Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements
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Published version
Accepted version
Author(s)
Kelwick, RJR
Webb, AJ
MacDonald, JT
Freemont, PS
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.
Date Issued
2016-09-30
Date Acceptance
2016-09-29
Citation
Metabolic Engineering, 2016, 38, pp.370-381
ISSN
1096-7184
Publisher
Elsevier
Start Page
370
End Page
381
Journal / Book Title
Metabolic Engineering
Volume
38
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/).
License URL
Sponsor
Engineering & Physical Science Research Council (E
Engineering & Physical Science Research Council (E
Grant Number
EP/J02175X/1
ERI-031108-EP/K034359/1
Subjects
Bacillus subtilis
Cell-free transcription-translation
Luciferase assay
Promoter library
Regulatory element prototyping
Synthetic biology
Biotechnology
1003 Industrial Biotechnology
Publication Status
Published