title: The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis.
abstract: 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, His10, cMyc, HA, StrepII), which can be translationally fused N- or C-terminally to any protein of choice.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department Biology I, AG Synthetic Microbiology, Ludwig-Maximilians-Universität München, Grosshaderner Str. 2-4, D-82152 Planegg-Martinsried, Germany.
authors.forename: Jara
authors.initials: J
authors.lastname: Radeck
authors.affiliation: Department Biology I, AG Synthetic Microbiology, Ludwig-Maximilians-Universität München, Grosshaderner Str. 2-4, D-82152 Planegg-Martinsried, Germany.
authors.forename: Korinna
authors.initials: K
authors.lastname: Kraft
authors.affiliation: Department Biology I, AG Synthetic Microbiology, Ludwig-Maximilians-Universität München, Grosshaderner Str. 2-4, D-82152 Planegg-Martinsried, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Bartels
authors.affiliation: Department Biology I, AG Synthetic Microbiology, Ludwig-Maximilians-Universität München, Grosshaderner Str. 2-4, D-82152 Planegg-Martinsried, Germany.
authors.forename: Tamara
authors.initials: T
authors.lastname: Cikovic
authors.affiliation: Department Biology I, AG Synthetic Microbiology, Ludwig-Maximilians-Universität München, Grosshaderner Str. 2-4, D-82152 Planegg-Martinsried, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Dürr
authors.affiliation: Department Biology I, AG Synthetic Microbiology, Ludwig-Maximilians-Universität München, Grosshaderner Str. 2-4, D-82152 Planegg-Martinsried, Germany.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Emenegger
authors.affiliation: Department Biology I, AG Synthetic Microbiology, Ludwig-Maximilians-Universität München, Grosshaderner Str. 2-4, D-82152 Planegg-Martinsried, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Kelterborn
authors.affiliation: Department Biology I, AG Synthetic Microbiology, Ludwig-Maximilians-Universität München, Grosshaderner Str. 2-4, D-82152 Planegg-Martinsried, Germany.|Present affiliation: Institute of Cell and Molecular Biosciences, Newcastle University, Centre for Bacterial Cell Biology, Richardson Road, NE2 4AX Newcastle upon Tyne, UK.
authors.forename: Christopher
authors.initials: C
authors.lastname: Sauer
authors.affiliation: Department Biology I, AG Synthetic Microbiology, Ludwig-Maximilians-Universität München, Grosshaderner Str. 2-4, D-82152 Planegg-Martinsried, Germany.|Ludwig-Maximilians-University Munich, Arnold Sommerfeld Center for Theoretical Physics, Theresienstr. 37, D-80333 München, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Fritz
authors.affiliation: Department Biology I, AG Synthetic Microbiology, Ludwig-Maximilians-Universität München, Grosshaderner Str. 2-4, D-82152 Planegg-Martinsried, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Gebhard
authors.affiliation: Department Biology I, AG Synthetic Microbiology, Ludwig-Maximilians-Universität München, Grosshaderner Str. 2-4, D-82152 Planegg-Martinsried, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Mascher