(Prototype team page) |
Lonely-boy (Talk | contribs) |
||
Line 1: | Line 1: | ||
{{CSU_CHINA}} | {{CSU_CHINA}} | ||
<html> | <html> | ||
+ | <body style="position: relative" data-spy="scroll" data-offset="300" data-target="#myScrollspy"> | ||
+ | <style> | ||
+ | .container{ | ||
+ | margin-bottom:5%; | ||
+ | margin-top:5%; | ||
+ | } | ||
+ | .Topbox{ | ||
+ | width: 100%; | ||
+ | min-height: 600px; | ||
+ | display: -webkit-flex; /* Safari */ | ||
+ | display: flex; | ||
+ | color:white; | ||
+ | background-image: url('https://static.igem.org/mediawiki/2019/d/d3/T--CSU_CHINA--TM_BC.jpg'); | ||
+ | background-size: cover; | ||
+ | background-repeat: no-repeat; | ||
+ | background-position: center; | ||
+ | justify-content: center; | ||
+ | align-items: center; | ||
+ | } | ||
+ | h2{ | ||
+ | margin-top: 30px; | ||
+ | } | ||
+ | p{ | ||
+ | font-family: Times New Roman; | ||
+ | text-align: justify; | ||
+ | font-size:1rem!important; | ||
+ | } | ||
+ | a:hover{ | ||
+ | cursor: pointer; | ||
+ | } | ||
+ | .container img{ | ||
+ | display: block; | ||
+ | margin: 0 auto; | ||
+ | box-shadow: 5px 5px 10px #888888; | ||
+ | } | ||
+ | .Teenav{ | ||
+ | background-color:#f3f3f3; | ||
+ | border-radius: 10px; | ||
+ | } | ||
+ | .flex-column{ | ||
+ | position: -webkit-sticky; /* Safari */ | ||
+ | position: sticky; | ||
+ | margin-top: 50px; | ||
+ | top: 25%; | ||
+ | } | ||
+ | .Teenav li{ | ||
+ | margin:10px 0px; | ||
+ | } | ||
+ | .Teenav a{ | ||
+ | color: #197399; | ||
+ | border-radius: 10px; | ||
+ | } | ||
+ | .Teenav a:hover{ | ||
+ | color: white; | ||
+ | background-color: #2194c5; | ||
+ | } | ||
+ | #toTOP{ | ||
+ | position:fixed; | ||
+ | right:2%; | ||
+ | bottom:2%; | ||
+ | border-radius:25%; | ||
+ | } | ||
+ | .ts { | ||
+ | margin-bottom: 0px; | ||
+ | margin-top: 5px; | ||
+ | text-align: center!important; | ||
+ | } | ||
+ | </style> | ||
+ | <div class="container-fluid Topbox" id="Top"> | ||
+ | <h1 class="display-3" style="color:white">Composite Part</h1> | ||
+ | </div> | ||
+ | <div class="container"> | ||
+ | <div class="row"> | ||
+ | <nav class="col-sm-2"id="myScrollspy"> | ||
+ | <ul class="nav flex-column Teenav"> | ||
+ | <li class="nav-item"> | ||
+ | <a class="nav-link" href="#Overview">Overview</a> | ||
+ | </li> | ||
+ | <li class="nav-item"> | ||
+ | <a class="nav-link" href="#Module1">Module1</a> | ||
+ | </li> | ||
+ | <li class="nav-item"> | ||
+ | <a class="nav-link" href="#Module2">Module2</a> | ||
+ | </li> | ||
+ | <li class="nav-item"> | ||
+ | <a class="nav-link" href="#Module3">Module3</a> | ||
+ | </li> | ||
+ | <li class="nav-item"> | ||
+ | <a class="nav-link" href="#References">References</a> | ||
+ | </li> | ||
+ | </ul> | ||
+ | </nav> | ||
+ | <div class="col-sm-10"> | ||
+ | <div id="mRNA" style="width: 100%; margin-bottom: 30px;"> | ||
+ | <div class="card"> | ||
+ | <div class="card-header"> | ||
+ | <a class="card-link" data-toggle="collapse" href="#mRNA1" style="color: black">Composite Part</a> | ||
+ | </div> | ||
+ | <div id="mRNA1" class="collapse" data-parent="#mRNA"> | ||
+ | <div class="card-body"> | ||
+ | <figure> | ||
+ | <center> | ||
+ | <div> | ||
