Team:TU-Eindhoven/LEC/LabTheory

From 2012.igem.org

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<h3>GECOs</h3>
<h3>GECOs</h3>
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<p>Real-time imaging of biochemical events inside living cells is important for understanding the molecular basis of physiological processes and diseases<html><a href="#ref_merkx” name=”text_merkx”><sup>[1]</sup></a></html>. Genetically encoded sensors based on fluorescent proteins (FPs) are frequently used for molecular recognition. In this iGEM project we use the fluorescent proteins for providing the light in our display.</p>
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<p>Real-time imaging of biochemical events inside living cells is important for understanding the molecular basis of physiological processes and diseases<html><a href="#ref_merkx" name="text_merkx"><sup>[1]</sup></a></html>. Genetically encoded sensors based on fluorescent proteins (FPs) are frequently used for molecular recognition. In this iGEM project we use the fluorescent proteins for providing the light in our display.</p>
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<p>A GECO is a protein which emits light in the presence of Ca<sup>2+</sup><html><a href="#ref_zhao" name=”text_zhao”><sup>[2]</sup></a></html>. There are two important classes of genetically encoded Ca<sup>2+</sup> indicators. One is called the Forster Resonance Energy Transfer (FRET)-based cameleon type<html><a href="#ref_miyawaki" name=”text_miyawaki”><sup>[3]</sup></a></html> and the other one is called the single Green Fluorescent Protein (GFP) type<html><a href="#ref_nakai" name=”text_nakai”><sup>[4]</sup></a></html>. The GECO protein belongs to the single GFP type. Research has shown that Ca<sup>2+</sup> indicators targeted to the E.coli periplasm can be shifted toward the Ca<sup>2+</sup>-free of Ca<sup>2+</sup> -bound states by manipulation of the environmental Ca<sup>2+</sup> concentration<html><a href="#ref_zhao" name="text_zhao"><sup>[2]</sup></a></html>. Robert E. Campbell et al. named those Ca<sup>2+</sup> indicators GECO’s. R-GECO, G-GECO and B-GECO emit respectively red, green or blue light with each another emission and excitation spectra (Fig. 1 and Fig2).</p>
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<p>A GECO is a protein which emits light in the presence of Ca<sup>2+</sup><html><a href="#ref_zhao" name="text_zhao"><sup>[2]</sup></a></html>. There are two important classes of genetically encoded Ca<sup>2+</sup> indicators. One is called the Forster Resonance Energy Transfer (FRET)-based cameleon type<html><a href="#ref_miyawaki" name="text_miyawaki"><sup>[3]</sup></a></html> and the other one is called the single Green Fluorescent Protein (GFP) type<html><a href="#ref_nakai" name="text_nakai"><sup>[4]</sup></a></html>. The GECO protein belongs to the single GFP type. Research has shown that Ca<sup>2+</sup> indicators targeted to the E.coli periplasm can be shifted toward the Ca<sup>2+</sup>-free of Ca<sup>2+</sup> -bound states by manipulation of the environmental Ca<sup>2+</sup> concentration<html><a href="#ref_zhao" name="text_zhao"><sup>[2]</sup></a></html>. Robert E. Campbell et al. named those Ca<sup>2+</sup> indicators GECO’s. R-GECO, G-GECO and B-GECO emit respectively red, green or blue light with each another emission and excitation spectra (Fig. 1 and Fig2).</p>
<p>The GECO has been implemented into the DNA of the yeast cells with the help of a YES3/CT plasmid (Fig. 3). After transcription and translation the protein emits light if there is enough Ca<sup>2+</sup> in the cytoplasm of the yeastcell. Light emission can only be established if the Ca<sup>2+</sup> threshold in the cytoplasm is exceeded.</p>  
<p>The GECO has been implemented into the DNA of the yeast cells with the help of a YES3/CT plasmid (Fig. 3). After transcription and translation the protein emits light if there is enough Ca<sup>2+</sup> in the cytoplasm of the yeastcell. Light emission can only be established if the Ca<sup>2+</sup> threshold in the cytoplasm is exceeded.</p>  
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<ul>
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<li><a href=#text_merkx” name=”ref_merkx”>[1]</a > Laurens Lindenburg and Maarten Merkx, ‘Colorful Calcium Sensors’, 2012</a></li>
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<li><a href="#text_merkx" name=”ref_merkx”>[1]</a > Laurens Lindenburg and Maarten Merkx, ‘Colorful Calcium Sensors’, 2012</a></li>
<li><a href=”#text_zhao” name=”ref_zhao”>[2]</a> Robert E. Campbell et al., ‘An Expanded Palette of Genetically Encoded Ca<sup>2+</sup> Indicators’, 2011</a></li>
<li><a href=”#text_zhao” name=”ref_zhao”>[2]</a> Robert E. Campbell et al., ‘An Expanded Palette of Genetically Encoded Ca<sup>2+</sup> Indicators’, 2011</a></li>
<li><a href=”#text_miyawaki” name=”ref_miyawaki”>[3]</a> A. Miyawaki et al., Nature 338, 1997</a></li>
<li><a href=”#text_miyawaki” name=”ref_miyawaki”>[3]</a> A. Miyawaki et al., Nature 338, 1997</a></li>

Revision as of 15:03, 24 September 2012