Synthetic chemical inducers and genetic decoupling enable orthogonal control of the rhaBAD promoter
File(s)
Accepted version
Published version
Author(s)
Heap, JT
Kelly, C
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.
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.
Date Issued
2016-05-31
Date Acceptance
2016-05-31
Citation
ACS Synthetic Biology, 2016, 5 (10), pp.1136-1145
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.
License URL
Sponsor
Biotechnology and Biological Sciences Research Council (BBSRC)
Grant Number
BB/M011321/1
Subjects
expression system
inducible promoter
mannose
nonmetabolized inducer
rhaBAD
rhamnose
Publication Status
Published