Difference between revisions of "Team:CSU CHINA/Part Collection"

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{{CSU_CHINA}}
 
{{CSU_CHINA}}
 
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     </style>
 
     </style>
 
     <div class="container-fluid Topbox" id="Top">
 
     <div class="container-fluid Topbox" id="Top">
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             <h1 class="display-3" style="color:white">Composite Part</h1>
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             <h1>Improved Part</h1>
 
     </div>
 
     </div>
 
     <div class="container">
 
     <div class="container">
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        <div class="row">
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            <div class="row">
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            <nav class="col-sm-2"id="myScrollspy">
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                    <div id="col-md-8 offset-md-2">
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                <ul class="nav flex-column Teenav">
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                             <div id="mRNA" style="width: 100%; margin-bottom: 30px;">
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                        <li class="nav-item">
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                                    <div class="card">
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                                <a class="nav-link" href="#Overview">Overview</a>
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                                        <div class="card-header">
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                             </li>
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                                            <a class="card-link" data-toggle="collapse" href="#mRNA1" style="color: black">Basic Part</a>
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                            <li class="nav-item">
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                                        </div>
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                              <a class="nav-link" href="#Module1">Module1</a>
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                                        <div id="mRNA1" class="collapse" data-parent="#mRNA">
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                            </li>
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                                            <div class="card-body">
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                            <li class="nav-item">
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                                                <figure>
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                              <a class="nav-link" href="#Module2">Module2</a>
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                                                    <center>
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                            </li>
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                    <div>
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                            <li class="nav-item">
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                        <table class="table table-hover">
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                              <a class="nav-link" href="#Module3">Module3</a>
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                            <thead class="thead-inverse">
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                            </li>
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                                <tr>
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                            <li class="nav-item">
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                                        <td>Section</td>
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                                    <a class="nav-link" href="#References">References</a>
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                                        <td>Name</td>
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                            </li>
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                                        <td>Type</td>
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                </ul>
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                                        <td>Description</td>
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            </nav>
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                                </tr>
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            <div class="col-sm-10">
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                                </thead>
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                <div id="mRNA" style="width: 100%; margin-bottom: 30px;">
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                                <tbody>
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                    <div class="card">
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                                    <tr>
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                            <div class="card-header">
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                                        <td>New part</td>
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                                <a class="card-link" data-toggle="collapse" href="#mRNA1" style="color: black">Composite Part</a>
+
                                        <td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2908676">BBa_K2908676</a></td>
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                            </div>
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                                        <td>DNA</td>
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                            <div id="mRNA1" class="collapse" data-parent="#mRNA">
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                                        <td>s(GATA3)p-GAD-miR101-BS</td>
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                                <div class="card-body">
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                                    </tr>
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                                    <figure>
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                                    <tr>
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                                        <center>
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                                        <td>Existing part</td>
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                                                <div>
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                                        <td><a href="http://parts.igem.org/Part:BBa_K2580666">BBa_K2580666</td>
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                                                        <table class="table table-hover table-responsive-sm">
+
                                        <td>Protein</td>
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                                                                <thead>
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                                        <td>CMV-Gal4-VP16</td>
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                                                                    <tr>
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                                    </tr>
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                                                                            <td>Section</td>
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                                </tbody>
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                                                                            <td>Name</td>
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                        </table>
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                                                                            <td>Type</td>
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                                                                            <td>Description</td>
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−
                                                                    </tr>
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                                                                    </thead>
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                                                                    <tbody>
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−
                                                                        <tr>
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−
                                                                            <th>Module1</th>
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                                                                            <td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2908671">BBa_K2908671</a></td>
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                                                                            <td>Plasmid</td>
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−
                                                                            <td>pLN431-s(GATA3)p-GAD-miR101-BS</td>
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                                                                        </tr>
+
−
                                                                        <tr>
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                                                                            <th rowspan="3">Module2</th>
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                                                                            <td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2908679">BBa_K2908679</a></td>
+
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                                                                            <td>Plasmid</td>
+
−
                                                                            <td>s(ESR1)p-miR141spe-EGFP</td>
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                                                                        </tr>
+
−
                                                                        <tr>
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−
                                                                            <td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2908680">BBa_K2908680</a></td>
+
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                                                                            <td>Plasmid</td>
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−
                                                                            <td>s(ESR1)p-miR148bspe-EGFP </td>
+
−
                                                                        </tr>
+
−
                                                                        <tr>
+
−
                                                                            <td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2908681">BBa_K2908681</a></td>
+
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                                                                            <td>Plasmid</td>
+
−
                                                                            <td>s(ESR1)p-miR101spe-EGFP</td>
+
−
                                                                        </tr>
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−
                                                                        <tr>
+
−
                                                                            <th>Module3</th>
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−
                                                                            <td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2908670">BBa_K2908670</a></td>
+
−
                                                                            <td>Plasmid</td>
+
−
                                                                            <td>pLN431-G8p-yCD-miR101BS</td>
+
−
                                                                        </tr>
+
−
                                                                    </tbody>
+
−
                                                            </table>
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                                                </div>
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−
                                        </center>
+
−
                                    </figure>
+
−
                                </div>
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−
                            </div>
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−
                        </div>
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                    <div id="Overview">
+
−
                        <h2>Overview</h2>
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−
                        <h3>√ Golden Gate assay to construct the plasmids</h3>
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−
                        <p>First step: Construction of pocket plasmid </p>
+
−
                        <p>Next: ‘Golden Gate’</p>
+
−
                        <p>In 2008, Engler etc. reported a kind of cloned strategy through IIS restriction enzymes , called IIS cloning,  namely ‘Golden Gate’ cloning. Type IIS restriction enzyme can specifically identify the target sites on double-stranded DNA, and non-specifically cut the DNA double-stranded downstream of the target sites to produce a cohesive end at the 5 'or 3' end of the DNA double-stranded [1]. </p>
+
−
                        <p>"Golden Gate" cloning method is in the same reaction system, using IIS type restriction endonuclease, cut beyond recognition site of DNA, DNA fragments containing cohesive end, at the same time by ligase connect several DNA fragments according to the established order, and spliced into excluding enzyme recognition site of DNA fragments, is like a linear puzzles are correctly spliced together, make the multiple objective DNA fragments in the order set to realize "seamless connection”.</p>                   
+
 
