Team:UESTC-China/result

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<a href="https://2014.igem.org/Team:UESTC-China/Lecture.html"><li>Lecture</li></a>
<a href="https://2014.igem.org/Team:UESTC-China/Lecture.html"><li>Lecture</li></a>
<a href="https://2014.igem.org/Team:UESTC-China/Communication.html"><li>Communication</li></a>
<a href="https://2014.igem.org/Team:UESTC-China/Communication.html"><li>Communication</li></a>
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<a href="https://2014.igem.org/Team:UESTC-China/Art"><li>Art</li></a>
</ul>
</ul>
</div>
</div>
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<div class="middle-photo-each">
<div class="middle-photo-each">
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<h1 class="SectionTitles" style="width:900px; ">Vectors Construction</h1><br/>
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<h1 style="color:#1b1b1b; position:relative; left:0px; padding:15 5px; font-size:40px; font-family: calibri, arial, helvetica, sans-serif; font-weight: bold;font-style: Italic; text-align:center; width:1140px;">Result</h1>
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<p style="color:#1b1b1b;">We have successfully constructed 2 backbones, <em>piGEM001</em> and <em>piGEM002</em>. And we have verified them using digestion (Fig.1) and sequencing.</p>
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<h1 class="SectionTitles" style="width:1100px; ">Vectors construction</h1><br/>
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<p style="color:#1b1b1b;">We have successfully constructed 2 backbones, piGEM001</em> and piGEM002. And we have verified them using digestion (Fig.1) and sequencing.</p>
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<div align="center">
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<div ><img style="width:50% ;" src="https://static.igem.org/mediawiki/2014/4/44/Result_1.png"></div>
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<div ><img style="width:50% ;left: 40px;position: relative;" src="https://static.igem.org/mediawiki/2014/4/44/Result_1.png"></div>
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<div ><p style="position:relative; left:0px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:650px; color:#1b1b1b;">
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<div ><p style="position:relative; left:0px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:1100px; color:#1b1b1b;">
<strong>Fig.1</strong> Verification of backbones using digestion
<strong>Fig.1</strong> Verification of backbones using digestion
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A: Digestion the plasmid piGEM001 with <i>Hpa</i>I and <i>Spe</i>I.  
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<strong>A.</strong> Digestion the plasmid piGEM001 with HpaI and SpeI.  
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M: DNA marker;
M: DNA marker;
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1: plasmid piGEM001 and its digestion product  
1: plasmid piGEM001 and its digestion product  
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<strong>B.</strong> Digestion the plasmid piGEM002 with HpaI and DraIII.  
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B: Digestion the plasmid piGEM002 with <i>Hpa</i>I and <i>Dra</i>III.  
M: DNA marker;
M: DNA marker;
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<div><br/>
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<p style="color:#1b1b1b;">Then we have successfully constructed 6 monogene expression vectors, from vector <em>piGEM003</em> to vector <em>piGEM008</em>. And we have verified all of them using digestion and sequcencing. Here we only show the result of vector <em>piGEM005</em> (Fig.2). You can browse notebook for more results.</p>
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<p style="color:#1b1b1b;">Then we have successfully constructed 6 monogene expression vectors, from vector piGEM003 to vector piGEM008. And we have verified all of them using digestion and sequcencing. Here we only show the result of vector piGEM005 (Fig.2). </p>
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<div align="center"><img style="width:20% ;" src="https://static.igem.org/mediawiki/2014/3/38/Result_2.png">
<div align="center"><img style="width:20% ;" src="https://static.igem.org/mediawiki/2014/3/38/Result_2.png">
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<p style="position:relative; left:0px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:650px; color:#1b1b1b;">
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<p style="position:relative; left:0px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:1100px; color:#1b1b1b;">
<strong>Fig.2</strong>
<strong>Fig.2</strong>
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Digestion the plasmid piGEM005 with EcoRI and SacI.  
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Digestion the plasmid piGEM005 with <i>EcoR</i>I and <i>Sac</i>I.  
M: DNA marker;
M: DNA marker;
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5: piGEM005 plasmid and TCP02-HPS-PHI fragment  
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1: piGEM005 plasmid and <i>TCP02-HPS-PHI</i> fragment  
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<p style="color:#1b1b1b;">In order to enhance the ability of tobacco to metabolize formaldehyde, we have successfully constructed 3 multigenge expression vectors, from vector <em>piGEM009</em> to vector <em>piGEM011</em>. We have verified all of them using digestion (Fig.3) and sequcencing.</p>
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<p style="color:#1b1b1b;">In order to enhance the ability of tobacco to metabolize formaldehyde, we have successfully constructed 3 multigenge expression vectors, from vector piGEM009 to vector piGEM011. We have verified all of them using digestion (Fig.3) and sequcencing.</p>
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<div align="center"><img style="width:50% ;" src="https://static.igem.org/mediawiki/2014/7/75/Result_3.png">
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<div align="center"><img style="width:60% ;" src="https://static.igem.org/mediawiki/2014/7/75/Result_3.png">
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<p style="position:relative; left:0px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:1100px; color:#1b1b1b;">
<strong>Fig.3</strong> Verification of multi-gene expression vectors using digestion
<strong>Fig.3</strong> Verification of multi-gene expression vectors using digestion
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<strong>A.</strong>Digestion the plasmid piGEM009 with XbaI and SalI.  
