Difference between revisions of "Team:CGU Taiwan/Proof"

 
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{{CGU_Taiwan}}
 
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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2016.igem.org/Judging/Medals">gold medal criterion for proof of concept</a>. </p>
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<p> Delete this box in order to be evaluated for this medal. See more information at <a href="https://2016.igem.org/Judging/Pages_for_Awards/Instructions"> Instructions for Pages for awards</a>.</p>
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<a name='anchor1'></a>
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<div class="top">
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<img style="margin-top:-2%;width:100%;height:140%;position:absolute;" src="https://static.igem.org/mediawiki/2016/0/0b/CGU_Taiwan--top.jpg">
 +
</div>
 
</div>
 
</div>
  
 +
<header class="top">
 +
<ul class="header-subnav">
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan">HOME</a></li>
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Achievements">ACHIEVEMENTS</a></li>
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Description">PROJECT</a></li>
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Software">MODELING</a></li>
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Human_Practices">HUMAN PRACTICES</a></li>
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Team">PEOPLE</a></li>
 +
<li class="dropdown"><a href="https://2016.igem.org/Team:CGU_Taiwan/Interlab">INTERLAB</a></li>
 +
<li class="dropdown"><a href="https://2016.igem.org/Team:CGU_Taiwan/Safety">SAFETY</a></li>
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Parts">PARTS</a></li>
 +
</ul>
 +
<div class="circle"><img style="position:absolute;z-index:+1;width:90%;height:95%;margin-left:3%;margin-top:2%;" src="https://static.igem.org/mediawiki/2016/8/8a/CGU_Taiwan--logo9.jpg"></div>
 +
</header>
  
 +
<div class="left">
 +
<a href="#anchor1"><section class="left-tab" style="top:240px;" id="myP" onmousedown="mouseDown()" onmouseup="mouseUp()"><b>TOP</b></section></a>
 +
<a href="https://2016.igem.org/Team:CGU_Taiwan/Description"><section class="left-tab" style="top:275px;left:50px;">Overview</section></a>
 +
<a href="https://2016.igem.org/Team:CGU_Taiwan/Design"><section class="left-tab" style="top:310px;left:50px;">Design</section></a>
 +
<a href="https://2016.igem.org/Team:CGU_Taiwan/Background"><section class="left-tab" style="top:345px;left:50px;">Background</section></a>
 +
<a href="https://2016.igem.org/Team:CGU_Taiwan/Results"><section class="left-tab" style="top:380px;left:50px;">Results</section></a>
 +
<a href="https://2016.igem.org/Team:CGU_Taiwan/Proof"><section class="left-tab" style="top:415px;left:50px;">Proof</section></a>
 +
<a href="https://2016.igem.org/Team:CGU_Taiwan/Notebook"><section class="left-tab" style="top:450px;left:50px;">Notebook</section></a>
 +
</div>
  
  
  
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<div class="column full_size">
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<div class="mid" style="top:180px;margin-left:15%;margin-top:6%;">
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<br>
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<div style="font-size:60px;color:#66B3FF;text-decoration:none;">
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Proof of Concept
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</div>
 +
<br><br>
  
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<b><font size="5px">1. We established stable Leishmania transfectants and <br>proved the function of
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promotor with hygromycin drug selection gene</font></b>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;margin-right:15%;"><br>
 +
The promoter and the ribosomal binding site of Leishmania genome has not
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been elucidated yet. We selected the 5'- untranslated region of a highly expressed
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gene, P36, to be the promoter, RBS binding site and other extra function needed for
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Leishmania protein expression. We also added a hygromycin resist gene to serve as a
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dual functional biobrick of regulatory and selection marker. We provide the user with
 +
the regulation of protein expression and also the drug selection system that is most
 +
commonly and effectively used in Leishmania experiments.
 +
The pSB1C3-5'HYG-OVA-3'UTR was transfected into Leishmania 12-DT strain,
 +
and the transfectants were selected by hygromycin. We obtained the
 +
hygromycin-resistant Leishmania transfectants (Figure 1. B, C). In the negative control
 +
group (Figure 1. A), we could see that almost all cells were dead and the shape of
 +
Leishmania was not normal. It showed that Leishmania transfectants were resistant
 +
to high concentration of hygromycin. The function of 5’HYG was assayed more
 +
precisely in figure 2.
 +
<img src="https://static.igem.org/mediawiki/2016/3/37/Proof1.jpeg" width=850px height=500px margin-right=15%></img><br><br>
 +
</div><br>
  
