The Effects of Gratuitous Heterologous Gene Expression on the Amplification of ColE1-related Plasmids in Cultures of Escherichia coli BL21 (DE3) in the Presence of Sub-inhibitory Levels of Chloramphenicol

Abstract

The use of chloramphenicol (CAM) in the amplification of ColE1-derived plasmids is commonly employed and its mechanisms of action are well documented. Plasmid amplification following inhibition of chromosomal DNA replication can also be achieved through IPTG-induced gratuitous over-expression of heterologous genes. In this experiment, the additive effects of IPTG induction of the fre gene and sub-inhibitory chloramphenicol treatments on plasmid pES1 amplification in Escherichia coli BL21 (DE3) were examined. Bacterial cultures were grown in sub-inhibitory concentrations of chloramphenicol alone or in combination with pre-treatment of IPTG. Total DNA was purified by phenol:chloroform:isoamyl alcohol extraction and plasmid concentration was determined using agarose gel assay and line densitometry. ColE1-related plasmid amplification was found to be enhanced 1-2 fold in cultures subjected to simultaneous treatment of chloramphenicol and IPTG compared to those treated with the antibiotic alone. No plasmid amplification was observed under low levels of IPTG treatment alone and the possible effects of active transcription due to IPTG induction on ColE1-related plasmid amplification was not observed.