Genetic programs constructed from layered logic gates in single cells.
Genetic programs constructed from layered logic gates in single cells.
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遗传程序的功能是整合环境传感器，实现信号处理算法和控制表达动力学。这些程序由集成的遗传电路组成，这些遗传电路单独执行从数字逻辑到动态电路的操作，并且它们已被用于各种细胞工程应用中，包括在代谢网络中执行过程控制和在人工组织中协调空间分化。一个关键的限制是，电路是基于在细胞的有限体积中发生的生物化学相互作用，因此程序的大小被限制在几个电路中。在这里，我们应用部分挖掘和定向进化建立一套转录与门大肠杆菌。每个与门整合两个启动子输入并控制一个启动子输出。这允许通过使上游电路的输出启动子用作下游电路的输入启动子来对门进行分层。每个门由一个转录因子组成，需要第二个伴侣蛋白来激活输出启动子。从不同菌株的III型分泌途径中鉴定出多种激活剂-伴侣蛋白对。应用定向进化来增加电路的动态范围和正交性。这些门以不同的排列方式连接形成程序，其中最大的是一个4输入与门，由3个电路组成，集成了4个诱导系统，因此需要11个调节蛋白。测量单个门的性能足以捕获整个程序的行为。由于延迟（故障），分层电路的一个常见问题，在输出中的错误，没有观察到。这项工作展示了正交逻辑门的成功分层，这种设计策略可以在单个单元中构建大型集成电路。
Genetic programs function to integrate environmental sensors, implement signal processing algorithms and control expression dynamics. These programs consist of integrated genetic circuits that individually implement operations ranging from digital logic to dynamic circuits, and they have been used in various cellular engineering applications, including the implementation of process control in metabolic networks and the coordination of spatial differentiation in artificial tissues. A key limitation is that the circuits are based on biochemical interactions occurring in the confined volume of the cell, so the size of programs has been limited to a few circuits. Here we apply part mining and directed evolution to build a set of transcriptional AND gates in Escherichia coli. Each AND gate integrates two promoter inputs and controls one promoter output. This allows the gates to be layered by having the output promoter of an upstream circuit serve as the input promoter for a downstream circuit. Each gate consists of a transcription factor that requires a second chaperone protein to activate the output promoter. Multiple activator–chaperone pairs are identified from type III secretion pathways in different strains of bacteria. Directed evolution is applied to increase the dynamic range and orthogonality of the circuits. These gates are connected in different permutations to form programs, the largest of which is a 4-input AND gate that consists of 3 circuits that integrate 4 inducible systems, thus requiring 11 regulatory proteins. Measuring the performance of individual gates is sufficient to capture the behaviour of the complete program. Errors in the output due to delays (faults), a common problem for layered circuits, are not observed. This work demonstrates the successful layering of orthogonal logic gates, a design strategy that could enable the construction of large, integrated circuits in single cells.