Difference between revisions of "Team:Peking/Design"

Design By SSY While people are constantly exploring the world, the greatest pursuit is to remould the world. While the ‘phase separation’ in cells is under investigation and in a research boom, the scientific community hopes the phenomenon ‘worth of millions of dollars’ to be artificially designed to enhance original functions and even acquire new functions. Our team, Peking iGEM 2018 go all out to overcome the challenge: fulfill phase separation in cells and synthesize membraneless organelles. 一、 Overall design 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 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 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 conclusion，multivalency drives protein’s self-assembly and interaction binds the parts together. It means, interaction can induce phase separation and multivalency can make larger assemblies, which are two essential elements in our design and ensure the formation of synthetic organelles. Figure 1 Overall design 二、 Multivalency To design multivalent modules, it is not ideal to use multiple repeat domains, which will not only make the protein extremely large and bring difficulty to molecular cloning, 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 HOTag (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. It has been proved by previous work of Shu Xiaokun’s lab that HOTag3 and HOTag6 are most robust in driving protein droplet formation over a wide range of protein concentrations, so we chose them. Figure 2 Coiled-coil assemblies and helical bundles. 三、 Interaction To design interaction modules, we tried a lot of components and we fused them to the N-terminus of HOTag3 or HOTag6. Some of them are spontaneous and some are inducible. And we can regulate them through various kinds of inducers and different intensities of promoters. (一) SUMO and SIM Post-translational modifications by the small ubiquitin-like modifier (SUMO) are crucial events in cellular response to radiation and a wide range of DNA-damaging agents. Previous studies have shown that SUMO mediates protein-protein interactions by binding to a SUMO-interacting motif (SIM) on receptor proteins. And recent studies have shown that a protein with ten repeats of human SUMO3 (polySUMO) and a protein with ten repeats of SIM (polySIM) can phase separate in vitro. Therefore, we chose SUMO3 and SIM as a pair of interaction modules and they can drive the formation of synthetic organelles spontaneously. For plasmid construction, in order to make synthetic organelles visible, we chose mCherry (red fluorescent protein) and yEGFP (yeast-enhanced green fluorescent protein) as reporters. Then, we fused mCherry between the C-terminus of SIM and the N-terminus of HOTag6. Similarly, we fused yEGFP between the C-terminus of SUMO and the N-terminus of HOTag3. We transformed them into yeast and proved that they can stably express. If it work, we will find red granules colocalize with green granules in cells under fluorescence microscope. There is a pattern diagram composing SUMO and SIM. Figure 3 Interaction of SUMO and SIM (二) FKBP and Frb The interaction between FKBP and FRB can be robustly induced by rapamycin. Rapamycin is a 31-membered macrolide antifungal antibiotic. It binds with high affinity (Kd=0.2nM) to the 12-kDa FK506 binding protein (FKBP), as well as to a 100-amino acid domain of the mammalian target of rapamycin (mTOR) known as FKBP-rapamycin binding domain (Frb). Thus, we chose them as a pair of interaction modules. And we assembled them on to yeast plasmid as the same as the construction of SUMO and SIM and transformed them into yeast. if we add rapamycin to the yeast, we will see red granules colocalize with green granules in cells under fluorescence microscope. Synthetic organelles come true! Figure 4 Interaction of FKBP and FRB induced by rapamycin (三) Optogenetic control All the interaction modules described above are irreversible. Now, this problem can be solved be the Arabidopsis red light-inducible phytochrome (PHYB-PIF) system, which comprises the phytochrome B (PHYB) protein and phytochrome interaction factor (PIF; PIF3 or PIF6). These two domains are induced to bind under far infrared light and the binding is reversed within seconds of exposure to infrared light but is otherwise stable for hours in the dark. What’s more, the phytochrome system has a 10-100 larger dynamic range than the cryptochrome and LOV-based systems, and the affinity of its light-gated interaction is tighter than others. Therefore, we chose PHYB and PIF6 as a pair of interaction modules. Then, we assembled them onto yeast plasmid in the same way as the construction of SUMO and SIM and transformed them into yeast. We suppose SPOT can form and disappear under the regulation of far infrared light. Figure 7 Interaction of PhyB and PIF (四) Two function sites Now, we have designed artificial membraneless organelles. But how can we fulfill intended functions with them? Here, we propose two ideas. We reserve two sites for implement functions, where function modules like enzymes in metabolism, proteins in signaling pathway and transcription factors in transcription can be loaded. Direct integration into the skeleton Just as we characterize synthetic organelles with fluorescent proteins, we can fuse function modules between the C-terminus of interaction modules and the N-terminus of HOTags. Then, the function modules can be fuse into S to fulfill intended functions. Figure 8 Integrate function modules to the skeleton Targeting GFP with nanobody We introduce a magic protein, anti-GFP nanobody, which is very small (only 13-kDa, 1.5nm 2.5nm) and a high-affinity (0.59nM) camelid antibody to GFP. So we can use its characteristic to improve our designs. We can fuse GFP between the C-terminus of interaction modules and the N-terminus of HOTags, and fuse function modules to the C-terminus of anti-GFP nanobodies. Then, with the help of interaction between anti-GFP nanobodies and GFP, SPOT can load function modules, expected functions can be realized. You may ask: How does anti-GFP nanobody improve the design? Firstly, it will not make the protein extremely large and will reduce the effect on the structure of function modules, which can ensure the quality of functions. Secondly, it can bring components not belonging to the original structure to synthetic organelles, which can extend the function of synthetic organelles. Thirdly, it is easy to regulate the expression of target proteins. So you can see, nanobodies may do better and give you a surprise! Figure 9 Interaction of anti-GFP nanobody and GFP 四、 Conclusion We fulfilled phase separation in vivo and synthesized membraneless organelles. And the main work to synthesize an organelle is to fulfill phase separation in a cell, so we stress the importance of interactions and multivalency. For these two aspects, we gave our ideas and the feasibility was analyzed. At last, we proposed two ideas to implement functions. We believe that in the near future, “millions of dollars” will no longer be a dream! References 1. Yin P, Fan H, Hao Q, et al. Structural insights into the mechanism of abscisic acid signaling by PYL proteins[J]. Nature Structural & Molecular Biology, 2009, 16(12):1230-1236. 2. Park S Y, Fung P, Nishimura N, et al. Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins[J]. Science, 2009, 324(5930):1068-1071. 3. 冯婵莹, 王永飞. 植物脱落酸PYR/PYL/RCAR受体[J]. 生命的化学, 2015(6):721-726. 4. Nambara E, Marion-Poll A. Abscisic acid biosynthesis and catabolism.[J]. Annual Review of Plant Biology, 2005, 56(56):165-185. 5. Hirano K, Ueguchi-Tanaka M, Matsuoka M. 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