Achievements
Experiment:
1. We constructed a part library that can drive protein phase separation. The library includes several dimerization modules and multivalent modules.
2. We rationally designed a synthetic organelle called SPOT (Synthetic Phase separation-based Organelle Platform) and used it to achieve multi-functions. We believed SPOT to be a useful platform for other iGEM teams to investigate protein phase separation and design synthetic organelles.
3. The synthetic organelles could form spontaneously in yeast. They could also be induced by rapamycin. We chose different platforms according to the different functions we wanted to achieve.
4. We set up a thermodynamic model and a kinetic model to characterize our system. The experimental results were consistent with the models.
5. We tested the strength of four different yeast promoters using flow cytometry.
6. We thoroughly tested some key parameters of the paired FKBP-HOTag3 and Frb-HOTag6. The thermodynamic properties and the kinetic properties were well characterized.
7. We explored the biophysical properties of the SUMO-SIM system. We proved the granules were liquid-like.
8. Three enzymes were essential for β-carotene producing in yeast. We loaded the three enzymes on the synthetic organelle and increased the reaction rate. We confirmed SPOT could function as a reaction crucible.
9. The inducible SPOT could sequester rapamycin with high efficiency. We used SPOT as a cure of the toxicity of rapamycin.
10. We verified the high sensibility of SPOT-based sensor system. We also built an ABA sensor.
manifested
Beyond experiment:
1. We submitted 29 high-quality Biobricks, including basic parts, such as multivalent HOTags, and composite parts that can robustly drive phase separation.
2. We developed a batch file to process large amounts of fluorescence images. It offered a high throughput solution to analyze experimental results derived from fluorescence microscope.
3. We did a statistical investigation of iGEM in the past decade. The academic background of the iGEMers provided evidence of the increasing diversity of iGEM.
4. We played an active part in preschool education and tried to integrate some biological knowledge into the games. We also devoted ourselves to promoting synthetic biology in high schools.
5. We focused on how to build a bridge between scientific research and the public. We exchanged ideas with experts from academia and developed a feasible idea to design a cheap fluorescence microscope affordable by the public.
6. We collaborated with many universities. We shared experimental equipment and helped each other with modeling as well as experiments. We also attended CCiC (Conference of China iGEMer Community) and got inspiration from other teams.
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.
We believe that our work has reached the medal requirements of demonstration as we have confirmed that our synthetic organelles can be formed in vivo and deliver a range of functions both for engineering and research due to their amazing properties. The concrete demonstration of the whole platform is shown below. You can see more details of experiments and modeling in our Data Page and Modeling
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.
（这里可能需要一张cartoon的设计图）
b, c fluorescence images of spontaneous organelles (SUMO-SIM based) and inducible synthetic organelles (FKBP-Frb based, after adding 10000 nM rapamycin)The formation of organelles has flexible but predictable properties and kinetics in different conditions
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.
Figure2 (a) Phase diagram of a phase separation system with three components(simulation). To fit our system, the x-axis and the y-axis stands for the two components in the granules. The asymmetry comes from the assumption that the two components have different interactions with water. (b) Fluorescence movies of different promoter combinations of FKBP-Frb mediated system after adding rapamycin. Only in specific combinations, synthetic organelles can be formed by phase separation. (c) The formation process of SUMO-SIM mediated synthetic organelles can be controlled by inducible promoters. While the expression of Tet07-SIM-mCherry-HoTag6 is induced by dox gradually, the granules will occur abruptly in some time.
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.
Figure3 (a) A simulation of organelle formation process in different interaction strength of components. (b) The speed of FKBP-Frb mediated organelle formation increases with the increasing concentration of rapamycin.
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.