                     </div>
 
                     </div>
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                    <div id="Module1">
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                </center>
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                         <h2 >Module1</h2>
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            </figure>
−
                         <img src="https://static.igem.org/mediawiki/2019/6/6a/T--CSU_CHINA--CP_Module1.png" class="mx-auto" style="width:60% ">
+
        </div>
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                         <p class="ts"><b>pLN431-s(GATA3)p-GAD-miR101BD (BBa_K2908671)</b></p>
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    </div>
−
                        <p>We use the pLN431[2] as the back-clone of the Module1, then we cut the vector and an insert sequence (lacZp+lacZ) with MluI and NotI to form the pocket plasmid.</p>
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    </div>
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                         <div class="row">
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</div>
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                                <div class="col-sm-6">
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−
                                 <img src="https://static.igem.org/mediawiki/2019/e/e0/T--CSU_CHINA--CP_Module1_KDXQ.png" style="width:90%;margin-top:100px;">
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−
                                <p class="ts"><b>Module1 口袋质粒构建</b></p>
+
                         <h2>Improved Part</h2>
−
                                </div>
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                        <br>
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                                <div class="col-sm-6">
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                        <h3>New part</h3>
−
                                <img src="https://static.igem.org/mediawiki/2019/8/8b/T--CSU_CHINA--CP_Module1_KD.png" class="mx-auto" style="width:90% ">
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                         <img src="./Part/improved part.png" class="mx-auto" style="width: 80% ">
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                                <p class="ts"><b>Module1 口袋质粒 详细</b></p>
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                         <p class="ts"><b>s(GATA3)p-GAD-miR101-BD (BBa_K2908676) [GAD is totally the same as Gal4-VP16]</b></p>
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                                </div> 
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                         <div class="d-flex justify-content-center">
 +
                                 <p style="color: grey; width: 80%; margin-bottom: 32px;text-align: center;">The part improved in the specificity in Triple negative cancer (TNBC). </p>
 
