Difference between revisions of "Team:EPF Lausanne/Notebook/Yeast"

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</style>
 
</style>
 
</head>
 
</head>
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<body>
 
  
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    <div class="page-h promotion">
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<div class="classy-bar">
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        <div class="container">
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    <div class="container">
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            <div class="row">
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        <div class="row">
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                <div class="col-md-12 text-center">
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            <div class="col-md-12 text-center">
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                    <h3>saccharomyces cerevisiae</h3>
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                <h3>saccharomyces cerevisiae</h3>
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                </div>
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             </div>
 
             </div>
 
         </div>
 
         </div>
 
     </div>
 
     </div>
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</div>
  
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    <body data-spy="scroll" data-target="#myScrollspy" data-offset="20">
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<!--Protocols have to be added here-->
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     <div class="prot-section">
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<body id="Content" data-spy="scroll" data-target=".scrollspy" data-offset="20">
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        <div class="row">
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            <div class="col col-md-3">
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<div class="prot-section">
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                <nav id="affix-nav" class="sidebar hidden-sm hidden-xs">
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    <div class="row">
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                    <ul class="nav nav-pills nav-stacked" data-spy="affix" data-offset-top="200" data-offset-bottom="600">
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        <div class="col col-md-3 scrollspy">
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                      <li class="active"><a href="#express_dCas9-VP64">Express dCas9-VP64</a></li>
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            <ul id="nav" class="nav hidden-xs hidden-sm" data-spy="affix">
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                      <ul>
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              <li class="active"><a href="#express_dCas9-VP64">Express dCas9-VP64</a>
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                          <li><a href="#integrate_pTPGI_dCas9_VP64">Integrate pTPGI_dCas9_VP64</a></li>
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                <ul class="nav">
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                          <li><a href="#wb_dCas9-VP64">Western Blot of dCas9-VP64</a></li>
+
                  <li><a href="#integrate_pTPGI_dCas9_VP64">Integrate pTPGI_dCas9_VP64</a></li>
−
                        </ul>
+
                  <li><a href="#wb_dCas9-VP64">Western Blot of dCas9-VP64</a></li>
 +
                </ul>
 +
                </li>
  
 +
                <li><a href="#integrate_reporter_plasmid">Integrate reporter plasmid</a>
 +
                <ul class="nav">
 +
                  <li><a href="#linearise_reporter_plasmid">Linearise the plasmid by PCR</a></li>
 +
                  <li><a href="#synthesize_promoters">Synthesize promoters</a></li>
 +
                  <li><a href="#PCRoverlaps_prom">Add Gibson overlaps to CYC promoters by PCR</a></li>
 +
                  <li><a href="#gibsonprom">Gibson assembly of promoters in reporter plasmid</a></li>
 +
                  <li><a href="#integrate_reporters_plasmids">Integrate the reporter plasmids</a></li>
 +
                </ul>
 +
                </li>
  
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                      <li><a href="#integrate_reporter_plasmid">Integrate reporter plasmid</a></li>
+
                <li><a href="#Integrate_and_express_gRNAs">Integrate and express gRNAs</a>
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                      <ul>
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                <ul class="nav">
−
                          <li><a href="#linearise_reporter_plasmid">Linearise the plasmid by PCR</a></li>
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                  <li><a href="#PCR-amplify_the_gRNAs_expressing_cassettes">PCR-amplify the gRNAs expressing cassettes</a></li>
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                          <li><a href="#synthesize_promoters">Synthesize promoters</a></li>
+
                  <li><a href="#PCR_out_DsRed2">PCR out DsRed2</a></li>
−
                          <li><a href="#PCRoverlaps_prom">Add Gibson overlaps to CYC promoters by PCR</a></li>
+
                  <li><a href="#PCRoverlaps_gRNAs">Add Gibson overlaps to gRNAs by PCR</a></li>
−
                          <li><a href="#gibsonprom">Gibson assembly of promoters in reporter plasmid</a></li>
+
                  <li><a href="#gibson_gRNAs">Gibson assembly of gRNAs</a></li>
−
                          <li><a href="#integrate_reporters_plasmids">Integrate the reporter plasmids</a></li>
+
                </ul>
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                        </ul>
+
                </li>
 +
            </ul>
  
