Difference between revisions of "Team:NAU-CHINA/Composite Part"

(Undo revision 486862 by Brunoinigem (talk))
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     <a href="https://2018.igem.org/Team:NAU-CHINA/Basic_Part">
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     <a href="https://2018.igem.org/Team:NAU-CHINA/Composite_Part">
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         <img id="icon1" class="guide-icon" src="https://static.igem.org/mediawiki/2018/c/c9/T--NAU-China--pa4.png" />
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         <img id="icon1" class="guide-icon" src="https://static.igem.org/mediawiki/2018/0/00/T--NAU-China--pa1.png" />
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     </a>
 
     </a>
 
     <a href="https://2018.igem.org/Team:NAU-CHINA/Improve">
 
     <a href="https://2018.igem.org/Team:NAU-CHINA/Improve">
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         <img id="icon2" class="guide-icon" src="https://static.igem.org/mediawiki/2018/8/8e/T--NAU-China--pa2.png" />
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     </a>
 
     </a>
 
     <a href="https://2018.igem.org/Team:NAU-CHINA/Part_Collection">
 
     <a href="https://2018.igem.org/Team:NAU-CHINA/Part_Collection">
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         <img id="icon3" class="guide-icon" src="https://static.igem.org/mediawiki/2018/a/ae/T--NAU-China--pa3.png" />
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<body>
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     <div class="topLine">
 
     <div class="topLine">
 
         <p class="top-title"> Parts</p>
 
         <p class="top-title"> Parts</p>
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         <p class="sec-title"> Basic Parts</p>
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         <p class="sec-title"> Composite Parts</p>
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     </div>
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     </div>  
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     <div class="main-content">
 
     <div class="main-content">
 
         <div class="textblock">
 
         <div class="textblock">
 
            
 
            
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             <h1>Our Best Basic Part:BBa_K2557001</h1>
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             <h1>Our Best Composite Part:BBa_K2557010</h1>
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             <p>This year, our team has selected a series of recombinases through literature searches and characterized a number of them. Here we present the best characterized recombinase - Bxb1 (BBa_K2557001) as our best basic part for the award.</p>
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            <figure>
 +
                    <img src="https://static.igem.org/mediawiki/2018/c/c6/T--NAU-China--teto_recombinase.gif">
 +
                </figure>
 +
             <p>We combined three promoters, miniCMV promoter, EF1α promoter and Ubc promoter, with three recombinases, Bxb1, PhiC31 and TP901, and identified nine combinations. In the end, we chose TetO-miniCMV promoter-Bxb1 as our best composite part.</p>
 
             <div class="section">
 
             <div class="section">
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                 <h2>Background</h2>
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                 <h2>Function In Our Genetic Circuit</h2>
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                <p>Bxb1, also known as Bxb1 gp35, is an recombinase comeing from Mycobacterium Phage Bxb1 and can bind to specific attB/P sites to catalyze DNA recombination. It helps the phage to integrate its genome into bacterial genome naturally. By constructing the attB/P sites in different directions, Bxb1 gp35 can catalyze the recombination of DNA between their sites, leading to inversion when attB/P are in opposite directions and excision when attB/P are in the same directions. The resulting attL and attR sequences cannot be recognized and bound by integrases alone, so the state after integration is stable. Bxb1 gp35 is widely used to construct combinational logic gate and synthetic biology.</p>
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                 <figure>
 
                 <figure>
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                     <img src="https://static.igem.org/mediawiki/2018/9/95/T--NAU-China--bxb1png.png" width="300"/>
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                     <img src="https://static.igem.org/mediawiki/2018/7/70/T--NAU-China--part_teto_anime.gif">
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                    <figcaption class="_table">Fig. 1 Bxb1 structure homology-model provided by SWISS-MODEL.</figcaption>
+
 