                         </div>
 
                         </div>
−
                         <p>Supplementary figure: </p>
+
                         <br>
−
                         <img src="https://static.igem.org/mediawiki/2019/2/26/T--CSU_CHINA--CP_lacZ.png" class="mx-auto" style="width: 60% ">
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                         <h3>Existing part</h3>
−
                        <p>We designed the BsmBI restriction digest location on the 5’ and 3’ top of the inserted sequence, the following figure shows the pattern of construction method.</p>
+
                         <img src="./Part/improved part.jpg" class="mx-auto" style="width: 80% ">
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                         <img src="https://static.igem.org/mediawiki/2019/9/9f/T--CSU_CHINA--CP_Module1-Golden_Gate.png" class="mx-auto" style="width: 60% ">
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                         <p class="ts"><b>CMV-Gal4-VP16 (BBa_K2580666)</b></p>
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                         <p class="ts"><b>Module1-Golden Gate</b></p>
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                        <div class="d-flex justify-content-center">
−
                    </div>
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                                <p style="color: grey; width: 80%; margin-bottom: 32px;text-align: center;">The part has low specificity in the TNBC cells compared to normal cells although the team members who created the part stated that it is a cancer-specific part.</p>
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                    <div  id="Module2">
+
                         </div>
−
                        <h2>Module2</h2>
+
                         <p>As for this new part we created this year, it contains the brand new synthetic promoter we designed (BBa_K2908000),  the existing sequence in the previous iGEM parts, GAD (Gal4-VP16), and the new designed part miR101-BD. </p>
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                         <img src="https://static.igem.org/mediawiki/2019/6/68/T--CSU_CHINA--CP_Module2-141spe.png" class="mx-auto" style="width: 60% ">
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                         <p>In our project, we wanted to make the expression of GAD to be specific in Triple negative breast cancer cells (TNBC cells). After communicating with the team creating the part who says that in breast cancer, this part has higher efficiency in cancer cells than normal cells, we tested the efficiency of GAD expression by co-transfecting the CMV-Gal4-VP16 plasmid and pGL4.22-G8p (pGL4.22 has a luciferase gene after G8p, a promoter can be driven by Gal4-VP16) into TNBC cells (cancer cells) and normal cells. A in the figure below shows the results.</p>
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                         <p class="ts"><b>p(ESR1)p-miR141spe-EGFP (BBa_K2908672)</b></p>
+
                         <p>Then, we <strong>exchange the promoter CMV into s(GATA3)p, and added a miR101-BD in the 3’UTR of the gene GAD (Gal4-VP16)</strong>, as miR101 is low expressed in the cancer cells while high in the normal cells [1] (the binding of miR101 to miR101-BD can inhibit the expression of GAD protein, then to down-regulate the efficiency of G8p). We used the same method to test the efficiency of our part, that is, co-transfecting the s(GATA3)p-GAD-miR101-BD plasmid and pGL4.22-G8p, and doing the luciferase assay [2]. The figure B shows the results. We also compared the specificity between CMV-Gal4-VP16 and s(GATA3)p-GAD-miR101-BD (C), which indicated that our part dramatically improved the function in specifically regulating the expression of GAD.</p>
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                         <img src="https://static.igem.org/mediawiki/2019/9/93/T--CSU_CHINA--CP_Module2-148bspe.png" class="mx-auto" style="width: 60% ">
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                         <p></p>
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                         <p class="ts"><b>p(ESR1)p-miR148bspe-EGFP (BBa_K2908674)</b></p>
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                         <img src="./Part/improved part.jpg" class="mx-auto" style="width: 80% ">
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                        <img src="https://static.igem.org/mediawiki/2019/b/bb/T--CSU_CHINA--CP_Module2-101spe.png" class="mx-auto" style="width: 60% ">
+
                         <div class="d-flex justify-content-center">
−
                        <p class="ts"><b>p(ESR1)p-miR101spe-EGFP (BBa_K2908675)</b></p>
+
                                <p style="color: grey; width: 80%; margin-bottom: 32px;text-align: center;">(A) The relative strength of previous part ‘CMV-Gal4-VP16’. (B) The relative strength of the new part ’s(GATA3)p-GAD-miR101-BD’. (C) The comparison of the specificity in cancer cells between new part and the previous part.</p>
−
                         <p>The construction method is the same as module1, only except the paired sequence of each inserted fragment, which shows in the following picture.</p>
+
                        </div>
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                         <img src="https://static.igem.org/mediawiki/2019/5/5b/T--CSU_CHINA--CP_Module2-Golden_Gate.png" class="mx-auto" style="width: 60% ">
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                         <h3>Appendix: Brief introduction of luciferase assay</h3>
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                         <p class="ts"><b>Module2-Golden Gate</b></p>
+
                         <p>The Luciferase Assay System is substantially improved over conventional assay methods in both sensitivity and simplicity. Light is produced by converting the chemical energy of luciferin oxidation through an electron transition, forming the product molecule oxyluciferin. Firefly luciferase, a monomeric 61kDa protein, catalyzes luciferin oxidation using ATP-Mg2+ as a cosubstrate. In the conventional assay for luciferase, a flash of light is generated that decays rapidly after the enzyme and substrates are combined. The Luciferase Assay System incorporates coenzyme A (CoA) for improved kinetics, allowing greater enzymatic turnover and resulting in increased light intensity that is nearly constant for at least 1 minute. The Luciferase Assay System yields linear results over at least eight orders of magnitude. Less than 10-20 moles of luciferase have been detected under optimal conditions. Generally, 100-fold greater sensitivity can be achieved over the chloramphenicol acetyltransferase (CAT) assay.</p>
−
                    </div>
+
−
                    <div id="Module3">
+
−
                         <h2>Module3</h2>
+
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                         <img src="https://static.igem.org/mediawiki/2019/f/f5/T--CSU_CHINA--CP_Module3.png" class="mx-auto" style="width: 60% ">
+
−
                        <p class="ts"><b>pLN431-G8p-yCD-miR101BD (BBa_K2908670)</b></p>
+
−
                    </div>  
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                     <div id="References">
 
