We fused part Frb (BBa_K209496) and FKBP (BBa_K209023) with yEGFP, which made it visible in the yeast under fluorescent microscope. Then we got part Frb-yEGFP (BBa_K2601007) and FKBP-yEGFP (BBa_K2601008) .
Then we characterized the expression of Frb-yEGFP and FKBP-yEGFP with 4 promoters pUra3, pTet07, pTEF1 and PDH3. The expression of these promoters was measured through flow cytometry (Fig. 1).
We made these improved prats:
Tet07-Frb-yEGFP (BBa_K2601021) ;
PDH3-Frb-yEGFP (BBa_K2601023) ;
Tet07-FKBP-yEGFP (BBa_K2601025) ;
TEF1-FKBP-yEGFP (BBa_K2601026) ;
PDH3-FKBP-yEGFP (BBa_K2601027) .
Figure. 1 The strength of three different yeast promoters tested through flow cytometry.
What's more, we fused Frb-yEGFP with HOtag6 and fused FKBP-yEGFP with HOtag3. These two parts can form phase separation in the presence of rapamycin (Fig. 2). The original part BBa_K209496 (Frb) and BBa_K209023 (FKBP) have not this function. We uploaded 2 parts: BBa_K2601010 (Frb-yEGFP-HOTag6) and BBa_K2601011 (FKBP-yEGFP-HOTag3).
Figure. 2A The structure of FKBP and Frb. Rapamycin can induce the interaction between them.
Figure. 2B 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 assemblies, which will be an organelle in cells.
Figure. 2C Granules in chemical-induced SPOT after adding rapamycin. Ura3-FKBP-HOTag3(mCherry) and PDH3-Frb-HOTag6 with yEGFP are transferred and expressed in S. cerevisiae. Fluorescence images in both GFP and mCherry channels of cells are taken after the addition of 10 μM rapamycin. Granules occurs in less than 20 minutes and the two fluorescence channels merge well.
Figure. 2D Growth curves of wild-type yeasts (black curve) and yeast strains with Ura3-FKBP-HOTag3 and PDH3-Frb-HOTag6 (red curve). They are measured after adding different concentration of rapamycin for 24 hours. Wild-type yeasts cannot grow and reproduce normally while engineered yeasts with FKBP-HOTag3 and Frb-HOTag6 can survive when exposed to rapamycin.
Then, we drive the expression of Frb-yEGFP-HOTag6 with 3 promoters pUra3, pTEF1 and PDH3. As our Modeling work predicts, the kinetics of a system depends on the concentration of the components and the interaction strength(Fig .3). We successfully uploaded 5 parts form them: BBa_K2601032 (pTet07-Frb-yEGFP-HOTag6), BBa_K2601033 (pTEF1-Frb-yEGFP-HOTag6), BBa_K2601034 (PDH3-Frb-yEGFP-HOTag6), BBa_K2601011 (pTEF1-FKBP-yEGFP-HOTag3) and BBa_K2601011 (PDH3-yEGFP-HOTag3).
Figure. 3B Flow Cytometry results of three promoters (Ura3, Tef2, and PDH3). The expression level of Ura3 is lowest while PDH3 is the strongest promoter.
Figure. 3C RapaSPOT of different promoter combinations after 10 μM rapamycin induction. Two axes stand for the expression level of components. After 3 hours, Only SPOT system with high level of Frb can be observed.
Figure. 3D Proportion of yeasts with granules after rapamycin induction. The rapamycin is from 1μM to 100 μM.