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Nucleic Acids Research 2005 33(18):e153; doi:10.1093/nar/gni154
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Published online 12 October 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Methods Online

Integrative elements for Bacillus subtilis yielding tetracycline-dependent growth phenotypes

Ralph Bertram, Martin Köstner, Judith Müller, José Vazquez Ramos and Wolfgang Hillen*

Lehrstuhl für Mikrobiologie, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg Staudtstrasse 5, 91058 Erlangen, Germany

*To whom correspondence should be addressed. Tel: +49 9131 85 28081; Fax: +49 9131 85 28082; Email: whillen{at}biologie.uni-erlangen.de

Received August 24, 2005. Revised September 20, 2005. Accepted September 20, 2005.

We describe the construction and application of elements for random insertion of promoter containing DNA into the genome of Bacillus subtilis. The outward-facing promoter of these integrative elements termed InsTetG+ is inducible by tetracycline so that conditional mutants are generated. We constructed three InsTetG+ variants using different regulatory windows. In the first, the regulator gene tetR is located within the element, allowing one-step mutagenesis. The second contains tetR in the chromosome and yields the best regulation efficiency. The third exploits xylose-dependent tetR expression from a plasmid, enabling induction of TetR synthesis so that distinct expression levels of an affected gene can be adjusted. We have obtained mutant strains with all three variants. For some of them, growth can be modulated by the presence of effectors. Most growth defects occur in the presence of inducers, presumably due to regulated expression of antisense RNA.


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