Difference between revisions of "Team:Mingdao/Composite Part"

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{{Mingdao}}
+
{{:Team:Mingdao/test9}}
−
<html>
+
<html lang="en">
 +
  <head>
 +
    <meta charset="UTF-8">
 +
    <link href='https://fonts.googleapis.com/css?family=Arizonia' rel='stylesheet'>
 +
    <link href='https://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet'>
 +
    <title>Description</title>
  
 +
<style type="text/css">
 +
* {
 +
  margin: 0;
 +
  padding: 0;
 +
  box-sizing: border-box; }
  
 +
body {
 +
  width: 100%;
 +
  margin: 0;
 +
  padding: 0;
 +
  font-family: 'Ubuntu' !important; }
  
−
<div class="column full_size judges-will-not-evaluate">
 
−
<h3>★  ALERT! </h3>
 
−
<p>This page is used by the judges to evaluate your team for the <a href="https://2018.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2018.igem.org/Judging/Awards"> award listed below</a>. </p>
 
−
<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2018.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
 
−
</div>
 
  
 +
.bg-container {
 +
  background-attachment: fixed;
 +
  overflow: hidden;
 +
  position: relative;
 +
  background-image: url(https://static.igem.org/mediawiki/2018/b/b7/T--Mingdao--ModelingBackground.jpg);
 +
  background-repeat: no-repeat;
 +
  background-size: 100%;
 +
  background-position: center;
  
−
<div class="clear"></div>
+
}
  
  
 +
.my-main-container {
 +
  width: 103.4%;
 +
  padding: 50px 7%;
 +
}
  
 +
.main-content {
 +
  background-color: rgba(255,255,255, .7);
 +
  width: 75%;
 +
  margin-left: 11%;
 +
  padding: 50px;
 +
  min-height: 180vh;
 +
margin-top:600px;
 +
}
  
 +
.text-area {
 +
  margin-bottom: 80px; }
 +
  .text-area h1 {
 +
    font-size: 130px;
 +
    font-weight: 700;
 +
    font-family: 'Arizonia' !important;
 +
    text-align: center;
 +
    margin-bottom: 1rem; }
 +
  .text-area p {
 +
    font-size: 22px;
 +
    font-weight: 500; }
  
−
<div class="column full_size">
+
.m-block img {
−
<h1>Composite Parts</h1>
+
  width: 100%; }
  
 +
.path-btns {
 +
z-index:0;
 +
  position: fixed;
 +
  width: 250px;
 +
  top: 200px;
 +
  left: 225px; }
  