                 </figure>
 
                 </figure>
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                <div class="divvideo">
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                <p>This part can convert the input signal of the upstream TEV into the output signal of the downstream RFP. When TEV is detached from the cell membrane, it functions as a protease, and the inhibition of the promoter by TetR is released, and the expression of Bxb1 recombinase is turned on. Finally, Bxb1 functions as a recombinase to initiate expression of RFP.</p>
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                <video src="https://static.igem.org/mediawiki/2018/a/ab/T--NAU-China--bxb1mp4.mp4"  controls="controls">
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                </video>      
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                </div>
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             </div>
 
             </div>
 
             <div class="section">
 
             <div class="section">
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                 <h2>Function In our Genetic Circuit</h2>
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                 <h2>Characterization</h2>
 +
               
 +
                <p><b>TetR can effectively repress Tet operator in HEK 293T</b></p>
 
                 <figure>
 
                 <figure>
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                     <img src="https://static.igem.org/mediawiki/2018/9/9e/T--NAU-China--partbxb1anime.gif">
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                     <img src="https://static.igem.org/mediawiki/2018/7/70/T--NAU-China--enhance_gfp.gif">
 
                 </figure>
 
                 </figure>
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                <p>In our genetic circuit, Bxb1 regulated by TetR. When the inhibition released by TEV protease, Bxb1 can bind to specific attB/P sites to catalyze DNA recombination, leading to the expression of RFP.</p>
 
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            </div>
 
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              <div class="section">
 
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                <h2>Characterization</h2>
 
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                <p>Method: Co-transfected with six different numbers of plasmids containing recombinase genes(miniCMV-Bxb1 and miniCMV-TP901) and fixed numbers of plasmids containing corresponding recombinase recognition sites. After 36 hours of plasmid co - transfection, the proportion of fluorescent cells and the average fluorescence intensity of cells were detected by flow cytometry. The experiment was repeated three times.</p>
 
−
                <p>Result:</p>
 
 
                 <figure>
 
                 <figure>
 +
                    <img src="https://static.igem.org/mediawiki/2018/d/d7/T--NAU-China--newpart1.jpg">
 +
                    <figcaption class="_table">Fig. 1 Inhibition of TetR on promoter with TetO sequence<br>
 +
              (A)Fluorescence microscope observation of HEK 293T only transfected with plasmids containing promoters with TetO sequence.<br>
 +
              (B)Fluorescence microscope observation of HEK 293T transfected with plasmids containing promoters with TetO sequence and tetR.<br>
 +
            </figcaption> </figure>
 +
            <p><b>Bxb1 works well in HEK 293T</b></p>
 +
            <figure>
 
                     <img src="https://static.igem.org/mediawiki/2018/f/f4/T--NAU-China--newpart7.jpg">
 
                     <img src="https://static.igem.org/mediawiki/2018/f/f4/T--NAU-China--newpart7.jpg">
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                     <figcaption class="_table">Fig.2 Function verification of reversal efficiency and threshold characteristics of different recombinase in HEK 293 T Cells.<br>(A)Fluorescence microscope observation of HEK 293T undergone different experimental treatments.<br>(B)The statistical chart of average fluorescence intensity of cells shows that the cells with Bxb1 recombinase have a higher fluorescence intensity than those with TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity.<br>(C)The statistics of the proportion of fluorescent cells show that the proportion of fluorescent cells has a sudden jump discontinuity between low concentration and high concentration of Bxb1 and TP901 recombinases. Similar results were obtained in all three repetitions.</figcaption>
+
                     <figcaption class="_table">Fig.2 Function verification of reversal efficiency and threshold characteristics of different recombinase in HEK 293 T Cells.<br>(A)Fluorescence microscope observation of HEK 293T undergone different experimental treatments.<br>(B)The statistical chart of average fluorescence intensity of cells shows that the cells with Bxb1 recombinase have a higher fluorescence intensity than those with TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity.<br>(C)The statistics of the proportion of fluorescent cells show that the proportion of fluorescent cells has a sudden jump discontinuity between low concentration and high concentration of Bxb1 and TP901 recombinases. Similar results were obtained in all three repetitions.</figcaption>
 
                 </figure>
 
                 </figure>
 
                  
 
                  
 
