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Noise-reducing optogenetic negative-feedback gene circuits in human cells

Gene autorepression is widely present in nature and is also employed in synthetic biology, partly to reduce gene expression noise in cells. Optogenetic systems have recently been developed for controlling gene expression levels in mammalian cells, but most have utilized activator-based proteins, neg...

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Autores principales: Guinn, Michael Tyler, Balázsi, Gábor
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698750/
https://www.ncbi.nlm.nih.gov/pubmed/31269201
http://dx.doi.org/10.1093/nar/gkz556
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author Guinn, Michael Tyler
Balázsi, Gábor
author_facet Guinn, Michael Tyler
Balázsi, Gábor
author_sort Guinn, Michael Tyler
collection PubMed
description Gene autorepression is widely present in nature and is also employed in synthetic biology, partly to reduce gene expression noise in cells. Optogenetic systems have recently been developed for controlling gene expression levels in mammalian cells, but most have utilized activator-based proteins, neglecting negative feedback except for in silico control. Here, we engineer optogenetic gene circuits into mammalian cells to achieve noise-reduction for precise gene expression control by genetic, in vitro negative feedback. We build a toolset of these noise-reducing Light-Inducible Tuner (LITer) gene circuits using the TetR repressor fused with a Tet-inhibiting peptide (TIP) or a degradation tag through the light-sensitive LOV2 protein domain. These LITers provide a range of nearly 4-fold gene expression control and up to 5-fold noise reduction from existing optogenetic systems. Moreover, we use the LITer gene circuit architecture to control gene expression of the cancer oncogene KRAS(G12V) and study its downstream effects through phospho-ERK levels and cellular proliferation. Overall, these novel LITer optogenetic platforms should enable precise spatiotemporal perturbations for studying multicellular phenotypes in developmental biology, oncology and other biomedical fields of research.
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spelling pubmed-66987502019-08-22 Noise-reducing optogenetic negative-feedback gene circuits in human cells Guinn, Michael Tyler Balázsi, Gábor Nucleic Acids Res Synthetic Biology and Bioengineering Gene autorepression is widely present in nature and is also employed in synthetic biology, partly to reduce gene expression noise in cells. Optogenetic systems have recently been developed for controlling gene expression levels in mammalian cells, but most have utilized activator-based proteins, neglecting negative feedback except for in silico control. Here, we engineer optogenetic gene circuits into mammalian cells to achieve noise-reduction for precise gene expression control by genetic, in vitro negative feedback. We build a toolset of these noise-reducing Light-Inducible Tuner (LITer) gene circuits using the TetR repressor fused with a Tet-inhibiting peptide (TIP) or a degradation tag through the light-sensitive LOV2 protein domain. These LITers provide a range of nearly 4-fold gene expression control and up to 5-fold noise reduction from existing optogenetic systems. Moreover, we use the LITer gene circuit architecture to control gene expression of the cancer oncogene KRAS(G12V) and study its downstream effects through phospho-ERK levels and cellular proliferation. Overall, these novel LITer optogenetic platforms should enable precise spatiotemporal perturbations for studying multicellular phenotypes in developmental biology, oncology and other biomedical fields of research. Oxford University Press 2019-08-22 2019-07-03 /pmc/articles/PMC6698750/ /pubmed/31269201 http://dx.doi.org/10.1093/nar/gkz556 Text en © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Synthetic Biology and Bioengineering
Guinn, Michael Tyler
Balázsi, Gábor
Noise-reducing optogenetic negative-feedback gene circuits in human cells
title Noise-reducing optogenetic negative-feedback gene circuits in human cells
title_full Noise-reducing optogenetic negative-feedback gene circuits in human cells
title_fullStr Noise-reducing optogenetic negative-feedback gene circuits in human cells
title_full_unstemmed Noise-reducing optogenetic negative-feedback gene circuits in human cells
title_short Noise-reducing optogenetic negative-feedback gene circuits in human cells
title_sort noise-reducing optogenetic negative-feedback gene circuits in human cells
topic Synthetic Biology and Bioengineering
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698750/
https://www.ncbi.nlm.nih.gov/pubmed/31269201
http://dx.doi.org/10.1093/nar/gkz556
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