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| - | {{:Team:TU-Eindhoven/Templates/header}}
| + | #REDIRECT [[Team:TU-Eindhoven/Parts]] |
| - | {{:Team:TU-Eindhoven/Templates/head|image=https://static.igem.org/mediawiki/2012/9/97/Ourbiobricks_ehv.jpg}}
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| - | <groupparts>iGEM012 TU-Eindhoven</groupparts>
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| - | <p>Our iGEM team prepared three different BioBricks: BBa_K881000 (R-GECO), BBa_K881001 (G-GECO), BBa_K881002 (B-GECO). These three GECO proteins, which are obtained from Zhao et al. 2011<a href="#ref_zhao" name="text_zhao"><sup>[1]</sup></a></html>,
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| - | The GECO’s, R-GECO (red), G-GECO (green) and B-GECO (blue) are the biobricks we will send to the registry. Those biobricks can be used in yeast and in E.coli cells. The protocol attached with the biobrick-library didn’t work for us. We used our own restriction and ligation protocols to develop the biobrick.</p>
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| - | <p>The permission we needed to add the GECO’s to registry library was not easy to get. You can read more about this struggle at the Considerations-page.</p>
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| - | [[Image:Growth_rate.JPG|400px|right|link=]] | + | |
| - | <h3>Characterization</h3>
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| - | <p>E. Coli strain BL21 transformed with Part BBa_K881000 and Part BBa_K881001 ligated into vector pET_28a were cultured in LB media containing 100 µg/ml kanamycin for ~4 hours at 37°C. The growth rate was followed by use of OD600 measurements (see figure on the right). Then the culture was induced at an OD600 of 0,82 absorption units with 100 µg/ml IPTG.</p>
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| - | <h3>Harvesting and Purification</h3>
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| - | <p>After induction for 15 hours, the cells were harvested and lysed with 5ml BugBuster containing 1µl/ml Benzonase. The supernatant of this suspension was put on a Ni-NTA column for purification and afterwards put on a PD10 column and washed with a TRIS buffer containing NaCl to remove all the calcium molecules. Then, an SDS-gel was prepared to check the purity of the GECO proteins (See figure below).
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| - | [[Image:SDS-gel.jpg|500px|left]]</p>
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| - | <h3>Calcium titration</h3>
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| - | <h3>References</h3>
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| - | <html>
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| - | <ul>
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| - | <li><a href="#text_miseta" name="ref_miseta">[1]</a> Zhao, Y. et al. ''An Expanded Palette of Genetically Encoded Ca2+ Indicators'' in ''Science'' 30 September 2011,
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| - | Vol. 333 no. 6051 pp. 1888-1891, DOI: <a href="http://dx.doi.org/10.1126/science.1208592">10.1126/science.1208592</a>)</li>
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| - | </ul>
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| - | </html>
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| - | {{:Team:TU-Eindhoven/Templates/footer}}
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