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Comparison between Effects of Retroactivity and Resource Competition upon Change in Downstream Reporter Genes of Synthetic Genetic Circuits
Reporter genes have contributed to advancements in molecular biology. Binding of an upstream regulatory protein to a downstream reporter promoter allows quantification of the activity of the upstream protein produced from the corresponding gene. In studies of synthetic biology, analyses of reporter...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463139/ https://www.ncbi.nlm.nih.gov/pubmed/30917535 http://dx.doi.org/10.3390/life9010030 |
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author | Moriya, Takefumi Yamaoka, Tomohiro Wakayama, Yuki Ayukawa, Shotaro Zhang, Zicong Yamamura, Masayuki Wakao, Shinji Kiga, Daisuke |
author_facet | Moriya, Takefumi Yamaoka, Tomohiro Wakayama, Yuki Ayukawa, Shotaro Zhang, Zicong Yamamura, Masayuki Wakao, Shinji Kiga, Daisuke |
author_sort | Moriya, Takefumi |
collection | PubMed |
description | Reporter genes have contributed to advancements in molecular biology. Binding of an upstream regulatory protein to a downstream reporter promoter allows quantification of the activity of the upstream protein produced from the corresponding gene. In studies of synthetic biology, analyses of reporter gene activities ensure control of the cell with synthetic genetic circuits, as achieved using a combination of in silico and in vivo experiments. However, unexpected effects of downstream reporter genes on upstream regulatory genes may interfere with in vivo observations. This phenomenon is termed as retroactivity. Using in silico and in vivo experiments, we found that a different copy number of regulatory protein-binding sites in a downstream gene altered the upstream dynamics, suggesting retroactivity of reporters in this synthetic genetic oscillator. Furthermore, by separating the two sources of retroactivity (titration of the component and competition for degradation), we showed that, in the dual-feedback oscillator, the level of the fluorescent protein reporter competing for degradation with the circuits’ components is important for the stability of the oscillations. Altogether, our results indicate that the selection of reporter promoters using a combination of in silico and in vivo experiments is essential for the advanced design of genetic circuits. |
format | Online Article Text |
id | pubmed-6463139 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-64631392019-04-22 Comparison between Effects of Retroactivity and Resource Competition upon Change in Downstream Reporter Genes of Synthetic Genetic Circuits Moriya, Takefumi Yamaoka, Tomohiro Wakayama, Yuki Ayukawa, Shotaro Zhang, Zicong Yamamura, Masayuki Wakao, Shinji Kiga, Daisuke Life (Basel) Article Reporter genes have contributed to advancements in molecular biology. Binding of an upstream regulatory protein to a downstream reporter promoter allows quantification of the activity of the upstream protein produced from the corresponding gene. In studies of synthetic biology, analyses of reporter gene activities ensure control of the cell with synthetic genetic circuits, as achieved using a combination of in silico and in vivo experiments. However, unexpected effects of downstream reporter genes on upstream regulatory genes may interfere with in vivo observations. This phenomenon is termed as retroactivity. Using in silico and in vivo experiments, we found that a different copy number of regulatory protein-binding sites in a downstream gene altered the upstream dynamics, suggesting retroactivity of reporters in this synthetic genetic oscillator. Furthermore, by separating the two sources of retroactivity (titration of the component and competition for degradation), we showed that, in the dual-feedback oscillator, the level of the fluorescent protein reporter competing for degradation with the circuits’ components is important for the stability of the oscillations. Altogether, our results indicate that the selection of reporter promoters using a combination of in silico and in vivo experiments is essential for the advanced design of genetic circuits. MDPI 2019-03-26 /pmc/articles/PMC6463139/ /pubmed/30917535 http://dx.doi.org/10.3390/life9010030 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Moriya, Takefumi Yamaoka, Tomohiro Wakayama, Yuki Ayukawa, Shotaro Zhang, Zicong Yamamura, Masayuki Wakao, Shinji Kiga, Daisuke Comparison between Effects of Retroactivity and Resource Competition upon Change in Downstream Reporter Genes of Synthetic Genetic Circuits |
title | Comparison between Effects of Retroactivity and Resource Competition upon Change in Downstream Reporter Genes of Synthetic Genetic Circuits |
title_full | Comparison between Effects of Retroactivity and Resource Competition upon Change in Downstream Reporter Genes of Synthetic Genetic Circuits |
title_fullStr | Comparison between Effects of Retroactivity and Resource Competition upon Change in Downstream Reporter Genes of Synthetic Genetic Circuits |
title_full_unstemmed | Comparison between Effects of Retroactivity and Resource Competition upon Change in Downstream Reporter Genes of Synthetic Genetic Circuits |
title_short | Comparison between Effects of Retroactivity and Resource Competition upon Change in Downstream Reporter Genes of Synthetic Genetic Circuits |
title_sort | comparison between effects of retroactivity and resource competition upon change in downstream reporter genes of synthetic genetic circuits |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463139/ https://www.ncbi.nlm.nih.gov/pubmed/30917535 http://dx.doi.org/10.3390/life9010030 |
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