Engineered gene circuits for basic science and biotechnology

用于基础科学和生物技术的工程基因电路

• 批准号: 9177553
• 负责人: Tal Danino
• 金额: $ 60.45万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9177553
• 关键词: Acute; Address; Animal Experiments; Animal Model; Animals; Antibiotics; Back; Bacteria; Bacterial Luciferases; Basic Science; Biotechnology; Cell Line; Cells; Cessation of life; Characteristics; Cloning; Colorectal Cancer; Computer Simulation; Custom; Cytolysis; DNA; Devices; Disease; Ecology; Effectiveness; Engineered Gene; Engineering; Environment; Enzymes; Evolution; Genes; Genetic; Genome; Genomics; Goals; Growth; Health; Human; Human Resources; Human body; Immunocompetent; In Vitro; Knowledge; Lead; Left; Libraries; Life; Measures; Microbe; Modeling; Molecular Biology; Molecular Biology Techniques; Monitor; Mus; Mutation; Optics; Plasmids; Population; Population Density; Population Dynamics; Population Growth; Postdoctoral Fellow; Prevalence; Production; Safety; Signal Transduction; Site; Solid Neoplasm; System; Technology; Testing; Therapeutic; Training; Weight; Work; base; cost; density; design; design and construction; graduate student; imaging system; in vivo; intercellular communication; killings; mathematical model; microbiota; model development; mouse model; novel therapeutics; pathogen; pressure; quorum sensing; response; safety engineering; safety testing; stability testing; subcutaneous; success; synthetic biology; technology development; time use; tool; tumor

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 a subset of the engineered bacterial strains as therapies in animal models. Single cell and colony dynamics will inform mathematical models that will be used to identify key design characteristics, which will then be rigorously tested using previously established molecular biology techniques. Eight graduate students and postdocs will work on multiple aspects of the project, while maintaining a particular focus on modeling or technology development for monitoring bacteria or in vivo characterization. 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 bene cial strains that are stable and safe over therapeutically relevant timescales. Accordingly, our Speci c Aims focus on stability (Aim 1), delivery (Aim 2), safety (Aim 3), and in vivo testing (Aim 4). Gene circuits inevitably generate mutations that are selected to decrease the additional burden created by the inserted genetic machinery. Our rst aim will develop strategies for extending the \lifetime" of gene circuits in bacteria before selective pressure disables their desired functionality. We will develop computational models and experimentally quantify how circuit redundancy increases circuit lifetime. We will use our experimental platform to monitor functionality across scales from single-cell to batch culture environments. Our second aim will primarily focus on engineering small bacterial ecologies. Here we will use modeling to guide the design of up to three interacting strains that can deliver therapies in a pre-determinted sequential order. In the third aim, we will build a safety circuit that triggers the death of all bacteria at a given threshold population density. The goal is to create an irreversible intracellular switch that rapidly and eciently kills all cells before mutations can compromise the safety strategy. In the nal aim, we will test the circuits designed in the rst three aims in animal models. We will engineer optical markers that enable characterization of the dynamics of bacterial colonies and tumor size in vivo. Importantly, the relative ease and low cost of bacterial cloning will inevitably lead to a bottleneck for the eld of Synthetic Biology, as therapeutic strains can be created at a rate that will far exceed the ability to test them. This highlights an acute need for quantitative models that have been thoroughly validated using in vitro technologies. Consequently, only a fraction of the circuits built in Aims 1-3 will be deemed worthy of in vivo testing. More generally, we anticipate that the computational models arising from these studies will be generally applicable across a wide range of emerging applications that employ bacteria.

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 在动物模型中测试作为疗法的工程细菌菌株的子集。单细胞和菌落 动力学将为数学模型提供信息，这些数学模型将用于确定关键的设计特性， 然后将使用先前建立的分子生物学技术进行严格测试。八毕业生 学生和博士后将致力于项目的多个方面，同时保持特别关注 用于监测细菌或体内表征的建模或技术开发。我们的记录 展示了我们以多学科方法培训人员的能力，这种方法导致了新的工具， 合成生物学，沿着对基因和信号网络的理解。 我们最近在动物模型中对细菌回路的表征突出了 在治疗相关的时间尺度上稳定和安全的贝内菌株。因此，我们的 具体目标侧重于稳定性（目标1）、输送（目标2）、安全性（目标3）和体内试验（目标4）。 基因回路不可避免地产生突变，这些突变被选择来减少所产生的额外负担。 被插入的基因机器所控制我们的第一个目标是制定战略， 在选择性压力使其所需功能失效之前，细菌中的基因电路。我们将开发 计算模型和实验量化电路冗余如何增加电路寿命。我们 将使用我们的实验平台来监测从单细胞到批量培养的各种规模的功能 环境.我们的第二个目标将主要集中在工程小细菌生态。这里我们 将使用建模来指导多达三种相互作用的菌株的设计，这些菌株可以在一个 预定的顺序。在第三个目标中，我们将建立一个安全电路， 在给定的阈值种群密度下所有细菌的数量。目标是在细胞内创造一个不可逆的 在突变可能危及安全策略之前，快速而有效地杀死所有细胞的开关。在 最后，我们将在动物模型中测试前三个目标中设计的电路。我们将工程师 光学标记物能够表征体内细菌菌落和肿瘤大小的动态。 重要的是，细菌克隆的相对容易和低成本将不可避免地导致细菌克隆的瓶颈。 合成生物学领域，因为治疗菌株可以以远远超过合成生物学能力的速度产生。 来测试它们这凸显了对经过彻底验证的定量模型的迫切需求 使用体外技术。因此，只有一小部分目标1-3中的电路将被视为 值得在体内测试。更一般地说，我们预计，计算模型产生于这些 研究将普遍适用于使用细菌的广泛的新兴应用。