Tools for Rapid Genetic Engineering of Vibrio fischeri

Applied and Environmental Microbiology
Karen L VisickVincent Mastrodomenico

Abstract

Vibrio fischeri is used as a model for a number of processes, including symbiosis, quorum sensing, bioluminescence, and biofilm formation. Many of these studies depend on generating deletion mutants and complementing them. Engineering such strains, however, is a time-consuming, multistep process that relies on cloning and subcloning. Here, we describe a set of tools that can be used to rapidly engineer deletions and insertions in the V. fischeri chromosome without cloning. We developed a uniform approach for generating deletions using PCR splicing by overlap extension (SOEing) with antibiotic cassettes flanked by standardized linker sequences. PCR SOEing of the cassettes to sequences up- and downstream of the target gene generates a DNA product that can be directly introduced by natural transformation. Selection for the introduced antibiotic resistance marker yields the deletion of interest in a single step. Because these cassettes also contain FRT (FLP recognition target) sequences flanking the resistance marker, Flp recombinase can be used to generate an unmarked, in-frame deletion. We developed a similar methodology and tools for the rapid insertion of specific genes at a benign site in the chromosome for purposes such as co...Continue Reading

References

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Citations

Oct 10, 2018·Molecular Microbiology·Cecilia M ThompsonKaren L Visick
Feb 21, 2019·Journal of Bacteriology·Ella R RotmanMark J Mandel
Jun 5, 2020·Current Protocols in Microbiology·David G Christensen, Karen L Visick
Oct 26, 2018·The ISME Journal·Clotilde Bongrand, Edward G Ruby
Jul 22, 2018·Applied and Environmental Microbiology·Cecilia M ThompsonKaren L Visick
Sep 23, 2020·Scientific Reports·Triana N DaliaAnkur B Dalia
Sep 26, 2020·Current Protocols in Microbiology·David G ChristensenKaren L Visick
Nov 15, 2020·Applied and Environmental Microbiology·Pat M FidopiastisKaren L Visick
Jan 6, 2021·Applied and Environmental Microbiology·Joshua J CohenKaren L Visick
Feb 27, 2021·Frontiers in Microbiology·Ilenne Del ValleLauren B Stadler
Mar 14, 2021·Applied and Environmental Microbiology·Cameron J LloydKarl E Klose
Jun 6, 2021·Nature Reviews. Microbiology·Karen L VisickEdward G Ruby
Jul 16, 2021·Frontiers in Microbiology·Courtney N DialKaren L Visick
Jul 3, 2021·Molecular Microbiology·Nathan P WasilkoTim I Miyashiro
Aug 15, 2021·Nature Reviews. Genetics·Julie Perreau, Nancy A Moran

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