−
                        <li><a href="#Integrate_and_express_gRNAs">Integrate and express gRNAs</a></li>
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            </nav>
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                        <ul>
+
        </div>  
−
                          <li><a href="#PCR-amplify_the_gRNAs_expressing_cassettes">PCR-amplify the gRNAs expressing cassettes</a></li>
+
        <div class="col-md-9">
−
                          <li><a href="#PCR_out_DsRed2">PCR out DsRed2</a></li>
+
<!--Integrate pDCAS9-->
−
                          <li><a href="#PCRoverlaps_gRNAs">Add Gibson overlaps to gRNAs by PCR</a></li>
+
            <section id="integrate_pTPGI_dCas9_VP64" class="panel">
−
                          <li><a href="#gibson_gRNAs">Gibson assembly of gRNAs</a></li>
+
                <h1><small>Express dCas9-VP64</small></br>Integrate pTPGI_dCas9_VP64</h1>
−
                      </ul>
+
                <p>We received plasmid pTPGI_dCas9_VP64 from Addgene. The plasmid was found from the article "Tunable and multifunctional eukaryotic transcription factors based on Crispr/Cas". After glycerol stocks and miniprep, we performed a restriction analysis to check the identity of our plasmid. We linearised the plasmid, in order to integrate it into yeast genome. For more details about our experiments, see <a target="_blank" href="https://static.igem.org/mediawiki/2015/3/30/EPF_Lausanne_Journal_integrate_dCas9-VP64.pdf">here</a>. Only our fourth trial to integrate the plasmid was successful.</p>
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                    </ul>
+
  
−
                 </nav>
+
                 <h2>Materials and methods</h2>
−
            </div>
+
−
           
+
−
            <div class="col-sm-9">
+
−
 
+
−
    <!--Integrate pDCAS9-->
+
−
    <div id="integrate_pTPGI_dCas9_VP64" class="panel">
+
−
    <h1><small>Express dCas9-VP64</small></br>Integrate pTPGI_dCas9_VP64</h1>
+
−
        <p><small>We received plasmid pTPGI_dCas9_VP64 from Addgene. The plasmid was found from the article "Tunable and multifunctional eukaryotic transcription factors based on Crispr/Cas". After glycerol stocks and miniprep, we performed a restriction analysis to check the identity of our plasmid. We linearised the plasmid, in order to integrate it into yeast genome. For more details about our experiments, see <a target="_blank" href="https://static.igem.org/mediawiki/2015/3/30/EPF_Lausanne_Journal_integrate_dCas9-VP64.pdf">here</a>. Only our fourth trial to integrate the plasmid was successful.
+
−
        </small></p>
+
−
 
+
−
            <h2>Materials and methods</h2>
+
 
                 <ul>
 
                 <ul>
 
                     <li>Glycerol stocks</li>
 
                     <li>Glycerol stocks</li>
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                 </ul>
 
                 </ul>
  
−
            <h2>Results</h2>
+
                <h2>Results</h2>
−
                 <div id="divleft1">
+
                 <div class="row">
−
                     <figure>
+
                     <div class="col-md-6">
−
                    <a href="https://static.igem.org/mediawiki/2015/3/3e/EPF_Lausanne_Gel_dCas9.png"><img src="https://static.igem.org/mediawiki/2015/3/3e/EPF_Lausanne_Gel_dCas9.png" alt="dCas9 gel" width="75%"></a>
+
                        <figure>
−
                    <figcaption>Fig.1 - Gel of the dCas9.</figcaption>
+
                        <a href="https://static.igem.org/mediawiki/2015/3/3e/EPF_Lausanne_Gel_dCas9.png"><img src="https://static.igem.org/mediawiki/2015/3/3e/EPF_Lausanne_Gel_dCas9.png" alt="dCas9 gel" width="75%"></a>
−
                    </figure>
+
                        <figcaption>Fig.1 - Gel of the dCas9.</figcaption>
 +
                        </figure>
 +
                    </div>
 +
                    <div class="col-md-6">
 +
                        <p>We used four different sets of enzymes for the restriction analysis. Linearized pTPGI_dCas9_VP64 is expected to be 10'987 bp. We observe that the gel (fig.1) corresponds to the <a target="_blank" href="https://static.igem.org/mediawiki/2015/2/2b/EPF_Lausanne_Expected_Gel_dCas9.pdf">expected one</a>. </br>
 +
                        The plasmid was linearised both with EagI HF and NotI HF prior to integration. We integrated each linearised plasmid to obtain two different yeast strains.
 +
                        </p>
 +
                    </div>
 
