Difference between revisions of "Team:Calgary/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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<h3>What should this page contain?</h3>
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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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<h3>Describe what your results mean </h3>
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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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<li> Show data, but remember all measurement and characterization data must be on part pages in the Registry. </li>
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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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<h3> Project Achievements </h3>
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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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<li>A list of linked bullet points of the successful results during your project</li>
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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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<h3>Inspiration</h3>
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<p>See how other teams presented their results.</p>
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<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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    <title>Team:Calgary/Results - 2018.igem.org</title>
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        <h1 style="transform: translateY(-50%)" class="infotitle">RESULTS</h1>
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        <br>
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        <div class="infosection">
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            <h3 class="infosubtitle">Results Overview</h3>
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            <br>
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            <p style="text-indent: 0px"> The Calgary iGEM team was able to compare the effectiveness of 7 different sgRNA candidates at directing CRISPR/Cas9 cleavage. These results are semi-quantitative, providing the relative rate of indel formation which can be used as a surrogate measure for cleavage activity and NHEJ. The CCR5-17R sgRNA candidate was found to be the most effective, with an indel formation rate of 11.1%. A more thorough explanation can be found on the <a href="https://2018.igem.org/Team:Calgary/CRISPR"> CRISPR <a/> page.</p>
  
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<img class="info-img" src="https://static.igem.org/mediawiki/2018/d/d4/T--Calgary--IndelEfficiencyComparison.png">
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            <br>
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            <p style="text-indent: 0px"> With respect to CRISPR knock-ins, the Calgary iGEM team was unable to sequence confirm the targeted insertion of an FRT site using CRISPR/Cas9. However, attempted integration showed significant indel formation, demonstrating that Cas9 was successfully cleaving the targeted DNA. The issue therefore lay with the incorporation of template DNA through homology directed repair. The attached graphs demonstrate indel activity of the attempted insertion. More information regarding these graphs can be found on the <a href="https://2018.igem.org/Team:Calgary/CRISPR"> CRISPR <a/> page. </p>
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                    <img style="display: block; margin: 0 auto;" class="info-img2-div" src="https://static.igem.org/mediawiki/2018/e/e4/T--Calgary--Alltransfectiongraph.jpg">
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                    <p class = "caption">Figure 11. Complete Transfection:  26.2%</p>
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                    <img class="info-img2-div" src="https://static.igem.org/mediawiki/2018/0/05/T--Calgary--NoFlpOgraph.jpg">
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                    <p class = "caption">Figure 12. No-FlpO Control:  26.2%</p>
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            </div>
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<br>
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<p> Despite no sequencing data confirming insertion, the amount of DNA sequenced and analyzed did not cover the entire cell population. This leaves the possibility that integration was successful, albeit at extremely low efficiency. To further support this hypothesis, we found that a small number of cells trasnfected with all components (CRISPR/Cas9 and sgRNA, FLPo-expressing plasmid, and plasmid with hybrid beta-resolvase: <a href="http://parts.igem.org/Part:BBa_K2605006" target="_blank">BBa_K2605006</a>) successfully grew in puromycin-containing cell media. Puromycin resistance was placed promoter-less on a plasmid which was designed to enter the genome through Flp recombinase. Thus, growth in puromycin indicates the possibility that not only did some cells successfully receive an FRT insert from CRISPR, but they also had our desired plasmid integrated into their genome using our recombinase system at the FRT site. A deeper explanation of this result can be found in the <a href="https://2018.igem.org/Team:Calgary/FLP-Beta"> Flp-Beta </a> page. We are optimistic about this qualitative measure of success and hope to expand upon these results in the brief time before the Jamboree.
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                    <img style="display: block; margin: 0 auto;" class="info-img2-div" src="https://static.igem.org/mediawiki/2018/d/d6/T--Calgary--LivingHEK.jpg">
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                    <p class = "caption">Living HEK cells, adhered to plate</p>
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                    <img class="info-img2-div" src="https://static.igem.org/mediawiki/2018/1/18/T--Calgary--DeadHEK.jpg">
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                    <p class = "caption">Dead HEK cells, no adherence to plate</p>
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<img class="info-img" src="https://static.igem.org/mediawiki/2018/3/33/T--Calgary--LoneHEK.jpg">
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<p class = "caption">Single HEK cell adhering to plate in puromycin antiobiotic, following transfection with all components</p>
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Latest revision as of 03:18, 18 October 2018

Team:Calgary/Results - 2018.igem.org

RESULTS


Results Overview


The Calgary iGEM team was able to compare the effectiveness of 7 different sgRNA candidates at directing CRISPR/Cas9 cleavage. These results are semi-quantitative, providing the relative rate of indel formation which can be used as a surrogate measure for cleavage activity and NHEJ. The CCR5-17R sgRNA candidate was found to be the most effective, with an indel formation rate of 11.1%. A more thorough explanation can be found on the CRISPR page.


With respect to CRISPR knock-ins, the Calgary iGEM team was unable to sequence confirm the targeted insertion of an FRT site using CRISPR/Cas9. However, attempted integration showed significant indel formation, demonstrating that Cas9 was successfully cleaving the targeted DNA. The issue therefore lay with the incorporation of template DNA through homology directed repair. The attached graphs demonstrate indel activity of the attempted insertion. More information regarding these graphs can be found on the CRISPR page.


Figure 11. Complete Transfection: 26.2%

Figure 12. No-FlpO Control: 26.2%


Despite no sequencing data confirming insertion, the amount of DNA sequenced and analyzed did not cover the entire cell population. This leaves the possibility that integration was successful, albeit at extremely low efficiency. To further support this hypothesis, we found that a small number of cells trasnfected with all components (CRISPR/Cas9 and sgRNA, FLPo-expressing plasmid, and plasmid with hybrid beta-resolvase: BBa_K2605006) successfully grew in puromycin-containing cell media. Puromycin resistance was placed promoter-less on a plasmid which was designed to enter the genome through Flp recombinase. Thus, growth in puromycin indicates the possibility that not only did some cells successfully receive an FRT insert from CRISPR, but they also had our desired plasmid integrated into their genome using our recombinase system at the FRT site. A deeper explanation of this result can be found in the Flp-Beta page. We are optimistic about this qualitative measure of success and hope to expand upon these results in the brief time before the Jamboree.

Living HEK cells, adhered to plate

Dead HEK cells, no adherence to plate

Single HEK cell adhering to plate in puromycin antiobiotic, following transfection with all components