                     <div id="References">
 
                             <h2>References</h2>
 
                             <h2>References</h2>
−
                             <p>[1] Waclaw Szybalski”, Sun C. Kim”, Noaman Hasan” and Anna J. Podhajskab, Class IIS restriction enzymes, a review.1991, Gene, 100: 13-26.</p>
+
                             <p>[1] H Guan et al.  Int J Mol Med (2016), MicroRNA-101 inhibits cell proliferation and induces apoptosisby targeting EYA1 in breast cancer.</p>
−
                             <p>[2] Lior Nissim, Ming-Ru Wu, Erez Pery, Adina Binder-Nissim, Hiroshi I. Suzuki, Doron Stupp, Claudia Wehrspaun, Yuval Tabach, Phillip A. Sharp, and Timothy K. Lu, Cell, (2017),Synthetic RNA-Based Immunomodulatory Gene Circuits for Cancer Immunotherapy.</p>
+
                             <p>[2] Solberg N1, Krauss S. , Methods Mol Biol. 2013;977:65-78. Luciferase assay to study the activity of a cloned promoter DNA fragment.</p>
 
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Revision as of 22:41, 21 October 2019

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Improved Part

Basic Part
Section Name Type Description
New part BBa_K2908676 DNA s(GATA3)p-GAD-miR101-BS
Existing part BBa_K2580666 Protein CMV-Gal4-VP16

Improved Part


New part

s(GATA3)p-GAD-miR101-BD (BBa_K2908676) [GAD is totally the same as Gal4-VP16]

The part improved in the specificity in Triple negative cancer (TNBC).


Existing part

CMV-Gal4-VP16 (BBa_K2580666)

The part has low specificity in the TNBC cells compared to normal cells although the team members who created the part stated that it is a cancer-specific part.

As for this new part we created this year, it contains the brand new synthetic promoter we designed (BBa_K2908000), the existing sequence in the previous iGEM parts, GAD (Gal4-VP16), and the new designed part miR101-BD.

In our project, we wanted to make the expression of GAD to be specific in Triple negative breast cancer cells (TNBC cells). After communicating with the team creating the part who says that in breast cancer, this part has higher efficiency in cancer cells than normal cells, we tested the efficiency of GAD expression by co-transfecting the CMV-Gal4-VP16 plasmid and pGL4.22-G8p (pGL4.22 has a luciferase gene after G8p, a promoter can be driven by Gal4-VP16) into TNBC cells (cancer cells) and normal cells. A in the figure below shows the results.

Then, we exchange the promoter CMV into s(GATA3)p, and added a miR101-BD in the 3’UTR of the gene GAD (Gal4-VP16), as miR101 is low expressed in the cancer cells while high in the normal cells [1] (the binding of miR101 to miR101-BD can inhibit the expression of GAD protein, then to down-regulate the efficiency of G8p). We used the same method to test the efficiency of our part, that is, co-transfecting the s(GATA3)p-GAD-miR101-BD plasmid and pGL4.22-G8p, and doing the luciferase assay [2]. The figure B shows the results. We also compared the specificity between CMV-Gal4-VP16 and s(GATA3)p-GAD-miR101-BD (C), which indicated that our part dramatically improved the function in specifically regulating the expression of GAD.

(A) The relative strength of previous part ‘CMV-Gal4-VP16’. (B) The relative strength of the new part ’s(GATA3)p-GAD-miR101-BD’. (C) The comparison of the specificity in cancer cells between new part and the previous part.

Appendix: Brief introduction of luciferase assay

The Luciferase Assay System is substantially improved over conventional assay methods in both sensitivity and simplicity. Light is produced by converting the chemical energy of luciferin oxidation through an electron transition, forming the product molecule oxyluciferin. Firefly luciferase, a monomeric 61kDa protein, catalyzes luciferin oxidation using ATP-Mg2+ as a cosubstrate. In the conventional assay for luciferase, a flash of light is generated that decays rapidly after the enzyme and substrates are combined. The Luciferase Assay System incorporates coenzyme A (CoA) for improved kinetics, allowing greater enzymatic turnover and resulting in increased light intensity that is nearly constant for at least 1 minute. The Luciferase Assay System yields linear results over at least eight orders of magnitude. Less than 10-20 moles of luciferase have been detected under optimal conditions. Generally, 100-fold greater sensitivity can be achieved over the chloramphenicol acetyltransferase (CAT) assay.

References

[1] H Guan et al.  Int J Mol Med (2016), MicroRNA-101 inhibits cell proliferation and induces apoptosisby targeting EYA1 in breast cancer.

[2] Solberg N1, Krauss S. , Methods Mol Biol. 2013;977:65-78. Luciferase assay to study the activity of a cloned promoter DNA fragment.

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