Abstract
Intrinsically disordered proteins (IDPs) lack a fixed structure and play important roles in organizing cellular biochemistry. Many membraneless organelles, such as nucleoli and RNA granules, arise via liquid-liquid phase separation (LLPS) of IDPs or intrinsically disordered regions (IDRs) within the proteins. These dynamic condensates can selectively concentrate specific biomolecules, enabling spatial control of processes like gene expression, signaling, and stress responses. Based on this distinct biochemical behavior, recent research has begun to engineer IDP-based systems that mimic and extend existing compartmentalization strategies to regulate the intracellular environments. Apart from this, synthetic biologists also explored the molecular grammar of IDPs and created synthetic IDPs (synIDPs) to precisely program the cellular functions. However, limited understanding of how molecular grammar and sequence-dependent interaction cooperativity relate to the functional impacts of synIDPs and synthetic condensates on endogenous cellular processes constrain the design space of this powerful capability. In this thesis, I first developed a platform that evolves synIDPs with desired properties, such as concentration- or temperature-dependent phase transition behaviors. Next, I applied the evolved synIDPs from the first part to demonstrate their applicability in synthetic biology. Specifically, these evolved IDPs were used as modular genetic parts to enhance protein solubility and reversibly control the ampicillin resistance of E. coli. Finally, based on the same evolutionary platform, I engineered binders derived from native sequences of natural condensate-forming proteins, such as FUS and TDP-43. These formation of these intracellular condensates was effectively inhibited by fusing the evolved binders with soluble IDPs. Altogether, these tools expand our understanding of IDP molecular grammar and enable precise engineering of cellular behaviors using evolved IDPs.
Committee Chair
Yifan Dai
Committee Members
Jianmin Cui; Joshua Yuan; Song Hu; Yinjie Tang
Degree
Doctor of Philosophy (PhD)
Author's Department
Energy, Environmental & Chemical Engineering
Document Type
Dissertation
Date of Award
12-9-2025
Language
English (en)
DOI
https://doi.org/10.7936/abb3-7x85
Recommended Citation
Ma, Yuefeng, "Biomolecular Engineering of Intrinsically Disordered Proteins" (2025). McKelvey School of Engineering Theses & Dissertations. 1314.
The definitive version is available at https://doi.org/10.7936/abb3-7x85