Schematic diagram
BBa_K2601010
We combined Frb, yEGFP and HOTag6 togethor in BBa_K2601010. Frb can interact with FKBP after adding rapamycin, which makes the RapaSPOT formation controllable. We constructed yeast strains with FKBP-mCherry-HOTag3 and Frb-yEGFP-HOTag6 and used 10 μM rapamycin to induce SPOT formation. Minutes after adding rapamycin, granules appeared and became larger gradually.
BBa_K2601011
To make our SPOT a reaction hub, we fused the enzymes of β-carotene synthase system, CrtI, CrtE and CrtYB into Frb-HOTag6 backbone. In the presence of rapamycin, FKBP-yEGFP-HOTag3 and Frb-enzyme-HOTag6 can condense into aggregates. And if the yeast contains SPOT, there will be more β-carotene producted. Meanwhile, we wondered if we can load enzymes onto SPOT mediated by indirected connection. We fused CFP to anti-GFP nanobody as the demonstration of our design and we observed the co-localization of the blue and green fluorescence as expected.
BBa_K2601012
In the biobrick BBa_K2601012, we fused SUMO, yEGFP and HOTag3 together. SUMO is the interaction module which can bind SIM spontaneously, while HOTag3 can introduce multivalence. These two features are necessary for SPOT formation. We expressed SUMO-yEGFP-HOTag3 and SIM-mCherry-HOTag6 in one yeast strain, and we observed the colocalization of yEGFP and mCherry in two different fluorescence channels, which confirms our hypothesis that the two components could form synthetic organelles.
| BBa_ | Part |
| K2601000 | Promoter-tet07 |
| K2601001 | Promoter-PDH3 |
| K2601002 | SUMO |
| K2601003 | SIM |
| K2601004 | HOTag3 |
| K2601005 | HOTag6 |
| K2601007 | Frb-yEGFP |
| K2601008 | FKBP-yEGFP |
| K2601010 | Frb-yEGFP-HOTag6 |
| K2601011 | FKBP-yEGFP-HOTag3 |
| K2601012 | SUMO-yEGFP-HOtag3 |
| K2601021 | Tet07-Frb-yEGFP |
| K2601023 | PDH3-Frb-yEGFP |
| K2601025 | Tet07-FKBP-yEGFP |
| K2601026 | TEF1-FKBP-yEGFP |
| K2601027 | PDH3-FKBP-yEGFP |
| K2601032 | Tet07-Frb-yEGFP-HOTag6 |
| K2601033 | TEF1-Frb-yEGFP-HOTag6 |
| K2601034 | PDH3-Frb-yEGFP-HOTag6 |
| K2601037 | TEF1-FKBP-yEGFP-HOTag3 |
| K2601038 | PDH3-FKBP-yEGFP-HOTag3 |
| K2601040 | Tet07-SUMO-yEGFP-HOtag3 |
| K2601041 | TEF1-SUMO-yEGFP-HOtag3 |
| K2601042 | PDH3-SUMO-yEGFP-HOtag3 |
| K2601054 | PDH3-Frb-crtE-HOTag6 |
| K2601056 | Tet07-Frb-crtYB-HOTag6 |
| K2601058 | PDH3-Frb-crtYB-HOTag6 |
| K2601060 | SIM-crtE-Hotag6 |
| K2601061 | SIM-crtYB-Hotag6 |
BBa_K2601010
For this year’s project, we chose BBa_K2601004（加超链接） which codes homo-oligomeric tag 3 (short as HOTag3) for the special award of the best Basic Part. HOTags are the biobricks that we used to introduce multivalence in our synthetic organelles. In natural process, phase separation occurred depending on multiple repeats protein domains, such as the interaction between Nephrin which three phosphotyrosine motifs and the SRC homology 2 (SH2) domain on Nck [1]. Using multiple repeat domains in a man-made design is not ideal, because it would not only make the scaffold extremely large but also be problematic for molecular cloning and making transgenic yeasts. Thus, instead of using multiple repeats, we turned to use the de novo-designed HOTags. These HOTags contain approximately 30 amino acids. HOTag3 has high stoichiometry, forming hexamer spontaneously[2].
We fused two sets of interaction module into the multivalence module. First, we fused SUMO to hexameric HOTag3 and SIM to tetrameric HOTag6. With the result of fluorescence imaging (Fig. 1), we confirmed that these two recombinant proteins can form granules successfully. Meanwhile we used FRAP to verify if the granule is liquid-like. After photobleaching, the fluorescence of granules quickly recovered, which indicated that the granules had rapid mass exchange with cytoplasm.
BBa_K2601010
2018 Peking iGEM team members devoted themselves to constructing a part collection that can drive phase separation. We have not only submitted all of new basic HOTag parts, but also provided multiple composite parts combining HOTags with other protein-protein interaction modules and fluorescent reporters. The basic part, HOTag3, is a homo-oligomeric short peptide containing only 30 amino acids. It has high stoichiometry, forming hexamer spontaneously. The HOTag3, together with another tetrameric HOTag6, can robustly drive phase separation upon protein-protein interaction. Protein-protein interaction is achieved by our dimerization parts, including FKBP/Frb and SUMO/SIM. Functions of all the parts were thoroughly tested. Some thermodynamic and kinetic properties of the parts were characterized as well. We believe the HOTag is useful tool for other iGEM teams to investigate protein phase separation and design synthetic organelles.