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A: Digestion the plasmid piGEM009 with <i>Xba</i>I and <i>Sal</i>I.  
M: DNA marker;
M: DNA marker;
1: piGEM009 plasmid and it's digestion product  
1: piGEM009 plasmid and it's digestion product  
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<strong>B.</strong>
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B:
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Digestion the plasmid piGEM010 with HindIII and SacI.  
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Digestion the plasmid piGEM010 with <i>Hind</i>III and <i>Sac</i>I.  
M: DNA marker;
M: DNA marker;
1: piGEM010 plasmid and it's digestion product  
1: piGEM010 plasmid and it's digestion product  
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<strong>C.</strong>
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C:
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Digestion of the plasmid piGEM011 with EcoRI and SacI.  
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Digestion of the plasmid piGEM011 with <i>EcoR</i>I and <i>Sac</i>I.  
M: DNA marker;
M: DNA marker;
6: plasmid piGEM011 and it's digestion production
6: plasmid piGEM011 and it's digestion production
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<h1 class="SectionTitles" style="width:900px; ">Plant transformation</h1><br/>
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<h1 class="SectionTitles" style="width:1100px; ">Tobacco transformation</h1><br/>
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<p style="color:#1b1b1b;">Tobacco was transformed essentially using the leaf disk co-cultivation protocol of Horsch. This protocol includes three stages, co-cultivation (Fig.4A), screening cultivation (Fig.4B and C) and rooting cultivation (Fig.4D). We have successfully transformed each vector into babacco and got positive transgenic seedlings (Fig.5). Table 1 is the statistical result of quantity of each transgenic line. And we have got PCR positive plantlet of every transgenic line.</p>
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<p style="color:#1b1b1b;">Tobacco was transformed essentially using the leaf disk co-cultivation protocol of Horsch. This protocol includes infection and co-cultivation (Fig.4A), selection (Fig.4B), regeneration and rooting (Fig.4C). We have successfully transformed each vector into babacco and got positive transgenic plantlets (Fig.5). Table 1 is the statistical result of quantity of each transgenic line. And we have got PCR positive plantlets of every transgenic line.</p>
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<div align="center"><img style="width:60% ;" src="https://static.igem.org/mediawiki/2014/d/d4/Result_fig4.jpg">
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<div align="center"><img style="width:80% ;" src="https://static.igem.org/mediawiki/2014/d/d4/Result_fig4.jpg">
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<p style="position:relative; left:0px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:700px; color:#1b1b1b;">
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<p style="position:relative; left:0px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:1100px; color:#1b1b1b;">
<strong>Fig.4</strong>
<strong>Fig.4</strong>
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Transform piGEM003, piGEM004, piGEM005, piGEM006, piGEM007, piGEM008, piGEM009, piGEM010 and piGEM011 into tobacco. Co-cultured for 48 hours(A); Screening cultivation for one month(B and
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Transform piGEM003, piGEM004, piGEM005, piGEM006, piGEM007, piGEM008, piGEM009, piGEM010 and piGEM011 into tobacco. Co-cultured for 48 hours (A); Selection for one month (B); Rooting for one month (C).
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C); Rooting cultivation for one month (D).
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<div align="center"><img style="width:60% ;" src="https://static.igem.org/mediawiki/2014/d/d7/Result_5.png">
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<div align="center"><img style="width:60% ;" src="https://static.igem.org/mediawiki/2014/8/83/Result_5.jpg">
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<p style="position:relative; left:0px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:700px; color:#1b1b1b;">
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<p style="position:relative; left:0px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:500px; color:#1b1b1b;">
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<strong>Fig.5</strong>
<strong>Fig.5</strong>
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positive tobacco seedlings of each transgenic lines.
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Positive tobacco plantlets of each transgenic line.
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<p style="position:relative; left:0px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:650px; color:#1b1b1b;"><strong>Table.1</strong>
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<p style="position:relative; left:0px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:800px; color:#1b1b1b;"><strong>Table.1</strong>
Statistical result of quantity of each transgenic line.
Statistical result of quantity of each transgenic line.
