used as vector tool in molecular biology. It has been observed that when Escherichia coli cells are co-transformed with pBR322 and pUC19 (another ColE1-type plasmid) pUC19 is retained and pBR322 is excluded. Several mechanisms have been proposed to explain this phenomenon, including the larger size of pBR322, absence of the rop gene in pUC19, and an existing point mutation in RNA II of pUC19 absent in pBR322. To examine the role of the repressor of primer (Rop) protein on the exclusion effect, previous studies have attempted to create a non-functional Rop protein by PCR-site directed mutagenesis but have been unsuccessful. The objective of this study was to create a Rop mutant by insertional inactivation of the rop gene. A 34-bp DNA insert was designed with (1) PvuII restriction digest sites flanking the ends; (2) one stop codon in each of six possible reading frames; and (3) a central EagI restriction site. This DNA was ligated into the rop gene of pBR322 so that Rop protein translation is terminated prematurely after the 50th amino acid, arginine. This disruption will result in a non-functional protein as it has been demonstrated that residues Phe-56 and Gly-57 are essential for Rop functionality. Restriction digests confirmed successful insertion of the DNA insert into the rop gene to create mutant pBR322. This study was the first to be successful in creating a mutant pBR322 plasmid with a disrupted rop gene, named pCAWK. This novel plasmid can be used to further explore the role of the Rop protein on pBR322 exclusion.