TL;DR
This chapter describes synthetic gene circuit-based methods for applying synthetic condensates in E. coli to regulate transcription and translation and demonstrates how engineering synthetic condensates offers a new layer of cellular control for synthetic biology.
Biomolecular condensates formed through phase separation have emerged as a central principle of cellular organization, enabling the dynamic regulation of gene expression, signaling, metabolism, and stress responses. While early conceptual advances in condensate biology have largely originated from animal and in vitro systems, plant cells present a unique set of biological and technical challenges, including rigid cell walls, turgor pressure, plastid autofluorescence, complex endomembrane organization, and acute environmental responsiveness. These distinctive features impede the direct transfer of existing methodologies and drive the development of heterogeneous experimental practices. In this community comment, we present a comprehensive methodological framework for studying biomolecular condensates in plants, spanning in silico prediction, in vitro reconstitution, molecular dynamics simulations, live-cell and super-resolution imaging, material property measurements, membrane-associated condensates, and synthetic condensate engineering. We highlight best practices, common pitfalls, and plant-specific considerations, emphasizing the need for orthogonal validation, quantitative interpretation, and physiological relevance. By consolidating current methodologies and articulating shared principles, this review aims to establish a foundation for rigorous, reproducible, and conceptually coherent research in condensate biology of plants and beyond, with emerging implications for crop genetic improvement and synthetic biology applications.
Liquid-liquid phase separation (LLPS), an interesting process in which a homogeneous solution demixes into solute-rich and solute-poor phases, serves as a ubiquitous mechanism for the formation of biomolecular condensates in living cells. Biomolecular condensates, also known as membraneless organelles, play critical roles in many fundamental physiological processes, including gene transcription, signal transduction, and stress responses. The intriguing LLPS phenomenon has inspired the development of artificial coacervates composed of biopolymers, synthetic macromolecules, or small molecules, driving exploration toward smart functional materials. For these synthetic coacervates, tailoring their structures and manipulating phase separation behaviors through external stimuli are essential to achieve precise functional control. This review summarizes recent advances in stimuli-responsive LLPS-based coacervates, focusing on structural design, formation mechanisms, stimuli-responsive characteristics, and functional applications. We thoroughly categorize the coacervates based on the diversity of stimulus sources used to regulate phase separation, with particular emphasis on the underlying molecular-level mechanisms. We highlight the applications of stimuli-responsive coacervates ranging from drug delivery and microreactors to biosensing. The prospects for stimuli-responsive LLPS systems for next-generation smart materials are also presented, aiming to elucidate their great potential in bridging the gap between the sophistication of biological coacervates and the performance of artificial smart materials.
Biomolecular self‐assembly is ubiquitous in nature, encompassing both ordered and disordered structures to create sophisticated superstructures essential for complex biological functions. Protein and peptide condensates formed via liquid–liquid phase separation (LLPS) are characterize by disordered assembly, gaining significant interest due to their crucial role in physiological events and potential applications from drug delivery to biosensing. Short peptides with ordered structures have been widely explored as building blocks for nanoarchitectured materials, but they lack the disordered features that endow biological systems with flexibility and adaptability. Here we introduce a minimalistic peptide sticker‐and‐spacer model that forms biomolecular condensates with core–shell structure through phase separation and spontaneous evaporation. The design allows to derive the guidelines for programming condensate's architecture from homogeneous to multiphasic state via the selection of sticker and spacer. Furthermore, we demonstrate control over compartmentalization driven by intrinsic redox chemistry and post‐assembly modification. The condensates efficiently encapsulate and protect small‐molecule payloads and function as microreactors. The evaporation‐induced spontaneous phase separation results in solidified condensates enriched with redox‐active tyrosine, which serve as novel nano‐bioreactors, promoting selective biomineralization and formation of uniform metal–peptide nanohybrids. Therefore, our study provides a framework for the artificial design of protocells mimetic multicompartmental condensates endowed with on‐demand functionality.
