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Background  Standardized and well-characterized genetic building blocks are a prerequisite for the convenient and reproducible assembly of novel genetic modules and devices. While numerous standardized parts exist for  Escherichia coli , such tools are still missing for the Gram-positive model organism  Bacillus subtilis . The goal of this study was to develop and thoroughly evaluate such a genetic toolbox.    Results  We developed five BioBrick-compatible integrative  B. subtilis  vectors by deleting unnecessary parts and removing forbidden restriction sites to allow cloning in BioBrick (RFC10) standard. Three empty backbone vectors with compatible resistance markers and integration sites were generated, allowing the stable chromosomal integration and combination of up to three different devices in one strain. In addition, two integrative reporter vectors, based on the  lacZ  and  luxABCDE  cassettes, were BioBrick-adjusted, to enable β-galactosidase and luciferase reporter assays, respectively. Four constitutive and two inducible promoters were thoroughly characterized by quantitative, time-resolved measurements. Together, these promoters cover a range of more than three orders of magnitude in promoter strength, thereby allowing a fine-tuned adjustment of cellular protein amounts. Finally, the  Bacillus  BioBrick Box also provides five widely used epitope tags (FLAG, His 10 , cMyc, HA, StrepII), which can be translationally fused N- or C-terminally to any protein of choice.    Conclusion  Our genetic toolbox contains three compatible empty integration vectors, two reporter vectors and a set of six promoters, two of them inducible. Furthermore, five different epitope tags offer convenient protein handling and detection. All parts adhere to the BioBrick standard and hence enable standardized work with  B. subtilis . We believe that our well-documented and carefully evaluated  Bacillus  BioBrick Box represents a very useful genetic tool kit, not only for the iGEM competition but any other BioBrick-based project in  B. subtilis .

The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis