Construction of pCXZ14W, a Novel pUC19-derived Plasmid Encoding the rop Gene

09/05/2015

Shary Chen, Ziyan Xu, Wenchen Zhao

Department of Microbiology and Immunology, University of British Columbia

Volume 19
Fall 2014 / Winter 2015

When the ColE1-derived cloning vectors pUC19 and pBR322 are co-transformed into Escherichia coli DH5α, pBR322 is excluded from the cells, meaning that overtime the plasmid copy number of pUC19 is higher than that of pBR322. This phenomenon has been attributed to the absence and modification of specific elements involved in the regulation of plasmid copy number, one of which is a protein called Repressor of Primer (Rop). Rop stabilizes the interaction between replication primer RNA II and its inhibitory RNA molecule, RNA I. In the absence of free RNA II plasmid copy number per cell decreases. To assess the role of Rop in the exclusion effect, several teams of researchers have attempted to construct a rop-deficient pBR322 plasmid. To date these efforts have been unsuccessful. The aim of this study was to create a novel plasmid, pCXZ14W, in which the rop gene is inserted into the pUC19 multi-cloning site (MCS). The construct can then be used to assess the effect of Rop on its copy number if Rop is expressed. Verified by both colony PCR and DNA sequencing, the rop gene was inserted into the pUC19 MCS without any mutation, though inadvertently being positioned at a distance of 52 nucleotides away from the Shine-Dalgarno sequence located in the lac operon. SDS-PAGE analysis showed no overexpression of Rop as expected. We next attempted to delete the sequence between the Rop start codon and the Shine-Dalgarno sequence to correctly position these elements for expression. 30 transformants were screened using colony PCR. However, clones bearing the deletion were not observed. We speculate that the deletion may have resulted in successful expression of the rop gene, driven from the upstream lac promoter, which downregulated plasmid copy number and rendered the desired transformant cells non-viable.