Genetic Manipulation of Vibrio fischeri

Abstract

Vibrio fischeri is a nonpathogenic organism related to pathogenic Vibrio species. The bacterium has been used as a model organism to study symbiosis in the context of its association with its host, the Hawaiian bobtail squid Euprymna scolopes. The genetic tractability of this bacterium has facilitated the mapping of pathways that mediate interactions between these organisms. The protocols included here describe methods for genetic manipulation of V. fischeri. Following these protocols, the researcher will be able to introduce linear DNA via transformation to make chromosomal mutations, to introduce plasmid DNA via conjugation and subsequently eliminate unstable plasmids, to eliminate antibiotic resistance cassettes from the chromosome, and to randomly or specifically mutagenize V. fischeri with transposons. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Transformation of V. fischeri with linear DNA

Basic Protocol 2: Plasmid transfer into V. fischeri via conjugation

Support Protocol 1: Removing FRT-flanked antibiotic resistance cassettes from the V. fischeri genome

Support Protocol 2: Eliminating unstable plasmids from V. fischeri

Alternate Protocol 1: Introduction of exogenous DNA using a suicide plasmid

Alternate Protocol 2: Site-specific transposon insertion using a suicide plasmid

Alternate Protocol 3: Random transposon mutagenesis using a suicide plasmid