Algorithmic Programming of Sequential Logic and Genetic Circuits for Recording Biochemical Concentration in a Probiotic Bacterium

Through the implementation of designable genetic circuits, engineered probiotic microorganisms could be used as noninvasive diagnostic tools for the gastrointestinal tract. For these living cells to report detected biomarkers or signals after exiting the gut, the genetic circuits must be able to record these signals by using genetically encoded memory. Complex memory register circuits could enable multiplex interrogation of biomarkers and signals. A theory-based approach to create genetic circuits containing memory, known as sequential logic circuits, was previously established for a model laboratory strain of Escherichia coli, yet how circuit component performance varies for nonmodel and clinically relevant bacterial strains is poorly understood. Here, we develop a scalable computational approach to design robust sequential logic circuits in probiotic strain Escherichia coli Nissle 1917 (EcN). In this work, we used TetR-family transcriptional repressors to build genetic logic gates that can be composed into sequential logic circuits, along with a set of engineered sensors relevant for use in the gut environment. Using standard methods, 16 genetic NOT gates and nine sensors were experimentally characterized in EcN. These data were used to design and predict the performance of circuit designs. We present a set of genetic circuits encoding both combinational logic and sequential logic and show that the circuit outputs are in close agreement with our quantitative predictions from the design algorithm. Furthermore, we demonstrate an analog-like concentration recording circuit that detects and reports three input concentration ranges of a biochemical signal using sequential logic.通过构建可设计的遗传回路（designable genetic circuits），工程化益生菌微生物（engineered probiotic microorganisms）可作为针对胃肠道（gastrointestinal tract）的非侵入式诊断工具（noninvasive diagnostic tools）。若要使这些活体细胞在离开肠道后仍能报告其检测到的生物标志物或信号，该遗传回路必须能够借助遗传编码记忆（genetically encoded memory）对上述信号进行记录。复杂记忆寄存器回路（complex memory register circuits）可实现对多类生物标志物与信号的多重检测（multiplex interrogation）。此前，针对实验室模式大肠杆菌菌株，已建立基于理论的方法以构建具备记忆功能的遗传回路，即时序逻辑回路（sequential logic circuits），但目前对于非模式且与临床相关的细菌菌株而言，回路组件的性能变化规律仍不甚明确。 本研究开发了一种可扩展的计算方法，用于在益生菌菌株大肠杆菌Nissle 1917（Escherichia coli Nissle 1917, 简称EcN）中设计稳健的时序逻辑回路。本研究采用TetR家族转录阻遏蛋白（TetR-family transcriptional repressors）构建可组合为时序逻辑回路的遗传逻辑门（genetic logic gates），同时搭配一套适配肠道环境的工程化传感器（engineered sensors）。通过标准实验方法，我们在EcN中完成了16个遗传非门（NOT gates）与9个传感器的实验表征。上述数据被用于设计并预测各类回路设计的性能表现。 我们构建了一系列兼具组合逻辑（combinational logic）与时序逻辑功能的遗传回路，并证实回路输出与我们通过设计算法得到的定量预测结果高度吻合。此外，我们还演示了一款类模拟浓度记录回路（analog-like concentration recording circuit），该回路可借助时序逻辑检测并报告一种生化信号的三类输入浓度区间。