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.
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Acknowledgments
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. [8].
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.
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Lobato-Márquez, D. (2020). Measuring Plasmid Stability in Gram-Negative Bacteria. In: de la Cruz, F. (eds) Horizontal Gene Transfer. Methods in Molecular Biology, vol 2075. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9877-7_16
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DOI: https://doi.org/10.1007/978-1-4939-9877-7_16
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