(556d) Programmable Control of Protein Activity Via Reversible Formation of Biomolecular Condensates in Bacteria

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 phenomena 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 and characterized its dynamics in cell-free systems. 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 an order of magnitude reduction in GFP expression relative to unfused transcription factor controls at elevated temperatures. More importantly, the threshold for coacervate formation and control of protein activity may be tuned through appropriate design of the ELP fusion partner. We also examine the reversibility and dynamic response of these methods. 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.