Team:Tsinghua/Project/Drug

From 2014.igem.org

(Difference between revisions)
 
(30 intermediate revisions not shown)
Line 62: Line 62:
     </ul>
     </ul>
   </nav>
   </nav>
-
<aside style="height: 260px;"></aside>
+
<aside style="height: 3150px;"></aside>
   <!-- end .sidebar1 --></div>
   <!-- end .sidebar1 --></div>
   <article class="content">
   <article class="content">
-
   <h1>AAV</h1>
+
   <h1>The Drug</h1>
<p style="text-align: right;">Return to: <a href="https://2014.igem.org/Team:Tsinghua/Project">Project</a></p>
<p style="text-align: right;">Return to: <a href="https://2014.igem.org/Team:Tsinghua/Project">Project</a></p>
 +
<img class="fltrt" style="border-radius:10px; margin-right:30px" src="https://static.igem.org/mediawiki/2014/e/ee/Tsinghua_Icon_Project_Drug.gif" height="250" />
 +
<p>In nature, P<sub>tet</sub> promoters expresses two proteins, one of which, TetR, acts as a repressor and the other, TetA, pumps tetracycline out of the cell.</p>
-
    <p>We choose <i><b> adeno-associated virus </b></i> as platform to construct plasmids, which could just insert into 19 chromosome. Then we designed the following experiments to prove it safe and practicable .</p>
+
<p>Based on this, Tet-On and Tet-Off systems are two types of tet-involved activation systems that help induce gene expression, and they are the most commonly used ones in eukaryotic cells. Transcription is up or down regulated when tetracycline or its derivatives are present. Tet-On and Tet-Off systems are different in that Tet-On activates transcription with doxycycline is present while Tet-Off does when dox is absent.</p>
-
  <p>1.  Add NotI cleavage sites on the both ends of mCherry gene. (the AAV vector we bought has NotI cleavage sites )</br>
+
-
  2. Insert mCherry gene into the AAV vector by restriction endonuclease NotI. Then we got the recombinant vector mCherry-AAV.</br>
+
-
    3. Transfect the <i><b> mCherry-AAV </b></i> into 293T cell line through calcium phosphate-based method. During this step the plasmids, pAAV-RC and pHelper, which express the shell of AAV are needed. (this kind of method that divide the virus into more than one expressing plasmids makes AAV safe)</br>
+
-
  4. Collect the virus through dry ice-ethanol bath and water bath in 37℃, and then add the virus into the 293T cell line.</br>
+
-
    5. After 48h, we can track the AAV in 293T cell lines by detecting the fluorescence of mCherry.
+
-
The results are as follows:
+
-
</br>
+
-
    </p>
+
-
<table align="center"><tr><td align="center"><img src="https://static.igem.org/mediawiki/2014/7/7a/2014_thu_aav_fig1.jpg" width="350" height="280"></br><span style="font-size:10px">Figure1. 293T cell under mcherry fluorescence</span></td><td align="center"><img src="https://static.igem.org/mediawiki/2014/5/52/2014_thu_aav_fig2.jpg" width="350" height="280"></br><span style="font-size:10px">Figure2. 293T cell under whitefield</span></td></tr></table>
+
-
<div align="center"><img src="https://static.igem.org/mediawiki/2014/6/63/2014_thu_aav_fig3.png" width="350" height="350"></br><span style="font-size:10px" >Figure3. 293T cell after merge</span></div>
+
-
<p>We can see from the figure that recombinant mcherry-AAV vectors express in the 293T cell successfully, which prove that the AAV system is practicable.</br>
+
-
However, the system is not exactly perfect. Although when 293T cells are producing virus we can detect 70%-80% positive rate, after virus infect the 293T cells, less than 1% positive rate can be observed. We have suspected that the pAAV-RC and pHelper plasmids are wrong, so we do an enzyme-cleavage experiment to test them, the results are as follows:</p>
+
-
<div align="center"><img src="https://static.igem.org/mediawiki/2014/f/fe/2014_thu_aav_fig4.jpg" width="350" height="280"></br><span style="font-size:10px" >Figure4. RC1 mono-enzyme cleave the pAAV-RC</span></div>
+
-
<p>The result match the DNA sequence we expect, so the plasmids are right.</br>
+
-
Another condition can’t be ignored that we use 293T as the virus-producing platform, but in fact, there are various 293 cell lines, such as 293F, 293A and etc. Therefore, 293T cell lines may not be the best cell line to produce the AAV virus.</br>
+
<p>Tet-Off was first developed and published in 1992, featuring a fusion protein, tTA, obtained from TetR and a virus protein. tTA responds to Tet0 sequences and its binding activates transcription, while presence of tetracycline or its derivatives would block its binding.</p>
   