                 <p>The results of image B show that the reverse efficiency of Bxb1 recombinase is higher than TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity. The results of the image C show that Bxb1 and TP901 recombinases have a threshold property. So, the proportion of fluorescent cells have a jump discontinuity between low concentration and high concentration of recombinase.</p>
 
                 <p>The results of image B show that the reverse efficiency of Bxb1 recombinase is higher than TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity. The results of the image C show that Bxb1 and TP901 recombinases have a threshold property. So, the proportion of fluorescent cells have a jump discontinuity between low concentration and high concentration of recombinase.</p>
−
                <p><b>In conclusion, Bxb1 works well in HEK 293T as expected, so we present Bxb1(BBa_K2557001) as our best basic part.</b></p>
+
            <p>Combining these data and, to our knowledge, we first constructed a TetO-regulated recombinase genetic circuit in mammalian cells, so we present BBa_K2557010 as the Our Best Composite Part.</p>
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             </div>
 
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         </div>
 
         </div>
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 +
                </div>
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                  <div class="main-content">
 
         <div class="textblock">
 
         <div class="textblock">
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            <h1>Basic Part List</h1>
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−
          </div></div>
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            <h1>Composite Part List</h1></div></div>
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<table style='width: 75%; margin: 0px 14% 20px 14%;'>
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            <table style='width: 75%; margin: 0px 14% 20px 14%;'>
 
           <thead>
 
           <thead>
 
             <tr>
 
             <tr>
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             <tbody>
 
             <tbody>
 
               <tr>
 
               <tr>
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               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557001">BBa_K2557001</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557010">BBa_K2557010</a></td>
−
               <td align='center'>Coding</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>Bxb1 recombinase</td>
+
               <td align='center'>TetO-miniCMV promoter-Bxb1</td>
−
               <td align='center'>1565</td>
+
               <td align='center'>1890</td>
 
             </tr>
 
             </tr>
 
                   <tr>
 
                   <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557000">BBa_K2557000</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557002">BBa_K2557002</a></td>
−
               <td align='center'>Coding</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>Anti-GFP-mnotch-TEV protease-NLS</td>
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               <td align='center'>Bxb1 attB-RFP-Bxb1 attP</td>
−
               <td align='center'>2409</td>
+
               <td align='center'>935</td>
 
             </tr>
 
             </tr>
 
               <tr>
 
               <tr>
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               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557003">BBa_K2557003</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557005">BBa_K2557005</a></td>
−
               <td align='center'>DNA</td>
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               <td align='center'>Composite</td>
−
               <td align='center'>Bxb1 attB-Bxb1 attP</td>
+
               <td align='center'>PhiC31 attB-mCherry-PhiC31 attP</td>
−
               <td align='center'>200</td>
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               <td align='center'>907</td>
 
             </tr>
 
             </tr>
 
               <tr>
 
               <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557004">BBa_K2557004</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557008">BBa_K2557008</a></td>
−
               <td align='center'>Coding</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>PhiC31 recombinase</td>
+
               <td align='center'>TP901 attB-mCherry-TP901 attP</td>
−
               <td align='center'>1881</td>
+
               <td align='center'>949</td>
 
             </tr>
 
             </tr>
 
               <tr>
 
               <tr>
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               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557006">BBa_K2557006</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557011">BBa_K2557011</a></td>
−
               <td align='center'>DNA</td>
+
               <td align='center'>Composite</td>
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               <td align='center'>PhiC31 attB-PhiC31 attP</td>
+
               <td align='center'>TetO-EF1α promoter-Bxb1</td>
−
               <td align='center'>204</td>
+
               <td align='center'>2070</td>
 
             </tr>
 
             </tr>
 
               <tr>
 
               <tr>
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               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557007">BBa_K2557007</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557012">BBa_K2557012</a></td>
−
               <td align='center'>Coding</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>TP901 recombinase</td>
+
               <td align='center'>TetO-Ubc promoter-Bxb1</td>
−
               <td align='center'>1604</td>
+
               <td align='center'>2258</td>
 