−
<p>
 
−
iGEM teams are great at making things work! We value teams not only doing an incredible job with theoretical models and experiments, but also in taking the first steps to make their project real.
 
−
</p>
 
  
 +
<br><br>
  
−
<h4> What should we do for our proof of concept? </h4>
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<b><font size="6px">2. Successfully expressed Hemagglutinin of H1N1</font></b>
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<p>  
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;margin-right:15%;"><br>
−
You can assemble a device from BioBricks and show it works. You could build some equipment if you're competing for the hardware award. You can create a working model of your software for the software award. Please note that this not an exhaustive list of activities you can do to fulfill the gold medal criterion. As always, your aim is to impress the judges!
+
Since we are performing immunology experiments, it is very important that the
−
</p>
+
antigen is correctly expressed. We used BL21 competent cell to express the HA
 +
sequence and detect the protein by Western blot analysis.<br>
 +
The pSB1C3-J04500-HA can is checked by Western blot analysis. We successfully
 +
recognize the HA protein from Western blot analysis (approximately 62.3kd) when
 +
induced by IPTG (Figure 3.).<br><br>
 +
<img src="https://static.igem.org/mediawiki/2016/4/4e/Proof2.jpeg" width=850px height=500px margin-right=15%></img><br><br>
 +
</div>
 +
<br><br>
  
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<b><font size="6px">3. The antibody production of Leijuvant in mice</font></b>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;margin-right:15%;"><br>
 +
The IgG1 antibody titer increased drastically after the second boost on the 15<sup>th</sup> day.
 +
And the antibody titer reached to peak on the 25<sup>th</sup> day <b>(Figure 4A.)</b>. Inactivated
 +
Leishmania can achieve over 60% of the antibody titer. As for OVA-expressing
 +
inactivated Leishmania, the antibody titer can reach to 70% on the 25<sup>th</sup> day. The
 +
IgG2a antibody titer of inactivated Leishmania increased extremely on the 20<sup>th</sup> day
 +
and is significantly higher compare to Alum on the 25<sup>th</sup> day<b>(Figure 4B.)</b>. <b>Leijuvant</b> is a
 +
potential <b>bi-pathway</b> adjuvant that can stimulate both Th1 and Th2 immune
 +
responses.<br><br>
 +
<img src="https://static.igem.org/mediawiki/2016/5/5a/CGU_Taiwan_result_final.png" width=850px height=500px margin-right=15%></img><br><br>
 +
<br>
 +
</div>
 
</div>
 
</div>
  
  
  
 +
<div class="end2" style="margin-top:220%;">
 +
<br>
 +
<ul class="list3" class="margin-left:1%;margin-top:2%;">
 +
<li><a href="#anchor1"><b style="color:white;font-size:28px;margin-left:7%;">TOP</a></li>
 +
</ul>
 +
<br>
 +
<div class="sitemap" style="margin-left:5%;">
 +
<br>
 +
<b style="color:white;font-size:28px;margin-top:5%;margin-left:4%;text-decoration:none;">SITE MAP</b><br>
 +
<br><br>
 +
<img style="background-color:#0D014D;margin-left:20%;border-radius:50%;border:6px solid white;padding:5px;width:40%;height:70%;position:absolute;margin-left:2%;" src="https://static.igem.org/mediawiki/2016/0/03/CGU_Taiwan--leishmania3.jpg">
 
