Quantitative Polymerase Chain Reaction Assay to Study the Role of Genetic Elements Involved in Regulating Plasmid Copy Number

09/15/2015

Sara Appleby, Hayley Bessette, Casey Jin, Cloé Solanki

Department of Microbiology and Immunology, University of British Columbia

Volume 19
Fall 2014 / Winter 2015

We have created a quantitative polymerase chain reaction (qPCR) assay that can measure relative plasmid copy number. The assay uses a calibrator plasmid, pCHCS, containing both β-lactamase (bla) and d-1-deoxyxylulose 5-phosphate synthase (dxs). bla is a single-copy plasmid-based target gene and dxs is a single-copy reference gene encoded in the Escherichia coli genome. We generated standard curves from the bla and dxs qPCR amplification of various dilutions of pCHCS. These standard curves were used to relate gene copy numbers of bla and dxs to qPCR Cq values. We demonstrated that our assay responds in a dose-dependent manner. Using this assay we attempted to evaluate the copy number of several ColE1- derived plasmids bearing mutations in genetic elements involved in regulating plasmid copy number. The results followed the trend of increasing plasmid copy numbers in E. coli DH5α cells harboring plasmids pBR322, pBART or pUC19. We discuss the utility and some of the remaining challenges associated with this assay for measuring plasmid copy number.