Difference between revisions of "Team:SIAT-SCIE/Results"

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<h1>Results</h1>
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<p>Here you can describe the results of your project and your future plans. </p>
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<h5>What should this page contain?</h5>
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<body style="width:1263px;margin-left:auto;margin-right:auto;">
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<ul>
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    <div id="body">
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<li> Clearly and objectively describe the results of your work.</li>
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<li> Future plans for the project. </li>
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<li> Considerations for replicating the experiments. </li>
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</ul>
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<h5>You should also describe what your results mean: </h5>
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        <!--default for floating navigation-->
  
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<ul>
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        <div id="comic">
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<li> Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for. </li>
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            <p>
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<li> Show data, but remember all measurement and characterization data must be on part pages in the Registry. </li>
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                <img src="https://static.igem.org/mediawiki/2017/c/cf/SIAT-SCIE_result.png" alt="comic" />
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<li> Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project. </li>
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            </p>
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</ul>
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        </div>
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      <div id="content">
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          <h2 style="font-family:'Avenir'; font-size:23px;">Vector Construction</h2>
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          <h4 style="font-family:'Avenir'; font-size:23px;"> -Overview:</h4>
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          <p style="font-family:'Avenir'; font-size:23px;">We are using pMD-19 plasmid backbone for CAHS and p15A plasmid backbone of DSUP.</p>
 +
          <p style="font-family:'Avenir'; font-size:23px;">
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              At the beginning, we tried to use pET28a for protein extraction, but due to the unknown region on the sequencing, we gave it up.
 +
          </p>
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          <p style="font-family:'Avenir'; font-size:23px;">
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              Here, we will show the construction of pMD-19-pTet-tetR-CAHS plasmids, since p15A-pTac-sfGFP-Dsup was constructed by Bluepha.
 +
          </p>
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          <p style="font-family:'Avenir'; font-size:23px;">
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              The assembly method we choose was similar to Gibson assembly, it requires overlaps on both ends, and use exonuclease to cut out the ends for ligation.
 +
          </p>
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          <br />
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          <h4 style="font-family:'Avenir'; font-size:23px;">-PCR:</h4>
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          <p style="font-family:'Avenir'; font-size:23px;">We used PCR to add the overlapping fragments on our DNA fragment. </p>
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          <img src="https://static.igem.org/mediawiki/2017/d/d4/SIAT-SCIE_result1.png" title="Result1" style="width:1000px;margin-left:130px;" />
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          <img src="https://static.igem.org/mediawiki/2017/3/32/SIAT-SCIE_result2.png" title="Result2" style="width:1260px;"/>
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          <p style="font-family:'Avenir'; font-size:23px;">The result of it is shown by gel electrophoresis.</p>
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          <img src="https://static.igem.org/mediawiki/2017/9/98/SIAT-SCIE_result3.png" title="Result3" style="width:600px;margin-left:350px;"/>
  
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</div>
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          <h4 style="font-family:'Avenir'; font-size:23px;">-Ligation</h4>
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          <p style="font-family:'Avenir'; font-size:23px;">After gel-extraction, we added 0.06 pmol of DNA fragments and 0.03 pmol of backbone, incubated for 30 minutes under 37°C in PCR machine, the product was cooled in ice for three minutes, and immediately transform it into the DH5α Chemical Component cell.</p>
 +
          <h4 style="font-family:'Avenir'; font-size:23px;">-Clone PCR</h4>
 +
          <p style="font-family:'Avenir'; font-size:23px;">After leaving the DH5α containing previous products in 37°C overnight, we carried out the Clone PCR to verify the ligation</p>
 +
          <p style="font-family:'Avenir'; font-size:23px;">The results are shown by gel electrophoresis.</p>
 +
          <img src="https://static.igem.org/mediawiki/2017/3/3b/SIAT-SCIE_result4.png" title="Result4"  style="width:800px;margin-left:250px;"/>
 +
          <p style="font-family:'Avenir'; font-size:23px;">According to this, we found that Dsup was not successfully inserted into the backbone. After several times, we decided to send it to Bluepha, for the construction of Dsup.</p>
 +
          <h4 style="font-family:'Avenir'; font-size:23px;">-Sequencing</h4>
 +
          <p style="font-family:'Avenir'; font-size:23px;">Samples were sent to BGI for sequencing, the results are complementary to our design.</p>
 +
          <img src="https://static.igem.org/mediawiki/2017/a/aa/SIAT-SCIE_result5.png" title="Result5" style="width:1000px;margin-left:130px;" />
  
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<div class="clear"></div>
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          <h2 style="font-family:'Avenir'; font-size:23px;">Protein Expression</h2>
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          <h4 style="font-family:'Avenir'; font-size:23px;">-Inducer Adding</h4>
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          <img src="https://static.igem.org/mediawiki/2017/7/71/SIAT-SCIE_result6.jpg" title="Result6" style="width:1000px;margin-left:130px;"/>
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          <h4 style="font-family:'Avenir'; font-size:23px;">SDS Page</h4>
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          <img src="https://static.igem.org/mediawiki/2017/0/03/SIAT-SCIE_result7.png" title="Result7"style="width:1000px;margin-left:130px;" />
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          <img src="https://static.igem.org/mediawiki/2017/e/ed/SIAT-SCIE_result7-1.jpg" title="Result7-1" style="width:600px;margin-left:350px;"/>
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          <p style="font-family:'Avenir'; font-size:23px;">Unfortunately, the SDS-PAGE experiments were done badly, there were always mistakes, including sample contamination, failure in protein expression, faulty in stain and decolor, so we did not get a very reliable result on this.</p>
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          <p style="font-family:'Avenir'; font-size:23px;">
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              BUT WE MADE IT!
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            </p>
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          <p style="font-family:'Avenir'; font-size:23px;">
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              DURING OUR LAST TRIAL ON OCTOBER 27TH
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            </p>
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              <img src=" https://static.igem.org/mediawiki/2017/2/2f/SIAT-SCIE_result16.jpg" alt="Result16"style="width:1000px;margin-left:130px;" />
  
