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Mutagenesis of Vibrio fischeri and Other Marine Bacteria Using Hyperactive Mini-Tn5 Derivatives

  • Julie L. Stoudenmire
  • Michael Black
  • Pat M. Fidopiastis
  • Eric V. StabbEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2016)

Abstract

Mutagenizing bacterial genomes with selectable transposon insertions is an effective approach for identifying the genes underlying important phenotypes. Specific bacteria may require different tools and methods for effective transposon mutagenesis, and here we describe methods to mutagenize Vibrio fischeri using an engineered mini-Tn5 transposon with synthetic “mosaic” transposon ends. The transposon is delivered by conjugation on a plasmid that cannot replicate in V. fischeri and that encodes a hyperactive transposase outside the transposon itself. The chromosomal location of insertions can be readily identified by cloning and/or PCR-based methods described here. Although developed in V. fischeri, these tools and methods have proven effective in some other bacteria as well.

Key words

Vibrio Photobacterium Transposon Mutagenesis Mini-Tn5 

Notes

Acknowledgments

The authors wish to thank Alecia Septer, Mark Mandel, Tim Miyashiro, and Karen Visick for helpful discussions, and Helen Dukes for technical assistance. This work was supported by NSF grants CAREER-MCB-0347317, IOS-0841480, IOS-1557964, IOS-0843317, and MCB-1716232.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Julie L. Stoudenmire
    • 1
  • Michael Black
    • 2
  • Pat M. Fidopiastis
    • 2
  • Eric V. Stabb
    • 1
    Email author
  1. 1.Department of MicrobiologyUniversity of GeorgiaAthensUSA
  2. 2.Biological Sciences DepartmentCalifornia Polytechnic UniversitySan Luis ObispoUSA

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