 +
  .path-btns .path-dot {
 +
    width: 25px;
 +
    height: 25px;
 +
    border-radius: 50%;
 +
    position: absolute;
 +
    background-color: #fff;
 +
    top: 5px;
 +
    left: 27px;
 +
    border: 5px solid #385e66;
 +
    cursor: pointer; }
 +
    .path-btns .path-dot.active {
 +
      background-color: #385e66; }
 +
    .path-btns .path-dot:hover {
 +
      transition: all .3s;
 +
      transform: scale(1.2); }
 +
 +
.path {
 +
  height: 100px;
 +
  display: flex; }
 +
  .path .pathSvg {
 +
    display: block; }
 +
  .path .pathWord {
 +
    padding-right: 10%; }
 +
  .path .path-btn {
 +
    cursor: pointer;
 +
    fill: #fff; }
 +
    .path .path-btn.path-active {
 +
      fill: #385e66; }
 +
  .path .path-word-sm {
 +
    font-size: 12px; }
 +
#HQ_page .path .pathWord p {
 +
      font-weight: 700;
 +
      text-align: left !important;
 +
      font-size: 16px;
 +
}
 +
#HQ_page .text-area p {
 +
  font-size: 22px;
 +
  font-family: 'Ubuntu';
 +
  font-weight: 500; }
 +
img.center {
 +
  display: block;
 +
  margin: 0 auto; }
 +
 +
.text-area h2{
 +
    text-align: center;
 +
    margin-bottom: 2rem !important;
 +
    margin-top: 2rem;
 +
    color: black !important;
 +
    font-size: 40px;
 +
    font-weight:bold;
 +
    font-family:serif;
 +
}
 +
  .text-area h3{
 +
  text-align: left;
 +
  margin-bottom: 2rem !important;
 +
  margin-top: 2rem;
 +
  color: #4682B4  !important;
 +
  font-size: 28px;
 +
  font-weight:bold;
 +
}
 +
  .text-area h4{
 +
  text-align: left;
 +
  margin-bottom: 2rem !important;
 +
  margin-top: 2rem;
 +
  color: #f9000c !important;
 +
  font-size: 30px;
 +
  font-weight:bold;
 +
}
 +
 +
.top {
 +
  position: fixed;
 +
  right: 50px;
 +
  bottom: 50px;
 +
  height: 50px;
 +
  width: 50px;
 +
  cursor: pointer; }
 +
  .top img {
 +
    width: 100%; }
 +
img.pic {
 +
  display: block !important;
 +
  margin: 40px auto !important;
 +
}
 +
 +
.top-picture{
 +
z-index:2;
 +
width:100%;
 +
position: absolute;}
 +
 +
video {
 +
            position:relative;
 +
            width: 100%;
 +
            height:auto;
 +
            margin: 20px auto !important;
 +
          }
 +
</style>
 +
  </head>
 +
 +
<body>
 +
    <img class="top-picture" src="https://static.igem.org/mediawiki/2018/0/08/T--Mingdao--Phil13-4.png">
 +
    <div class="bg-container" style="max-height:none;">
 +
      <div class="my-main-container">
 +
        <div class="main-content">
 +
          <div class="text-area">
 +
            <h2 id = "d-ac5"></h2>
 +
<br />
 +
            <img class="center" src="https://static.igem.org/mediawiki/2018/5/5a/T--Mingdao--phil13M11.png" alt="" style="width: 80%; margin-bottom: 20px;">
 +
  <br /><br /><br />         
 +
<p style="text-indent:2em">
 +
HIV is a huge epidemic around the world which can cause AIDS in infected people. To identify HIV is very difficult for people in limited-resource countries and individuals who wants privacy. An easy-to-use, cheap and portable testing device is urgently need around the world.
 +
</p>
 +
<br />
 +
<p style="text-indent:2em">
 +
To further engineer the mosquito to recognize HIV, we designed and created a synthetic HIV-specific receptor composed of <a href=https://www.uniprot.org/uniprot/P01730>human CD4</a> extracellular domain (1-396 aa) and <a href=https://www.uniprot.org/uniprot/P08953>Drosophila Toll</a> transmembrane and intracellular domains (808-828 aa and 829-1097 aa, respectively) based on UniProt protein database.
 +
</p>
 +
<br />
 +
<p style="text-indent:2em">
 +
The DNA fragments of human CD4 and Drosophila Toll domains were synthesized by Integrated DNA Technologies, Inc. (IDT). The DNAs were cloned onto pSB1C3 as BioBrick basic parts and confirmed by sequencing. The CD4 and Toll was further assembled with Ac5 promoter and polyA to become BioBrcik composite parts. Finally, the fusion protein was made as Ac5-hCD4-dToll-polyA / pSB1C3 (<a href=http://parts.igem.org/Part:BBa_K2543010>BBa_K2543010</a>)
 +
</p>
 +
<br />
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/8/8e/T--Mingdao--sam200-1.png" alt="" style="width: 60%; margin-bottom: 20px;"><br />
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/7/7b/T--Mingdao--sam200-2.png" alt="" style="width: 60%; margin-bottom: 20px;"><br />
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/c/c4/T--Mingdao--sam200-3.png" alt="" style="width: 60%; margin-bottom: 20px;"><br />
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/2/23/T--Mingdao--sam200-4.png" alt="" style="width: 60%; margin-bottom: 20px;"><br />
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/3/3f/T--Mingdao--sam200-5.png" alt="" style="width: 80%; margin-bottom: 20px;"><br /><br />
 +
            <h4> Ac5 promoter with strong activity </h4>
 +
<p style="text-indent:2em">
 +
To test the gene expression driven by Ac5 promoter, we cultured a mosquito Aedes albopictus C6/36 cell line and transfected cells with the plasmid of Ac5-GFP-polyA (K2543004). GFP positive cells and intensity were analyzed 2 days after transfection.
 +
</p>
 +
<br />
 +
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/a/ad/T--Mingdao--phil13M13.png" alt="" style="width: 80%; margin-bottom: 20px;">
 +
 +
            <h3>EXPERIMENT</h3>
 +
    <p>
 +
&#8595; C6/36 cells (1.8 x 10<sup>5</sup> cells/well in a 96-well plate)<br />
 +
&#8595; Liposome-mediated transfection and culture for 2 more days<br />
 +
&#8595; Read fluorescence intensity at Ex/Em = 480/520 nm with a microplate reader<br />
 +
&#8595; Observe GFP+ cells under a fluorescence microscope<br />
 +
<br />
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/2/20/T--Mingdao--phil13M14.png" alt="" style="width: 80%; margin-bottom: 20px;">
 +
              <h3>RESULT</h3>
 +
<p style="text-indent:2em">
 +
As data shown here, Ac5 is a strong and constitutive promoter which can drive GFP to high expression level in mosquito cells. And we can transfect more than 50% of GFP positive cell with liposome-mediated DNA delivery.
 +
</p>
 +
<br /><br />
 +
            <h4 id="d-cd4"> Signal driven by CD4-Toll chimera and blocked by HIV </h4>
 +
<p style="text-indent:2em">
 +
To test feasibility of fusion CD4-Toll chimera, we acquired the plasmid of Drosomycin promoter-luciferase from world-renowned insect geneticist, Dr. Jean-Luc Imler and conducted the luc reporter assay with Ac5-CD4-Toll-polyA in the mosquito cells.
 +
</p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/a/ab/T--Mingdao--phil13M15.png" alt="" style="width: 30%; margin-bottom: 20px;">
 +
<br />
 +
            <h3>EXPERIMENT</h3>
 +
    <p>
 +
&#8595; C6/36 cells (1.8 x 10<sup>5</sup> cells/well in a 96-well plate)<br />
 +
&#8595; Liposome-mediated transfection and culture for 2 more days<br />
 +
&#8595; Add gp120 of HIV (1 μg/ml*) or not and incubate for 24 hours<br />
 +
&#8595; Cell lysis and luciferase assay<br />
 +
*The concentration of gp120 in the serum of HIV-infected people is between 0.12~1 μg/ml.
 +
<br /><br />
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/6/63/T--Mingdao--phil13M16.png" alt="" style="width: 90%; margin-bottom: 20px;">
 +
              <h3>RESULT</h3>
 +
<p style="text-indent:2em">
 +
The result indicated that luciferase activity driven by Drosomycin promoter can be triggered by CD4-Toll chimera. The activity was decreased in the presence of gp120 of HIV. The finding demonstrates the possibility that GE mosquito created by our project could be applied to detect HIV virus in infected human blood. 
 +
</p>
 +
<br /> <br /><br />
 +
            <h4 id="d-design"> Design principle of GE mosquito</h4>
 +
          <video playinline controls="true">
 +
<source src="https://static.igem.org/mediawiki/2018/5/54/T--Mingdao--HomePage_BriefIntro.mp4" type="video/mp4" >
 +
</video>
 +
              <h2>Reference</h2>
 