             </tr>
 
             </tr>
 
                   <tr>
 
                   <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557009">BBa_K2557009</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557013">BBa_K2557013</a></td>
−
               <td align='center'>DNA</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>TP901 attB-TP901 attP</td>
+
               <td align='center'>TetO-miniCMV-PhiC31</td>
−
               <td align='center'>210</td>
+
               <td align='center'>2212</td>
 
             </tr>
 
             </tr>
 
               <tr>
 
               <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557032">BBa_K2557032</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557014">BBa_K2557014</a></td>
−
               <td align='center'>promoter</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>miniCMV promoter</td>
+
               <td align='center'>TetO-EF1α-PhiC31</td>
−
               <td align='center'>38</td>
+
               <td align='center'>2386</td>
 
             </tr>
 
             </tr>
 
               <tr>
 
               <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557033">BBa_K2557033</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557015">BBa_K2557015</a></td>
−
               <td align='center'>promoter</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>EF1α promoter</td>
+
               <td align='center'>TetO-Ubc promoter-PhiC31</td>
−
               <td align='center'>212</td>
+
               <td align='center'>2574</td>
 
             </tr>
 
             </tr>
 
               <tr>
 
               <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557034">BBa_K2557034</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557016">BBa_K2557016</a></td>
−
               <td align='center'>promoter</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>Ubc promoter</td>
+
               <td align='center'>TetO-miniCMV promoter-TP901</td>
−
               <td align='center'>400</td>
+
               <td align='center'>1935</td>
 
             </tr>
 
             </tr>
 
               <tr>
 
               <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557035">BBa_K2557035</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557017">BBa_K2557017</a></td>
−
               <td align='center'>Reporter</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>TagRFP</td>
+
               <td align='center'>TetO-EF1α promoter-TP901</td>
−
               <td align='center'>711</td>
+
               <td align='center'>2109</td>
 
             </tr>
 
             </tr>
 
                   <tr>
 
                   <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557036">BBa_K2557036</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557018">BBa_K2557018</a></td>
−
               <td align='center'>Reporter</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>mCherry</td>
+
               <td align='center'>TetO-Ubc promoter-TP901</td>
−
               <td align='center'>708</td>
+
               <td align='center'>2297</td>
 
             </tr>
 
             </tr>
 
               <tr>
 
               <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557037">BBa_K2557037</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557019">BBa_K2557019</a></td>
−
               <td align='center'>Reporter</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>EGFP</td>
+
               <td align='center'>miniCMV promoter-Bxb1-RDF</td>
−
               <td align='center'>738</td>
+
               <td align='center'>2391</td>
 
             </tr>
 
             </tr>
 
               <tr>
 
               <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557038">BBa_K2557038</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557020">BBa_K2557020</a></td>
−
               <td align='center'>DNA</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>TetO</td>
+
               <td align='center'>EF1α promoter-Bxb1-RDF</td>
−
               <td align='center'>271</td>
+
               <td align='center'>2565</td>
 
             </tr>
 
             </tr>
 
               <tr>
 
               <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557041">BBa_K2557041</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557021">BBa_K2557021</a></td>
−
               <td align='center'>DNA</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>Bxb1 attB</td>
+
               <td align='center'>Ubc promoter-Bxb1-RDF</td>
−
               <td align='center'>96</td>
+
               <td align='center'>2753</td>
 
             </tr>
 
             </tr>
 
               <tr>
 
               <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557042">BBa_K2557042</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557022">BBa_K2557022</a></td>
−
               <td align='center'>DNA</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>Bxb1 attP</td>
+
               <td align='center'>miniCMV promoter-PhiC31-RDF</td>
−
               <td align='center'>112</td>
+
               <td align='center'>2676</td>
 
             </tr>
 
             </tr>
 
                   <tr>
 
                   <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557043">BBa_K2557043</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557023">BBa_K2557023</a></td>
−
               <td align='center'>DNA</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>PhiC31 attB</td>
+
               <td align='center'>EF1α promoter-PhiC31-RDF</td>
−
               <td align='center'>45</td>
+
               <td align='center'>2850</td>
 