                 </div>
 
                 </div>
−
                <div id="divright1">
+
            </section>
−
                    <p><small>We used four different sets of enzymes for the restriction analysis. Linearized pTPGI_dCas9_VP64 is expected to be 10'987 bp. We observe that the gel (fig.1) corresponds to the <a target="_blank" href="https://static.igem.org/mediawiki/2015/2/2b/EPF_Lausanne_Expected_Gel_dCas9.pdf">expected one</a>. </br>
+
−
                    The plasmid was linearised both with EagI HF and NotI HF prior to integration. We integrated each linearised plasmid to obtain two different yeast strains.
+
−
                    </small></p>
+
−
                </div>
+
−
    </div>
+
 
      
 
      
  
−
    <!--Western Blot pDCAS9-->
+
<!--Western Blot pDCAS9-->
−
    <div id="wb_dCas9-VP64" class="panel">
+
            <section id="wb_dCas9-VP64" class="panel">
−
    <h1><small>Express dCas9-VP64</small></br>Western Blot of dCas9-VP64</h1>
+
                <h1><small>Express dCas9-VP64</small></br>Western Blot of dCas9-VP64</h1>
−
        <p><small>The Western Blot allows to check the expression of dCas9.
+
                <p>The Western Blot allows to check the expression of dCas9.</p>
−
        </small></p>
+
  
−
            <h2>Materials and methods</h2>
+
                <h2>Materials and methods</h2>
 
                 <ul>
 
                 <ul>
 
                     <li>Western Blot A AJOUTER SUR PROTOCOLS</li>
 
                     <li>Western Blot A AJOUTER SUR PROTOCOLS</li>
 
                 </ul>
 
                 </ul>
  
−
            <h2>Results</h2>
+
                <h2>Results</h2>
−
                 <div id="divleft2">
+
                 <div class="row">
−
                     <img src="" style="width:75%">
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                     <div class="col-md-6">
 +
                        <figure>
 +
                        <a href="Link"><img src="image" alt="name the image" width="75%"></a>
 +
                        <figcaption>Insert legend here</figcaption>
 +
                        </figure>
 +
                    </div>
 +
                    <div class="col-md-6">
 +
                        <p>Results of Western Blot.</p>
 +
                    </div>
 
                 </div>
 
                 </div>
−
                <div id="divright2">
+
            </section>
−
                    <p><small>Results of Western Blot.
+
−
                    </small></p>
+
−
                </div>
+
−
    </div>
+
  
 
<!--INTEGRATE REPORTER PLASMID - LINEARISE REPORTER PLASMID-->
 
<!--INTEGRATE REPORTER PLASMID - LINEARISE REPORTER PLASMID-->
−
    <div id="linearise_reporter_plasmid" class="panel">  
+
            <section id="linearise_reporter_plasmid" class="panel">  
−
    <h1><small>Integrate reporter plasmid</small></br>Linearise reporter plasmid</h1>
+
                <h1><small>Integrate reporter plasmid</small></br>Linearise reporter plasmid</h1>
−
        <p><small>We received the plasmid pCYC1m_yeGFP from Addgene. The plasmid was found from the article "Tunable and multifunctional eukaryotic transcription factors based on Crispr/Cas". After glycerol stocks and minipreps. We linearised the plasmid by PCR according to the following <a target="_blank" href="https://static.igem.org/mediawiki/2015/d/d3/EPF_Lausanne_Linearise_pCYC_yeGFP.pdf">protocol</a>.
+
                <p>>We received the plasmid pCYC1m_yeGFP from Addgene. The plasmid was found from the article "Tunable and multifunctional eukaryotic transcription factors based on Crispr/Cas". After glycerol stocks and minipreps. We linearised the plasmid by PCR according to the following <a target="_blank" href="https://static.igem.org/mediawiki/2015/d/d3/EPF_Lausanne_Linearise_pCYC_yeGFP.pdf">protocol</a>.</p>
−
        </small></p>
+
  
−
            <h2>Materials and methods</h2>
+
                <h2>Materials and methods</h2>
 
