BME PhD Defense Announcement for Mrugesh Krishna Parasa (Dr. Kevin Solomon, advisor) Everyone is invited to attend the public presentation beginning at 3:00 PM. Title of the Thesis Research: PROGRAMMABLE CONTROL OF PROTEIN ACTIVITY VIA FORMATION OF BIOMOLECULAR CONDENSATES IN BACTERIA Date: May 23, 2023 Time: 3:00 PM Place: https://purdue-edu.zoom.us/j/99880181010 Thesis Committee members: Dr. Kevin Solomon, Dr. Tamara Kinzer-Ursem, Dr. David Thompson, Dr. Leopold Green Abstract: Biomolecular condensates or membraneless organelles are phase separated proteins and/or other biomolecules that are ubiquitous in eukaryotic cells. While these condensates may be liquid with exchange and diffusion of their components with the rest of the cell (e.g. cytoplasm), they locally concentrate their constituent biomolecules altering their interactions in normal cellular processes. Here, we exploit this phenomenon via reversible coacervate formation to control the activity of cellular proteins in E. coli. To induce liquid-liquid phase separation, we fuse proteins to elastin-like polypeptides (ELP) that reversibly aggregate in response to increases in temperature and/or changes in intracellular pH. In so doing, we sequester their fusion partners from the cytoplasm, limiting their ability to participate in cytoplasmic reactions. We have demonstrated this concept in vivo with enzymes and transcription factors for switchable control of protein activity with temperature. For example, I-SceI mediated cleavage of a host genome can be inhibited by increasing the cultivation temperature, creating a simple temperature-sensitive kill switch; accidental release will lower the culture temperature leading to cell death. Similarly, coupled transcription factors exhibit a 2-fold increase in transcription in response to temperature. More importantly, the threshold for coacervate formation and control of protein activity may be tuned through appropriate design of the ELP fusion partner. Our results introduce a simple yet effective, rapid and tunable approach to control protein activity via induction of coacervate formation that may form a powerful new tool for synthetic biology.