Comparison between effects of retroactivity and resource competition upon change in downstream reporter genes of synthetic genetic circuits

Takefumi Moriya, Tomohiro Yamaoka, Yuki Wakayama, Shotaro Ayukawa, Zicong Zhang, Masayuki Yamamura, Shinji Wakao, Daisuke Kiga

Research output: Contribution to journalArticle

1 Citation (Scopus)

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.

Original languageEnglish
Article number30
JournalLife
Volume9
Issue number1
DOIs
Publication statusPublished - 2019 Mar 1

Keywords

  • Protein-binding site
  • Reporter gene
  • Resource competition
  • Retroactivity
  • Synthetic biology
  • Systems biology

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Biochemistry, Genetics and Molecular Biology(all)
  • Space and Planetary Science
  • Palaeontology

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