title: Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements.
abstract: 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 0.8µM GFP, which gave a ~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 Pgrac(σA) 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.
authors.affiliation: Centre for Synthetic Biology and Innovation, Imperial College London, London SW7 2AZ, UK; Section of Structural Biology, Department of Medicine, Imperial College London, London SW7 2AZ, UK.
authors.forename: Richard
authors.initials: R
authors.lastname: Kelwick
authors.affiliation: Centre for Synthetic Biology and Innovation, Imperial College London, London SW7 2AZ, UK; Section of Structural Biology, Department of Medicine, Imperial College London, London SW7 2AZ, UK.
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Webb
authors.affiliation: Centre for Synthetic Biology and Innovation, Imperial College London, London SW7 2AZ, UK; Section of Structural Biology, Department of Medicine, Imperial College London, London SW7 2AZ, UK.
authors.forename: James T
authors.initials: JT
authors.lastname: MacDonald
authors.affiliation: Centre for Synthetic Biology and Innovation, Imperial College London, London SW7 2AZ, UK; Section of Structural Biology, Department of Medicine, Imperial College London, London SW7 2AZ, UK. Electronic address: p.freemont@imperial.ac.uk.
authors.forename: Paul S
authors.initials: PS
authors.lastname: Freemont
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics*; D002474:Cell-Free System / Q000502:physiology; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D024861:Genetic Enhancement / Q000379:methods; D060847:Metabolic Engineering / Q000379:methods; D053858:Metabolic Networks and Pathways / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics*; D050436:Regulatory Elements, Transcriptional / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins
keywords: Bacillus subtilis; Cell-free transcription-translation; Luciferase assay; Promoter library; Regulatory element prototyping; Synthetic biology