Amplification of lambda plasmids in Escherichia coli relA mutants

Abstract

It was previously demonstrated that, contrary to wild-type stringent (rel+) strains of Escherichia coli, in amino acid-starved relaxed (relA) mutants the replication of lambda plasmid proceeds for several hours. The replication leads to amplification of lambda plasmid DNA. Here, the conditions for this amplification have been optimized. The amplification efficiency depends on the temperature as well as on the nature of amino acid starvation, but it is only little or totally not dependent on the pH value of the medium in a range from 6.0 to 8.0. It seems that the most efficient amplification can be achieved by overnight cultivation of E. coli relA arg strain harbouring lambda plasmid at 36-39 degrees C in minimal medium containing Casamino acids. Under these conditions, the copy number of lambda plasmid increases from about 40 to about 300 per cell giving greater than 7-fold amplification.