Expanding the utility of transcriptional bacterial computing

对于许多胃肠道疾病，一种有前途的新治疗概念使用工程微生物， 原位合成治疗剂。为了实现这种方法，合成必须是可靠的，可控的， 可调节。对于工程菌，这可以通过基因调控蛋白质的表达来实现 通过由配体诱导型转录因子控制的启动子来产生治疗剂所需要的。许多 天然转录因子的特征在于，它们通过广泛的免疫反应来响应它们的外部环境。 一系列小分子信号。然而，大多数合成基因电路都是由一个 几个组件。为了扩展这些能力，我们需要新的方法来构建、调整和控制 转录调控网络理想情况下，这些方法将提供“即插即用”的组件， 用于构建复杂的电路，并将对患者饮食中的信号分子做出反应。 为此，我们将构建和整合两个互补文库，包括：（1）工程化的 响应于大范围的可食用化学诱导物的转录因子;和（2）高响应性和 受工程化转录因子调控的可调启动子。我们的关键创新在于 其中我们使用了嵌合转录因子。这些模块化蛋白各自含有配体结合蛋白， DNA结合域（DBD）和DNA结合域（LBD），可以混合和匹配以产生新的功能。 值得注意的是，具有相同DBD的嵌合体将独立地且同时地通过转录调节子调控相同的启动子。 单个操作员站点。以这种方式，双输入逻辑门控由组合两个嵌合体产生，所述嵌合体响应于 不同的配体;多输入门控源于同时使用三个或更多个嵌合体。 在目标1中，我们将从嵌合转录阻遏物中设计AND门，从嵌合转录阻遏物中设计OR门。 嵌合转录激活因子。我们的初步数据表明，至少有四种嵌合阻遏物可以共同作用， 表示以创建可预测的与门。额外的蛋白质工程将用于调整配体 生理反应所需的浓度。在目标2中，我们将整合嵌合转录 因子与新的杂合启动子工程改造具有可调的-10和-35位点。这将允许我们指定 启动子的泄漏（在没有诱导物的情况下）和其最大输出（当完全诱导时）。我们 将使用这些电路的输出来开发一个数学模型， 未来基于Chimera的电路的性能。最后，在目标3中，我们将测试我们的假设， 转录因子可用于可靠地增加合成基因回路的复杂性和可调性。到 最后，我们将构建原理验证系统，该系统使用嵌合转录因子来偶联各种 电路模块，如脉冲发生器和遗传振荡器。总的来说，拟议的工作将大大 扩展可用于工程复杂合成基因电路的组件，用于治疗 微生物对患者饮食中的化学信号做出反应。

For many gastrointestinal diseases, a promising new treatment concept uses engineered microbes to synthesize therapeutics in situ. To implement this approach, synthesis must be reliable, controllable, and adjustable. With engineered bacteria, this can be accomplished by genetically regulating expression of proteins required to create the therapeutic via promoters controlled by ligand-inducible transcription factors. Many natural transcription factors have been characterized that respond to their external environments via a wide range of small molecule signals. However, the majority of synthetic gene circuits have been built with just a handful of components. To expand these capabilities, we need new ways to construct, tune, and control transcriptional regulatory networks. Ideally, these methods will provide “plug-and-play” components that can be used to build complex circuits, and will respond to signaling molecules incorporated into the patient's diet. To that end we will construct and integrate two complementary libraries, comprising: (1) engineered transcription factors that respond to a large range of edible chemical inducers; and (2) highly responsive and tunable promoters that are regulated by the engineered transcription factors. Our key innovation is the manner in which we employ chimeric transcription factors. These modular proteins each contain a ligand-binding domain (LBD) and a DNA-binding domain (DBD) that can be mixed and matched to create new functionalities. Notably, chimeras with the same DBD will independently and simultaneously regulate the same promoter via a single operator site. In this manner, two-input logic gating arises from combining two chimeras that respond to different ligands; multi-input gating arises from the simultaneous use of three or more chimeras. In Aim 1, we will engineer AND gates from chimeric transcription repressors and OR gates from chimeric transcription activators. Our preliminary data show that at least four chimeric repressors can be co- expressed to create a predictable AND gate. Additional protein engineering will be used to tune the ligand concentration that is required for physiological response. In Aim 2, we will integrate the chimeric transcription factors with novel hybrid promoters engineered to have tunable -10 and -35 sites. This will allow us to specify both the leakiness of a promoter (in the absence of inducer) and its maximal output (when fully induced). We will use the output from these circuits to develop a mathematical model that will predict the capacity and performance of future chimera-based circuits. Finally, in Aim 3, we will test our hypothesis that chimeric transcription factors can be used to reliably increase the complexity and tunability of synthetic gene circuits. To that end, we will construct proof-of-principle systems that use chimeric transcription factors to couple various circuit modules, such as pulse generators and genetic oscillators. Overall, the proposed work will greatly expand the components available for engineering complex synthetic gene circuits for use in therapeutic microbes that respond to chemical signals incorporated into patient diet.