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Comparison between Effects of Retroactivity and Resource Competition upon Change in Downstream Reporter Genes of Synthetic Genetic Circuits

1
Department of Computational Intelligence and Systems Science, Tokyo Institute of Technology, Yokohama, Kanagawa 226-8503, Japan
2
Department of Electrical Engineering and Bioscience, Waseda University, Shinjuku, Tokyo 169-8050, Japan
3
Waseda Research Institute for Science and Engineering, Waseda University, Shinjuku, Tokyo 169-8050, Japan
*
Author to whom correspondence should be addressed.
Received: 10 October 2018 / Revised: 20 March 2019 / Accepted: 22 March 2019 / Published: 26 March 2019
(This article belongs to the Special Issue Approaches toward Artificial Cell Construction and Applications)
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Abstract

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. View Full-Text
Keywords: synthetic biology; systems biology; retroactivity; resource competition; reporter gene; protein-binding site synthetic biology; systems biology; retroactivity; resource competition; reporter gene; protein-binding site
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Moriya, T.; Yamaoka, T.; Wakayama, Y.; Ayukawa, S.; Zhang, Z.; Yamamura, M.; Wakao, S.; Kiga, D. Comparison between Effects of Retroactivity and Resource Competition upon Change in Downstream Reporter Genes of Synthetic Genetic Circuits. Life 2019, 9, 30.

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