Difference between revisions of "Team:Sorbonne U Paris/Demonstrate"

 
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        <meta name="description" content="Sugar is the main source of energy in synthetic biology. To bring a solution to the dramatical environmental impacts of its massive production this year iGEM Sorbonne U Paris wants to engineer green microalgae, Chlamydomonas Reinhardtti, to allow an ecofriendly sugar production within marin water. In order to spread the use of microalgae we will create a toolkit for C. reinhardtti with a synthetic retrotransposon to generate in vivo continuous directed evolution ">
 
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<h1>Demonstrate</h1>
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<h3>Gold Medal Criterion #4</h3>
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<div class="container"><article class="col-md-10"><h1>Demonstrate</h1>
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<p class="introduction-text">Our aim on part 1 is to show how we determined whether we succeed a cloning or not. Then
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on part 2 we will explain how we intend to demonstrate that our SugaRevolution system does
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work. </p></article></div>
  
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Teams that can show their system working under real world conditions are usually good at impressing the judges in iGEM. To achieve gold medal criterion #4, convince the judges that your project works. There are many ways in which your project working could be demonstrated, so there is more than one way to meet this requirement. This gold medal criterion was introduced in 2016, so check our what 2016 teams did to achieve their gold medals!
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<h2 class="title-h2">1. Determination of cloning succes  </h2>
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Please see the <a href="https://2018.igem.org/Judging/Medals">2018 Medals Page</a> for more information.
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<p>Here is an example of our cloning experiments. You can see below the photo of  the gel on which all our first cloned and digested fragments have migrated.  </p>
  
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    <figcaption style="padding: 10px;"><b>Figure 1 : Migration of checking ligation. </b>This two pictures above come from the same gel, but the black one is overexposed to show the tiniest band.
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<p> After we have made our first level 0 cloning, we digested them with the adequate digestion enzyme, cutting next to the insert sites, here BsaI. If the product of the digestion had the expected length we would have collected them, using gel to pcr DNA purification, and would have sent our DNA samples to sequencing. In our case the DNA was not of the correct length, probably because the sequence cloned was not the right one or because something went wrong while cloning.  Then, we performed the cloning procedure once until it works.. </p>
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<p>In the picture above (figure 1) you can see that Paro1 (0.8kbp), Paro 2 (0.8kbp), 3’LTR (0.02kbp), 5’3 LTR  (0.07kpb) having the expected length. We sent 3’2 and 3’4 (3’LTR), paro 1, paro 2 and 5’3 LTR to sequencing. Gag/Pol 3 and 5 were the only Gag/Pol subsequences that had the expected length (4.3kbp). These were also sent to sequencing.
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</p>
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<p> To have an otherview of our progression in cloning, please refer to the results page. Precision on parts that were cloned and on their sequence quality are explained there.</p>
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<p>The project is still now on progress. Indeed, cloning experiments and assessment of our retrotransposon activity in Chlamydomonas are conceived and planned for weeks to come. </p>
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Latest revision as of 02:24, 18 October 2018

Demonstrate

Our aim on part 1 is to show how we determined whether we succeed a cloning or not. Then on part 2 we will explain how we intend to demonstrate that our SugaRevolution system does work.

1. Determination of cloning succes

Here is an example of our cloning experiments. You can see below the photo of the gel on which all our first cloned and digested fragments have migrated.

Demonstrate
Figure 1 : Migration of checking ligation. This two pictures above come from the same gel, but the black one is overexposed to show the tiniest band.

After we have made our first level 0 cloning, we digested them with the adequate digestion enzyme, cutting next to the insert sites, here BsaI. If the product of the digestion had the expected length we would have collected them, using gel to pcr DNA purification, and would have sent our DNA samples to sequencing. In our case the DNA was not of the correct length, probably because the sequence cloned was not the right one or because something went wrong while cloning. Then, we performed the cloning procedure once until it works..

In the picture above (figure 1) you can see that Paro1 (0.8kbp), Paro 2 (0.8kbp), 3’LTR (0.02kbp), 5’3 LTR (0.07kpb) having the expected length. We sent 3’2 and 3’4 (3’LTR), paro 1, paro 2 and 5’3 LTR to sequencing. Gag/Pol 3 and 5 were the only Gag/Pol subsequences that had the expected length (4.3kbp). These were also sent to sequencing.

To have an otherview of our progression in cloning, please refer to the results page. Precision on parts that were cloned and on their sequence quality are explained there.

The project is still now on progress. Indeed, cloning experiments and assessment of our retrotransposon activity in Chlamydomonas are conceived and planned for weeks to come.

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