Part:BBa_K2601011:Design
FKBP-yEGFP-HOTag3
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 337
Illegal BamHI site found at 358 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 79
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1004
Design Notes
FKBP-yEGFP-HOTag3
Some membrane-less organelles, such as stress granules and P bodies, have been discovered in recent years. Proteins condense into droplets and assemble these organelles through a process called phase separation. In order to rationally design a synthetic organelle based on protein phase separation, we needed a multivalent module and a protein-protein interaction module. The paired FKBP and Frb was one of the bioparts that we chose to introduce protein-protein interaction. FKBP and Frb could dimerize upon adding rapamycin. As for the multivalent module, we turned to de novo-designed homo-oligomeric short peptides. These short peptides are called HOTags (homo-oligomeric tags). HOTags contain approximately 30 amino acids. They have high stoichiometry, forming hexamer or tetrameric spontaneously. The hexameric HOTag3, together with the tetrameric HOTag6, could robustly drive protein phase separation upon protein interaction (achieved by FKBP-Frb module). To verify the feasibility of the system, we fused two fluorescence proteins with the two components of synthetic organelles. Thus, we could observe the self-organization of components and the formation of organelles under fluorescence microscope.
Source
FKBP-yEGFP-HOTag3