                 <ul>
 
                 <ul>
 
                     <li>Polymerase Chain Reaction</li>
 
                     <li>Polymerase Chain Reaction</li>
 
                 </ul>
 
                 </ul>
  
−
            <h2>Results</h2>
+
                <h2>Results</h2>
−
                 <div id="divleft1">
+
                 <div class="row">
−
                     <img src="" style="width:80%">
+
                     <div row="col-md-6">
 +
                        <figure>
 +
                        <a href="Link"><img src="image" alt="name the image" width="75%"></a>
 +
                        <figcaption>Insert legend here</figcaption>
 +
                        </figure>
 +
                    </div>
 +
                    <div row="col-md-6">
 +
                        <p>Linearized pCYC1m_yeGFP is expected to be 5'485 bp. Running an agarose gel electrophoresis allowed to verify that we had linearised the plasmid.</p>
 +
                    </div>
 
                 </div>
 
                 </div>
−
                <div id="divright1">
+
            </section>
−
                    <p><small>Linearized pCYC1m_yeGFP is expected to be 5'485 bp. Running an agarose gel electrophoresis allowed to verify that we had linearised the plasmid.</br>
+
−
                    </small></p>
+
−
                </div>
+
−
    </div>
+
  
  
  
 
<!--INTEGRATE REPORTER PLASMID - SYNTHESIZE PROMOTERS-->
 
<!--INTEGRATE REPORTER PLASMID - SYNTHESIZE PROMOTERS-->
−
    <div id="synthesize_promoters" class="panel">  
+
            <section id="synthesize_promoters" class="panel">  
−
    <h1><small>Integrate reporter plasmid</small></br>Synthesize promoters</h1>
+
            <h1><small>Integrate reporter plasmid</small></br>Synthesize promoters</h1>
−
        <p><small>From the article "Tunable and multifunctional eukaryotic transcription factors based on Crispr/Cas", we learnt that different regions on the promoters could lead to activation or inhibition when dCas9-VP64 was bound. We chose to modify the region of strongest activation, named c3, and the two regions that lead to the strongest inhibition, c6 and c7. We synthesized promoters CYC_0, CYC_1, CYC_2 and CYC_3. The only differences between them are the three regions c3, c6 and c7. The promoter CYC_0 is the original promoter, already present in the plasmid pCYC1m_yeGFP.
+
            <p>From the article "Tunable and multifunctional eukaryotic transcription factors based on Crispr/Cas", we learnt that different regions on the promoters could lead to activation or inhibition when dCas9-VP64 was bound. We chose to modify the region of strongest activation, named c3, and the two regions that lead to the strongest inhibition, c6 and c7. We synthesized promoters CYC_0, CYC_1, CYC_2 and CYC_3. The only differences between them are the three regions c3, c6 and c7. The promoter CYC_0 is the original promoter, already present in the plasmid pCYC1m_yeGFP.</p>
−
        </small></p>
+
  
 
             <h2>Materials and methods</h2>
 
             <h2>Materials and methods</h2>
−
                <ul>
+
            <ul>
−
                    <li>Synthesis ??</li>
+
                <li>Synthesis ??</li>
−
                </ul>
+
            </ul>
  
 
             <h2>Results</h2>
 
             <h2>Results</h2>
−
                 <div id="divleft1">
+
            <div class="row">
 +
                 <div class="col-md-6">
 
                     <img src="https://static.igem.org/mediawiki/2015/7/72/EPF_Lausanne_CYC_0_IDT.PNG" style="width:80%">
 
                     <img src="https://static.igem.org/mediawiki/2015/7/72/EPF_Lausanne_CYC_0_IDT.PNG" style="width:80%">
 
                     <img src="https://static.igem.org/mediawiki/2015/9/91/EPF_Lausanne_CYC_1_IDT.PNG" style="width:80%">
 
                     <img src="https://static.igem.org/mediawiki/2015/9/91/EPF_Lausanne_CYC_1_IDT.PNG" style="width:80%">
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                     <img src="https://static.igem.org/mediawiki/2015/c/c6/EPF_Lausanne_CYC_3_IDT.PNG" style="width:80%">
 
                     <img src="https://static.igem.org/mediawiki/2015/c/c6/EPF_Lausanne_CYC_3_IDT.PNG" style="width:80%">
 
