Contraintes Research Group, Institut National de Recherche en Informatique et en Automatique, INRIA Paris-Rocquencourt, 78150 Rocquencourt, France.
Proc Natl Acad Sci U S A. 2012 Aug 28;109(35):14271-6. doi: 10.1073/pnas.1206810109. Epub 2012 Aug 14.
Gene expression plays a central role in the orchestration of cellular processes. The use of inducible promoters to change the expression level of a gene from its physiological level has significantly contributed to the understanding of the functioning of regulatory networks. However, from a quantitative point of view, their use is limited to short-term, population-scale studies to average out cell-to-cell variability and gene expression noise and limit the nonpredictable effects of internal feedback loops that may antagonize the inducer action. Here, we show that, by implementing an external feedback loop, one can tightly control the expression of a gene over many cell generations with quantitative accuracy. To reach this goal, we developed a platform for real-time, closed-loop control of gene expression in yeast that integrates microscopy for monitoring gene expression at the cell level, microfluidics to manipulate the cells' environment, and original software for automated imaging, quantification, and model predictive control. By using an endogenous osmostress responsive promoter and playing with the osmolarity of the cells environment, we show that long-term control can, indeed, be achieved for both time-constant and time-varying target profiles at the population and even the single-cell levels. Importantly, we provide evidence that real-time control can dynamically limit the effects of gene expression stochasticity. We anticipate that our method will be useful to quantitatively probe the dynamic properties of cellular processes and drive complex, synthetically engineered networks.
基因表达在细胞过程的协调中起着核心作用。使用诱导型启动子来改变基因的表达水平，使其超出生理水平，这对理解调控网络的功能有很大的帮助。然而，从定量的角度来看，它们的使用仅限于短期的、基于群体的研究，以平均细胞间的变异性和基因表达噪声，并限制内部反馈回路的不可预测的影响，这些影响可能会拮抗诱导剂的作用。在这里，我们展示了通过实现外部反馈回路，可以在多个细胞世代中以定量精度紧密控制基因的表达。为了实现这一目标，我们开发了一个用于酵母中基因表达的实时、闭环控制平台，该平台集成了用于监测细胞水平基因表达的显微镜、用于操纵细胞环境的微流控技术以及用于自动成像、定量和模型预测控制的原始软件。通过使用内源性渗透压响应启动子并调整细胞环境的渗透压，我们证明了长期控制确实可以在群体水平甚至单细胞水平上实现对时不变和时变目标曲线的控制。重要的是，我们提供了证据表明实时控制可以动态地限制基因表达随机性的影响。我们预计，我们的方法将有助于定量研究细胞过程的动态特性，并驱动复杂的、合成工程网络。
