Team:Freiburg/Highlights/2
From 2013.igem.org
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- | <p id="h4"><font size="5"><i style="margin-left: | + | <p id="h4"><font size="5"><i style="margin-left:85px;">In the last months we were able to ...</i></font></p> |
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<li> ... construct a catalytically inactive version of <b>Cas9</b> and thus generate a <b>DNA binding protein</b>.</li> | <li> ... construct a catalytically inactive version of <b>Cas9</b> and thus generate a <b>DNA binding protein</b>.</li> | ||
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6 opportunities with our uniCAS toolkit | 6 opportunities with our uniCAS toolkit | ||
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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! | 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! | ||
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+ | <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;"> | ||
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dCas9 - The Heart of our toolkit | dCas9 - The Heart of our toolkit | ||
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<!--simple DNA binding protein is the foundation of our project and all effectors used in this toolkit are fused to it.--> | <!--simple DNA binding protein is the foundation of our project and all effectors used in this toolkit are fused to it.--> | ||
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+ | <div class="slide-right-col" style="margin-top:-450px"> | ||
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+ | <!-- Cas video --> | ||
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+ | Activation | ||
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+ | 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. | ||
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+ | Repression | ||
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+ | The fusion of the transcriptional repressor domain KRAB leads to synthetic repression of gene expression. With this construct a strong repression could be observed. | ||
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<!-- <img src="https://static.igem.org/mediawiki/2013/2/20/Freiburg2013-Highlights-KRAB3.png" style="width:500px"> --> | <!-- <img src="https://static.igem.org/mediawiki/2013/2/20/Freiburg2013-Highlights-KRAB3.png" style="width:500px"> --> | ||
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+ | 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. | ||
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<!-- <img style="width:500px" src="https://static.igem.org/mediawiki/2013/6/6d/Freiburg2013-Highlights-Epigenetik2.png"> --> | <!-- <img style="width:500px" src="https://static.igem.org/mediawiki/2013/6/6d/Freiburg2013-Highlights-Epigenetik2.png"> --> | ||
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+ | Multiple Targeting | ||
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+ | 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! | ||
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+ | uniBAss | ||
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+ | 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. | ||
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<!--<img src="https://static.igem.org/mediawiki/2013/f/f6/UniBASS_Freiburg_2013.JPG" style="width:500px">--> | <!--<img src="https://static.igem.org/mediawiki/2013/f/f6/UniBASS_Freiburg_2013.JPG" style="width:500px">--> | ||
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- | <td style="height:30px | + | <td style="height:30px; "> <b>Figure 5: uniBAss - universal Binding Assay</b><br> |
First step ... | First step ... | ||
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Latest revision as of 16:50, 4 October 2013
HIGHLIGHTS