Generation of a large family of genetic logic gates for applications in biosensing and information processing

电子逻辑门是我们所依赖的所有数字设备（如计算机和智能手机）的基石。单个逻辑门接受一个或多个数字（ON或OFF）输入，执行逻辑运算并产生单个数字输出。例如，与门的输出只有在其两个输入都为ON时才为ON，而或门的输出只有在其中一个（或两个）输入都为ON时才为ON。电子逻辑门可以连接成电路，可以存储数据，加减数字，计数，并进行任何其他逻辑或数学计算。典型的现代数字计算机的核心是微处理器，它有数千万个逻辑门，所有这些逻辑门都连接在一起，形成复杂的电路。研究简单细菌细胞的科学家们已经利用它们创建了可以复制单个逻辑门行为的遗传网络。就像它们的电子对应物一样，这些生物逻辑门可以接受一个或多个输入，执行逻辑操作并提供输出。例如，细菌与门被设计成从细菌细胞中检测环境中不同糖的两个天然传感器获取输入。逻辑门将这两种输入结合起来，只有在两种糖都存在的情况下，才会以荧光蛋白的形式产生输出。希望有一天，这些生物逻辑门可以被设计成更复杂的电路，可以执行多种逻辑操作，存储信息，做出复杂的预编程决策，并有多种输出。例如，可以设计一个细胞来检测污染物，并采取行动来中和存在的污染物的确切混合物，或者经历一系列复杂的代谢步骤，将农业废物转化为有价值的化学原料或制药产品。每个生物逻辑门都必须由转录因子和启动子等遗传成分单独构建。转录因子和启动子被细胞用来控制自身的基因表达，以响应其环境。为了确保各个逻辑门在具有多个逻辑门的电路中相互独立地工作，必须为每个逻辑门使用不同的转录因子。到目前为止，转录因子和启动子必须从自然系统中获取并逐一表征，缺乏大量不同的、具有良好表征的转录因子严重限制了可以构建的数字遗传电路的复杂性。在这里提出的研究中，我们将开发一种新的方法来产生大量互不干扰的转录因子和启动子。与迄今为止用于构建遗传逻辑门的其他转录因子不同，这些转录因子不会通过改变其细菌宿主的基因表达而引起不必要的副作用。因此，我们生成的转录因子集将是构建包含多个逻辑门的大型遗传电路的理想选择，以执行复杂的数据处理和存储操作。生物逻辑电路在现场检测污染物、有毒物质、病原体、水污染物、爆炸物等方面的应用将取决于其输出的小型化可靠探测器。我们将开发一种微型设备来检测作为细菌遗传逻辑电路输出的荧光蛋白，并将使用由我们的新转录因子构建的遗传电路来测试该设备。这将为便携式、易于使用的生物传感器和处理器的发展铺平道路。

Electronic logic gates are the building blocks of all the digital devices, such as computers and smart phones, on which we have come to rely. Individual logic gates take one or more digital (ON or OFF) inputs, perform a logical operation and produce a single digital output. For example, the output of an AND gate is ON only if both of its inputs are ON, whereas the output of an OR gate is ON if either (or both) of its inputs is ON. Electronic logic gates can be connected together into circuits that can store data, add and subtract numbers, count, and carry out any other logical or mathematical calculation. The microprocessor at the heart of a typical modern digital computer has tens of millions of logic gates, all connected together into complex circuits.Scientists working on simple bacterial cells have used them to create genetic networks that can reproduce the behaviour of individual logic gates. Just like their electronic counterparts, these biological logic gates can take one or more inputs, perform a logical operation and provide an output. For example, a bacterial AND gate has been engineered to take its input from two natural sensors in the bacterial cell that detect different sugars in the environment. The logic gate combines these two inputs and produces an output, in the form of a fluorescent protein, only when both sugars are present. The hope is that one day these biological logic gates can be engineered into more complex circuits that can perform multiple logical operations, store information, make complex preprogrammed decisions, and have multiple outputs. For instance, a cell could be engineered to detect pollutants and to take action to neutralize the exact cocktail of pollutants present, or to undergo a complex series of metabolic steps to turn agricultural waste into valuable chemical feedstocks or pharmaceutical products.Each biological logic gate has to be individually built from genetic components known as transcription factors and promoters. Transcription factors and promoters are used by cells to control their own gene expression in response to their environment. To ensure that individual logic gates operate independently of each other in a circuit with multiple logic gates, a different transcription factor must be used for each logic gate. Up until now, transcription factors and promoters had to be sourced from natural systems and characterised one by one, and the lack of a large number of different, well characterised transcription factors has severely limited the complexity of digital genetic circuits that can be built.In the research proposed here, we will develop a new way to produce a large set of transcription factors and promoters that do not interfere with each other's action. Unlike other transcription factors that have been used to build genetic logic gates to date, these transcription factors will not cause unwanted side effects by altering gene expression in their bacterial host. Therefore, the set of transcription factors we generate will be ideal for building large genetic circuits, containing multiple logic gates, to carry out complex data processing and storage operations.Application of biological logic circuits for in-the-field detection of pollutants, toxic agents, pathogens, water contaminants, explosives etc. will depend on miniaturised reliable detectors of their outputs. We will develop a miniature device to detect fluorescent proteins produced as outputs of bacterial genetic logic circuits, and will test this device using a genetic circuit built from our new transcription factors. This should pave the way towards the development of portable, easy to use biological sensors and processors for a whole host of applications.