Format

Send to

Choose Destination
Nucleic Acids Res. 2016 Sep 6;44(15):7495-508. doi: 10.1093/nar/gkw624. Epub 2016 Jul 8.

A part toolbox to tune genetic expression in Bacillus subtilis.

Author information

1
Centre de Biochimie Structurale, INSERM U1054, CNRS UMR5048, University of Montpellier, France.
2
Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France.
3
Centre de Biochimie Structurale, INSERM U1054, CNRS UMR5048, University of Montpellier, France jerome.bonnet@inserm.fr.

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 ∼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 μM. This toolbox of regulatory components will support many research and engineering applications in B. subtilis.

PMID:
27402159
PMCID:
PMC5009755
DOI:
10.1093/nar/gkw624
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Silverchair Information Systems Icon for PubMed Central
Loading ...
Support Center