Engineered Gene Circuits for Basic Science and Biotechnology

项目摘要 我们将继续设计，构建和表征遗传电路。我们将使用微型工具 在精确控制的环境条件下生长和观察单细胞和菌落，我们将 在肿瘤球体中检测工程菌株。我们将描述电路-主机相互作用 开发新的设计原则。在多个实验中表征细胞行为 平台将提供数学模型，用于确定关键设计特征， 然后将使用先前建立的技术进行严格测试。两个博士后，两个研究阶段 科学家和一名研究生研究员将与Hasty和Tsimring博士在多个方面合作 项目以综合的方式。我们的业绩证明我们有能力培训人员， 一种多学科的方法，导致了合成生物学的新工具，沿着增加了 一般理解基因和信号网络。 我们最近在动物模型中对细菌回路的表征突出了以下需要： 更好地了解工程细菌如何在肿瘤环境中发挥作用。因此，我们 具体目标侧重于开发小生态系统中的输送回路（目标1）， 目的2：研究工程菌在肿瘤球体中的作用，目的3：研究工程菌与宿主的相互作用。 我们的第一个目标是开发小型生态传递系统， 在肿瘤环境中。一个系统将产生规则的异相输送序列，而第二个系统将产生规则的异相输送序列。 将被设计用于混沌动力学，这可能有助于逃避肿瘤适应的治疗。我们 将开发计算模型，并通过实验量化电路在微环境中的行为 装置.虽然这些数学模型在预测种群动态方面是有效的， 当工程细菌在隔离中生长时，肿瘤的复杂环境并不代表肿瘤的生长环境。 我们现有理解的简单扩展。动物模型的实验周期太长， 这对于开发基于工程的电路设计方法是昂贵的。我们的第二个目标是 使用肿瘤球体平台开发工程细菌的数学建模， 存在于肿瘤中。我们将使用ndings来识别计算建模的基本修改。 最后，基因电路通常是用假设与宿主隔离的模型方程设计的。 第三个目标是将联合收割机集成电路-主机建模与高通量微处理器平台相结合 定量表征工程基因电路与其宿主之间的双向耦合。 我们将探索肿瘤中存在的环境成分的影响，并评估 回路基因组对肿瘤球状体裂解物的反应（来自Aim 2）。这个目标的目的是推断出 改进基因电路的设计-构建-测试-修复过程的基本原则。

Project Summary We will continue to design, construct and characterize genetic circuits. We will use micro uidic tools to grow and observe single cells and colonies in precisely controlled environmental conditions, and we will test the engineered bacterial strains in tumor spheroids. We will characterize circuit-host interactions and develop new design principles. The characterization of cellular behavior across multiple experimental platforms will inform mathematical models that will be used to identify key design characteristics, which will then be rigorously tested using previously established techniques. Two Postdocs, two Sta Research Scientists, and a Graduate Student Researcher will work with Drs. Hasty and Tsimring on multiple aspects of the project in an integrated manner. Our track record demonstrates our ability to train personnel in a multi-disciplinary approach that has led to new tools for synthetic biology, along with an increased understanding of gene and signaling networks generally. Our recent characterization of bacterial circuits in animal models has served to highlight the need for a better understanding of how engineered bacteria function in a tumor environment. Accordingly, our Speci c Aims focus on the development of delivery circuits in small ecologies (Aim 1), the characterization of engineered bacteria in tumor spheroids (Aim 2), and the interaction of circuits with their hosts (Aim 3). Our rst aim is to develop small ecological delivery systems consisting of bacterial strains that can be found in the tumor environment. One system will generate regular out-of-phase delivery sequences, while a second will be designed for chaotic dynamics that could be useful for therapies that evade tumor adaptation. We will develop computational models and experimentally quantify how the circuits behave in micro uidic devices. While such mathematical models are generally e ective in predicting the population dynamics of engineered bacteria when grown in isolation, the complex environment of a tumor does not represent a simple extension of our existing understanding. The experimental cycle for animal models is too long and costly for the development of an engineering-based approach to circuit design. Our second aim will be to use a tumor spheroid platform for the development of mathematical modeling for engineered bacteria that reside in tumors. We will use the ndings to identify essential modi cations to the computational modeling. Finally, gene circuits are typically engineered with model equations that assume isolation from the host. The third aim will combine integrative circuit-host modeling with a high-throughput micro uidic platform to quantitatively characterize the bidirectional coupling between engineered gene circuits and their hosts. We will explore the e ects of environmental constituents that are present in sold tumors and evaluate the circuit-genome response to tumor spheroid lysate (from Aim 2). The goal of this aim is to deduce fundamental principles that improve the design-build-test-re ne process for gene circuits.