The Bacillus BioBrick Box (B4): Generation and Evaluation of Essential Genetic Building Blocks for the Standardized Work with Bacillus subtilis

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Jara Radeck, Korinna Kraft, Julia Bartels, Tamara Cikovic, Franziska Dürr, Jennifer Emenegger, Simon Kelterborn, Christopher Sauer, Georg Fritz, Susanne Gebhard, and Thorsten Mascher

Ludwigs-Maximilians-Unitersitt, Germany

Standardized and well-characterized genetic building blocks allow the convenient and reproducible assembly of novel genetic modules and devices. During the iGEM competition 2012, the team LMU-Munich initiated the development of the BacillusBioBrick Box (B4) – containing vectors, promoters, reporter genes and epitope tags for the Gram-positive model bacterium Bacillus subtilis – which has now been fully evaluated. We developed five BioBrick-compatible 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 or luxABCDE cassettes, were BioBrick-adjusted, to enable -galactosidase and luciferase reporter assays, respectively. Six constitutive and two inducible promoters were thoroughly characterized by quantitative, time-resolved measurements. Together, these promoters cover a range of four orders of magnitudes in promoter strength, thereby allowing a fine-tuned adjustment of cellular protein amounts. The dynamic range of four codon-optimized reporter genes (gfp, mKate2, lacZ and luc+) was evaluated with inducible promoters. Moreover, the suitability of GFP and mKate2 as protein tags was verified by N- and C-terminal protein fusions that were analyzed by fluorescence microscopy. Finally, the BacillusBioBrick Box also provides five widely used epitope tags (FLAG-, His10-, cMyc, HA-, Strep-), which can be translationally fused N- or C-terminally to any gene of choice. Three reporter genes (gfp, mKate2, and lacZ) and the tags are provided in BioBrick (RFC10) and Freiburg (RFC25) standard, to allow the construction of transcriptional and N-/C-terminal translational fusions. For N-terminal fusions, these parts are provided with optimized ribosome-binding sites. We believe that our well-document and carefully evaluated BacillusBioBrick Box represents a very useful genetic tool kit, for iGEM and beyond.