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Team:TU-Eindhoven/LEC/Lab
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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 these channels. (''Iida, K. et al. 2007'') Light will be emitted through the fluorescence of the GECO protein (''Zhao et al. 2011''), a calcium sensor that is expressed from a genetically engineered plasmid. When the calcium concentration is high, the GECO protein will fluoresce, but when it is low, the protein will not fluoresce. Electrical stimulation of the cell will allow calcium to enter into the cytoplasm and the 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. Challenges in the laboratory can be found in creating yeast cells with both GECO proteins and a sufficient number of calcium channels incorporated. | 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 these channels. (''Iida, K. et al. 2007'') Light will be emitted through the fluorescence of the GECO protein (''Zhao et al. 2011''), a calcium sensor that is expressed from a genetically engineered plasmid. When the calcium concentration is high, the GECO protein will fluoresce, but when it is low, the protein will not fluoresce. Electrical stimulation of the cell will allow calcium to enter into the cytoplasm and the 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. Challenges in the laboratory can be found in creating yeast cells with both GECO proteins and a sufficient number of calcium channels incorporated. | ||
| - | [[File:Plasmids.jpg]] | + | [[File:Plasmids.jpg|710px]] |
Revision as of 13:14, 25 September 2012
Biological work
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 these channels. (Iida, K. et al. 2007) Light will be emitted through the fluorescence of the GECO protein (Zhao et al. 2011), a calcium sensor that is expressed from a genetically engineered plasmid. When the calcium concentration is high, the GECO protein will fluoresce, but when it is low, the protein will not fluoresce. Electrical stimulation of the cell will allow calcium to enter into the cytoplasm and the 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. Challenges in the laboratory can be found in creating yeast cells with both GECO proteins and a sufficient number of calcium channels incorporated.
References
- Iida, K. et al. ''Essential, Completely Conserved Glycine Residue in the Domain III S2S3 Linker of Voltage-gated Calcium Channel α1 Subunits in Yeast and Mammals'' in ''Journal of Biological Chemistry'' August 31 2007, Vol. 282, issue 35 pp. 25659-25667, DOI: 10.1074/jbc.M703757200
- Zhao, Y. et al. ''An Expanded Palette of Genetically Encoded Ca2+ Indicators'' in ''Science'' 30 September 2011, Vol. 333 no. 6051 pp. 1888-1891, DOI: 10.1126/science.1208592
