Overall design

During humanity’s constant exploration of the world, the greatest pursuit is to remodel it to improve human life. While ‘phase separation’ in cells is under intense investigation, experiencing a research boom, the scientific community hopes that this phenomenon ‘worth millions of dollars’ can be artificially designed to enhance native functions and even acquire new ones. Our team, Peking iGEM 2018, went all out to overcome the challenge: artificially design Synthetic Phase separation-based Organelle Platform (SPOT).

Then we put forward two questions: Why can phase separation in cells produce membrane-less organelles? And how can we design our system to implement its intended functions?

Like oil in water, the contents of cells can separate into droplets. According to physical principles, the process where materials 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 are no longer distributed uniformly but form granules locally, which can act as organelles in the cell.

Thus, the main work to synthesize an organelle is to implement phase separation in a cell. Then, how can we do it? Composition can switch rapidly through multivalence. Our design was inspired by recent studies showing that multivalence drives protein phase separation and formation of synthetic organelles. What’s more, we take our inspiration from existing living systems and previous work. For example, intrinsic disordered regions are an indicator of large-scale phase separation in the cell. They interact with each other through van der Waals forces, hydrophobic effects and electrostatic attraction. 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 the self-assembly of our designed organelles and regulate them in various ways.

In conclusion, multivalence drives the self-assembly of proteins and interaction binds the parts together. Therefore, interaction can induce phase separation and multivalence can make larger assemblies, which are two essential elements in our design that ensure the formation of synthetic organelles.

Figure. 1 Overall design


To design multivalent modules, it is not ideal to use multiple repeat domains, which not only makes the protein extremely large but also brings difficulties to DNA recombination. Thus, instead of using multiple repeats, we turned to de novo-designed homo-oligomeric coiled coils, namely HOTags (homo-oligomeric tags), designed by Prof. Shu Xiaokun[1]. They are short peptides with approximately 30 amino acids, therefore are ideal to introduce multivalence. There are seven coiled coils previously characterized in protein de novo design studies, among which HOTag3 and HOTag6 are the most robust in driving protein droplet formation over a wide range of protein concentrations, so we utilize them to design our SPOT.

Figure. 2 Coiled-coil assemblies and helical bundles[2].


To design interaction modules, we tested many components and fused them to the N-termini of HOTag3 or HOTag6. Some of them spontaneously self-assemble and some are inducible. Moreover, we can regulate them using various different inducers and promoters with different intensities.



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. Furthermore, 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[3]. Therefore, we chose SUMO3 and SIM as a pair of interaction modules that 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 used the resulting constructs to transform yeast and proved that they can be stably co-expressed. If the system works, we will find red granules co-localized with green granules in cells under a fluorescence microscope.

Figure. 3A Structure of SUMO3 and SIM. Modification of proteins by SUMO are recognized by SUMO-interacting motifs termed SIMs. In natural process, polySUMOylation recruits distinct interaction partners, such as E3 ubiquitin ligases, that bind to polySUMO chains through tandem SIMs. SIMs bind to a surface patch between the α-helix and a β-sheet of the SUMO protein and extend the β-sheet of SUMO by one additional strand. the SIM either attaches as a parallel or an antiparallel strand to the SUMO β-sheet. Binding is primarily mediated by a stretch of four residues containing 3–4 hydrophobic amino acids (I, V, or L). This core interaction motif is a common property of all SIMs[4]. Figure. 3B Pattern diagram of SUMO-yeGFP and SIM-mCherry. YeGFP is fused to the C-terminus of SUMO and to the N-terminus of HOTag3, mCherry is fused to the C-terminus of SIM and to the N-terminus of HOTag6.

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=0.2 nM) 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)[5]. Thus, we chose these two proteins as a pair of interaction modules and assembled them onto a yeast plasmid the same as in the construction of SUMO and SIM and introduced them into yeast. If we add rapamycin to the yeast, we will see red granules co-localized with green granules in cells under a fluorescence microscope. Synthetic organelles become real!