</div>
 
</div>
 +
<br>
 +
 +
<div class="sitemap" style="margin-left:22%;margin-top:-1%;">
 +
<ul class="list1" class="margin-left:5%;">
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan"><b style="color:white;font-size:18px;margin-left:20%;">HOME</li></a>
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Achievements"><b style="color:white;font-size:18px;margin-left:20%;">ACHIEVEMENTS</li></a>
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Interlab"><b style="color:white;font-size:18px;margin-left:20%;">INTERLAB</li></a>
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Safety"><b style="color:white;font-size:18px;margin-left:20%;">SAFETY</li></a>
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Parts"><b style="color:white;font-size:18px;margin-left:20%;">PARTS</li></a>
 +
</ul>
 +
</div>
 +
 +
<div class="sitemap" style="margin-left:40%;margin-top:-2%;">
 +
<ul class="list1" class="margin-left:5%;">
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Description"><b style="color:white;font-size:18px;margin-left:20%;">PROJECT</li></a>
 +
<br><br>
 +
<ul class="list2" class="margin-left:6%;margin-top:50%;">
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Design"><b style="color:white;font-size:17px;margin-left:10%;">Design</li></a>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Background"><b style="color:white;font-size:17px;margin-left:10%;">Background</li></a>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Results"><b style="color:white;font-size:17px;margin-left:10%;">Results</li></a>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Proof"><b style="color:white;font-size:17px;margin-left:10%;">Proof</li></a>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Notebook"><b style="color:white;font-size:17px;margin-left:10%;">Notebook</li></a>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Human_Practices_Survey"><b style="color:white;font-size:17px;margin-left:10%;">Survey</li></a>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Human_Practices_Education"><b style="color:white;font-size:17px;margin-left:10%;">Education</li></a>
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<li><a href="http://163.25.92.36/igemcgu/igemwebhome.htm"><b style="color:white;font-size:17px;margin-left:10%;">McHug</li></a>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Team"><b style="color:white;font-size:18px;margin-left:20%;">OUR TEAM</li></a>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Collaborations"><b style="color:white;font-size:17px;margin-left:10%;">Collaborations</li></a>
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Latest revision as of 06:01, 2 December 2016

TOP
Overview
Design
Background
Results
Proof
Notebook

Proof of Concept


1. We established stable Leishmania transfectants and
proved the function of promotor with hygromycin drug selection gene

The promoter and the ribosomal binding site of Leishmania genome has not been elucidated yet. We selected the 5'- untranslated region of a highly expressed gene, P36, to be the promoter, RBS binding site and other extra function needed for Leishmania protein expression. We also added a hygromycin resist gene to serve as a dual functional biobrick of regulatory and selection marker. We provide the user with the regulation of protein expression and also the drug selection system that is most commonly and effectively used in Leishmania experiments. The pSB1C3-5'HYG-OVA-3'UTR was transfected into Leishmania 12-DT strain, and the transfectants were selected by hygromycin. We obtained the hygromycin-resistant Leishmania transfectants (Figure 1. B, C). In the negative control group (Figure 1. A), we could see that almost all cells were dead and the shape of Leishmania was not normal. It showed that Leishmania transfectants were resistant to high concentration of hygromycin. The function of 5’HYG was assayed more precisely in figure 2.




2. Successfully expressed Hemagglutinin of H1N1

Since we are performing immunology experiments, it is very important that the antigen is correctly expressed. We used BL21 competent cell to express the HA sequence and detect the protein by Western blot analysis.
The pSB1C3-J04500-HA can is checked by Western blot analysis. We successfully recognize the HA protein from Western blot analysis (approximately 62.3kd) when induced by IPTG (Figure 3.).





3. The antibody production of Leijuvant in mice

The IgG1 antibody titer increased drastically after the second boost on the 15th day. And the antibody titer reached to peak on the 25th day (Figure 4A.). Inactivated Leishmania can achieve over 60% of the antibody titer. As for OVA-expressing inactivated Leishmania, the antibody titer can reach to 70% on the 25th day. The IgG2a antibody titer of inactivated Leishmania increased extremely on the 20th day and is significantly higher compare to Alum on the 25th day(Figure 4B.). Leijuvant is a potential bi-pathway adjuvant that can stimulate both Th1 and Th2 immune responses.







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