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

 
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<a name='anchor1'></a>
 
<div class="top">
 
<div class="top">
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<img style="position:absolute;left:1170px;top:35px;z-index:+1;width:155px;height:180px;" src="https://static.igem.org/mediawiki/2016/8/8a/CGU_Taiwan--logo9.jpg">
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<a name='anchor0'></a>
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<header class="top">
 
<header class="top">
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<h1 class="headline">Leijuvant <small></small></h1>
 
 
<ul class="header-subnav">
 
<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">HOME</a></li>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Parts">PARTS</a></li>
 
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Parts">PARTS</a></li>
 
</ul>
 
</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>
 
</header>
  
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<div class="mid" style="top:180px;">
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<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>
 +
 
 +
 
 +
 
 +
<div class="mid" style="top:180px;margin-left:15%;margin-top:6%;">
 
<br>
 
<br>
 
<div style="font-size:60px;color:#66B3FF;text-decoration:none;">
 
<div style="font-size:60px;color:#66B3FF;text-decoration:none;">
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<br><br>
 
<br><br>
  
−
<b><font size="5px">We established stable Leishmania transfectants and <br>proved the function of
+
<b><font size="5px">1. We established stable Leishmania transfectants and <br>proved the function of
 
promotor with hygromycin drug selection gene</font></b>
 
promotor with hygromycin drug selection gene</font></b>
−
<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
+
<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
 
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
 
been elucidated yet. We selected the 5'- untranslated region of a highly expressed
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to high concentration of hygromycin. The function of 5’HYG was assayed more
 
to high concentration of hygromycin. The function of 5’HYG was assayed more
 
precisely in figure 2.
 
precisely in figure 2.
−
<img src="https://static.igem.org/mediawiki/2016/3/37/Proof1.jpeg" width=850px height=500px></img><br><br>
+
<img src="https://static.igem.org/mediawiki/2016/3/37/Proof1.jpeg" width=850px height=500px margin-right=15%></img><br><br>
 
</div><br>
 
</div><br>
  
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<br><br>
 
<br><br>
  
−
<b><font size="6px">Successfully expressed Hemagglutinin of H1N1</font></b>
+
<b><font size="6px">2. Successfully expressed Hemagglutinin of H1N1</font></b>
−
<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
+
<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;margin-right:15%;"><br>
 
Since we are performing immunology experiments, it is very important that the
 
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
 
antigen is correctly expressed. We used BL21 competent cell to express the HA
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recognize the HA protein from Western blot analysis (approximately 62.3kd) when
 
recognize the HA protein from Western blot analysis (approximately 62.3kd) when
 
induced by IPTG (Figure 3.).<br><br>
 
induced by IPTG (Figure 3.).<br><br>
−
<img src="https://static.igem.org/mediawiki/2016/4/4e/Proof2.jpeg" width=850px height=500px></img><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>
 
</div>
 
<br><br>
 
<br><br>
  
−
<b><font size="6px">The immune response of Leijuvant in mice</font></b>
+
<b><font size="6px">3. The antibody production of Leijuvant in mice</font></b>
−
<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
+
<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 15th day.
+
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 25th day (Figure 4A.). Inactivated
+
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
 
Leishmania can achieve over 60% of the antibody titer. As for OVA-expressing
−
inactivated Leishmania, the antibody titer can reach about 80% on the 25th day. The
+
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 20th day
+
IgG2a antibody titer of inactivated Leishmania increased extremely on the 20<sup>th</sup> day
−
and is significantly higher compare to Alum on the 25th day(Figure 4A.). Leijuvant is a
+
and is significantly higher compare to Alum on the 25<sup>th</sup> day<b>(Figure 4B.)</b>. <b>Leijuvant</b> is a
−
potential bi-pathway adjuvant that can stimulate both Th1 and Th2 immune  
+
potential <b>bi-pathway</b> adjuvant that can stimulate both Th1 and Th2 immune  
 
responses.<br><br>
 
responses.<br><br>
−
<img src="https://static.igem.org/mediawiki/2016/1/19/Proof3.jpeg" width=850px height=500px></img><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>
 
<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>
 
<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>
 +
<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%;">
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Design"><b style="color:white;font-size:17px;margin-left:10%;">Design</li></a>
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Background"><b style="color:white;font-size:17px;margin-left:10%;">Background</li></a>
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Results"><b style="color:white;font-size:17px;margin-left:10%;">Results</li></a>
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Proof"><b style="color:white;font-size:17px;margin-left:10%;">Proof</li></a>
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Notebook"><b style="color:white;font-size:17px;margin-left:10%;">Notebook</li></a>
 +
<br><br>
 +
</ul>
 +
</ul>
 
</div>
 
</div>
 +
 +
<div class="sitemap" style="margin-left:55%;margin-top:-3.5%;">
 +
<ul class="list1" class="margin-left:5%;">
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Human_Practices"><b style="color:white;font-size:18px;margin-left:20%;">HUMAN PRACTICES</li></a>
 +
<br><br>
 +
<ul class="list2" class="margin-left:6%;margin-top:50%;">
 +
<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>
 +
<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>
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Human_Practices_Communication"><b style="color:white;font-size:17px;margin-left:10%;">Communication</li></a>
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Entrepreneurship"><b style="color:white;font-size:17px;margin-left:10%;">Entrepreneurship</li></a>
 +
</ul>
 +
</ul>
 +
</div>
 +
 +
<div class="sitemap" style="margin-left:75%;margin-top:-3.5%;">
 +
<ul class="list1" class="margin-left:5%;">
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Software"><b style="color:white;font-size:18px;margin-left:20%;">MODELING</li></a>
 +
<br><br>
 +
<ul class="list2" class="margin-left:6%;">
 +
<li><a href="http://163.25.92.36/igemcgu/igemwebhome.htm"><b style="color:white;font-size:17px;margin-left:10%;">McHug</li></a>
 +
</ul>
 +
</ul>
 +
<ul class="list1" class="margin-left:1%;margin-top:-10%;">
 +
<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>
 +
<br><br>
 +
<ul class="list2" class="margin-left:6%;margin-top:-5%;">
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Collaborations"><b style="color:white;font-size:17px;margin-left:10%;">Collaborations</li></a>
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Attributions"><b style="color:white;font-size:17px;margin-left:10%;">Attributions</li></a>
 +
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Team#anchor2"><b style="color:white;font-size:17px;margin-left:10%;">Sponsors</li></a>
 +
</ul>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
  
 
</body>
 
</body>
 
</html>
 
</html>

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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