CAREER: Complex Coacervation in Cells

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Non-technical AbstractCompartmentalization allows incompatible processes to occur simultaneously and can improve efficiency. For example, having separate washer and dryer appliances allows clothes to be cleaned and dried simultaneously. While all-in-one washer/dryer appliances exist, they are less efficient, limiting the overall amount of laundry that can be done at one time. Cells use this same compartmentalization strategy to facilitate the hundreds of different processes that must take place concurrently to maintain life. However, cells must also be able to adapt to changing environments and growth conditions. To address this challenge, cells have certain compartments that responsively appear and disappear. These dynamic compartments form via a phase transition of cell contents - a process akin to liquid water freezing to form solid ice cubes. This project will engineer these dynamic compartments from the ground-up. Using inspiration from biological systems and guidance from polymer science, new dynamic compartments will be built in bacteria. A library of new biomaterials will be prepared and their phase transitions ("water freezing") will be evaluated in test tubes and living cells. These efforts will provide a fundamental understanding of how cells form these responsive compartments and will enable us to efficiently engineer new processes in cells. This project also aims to educate and train a diverse set of students for careers in science and engineering. To do this, tutoring in STEM and a short course on the "Art of Engineering" will be developed for local incarcerated students.Technical AbstractCells organize their contents from the molecular to the sub-cellular scale. The liquid-liquid phase separation of biomacromolecules has recently been appreciated as a mechanism for cellular subcompartmentalization. These phase separated compartments are termed membraneless organelles or biomolecular condensates. This biological phase separation is driven by weak, multivalent interactions and shares many features with the well-studied phase separation of synthetic macromolecules (polymers). In particular, many membraneless organelles form via complex coacervation of nucleic acids and proteins - or the liquid-liquid phase separation of oppositely charged polyelectrolytes. The goal of this project is to use the process of complex coacervation to artificially promote globular protein phase separation in E. coli. The design parameters for the in vivo formation of both single and multi-protein condensates will be determined. Model fluorescent proteins will be engineered with altered charge and charge distribution to create a library of supercharged protein polyions. The phase behavior of these engineered proteins with oppositely charged biomolecules (nucleic acids, proteins) will be characterized in vitro and in vivo. The insights from these experiments will enable the creation of synthetic membraneless organelles with potential applications in metabolic engineering and synthetic biology. These research objectives will be coupled with educational goals to improve the scientific and engineering literacy of incarcerated students. Following STEM tutoring of students inside via existing outreach programs, a short course will be created on the "Art of Engineering" to introduce engineering concepts to these students and generate excitement about potential careers in STEM.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.