The aim of our project is to build a synthetic organelle based on phase separation as a multifunctional platform. Based on the principle of multivalence and interaction, we fused interactional modules into homo-oligomeric tags (HOtags) to form granules in S. cerevisiae.

We have built spontaneous and induced synthetic organelles by specific interaction modules, so that we can control the formation process by different ways for demands in biological engineering. Then we characterized the kinetics and properties of synthetic organelles theoretically and experimentally. These results confirm the potential of synthetic organelles in synthetic biology.

It inspired us to propose some specific applications of our synthetic organelles, including organization hub, sensor, and metabolism regulator. We have verified the feasibility of them by loading GFP-nanobody module, NAD+ sensor module and carotene production module to the whole system.

Ever since the beginning of life, compartment has been playing a crucial rule in biological systems. The famous Miller-Urey experiment shows that inorganic molecules can transform into organic substances under extreme conditions, for example lightening. However, homogeneously distributed organic matters are not enough for life to emerge. It is almost impossible that all conditions are proper in the entire primordial soup.

That is where the compartment comes in.

Only after coacervate droplet forms and organic molecules condense inside, a completely different environment can be attained within, thus enabling the emergence of bio-macromolecules, or in other word, making life possible.

In cells, compartmentalization is mainly achieved be all sorts of organelles, for instance, mitochondrion, chloroplast, lysosome etc. They take up three major roles: A, B, C

Intuitively, for a organelle to sustain a stable compartment, it seems necessary to require a material boundary, more precisely, a membrane. Membrane-bound organelles are indeed common and stable, but from the perspective of synthesis, it is way too complicated. However, there are also non-membrane-bound organelles, for instance, stress granule, P granule and nucleolus. More importantly, their formation is guided by simple physical principals.

Then came the question that how can we synthase membraneless organelles. The process where material self-assemble into organelles is described as ‘phase separation’ according to physical chemistry, which is the conversion of a single-phase system into a multiphase system, much like how oil and water will demix from each other. In general, materials flow to regions with low chemical potential instead of low concentration. Finally, the components no longer distribute uniformly but form granules locally which are organelles in the cell.

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(动图3) That is to say, the main work to synthase an organelle is to fulfill phase separation in a cell. We take our inspiration from existing life systems. For example, stress granules and P bodies are formed by the interaction between mRNA and proteins. RNA and protein play a significant part in the phase separation in cells. IDR(Intrinsic Disordered Regions) are the symbol of massive phase separation in the cell. IDR interact with each other through the van der Waals force, electrostatic effect and hydrophobic effect between the residues of amino acids, while RNA get together with proteins through massive bases and ribose. Previous work has been done to reproduce natural phase separation by connecting interaction modules like SUMO/SIM, SH3/PRM, constructing granules in the cell.

(图片4) Summarizing these examples and according to physical principles, interaction between modules and multivalence are essential for phase separation. In general, interaction binds the parts together and multivalence makes larger assemblies, which are two guidance of our design. (图片5 )