RoL: EAGER: DESYN-C3 Membraneless organelles by design: a biomimetic approach

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The cell has to organize a large number of reactions. It accomplishes this by isolating certain reactions and molecules inside of compartments known as organelles. Sometimes these organelles exist within membranes. Sometimes they exist simply as a distinct liquid phase. Controlling organelle formation and composition is central to creating a synthetic cell. Synthetic cells could be used in the biomanufacturing of chemicals, biofuels, and therapeutics, among potential applications. This project will investigate whether synthetic organelles without membranes can house complex reaction sequences. High school students will be involved in this project directly, through hands-on training in synthetic biology and participation in the international genetically engineered machine (iGEM) competition. Their challenge will be to develop protein tags that can trigger formation of the liquid phase organelles. They will also share their experiences on a blog. This will hopefully increase their enthusiasm and interest in pursuing careers in STEM-related fields.Liquid-liquid phase separation (LLPS) of proteins is a fundamental process in living cells. It underpins the formation of functional, non-membrane bound, liquid-like compartments involved in cell function and development. Examples include the nucleolus, Cajal bodies, nuclear speckles, germ granules, processing bodies, stress granules, and cell signaling compartments. These membraneless organelles can separate and concentrate specific sets of molecules in a highly dynamic manner. They also exchange components with their microenvironments rapidly. Within the liquid droplet, proteins maintain or acquire a folded structure, and conserve functions such as specific protein recognition. Increasingly, these structures are being implicated in diseases caused by protein aggregation. In this project, we will test whether LLPS can be exploited to enhance catalysis. An organelle that brings together elements of an enzyme cascade will be constructed and evaluated. These membraneless organelles will be evaluated by expressing the modular proteins in vivo and visualizing the formation of LLPS droplets by microscopy. Further, we will extend this approach to enable enzymatic cascades that include artificial metalloenzymes. If successful, this proposal will establish an innovative, simple method to generate organelles for synthetic biology, complementing existing methods such as encapsulation by protein shells or by vesicles.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.