RoL: RAISE: DESYN-C3: Membrane-based cell-free systems for scalable production of plant natural products

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Making something that works signifies that one understands how it works at a fundamental level and that one can add features to study their effects upon performance. For example, to a working airplane model one can add a curve to the wings, up-notched wingtips, and a whole host of modifications to see how they affect the performance of the plane. The same design paradigm holds true in biology. If one can make a cell from scratch that works, then one can start adding pieces to it and engineering functions into the system. This research project aims to create a synthetic cell system that mimics the membrane of a plant cell. Once a functioning system is achieved, different proteins that normally are found in plant cell membranes will be added to the synthetic cell system to determine what functions they display. The researchers will primarily focus on model set of proteins that produce an Alzheimer's drug. To further enhance the impact of the research project, the researchers will also develop new educational material about synthetic cells, train graduate and undergraduate students, and perform outreach to the general public to generate and further understanding about the idea of synthetic cells and their potential uses. To date, it has not been possible to create self-assembling, membrane-based cell-free systems. The primary hypothesis of this research project is that success can be achieved through regulated control over the timing and expression levels of enzymes for phospholipid vesicle generation. To achieve this goal, the researchers will integrate a CRISPR-based genetic control system with phospholipid production pathways. Specifically, they will construct a highly-efficient membrane-based gene expression system for plant transmembrane proteins. The intended plant natural product to be synthesized is the Alzheimer's drug, Galantamine. If successful in producing Galantamine, the researchers will expand the system to a variety of other transmembrane proteins, emulating plant cell environments. As a result, this research project will expand cell-free bioengineering capabilities and will provide new methods to prepare synthetic cell systems with complex membrane compositions. These systems will enable scientists to better understand the relationship between membrane biophysics and enzymatic activity.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.