期刊
NUCLEIC ACIDS RESEARCH
卷 44, 期 15, 页码 7495-7508出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkw624
关键词
-
资金
- French Institut National de la Sante et de la Recherche Medicale (INSERM)
- Centre National pour la Recherche Scientifique (CNRS)
- Institut National de la Recherche Agronomique (INRA)
- French Ministry of Research
- INSERM Avenir program
- Bettencourt-Schueller Foundation
- ERC
- INSERM (Atip-Avenir program)
Libraries of well-characterised components regulating gene expression levels are essential to many synthetic biology applications. While widely available for the Gram-negative model bacterium Escherichia coli, such libraries are lacking for the Gram-positive model Bacillus subtilis, a key organism for basic research and biotechnological applications. Here, we engineered a genetic toolbox comprising libraries of promoters, Ribosome Binding Sites (RBS), and protein degradation tags to precisely tune gene expression in B. subtilis. We first designed a modular Expression Operating Unit (EOU) facilitating parts assembly and modifications and providing a standard genetic context for gene circuits implementation. We then selected native, constitutive promoters of B. subtilis and efficient RBS sequences from which we engineered three promoters and three RBS sequence libraries exhibiting similar to 14 000-fold dynamic range in gene expression levels. We also designed a collection of SsrA proteolysis tags of variable strength. Finally, by using fluorescence fluctuation methods coupled with two-photon microscopy, we quantified the absolute concentration of GFP in a subset of strains from the library. Our complete promoters and RBS sequences library comprising over 135 constructs enables tuning of GFP concentration over five orders of magnitude, from 0.05 to 700 mu M. This toolbox of regulatory components will support many research and engineering applications in B. subtilis.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据