Determination of Temperature Sensitive Plasmid Copy Number in Escherichia coli by Absolute and Relative Real Time Quantitation PCR

Background: Temperature sensitive plasmid pBBR1MCS2-Ts, mutated from pBBR1MCS and derived from pBBR1, is a broad host range plasmid and it is especially useful for gene targeting and integration in various hosts. The plasmid copy number (PCN) of temperature sensitive plasmid in host mainly depends on the stability of plasmid. Objectives: The present study aimed at investigating the PCN of pBBR1MCS2-Ts and pBBR1MCS2 at permissive (30°C) or nonpermissive (42°C) temperatures. Methods: The rep gene in the plasmid and the dxs gene in the Escherichia coli genome were used as target and reference gene. A standard plasmid pLB1k-dxs-rep was constructed for real time PCR calibration. The PCNs were calculated by absolute and relative quantitation. Total DNA of E. coli T1 harboring plasmid pBBR1MCS2 or pBBR1MCS2-Ts were extracted and real time qPCR were performed in triplicate, with 2 independent biological replicates. Results: The primer setsQrep andQdxs produced specific products and could be used to detect the target plasmid and chromosomal DNA, respectively. The PCN determined by the absolute and relative quantitation PCR were similar and reproducible. The PCN of pBBR1MCS2 in E. coli was about 19 when cultured at 30°C and about 10 when cultured at 42°C, and the PCN of pBBR1MCS2-Ts was about 6 when cultured at 30°C and nearly zero when cultured at 42°C. Compared with pBBR1MCS2, the temperature shift from 30°C to 42°C caused a significant decrease in the PCN of temperature sensitive plasmid pBBR1MCS2-Ts. Conclusions: ThePCNof temperature sensitive plasmid was very low at 42°C and temperature sensitivity of the plasmid was mainly caused by the mutation of rep ORF, which subsequently affected the plasmid replication and stability.