Journal
METABOLIC ENGINEERING
Volume 38, Issue -, Pages 370-381Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2016.09.008
Keywords
Synthetic biology; Bacillus subtilis; Cell-free transcription-translation; Regulatory element prototyping; Promoter library; Luciferase assay
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Funding
- Engineering and Physical Science Research Council (EPSRC) [EP/K034359/1, EP/J02175X/1]
- BBSRC [BB/L027852/1] Funding Source: UKRI
- EPSRC [EP/L011573/1, EP/M006700/1, EP/J02175X/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/L027852/1] Funding Source: researchfish
- Engineering and Physical Sciences Research Council [EP/L011573/1, EP/M006700/1, EP/J02175X/1] Funding Source: researchfish
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Cell-free transcription-translation systems were originally applied towards in vitro protein production. More recently, synthetic biology is enabling these systems to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and biosynthetic pathways. The Gram-positive soil bacterium, Bacillus subtilis, is an established model organism of industrial importance. To this end, we developed several B. subtilis-based cell-free systems. Our improved B. subtilis WB800N-based system was capable of producing 0.8 mu M GFP, which gave a similar to 72x fold-improvement when compared with a B. subtilis 168 cell-free system. Our improved system was applied towards the prototyping of a B. subtilis promoter library in which we engineered several promoters, derived from the wild-type P-grac (sigma A) promoter, that display a range of comparable in vitro and in vivo transcriptional activities. Additionally, we demonstrate the cell-free characterisation of an inducible expression system, and the activity of a model enzyme - renilla luciferase.
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