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Quantitative characterization of translational riboregulators using an in vitro transcription–translation system

AuthorsSenoussi, Anis; Lee Tin Wah, Jonathan; Shimizu, Yoshihiro; Robert, Jérôme; Jaramillo, Alfonso; Findeiss, Sven; Axmann, Ilka M.; Estevez-Torres, André
KeywordsRNA translational riboregulator
Cell-free protein synthesis
In vitro synthetic biology
Issue Date2018
PublisherAmerican Chemical Society
CitationACS Synthetic Biology 7(5): 1269-1278 (2018)
AbstractRiboregulators 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.
Publisher version (URL)https://doi.org/10.1021/acssynbio.7b00387
Appears in Collections:(I2SysBio) Artículos
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