CBET-EPSRC: Developing standardized cell-free systems for prototyping complex synthetic biology circuits and pathways

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This research was funded in response to the NSF Engineering - UKRI Engineering and Physical Sciences Research Council Opportunity NSF 18-067. The capability exists to design and implement functioning circuits and reaction pathways inside cells. This project makes use of cell-free systems to overcome some of the difficulties associated with these activities in living cells. The necessity to test performance inside cells limits the possible pathways to those that are not toxic to the host cell. Unfortunately, the performance of cell-free systems has proven to be erratic, exhibiting poor reliability. This project will establish a reliable, reproducible, cell-free platform. The design-build-test cycle could be shortened dramatically if it could be accomplished in a cell-free environment. This would have a beneficial impact on scientific research, engineered devices, and STEM education. As a collaboration with Imperial College London, this project will also promote research exchanges, providing US students with international research experiences. The primary goal of this project is to standardize cell-free systems from engineered E. coli and other organisms. Such systems will be used to explore the boundaries of cell-free prototyping and enable more detailed understanding of key mechanisms. This could accelerate the usage and broader utility of cell-free systems in industry and academia. The long term vision is to establish cell-free systems as a platform for implementation of synthetic biological circuits, pathways, and systems, where modular and complex biomolecular systems can be engineered in a systematic fashion. This project seeks to overcome some of the current limitations of cell-free systems through a combination of experimental characterization and computational modeling. If successful, this project will expand the utility of cell-free systems to a broader class of circuits and pathways. The specific objectives of the project are (1) the development of well-understood cell-free reaction systems suitable for prototyping circuits and pathways for a variety of cells; (2) characterization and modeling of complex synthetic biology components and core mechanisms using cell-free extracts; and (3) development of new biochemical indicator modules for monitoring and characterizing circuit performance.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.