title: A part toolbox to tune genetic expression in Bacillus subtilis.
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.
authors.affiliation: Centre de Biochimie Structurale, INSERM U1054, CNRS UMR5048, University of Montpellier, France.
authors.forename: Sarah
authors.initials: S
authors.lastname: Guiziou
authors.affiliation: Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Sauveplane
authors.affiliation: Centre de Biochimie Structurale, INSERM U1054, CNRS UMR5048, University of Montpellier, France.
authors.forename: Hung-Ju
authors.initials: HJ
authors.lastname: Chang
authors.affiliation: Centre de Biochimie Structurale, INSERM U1054, CNRS UMR5048, University of Montpellier, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Clerté
authors.affiliation: Centre de Biochimie Structurale, INSERM U1054, CNRS UMR5048, University of Montpellier, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Declerck
authors.affiliation: Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Jules
authors.affiliation: Centre de Biochimie Structurale, INSERM U1054, CNRS UMR5048, University of Montpellier, France jerome.bonnet@inserm.fr.
authors.forename: Jerome
authors.initials: J
authors.lastname: Bonnet
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics*; D005453:Fluorescence; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D017930:Genes, Reporter / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D017785:Photons; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014176:Protein Biosynthesis; D059748:Proteolysis; D012270:Ribosomes / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D049452:Green Fluorescent Proteins