Difference between revisions of "Team:TP-CC San Diego/Results"

 
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<body>
 
<body>
  
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<h2 class = "main-title"> Results </h2>
 
  
 +
<h2 class = "main-title fade-in"> Results </h2>
 
<div class = "text-wrapper">
 
<div class = "text-wrapper">
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    <h3 class = "day"> DAY 1 </h3>
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    <div class = "description">  
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<h3 class ="day"> Effects of ecDNA </h3>
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        <p>  
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<p style="padding: 2% 11%;font-size:16px;">There is a significantly depressed rate of cell growth due to the treatment. New cell growth, measured in the increased of density, was significantly retarded in the induced group. Based on the single factor analysis of variance test, our data after testing declares a p-value deemed low enough to reject the null hypothesis.</p>
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            <b>SUMMARY of July 17th: </b>  
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<div class="cellcontainer">
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         </p>
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<img src="https://static.igem.org/mediawiki/2017/b/ba/T--TP-CC_San_Diego--celldenovertime.png">
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         <ul>
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<img src="https://static.igem.org/mediawiki/2017/6/6f/T--TP-CC_San_Diego--analysisofvariance.png">
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            <li>DNA extraction from GBM39 cells </li>
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</div>
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            <li>Agarose gel electrophoresis to confirm DNA </li>
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<h3 class ="day">Parts</h3>
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         </ul>
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<div class="rowfirst">
 +
<div class="item1">
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         <img src="https://static.igem.org/mediawiki/2017/archive/d/d5/20171102014221%21T--TP-CC_San_Diego--k2369000.png">
 +
         <span class="caption">BBa_K2369000</span>
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</div>
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<div class="item2">
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<img src="https://static.igem.org/mediawiki/2017/archive/7/7b/20171102014242%21T--TP-CC_San_Diego--k2369001.png">
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<span class="caption">BBa_K2369001</span>
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</div>
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<div class="item3">
 +
<img src="https://static.igem.org/mediawiki/2017/archive/f/f7/20171102014255%21T--TP-CC_San_Diego--k2369002.png">
 +
<span class="caption">BBa_K2369002</span>
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</div>
 +
</div>
 +
 
 +
<h3 class ="day">Images</h3>
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<h3 class = "text-title"> EGFR Testing </h3>
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<center>
 +
<div class="containertop2">
 +
    <div class="item">
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         <img src="https://static.igem.org/mediawiki/2017/a/a1/T--TP-CC_San_Diego--EGFR.png" width="400">
 +
        <span class="caption"> EGFR vs. Normalized Vector Expansion</span>
 
     </div>
 
     </div>
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     <h3 class = "day"> DAY 2 </h3>
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     <div class="item">
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        <img src="https://static.igem.org/mediawiki/2017/e/e2/T--TP-CC_San_Diego--GBM39.png" width="500">
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    <div class = "description">
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         <span class="caption"> E1G4</span>
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        <p>
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            <b>SUMMARY of July 17th: </b>
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−
        </p>
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−
         <ul>
+
−
            <li>DNA extraction from GBM39 cells </li>
+
−
            <li>Agarose gel electrophoresis to confirm DNA </li>
+
−
        </ul>
+
 
