Flow Cytometry Analysis of Plasmid Amplification in Escherichia coli

Large quantities of cloned DNAs for use in characterization, or in subsequent recombinant plasmid construction, may be obtained by plasmid amplification, which is the increase of cellular plasmid DNA content relative to the amount of host cell chromosomal DNA. Plasmid amplification has been investigated with flow cytometry in chloramphenicol (Cm) treated cell populations of Escherichia coli HB101 carrying plasmids with pMBI replication origins at different copy number levels ranging from 12 to 122. The flow cytometry method is based on the determination of the total cellular DNA content after staining with mithramycin, a DNA-specific fluororescent dye. This method permits direct measurement of the rate of plasmid amplification and classification of cell populations according to chromosomal replication forks at the time of Cm treatment. Both initial rates of plasmid amplification and final contents of amplified plasmids were found to be proportional to the average initial plasmid copy numbers. The maximum degree of amplification, defined as the ratio of plasmid content after eighteen hours incubation to the preamplification plasmid content at steady-state growth, was approximately equal for the strains studied.Larger cells, with four active chromosomal replication forks at Cm addition, exhibit faster plasmid replication rates than cells initially at earlier cell cycle states. A kinetic model was proposed that agrees well with the present and previous experimental observations. This work clearly demonstrates the advantage of flow cytometry for experimental analysis of plasmid propagation in recombinant organisms.