Measuring Plasmid Stability in Gram-Negative Bacteria
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.
Key wordsPlasmid stability aph-parE Rhamnose Gram-negative Toxin–antitoxin Salmonella
I thank Alexandra Willis, Gloria del Solar Dongil, and Ramon Díaz Orejas for their critical review of the manuscript.
The protocol described in this chapter was originally described in Lobato-Márquez et al. .
D.L.-M. is funded by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. H2020-MSCA-IF-2016-752022.
- 8.Lobato-Márquez D, Molina-García L, Moreno-Córdoba I, García-del Portillo F, Díaz-Orejas R (2016) Stabilization of the virulence plasmid pSLT of Salmonella Typhimurium by three maintenance systems and its evaluation by using a new stability test. Front Mol Biosci 3:66Google Scholar
- 9.Lobato-Márquez D, Molina-García L (2017) Evaluation of plasmid stability by negative selection in Gram-negative bacteria. BioProtocol 7(9)Google Scholar