     </div>
 
     </div>
 +
</div>
 +
</center>
 +
 +
<h3 class = "text-title"> References </h3>
 +
<center>
 +
<div class="containertop2">
 +
    <div class="item">
 +
        <img src="https://static.igem.org/mediawiki/2017/f/f5/T--TP-CC_San_Diego--finalplasmid.png" height="300">
 +
        <span class="caption"> Guide RNA plasmid design  </span>
 +
    </div>
 +
    <div class="item">
 +
        <img src="https://static.igem.org/mediawiki/2017/6/6c/T--TP-CC_San_Diego--100bpLadder.png" height="300">
 +
        <span class="caption"> 100 Basepair ladder</span>
 +
    </div>
 +
</div>
 +
</center>
 +
 +
<h3 class = "text-title">guide RNAs </h3>
 +
<center>
 +
<div class="containertop">
 +
    <div class="item">
 +
        <img class = "pictures" src="https://static.igem.org/mediawiki/2017/f/f2/T--TP-CC_San_Diego--e1g2.jpg" width="500">
 +
        <span class="caption"> E1G2</span>
 +
    </div>
 +
    <div class="item">
 +
        <img class = "pictures" src="https://static.igem.org/mediawiki/2017/8/8c/T--TP-CC_San_Diego--e1g4.jpg" width="500">
 +
        <span class="caption"> E1G4</span>
 +
    </div>
 +
    <div class="item">
 +
    <img class = "pictures" src="https://static.igem.org/mediawiki/2017/2/2b/T--TP-CC_San_Diego--e8g2.jpg" width="500">
 +
        <span class="caption"> E8G2</span>
 +
    </div>
 +
    <div class="item">
 +
    <img class = "pictures" src="https://static.igem.org/mediawiki/2017/9/94/T--TP-CC_San_Diego--e8g4.jpg" width="500">
 +
        <span class="caption"> E8G3 </span>
 +
    </div>
 +
</div>
 +
</center>
 +
 +
<h3 class = "text-title"> Gels </h3>
 +
<center>
 +
<div class="containertop3">
 +
    <div class="item">
 +
        <img src="https://static.igem.org/mediawiki/2017/f/f5/T--TP-CC_San_Diego--TLCV2.png" width="400">
 +
        <span class="caption"> Digested TLCV2 Vector </span>
 +
    </div>
 +
    <div class="item">
 +
        <img src="https://static.igem.org/mediawiki/2017/8/83/T--TP-CC_San_Diego--pcr.png" width="280">
 +
        <span class="caption"> PCR </span>
 +
    </div>
 +
    <div class="item">
 +
        <img src="https://static.igem.org/mediawiki/2017/0/0f/T--TP-CC_San_Diego--Gel%281%29.jpg" width="350">
 +
        <span class="caption"> GBM39 Genomic DNA </span>
 +
    </div>
 +
</div>
 +
</center>
 +
 +
 +
<h3 class ="day">Summary</h3>
 +
<p style="padding: 0 11% 4% 11%;font-size: 16px;">
 +
We were able to confirm that the GBM39 cell line had a substantial copy number of the EGFR gene. From there, we designed 4 guide RNAs. Two from exon 1 and two from exon 8. We were unable to design any gRNA using exons 2-7 because in the GBM-39 cell line, that portion of the EGFR gene is mutated. Using the TLCV2 backbone, we removed the stuffer sequence and inserted the guideRNAs in its place.  We were successful with constructing 3 gRNAs but the 4th failed to clone twice. After Sequencing our plasmids, we transfected the guideRNAs into GBM39 cells. Currently, we are monitoring the growth of the cells, and confluency and growth of the cancerous cells has been, over the past 2 months, negatively affected in a statistically significant manner - with over a reduction in total confluency growth of over 50% when compared to the control, suggesting that our treatment has an inhibitory effect upon cancer cell growth.
 +
</p>
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</div>
 
</div>
 
</body>
 
</body>
 
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</html>

Latest revision as of 03:40, 2 November 2017

Results

Effects of ecDNA

There is a significantly depressed rate of cell growth due to the treatment. New cell growth, measured in the increased of density, was significantly retarded in the induced group. Based on the single factor analysis of variance test, our data after testing declares a p-value deemed low enough to reject the null hypothesis.

Parts

BBa_K2369000
BBa_K2369001
BBa_K2369002

Images

EGFR Testing

EGFR vs. Normalized Vector Expansion
E1G4

References

Guide RNA plasmid design
100 Basepair ladder

guide RNAs

E1G2
E1G4
E8G2
E8G3

Gels

Digested TLCV2 Vector
PCR
GBM39 Genomic DNA

Summary

We were able to confirm that the GBM39 cell line had a substantial copy number of the EGFR gene. From there, we designed 4 guide RNAs. Two from exon 1 and two from exon 8. We were unable to design any gRNA using exons 2-7 because in the GBM-39 cell line, that portion of the EGFR gene is mutated. Using the TLCV2 backbone, we removed the stuffer sequence and inserted the guideRNAs in its place. We were successful with constructing 3 gRNAs but the 4th failed to clone twice. After Sequencing our plasmids, we transfected the guideRNAs into GBM39 cells. Currently, we are monitoring the growth of the cells, and confluency and growth of the cancerous cells has been, over the past 2 months, negatively affected in a statistically significant manner - with over a reduction in total confluency growth of over 50% when compared to the control, suggesting that our treatment has an inhibitory effect upon cancer cell growth.