                 </div>
 
                 </div>
−
                 <div id="divright1">
+
                 <div class="col-md-6">
−
                     <p><small>Four different promoters according to fig...
+
                     <p>Four different promoters according to fig...</p>
−
                    </small></p>
+
 
                 </div>
 
                 </div>
−
    </div>
+
            </div>
 +
            </section>
  
  
  
 
<!--INTEGRATE REPORTER PLASMID - CYC Gibson overlaps-->
 
<!--INTEGRATE REPORTER PLASMID - CYC Gibson overlaps-->
−
    <div id="synthesize_promoters" class="panel">  
+
            <section id="synthesize_promoters" class="panel">  
−
    <h1><small>Integrate reporter plasmid</small></br>Add Gibson overlaps by PCR</h1>
+
            <h1><small>Integrate reporter plasmid</small></br>Add Gibson overlaps by PCR</h1>
−
        <p><small>We amplified the promoters CYC_0, 1, 2, 3 by PCR adding the Gibson overlaps in order to assemble each fragment in the linearised plasmid pCYC1m_yeGFP.The PCR was performed according to the following <a target="_blank" href="https://static.igem.org/mediawiki/2015/7/74/EPF_Lausanne_Add_Gibson_overlaps_to_CYC-1%2C2%2C3.pdf">protocol</a>.
+
            <p>We amplified the promoters CYC_0, 1, 2, 3 by PCR adding the Gibson overlaps in order to assemble each fragment in the linearised plasmid pCYC1m_yeGFP.The PCR was performed according to the following <a target="_blank" href="https://static.igem.org/mediawiki/2015/7/74/EPF_Lausanne_Add_Gibson_overlaps_to_CYC-1%2C2%2C3.pdf">protocol</a>.</p>
−
        </small></p>
+
  
 
             <h2>Materials and methods</h2>
 
             <h2>Materials and methods</h2>
−
                <ul>
+
            <ul>
−
                    <li>Polymerase Chain Reaction</li>
+
                <li>Polymerase Chain Reaction</li>
−
                </ul>
+
            </ul>
  
 
             <h2>Results</h2>
 
             <h2>Results</h2>
−
                 <div id="divleft1">
+
            <div class="row">
 +
                 <div class="col-md-6">
 +
                    <figure>
 +
                        <a href="https://static.igem.org/mediawiki/2015/f/fd/EPF_Lausanne_CYC_promoters_with_gbs_overlaps.png"><img src="https://static.igem.org/mediawiki/2015/f/fd/EPF_Lausanne_CYC_promoters_with_gbs_overlaps.png" alt="CYC promoters" width="75%"></a>
 +
                        <figcaption>Fig.x - CYC promoters with gbs</figcaption>
 +
                    </figure>
 
                     <img src="https://static.igem.org/mediawiki/2015/f/fd/EPF_Lausanne_CYC_promoters_with_gbs_overlaps.png" style="width:80%">
 
                     <img src="https://static.igem.org/mediawiki/2015/f/fd/EPF_Lausanne_CYC_promoters_with_gbs_overlaps.png" style="width:80%">
 
                 </div>
 
                 </div>
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                 <div class="col-md-6">
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                     <p><small>On the gel electrophoresis that we ran after PCR, we observe the three CYC fragments at the right height. These fragments are then used for the Gibson assembly in the plasmid pCYC_yeGFP.
+
                     <p>On the gel electrophoresis that we ran after PCR, we observe the three CYC fragments at the right height. These fragments are then used for the Gibson assembly in the plasmid pCYC_yeGFP.</p>
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                    </small></p>
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                 </div>
 
                 </div>
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            </div>
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            </section>
  
  
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<!--Integrate and express gRNAs -->
 