   
-
Then we sort the 293T cells which have mcherry fluorescence, and after incubate the cell as many as enough, we can test the expression of mcherry on the  mRNA level by qPCR.</p>
+
<img class="center" src="https://static.igem.org/mediawiki/2014/a/a6/Tsinghua_Tetoff.png" width="700" />
 +
<p class="center">Fig. 1 Tet-Off system schematic</p>
 +
 
 +
<p>Tet-On system works similarly to Tet-Off system. The corresponding protein, rtTA, binds to the operator with Tet or its derivatives present.</p>
 +
 +
<img class="center" src="https://static.igem.org/mediawiki/2014/b/bd/Tsinghua_TetOn.png" width="700" />
 +
<p class="center">Fig. 2 Tet-On system schematic</p>
 +
 
 +
<p>We get Tet-On system from 2013 Tsinghua team. At first, we design a test plasmid to determine the feasibility of applying Tet-On system in our system. </p>
 +
<p>We constructed an mCherry gene after a CYC1 TATA region (TATA region) which is regulated by a tetracycline response element on its upstream. After mCherry gene, a CYC1 terminator is used to terminate the transcription of mRNA.</p> 
 +
<img class="center" src = "https://static.igem.org/mediawiki/2014/6/6a/Tsinghua_CM252%2BmCherry.png" width="400" />
 +
<p class="center">Fig. 3 CM252-mCherry map</p>
 +
 
 +
<p>After construction of this plasmid, we transfect it into HEK293T cell to see if the mCherry gene can be expressed under the regulation of the DOX (tetracycline analogue).</p>
 +
 
 +
<p>Then we do the PCR of the Tet-On system with mCherry to bring in two restrition sites at the terminals. After purification of the PCR products, we cut the vector and the tet on system and ligate them. The plasmid which has AAV vector that we bought from a company has two the same restrition sites(NotI) in its insert rigion. So, to aviod the vector’s self-ligation, we dispose the digestion product with CIAP to remove the phosphate group at the sticky end of the vector.</p>
 +
 +
<img class="center" src = "https://static.igem.org/mediawiki/2014/2/2a/Tsinghua_PAAV%2BTet_on%2BmCherry.png" width="400" />
 +
<p class="center">Fig. 4 CM252-pAAV+Tet-On+mCherry map</p>
 +
 
 +
<p>After construction of this plasmid, we want to replace this mCherry gene with insulin. Since we want to transfect the AAV vector into somatic cells, we must make sure that the insulin can be processed in these cells. In beta cells, proinsulin was processed by cellular endopeptidases known as prohormone convertases (PC1 and PC2), as well as the exoprotease carboxypeptidase. However, in non-endocrine cells, some of these enzyme are lacking. So, if we want to get mature insulin, we must alter the insulin gene sequence. In non-endocrine cells, an endoprotease called furin which can cleave the concensus processing site, -Arg<sup>-4</sup>-X<sup>-3</sup>-Lys/Arg<sup>-2</sup>-Arg<sup>-1</sup>. So, we add two 6bp nucleotides into two cleavage sites in insulin CDS to get the furin endoprotease sites.</p>
 +
<p>To get the insulin gene, firstly we want to PCR every exons of its in human genome and then overlap them. However, the sequence of insulin is in a high GC region in the genome. So, our first trial was not successful. Fortunately, the insulin gene is very small. So, we design 12 overlap primers and do one reaction to ligate these oligos. In that step, we insert these two furin sites into our sequence.</p>
 +
<p class="center"><img src="https://static.igem.org/mediawiki/2014/0/0a/Tsinghua_insulin-band.png" height="300" /></p>
 +
<p class="center">Fig. 5 insulin electrophoresis gel image</p>
 +
 
