Improved Method for Transformation of Vibrio vulnificus by Electroporation.
Vibrio vulnificus
electroporation
nuclease
sucrose
transformation
Journal
Current protocols in microbiology
ISSN: 1934-8533
Titre abrégé: Curr Protoc Microbiol
Pays: United States
ID NLM: 101257113
Informations de publication
Date de publication:
09 2020
09 2020
Historique:
entrez:
3
7
2020
pubmed:
3
7
2020
medline:
26
5
2021
Statut:
ppublish
Résumé
Vibrio vulnificus, an emergent human pathogen, causes fulminant septicemia with a mortality rate of over 50%. Unlike for other pathogenic Vibrio species, the factors to conclusively indicate the virulence potential of V. vulnificus strains remain largely unknown. Understanding the pathogenesis of this bacterium at a molecular level is severely hindered by inefficiencies in transformation, for instance, due to the presence of a periplasmic nuclease, Vvn. Currently, successful transformation of V. vulnificus is nearly impossible due to lack of mobilizable plasmids for the bacterium, requiring (i) very high DNA concentrations, (ii) plasmid linearization, (iii) development of novel V. vulnificus-derived plasmids, or (iv) time-consuming conjugation-based methods. To overcome these limitations, we describe a rapid, efficient, and reproducible electroporation protocol to effectively transform widely available plasmids, with different copy numbers and antibiotic resistances, into phylogenetically distant strains of V. vulnificus. Cells are made competent in high concentrations of sucrose devoid of cations and recovered from electroporation using a high-salinity recovery medium. Compared to existing methods for transformation of V. vulnificus, significantly higher efficiencies are obtained using this improved protocol. Rapid and effective transformations can markedly improve molecular analyses of V. vulnificus leading to a greater understanding of its virulence potential. This is crucial to develop rapid detection methods which have the potential to prevent future outbreaks. The electroporation protocol described here may be particularly useful for optimizing transformation of other nuclease-producing bacteria. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of competent cells Basic Protocol 2: Transformation of cells by electroporation.
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
e106Informations de copyright
© 2020 Wiley Periodicals LLC.
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