<p>
 
<p>
−
A composite part is a functional unit of DNA consisting of two or more basic parts assembled together. <a href="http://parts.igem.org/wiki/index.php/Part:BBa_I13507">BBa_I13507</a> is an example of a composite part, consisting of an RBS, a protein coding region for a red fluorescent protein, and a terminator.
+
1. <a href=https://www.uniprot.org/uniprot/P01730>UniProtKB - P01730 (CD4_HUMAN)</a> <br />
 +
<p>
 +
2. <a href=https://www.uniprot.org/uniprot/P08953> UniProtKB - P08953 (TOLL_DROME)</a> <br />
 +
<p>
 +
3. <a href=https://www.ncbi.nlm.nih.gov/pubmed/2449285> Cell. (1988) The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein.
 +
</a> <br />
 +
<p>
 +
4. <a href=https://www.ncbi.nlm.nih.gov/pubmed/16709847>J Immunol. (2006) Evidence for a domain-swapped CD4 dimer as the coreceptor for binding to class II MHC.</a> <br />
 +
<p>
 +
5. <a href=https://www.ncbi.nlm.nih.gov/pubmed/16622011>J Immunol. (2006) Triggering of T cell activation via CD4 dimers.
 +
</a> <br />
 +
<p>
 +
6. <a href=https://www.ncbi.nlm.nih.gov/pubmed/16917510>Nat Rev Immunol. (2006) Toll-like receptors as molecular switches.
 +
</a> <br />
 +
<p>
 +
7. <a href=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1524797>Retrovirology. (2006) Association between disruption of CD4 receptor dimerization and increased human immunodeficiency virus type 1 entry
 +
</a> <br />
 +
<p>
 +
8. <a href=https://www.ncbi.nlm.nih.gov/pubmed/21209287>J Immunol. (2011) The Drosophila Toll signaling pathway.</a> <br />
 +
<p>
 +
9. <a href=https://www.ncbi.nlm.nih.gov/pubmed/24550395>J Biol Chem. (2014) Disulfide reduction in CD4 domain 1 or 2 is essential for interaction with HIV glycoprotein 120 (gp120), which impairs thioredoxin-driven CD4 dimerization. </a> <br />
 +
<p>
 +
 
