Reconstitution of an Ultradian Oscillator in Mammalian Cells by a Synthetic Biology Approach,ACS Synthetic Biology

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Reconstitution of an Ultradian Oscillator in Mammalian Cells by a Synthetic Biology Approach
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-05-04 00:00:00 , DOI: 10.1021/acssynbio.8b00083
Marco Santorelli ₁ , Daniela Perna ₁ , Akihiro Isomura ₂ , Immacolata Garzilli ₁ , Francesco Annunziata ₁ , Lorena Postiglione ₁ , Barbara Tumaini ₁ , Ryoichiro Kageyama ₂ , Diego di Bernardo _{1,

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Affiliation

The Notch effector gene Hes1 is an ultradian clock exhibiting cyclic gene expression in several progenitor cells, with a period of a few hours. Because of the complexity of studying Hes1 in the endogenous setting, and the difficulty of imaging these fast oscillations in vivo, the mechanism driving oscillations has never been proven. Here, we applied a “build it to understand it” synthetic biology approach to construct simplified “hybrid” versions of the Hes1 ultradian oscillator combining synthetic and natural parts. We successfully constructed a simplified synthetic version of the Hes1 promoter matching the endogenous regulation logic. By mathematical modeling and single-cell real-time imaging, we were able to demonstrate that Hes1 is indeed able to generate stable oscillations by a delayed negative feedback loop. Moreover, we proved that introns in Hes1 contribute to the transcriptional delay but may not be strictly necessary for oscillations to occur. We also developed a novel reporter of endogenous Hes1 oscillations able to amplify the bioluminescence signal 5-fold. Our results have implications also for other ultradian oscillators.

中文翻译：

通过合成生物学方法重建哺乳动物细胞中的超摆动子。

Notch效应基因Hes1是一个超时钟，在几个祖细胞中表现出循环基因表达，周期为几个小时。由于在内源性环境中研究Hes1的复杂性，以及在体内对这些快速振荡进行成像的难度，驱动振荡的机制尚未得到证实。在这里，我们采用了一种“了解它”的合成生物学方法，来构造结合了合成部分和天然部分的Hes1超电子振荡器的简化“混合”版本。我们成功构建了Hes1的简化合成版本与内源调节逻辑匹配的启动子。通过数学建模和单细胞实时成像，我们能够证明Hes1确实能够通过延迟的负反馈回路产生稳定的振荡。此外，我们证明了Hes1中的内含子有助于转录延迟，但对于发生振荡可能不是严格必需的。我们还开发了内源性Hes1振荡的新型报告基因，能够将生物发光信号放大5倍。我们的结果对其他超弧度振荡器也有影响。

更新日期：2018-05-04

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