Creation of a Rop- Plasmid Using Site-Directed Mutagenesis

Abstract

pBR322 is a pColE1-derived plasmid that is widely used in molecular biology. Of particular interest is the observation that when pBR322 is co-transformed with pUC19, pBR322 is excluded from the cell. While many factors may contribute to this, it is the authors’ opinion that the pBR322-encoded Rop protein is a major factor in this exclusion. Rop is a small protein that stabilizes the interaction between RNA I and RNA II, which in turns represses replication of pBR322. This study attempted to create a pBR322( ∆ Rop ) by inserting ochre codons into the rop reading frame, thus creating a non-functional rop, which would, theoretically, result in a higher copy number of pBR322. After the mutagenic PCR, ligation and transformation into DH5 α cells, two clones appeared to possess the mutated plasmid, indicating that the mutagenic rop had correctly ligated into pBR322. Unfortunately, sequencing showed the two mutated clones had identical sequence to the wild-type rop, showing that the mutagenesis was unsuccessful.