MO
Mary X. O’Riordan
  • Department of Microbiology and Immunology, University of Michigan Medical School, USA
Research fields
  • Immunology
Evaluation of Plasmid Stability by Negative Selection in Gram-negative Bacteria
Authors:  Damián Lobato Márquez and Laura Molina García, date: 05/05/2017, view: 11575, Q&A: 0
Plasmid stability can be measured using antibiotic-resistance plasmid derivatives by positive selection. However, highly stable plasmids are below the sensitivity range of these assays. To solve this problem we describe a novel, highly sensitive method to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells. The assay proposed here is based on an aph-parE cassette. When synthesized in the cell, the ParE toxin induces cell death. ParE synthesis is controlled by a rhamnose-inducible promoter. When bacteria carrying the aph-parE module are grown in media containing rhamnose as the only carbon source, ParE is synthesized and plasmid-containing cells are eliminated. Kanamycin resistance (aph) is further used to confirm the absence of the plasmid in rhamnose grown bacteria.
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