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Quantitative characterization of translational riboregulators using an in vitro transcription–translation system
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Senoussi, Anis, Lee Tin Wah, Jonathan, Shimizu, Yoshihiro, Robert, Jérôme, Jaramillo, Alfonso, Findeiss, Sven, Axmann, Ilka M. and Estevez-Torres, André (2018) Quantitative characterization of translational riboregulators using an in vitro transcription–translation system. ACS Synthetic Biology, 7 (5). pp. 1269-1278. doi:10.1021/acssynbio.7b00387 ISSN 2161-5063.
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Official URL: http://dx.doi.org/10.1021/acssynbio.7b00387
Abstract
Riboregulators are short RNA sequences that, upon binding to a ligand, change their secondary structure and influence the expression rate of a downstream gene. They constitute an attractive alternative to transcription factors for building synthetic gene regulatory networks because they can be engineered de novo. However, riboregulators are generally designed in silico and tested in vivo, which provides little quantitative information about their performances, thus hindering the improvement of design algorithms. Here we show that a cell-free transcription-translation (TX–TL) system provides valuable information about the performances of in silico designed riboregulators. We first propose a simple model that provides a quantitative definition of the dynamic range of a riboregulator. We further characterize two types of translational riboregulators composed of a cis-repressed (cr) and a trans-activating (ta) strand. At the DNA level we demonstrate that high concentrations of taDNA poisoned the activator until total shut off, in agreement with our model, and that relative dynamic ranges of riboregulators determined in vitro are in agreement with published in vivo data. At the RNA level, we show that this approach provides a fast and simple way to measure dissociation constants of functional riboregulators, in contrast to standard mobility-shift assays. Our method opens the route for using cell-free TX–TL systems for the quantitative characterization of functional riboregulators in order to improve their design in silico.
Item Type: | Journal Article | ||||||
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Subjects: | Q Science > QH Natural history > QH426 Genetics | ||||||
Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||||
Library of Congress Subject Headings (LCSH): | Gene expression, RNA, Synthetic biology, Ligands | ||||||
Journal or Publication Title: | ACS Synthetic Biology | ||||||
Publisher: | American Chemical Society | ||||||
ISSN: | 2161-5063 | ||||||
Official Date: | 4 April 2018 | ||||||
Dates: |
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Volume: | 7 | ||||||
Number: | 5 | ||||||
Page Range: | pp. 1269-1278 | ||||||
DOI: | 10.1021/acssynbio.7b00387 | ||||||
Status: | Peer Reviewed | ||||||
Publication Status: | Published | ||||||
Access rights to Published version: | Restricted or Subscription Access | ||||||
Date of first compliant deposit: | 8 June 2018 | ||||||
Date of first compliant Open Access: | 4 April 2019 | ||||||
RIOXX Funder/Project Grant: |
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