Part:BBa_K2601004:Design
HOTag3 (Homo-Oligomeric Tag3)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
HOTag3
When we wanted to rationally design a synthetic organelle based on phase separation and used it as a platform to achieve multi-functions, some design principles had to be followed. Interaction can bind the parts together while multivalence can make larger assemblies. In order to drive protein phase separation, we needed a multivalent module and a protein-protein interaction module. HOTag is the biobrick that we used to introduce multivalence. In natural process, such as phase separation occurred during T cell signal transduction, multivalency depends on multiple repeats protein domains. But it was not ideal to use multiple repeat domains in our design, 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 de novo-designed homo-oligomeric short peptides. These short peptides are called HO-Tag (homo-oligomeric tag). HOTags contain approximately 30 amino acids. HOTag3 has high stoichiometry, forming hexamer spontaneously. As for the protein-protein interaction part, we chose two sets of dimerization modules. The first pair was SUMO and SIM, which can dimerize spontaneously. The second one was chemically inducible FKBP and Frb. Rapamycin was the inducer of dimerization. We chose different modules according to the different functions we wanted to achieve. The hexameric HOTag3, together with another tetrameric HOTag (HOTag6), could robustly drive protein phase separation upon protein interaction (achieved by the protein-protein interaction module). Thus, HOTag3/6 pair is a useful tool to investigate protein phase separation and design a synthetic organelle. To verify the feasibility of the system, we fused two fluorescence proteins with the two components of synthetic organelles. We could observe the self-organization of components and the formation of organelles under fluorescence microscope. We named our system SPOT (Synthetic Phase separation-based Organelle Platform) because it could form granules (fluorescent spots) in yeast. Here is a demonstration of our overall design.
Source
HOTag3