Multiplexing Autonomous Metabolite Valves

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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.