<!--Integrate and express gRNAs -->
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    <div id="PCR-amplify_the_gRNAs_expressing_cassettes" class="panel">
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            <section id="PCR-amplify_the_gRNAs_expressing_cassettes" class="panel">
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    <h1><small>Integrate and express gRNAs</small></br>PCR-amplify the gRNA expressing cassettes</h1>
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                <h1><small>Integrate and express gRNAs</small></br>PCR-amplify the gRNA expressing cassettes</h1>
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        <p><small>The gRNA expressing cassettes c3_0, c3_1, c3_2, c3_3 (activating sequences), c6_0, c6_1, c6_2, c6_3, (inhibiting sequences), c7_0, c7_1, c7_2, c7_3 (inhibiting sequences) were ordered together with the promoters CYC_0, CYC_1, CYC_2, CYC_3 promoters. The c3 gRNA expressing cassettes were synthesized along with their corresponding CYC promoter as individual G-Blocks, all other gRNAs were synthesized as individual G-Blocks. Four primers were used in order to PCR out or amplify the gRNAs and the promoters: f_IDT_tri, f_IDT_squ, r_IDT_dia and r_IDT_cir. For detailed reaction mixes, click here LIEN A INSERER
+
                <p>The gRNA expressing cassettes c3_0, c3_1, c3_2, c3_3 (activating sequences), c6_0, c6_1, c6_2, c6_3, (inhibiting sequences), c7_0, c7_1, c7_2, c7_3 (inhibiting sequences) were ordered together with the promoters CYC_0, CYC_1, CYC_2, CYC_3 promoters. The c3 gRNA expressing cassettes were synthesized along with their corresponding CYC promoter as individual G-Blocks, all other gRNAs were synthesized as individual G-Blocks. Four primers were used in order to PCR out or amplify the gRNAs and the promoters: f_IDT_tri, f_IDT_squ, r_IDT_dia and r_IDT_cir. For detailed reaction mixes, click here LIEN A INSERER</p>
−
        </small></p>
+
  
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            <h2>Materials and methods</h2>
+
                <h2>Materials and methods</h2>
 
                 <ul>
 
                 <ul>
 
                     <li>Polymerase Chain Reaction</li>
 
                     <li>Polymerase Chain Reaction</li>
 
                 </ul>
 
                 </ul>
  
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            <h2>Results</h2>
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                <h2>Results</h2>
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                     <img src="https://static.igem.org/mediawiki/2015/f/f6/EPF_Lausanne_C6%2C_CYC_0_1_2.png" style="width:75%">
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                    <img src="https://static.igem.org/mediawiki/2015/1/1c/EPF_Lausanne_C7_0_1_2.png" style="width:75%">
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                        <img src="https://static.igem.org/mediawiki/2015/f/f6/EPF_Lausanne_C6%2C_CYC_0_1_2.png" style="width:75%">
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                    <img src="https://static.igem.org/mediawiki/2015/1/10/EPF_Lausanne_C7_3.png" style="width:75%">  
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                        <img src="https://static.igem.org/mediawiki/2015/1/1c/EPF_Lausanne_C7_0_1_2.png" style="width:75%">
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                    <img src="https://static.igem.org/mediawiki/2015/d/dc/EPF_Lausanne_CYC_3_c3_1_2_3.png" style="width:75%">  
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                        <img src="https://static.igem.org/mediawiki/2015/1/10/EPF_Lausanne_C7_3.png" style="width:75%">  
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                    <img src="https://static.igem.org/mediawiki/2015/6/6a/EPF_Lausanne_C3_0.png" style="width:75%">
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                        <img src="https://static.igem.org/mediawiki/2015/d/dc/EPF_Lausanne_CYC_3_c3_1_2_3.png" style="width:75%">  
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                        <img src="https://static.igem.org/mediawiki/2015/6/6a/EPF_Lausanne_C3_0.png" style="width:75%">
  
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                </div>
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                    </div>
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                <div id="divright1">
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                    <div class="col-md-6">
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                    <p><small> Each amplicon (gRNA expressing cassette or CYC promoter) is 250 bp long (the exact length can vary by max two bp depending on the primers used). Several PCRs were required for amplification, we only show the gels corresponding to the PCR products we kept.</br>
+
                        <p>Each amplicon (gRNA expressing cassette or CYC promoter) is 250 bp long (the exact length can vary by max two bp depending on the primers used). Several PCRs were required for amplification, we only show the gels corresponding to the PCR products we kept.</br>
  
