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Improved Method for Transformation of Vibrio vulnificus by Electroporation.
Curr Protoc Microbiol. 2020 Sep; 58(1):e106.CP

Abstract

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.

Authors+Show Affiliations

Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida.Institute for Postharvest and Food Sciences, Volcani Research Center, Rishon LeZion, Israel.Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32614522

Citation

Jayakumar, Jane M., et al. "Improved Method for Transformation of Vibrio Vulnificus By Electroporation." Current Protocols in Microbiology, vol. 58, no. 1, 2020, pp. e106.
Jayakumar JM, Shapiro OH, Almagro-Moreno S. Improved Method for Transformation of Vibrio vulnificus by Electroporation. Curr Protoc Microbiol. 2020;58(1):e106.
Jayakumar, J. M., Shapiro, O. H., & Almagro-Moreno, S. (2020). Improved Method for Transformation of Vibrio vulnificus by Electroporation. Current Protocols in Microbiology, 58(1), e106. https://doi.org/10.1002/cpmc.106
Jayakumar JM, Shapiro OH, Almagro-Moreno S. Improved Method for Transformation of Vibrio Vulnificus By Electroporation. Curr Protoc Microbiol. 2020;58(1):e106. PubMed PMID: 32614522.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Improved Method for Transformation of Vibrio vulnificus by Electroporation. AU - Jayakumar,Jane M, AU - Shapiro,Orr H, AU - Almagro-Moreno,Salvador, PY - 2020/7/3/entrez PY - 2020/7/3/pubmed PY - 2020/7/3/medline KW - Vibrio vulnificus KW - electroporation KW - nuclease KW - sucrose KW - transformation SP - e106 EP - e106 JF - Current protocols in microbiology JO - Curr Protoc Microbiol VL - 58 IS - 1 N2 - 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. SN - 1934-8533 UR - https://www.unboundmedicine.com/medline/citation/32614522/Improved_Method_for_Transformation_of_Vibrio_vulnificus_by_Electroporation L2 - https://doi.org/10.1002/cpmc.106 DB - PRIME DP - Unbound Medicine ER -
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