Biomolecular condensates (BMCs) organize cellular biochemistry by concentrating selected molecules into dynamic membrane-free compartments. Yet the molecular parameters that determine not only whether condensates form, but also how they behave and what they do, remain poorly defined. Here we show that scaffold binding affinity (Kd) is a quantitative determinant of condensate phase behavior, internal dynamics and biochemical output. Using a modular SUMO-SIM system in which scaffold valency was held constant while binding affinity was systematically varied, we found that affinity governs the phase boundary, resistance to chemical perturbation, and molecular mobility of condensates in vitro and in human cells. In multicomponent mixtures, the highest-affinity scaffold dominated dense-phase composition and dynamics, revealing a hierarchical rule for condensate organization. Finally, affinity-dependent changes in condensate dynamics translated into tunable enzyme activity, establishing binding energetics as an engineerable parameter for programming condensate biochemistry.
The formation of active and catalytic biomolecular condensates is critical to orchestrate intracellular biochemical reactions and cellular functions. Synthetic analogues that mimic such behaviors are typically constructed via liquid-liquid phase separation that generates liquid coacervate-based droplets, which promote reaction efficiency through molecular confinement. Such a type of confined reaction is usually achieved by partitioning and sequestrating active species such as biological enzymes or their mimics (such as metalloenzymes), which can be easily affected by encapsulation efficiency and sensitivity to the local environment. Engineering coacervate-based compartments that can display inherent catalytic functionality remains a significant challenge. Here, we report a bioinspired strategy to construct programmable peptide-based coacervates with inherent enzyme-like catalytic activity via the co-assembly of short peptides. We find that mixing the histidine-tagged short peptides with triphenylalanine-based peptides leads to the formation of a stable coacervate phase, in contrast to the rigid aggregates formed by each individual component. This cooperative assembly enables the generation of functional coacervate compartments with built-in catalytic capability. The resulting peptide coacervates exhibit selective partitioning and sequestration of hydrophobic substrates, thereby enhancing local substrate concentration and promoting catalytic hydrolysis reactions. Our results demonstrate that catalytic activity can be encoded directly into coacervate-forming building blocks through rational peptide design, providing a versatile platform for programming LLPS behavior and constructing biomimetic active materials with potential applications in synthetic biology.
Synthetic cells are compartments designed to mimic the functions and characteristics of living cells. By constructing synthetic cells from abiotic, basic components (bottom-up), it is possible to investigate the minimal requirements for life and gain insights into fundamental principles of biology. Among the available platforms, complex coacervates are particularly attractive, due to their potential to encapsulate a wide range of biomolecules and other cargo, enabling genotype-phenotype mapping. By coupling coacervate formation to a fueled chemical reaction cycle, the synthetic cells become fuel-dependent, growing in the presence of fuel and decaying in its absence, resembling biological cells. However, constructing cellular substructures for these fuel-dependent synthetic cells, such as cytoskeletons, has remained an unresolved challenge. Here, we show that supramolecular (co)polymers can act as cytoskeletons for the synthetic cells, depending on their condenophilicity (their affinity for the droplet phase). By using two distinct supramolecular building blocks, the condenophilicity can be modulated. Supramolecular copolymers where only some of the monomers bind to the complex coacervate result in the formation of a protruding cytoskeleton in and around the droplet. However, as condenophilicity increases, the polymers partition strongly into the coacervates, leading to the formation of a fully encapsulated cytoskeleton. We found that these internal structures influence the synthetic cell properties, such as morphology and lifespan. Moreover, the synthetic cells can dynamically reconstitute the supramolecular fibers, creating distinct populations within the cells and the surrounding dilute phase. Our results demonstrate that orthogonally assembled structures can serve as cellular substructures for active complex coacervate-based synthetic cells, broadening the existing arsenal of tools to bestow these rudimentary synthetic cells with more life-like properties.
We use cookies to run the site and, with your consent, for analytics and to show ads. See our Cookie Policy.
