The CRISPR toolbox for the gram-positive model bacterium Bacillus subtilis.

CRISPR has revolutionized the way we engineer genomes. Its simplicity and modularity have enabled the development of a great number of tools to edit genomes and to control gene expression. This powerful technology was first adapted to Bacillus subtilis in 2016 and has been intensely upgraded since then. Many tools have been successfully developed to build a CRISPR toolbox for this Gram-positive model and important industrial chassis. The toolbox includes tools, such as double-strand and single-strand cutting CRISPR for point mutation, gene insertion, and gene deletion up to 38 kb. Moreover, catalytic dead Cas proteins have been used for base editing, as well as for the control of gene expression (CRISPRi and CRISPRa). Many of these tools have been used for multiplex CRISPR with the most successful one targeting up to six loci simultaneously for point mutation. However, tools for efficient multiplex CRISPR for other functionalities are still missing in the toolbox. CRISPR engineering has already resulted in efficient protein and metabolite-producing strains, demonstrating its great potential. In this review, we cover all the important additions made to the B. subtilis CRISPR toolbox since 2016, and strain developments fomented by the technology.

Dataset for supporting a modular autoinduction device for control of gene expression in Bacillus subtilis.

Modular and tuneable genetic tools for Metabolic Engineering fuels the development of chassis for the efficient production of biocompounds at industrial scale. We have constructed an autoinduction device for gene expression in Bacillus subtilis based on the LuxR/I quorum sensing system [1]. Here, we present raw and processed data regarding to B. subtilis growth measured as OD600, performed in three different scales: microcultivation on 96-well plates (200 µL), test tubes (12 mL), and Erlenmeyer flasks (50 mL). We also present raw and processed data on gene expression measured as GFP fluorescence (485/535 nm), luminescence and riboflavin production. Measurements were performed on a microplate reader Tecan 200 PRO (iControl software) and on spectrophotometer (Thermo Fisher Scientific GENESYS 10S UV-Vis). Processed data are presented as product/OD600, maximum and minimum promoter activity, fold of induction, and the induction OD600.

A modular autoinduction device for control of gene expression in Bacillus subtilis.

Intense synthesis of proteins and chemicals in engineered microbes impose metabolic burden, frequently leading to reduced growth and heterogeneous cell population. Thus, the correct balance between growth and production is important. Such balance can be engineered through dynamic control of pathways, but few broadly applicable tools are available to achieve this. We present an autonomous control of gene expression mediated by quorum sensing in Bacillus subtilis, able to self-monitor and induce expression without human supervision. Two variations of the induction module and seven of the response module were engineered generating a range of induction folds and strengths for gene expression control. Our strongest response promoter is 2.5 and 3.2 times stronger than the well-characterized promoters PsrfA and Pveg, respectively. We applied our strongest autoinduction device for the production of the vitamin B2. This study presents a toolbox of autoinduction modules for B. subtilis that is modular and tunable.