Difference between revisions of "Team:SZU China/Achievement"

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         <div class="col-sm-8 blog-main" style="width:960px;">
 
         <div class="col-sm-8 blog-main" style="width:960px;">
  
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             <h3><stromg>Renilla Luciferase assay in three plasmids system</strong></h3>
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             <h3><strong>Judging Criteria</strong></h3>
 
            
 
            
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             <p>Three recombinant plasmids were generated and transiently transfected into a human bladder cancer cell line T24.
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             <p>In the past 9 months, we have gone through brainstorming, experimental design, biobrick combination, system verification, human practice and wiki making, ect. Through our hard work, our project has been greatly developed and we are fully prepared for the Giant Jamboree. We have submitted 12 biobricks and they are transiently transfected into different cell lines to verify the working efficiency of our system. What’s more, we performed our human practice with Cancer Mutual Aid Association, Shenzhen Sencond People’s Hospital and Moonbay Primary School. We also cooperate with Nankai and LZU-China. We believe we deserve Gold Medal since we have met all the requirements for it.</p>
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<br>
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          <h3><stromg>Judging Criteria</strong></h3>
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To test the working efficiency of our orthogonal system, we devided the cells into two groups. One has Ack in the culture medium and another does not.
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            <p>We have designed and constructed 12 parts, including 6 basic parts and 6 composite parts. Among all this parts, three of them are improved from previously existing Biobrick Part or Device. All of these parts were well documented on the part registry pages. They are listed as follows:</p>
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<br>
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<table class="table-hover table-borderd">
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Luciferase assays were performed using luminometer. Since Ack is essential for our system, we speculated that Renilla Luciferase(Rluc), the output gene in the circuits can be selectively expressed in Experiment group with Ack. As shown in Fig. 1, the activity of Renilla Luciferase(RLUC) varied widely between the two groups, and the circuit only demonstrated significant activity in the experimental group as expected. Therefore, our orthogonal system is verified to be in good condition and can work efficiently.</p>
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    <tbody><tr>
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<br>
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        <th>Part Number</th>
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<img src="https://static.igem.org/mediawiki/2015/6/6b/SZU_China_RESULT_1.jpg">
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        <th>Nickname</th>
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<br>
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        <th>Part Type</th>
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    </tr>
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            <h3><stromg>Luciferase assay in two plasmids system</strong></h3>
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    <tr>
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            <p>hTERT and hUPII promoters are cancer specific promoter and bladder specific promoter, respectively. By combining the two promoters into the design, we supposed that the constructed circuits have the ability to selectively identify bladder cancer cells, in which telomerase and uroplakins are both expressed.<br>
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        <td>BBa_K1722000</td>
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        <td>hUPII</td>
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To test this hypothesis, we used the luciferase reporter gene as the output of the circuit and tested the luciferase activity in HFC, which is short for human fiber epithelial cell, Hela, a cervical carcinoma cell line and 5637, a bladder cancer cell line. Also, we reconstructed our system in two plasmids to test if two plasmids system can perform higher efficiency.<br>
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        <td>Basic Part</td>
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    </tr>
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The result (Fig.2) shows that the activity of LUC in 5637 with Ack was about two times as high as that in 5637 without Ack and three times as high as that in Hela, while it could not be measured in HFC. We can learn from this result that this system is perfectly safe for normal cells, with no luciferase being detected in HFC. However, compared with three plasmids system, whose RLUC activity of experimental group is about 7 times as high as that of control group, the working efficiency of our orthogonal system in two plasmids is much lower. So we had better construct our system in two plasmids to express higher level of therapeutic gene in bladder cancer cells and reduce its expression in other cell types to increase its specificity.</p>
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    <tr>
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<br>
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        <td>BBa_K1722001</td>
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<img src="https://static.igem.org/mediawiki/2015/2/2f/SZU_China_RESULT_2.jpg">
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        <td>shTERT</td>
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<br>
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        <td>Basic Part(Improved)</td>
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    </tr>
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    <h3><stromg>Green fluorescent light measurement in two and three plasmids system</strong></h3>
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    <tr>
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        <td>BBa_K1722002</td>
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        <td>hTERT</td>
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        <td>Basic Part</td>
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    </tr>
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    <tr>
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        <td>BBa_K1722004</td>
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        <td>tRNA</td>
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        <td>Basic Part</td>
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    </tr>
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    <tr>
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        <td>BBa_K1722005</td>
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        <td>Rluc</td>
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        <td>Basic Part(Improved)</td>
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    </tr>
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    <tr>
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        <td>BBa_K1722006</td>
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        <td>SV40(En)</td>
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        <td>Basic Part(Improved)</td>
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    </tr>
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    <tr>
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        <td>BBa_K1722007</td>
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        <td>hUPII+AckRS</td>
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        <td>Composite Part</td>
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    </tr>
 +
    <tr>
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        <td>BBa_K1722009</td>
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        <td>hTERT+GFP</td>
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        <td>Composite Part</td>
 +
    </tr>
 +
    <tr>
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        <td>BBa_K1722010</td>
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        <td>hTERT+tRNA</td>
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        <td>Composite Part</td>
 +
    </tr>
 +
    <tr>
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        <td>BBa_K1722011</td>
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        <td>shTERT+tRNA</td>
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        <td>Composite Part</td>
 +
    </tr>
 +
    <tr>
 +
        <td>BBa_K1722011</td>
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        <td>shTERT+tRNA</td>
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        <td>Composite Part</td>
 +
    </tr>
 +
    <tr>
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        <td>BBa_K1722013</td>
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        <td>shTERT+tRNA</td>
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        <td>Composite Part</td>
 +
    </tr>
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    </tbody></table>
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    <p>hUPII(BBa_K1722000) is well documented in the registry and accepted. It is the highest quality basic part of our team and it deserves the Best Basic Part.</p>
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  <p>hUPII+AckRS(BBa_K1722007) is well documented in the registry and accepted. It is the highest quality composite part of our team and it deserves the Best Composite Part.</p>
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       <h3>Human practice outside the lab</h3>
  