             </tr>
 
             </tr>
 
               <tr>
 
               <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557044">BBa_K2557044</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557024">BBa_K2557024</a></td>
−
               <td align='center'>DNA</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>PhiC31 attP</td>
+
               <td align='center'>Ubc promoter--PhiC31-RDF</td>
−
               <td align='center'>135</td>
+
               <td align='center'>3038</td>
 
             </tr>
 
             </tr>
 
               <tr>
 
               <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557045">BBa_K2557045</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557025">BBa_K2557025</a></td>
−
               <td align='center'>DNA</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>TP901 attB</td>
+
               <td align='center'>miniCMV promoter-TP901-RDF</td>
−
               <td align='center'>113</td>
+
               <td align='center'>1842</td>
 
             </tr>
 
             </tr>
 
               <tr>
 
               <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557046">BBa_K2557046</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557026">BBa_K2557026</a></td>
−
               <td align='center'>DNA</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>TP901 attP</td>
+
               <td align='center'>EF1α promoter--TP901-RDF</td>
−
               <td align='center'>112</td>
+
               <td align='center'>2016</td>
 
             </tr>
 
             </tr>
 
               <tr>
 
               <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557047">BBa_K2557047</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557027">BBa_K2557027</a></td>
−
               <td align='center'>Coding</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>Bxb1-RDF</td>
+
               <td align='center'>Ubc promoter--TP901-RDF</td>
−
               <td align='center'>2345</td>
+
               <td align='center'>2204</td>
 
             </tr>
 
             </tr>
 
                   <tr>
 
                   <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557048">BBa_K2557048</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557028">BBa_K2557028</a></td>
−
               <td align='center'>Coding</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>PhiC31-RDF</td>
+
               <td align='center'>TetO-miniCMV promoter- EGFP</td>
−
               <td align='center'>2630</td>
+
               <td align='center'>1063</td>
 
             </tr>
 
             </tr>
 
               <tr>
 
               <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557049">BBa_K2557049</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557029">BBa_K2557029</a></td>
−
               <td align='center'>Coding</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>TP901-RDF</td>
+
               <td align='center'>TetO-EF1α promoter -EGFP</td>
−
               <td align='center'>1796</td>
+
               <td align='center'>1237</td>
 
             </tr>
 
             </tr>
 
               <tr>
 
               <tr>
−
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557050">BBa_K2557050</a></td>
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557030">BBa_K2557030</a></td>
−
               <td align='center'>Coding</td>
+
               <td align='center'>Composite</td>
−
               <td align='center'>TetR with TEV cleavage site</td>
+
               <td align='center'>TetO-Ubc promoter- EGFP</td>
−
               <td align='center'>651</td>
+
               <td align='center'>1425</td>
 
             </tr>
 
             </tr>
−
                
+
               <tr>
−
                
+
               <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557031">BBa_K2557031</a></td>
 +
              <td align='center'>Composite</td>
 +
              <td align='center'>miniCMV promoter-Anti-GFP-mnotch-TEV protease-NLS</td>
 +
              <td align='center'>2455</td>
 +
            </tr>
 +
              <tr>
 +
              <td align='center'><a href="http://parts.igem.org/Part:BBa_K2557051">BBa_K2557051</a></td>
 +
              <td align='center'>Composite</td>
 +
              <td align='center'>EF1α promoter-TetR</td>
 +
              <td align='center'>1871</td>
 +
              </tr>
 +
 
 +
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 +
         
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<p><a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2018&group=NAU-CHINA">Click here to view Part List</a></p>
 
<p><a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2018&group=NAU-CHINA">Click here to view Part List</a></p>
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Revision as of 03:23, 18 October 2018

Template:2018_NAU-CHINA

header
Composite Parts

Parts

Composite Parts

Our Best Composite Part:BBa_K2557010

We combined three promoters, miniCMV promoter, EF1α promoter and Ubc promoter, with three recombinases, Bxb1, PhiC31 and TP901, and identified nine combinations. In the end, we chose TetO-miniCMV promoter-Bxb1 as our best composite part.

