Team:Potsdam/Demonstrate
Demonstrate - Gold
We have built a proof of concept system, where we could demonstrate that our system is working under real world conditions. We could show that the formation of Ddx4 droplets is possible not only in mammalian cells (HeLa cells1) but also in yeast cells (S. cerevisiae). This finding provides the basis for our experiments to achieve metabolic channeling.
The experiment was then conducted with the following steps: First, Ddx4-YFP was synthesized by IDT with overhangs to the vector. In a next step Ddx4-YFP was cloned into the vector using TAR. This lead to the following final construct in the yeast cells, as depicted in figure 1.
The experiment was then conducted with the following steps: First, Ddx4-YFP was synthesized by IDT with overhangs to the vector. In a next step Ddx4-YFP was cloned into the vector using TAR. This lead to the following final construct in the yeast cells, as depicted in figure 1.
Figure 2: Final construct of Ddx4-YFP in pYES2-CT.
It was then tested, if droplet formation due to the Ddx4-YFP was possible in the yeast cells. For that, fluorescence intensity images and bright field images were taken from yeast cells expressing the Ddx4-YFP and wild type yeast cells (YPH500 strain). The results are shown in figure 2. In the fluorescence intensity image of the yeast cells expressing Ddx4-YFP (induction of protein expression with galactose) bright dots can be seen, indicating the formation of fluorescing droplets. A comparison to the corresponding bright field image shows, that those droplets formed in the cytoplasm of the yeast cells. The wild type yeast cells show a small amount of background fluorescence, but no significant droplet like structures could be seen. It was therefore shown, that droplets do form in the yeast cells in which the Ddx4-YFP gene was inserted via TAR.
Figure 3: Fluorescence intensity images and bright field images of yeast cells expressing Ddx4-YFP (induction of protein expression with galactose) and wild type yeast cells. Significant droplet formation can be seen in the cytoplasm of yeast cells expressing Ddx4-YFP.
[1]Timothy J. Nott et al., “Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles”, Molecular Cell 57, March 5, 2015, 936–947.
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