Transcription factors and their target promoters are central to synthetic biology. By arranging these components into novel gene regulatory circuits, synthetic biologists have been able to create a wide variety of phenotypes, including bistable switches, oscillators, and logic gates. However, transcription factors (TFs) do not instantaneously regulate downstream targets. After the gene encoding a TF is turned on, the gene must first be transcribed, the transcripts must be translated, and sufficient TF must accumulate in order to bind operator sites of the target promoter. The time to complete this process, here called the “signaling time,” is a critical aspect in the design of dynamic regulatory networks, yet it remains poorly characterized. In this work, we measured the signaling time of two TFs in Escherichia coli commonly used in synthetic biology: the activator AraC and the repressor LacI. We found that signaling times can range from a few to tens of minutes, and are affected by the expression rate of the TF. Our single-cell data also show that the variability of the signaling time increases with its mean. To validate these signaling time measurements, we constructed a two-step genetic cascade, and showed that the signaling time of the full cascade can be predicted from those of its constituent steps. These results provide concrete estimates for the time scales of transcriptional regulation in living cells, which are important for understanding the dynamics of synthetic transcriptional gene circuits.

中文翻译：

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合成基因电路中转录调控的时机

转录因子及其靶启动子对合成生物学至关重要。通过将这些成分安排在新颖的基因调节电路中，合成生物学家已经能够创建各种各样的表型，包括双稳态开关，振荡器和逻辑门。但是，转录因子（TFs）不能即时调节下游目标。在打开编码TF的基因后，必须首先转录该基因，必须翻译转录本，并且必须积累足够的TF才能结合靶标启动子的操纵位点。完成此过程的时间，这里称为“信号时间”，是动态监管网络设计中的关键方面，但其特征仍然很差。在这项工作中，我们测量了大肠杆菌中两个TF的信号传导时间合成生物学中常用的：活化剂AraC和阻遏物LacI。我们发现信号转导时间可能在几分钟到几十分钟之间，并且受TF表达率的影响。我们的单细胞数据还表明，信号传递时间的变异性随其平均值的增加而增加。为了验证这些信号转导时间的测量，我们构建了一个两步遗传级联，并表明可以从其组成步骤中预测整个级联的信号转导时间。这些结果为活细胞中转录调节的时间尺度提供了具体的估计，这对于理解合成转录基因电路的动力学非常重要。