Uhlendorf Jannis, Miermont Agnès, Delaveau Thierry, Charvin Gilles, Fages François, Bottani Samuel, Hersen Pascal, Batt Gregory
INRIA Paris-Rocquencourt, Domaine de Voluceau, Rocquencourt - BP 105, 78153, Le Chesnay, France.
Methods Mol Biol. 2015;1244:277-85. doi: 10.1007/978-1-4939-1878-2_13.
By implementing an external feedback loop one can tightly control the expression of a gene over many cell generations with quantitative accuracy. Controlling precisely the level of a protein of interest will be useful to probe quantitatively the dynamical properties of cellular processes and to drive complex, synthetically-engineered networks. In this chapter we describe 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 osmo-stress responsive promoter and playing with the osmolarity of the cells environment, we demonstrate that long-term control can indeed be achieved for both time-constant and time-varying target profiles, at the population level, and even at the single-cell level.
通过实施外部反馈回路，可以在多个细胞世代中以定量精度严格控制基因的表达。精确控制目标蛋白质的水平将有助于定量探究细胞过程的动态特性，并驱动复杂的合成工程网络。在本章中，我们描述了一个用于酵母基因表达实时闭环控制的平台，该平台集成了用于在细胞水平监测基因表达的显微镜、用于操纵细胞环境的微流体技术，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应激响应启动子并改变细胞环境的渗透压，我们证明了在群体水平甚至单细胞水平上，对于时间常数和时变目标曲线都确实可以实现长期控制。
