Cargando…
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...
Autores principales: | , |
---|---|
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 |
_version_ | 1783444607370002432 |
---|---|
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. |
format | Online Article Text |
id | pubmed-6698750 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT guinnmichaeltyler noisereducingoptogeneticnegativefeedbackgenecircuitsinhumancells AT balazsigabor noisereducingoptogeneticnegativefeedbackgenecircuitsinhumancells |