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<div class="column half_size" >
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              <h2 style="font-family:'Avenir'; font-size:23px;">
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                  Desiccation
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              </h2>
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<h5> Project Achievements </h5>
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              <h4 style="font-family:'Avenir'; font-size:23px;">Dilution</h4>
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<p style="font-family:'Avenir'; font-size:23px;">In order to find out the survival rate, we had to find out the change of CFU, the number of bacteria is too large in protein expressed broth, since they are in the stationary phase, so we had to do gradient dilution.</p>
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
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          <img src="https://static.igem.org/mediawiki/2017/9/9c/SIAT-SCIE_result8.jpg" title="Result8" style="width:600px;margin-left:330px;transform: rotate(270deg);margin-top:-200px;"/>
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          <img src="https://static.igem.org/mediawiki/2017/1/15/SIAT-SCIE_result9.jpg" title="Result9" style="width:600px;margin-left:330px;transform: rotate(270deg);margin-top:-450px;"/>
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<ul>
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          <img src="https://static.igem.org/mediawiki/2017/3/3c/SIAT-SCIE_result9-1.jpg" title="Result9-1"style="width:600px;margin-left:330px;transform: rotate(270deg);margin-top:-310px;" />
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<li>A list of linked bullet points of the successful results during your project</li>
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          <img src="https://static.igem.org/mediawiki/2017/5/51/SIAT-SCIE_result10.jpg" title="Result10" style="width:600px;margin-left:330px;transform: rotate(270deg);margin-top:-320px;"/>
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<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
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          <img src="https://static.igem.org/mediawiki/2017/e/ee/SIAT-SCIE_result11.jpg" title="Result11" style="width:600px;margin-left:330px;transform: rotate(270deg);margin-top:-430px;margin-bottom:-240px"/>
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</ul>
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          <p style="font-family:'Avenir'; font-size:23px;">And then spread the diluted broth to the petri dish, showed that when the broth was diluted to 10^13 times, we could count the cfu.</p>
 +
    <h4 style="font-family:'Avenir'; font-size:23px;">Desiccation</h4>
 +
    <p style="font-family:'Avenir'; font-size:23px;">We used centrifuge machine and concentrator to remove the water.</p>
 +
          <img src="https://static.igem.org/mediawiki/2017/c/c4/SIAT-SCIE_result12.jpg" title="Result12" style="width:800px;margin-left:250px;"/>
 +
   
 +
          <h2 style="font-family:'Avenir'; font-size:23px;">Irradiation</h2>
 +
          <p style="font-family:'Avenir'; font-size:23px;">More information, please go to our <a href="https://2017.igem.org/Team:SIAT-SCIE/Demonstrate" alt="demostrate">demonstration page.</a></p>
 +
          <img src="https://static.igem.org/mediawiki/2017/7/75/SIAT-SCIE_result13.jpg" title="Result13"  style="width:800px;margin-left:250px;"/>
  
 
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</div>
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<div class="column half_size" >
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<h5>Inspiration</h5>
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<p>See how other teams presented their results.</p>
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<ul>
+
−
<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
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−
<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
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</ul>
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</div>
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{{SIAT-SCIE footer}}

Latest revision as of 23:00, 1 November 2017

comic

Vector Construction

-Overview:

We are using pMD-19 plasmid backbone for CAHS and p15A plasmid backbone of DSUP.

At the beginning, we tried to use pET28a for protein extraction, but due to the unknown region on the sequencing, we gave it up.

Here, we will show the construction of pMD-19-pTet-tetR-CAHS plasmids, since p15A-pTac-sfGFP-Dsup was constructed by Bluepha.

The assembly method we choose was similar to Gibson assembly, it requires overlaps on both ends, and use exonuclease to cut out the ends for ligation.


-PCR:

We used PCR to add the overlapping fragments on our DNA fragment.

The result of it is shown by gel electrophoresis.

-Ligation

After gel-extraction, we added 0.06 pmol of DNA fragments and 0.03 pmol of backbone, incubated for 30 minutes under 37°C in PCR machine, the product was cooled in ice for three minutes, and immediately transform it into the DH5α Chemical Component cell.

-Clone PCR

After leaving the DH5α containing previous products in 37°C overnight, we carried out the Clone PCR to verify the ligation

The results are shown by gel electrophoresis.

According to this, we found that Dsup was not successfully inserted into the backbone. After several times, we decided to send it to Bluepha, for the construction of Dsup.

-Sequencing

Samples were sent to BGI for sequencing, the results are complementary to our design.

Protein Expression

-Inducer Adding

SDS Page

Unfortunately, the SDS-PAGE experiments were done badly, there were always mistakes, including sample contamination, failure in protein expression, faulty in stain and decolor, so we did not get a very reliable result on this.

BUT WE MADE IT!

DURING OUR LAST TRIAL ON OCTOBER 27TH

Result16

Desiccation

Dilution

In order to find out the survival rate, we had to find out the change of CFU, the number of bacteria is too large in protein expressed broth, since they are in the stationary phase, so we had to do gradient dilution.

And then spread the diluted broth to the petri dish, showed that when the broth was diluted to 10^13 times, we could count the cfu.

Desiccation

We used centrifuge machine and concentrator to remove the water.

Irradiation

More information, please go to our demonstration page.

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