Difference between revisions of "Team:UPMC PARIS/Experiments"

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<h1 class="section-heading content-title">Collaborations</h1>
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<h1 class="section-heading content-title">Experiments</h1>
 
 
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<p class="text-left">Collaboration between teams has been an important part of our project. Indeed, the data sharing and the joint work are not only necessary in this competition but more generally in the scientific field.
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So, we were lucky to collaborate with different teams around the world. We have discussed about our projects and about collaborative work on common themes. The exchanges and discussions were mostly done during meetings, through e-mail and skype session.</p>
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<h3 class="content-title">Collaboration with Evry-Paris Saclay Team</h3>
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<h3 class="content-title">Part IV - Determination of the best purification system</h3>
  
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<p class="text-left">We invited Evry-Paris Saclay iGEM team to join us to share with the pupils their knowledge, their lab skills and their vision of iGEM experience. Thus, we collaborated with two members of their team. Rose attended the practical class with us (1st session). She was very patient with the pupils and helped us in the long process preparation of the material and solutions on the day of the class. Yanis was here for the 2nd session (Interpretation of results, introduction to synthetic biology, Presentation of iGEM competition and our projects, overview of biology at university). He has an actual pedagogical talent and the pupils immediately appreciated him. This was a great illustration for the kids that scientific research is based on collaboration and team work. 
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<h4>Introduction</h4>
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We also collaborated on another side of the Human Practices: law. We met Evry team’s jurists and we had the opportunity to ask our question and learn a lot about intellectual property and legal issues. 
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Thank you very much Evry-Paris Saclay, we really had a smashing time working with you.</p>
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<p class="text-left">The goal is to test and compare the Gluthation purification system and the hisTrap one to systems to see which one would be the most effective for our box.
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To do so, we have purified GST-10XHIS proteins with both of the purification system. We have determined the GST enzymatic activity before and after the purifications to obtain the purification yields. Finally, we have done SDS-PAGE chromatographies to semi-quantify the proteins and compare the results with those obtained with the enzymatic activity.
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</p>
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<h4>Results</h4>
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<p>
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We cultured DH5α containing plasmid pet21 GST-10XHIS. We measured the OD every hour in order to follow the turbidity and thus determine the time corresponding to the exponential phase of growth(fig.11). 
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<p>
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Figure.11: Growth curve of the DH5α containing the PET 21 GST-10HIS
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<br>
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All the points data come from a mean done by data collected from 5 cultures. Cultures were incubated in LB at37°C. The DO(600nm) was calculated every hour.
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</p>
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<h4>Analysis of GST enzymatic activity: </h4>
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<p>
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We calculated the GST activity of the sample taken before and after the purification. We took 5µL of the aliquotes to do the reaction. We then calculated the total reaction for the all volume before and after lysis that can be found in table.1. 
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The table.2 presents the the activities and purification yields obtained for both of the purification.
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<br>
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For the HisTrap column, we observe a purification yield equal to 72.1 %.
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<br>
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For the Gluthation Agarose Resin we observe a purification yield equal to 32%.
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<br>
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<table>
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        <thead>
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          <tr>
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              <th>Fraction</th>
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              <th>Enzymatic activity From 5 µL (Δabs/min) </th>
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              <th>Total enzymatic activity (Δabs/min) </th>
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              <th>Purification Yield  </th>
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          </tr>
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        </thead>
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        <tbody>
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          <tr>
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            <td>Alvin</td>
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            <td>Eclair</td>
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            <td>$0.87</td>
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            <td>$0.87</td>
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          </tr>
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          <tr>
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            <td>Alan</td>
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            <td>Jellybean</td>
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            <td>$3.76</td>
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            <td>$0.87</td>
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          </tr>
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          <tr>
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            <td>Jonathan</td>
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            <td>Lollipop</td>
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            <td>$7.00</td>
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            <td>$0.87</td>
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          </tr>
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 +
          <tr>
 +
            <td>Jonathan</td>
 +
            <td>Lollipop</td>
 +
            <td>$7.00</td>
 +
            <td>$0.87</td>
 +
          </tr>
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        </tbody>
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      </table>
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</p>
  
 
<h3 class="content-title">Collaboration with Team Harvard</h3>
 
<h3 class="content-title">Collaboration with Team Harvard</h3>
 
 
 
<p class="text-left">Harvard team had the idea to gather ethical, academic and political issues concerning projects of 6 teams: Harvard, Linkoping, AQA_Unesp, Aalto-Helsinki, Hamburg and ours. Indeed all the teams’ projects have relevance to biomanufacturing and to creating  biologically synthesized useful substances at a commercial scale. Thus, gathering perspectives from people involved in these fields will enable us to better understand our subject as it is complex and controversial.
 