+ | <table class="table table-hover table-responsive-sm"> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <td>Section</td> | ||
+ | <td>Name</td> | ||
+ | <td>Type</td> | ||
+ | <td>Description</td> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <th>Module1</th> | ||
+ | <td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2908671">BBa_K2908671</a></td> | ||
+ | <td>Plasmid</td> | ||
+ | <td>pLN431-s(GATA3)p-GAD-miR101-BS</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th rowspan="3">Module2</th> | ||
+ | <td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2908679">BBa_K2908679</a></td> | ||
+ | <td>Plasmid</td> | ||
+ | <td>s(ESR1)p-miR141spe-EGFP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2908680">BBa_K2908680</a></td> | ||
+ | <td>Plasmid</td> | ||
+ | <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> | ||
+ | <td>Plasmid</td> | ||
+ | <td>s(ESR1)p-miR101spe-EGFP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Module3</th> | ||
+ | <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> | ||
+ | </div> | ||
+ | </center> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="Overview"> | ||
+ | <h2>Overview</h2> | ||
+ | <h3>√ Golden Gate assay to construct the plasmids</h3> | ||
+ | <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 id="Module1"> | ||
+ | <h2 >Module1</h2> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/6/6a/T--CSU_CHINA--CP_Module1.png" class="mx-auto" style="width:60% "> | ||
+ | <p class="ts"><b>pLN431-s(GATA3)p-GAD-miR101BD (BBa_K2908671)</b></p> | ||
+ | <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> | ||
+ | <div class="row"> | ||
+ | <div class="col-sm-6"> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/e/e0/T--CSU_CHINA--CP_Module1_KDXQ.png" style="width:90%;margin-top:100px;"> | ||
+ | <p class="ts"><b>Module1 口袋质粒构建</b></p> | ||
+ | </div> | ||
+ | <div class="col-sm-6"> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/8/8b/T--CSU_CHINA--CP_Module1_KD.png" class="mx-auto" style="width:90% "> | ||
+ | <p class="ts"><b>Module1 口袋质粒 详细</b></p> | ||
+ | </div> | ||
+ | </div> | ||
+ | <p>Supplementary figure: </p> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/2/26/T--CSU_CHINA--CP_lacZ.png" class="mx-auto" style="width: 60% "> | ||
+ | <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="https://static.igem.org/mediawiki/2019/9/9f/T--CSU_CHINA--CP_Module1-Golden_Gate.png" class="mx-auto" style="width: 60% "> | ||
+ | <p class="ts"><b>Module1-Golden Gate</b></p> | ||
+ | </div> | ||
+ | <div id="Module2"> | ||
+ | <h2>Module2</h2> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/6/68/T--CSU_CHINA--CP_Module2-141spe.png" class="mx-auto" style="width: 60% "> | ||
+ | <p class="ts"><b>p(ESR1)p-miR141spe-EGFP (BBa_K2908672)</b></p> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/9/93/T--CSU_CHINA--CP_Module2-148bspe.png" class="mx-auto" style="width: 60% "> | ||
+ | <p class="ts"><b>p(ESR1)p-miR148bspe-EGFP (BBa_K2908674)</b></p> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/b/bb/T--CSU_CHINA--CP_Module2-101spe.png" class="mx-auto" style="width: 60% "> | ||