Chemical-induced SPOT can be formed by using rapamycin to induce FKBP-Frb interacitons Figure. 4A The sturcture of FKBP and Frb. Rapamycin can induce the interaction between them. Figure. 4B Design of RapaSPOT. FKBP is fused with mCherry and HOTag3 while Frb is fused with yEGFP and HOTag6. After adding rapamycin, they are expected to self-organize to form large assemblys, which will be an organelle in cells.


As mentioned above, interactions can be formed not only by inducers such as rapamycin, but also spontaneously, just as SUMO and SIM. So can we combine these two ways of interactions? To solve this problem, we did further studies about phytohormone and found ABA. Abscisic acid (ABA) is an important phytohormone that regulates plant stress responses. Proteins from the PYR-PYL-PCAR family were identified as ABA receptors[6]. Upon binding to ABA, a PYL protein associates with type 2C protein phosphatases (PP2Cs) such as ABI1 and ABI2, inhibiting their activity[7]. Previous structural and biochemical observations have provided insight into PYL-mediated ABA signaling and given rise to a working model. In the absence of ABA signaling, PP2Cs are fully active and PYLs exist as inactive homodimers in cells, unable to bind or inhibit PP2Cs, mainly due to the incompatible conformation of CL2loop[7]. In response to ABA binding, the CL2 loop undergoes a conformational rearrangement to close onto the ABA-bound pocket, then, the interaction between PYLs and PP2Cs can be formed. Here we chose PYL1 and ABI1 as a pair of interaction modules. Then, we assembled them onto yeast plasmid as the same as the construction of FKBP and Frb and transformed them into yeast. Based on the interaction of PYL1 and ABI1, we can get a wonderful scene: In the absence of ABA, the synthetic organelles composed only of PYL1 appear, because of the homodimers of PYL1. And after we add ABA into yeast, ABI1 can enter the organelles with the interaction of ABI1 and PYL1, and we can see red droplets colocalize with green droplets in cells through fluorescence microscope. In this way, new components can enter the original organelles and the time of occurrence can be regulated as it is inducer-mediated regulation. So it give our designs and functions more possibilities.

Figure. 5 Interaction of PYL and PP2C[7]
Two function sites

Now, we have artificially designed phase separation in cells and synthesized membraneless organelles. But how can we fulfill intended functions with synthetic organelles? Here, we propose two ideas. We reserve two sites to implement functions, which means function modules, such as enzymes in metabolism, proteins in signaling pathway, transcription factors in transcription and so on, have two sites in our designs.


Direct integration into the skeleton

Just as we characterized synthetic organelles with fluorescent proteins, we can fuse function modules between the C-termini of the interaction modules and the N-termini of HOTags. Then, the function modules can be fused into S to implement intended functions.

Figure. 6 Integrate function modules to the skeleton

Targeting GFP with nanobody

We introduced a specific protein, an anti-GFP nanobody, which is very small (only 13 kDa, 1.5nm-2.5nm) and has a high affinity (0.59 nM). It is a camelid antibody against GFP[8]. We can fuse GFP between the C-termini of the interaction modules and the N-termini of HOTags, and fuse function modules to the C-terminus of the anti-GFP nanobody. Then, with the help of the interaction between the anti-GFP nanobody and GFP, SPOT can load function modules, and targeted functions can be realized. You may ask: How does an anti-GFP nanobody improve the design? Firstly, it will not make the protein very large and will reduce its effect on the structure of the function modules, which can ensure the quality of the designed functions. Secondly, it can bring components not belonging to the original structure to synthetic organelles, which can extend their function. Thirdly, it is easy to regulate the expression of target proteins. Thus, nanobodies may have a surprise in store!

Figure. 7 Interaction of anti-GFP nanobody and GFP

We implemented phase separation in vivo and synthesized artificial membrane-less organelles. The main challenge in synthesizing an organelle is to implement phase separation in a cell, so we stress the importance of interactions and multivalence. For these two aspects, we offered our ideas and their feasibility was analyzed. Finally, we proposed two approaches to implement that target functions. We believe that in the near future, making “millions of dollars” by harnessing phase separation in cells will no longer be a dream!


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