Phage Transposon Mutagenesis

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 465)

Abstract

Phage transduction is an attractive method of genetic manipulation in mycobacteria. ΦMycoMarT7 is well suited for transposon mutagenesis as it is temperature sensitive for replication and contains T7 promoters that promote transcription, a highly active transposase gene, and an Escherichia coli oriR6 K origin of replication. Mycobacterial transposon mutant libraries produced by ΦMycoMarT7 transduction are amenable to both forward and reverse genetic studies. In this protocol, we detail the preparation of ΦMycoMarT7, including a description of the phage, reconstitution of the phage, purification of plaques, preparation of phage stock, and titering of phage stock. We then describe the transduction procedure and finally outline the isolation of individual transposon mutants.

Keywords

library mutagenesis mycobacteria phage transduction transposon 

Notes

Acknowledgments

The authors thank Dr. C. Sassetti, J. Lane, Dr. S. Fortune, and A. Garces for method development. We also thank J. Murry and Dr. M. Unnikrishnan for critical reading of this manuscript.

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Department of Immunology and Infectious DiseasesHarvard School of Public HealthBostonUSA
  2. 2.HSPH, Immunology & Infectious DiseaseBostonUSA

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