Team:TU-Eindhoven/LEC/BioBricks

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Latest revision as of 18:27, 26 September 2012

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-{{:Team:TU-Eindhoven/Templates/head|image=https://static.igem.org/mediawiki/2012/9/97/Ourbiobricks_ehv.jpg}}
-<p>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>
-<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>
-[[Image:Growth_rate.JPG|400px|right|link=]]
-<h3>Characterization</h3>
-<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>
-<h3>Harvesting and Purification</h3>
-<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).
-[[Image:SDS-gel.jpg|500px|left]]</p>
-<h3>Calcium titration</h3>
-{{:Team:TU-Eindhoven/Templates/footer}}