Moriya Takefumi, Yamaoka Tomohiro, Wakayama Yuki, Ayukawa Shotaro, Zhang Zicong, Yamamura Masayuki, Wakao Shinji, Kiga Daisuke
Department of Computational Intelligence and Systems Science, Tokyo Institute of Technology, Yokohama, Kanagawa 226-8503, Japan.
Department of Electrical Engineering and Bioscience, Waseda University, Shinjuku, Tokyo 169-8050, Japan.
Life (Basel). 2019 Mar 26;9(1):30. doi: 10.3390/life9010030.
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.
报告基因推动了分子生物学的发展。上游调控蛋白与下游报告基因启动子的结合能够对相应基因产生的上游蛋白的活性进行定量分析。在合成生物学研究中，对报告基因活性的分析可确保通过合成基因回路对细胞进行控制，这是通过计算机模拟实验和体内实验相结合来实现的。然而，下游报告基因对上游调控基因的意外影响可能会干扰体内观察结果。这种现象被称为追溯性。通过计算机模拟实验和体内实验，我们发现下游基因中调控蛋白结合位点的拷贝数不同会改变上游动态，这表明在这个合成基因振荡器中报告基因存在追溯性。此外，通过分离追溯性的两种来源（成分的滴定和降解竞争），我们表明，在双反馈振荡器中，荧光蛋白报告基因与回路成分竞争降解的水平对振荡的稳定性很重要。总之，我们的结果表明，结合计算机模拟实验和体内实验来选择报告基因启动子对于基因回路的先进设计至关重要。