<p class="text-left">Harvard team had the idea to gather ethical, academic and political issues concerning projects of 6 teams: Harvard, Linkoping, AQA_Unesp, Aalto-Helsinki, Hamburg and ours. Indeed all the teams’ projects have relevance to biomanufacturing and to creating  biologically synthesized useful substances at a commercial scale. Thus, gathering perspectives from people involved in these fields will enable us to better understand our subject as it is complex and controversial.
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<br>
 
To do so, they created a document where the 5 teams could add their questions, complete others issues and had their opinion. From all these questions, they created a Global Perspective Outreach survey that we shared on our Facebook page. Finally, we were sent the collected data which we used for our Human Practices.
 
To do so, they created a document where the 5 teams could add their questions, complete others issues and had their opinion. From all these questions, they created a Global Perspective Outreach survey that we shared on our Facebook page. Finally, we were sent the collected data which we used for our Human Practices.
 
<br><br>
 
<br><br>

Revision as of 16:44, 5 December 2017

Impact UPMC

UPMC PARIS


Experiments


Part IV - Determination of the best purification system

Introduction

The goal is to test and compare the Gluthation purification system and the hisTrap one to systems to see which one would be the most effective for our box. To do so, we have purified GST-10XHIS proteins with both of the purification system. We have determined the GST enzymatic activity before and after the purifications to obtain the purification yields. Finally, we have done SDS-PAGE chromatographies to semi-quantify the proteins and compare the results with those obtained with the enzymatic activity.

Results

We cultured DH5α containing plasmid pet21 GST-10XHIS. We measured the OD every hour in order to follow the turbidity and thus determine the time corresponding to the exponential phase of growth(fig.11).

Figure.11: Growth curve of the DH5α containing the PET 21 GST-10HIS
All the points data come from a mean done by data collected from 5 cultures. Cultures were incubated in LB at37°C. The DO(600nm) was calculated every hour.

Analysis of GST enzymatic activity:

We calculated the GST activity of the sample taken before and after the purification. We took 5µL of the aliquotes to do the reaction. We then calculated the total reaction for the all volume before and after lysis that can be found in table.1. The table.2 presents the the activities and purification yields obtained for both of the purification.
For the HisTrap column, we observe a purification yield equal to 72.1 %.
For the Gluthation Agarose Resin we observe a purification yield equal to 32%.

Fraction Enzymatic activity From 5 µL (Δabs/min) Total enzymatic activity (Δabs/min) Purification Yield
Alvin Eclair $0.87 $0.87
Alan Jellybean $3.76 $0.87
Jonathan Lollipop $7.00 $0.87
Jonathan Lollipop $7.00 $0.87

Collaboration with Team Harvard

Harvard team had the idea to gather ethical, academic and political issues concerning projects of 6 teams: Harvard, Linkoping, AQA_Unesp, Aalto-Helsinki, Hamburg and ours. Indeed all the teams’ projects have relevance to biomanufacturing and to creating biologically synthesized useful substances at a commercial scale. Thus, gathering perspectives from people involved in these fields will enable us to better understand our subject as it is complex and controversial.
To do so, they created a document where the 5 teams could add their questions, complete others issues and had their opinion. From all these questions, they created a Global Perspective Outreach survey that we shared on our Facebook page. Finally, we were sent the collected data which we used for our Human Practices.

Global Perspective Outreach Question Sheet:
https://docs.google.com/document/d/1rtxv2tz-lPitS1TWYdmaYDiCWiK-a4v5lEd77xOydEA/edit?usp=sharing

Global Perspective Outreach Survey:
https://docs.google.com/forms/d/1hdlfDTzIzjLxdI5Obn35zn1U2sPNzRSJan_CCWNGmao/edit?usp=sharing

Collected Data:
https://docs.google.com/spreadsheets/d/1cTS0aEbsyruC1wJYp0EqW3nwyheSbChfm_HPTcNLbQY/edit?usp=sharing


Collaboration with Team Dusseldorf-Köln

Both our teams met at the meet-up organized by Delft team in Delft in The Netherland. We talked together about our projects and realized they had something in common: they both include an optogenetic system. After that we kept being in touch through e-mails and skype sessions. As our project went on, we had a hard time finding a way to emit the specific wave length we needed. Therefore, we asked Düsselforf-Köhln team for help and asked them how they managed to do it. They generously gave us the schematics for 3D-printing their lightning box so that we could 3D print our lightning system.