Team:TU-Eindhoven/LEC/GECO

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In the lab we will make living cells that emit light in response to an electric stimulus. This can be achieved by genetic modification of yeast cells, through the introduction of fluorescent calcium sensors and calcium channels. The plasma membrane of the brewer's yeast Saccharomyces cerevisiae contains the Cch1-Mid1 channel that is homologous to mammalian Voltage Gated Calcium Channels (VGCCs). It is hypothesized that upon depolarization of the plasma membrane calcium ions selectively enter the cytoplasm through Cch1-Mid1.^[1]. Light will be emitted through the fluorescence of the GECO protein, a calcium sensor that is expressed from a genetically engineered plasmid. When the calcium concentration is high the GECO protein is fluorescent, when the calcium concentration is low the GECO protein is not fluorescent. So upon electrical stimulation cells will let calcium into the cytoplasm and GECO proteins will start to fluoresce. After a while the calcium concentration will drop to homeostatic levels through active transport of calcium ions to the yeast's vacuole and fluorescence will cease. The time required to switch the light on and off should be as short as possible. Therefore, we will try to increase the number of calcium channels through over expression of the channel proteins so that calcium can enter the cell faster upon electrical stimulation. All this calcium that enters the cell has to be pumped out eventually to maintain a healthy cell. It seems that calcium is stored in the cell's vacuole before being excreted. The capacity for storage of calcium ions in the vacule is unknown, as is the rate a which cells can excrete calcium. There may be unexpected side-effects that have to be investigated.

References

[1] Kazuko Iida, Jinfeng Teng, Tomoko Tada, Ayaka Saka, Masumi Tamai, Hiroko Izumi-Nakaseko, Satomi Adachi-Akahane, and Hidetoshi Iida. Essential, Completely Conserved Glycine Residue in the Domain III S2–S3 Linker of Voltage-gated Calcium Channel α₁ Subunits in Yeast and Mammals. Journal of Biological Chemistry 282(35)