Determination of the molecular mass of bacterial genomic DNA and plasmid copy number by high-pressure liquid chromatography

Abstract

Relatively rapid methods for the determination of relative genome molecular mass (Mr) and the estimation of plasmid copy number have been developed. These methods are based on the ability of the Bio-Rad high-pressure liquid chromatography hydroxylapatite column to separate and quantify single-stranded DNA, double-stranded DNA, and plasmid DNA. Genome Mr values were calculated from reassociation kinetics of single-stranded DNA as measured with the hydroxylapatite column. Bacteriophage T4 DNA was used to establish a Cot (moles of nucleotides times seconds per liter), or standard reassociation value. From this Cot value, Cot values for Escherichia coli B, Beggiatoa alba B18LD, and Streptomyces coelicolor were determined by comparative calculations. From those calculated Cot values, the Mr values of 1.96 x 109 for E. coli, 2.02 x 109 for B. alba, and 3.28 x 109 for S. coelicolor were estimated. Plasmid concentration was determined from

Overproduction of recombinant ribulose 1,5-bisphosphate carboxylase/oxygenase from Synechococcus sp. strain PCC6301 in glucose-controlled high-cell-density fermentations by Escherichia coli K-12

“…Plasmid pANT900 contains lacI q , a lacI promoter up-mutation that yields about 10-fold oversynthesis of the repressor (22), as well as a strong transcription terminator downstream of rbcS, whereas pBGL710 does not contain lacI and lacks known vector terminators after the inserted genes. Both plasmids contain the pBR322 origin of replication, indicating that their copy numbers should be approximately 30 (8). E. coli K-12 strain ATCC 10798, transformed with either pBGL710 or pANT900 by standard procedures (21), was used for these studies.…”