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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...

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Autores principales: Moriya, Takefumi, Yamaoka, Tomohiro, Wakayama, Yuki, Ayukawa, Shotaro, Zhang, Zicong, Yamamura, Masayuki, Wakao, Shinji, Kiga, Daisuke
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
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.
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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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