Engineered Gene Circuits for Basic Science and Biotechnology

项目摘要 我们将继续设计、建造和表征基因电路。我们将使用微电子工具 在精确控制的环境条件下生长和观察单个细胞和克隆，我们将 在肿瘤球体中检测工程菌菌株。我们将描述电路与主机之间的相互作用 并制定新的设计原则。跨多个实验的细胞行为的特征 平台将通知数学模型，这些模型将用于确定关键设计特征，其中 然后将使用先前建立的技术进行严格的测试。两个博士后，两个阶段研究 科学家和一名研究生研究人员将在多个方面与哈蒂博士和齐姆林博士合作 以一种综合的方式对项目进行管理。我们的记录表明我们有能力在以下方面培训人员 一种多学科的方法导致了合成生物学的新工具，以及更多的 对基因和信号网络的一般理解。 我们最近在动物模型中对细菌回路的表征强调了 更好地理解工程菌如何在肿瘤环境中发挥作用。因此，我们的 SPECI c的目标是发展小型生态系统中的传递回路(目标1)，其特征 肿瘤球体中工程化细菌的研究(目标2)，以及电路与宿主的相互作用(目标3)。 我们的首要目标是开发由可以找到的细菌菌株组成的小型生态递送系统。 在肿瘤环境中。一个系统将生成常规的异相传送序列，而另一个系统将生成常规的异相传送序列 将为混沌动力学而设计，这可能对逃避肿瘤适应的治疗有用。我们 将开发计算模型，并通过实验量化电路在微指令中的行为 设备。虽然这样的数学模型通常对预测人口动态是有效的 工程菌在隔离生长时，肿瘤的复杂环境并不代表 只是对我们现有理解的简单扩展。动物模型的实验周期太长， 以工程为基础的电路设计方法的开发成本高昂。我们的第二个目标是 使用肿瘤球体平台为工程菌开发数学模型， 栖息在肿瘤中。我们将使用NDINS来确定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来设计的。 第三个目标是将集成电路-主机建模与高通量微处理器平台相结合 定量表征基因工程电路与宿主之间的双向耦合。 我们将探索出售的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体裂解物的反应(摘自AIM 2)。这一目标的目的是演绎 改进基因电路设计-制造-测试过程的基本原则。

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.