Determination of Plasmid Stability in Hydrogen-Producing Recombinant Clostridium tyrobutyricum JM1 by Real-Time PCR Quantification

Abstract

Real-time quantitative PCR (qPCR) was applied to determine plasmid stability in recombinant Clostridium tyrobutyricum harboring pJIR418 shuttle vector (ATCC 77387). For the detection of the plasmid and the host chromosomal DNA, two primer sets were designed specifically for the plasmid chloramphenicol-resistant gene (cml) and for the 16S rRNA gene (16S), respectively. The plasmid copy number can be determined as the copy ratio of cml to 16S. The qPCR assay was able to evaluate the plasmid stability with good reproducibility and high sensitivity in non-selective conditions during a continuous fermentation.