Team:ITESM-CEM/Project/Data

From 2014.igem.org

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       <sub2><a href="#Two" style="color: #FFF;">Two</a></sub2>
       <sub2><a href="#Two" style="color: #FFF;">Two</a></sub2>
       <sub2><a href="#Three" style="color: #FFF;">Three</a></sub2>
       <sub2><a href="#Three" style="color: #FFF;">Three</a></sub2>
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       <sub2><a href="#Four" style="color: #FFF;">Four</a></sub2>
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       <sub2><a href="#Four" style="color: #FFF;">Protein Expression</a></sub2>
       <sub2><a href="#Five" style="color: #FFF;">NeoR</a></sub2>
       <sub2><a href="#Five" style="color: #FFF;">NeoR</a></sub2>
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<a name="Four"><h2>Experiment Four</h2></a>  
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<a name="Four"><h2>Recombinant Protein Expression</h2></a>  
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       <p style="text-align: justify; text-justify: inter-word;">The samples were loaded in a 15% acrylamide gel, using Precision Plus Protein TM Dual Color Standards, for 20 minutes/90 V for the stacking gel and 60 minutes/150V for the resolving gel. The results are now presented:<br></p>
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<p class="centeredImage"><img src="https://static.igem.org/mediawiki/2014/2/20/Gel_1.jpg" width="530" height="408" hspace="20" BORDER=10></p><br>
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<p style="text-align: justify; text-justify: inter-word;">Only the samples shown in the image before were the ones that presented notable bands that represent our protein of interest. As expected, the most remarked band is the one of the time 3, which means that inductions was taken correctly and more protein was produced, in other words, the protein was overexpressing. The band marked with the arrow represents a protein that weights approximately 34 kDa, which corresponds to the molecular weight of oxoacyl reductase according to ExPASy’s Compute pI/MW tool.</p><br><br>
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<p style="text-align: justify; text-justify: inter-word;">7-dehydratase was analyzed by SDS-PAGE in a 15% acrylamide gel using Precision Plus Protein TM Unstained Standards, for 20 minutes/90 V for the stacking gel and 90 minutes/110V for the resolving gel. Four samples were taken, including one before and after induction with IPTG, one from the soluble phase and one from the inclusion bodies; all prepared with Laemmli buffer. The results are shown in the image below.<br><br> </p>
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<p class="centeredImage"><img src="https://static.igem.org/mediawiki/2014/f/f5/SDS_dehidratasa.jpg" width="530" height="408" hspace="10" BORDER=10></p><br>
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<p style="text-align: justify; text-justify: inter-word;">No analysis of solubility was realized due to the quantity of protein. It was supposed to be done exactly the same than oxoacyl reductase, as the protein was found in a notable way in the inclusion bodies as shown in the lane 5.</p><br>
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<p style="text-align: justify; text-justify: inter-word;">For both enzymes no further work was done. After the identification of each of them, and after the analysis of solubility, the proteins have to be purified by affinity chromatography with a Invitrogen Ni-NTA Agarose column, taking the advantage of the histidine tag added to the protein. After the purification, enzymatic parameters would be determined by the interaction of the enzymes with the substrate; 7β-Hydroxycholesterol for cholesterol oxidase, and 5-Cholesten-3β-ol-7-one for 7-dehydratase and oxoacyl reductase. </p><br><br>
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Revision as of 21:43, 17 October 2014

TEC-CEM | Project

ITESM-CEM | Enzy7-K me

Project 3014
 

Results & Discussion

If you choose to create a model during your project, please write about it here. Modeling is not an essential part of iGEM, but we encourage any and all teams to model some aspect of their project. See previous "Best Model" awards for more information.

Experiment One

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Vivamus fringilla porta nisi sed dictum. Aliquam at rutrum nisl. Ut eros quam, condimentum sed pretium et, euismod vitae tellus. Curabitur eu blandit massa. Vestibulum at euismod purus. Sed quis pretium ligula. Cras non tristique nulla. Nunc finibus risus non purus malesuada viverra. Cras bibendum augue eu lorem faucibus, nec auctor mi iaculis. Vivamus sagittis, mi non gravida accumsan, odio dui pellentesque leo, a varius dui mauris eu diam. Maecenas laoreet tellus sed porta efficitur.

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Experiment Two

Etiam tempus mi pulvinar purus iaculis bibendum. Vivamus vel risus eu enim volutpat finibus. Nullam bibendum est sit amet arcu lobortis, id laoreet ex vestibulum. Curabitur fringilla eleifend lacus, nec ornare nibh imperdiet sed. Maecenas vel velit consectetur, tempus libero quis, consectetur ex. Nulla porttitor pharetra velit. Curabitur tristique, dolor ut sodales euismod, diam diam ultricies arcu, at tincidunt tellus neque in felis. Etiam ut tempor ligula. Sed at dui sapien.

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Experiment Three

Proin aliquam nibh id elementum pellentesque. Suspendisse mollis est ut felis sagittis mollis. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Etiam accumsan ex ante, quis lobortis erat fermentum ac. Sed et egestas libero. Donec id diam vitae leo consequat interdum. Ut in sem in quam pretium finibus vitae non lectus.

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Recombinant Protein Expression

The samples were loaded in a 15% acrylamide gel, using Precision Plus Protein TM Dual Color Standards, for 20 minutes/90 V for the stacking gel and 60 minutes/150V for the resolving gel. The results are now presented:


Only the samples shown in the image before were the ones that presented notable bands that represent our protein of interest. As expected, the most remarked band is the one of the time 3, which means that inductions was taken correctly and more protein was produced, in other words, the protein was overexpressing. The band marked with the arrow represents a protein that weights approximately 34 kDa, which corresponds to the molecular weight of oxoacyl reductase according to ExPASy’s Compute pI/MW tool.



7-dehydratase was analyzed by SDS-PAGE in a 15% acrylamide gel using Precision Plus Protein TM Unstained Standards, for 20 minutes/90 V for the stacking gel and 90 minutes/110V for the resolving gel. Four samples were taken, including one before and after induction with IPTG, one from the soluble phase and one from the inclusion bodies; all prepared with Laemmli buffer. The results are shown in the image below.


No analysis of solubility was realized due to the quantity of protein. It was supposed to be done exactly the same than oxoacyl reductase, as the protein was found in a notable way in the inclusion bodies as shown in the lane 5.


For both enzymes no further work was done. After the identification of each of them, and after the analysis of solubility, the proteins have to be purified by affinity chromatography with a Invitrogen Ni-NTA Agarose column, taking the advantage of the histidine tag added to the protein. After the purification, enzymatic parameters would be determined by the interaction of the enzymes with the substrate; 7β-Hydroxycholesterol for cholesterol oxidase, and 5-Cholesten-3β-ol-7-one for 7-dehydratase and oxoacyl reductase.



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NeoR Results

Results obtained from experimentation.









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