Team:Freiburg/Highlights/2
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<body> | <body> | ||
+ | |||
+ | <div id="content_main"> | ||
+ | <p id="h1"> | ||
+ | HIGHLIGHTS | ||
+ | </p> | ||
- | |||
<div class="flexslider"> | <div class="flexslider"> | ||
<ul class="slides"> | <ul class="slides"> | ||
<li> | <li> | ||
<div class="frontpage-slide"> | <div class="frontpage-slide"> | ||
- | < | + | |
- | + | ||
- | + | <div id="preample"> | |
- | + | <p id="h4"><font size="5"><i style="margin-left:85px;">In the last months we were able to ...</i></font></p> | |
- | + | ||
- | + | <ul style="font-size:18px; margin-left:100px;"> | |
- | + | ||
- | + | <li> ... construct a catalytically inactive version of <b>Cas9</b> and thus generate a <b>DNA binding protein</b>.</li> | |
- | </div> | + | <li> ... combine this modified dCas9 with different transcriptional <b>effectors</b>.</li> |
+ | <li> ... express this fusion proteins in various <b>mammalian</b> cell lines.</li> | ||
+ | <li> ... <b>control</b> mammalian gene expression via our modified CRISPR/Cas fusion proteins.</li> | ||
+ | <li> ... build devices for controling gene expression by <b>light stimulus</b>.</li> | ||
+ | <li> ... provide an RNA plasmid for easily inserting sequences for crRNAs which target every desired target.</li> | ||
+ | <li> ... build an online tool that generates customized <b>manuals</b> for using our toolkit</li> | ||
+ | <li> ... develop a method to assess the <b>DNA binding capacity</b> of our dCas9-fusion proteins.</li> | ||
+ | <li>... make our dCas9 <b>accessible</b> to the whole iGEM community by mutating illegal iGEM restriction sites</b>.</li> | ||
+ | <li><i> ... In summary, we can now offer a universally applicable <b>toolkit</b> for gene regulation.</i></li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <div class=clear></div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li> | ||
+ | <div class="frontpage-slide"> <div id="left_column"> | ||
+ | |||
+ | <p id="headline"> | ||
+ | 6 opportunities with our uniCAS toolkit | ||
+ | </p> | ||
+ | <p> | ||
+ | We provide 3 different effectors, 2 methods & 1 effector controller! Using our toolkit it's possible to efficiently activate or repress genes. We also provide devices for effector controling by light. Use our custom-tailored <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/toolkit"> Manual Tool </a> to generate your individual manual for your needs of gene regulation. Further it's possible to target not only one, but multiple genes of interest! And we established <a id="link" href="">uniBAss</a> - our universal Binding Assay. Best of all: It's open source and in iGEM standard! | ||
+ | </p> | ||
+ | </div> | ||
+ | <div class="slide-right-col"><div id="right_column"> | ||
+ | <img id="main_images" src="https://static.igem.org/mediawiki/2013/f/f5/Freiburg2013-toolkit-for-highlight2.png" style="width:280px; margin-left:100px; margin-top:-10px;"> | ||
+ | |||
+ | </div></div> | ||
<div class=clear></div> | <div class=clear></div> | ||
</div> | </div> | ||
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<li> | <li> | ||
<div class="frontpage-slide"> | <div class="frontpage-slide"> | ||
- | < | + | <div id="left_column"> <p id="headline"> |
+ | dCas9 - The Heart of our toolkit | ||
+ | </p> | ||
+ | <p> | ||
+ | We started by mutating the DNA cleavage site in the Cas9 protein and generated a sequence specific DNA binding protein that is relying on a protein-RNA-DNA interaction. We are now able to influence the DNA binding locus and can direct the protein to requested DNA targets.<br> | ||
+ | This is the heart of our toolkit. A protein that allows multiple and sequence specifid DNA binding.<br><br> | ||
+ | For simple and flexible regulation we do need now effectors, that can be fused to the protein.<br> | ||
+ | |||
+ | |||
+ | <!--simple DNA binding protein is the foundation of our project and all effectors used in this toolkit are fused to it.--> | ||
+ | |||
+ | |||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | <div class="slide-right-col" style="margin-top:-450px"> | ||
+ | <div id="right_column"> <div style="text-align: center;"> | ||
+ | |||
+ | <!-- Cas video --> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | <div class=clear></div> | ||
+ | </div> | ||
+ | </li> | ||
+ | <li> | ||
+ | <div class="frontpage-slide"><div id="left_column"> | ||
+ | <p id="headline"> | ||
+ | Activation | ||
+ | </p> | ||
+ | <p> | ||
+ | |||
+ | For activation we choosed VP-16 as effector. Through the transactivating function of VP-16 the expression of these genes will be enhanced.<br> | ||
+ | We achieved up to 30-fold activation. | ||
+ | </p> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | |||
<div class="slide-right-col"> | <div class="slide-right-col"> | ||
- | + | <div id="right_column"> | |
- | + | <div> | |
- | + | <table class="imgtxt"> | |
- | + | <tbody><tr> | |
- | + | <td> <img src="https://static.igem.org/mediawiki/2013/5/5b/Freiburg2013-Highlights-VP16.png"> </td> | |
- | + | </tr> | |
- | + | <tr> | |
+ | <td style="height:10px"> <b>Figure 1:</b> Different error values plotted in increasing order.<br> </td> | ||
+ | </tr> | ||
+ | </tbody></table> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
<div class=clear></div> | <div class=clear></div> | ||
