Department of Biological Engineering, Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA, USA.
Department of Biological Engineering, Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA, USA
Mol Syst Biol. 2018 Nov 27;14(11):e8605. doi: 10.15252/msb.20188605.
Controlling gene expression during a bioprocess enables real-time metabolic control, coordinated cellular responses, and staging order-of-operations. Achieving this with small molecule inducers is impractical at scale and dynamic circuits are difficult to design. Here, we show that the same set of sensors can be integrated by different combinatorial logic circuits to vary when genes are turned on and off during growth. Three sensors that respond to the consumption of feedstock (glucose), dissolved oxygen, and by-product accumulation (acetate) are constructed and optimized. By integrating these sensors, logic circuits implement temporal control over an 18-h period. The circuit outputs are used to regulate endogenous enzymes at the transcriptional and post-translational level using CRISPRi and targeted proteolysis, respectively. As a demonstration, two circuits are designed to control acetate production by matching their dynamics to when endogenous genes are expressed ( or ) and respond by turning off the corresponding gene. This work demonstrates how simple circuits can be implemented to enable customizable dynamic gene regulation.
在生物过程中控制基因表达可以实现实时代谢控制、协调细胞反应和操作顺序。在大规模生产中，使用小分子诱导剂来实现这一目标是不切实际的，而且动态电路也很难设计。在这里，我们展示了相同的一组传感器可以通过不同的组合逻辑电路进行集成，从而改变基因在生长过程中何时开启和关闭。构建并优化了三个响应原料（葡萄糖）消耗、溶解氧和副产物积累（乙酸盐）的传感器。通过集成这些传感器，逻辑电路可以在 18 小时的时间内实现时间控制。然后，使用 CRISPRi 和靶向蛋白水解分别在转录和翻译后水平上调节内源性酶。作为一个演示，设计了两个电路来控制乙酸盐的产生，其动态与内源性基因表达（或）相匹配，并通过关闭相应的基因来做出响应。这项工作展示了如何实现简单的电路来实现可定制的动态基因调控。
