2024 AIChE Annual Meeting
Using Supercharged Dark GFP As a Tool to Engineer Protein Translation Droplets
Cells, as the most basic units of life, have evolved to be self-sustaining systems composed of thousands of biomolecules and numerous biochemical reactions. The depth of our understanding of these living systems is reflected in our ability to engineer similar life-like properties in synthetic cells. An important challenge of engineering artificial cells lies in replicating the complex spatiotemporal organization and compartmentalization of cells while integrating key functions. Much of the research in this area has focused on using membrane-bound microcompartments such as liposomes, polymersomes, and proteinosomes. However, this fails to emulate the crowded, molecularly dense milieu of cells, which is essential for many biological functions. To address this, we explored complex coacervation as an alternative, membrane-free compartmentalization approach to mimic the crowded, viscoelastic, and highly charged nature of cellular cytoplasm, using bacteria as an example. Previously, our group overexpressed a panel of globular cationic proteins in E. coli cells and found that the protein net charge was a key determinant of phase separation. Here we report the use of E. coli lysate as a model in vitro system. Using this cell free model, we have characterized the phase separation of endogenous biomolecules with engineered cationic scaffold proteins. Specifically, starting from superfolder GFP(+12) as a model scaffold protein, we mutated the chromophore (T65G, Y66G) to engineer a “DarkGFP” that exhibited the same structure and phase separation behavior but lacked fluorescence. As expected, in subsequent phase separation assays DarkGFP formed coacervates, but also reduced the spectral overlap between the scaffold protein and other fluorophores in the system, including an mScarlet-tagged ribosomal protein in the cell lysate. Overall, both in-cell and in vitro experiments showed that phase separation is primarily driven by heterotypic interactions between anionic RNA and cationic scaffold proteins. Notably, the cationic scaffold protein significantly concentrated macromolecules necessary for gene expression such as RNA and the large ribosomal subunit protein, bL9. Future work will focus on replicating life-like biochemical processes such as cell-free gene expression and understanding phase separation dynamics in response to internal and external perturbations.