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.
Moreover, we fused Frb-yEGFP with HOtag6 and FKBP-yEGFP with HOtag3. These parts can form phase separation in the presence of rapamycin (Fig. 2). The original part Frb (BBa_K209496) and FKBP (BBa_K209023) doesn't have this function.
We uploaded 2 parts:
Frb-yEGFP-HOTag6 (BBa_K2601010) ;
FKBP-yEGFP-HOTag3 (BBa_K2601011) .
Figure. 2 The improvement of Frb and FKBP fused with yEGFP.
Figure. 2A The structure of Frb and FKBP. Rapamycin induces 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 self-organizing to form large assemblies, which will be organelles in cells.
Figure. 2C Granules formed in chemical-induced SPOT after adding rapamycin. Ura3-FKBP-HOTag3 with 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 adding 10 μM Figure. 2D Growth curves of wild-type yeast (black curve) and yeast strains of Ura3-FKBP-HOTag3 and PDH3-Frb-HOTag6 (red curve). They are measured after adding different concentration of rapamycin for 24 hours. Wild-type yeast cannot grow and reproduce normally while recombination yeast strains of FKBP-HOTag3 and Frb-HOTag6 can survive when exposed to rapamycin.
Then we characterized 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:
pTet07-Frb-yEGFP-HOTag6 (BBa_K2601032) ;
pTEF1-Frb-yEGFP-HOTag6 (BBa_K2601033) ;
PDH3-Frb-yEGFP-HOTag6 (BBa_K2601034) ;
pTEF1-FKBP-yEGFP-HOTag3 (BBa_K2601011) ;
PDH3-yEGFP-HOTag3 (BBa_K2601011) .
Figure. 3A The formation of SPOT can be described as a phase separation process of three components. Two important variables, the concentration of components and the interaction strength are marked in the figure.
Figure. 3B Flow cytometry results of three promoters (pUra3, pTEF2, and PDH3). The expression level of Ura3 is the 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 yeast with granules after rapamycin induction. The rapamycin concentration range is from 1μM to 100 μM.