A part toolbox to tune genetic expression in Bacillus subtilis.
Nucleic Acids Res
; 44(15): 7495-508, 2016 09 06.
Article
em En
| MEDLINE
| ID: mdl-27402159
ABSTRACT
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 â¼14 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 µM. This toolbox of regulatory components will support many research and engineering applications in B. subtilis.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Ribossomos
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Bacillus subtilis
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Engenharia Genética
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Regulação Bacteriana da Expressão Gênica
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Regiões Promotoras Genéticas
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Proteólise
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Nucleic Acids Res
Ano de publicação:
2016
Tipo de documento:
Article
País de afiliação:
França