Our team seeks to synthesize membrane-less organelles and turn it into a multi-functional toolbox for synthetic biology based on basic phase separation principles, which is a rather fundamental field in condensed matter physics. Therefore, it’s not really a reality application so far. Nonetheless, it’s definitely not the reason that we are confined in the laboratory coping with experiments and mathematical models without making a difference to the society directly. Meanwhile, we need to get to know about the demand of engineers and consumers. Thus we did an integrated human practice in several different ways. Inside the iGEM community, we made statistics of the education background and numbers of igemers each year in order to investigate how iGEM has been broadcasted internationally and how the field of synthetic biology has changed over the last 14 years. We noticed that most iGEM teams are becoming more and more diverse, which promotes the development of iGEM community but make it more challenging for team members to communicate. This can also be read as more people from different disciplines especially mathematics and physics have been devoted to systems and synthetic biology, which are interdisciplinary sciences needing various knowledge while on the same time, they can feed back to enrich the individual scientific disciplines and biology-based solutions for societal problems can be worked out. We also tried to play an active part in public engagement. We communicated with people from various backgrounds in universities, high schools, kindergartens and on the internet. We realized that there has always been a gap between the achievements in scientific research and reality application. People from academic world and industrial world barely know each others’ requirements most of the time. Thus we discussed this topic in detail using fluorescence microscope as an example. Our human practice reinforced our team construction creating more chance for the team members to communicate and collaborate with each other. We tried to make synthetic biology accessible for as many people as possible and we do expect our efforts may make a difference. Meanwhile, we’d be more than glad if our work may give the synthetic biology community some inspiration. To gain a deeper understanding of biology in the 21st century, we need to integrate knowledge from various disciplines while biology-based solutions to societal problems can influence the world more profoundly. In the following sections, you will go through our human practice in details. Investigation on the education background of iGEMers

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

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.