 
</p>
 
</p>
  
−
<p>New composite BioBrick devices can be made by combining existing BioBrick Parts (like Inverters, Amplifiers, Smell Generators, Protein Balloon Generators, Senders, Receivers, Actuators, and so on).</p>
+
          </div>
−
</div>
+
        </div>
 +
      </div>
 +
    </div>
 +
 
 +
    <div class="path-btns" style="left:0;">
 +
      <div class="path">
 +
        <div class="pathSvg">
 +
          <svg width="80" height = "100">
 +
            <rect x ="36" y="20" width="6" height="80" style="fill:#385e66"/>
 +
          </svg>
 +
        </div>
 +
        <div id="ac5-btn" class="path-dot"></div>
 +
        <div class="pathWord path-word-sm">
 +
          <p>Ac5 promoter</p>
 +
        </div>
 +
      </div>
 +
 
 +
      <div class="path">
 +
        <div class="pathSvg">
 +
          <svg width="80" height = "100">
 +
            <rect x ="36" y="20" width="6" height="80" style="fill:#385e66"/>
 +
          </svg>
 +
        </div>
 +
        <div id="cd4-btn" class="path-dot" style="top: 100px"></div>
 +
        <div class="pathWord path-word-sm">
 +
          <p>CD4-Toll chimera</p>
 +
        </div>
 +
      </div>
 +
 
 +
      <div class="path">
 +
        <div class="pathSvg">
 +
          <svg width="80" height = "100">
 +
            <rect x ="36" y="20" width="0" height="80" style="fill:#385e66"/>
 +
          </svg>
 +
        </div>
 +
        <div id="design-btn" class="path-dot" style="top: 200px"></div>
 +
        <div class="pathWord path-word-sm">
 +
          <p>Design principle</p>
 +
        </div>
 +
      </div>
 +
 
 +
    </div>
  
−
<div class="column full_size">
+
    <div class="top">
−
<div class="highlight decoration_background">
+
      <img class="center" src="https://static.igem.org/mediawiki/2018/5/58/T--Mingdao--go_to_top.jpg" alt="">
−
<h3>Note</h3>
+
    </div>
−
<p>This page should list all the composite parts your team has made during your project. You must add all characterization information for your parts on the Registry. You should not put characterization information on this page. Remember judges will only look at the first part in the list for the Best Composite Part award, so put your best part first!</p>
+
−
</div>
+
−
</div>
+
  
 +
</body>
  
 +
<script type="text/javascript">
 +
    $("#ac5-btn").click(function() {
 +
      $('html, body').animate({
 +
          scrollTop: $("#d-ac5").offset().top
 +
      }, 400);
 +
    });
 +
    $("#cd4-btn").click(function() {
 +
      $('html, body').animate({
 +
          scrollTop: $("#d-cd4").offset().top
 +
      }, 400);
 +
    });
 +
    $("#design-btn").click(function() {
 +
      $('html, body').animate({
 +
          scrollTop: $("#d-design").offset().top
 +
      }, 400);
 +
    });
  
−
<div class="column full_size">
+
    $(document).ready(function(){
−
<h3>Best Composite Part Special Prize</h3>
+
      $('.top').on('click', function(){
 +
        $('html, body').animate({scrollTop: '0px'}, 400);
 +
      });
 +
        $("#ac5-btn").css('background-color', '#385e66');
 +
        var scroll_pos = 0;
 +
        $(document).scroll(function() {
 +
            scroll_pos = $(this).scrollTop();
  
−
<p>To be eligible for this award, this part must adhere to <a href="http://parts.igem.org/DNA_Submission">Registry sample submission guidelines</a> and have been sent to the Registry of Standard Biological Parts. If you have a part you wish to nominate your team for this <a href="https://2018.igem.org/Judging/Awards">special prize</a>, make sure you add your part number to your <a href="https://2018.igem.org/Judging/Judging_Form">judging form</a> and delete the box at the top of this page.
+
            d_ac5_pos = $("#d-ac5").offset().top -100
 +
            d_cd4_pos = $("#d-cd4").offset().top -100
 +
            d_design_pos= $("#d-design").offset().top -100
  
