2026 Theses Doctoral

Engineering Functional Biomolecular Condensates Driven by Gene Expression Across Bacterial, Reconstituted, and Cell-Free Systems

Liao, Jane

Biomolecular condensates are membraneless compartments that concentrate biomolecules like protein and RNA and play essential roles in cellular organization and gene regulation. Many condensates form through associative phase separation of oppositely charged biomacromolecules, and concepts from polymer physics have proven useful for understanding the parameters governing their assembly. Properties such as net charge, charge patterning, and macromolecular concentration have emerged as key determinants of condensate formation, composition, and dynamics. Beyond equilibrium principles, a central challenge is to understand how active processes such as gene expression both drive and respond to phase separation in cells.

In this dissertation, we aim to understand the determinants, dynamics, and functions of biomolecular condensates by engineering synthetic condensates across bacterial, reconstituted, and cell-free systems. Specifically, we engineer electrostatic interactions between cationic protein scaffolds and anionic RNA to drive condensate formation and interrogate their dynamics and functions. In Chapter 2, we express a library of supercharged GFP variants fused to disordered cationic peptides in E. coli and demonstrate that condensate formation requires a net charge threshold of approximately +6, with charge patterning and intracellular protein concentration together affecting condensate size, partitioning, and reversibility over time. Modular cationic peptide tags further enable co-recruitment of multiple proteins into engineered condensates with tunable composition.

Building on these design rules, we next ask how RNA transcription can drive condensate dynamics in a minimal reconstituted system. In Chapter 3, we reconstitute the dynamics of active transcription-driven condensates in vitro using RNA polymerase to drive phase separation with a cationic protein scaffold. We demonstrate that RNA flux governs condensate nucleation, growth, and dissolution along defined paths through the phase diagram. These functional active condensates exhibit rapid nucleation kinetics distinct from passive coarsening, regulate transcription through RNA-mediated positive and negative feedback, and can be cyclically renucleated by enzymatic RNA degradation.

In Chapter 4, we extend this framework to a coupled transcription and translation-driven system in which we develop a minimal defined buffer for cell-free protein synthesis, identify competition between transcription and translation as a key constraint on protein yield, and demonstrate that cationic scaffold proteins can undergo electrostatically driven phase separation with total cellular RNA in the complex mixture of cell lysate. Together, this work across different model systems establishes a framework connecting net charge, biomolecular concentration, and gene expression reactions to condensate properties, and lays the groundwork for understanding the role of biomolecular condensates in gene expression.