Synthetic chemical inducers and genetic decoupling enable orthogonal control of the rhaBAD promoter

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Title: Synthetic chemical inducers and genetic decoupling enable orthogonal control of the rhaBAD promoter
Author(s): Heap, JT
Kelly, C
Item Type: Journal Article
Abstract: External control of gene expression is crucial in synthetic biology and biotechnology research and applications, and is commonly achieved using inducible promoter systems. The E. coli rhamnose-inducible rhaBAD promoter has properties superior to more commonly-used inducible expression systems, but is marred by transient expression caused by degradation of the native inducer, L-rhamnose. To address this problem, 35 analogs of L-rhamnose were screened for induction of the rhaBAD promoter, but no strong inducers were identified. In the native configuration, an inducer must bind and activate two transcriptional activators, RhaR and RhaS. Therefore, the expression system was reconfigured to decouple the rhaBAD promoter from the native rhaSR regulatory cascade so that candidate inducers need only activate the terminal transcription factor RhaS. Re-screening the 35 compounds using the modified rhaBAD expression system revealed several promising inducers. These were characterised further to determine the strength, kinetics and concentration-dependence of induction; whether the inducer was used as a carbon source by E. coli; and the modality (distribution) of induction among populations of cells. L-Mannose was found to be the most useful orthogonal inducer, providing an even greater range of induction than the native inducer Lrhamnose, and crucially, allowing sustained induction instead of transient induction. These findings address the key limitation of the rhaBAD expression system, and suggest it may now be the most suitable system for many applications.
Publication Date: 31-May-2016
Date of Acceptance: 31-May-2016
URI: http://hdl.handle.net/10044/1/33313
DOI: https://dx.doi.org/10.1021/acssynbio.6b00030
ISSN: 2161-5063
Publisher: American Chemical Society
Start Page: 1136
End Page: 1145
Journal / Book Title: ACS Synthetic Biology
Volume: 5
Issue: 10
Copyright Statement: This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.
Sponsor/Funder: Biotechnology and Biological Sciences Research Council (BBSRC)
Funder's Grant Number: BB/M011321/1
Keywords: expression system
inducible promoter
mannose
nonmetabolized inducer
rhaBAD
rhamnose
Publication Status: Published
Appears in Collections:Faculty of Natural Sciences



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