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枯草芽孢杆菌中四环素依赖性条件基因敲除

Tetracycline-dependent conditional gene knockout in Bacillus subtilis.

作者信息

Kamionka Annette, Bertram Ralph, Hillen Wolfgang

机构信息

Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.

出版信息

Appl Environ Microbiol. 2005 Feb;71(2):728-33. doi: 10.1128/AEM.71.2.728-733.2005.

DOI:10.1128/AEM.71.2.728-733.2005
PMID:15691923
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC546824/
Abstract

Reversible tetracycline-dependent gene regulation allows induction of expression with the tetracycline repressor (TetR) or gene silencing with the newly developed reverse mutant revTetR. We report here the implementation of both approaches with full regulatory range in gram-positive bacteria as exemplified in Bacillus subtilis. A chromosomally located gene is controlled by one or two tet operators. The precise adjustment of regulatory windows is accomplished by adjusting tetR or revtetR expression via different promoters. The most efficient induction was 300-fold in the presence of 0.4 microM anhydrotetracycline obtained with a Pr-xylA-tetR fusion. Reversible 500-fold gene knockouts were obtained in B. subtilis after adjusting expression of revTetR by synthetically designed promoters. We anticipate that these tools will also be useful in many other gram-positive bacteria.

摘要

可逆的四环素依赖性基因调控可通过四环素阻遏物（TetR）诱导基因表达，或利用新开发的反向突变体revTetR实现基因沉默。我们在此报告了这两种方法在革兰氏阳性菌中具有完整调控范围的实施情况，以枯草芽孢杆菌为例。一个位于染色体上的基因由一个或两个四环素操纵子控制。通过不同启动子调节tetR或revtetR的表达，可实现调控窗口的精确调整。使用Pr-xylA-tetR融合体在0.4微摩尔脱水四环素存在的情况下，最有效的诱导倍数为300倍。通过合成设计的启动子调节revTetR的表达后，在枯草芽孢杆菌中实现了500倍的可逆基因敲除。我们预计这些工具在许多其他革兰氏阳性菌中也将有用。

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