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.
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.
We should reproduce all of the experiments that we have done this summer to make sure the results are credible.
We will optimize the whole strategy to enhance the adsorption efficiency by changing pH, temperature, reaction time of crosslinking and clearance. (The efficiency is only about 60% without further optimization)
According to the results for the adsorption of 13nM uranyl, the polymer network exhibited a good ability in a simulated seawater environment. We could thus also look into other usage scenarios of Uranium Reaper, such as bio-mining and uranium enrichment.
Exchange of the SUP module for other functional proteins. For example, we could integrate proteins which could bind other heavy metals such as mercury so that the polymer network could be used to treat other kinds of pollution as well.
We could assemble enzyme systems behind the SpyTag backbone to create a production plant in vitro. In the protein polymeric network, the concentration of enzymes could be increased and the efficiency of biocatalysis may consequently also be enhanced.
If we optimize the number of SpyTag or SpyCatcher modules per protein monomer, as well as the working concentrations of proteins, we may make protein-3D printing using the Spy Crosslinking Network come true.