An efficient stress- free strategy to displace stable bacterial plasmids

Research output: Contribution to journalArticle

Authors

Colleges, School and Institutes

Abstract

A key stage in determining the phenotype(s) conferred by a plasmid is its displacement, or 'curing,' to create a plasmid-free strain. However, many plasmids are very stable, not only because they contain multiple replicons, but also because they can encode post-segregational killing systems that reduce the viability of plasmid-free segregants. We have developed an efficient curing strategy that involves combining key regions of the replicons and the post-segregational killing loci into an unstable cloning vector carrying sacB, which confers sensitivity to sucrose. Targeting plasmids of both the F family of Escherichia coli and the broad-host-range IncP-1 family, we demonstrated displacement of susceptible resident plasmids from all clones tested. Growth on sucrose allowed the isolation of many clones without either plasmid. This strategy is highly efficient and avoids the stress of inducing and surviving the effects of post-segregational killing systems or other lethal gene products.

Details

Original languageEnglish
Pages (from-to)223-228
Number of pages6
JournalBioTechniques
Volume48
Issue number3
Publication statusPublished - 1 Mar 2010

Keywords

  • replication, post-segregational killing, plasmid curing, antibiotic resistance, virulence, mobile genetic elements