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Quantitative modeling of transcription and translation of an all-E. coli cell-free system

Cell-free transcription-translation (TXTL) is expanding as a polyvalent experimental platform to engineer biological systems outside living organisms. As the number of TXTL applications and users is rapidly growing, some aspects of this technology could be better characterized to provide a broader d...

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Autores principales: Marshall, Ryan, Noireaux, Vincent
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6700315/
https://www.ncbi.nlm.nih.gov/pubmed/31427623
http://dx.doi.org/10.1038/s41598-019-48468-8
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author Marshall, Ryan
Noireaux, Vincent
author_facet Marshall, Ryan
Noireaux, Vincent
author_sort Marshall, Ryan
collection PubMed
description Cell-free transcription-translation (TXTL) is expanding as a polyvalent experimental platform to engineer biological systems outside living organisms. As the number of TXTL applications and users is rapidly growing, some aspects of this technology could be better characterized to provide a broader description of its basic working mechanisms. In particular, developing simple quantitative biophysical models that grasp the different regimes of in vitro gene expression, using relevant kinetic constants and concentrations of molecular components, remains insufficiently examined. In this work, we present an ODE (Ordinary Differential Equation)-based model of the expression of a reporter gene in an all E. coli TXTL that we apply to a set of regulatory elements spanning several orders of magnitude in strengths, far beyond the T7 standard system used in most of the TXTL platforms. Several key biochemical constants are experimentally determined through fluorescence assays. The robustness of the model is tested against the experimental parameters, and limitations of TXTL resources are described. We establish quantitative references between the performance of E. coli and synthetic promoters and ribosome binding sites. The model and the data should be useful for the TXTL community interested either in gene network engineering or in biomanufacturing beyond the conventional platforms relying on phage transcription.
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spelling pubmed-67003152019-08-21 Quantitative modeling of transcription and translation of an all-E. coli cell-free system Marshall, Ryan Noireaux, Vincent Sci Rep Article Cell-free transcription-translation (TXTL) is expanding as a polyvalent experimental platform to engineer biological systems outside living organisms. As the number of TXTL applications and users is rapidly growing, some aspects of this technology could be better characterized to provide a broader description of its basic working mechanisms. In particular, developing simple quantitative biophysical models that grasp the different regimes of in vitro gene expression, using relevant kinetic constants and concentrations of molecular components, remains insufficiently examined. In this work, we present an ODE (Ordinary Differential Equation)-based model of the expression of a reporter gene in an all E. coli TXTL that we apply to a set of regulatory elements spanning several orders of magnitude in strengths, far beyond the T7 standard system used in most of the TXTL platforms. Several key biochemical constants are experimentally determined through fluorescence assays. The robustness of the model is tested against the experimental parameters, and limitations of TXTL resources are described. We establish quantitative references between the performance of E. coli and synthetic promoters and ribosome binding sites. The model and the data should be useful for the TXTL community interested either in gene network engineering or in biomanufacturing beyond the conventional platforms relying on phage transcription. Nature Publishing Group UK 2019-08-19 /pmc/articles/PMC6700315/ /pubmed/31427623 http://dx.doi.org/10.1038/s41598-019-48468-8 Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Marshall, Ryan
Noireaux, Vincent
Quantitative modeling of transcription and translation of an all-E. coli cell-free system
title Quantitative modeling of transcription and translation of an all-E. coli cell-free system
title_full Quantitative modeling of transcription and translation of an all-E. coli cell-free system
title_fullStr Quantitative modeling of transcription and translation of an all-E. coli cell-free system
title_full_unstemmed Quantitative modeling of transcription and translation of an all-E. coli cell-free system
title_short Quantitative modeling of transcription and translation of an all-E. coli cell-free system
title_sort quantitative modeling of transcription and translation of an all-e. coli cell-free system
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6700315/
https://www.ncbi.nlm.nih.gov/pubmed/31427623
http://dx.doi.org/10.1038/s41598-019-48468-8
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