Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli.
Real-time QPCR based methods for determination of plasmid copy number in recombinant Escherichia coli cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant E. coli DH5alpha harboring pBR322, which is a common bacterial cloning vector. The separate detection of the plasmid and the host chromosomal DNA was achieved using two separate primer sets, specific for the plasmid beta-lactamase gene (bla) and for the chromosomal d-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. Since both bla and dxs are single-copy genes of pBR322 and E. coli chromosomal DNA, respectively, the plasmid copy number can be determined as the copy ratio of bla to dxs. These methods were successfully applied to determine the plasmid copy number of pBR322 of E. coli host cells. The results of the absolute and relative analyses were identical and highly reproducible with coefficient of variation (CV) values of 2.8-3.9% and 4.7-5.4%, respectively. The results corresponded to the previously reported values of pBR322 copy number within E. coli host cells, 15-20. The methods introduced in this study are convenient to perform and cost-effective compared to the traditionally used Southern blot method. The primer sets designed in this study can be used to determine plasmid copy number of any recombinant E. coli with a plasmid vector having bla gene.
Lee
Changsoo
C
School of Environmental Science and Engineering, Pohang University of Science and Technology, San 31, Hyoja-dong, Nam-gu, Pohang, Kyungbuk 790-784, Republic of Korea.
Kim
Jaai
J
Shin
Seung Gu
SG
Hwang
Seokhwan
S
D000465:Algorithms; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D018628:Gene Dosage / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*
D004269:DNA, Bacterial