+ | <p class="ts"><b>p(ESR1)p-miR101spe-EGFP (BBa_K2908675)</b></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> | ||
+ | <img src="https://static.igem.org/mediawiki/2019/5/5b/T--CSU_CHINA--CP_Module2-Golden_Gate.png" class="mx-auto" style="width: 60% "> | ||
+ | <p class="ts"><b>Module2-Golden Gate</b></p> | ||
+ | </div> | ||
+ | <div id="Module3"> | ||
+ | <h2>Module3</h2> | ||
+ | <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> | ||
+ | <div id="References"> | ||
+ | <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>[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> | ||
+ | </div> | ||
+ | </div> | ||
− | + | </div> | |
− | + | </div> | |
− | + | <a href="#Top" class="btn btn-info" id="toTOP" role="button">▲</a> | |
− | + | </body> | |
− | + | ||
− | + | ||
− | </div> | + | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</html> | </html> |
Revision as of 08:20, 20 October 2019
![](https://static.igem.org/mediawiki/2019/8/8a/T--CSU_CHINA--SGoldMedal.png)
loading……
Composite Part
Section | Name | Type | Description |
Module1 | BBa_K2908671 | Plasmid | pLN431-s(GATA3)p-GAD-miR101-BS |
---|---|---|---|
Module2 | BBa_K2908679 | Plasmid | s(ESR1)p-miR141spe-EGFP |
BBa_K2908680 | Plasmid | s(ESR1)p-miR148bspe-EGFP | |
BBa_K2908681 | Plasmid | s(ESR1)p-miR101spe-EGFP | |
Module3 | BBa_K2908670 | Plasmid | pLN431-G8p-yCD-miR101BS |
Overview
√ Golden Gate assay to construct the plasmids
First step: Construction of pocket plasmid
Next: ‘Golden Gate’
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].
"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”.
Module1
![](https://static.igem.org/mediawiki/2019/6/6a/T--CSU_CHINA--CP_Module1.png)
pLN431-s(GATA3)p-GAD-miR101BD (BBa_K2908671)
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.
![](https://static.igem.org/mediawiki/2019/e/e0/T--CSU_CHINA--CP_Module1_KDXQ.png)
Module1 口袋质粒构建
![](https://static.igem.org/mediawiki/2019/8/8b/T--CSU_CHINA--CP_Module1_KD.png)
Module1 口袋质粒 详细
Supplementary figure:
![](https://static.igem.org/mediawiki/2019/2/26/T--CSU_CHINA--CP_lacZ.png)
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.
![](https://static.igem.org/mediawiki/2019/9/9f/T--CSU_CHINA--CP_Module1-Golden_Gate.png)
Module1-Golden Gate
Module2
![](https://static.igem.org/mediawiki/2019/6/68/T--CSU_CHINA--CP_Module2-141spe.png)
p(ESR1)p-miR141spe-EGFP (BBa_K2908672)
![](https://static.igem.org/mediawiki/2019/9/93/T--CSU_CHINA--CP_Module2-148bspe.png)
p(ESR1)p-miR148bspe-EGFP (BBa_K2908674)
![](https://static.igem.org/mediawiki/2019/b/bb/T--CSU_CHINA--CP_Module2-101spe.png)
p(ESR1)p-miR101spe-EGFP (BBa_K2908675)
The construction method is the same as module1, only except the paired sequence of each inserted fragment, which shows in the following picture.
![](https://static.igem.org/mediawiki/2019/5/5b/T--CSU_CHINA--CP_Module2-Golden_Gate.png)
Module2-Golden Gate
Module3
![](https://static.igem.org/mediawiki/2019/f/f5/T--CSU_CHINA--CP_Module3.png)
pLN431-G8p-yCD-miR101BD (BBa_K2908670)
References
[1] Waclaw Szybalski”, Sun C. Kim”, Noaman Hasan” and Anna J. Podhajskab, Class IIS restriction enzymes, a review.1991, Gene, 100: 13-26.
[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.