A useful gene cassette for conditional knock-down of essential genes by targeted promoter replacement in Mycobacteria

BioTechniques
Pauline TexierPierre Genevaux

Abstract

A direct method to study essential genes is to construct conditional knock-down mutants by replacement of their native promoter by an inducible one. In Mycobacteria, replacement of an essential gene promoter with an anhydrotetracycline inducible one was successfully used but required a multi-step approach. In this work, we describe a gene cassette for the engineering of a conditional knock-down mutant, which allows the one-step targeted replacement of mycobacterial promoters by an anhydrotetracycline-inducible promoter. The functionality of this cassette was successfully tested by engineering conditional clpP and SecA1 mutants of Mycobacterium smegmatis.

References

Apr 25, 1991·Proceedings of the National Academy of Sciences of the United States of America·M H LeeG F Hatfull
Feb 3, 2005·Nucleic Acids Research·Sabine EhrtDirk Schnappinger
Dec 21, 2006·Nature Methods·Julia C van Kessel, Graham F Hatfull
May 8, 2007·Journal of Bacteriology·Xinzheng V GuoDirk Schnappinger
Jan 29, 2009·Nucleic Acids Research·Marcus KlotzscheDirk Schnappinger
Mar 18, 2015·Methods in Molecular Biology·Dirk SchnappingerSabine Ehrt

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Datasets Mentioned

BETA
MG674207

Methods Mentioned

BETA
PCR

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