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Standardized and well-characterized genetic building blocks allow the convenient assembly of novel genetic modules and devices, ensuring reusability of parts and reproducibility of experiments. In the first  Bacillus subtilis -specific toolbox using the BioBrick standard, we presented integrative vectors, promoters, reporter genes and epitope tags for this Gram-positive model bacterium. With the  Bacillus  BioBrick Box 2.0, we significantly expand the range of our toolbox by providing new integrative vectors, introducing novel tools for fine-tuning protein expression, and carefully evaluating codon-adapted fluorescence proteins in  B. subtilis , which cover the whole spectrum of visible light. Moreover, we developed new reporter systems to allow evaluating the strength of promoters and ribosome binding sites. This well-evaluated extension of our BioBrick-based toolbox increases the accessibility of  B. subtilis  and will therefore promote the use of this model bacterium and biotechnological workhorse as a host for fundamental and applied Synthetic Biology projects.

The Bacillus BioBrick Box 2.0: expanding the genetic toolbox for the standardized work with Bacillus subtilis