Gene networks and signaling pathways display complex topologies and, as a result, complex nonlinear behaviors. Accumulating evidence shows that both static (concentration) and dynamical (rate-of-change) features of transcription factors, ligands and environmental stimuli control downstream processes and ultimately cellular functions. Currently, however, methods to generate stimuli with the desired features to probe cell response are still lacking. Here, combining tools from Control Engineering and Synthetic Biology (cybergenetics), we propose a simple and cost-effective microfluidics-based platform to precisely regulate gene expression and signaling pathway activity in mammalian cells by means of real-time feedback control. We show that this platform allows (i) to automatically regulate gene expression from inducible promoters in different cell types, including mouse embryonic stem cells; (ii) to precisely regulate the activity of the mTOR signaling pathway in single cells; (iii) to build a biohybrid oscillator in single embryonic stem cells by interfacing biological parts with virtual in silico counterparts. Ultimately, this platform can be used to probe gene networks and signaling pathways to understand how they process static and dynamic features of specific stimuli, as well as for the rapid prototyping of synthetic circuits for biotechnology and biomedical purposes.

中文翻译：

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自动化的微流控反馈控制哺乳动物细胞中的基因表达和信号通路活性。

基因网络和信号通路显示出复杂的拓扑结构，并因此显示出复杂的非线性行为。越来越多的证据表明，转录因子，配体和环境刺激的静态（浓度）和动态（变化率）特征均可控制下游过程，最终控制细胞功能。然而，目前，仍然缺乏产生具有期望特征以探测细胞应答的刺激物的方法。在这里，结合控制工程和合成生物学（cybergenetics）的工具，我们提出了一个简单且具有成本效益的基于微流体的平台，可以通过实时反馈控制精确调节哺乳动物细胞中的基因表达和信号通路活性。我们证明了该平台允许（i）自动调节来自不同细胞类型（包括小鼠胚胎干细胞）的诱导型启动子的基因表达；（ii）精确调节单个细胞中mTOR信号通路的活性；（iii）通过将生物部分与虚拟界面连接，在单个胚胎干细胞中构建生物杂交振荡器在计算机同行中。最终，该平台可用于探测基因网络和信号通路，以了解它们如何处理特定刺激的静态和动态特征，以及用于生物技术和生物医学目的的合成电路的快速原型制作。