</div> | </div> | ||
- | </li> | + | </li> |
- | + | <li> | |
<div class="frontpage-slide"> | <div class="frontpage-slide"> | ||
- | + | <div id="left_column"> | |
+ | |||
+ | <p id="headline"> | ||
+ | Repression | ||
+ | </p> | ||
+ | <p> | ||
+ | The fusion of the transcriptional repressor domain KRAB leads to synthetic repression of gene expression. With this construct a strong repression could be observed. | ||
+ | </p> | ||
+ | |||
+ | |||
+ | |||
+ | </div> | ||
<div class="slide-right-col"> | <div class="slide-right-col"> | ||
- | <div class=" | + | <div id="right_column"> |
- | + | ||
- | + | <!-- <img src="https://static.igem.org/mediawiki/2013/2/20/Freiburg2013-Highlights-KRAB3.png" style="width:500px"> --> | |
- | <p><a href=" | + | |
- | + | <div> | |
+ | <table id="Fig8" class="imgtxt"> | ||
+ | <tbody><tr> | ||
+ | <td> <img src="https://static.igem.org/mediawiki/2013/2/20/Freiburg2013-Highlights-KRAB3.png"> </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="height:10px"> <b>Figure 2:</b> Different error values plotted in increasing order.<br> </td> | ||
+ | </tr> | ||
+ | </tbody></table> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | <div class=clear></div> | ||
+ | </div> | ||
+ | </li> | ||
+ | <li> | ||
+ | <div class="frontpage-slide"> | ||
+ | <div id="left_column"> | ||
+ | <p id="headline"> | ||
+ | Chromatin modification (Repression) | ||
+ | </p> | ||
+ | <p > | ||
+ | Specific chromatin modification was achieved by fusing the histone methyltransferase G9a to dCas9. With this protein we are able to specifically repress endogenous gene expression. | ||
+ | </p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </div> | ||
+ | |||
+ | <div class="slide-right-col"> | ||
+ | <div id="right_column"> | ||
+ | |||
+ | <!-- <img style="width:500px" src="https://static.igem.org/mediawiki/2013/6/6d/Freiburg2013-Highlights-Epigenetik2.png"> --> | ||
+ | |||
+ | <div> | ||
+ | <table id="Fig8" class="imgtxt"> | ||
+ | <tbody><tr> | ||
+ | <td> <img src="https://static.igem.org/mediawiki/2013/6/6d/Freiburg2013-Highlights-Epigenetik2.png"> </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="height:10px"> <b>Figure 3:</b> Different error values plotted in increasing order.<br> </td> | ||
+ | </tr> | ||
+ | </tbody></table> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | <div class=clear></div> | ||
+ | </div> | ||
+ | </li> | ||
+ | <li> | ||
+ | <div class="frontpage-slide"> | ||
+ | <div id="left_column"> | ||
+ | |||
+ | <p id="headline" > | ||
+ | Multiple Targeting | ||
+ | </p> | ||
+ | <p> | ||
+ | One of the biggest advantages of the CRISPR/Cas9 system is that only one protein is required for targeting several DNA sites: For a new target there has to be just another guiding RNA. We designed an RNA plasmid, "<a id="link" href="https://2013.igem.org/Team:Freiburg/Project/crrna#multiple_targeting">RNAimer</a>", containing this RNA. For multiple targeting different RNAimers can be easily combined using the iGEM BioBrick system.<br><br> | ||
+ | And as the results show, multiple targeting is possible and even better! | ||
+ | </p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </div> | ||
+ | <div class="slide-right-col"> | ||
+ | <div id="right_column"> | ||
+ | |||
+ | <div> | ||
+ | <table id="Fig8" class="imgtxt"> | ||
+ | <tbody><tr> | ||
+ | <td> <img src="https://static.igem.org/mediawiki/2013/6/6b/Freiburg2013-Highlights-Multiple2.png"> </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="height:10px"> <b>Figure 4:</b> Different error values plotted in increasing order.<br> </td> | ||
+ | </tr> | ||
+ | </tbody></table> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | <div class=clear></div> | ||
+ | </div> | ||
+ | </li> | ||
+ | <li> | ||
+ | <div class="frontpage-slide"> <div id="left_column"> | ||
+ | |||
+ | <p id="headline"> | ||
+ | uniBAss | ||
+ | </p> | ||
+ | <p> | ||
+ | We developed a novel and innovative ELISA based method to quantify the binding efficiency of our proteins. We called this binding assay uniBAss. This is a powerful tool for characterizing the modified dCas9 by assessing its DNA binding capacity with high throughput capabilities. | ||
+ | </p> | ||
+ | |||
+ | |||
+ | </div< | ||
+ | |||
+ | |||
+ | |||
+ | <div class="slide-right-col"> | ||
+ | <div id="right_column" style="margin-top:-300px; margin-left:480px;"> | ||
+ | |||
+ | <!--<img src="https://static.igem.org/mediawiki/2013/f/f6/UniBASS_Freiburg_2013.JPG" style="width:500px">--> | ||
+ | |||
+ | <div> | ||
+ | <table class="imgtxt" width="500px"> | ||
+ | <tbody><tr> | ||
+ | <td> | ||
+ | <div> | ||
+ | <img style="width:400px; margin-left:-20px; margin-top:10px;" src="https://static.igem.org/mediawiki/2013/f/f6/UniBASS_Freiburg_2013.JPG" > | ||
+ | </div></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="height:30px; "> <b>Figure 5: uniBAss - universal Binding Assay</b><br> | ||
+ | First step ... | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody></table> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
+ | </div> | ||
</div> | </div> | ||
<div class=clear></div> | <div class=clear></div> | ||
</div> | </div> | ||
- | </li> | + | </li> |
+ | |||
</ul> | </ul> | ||
</div> | </div> |
Latest revision as of 16:50, 4 October 2013
HIGHLIGHTS