 +
<p>In this figure we can see the 12 overlap primers extension to the insulin. We cut the gel, do the gel purification and use the other two primers to do the PCR and get more insulin molecules. </p>
 +
 +
<p class="center"><img src = "https://static.igem.org/mediawiki/2014/c/c9/Tsinghua_Insulin.png" height="300" /></p>
 +
<p class="center">Fig. 6 insulin map</p>
 +
<p>Then, we replace mCherry gene in tet system by this insulin gene, and transfect it into HEK293T cells.</p>
 +
 +
<img class="center" src = "https://static.igem.org/mediawiki/2014/5/5d/Tsinghua_PAAV%2BTet_on%2Binsulin.png" width="300" />
 +
<p class="center">Fig. 7 pAAV+Tet-On+insulin map</p>
 +
 
<p style="text-align: right;">Return to: <a href="https://2014.igem.org/Team:Tsinghua/Project">Project</a></p>
<p style="text-align: right;">Return to: <a href="https://2014.igem.org/Team:Tsinghua/Project">Project</a></p>
<!-- end .content --></article>
<!-- end .content --></article>

Latest revision as of 23:29, 17 October 2014

The Drug

Return to: Project

In nature, Ptet promoters expresses two proteins, one of which, TetR, acts as a repressor and the other, TetA, pumps tetracycline out of the cell.

Based on this, Tet-On and Tet-Off systems are two types of tet-involved activation systems that help induce gene expression, and they are the most commonly used ones in eukaryotic cells. Transcription is up or down regulated when tetracycline or its derivatives are present. Tet-On and Tet-Off systems are different in that Tet-On activates transcription with doxycycline is present while Tet-Off does when dox is absent.

Tet-Off was first developed and published in 1992, featuring a fusion protein, tTA, obtained from TetR and a virus protein. tTA responds to Tet0 sequences and its binding activates transcription, while presence of tetracycline or its derivatives would block its binding.

Fig. 1 Tet-Off system schematic

Tet-On system works similarly to Tet-Off system. The corresponding protein, rtTA, binds to the operator with Tet or its derivatives present.

Fig. 2 Tet-On system schematic

We get Tet-On system from 2013 Tsinghua team. At first, we design a test plasmid to determine the feasibility of applying Tet-On system in our system.

We constructed an mCherry gene after a CYC1 TATA region (TATA region) which is regulated by a tetracycline response element on its upstream. After mCherry gene, a CYC1 terminator is used to terminate the transcription of mRNA.

Fig. 3 CM252-mCherry map

After construction of this plasmid, we transfect it into HEK293T cell to see if the mCherry gene can be expressed under the regulation of the DOX (tetracycline analogue).

Then we do the PCR of the Tet-On system with mCherry to bring in two restrition sites at the terminals. After purification of the PCR products, we cut the vector and the tet on system and ligate them. The plasmid which has AAV vector that we bought from a company has two the same restrition sites(NotI) in its insert rigion. So, to aviod the vector’s self-ligation, we dispose the digestion product with CIAP to remove the phosphate group at the sticky end of the vector.

Fig. 4 CM252-pAAV+Tet-On+mCherry map

After construction of this plasmid, we want to replace this mCherry gene with insulin. Since we want to transfect the AAV vector into somatic cells, we must make sure that the insulin can be processed in these cells. In beta cells, proinsulin was processed by cellular endopeptidases known as prohormone convertases (PC1 and PC2), as well as the exoprotease carboxypeptidase. However, in non-endocrine cells, some of these enzyme are lacking. So, if we want to get mature insulin, we must alter the insulin gene sequence. In non-endocrine cells, an endoprotease called furin which can cleave the concensus processing site, -Arg-4-X-3-Lys/Arg-2-Arg-1. So, we add two 6bp nucleotides into two cleavage sites in insulin CDS to get the furin endoprotease sites.

To get the insulin gene, firstly we want to PCR every exons of its in human genome and then overlap them. However, the sequence of insulin is in a high GC region in the genome. So, our first trial was not successful. Fortunately, the insulin gene is very small. So, we design 12 overlap primers and do one reaction to ligate these oligos. In that step, we insert these two furin sites into our sequence.

Fig. 5 insulin electrophoresis gel image

In this figure we can see the 12 overlap primers extension to the insulin. We cut the gel, do the gel purification and use the other two primers to do the PCR and get more insulin molecules.

Fig. 6 insulin map

Then, we replace mCherry gene in tet system by this insulin gene, and transfect it into HEK293T cells.

Fig. 7 pAAV+Tet-On+insulin map

Return to: Project