The Bacillus BioBrick Box

Generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis

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

Research output: Contribution to journalArticle

72 Citations (Scopus)
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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.

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.
Original languageEnglish
Pages (from-to)29
JournalJournal of Biological Engineering
Volume7
Issue number1
DOIs
Publication statusPublished - 1 Jan 2013

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Bacilli
Bacillus subtilis
Bacillus
Epitopes
Proteins
Equipment and Supplies
beta-Galactosidase
Luciferases
Cloning
Organism Cloning
Time measurement
Escherichia coli
Assays

Cite this

The Bacillus BioBrick Box : Generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. / Radeck, Jara; Kraft, Korinna; Bartels, Julia; Cikovic, Tamara; Dürr, Franziska; Emenegger, Jennifer; Kelterborn, Simon; Sauer, Christopher; Fritz, Georg; Gebhard, Susanne; Mascher, Thorsten.

In: Journal of Biological Engineering, Vol. 7, No. 1, 01.01.2013, p. 29.

Research output: Contribution to journalArticle

Radeck, J, Kraft, K, Bartels, J, Cikovic, T, Dürr, F, Emenegger, J, Kelterborn, S, Sauer, C, Fritz, G, Gebhard, S & Mascher, T 2013, 'The Bacillus BioBrick Box: Generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis', Journal of Biological Engineering, vol. 7, no. 1, pp. 29. https://doi.org/10.1186/1754-1611-7-29
Radeck, Jara ; Kraft, Korinna ; Bartels, Julia ; Cikovic, Tamara ; Dürr, Franziska ; Emenegger, Jennifer ; Kelterborn, Simon ; Sauer, Christopher ; Fritz, Georg ; Gebhard, Susanne ; Mascher, Thorsten. / The Bacillus BioBrick Box : Generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. In: Journal of Biological Engineering. 2013 ; Vol. 7, No. 1. pp. 29.
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T2 - Generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis

AU - Radeck, Jara

AU - Kraft, Korinna

AU - Bartels, Julia

AU - Cikovic, Tamara

AU - Dürr, Franziska

AU - Emenegger, Jennifer

AU - Kelterborn, Simon

AU - Sauer, Christopher

AU - Fritz, Georg

AU - Gebhard, Susanne

AU - Mascher, Thorsten

PY - 2013/1/1

Y1 - 2013/1/1

N2 - BackgroundStandardized 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.ResultsWe 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.ConclusionOur 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.

AB - BackgroundStandardized 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.ResultsWe 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.ConclusionOur 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.

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DO - 10.1186/1754-1611-7-29

M3 - Article

VL - 7

SP - 29

JO - Journal of Biological Engineering

JF - Journal of Biological Engineering

SN - 1754-1611

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