1] Department of Bioengineering, University of California, San Diego, La Jolla, California 92093, USA [2].
Department of Bioengineering, University of California, San Diego, La Jolla, California 92093, USA.
Nature. 2014 Apr 17;508(7496):387-91. doi: 10.1038/nature13238. Epub 2014 Apr 9.
One promise of synthetic biology is the creation of genetic circuitry that enables the execution of logical programming in living cells. Such 'wet programming' is positioned to transform a wide and diverse swathe of biotechnology ranging from therapeutics and diagnostics to water treatment strategies. Although progress in the development of a library of genetic modules continues apace, a major challenge for their integration into larger circuits is the generation of sufficiently fast and precise communication between modules. An attractive approach is to integrate engineered circuits with host processes that facilitate robust cellular signalling. In this context, recent studies have demonstrated that bacterial protein degradation can trigger a precise response to stress by overloading a limited supply of intracellular proteases. Here we use protease competition to engineer rapid and tunable coupling of genetic circuits across multiple spatial and temporal scales. We characterize coupling delay times that are more than an order of magnitude faster than standard transcription-factor-based coupling methods (less than 1 min compared with ∼20-40 min) and demonstrate tunability through manipulation of the linker between the protein and its degradation tag. We use this mechanism as a platform to couple genetic clocks at the intracellular and colony level, then synchronize the multi-colony dynamics to reduce variability in both clocks. We show how the coupled clock network can be used to encode independent environmental inputs into a single time series output, thus enabling frequency multiplexing (information transmitted on a common channel by distinct frequencies) in a genetic circuit context. Our results establish a general framework for the rapid and tunable coupling of genetic circuits through the use of native 'queueing' processes such as competitive protein degradation.
合成生物学的一个承诺是创建能够在活细胞中执行逻辑编程的遗传电路。这种“湿编程”有望改变从治疗和诊断到水处理策略等广泛而多样的生物技术领域。尽管遗传模块库的开发取得了快速进展，但将它们集成到更大的电路中的主要挑战是在模块之间产生足够快和精确的通信。一种有吸引力的方法是将工程电路与宿主过程集成，以促进稳健的细胞信号传递。在这种情况下，最近的研究表明，细菌蛋白降解可以通过使有限的细胞内蛋白酶供应过载来触发对压力的精确响应。在这里，我们使用蛋白酶竞争来设计在多个时空尺度上快速且可调谐的遗传电路耦合。我们描述的耦合延迟时间比基于标准转录因子的耦合方法快一个数量级以上（不到 1 分钟，而标准方法约为 20-40 分钟），并且通过操纵蛋白质与其降解标记之间的连接体来实现可调谐性。我们使用这种机制作为平台来在细胞内和菌落水平上耦合遗传时钟，然后同步多菌落动力学以减少两个时钟的变异性。我们展示了如何将耦合时钟网络用于将独立的环境输入编码为单个时间序列输出，从而在遗传电路上下文中实现频率复用（通过不同频率在公共通道上传输的信息）。我们的结果通过使用竞争蛋白降解等本地“排队”过程为遗传电路的快速和可调谐耦合建立了一个通用框架。
