Then we put forward two questions: Why phase separation in cells can produce membraneless organelles? And how can we design our system to fulfill its intended functions? Like oil in water, the contents of cells can separate into droplets. According to physical principles, the process where material self-assemble into organelles is described as ‘phase separation’, which is the conversion of a single-phase system into a multiphase system. 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. That is to say, the main work to synthesize an organelle is to fulfill phase separation in a cell. Then, how can we do it? Composition can switch rapidly through changes in scaffold concentration or multivalency. And our design was inspired by recent works showing that multivalency drives protein phase separation and formation of synthetic organelles. What’s more, we take our inspiration from existing life systems and previous works. For example, Intrinsic Disordered Regions are the symbol of massive phase separation in the cell. They interact with each other through the van der Waals force, hydrophobic effect and electrostatic attraction. And there are many interactions like this in nature, such as FKBP and FRB, SUMO and SIM, SH3 and PRM, phyB and PIF6. Thus, we can make good use of them to induce our designed organelles and regulate them variously.! In a conclusion，multivalency drives protein’s -self-assemblyies and interaction binds the parts together. It means, interaction can induce phase separation and multivalency can make larger assemblies, which are two essential elementmodules in our design and ensure the formation of synthetic organelles.

To design multivalent modules, it is not ideal to use multiple repeat domains, which not only will make the protein extremely large and bring difficulties to DNA recombination, but also may be problematic for making transgenic animals. Thus, instead of using multiple repeats, we turned to de novo-designed homo-oligomeric coiled coils. And we named these coiled coils as HO-Tag (homo-oligomeric tag). They are short peptides, ~30 amino acids, therefore they are ideal tags to introduce multivalency. There are seven coiled coils previously characterized in protein de novo design studies. They have been proved by previous work of Shu Xiaokun’s lab, and according to their work, HOTag3 and HOTag6 are most robust in driving protein droplet formation over a wide range of protein concentrations, so we choose them.

Spontaneous and induced synthetic organelles can be formed by phase separation

Our basic system consists of two components of synthetic organelles. Either of them has a specific HOtag to form homo-oligomers. We expect that they are able to form synthetic organelles due to the principles of phase separation. To verify the feasibility of the design, we fused two fluorescence proteins with the two components of synthetic organelles (Figure1.a) so that we can observe the self-organization of components and the formation of granules under fluorescence microscope.

We used SUMO-SIM interaction module to build a spontaneous organelle. When two components are expressed in yeasts, granules with the two fluorescence proteins can be observed in vivo (Figure1.b).

Meanwhile, by rapamycin induced interaction module, FKBP-Frb, we have built an inducible organelle. We can see granules occurs in yeasts within minutes after adding the inducer.

The formation of organelles has flexible but predictable properties and kinetics in different conditions

Then we combined modeling of phase separation and experiment to research the kinetics of the organelles formation process expecting that a well-characterized system can reach its whole potential in complex applications.

As the model predicts, the concentration of components and the interaction strength affect the kinetics of phase separation. First we controlled the expression levels of components by using several stable or inducible promoters and observe the system's behavior. We found that the formation of organelles happened in specific promoter combinations and can be controlled by inducible promoters. The analysis result does not only fit well with the simulation, but provides potential methods to control the organelles in applications.

The strength of interaction modules can be also controlled. In the rapamycin-induced organelle system, changing the concentration of rapamycin will affect the apparent value of K, a parameter reflecting the interaction strength in our model. In a gradient rapamycin-inducing experiment, the delay time from adding inducer to granules formation was found to be shorter when concentration of rapamycin increases. So we have confirmed the influence of two parameters in models and increased the flexibility of our synthetic organelles.

We also tried to characterized other properties, like the liquid-like property of the synthetic organelles, as they may affect the functions. See more details about our characterizations in DataPage Phase separation.

Since SPOT can form in the cell and be controlled, we go further to consider the functions of SPOT. The functions of SPOT can be descripted in three catalogs: Spatial segmentation, Sensor and metabolic regulation. We verified the spatial segmentation with the condensation of substrates, also we can load the protein we want by fusing it with nanobody. We then verified the sensor with detecting rapamycin and ABA, which shows strong relativity between the concentration and the proportion of yeasts with SPOT. To find the law behind metabolism in the SPOT, we fuse the enzymes that can produce β-carotene into SPOT and measure the difference between with or without SPOT in produce of β-carotene.

SPOT has been well verified and has various functions. And in the future, this modular system will have great potential in science and practice using. SPOT can change the modules to gain more different properties like diverse inducing method, we can also use it as a platform and then load other protein with some interactions like the interaction between nanobody and GFP. What’s more, we might have the ability to form differernt SPOTs in the cell and regulate them respectively. The functions of SPOT can also diverse. We can build a real time sensor for molecule in living cells to monitoring the concentration changing in environment or in cells. More metabolism pathway can be test in SPOT and we will find some laws of the function of regulate the metabolism. To be summary, more achievement is coming true with SPOT.