 
             <p>To further verifying the working efficiency of the unnatural amino acid orthogonal system, we replaced the amber mutated output gene in former plasmids to amber mutated GFP. The plasmid psiCHECKTM2-CMV-GFP was used as a positive control to monitor transfection and expression efficiency. The two plasmids and three plasmids system being constructed by GFP recombinant plasmids were transiently transfected into 5637 and T24, both of which are bladder cancer cell line.
 
             <p>To further verifying the working efficiency of the unnatural amino acid orthogonal system, we replaced the amber mutated output gene in former plasmids to amber mutated GFP. The plasmid psiCHECKTM2-CMV-GFP was used as a positive control to monitor transfection and expression efficiency. The two plasmids and three plasmids system being constructed by GFP recombinant plasmids were transiently transfected into 5637 and T24, both of which are bladder cancer cell line.

Revision as of 17:35, 18 September 2015



Achievement




Judging Criteria

In the past 9 months, we have gone through brainstorming, experimental design, biobrick combination, system verification, human practice and wiki making, ect. Through our hard work, our project has been greatly developed and we are fully prepared for the Giant Jamboree. We have submitted 12 biobricks and they are transiently transfected into different cell lines to verify the working efficiency of our system. What’s more, we performed our human practice with Cancer Mutual Aid Association, Shenzhen Sencond People’s Hospital and Moonbay Primary School. We also cooperate with Nankai and LZU-China. We believe we deserve Gold Medal since we have met all the requirements for it.

Judging Criteria

We have designed and constructed 12 parts, including 6 basic parts and 6 composite parts. Among all this parts, three of them are improved from previously existing Biobrick Part or Device. All of these parts were well documented on the part registry pages. They are listed as follows:

Part Number Nickname Part Type
BBa_K1722000 hUPII Basic Part
BBa_K1722001 shTERT Basic Part(Improved)
BBa_K1722002 hTERT Basic Part
BBa_K1722004 tRNA Basic Part
BBa_K1722005 Rluc Basic Part(Improved)
BBa_K1722006 SV40(En) Basic Part(Improved)
BBa_K1722007 hUPII+AckRS Composite Part
BBa_K1722009 hTERT+GFP Composite Part
BBa_K1722010 hTERT+tRNA Composite Part
BBa_K1722011 shTERT+tRNA Composite Part
BBa_K1722011 shTERT+tRNA Composite Part
BBa_K1722013 shTERT+tRNA Composite Part

hUPII(BBa_K1722000) is well documented in the registry and accepted. It is the highest quality basic part of our team and it deserves the Best Basic Part.

hUPII+AckRS(BBa_K1722007) is well documented in the registry and accepted. It is the highest quality composite part of our team and it deserves the Best Composite Part.

Human practice outside the lab

To further verifying the working efficiency of the unnatural amino acid orthogonal system, we replaced the amber mutated output gene in former plasmids to amber mutated GFP. The plasmid psiCHECKTM2-CMV-GFP was used as a positive control to monitor transfection and expression efficiency. The two plasmids and three plasmids system being constructed by GFP recombinant plasmids were transiently transfected into 5637 and T24, both of which are bladder cancer cell line.
Pictures of green fluorescent light produced by target cells were taken by fluorescent microscope. From these pictures (Fig. 3), we can see there is no expression of GFP being detected in no Ack groups while the light intensities are rather high in with Ack groups no matter in T24 or 5637 cell lines. These results indicate that the output gene is expressed only in cells which have Ack in the culture medium. These findings suggest that the constructed circuit based on AND GATE and UAA orthogonal system can be used to specifically identify bladder cancer cells, and may yield a new therapeutic approach for bladder cancer.