Team:TU-Eindhoven/LEC/BioBricks

From 2012.igem.org

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<p>Although, these BioBricks are prepared in a more efficient way using our own protocol, the permission we needed to apply the GECO BioBricks to registry library was not easy to obtain. You can read more about this struggle at the 'Considerations' page.</p>
<p>Although, these BioBricks are prepared in a more efficient way using our own protocol, the permission we needed to apply the GECO BioBricks to registry library was not easy to obtain. You can read more about this struggle at the 'Considerations' page.</p>
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[[Image:Growth_rate.JPG|400px|right|link=]]
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<h3>Characterization</h3>
<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>
<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>
<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).  
<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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[[File:SDS-gel.jpg|500px|left]]</p>
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<h3>Calcium titration</h3>
<h3>Calcium titration</h3>
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<h3>References</h3>
<h3>References</h3>
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Revision as of 09:36, 26 September 2012