−
<br><br>
+
            // introduction
−
<b>Please note:</b> Judges will only look at the first part number you list, so please only enter ONE (1) part number in the judging form for this prize. </p>
+
            if(scroll_pos < d_cd4_pos) {
 +
                $(".path-dot").css('background-color', '#fff')
 +
                $("#ac5-btn").css('background-color', '#385e66');}
 +
            // model1
 +
            else if(scroll_pos < d_design_pos){
 +
              if(scroll_pos >= d_cd4_pos){
 +
                $(".path-dot").css('background-color', '#fff')
 +
                $("#cd4-btn").css('background-color', '#385e66');}}
  
−
</div>
+
            // conclusion
 +
            else if( scroll_pos >= d_design_pos) {
 +
                $(".path-dot").css('background-color', '#fff')
 +
                $("#design-btn").css('background-color', '#385e66');
 +
            }
 +
        });
 +
    });
 +
  </script>
  
 
</html>
 
</html>
 +
{{:Team:Mingdao/test6}}

Latest revision as of 03:10, 18 October 2018

HOME
Description





HIV is a huge epidemic around the world which can cause AIDS in infected people. To identify HIV is very difficult for people in limited-resource countries and individuals who wants privacy. An easy-to-use, cheap and portable testing device is urgently need around the world.


To further engineer the mosquito to recognize HIV, we designed and created a synthetic HIV-specific receptor composed of human CD4 extracellular domain (1-396 aa) and Drosophila Toll transmembrane and intracellular domains (808-828 aa and 829-1097 aa, respectively) based on UniProt protein database.


The DNA fragments of human CD4 and Drosophila Toll domains were synthesized by Integrated DNA Technologies, Inc. (IDT). The DNAs were cloned onto pSB1C3 as BioBrick basic parts and confirmed by sequencing. The CD4 and Toll was further assembled with Ac5 promoter and polyA to become BioBrcik composite parts. Finally, the fusion protein was made as Ac5-hCD4-dToll-polyA / pSB1C3 (BBa_K2543010)








Ac5 promoter with strong activity

To test the gene expression driven by Ac5 promoter, we cultured a mosquito Aedes albopictus C6/36 cell line and transfected cells with the plasmid of Ac5-GFP-polyA (K2543004). GFP positive cells and intensity were analyzed 2 days after transfection.


EXPERIMENT

↓ C6/36 cells (1.8 x 105 cells/well in a 96-well plate)
↓ Liposome-mediated transfection and culture for 2 more days
↓ Read fluorescence intensity at Ex/Em = 480/520 nm with a microplate reader
↓ Observe GFP+ cells under a fluorescence microscope

RESULT

As data shown here, Ac5 is a strong and constitutive promoter which can drive GFP to high expression level in mosquito cells. And we can transfect more than 50% of GFP positive cell with liposome-mediated DNA delivery.



Signal driven by CD4-Toll chimera and blocked by HIV

To test feasibility of fusion CD4-Toll chimera, we acquired the plasmid of Drosomycin promoter-luciferase from world-renowned insect geneticist, Dr. Jean-Luc Imler and conducted the luc reporter assay with Ac5-CD4-Toll-polyA in the mosquito cells.


EXPERIMENT

↓ C6/36 cells (1.8 x 105 cells/well in a 96-well plate)
↓ Liposome-mediated transfection and culture for 2 more days
↓ Add gp120 of HIV (1 μg/ml*) or not and incubate for 24 hours
↓ Cell lysis and luciferase assay
*The concentration of gp120 in the serum of HIV-infected people is between 0.12~1 μg/ml.

RESULT

The result indicated that luciferase activity driven by Drosomycin promoter can be triggered by CD4-Toll chimera. The activity was decreased in the presence of gp120 of HIV. The finding demonstrates the possibility that GE mosquito created by our project could be applied to detect HIV virus in infected human blood.




Design principle of GE mosquito

Reference

1. UniProtKB - P01730 (CD4_HUMAN)

2. UniProtKB - P08953 (TOLL_DROME)

3. Cell. (1988) The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein.

4. J Immunol. (2006) Evidence for a domain-swapped CD4 dimer as the coreceptor for binding to class II MHC.

5. J Immunol. (2006) Triggering of T cell activation via CD4 dimers.

6. Nat Rev Immunol. (2006) Toll-like receptors as molecular switches.

7. Retrovirology. (2006) Association between disruption of CD4 receptor dimerization and increased human immunodeficiency virus type 1 entry

8. J Immunol. (2011) The Drosophila Toll signaling pathway.

9. J Biol Chem. (2014) Disulfide reduction in CD4 domain 1 or 2 is essential for interaction with HIV glycoprotein 120 (gp120), which impairs thioredoxin-driven CD4 dimerization.

Ac5 promoter

CD4-Toll chimera

Design principle