Our SPOT contains two main applications. One is to use the SPOT as a reaction hub to regulate various biochemical reactions. We have demonstrated that if we integrated three enzymes which could synthesize β-carotene into SPOT system, the production of β-carotene in yeasts will be enhanced if phase separation happens. In the future, if we use the yeasts to manufacture β-carotene in a large scale, we can sequester the yeasts in fermenters to prevented recombinant yeasts or recombinant DNA from flowing out into the environment. The other is to use our SPOT as a sensor. We achieved that if there are ABA or Rapamycin molecules in the environment, phase separation would happen. If we want to use the sensor in reality, we can package the yeast cells into resins. It will help us to observe the SPOT formation by microscopy, and limit yeast escape. In both two reality scenes, escape of microbes is prohibited.
In our experiments, we chose E. coli as the chassis for molecular cloning, and S. cerevisiae as SPOT formation chassis. These two species are well known and non-pathogenic. Our bioparts are constructed by collecting segments from safe plasmid by which means we avoid using unknown segments. Those parts which have potential to cause environment problem, animal and plant disease, ecosystem changing are strictly prohibited.
As the iGEM safety policy requires, we didn’t do any dangerous experiments in daily bench work or faced any unusual safety issues. The bench work followed some basic regulations as below: 1.Duplicating a key to the laboratory without permission was strictly prohibited. 2.All involved participants needed to understand the experiment completely before commencing work. 3.All experimenters had to wear rubber gloves. 4.Any steps involving potential release of live microorganisms were performed in a bio-safety cabinet. 5.Appliances such as Bunsen burners, electric heaters and microwave ovens were not left unattended while in use. 6.All liquid and solid waste potentially containing living organism was sterilized. 7.The entire lab was sterilized using UV-light every week. 8.The last person to leave the lab always made sure that water, electricity, gas, and the air conditioner were shut down, and doors and windows have been locked before leaving.
In the process of shipment, the DNA parts are absolutely safe because they encode non-hazardous proteins like HOtags and enzymes for carotene synthesis. The DNA parts were safely confined within 96-well plate as the Parts Registry requires.
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.