Development of bio-orthogonal synthetic organelles via engineered protein phase separation

Protein de-mixing has recently been implicated in the organization of cellular components. These phase separated membraneless organelles create distinct environments that are essential to cellular processes ranging from signaling to genome organization and gene expression. Despite numerous publications over the last decade investigating the structure and function of membraneless organelles, our understanding of this nascent field is limited; consequently, engineering the a priori formation and dissolution of membraneless organelles in vivo remains a challenge. The proposed research will explore the use of electrostatic interactions to drive protein phase separation in E. coli in order to begin to develop design rules for bio-orthogonal membraneless organelles. The proposed work will focus on developing ionic polypeptide tags that promote the intracellular phase separation of proteins of interest. The project will primarily focus on engineering phase separation in E. coli, but will also explore if these results can be translated to the model unicellular eukaryote, S. cerevisiae. The complex coacervation of multiple proteins with RNA will be evaluated in vitro at physiological conditions. Phase separation will be monitored using turbidimetric titrations and the formation of a liquid coacervate phase will be determined by optical microscopy. Following in vitro characterization, the intracellular phase behavior will be monitored by fluorescence microscopy. Image analysis using MicrobeJ and custom scripts will be implemented to quantitatively analyze altered protein distribution profiles. Additionally, the ionic tags are expected to minimally impact the activity of the engineered proteins of interest. The relative brightness of the engineered fluorescent proteins will be used as an indicator of how the ionic tags influence “activity” as compared to proteins with equivalent charge distributed on the protein surface.