−
                    Fig...: c6_0, c6_1, c6_2, c6_3, CYC_0, CYC_1, CYC_2</br>
+
                        Fig...: c6_0, c6_1, c6_2, c6_3, CYC_0, CYC_1, CYC_2</br>
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                    Fig...: c7_0, c7_1, c7_2</br>
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                        Fig...: c7_0, c7_1, c7_2</br>
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                    Fig...: c7_3</br>
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                        Fig...: c7_3</br>
−
                    Fig...: CYC_3, c3_1, c3_2, c3_3</br>
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                        Fig...: CYC_3, c3_1, c3_2, c3_3</br>
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                    Fig...: c3_0</br>
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                        Fig...: c3_0</br></p>
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                    </small></p>
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                    </div>
 
                 </div>
 
                 </div>
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    </div>
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            </section>
  
  
 
<!--PCR out DsRed2 -->
 
<!--PCR out DsRed2 -->
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    <div id="PCR_out_DsRed2" class="panel">
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            <section id="PCR_out_DsRed2" class="panel">
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    <h1><small>Integrate and express gRNAs</small></br>PCR out DsRed2</h1>
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                <h1><small>Integrate and express gRNAs</small></br>PCR out DsRed2</h1>
−
        <p><small>In order to verify the expression of the gRNAs cassettes, we used fluorescent protein DsRed2 as a reporter gene. It was PCRed out from the plasmid CMVp-dsRed2-Triplex-HHRibo-gRNA1-HDVRibo-pA. A synthetic polyA tail followed by a part of the Hammerhead ribozyme sequence was added on the reverse primer.
+
                <p>In order to verify the expression of the gRNAs cassettes, we used fluorescent protein DsRed2 as a reporter gene. It was PCRed out from the plasmid CMVp-dsRed2-Triplex-HHRibo-gRNA1-HDVRibo-pA. A synthetic polyA tail followed by a part of the Hammerhead ribozyme sequence was added on the reverse primer.</p>
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        </small></p>
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−
            <h2>Materials and methods</h2>
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                <h2>Materials and methods</h2>
 
                 <ul>
 
                 <ul>
 
                     <li>Miniprep</li>
 
                     <li>Miniprep</li>
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                 </ul>
 
                 </ul>
  
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                <h2>Results</h2>
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                     <img src="https://static.igem.org/mediawiki/2015/5/5f/EPF_Lausanne_DsRed2_polyA_HH1.png" style="width:75%">
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                     <div class="col-md-6">
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                        <figure>
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                            <a href="https://static.igem.org/mediawiki/2015/5/5f/EPF_Lausanne_DsRed2_polyA_HH1.png"><img src="https://static.igem.org/mediawiki/2015/5/5f/EPF_Lausanne_DsRed2_polyA_HH1.png" alt="DsRed2 polyA" width="75%"></a>
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                            <figcaption>Fig.x - DsRed2 polyA HH1</figcaption>
 +
                        </figure> 
 +
                    </div>
 +
                    <div class="col-md-6">
 +
                        <p><small> The size of the DsRed2_polyA_HH1 amplicon is 750 bp. The plasmid on the gel is CMVp-dsRed2-Triplex-HHRibo-gRNA1-HDVRibo-pA.</br>
 +
                        </small></p>
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                 </div>
 
                 </div>
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            </section>
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                    <p><small> The size of the DsRed2_polyA_HH1 amplicon is 750 bp. The plasmid on the gel is CMVp-dsRed2-Triplex-HHRibo-gRNA1-HDVRibo-pA.</br>
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Revision as of 08:34, 20 August 2015

EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits

saccharomyces cerevisiae

Express dCas9-VP64
Integrate pTPGI_dCas9_VP64

We received plasmid pTPGI_dCas9_VP64 from Addgene. The plasmid was found from the article "Tunable and multifunctional eukaryotic transcription factors based on Crispr/Cas". After glycerol stocks and miniprep, we performed a restriction analysis to check the identity of our plasmid. We linearised the plasmid, in order to integrate it into yeast genome. For more details about our experiments, see here. Only our fourth trial to integrate the plasmid was successful.

Materials and methods

  • Glycerol stocks
  • Miniprep
  • Restriction analysis
  • Polymerase Chain Reaction
  • Yeast integration A AJOUTER SUR PROTOCOLS

Results

dCas9 gel
Fig.1 - Gel of the dCas9.

We used four different sets of enzymes for the restriction analysis. Linearized pTPGI_dCas9_VP64 is expected to be 10'987 bp. We observe that the gel (fig.1) corresponds to the expected one.
The plasmid was linearised both with EagI HF and NotI HF prior to integration. We integrated each linearised plasmid to obtain two different yeast strains.

