Multiplexing Autonomous Metabolite Valves

生物技术有望提供许多社会需要的东西，如药品、材料和可再生资源的燃料。该项目旨在通过提供使微生物生物生产更有效的工具来加速这些努力。微生物和其他生物系统自然产生各种各样的化合物，尽管并非所有化合物都对人类的需要有用。这些化合物中的许多是微生物生长所必需的，它们的产生减少了目标分子的合成。在这个项目中开发的工具将允许动态控制对生长至关重要的途径，并导致目标分子。这些控制机制不需要人对系统进行任何改变来切换路径功能，因此系统将自动运行。这项研究将显著提高动态控制代谢途径的能力。除了科学进步之外，该项目还将促进一名研究生和至少一名本科生的博士学习。PI还将与K-12学生、麻省理工学院夏季研究项目的本科生、“教授之路”项目的女研究生和博士后开展外展活动。该项目的目标是开发多重代谢物阀，使独立控制细胞中的两个或多个基因，而不需要外源诱导剂。耦合和独立的群体感应系统将用于构建电路。多路复用电路最初将使用荧光报告器进行表征，并且将开发能够实现OFF-to-ON和ON-to-OFF开关的配置。已经确定了验证方法的两个目标路径，提供了必要的测试用例来演示构建的设备的广泛效用。最后，这些电路将与CRISPRi驱动相连接，以实现更高层次的控制。这些阀门将有助于在代谢工程和合成生物学的交叉努力的进步。该设备工具包将(i)在微生物培养中使用的常见变量方面具有良好的特征，（ii）可在许多途径中推广。独立和自主控制两个或多个基因的阀门的发展将代表代谢工程合成基因电路设计的一个新的复杂性水平。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Biotechnology promises to provide access to many of the things that society needs, such as medicines, materials and fuels from renewable resources. This project aims to accelerate these efforts by providing tools to make biological production from microbes more efficient. Microbes and other biological systems naturally produce a wide range of chemical compounds, though not all are useful for human needs. Many of these compounds are essential for microbial growth, and their production decreases synthesis of target molecules of interest. The tools developed in this project will allow for the dynamic control of pathways that are essential for growth and that lead to target molecules. These control mechanisms will not require a person to make any alterations to the system to switch pathway function, so the system will operate autonomously. This research will significantly advance the ability to dynamically control metabolic pathways. In addition to the scientific advancements, this project will facilitate training of one graduate student through the end of her doctoral studies along with at least one undergraduate student. The PI will also engage in outreach activities with K-12 students, undergraduates in summer research programs in residence at MIT, and female graduate students and post-docs in the "Path of Professorship" program. The goal of this project is to develop multiplexed Metabolite Valves to enable the independent control of two or more genes in a cell without the need for exogenous inducers. Coupled and independent quorum-sensing systems will be used to construct the circuits. Multiplexed circuits will initially be characterized using fluorescent reporters, and configurations will be developed that enable both OFF-to-ON and ON-to-OFF switching. Two target pathways have been identified for validating the approach, providing the test cases necessary to demonstrate broad utility of the constructed devices. Finally, the circuits will be interfaced with CRISPRi actuation to achieve an even higher order of control. These valves will contribute to the advancement of efforts at the intersection of metabolic engineering and synthetic biology. The device toolkit will be (i) well-characterized with respect to common variables employed in microbial cultivation and (ii) generalizable across many pathways. The development of valves to both independently and autonomously control two or more genes will represent a new level of complexity in the design of synthetic gene circuits for metabolic engineering.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.