Measuring Plasmid Stability in Gram-Negative Bacteria

Abstract

In this chapter, a highly sensitive method to measure plasmid stability in Gram-negative bacteria is described. This procedure is based on the counterselection of plasmid-containing cells using an aph-parE cassette. When bacteria carrying the aph-parE module in the plasmid of interest are grown in media containing rhamnose as the only carbon source, the PparE promoter is induced, ParE is synthesized, and plasmid-containing cells are eliminated; bacteria that have lost the plasmid survive. The absence of the kanamycin resistance marker (aph) can be used to confirm the loss of the plasmid in rhamnose grown bacteria.