Express dCas9-VP64
Western Blot of dCas9-VP64

The Western Blot allows to check the expression of dCas9.

Materials and methods

  • Western Blot A AJOUTER SUR PROTOCOLS

Results

name the image
Insert legend here

Results of Western Blot.

Integrate reporter plasmid
Linearise reporter plasmid

>We received the plasmid pCYC1m_yeGFP from Addgene. The plasmid was found from the article "Tunable and multifunctional eukaryotic transcription factors based on Crispr/Cas". After glycerol stocks and minipreps. We linearised the plasmid by PCR according to the following protocol.

Materials and methods

  • Polymerase Chain Reaction

Results

name the image
Insert legend here

Linearized pCYC1m_yeGFP is expected to be 5'485 bp. Running an agarose gel electrophoresis allowed to verify that we had linearised the plasmid.

Integrate reporter plasmid
Synthesize promoters

From the article "Tunable and multifunctional eukaryotic transcription factors based on Crispr/Cas", we learnt that different regions on the promoters could lead to activation or inhibition when dCas9-VP64 was bound. We chose to modify the region of strongest activation, named c3, and the two regions that lead to the strongest inhibition, c6 and c7. We synthesized promoters CYC_0, CYC_1, CYC_2 and CYC_3. The only differences between them are the three regions c3, c6 and c7. The promoter CYC_0 is the original promoter, already present in the plasmid pCYC1m_yeGFP.

Materials and methods

  • Synthesis ??

Results

Four different promoters according to fig...

Integrate reporter plasmid
Add Gibson overlaps by PCR

We amplified the promoters CYC_0, 1, 2, 3 by PCR adding the Gibson overlaps in order to assemble each fragment in the linearised plasmid pCYC1m_yeGFP.The PCR was performed according to the following protocol.

Materials and methods

  • Polymerase Chain Reaction

Results

CYC promoters
Fig.x - CYC promoters with gbs

On the gel electrophoresis that we ran after PCR, we observe the three CYC fragments at the right height. These fragments are then used for the Gibson assembly in the plasmid pCYC_yeGFP.

Integrate and express gRNAs
PCR-amplify the gRNA expressing cassettes

The gRNA expressing cassettes c3_0, c3_1, c3_2, c3_3 (activating sequences), c6_0, c6_1, c6_2, c6_3, (inhibiting sequences), c7_0, c7_1, c7_2, c7_3 (inhibiting sequences) were ordered together with the promoters CYC_0, CYC_1, CYC_2, CYC_3 promoters. The c3 gRNA expressing cassettes were synthesized along with their corresponding CYC promoter as individual G-Blocks, all other gRNAs were synthesized as individual G-Blocks. Four primers were used in order to PCR out or amplify the gRNAs and the promoters: f_IDT_tri, f_IDT_squ, r_IDT_dia and r_IDT_cir. For detailed reaction mixes, click here LIEN A INSERER

Materials and methods

  • Polymerase Chain Reaction

Results

Each amplicon (gRNA expressing cassette or CYC promoter) is 250 bp long (the exact length can vary by max two bp depending on the primers used). Several PCRs were required for amplification, we only show the gels corresponding to the PCR products we kept.
Fig...: c6_0, c6_1, c6_2, c6_3, CYC_0, CYC_1, CYC_2
Fig...: c7_0, c7_1, c7_2
Fig...: c7_3
Fig...: CYC_3, c3_1, c3_2, c3_3
Fig...: c3_0

Integrate and express gRNAs
PCR out DsRed2

In order to verify the expression of the gRNAs cassettes, we used fluorescent protein DsRed2 as a reporter gene. It was PCRed out from the plasmid CMVp-dsRed2-Triplex-HHRibo-gRNA1-HDVRibo-pA. A synthetic polyA tail followed by a part of the Hammerhead ribozyme sequence was added on the reverse primer.

Materials and methods

  • Miniprep
  • Polymerase Chain Reaction

Results

DsRed2 polyA
Fig.x - DsRed2 polyA HH1

The size of the DsRed2_polyA_HH1 amplicon is 750 bp. The plasmid on the gel is CMVp-dsRed2-Triplex-HHRibo-gRNA1-HDVRibo-pA.

EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits

NOT PROOFREAD