Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements

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Title: Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements
Author(s): Kelwick, RJR
Webb, AJ
MacDonald, JT
Freemont, PS
Item Type: Journal Article
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 0.8 µM 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.
Publication Date: 30-Sep-2016
Date of Acceptance: 29-Sep-2016
URI: http://hdl.handle.net/10044/1/41096
DOI: https://dx.doi.org/10.1016/j.ymben.2016.09.008
ISSN: 1096-7184
Publisher: Elsevier
Start Page: 370
End Page: 381
Journal / Book Title: Metabolic Engineering
Volume: 38
Sponsor/Funder: Engineering & Physical Science Research Council (E
Engineering & Physical Science Research Council (E
Funder's Grant Number: EP/J02175X/1
ERI-031108-EP/K034359/1
Copyright Statement: © 2016 The Authors. Published by Elsevier Inc. on behalf of International Metabolic Engineering Society. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: Bacillus subtilis
Cell-free transcription-translation
Luciferase assay
Promoter library
Regulatory element prototyping
Synthetic biology
Biotechnology
1003 Industrial Biotechnology
Publication Status: Published
Appears in Collections:Department of Medicine
Faculty of Medicine



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