Function In Our Genetic Circuit

This part can convert the input signal of the upstream TEV into the output signal of the downstream RFP. When TEV is detached from the cell membrane, it functions as a protease, and the inhibition of the promoter by TetR is released, and the expression of Bxb1 recombinase is turned on. Finally, Bxb1 functions as a recombinase to initiate expression of RFP.

Characterization

TetR can effectively repress Tet operator in HEK 293T

Fig. 1 Inhibition of TetR on promoter with TetO sequence
(A)Fluorescence microscope observation of HEK 293T only transfected with plasmids containing promoters with TetO sequence.
(B)Fluorescence microscope observation of HEK 293T transfected with plasmids containing promoters with TetO sequence and tetR.

Bxb1 works well in HEK 293T

Fig.2 Function verification of reversal efficiency and threshold characteristics of different recombinase in HEK 293 T Cells.
(A)Fluorescence microscope observation of HEK 293T undergone different experimental treatments.
(B)The statistical chart of average fluorescence intensity of cells shows that the cells with Bxb1 recombinase have a higher fluorescence intensity than those with TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity.
(C)The statistics of the proportion of fluorescent cells show that the proportion of fluorescent cells has a sudden jump discontinuity between low concentration and high concentration of Bxb1 and TP901 recombinases. Similar results were obtained in all three repetitions.

The results of image B show that the reverse efficiency of Bxb1 recombinase is higher than TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity. The results of the image C show that Bxb1 and TP901 recombinases have a threshold property. So, the proportion of fluorescent cells have a jump discontinuity between low concentration and high concentration of recombinase.

Combining these data and, to our knowledge, we first constructed a TetO-regulated recombinase genetic circuit in mammalian cells, so we present BBa_K2557010 as the Our Best Composite Part.

Composite Part List

Click here to view Part List

Name Type Description Length
BBa_K2557010 Composite TetO-miniCMV promoter-Bxb1 1890
BBa_K2557002 Composite Bxb1 attB-RFP-Bxb1 attP 935
BBa_K2557005 Composite PhiC31 attB-mCherry-PhiC31 attP 907
BBa_K2557008 Composite TP901 attB-mCherry-TP901 attP 949
BBa_K2557011 Composite TetO-EF1α promoter-Bxb1 2070
BBa_K2557012 Composite TetO-Ubc promoter-Bxb1 2258
BBa_K2557013 Composite TetO-miniCMV-PhiC31 2212
BBa_K2557014 Composite TetO-EF1α-PhiC31 2386
BBa_K2557015 Composite TetO-Ubc promoter-PhiC31 2574
BBa_K2557016 Composite TetO-miniCMV promoter-TP901 1935
BBa_K2557017 Composite TetO-EF1α promoter-TP901 2109
BBa_K2557018 Composite TetO-Ubc promoter-TP901 2297
BBa_K2557019 Composite miniCMV promoter-Bxb1-RDF 2391
BBa_K2557020 Composite EF1α promoter-Bxb1-RDF 2565
BBa_K2557021 Composite Ubc promoter-Bxb1-RDF 2753
BBa_K2557022 Composite miniCMV promoter-PhiC31-RDF 2676
BBa_K2557023 Composite EF1α promoter-PhiC31-RDF 2850
BBa_K2557024 Composite Ubc promoter--PhiC31-RDF 3038
BBa_K2557025 Composite miniCMV promoter-TP901-RDF 1842
BBa_K2557026 Composite EF1α promoter--TP901-RDF 2016
BBa_K2557027 Composite Ubc promoter--TP901-RDF 2204
BBa_K2557028 Composite TetO-miniCMV promoter- EGFP 1063
BBa_K2557029 Composite TetO-EF1α promoter -EGFP 1237
BBa_K2557030 Composite TetO-Ubc promoter- EGFP 1425
BBa_K2557031 Composite miniCMV promoter-Anti-GFP-mnotch-TEV protease-NLS 2455
BBa_K2557051 Composite EF1α promoter-TetR 1871

Click here to view Part List

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