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<img style="width:60% ;" src="https://static.igem.org/mediawiki/2014/9/93/Result2.png">
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<img style="width:70% ;" src="https://static.igem.org/mediawiki/2014/a/a5/Table.png">
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<p style="position:relative; left:0px; padding:15 5px; font-size:17px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:700px; color:#1b1b1b;">
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* Rate of  Kan-resistant plantlet = N0.of Kan-resistant plantlet / No. of transformed tobacco Leaf disc<br/>
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**Rate of positive seedlings =No. of PCR positive plantlet / N0.of Kan-resistant plantlet
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<h1 class="SectionTitles" style="width:900px; ">Expression of four key enzymes in tobacco</h1><br/>
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<h1 class="SectionTitles" style="width:1100px; ">Expression of four key enzymes in tobacco</h1><br/>
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<p style="color:#1b1b1b;">We extracted DNA from tobacco plantlets. And then we used specific primers to amplify the target gene to verify the kan-resistant seedlings (Fig.6). Next we extracted RNA from tobacco leaves which are PCR positive. We used  RT-PCR to detect whether target gene was expressed (Fig.7).</p>
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<p style="color:#1b1b1b;">We extracted DNA from tobacco plants. And then we used specific primers to amplify the target gene to verify the kan-resistant plants (Fig.6). Next we extracted RNA from tobacco leaves which are PCR positive. We used  RT-PCR to detect whether target gene was expressed (Fig.7).</p>
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<div align="center"><img style="width:50% ;" src="https://static.igem.org/mediawiki/2014/5/58/10.png">
<div align="center"><img style="width:50% ;" src="https://static.igem.org/mediawiki/2014/5/58/10.png">
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<p style="position:relative; left:0px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:650px; color:#1b1b1b;"><strong>Fig.6</strong>
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<p style="position:relative; left:0px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:1100px; color:#1b1b1b;"><strong>Fig.6</strong>
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PCR identification of kan-resistant tobacco seedlings (piGEM010 transgenic line).
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PCR identification of kan-resistant tobacco plants (piGEM010 transgenic line).
M: DNA marker;
M: DNA marker;
WT: widetype control;
WT: widetype control;
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<div align="center"><img style="width:45% ;" src="https://static.igem.org/mediawiki/2014/a/af/RNA%2BACTINJPG.JPG">
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<div align="center"><img style="width:45% ;" src="https://static.igem.org/mediawiki/2014/4/46/Fig_7.jpg"><br/>
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<p style="position:relative; left:0px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:650px; color:#1b1b1b;">
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<p style="position:relative; left:40px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:600px; color:#1b1b1b;">
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<strong>Fig.7</strong> RT-PCR verification of positive transgenic tobacco seedlings.<br/>
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<strong>Fig.7</strong> RT-PCR verification of positive transgenic tobacco plants.
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M: DNA marker; 1-8: 6 individual lines; 9: plasmid
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<h1 class="SectionTitles" style="width:900px; ">Enhanced HCHO Tolerance </h1><br/>
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<h1 class="SectionTitles" style="width:1100px; ">Enhanced formaldehyde tolerance </h1><br/>
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<p style="color:#1b1b1b;">The transgenic plants and wildtype, which had been grown separately in sealed boxes, were exposed to HCHO evaporated from a micro tube (0.5ml) containing HCHO solution (37%, 50ul) (Fig.8). One week later we observed the phenotype of transgeneic plants and widetype (Fig.9). We found that the transgenetic seedling is stronger than wildtype after formaldehyde exposure.This indicates that production of HPS/PHI, Faldh and FDH enhanced HCHO tolerance of transgenic seedlings to some extent.</p>
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<p style="color:#1b1b1b;">The transgenic and wildtype plants, which had been grown separately in sealed boxes, were exposed to formaldehyde evaporated from a micro tube (0.5ml) containing formaldehyde solution (37%, 10μl) (Fig.8). Two weeks later we observed the phenotype of transgeneic and widetype plants (Fig.9). We found that the transgenic plant is stronger than wildtype after formaldehyde exposure. This indicates that production of <i>HPS/PHI</i>, <i>FALDH</i> and <i>FDH</i> enhanced formaldehyde tolerance of transgenic plant.</p>
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<div align="center"><img style="width:50% ;" src="https://static.igem.org/mediawiki/2014/9/9e/12.png">
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<div align="center"><img style="width:50% ;" src="https://static.igem.org/mediawiki/2014/a/a5/Uestcresutl8.png">
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<p style="position:relative; left:0px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:1100px; color:#1b1b1b;">
<strong>Fig.8</strong>
<strong>Fig.8</strong>
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The transgenic plants (A) and wildtype (B), which had been grown separately in sealed boxes, were exposed to HCHO evaporated from a micro tube (0.5ml) containing HCHO solution (37%,50ul)
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The transgenic (A) and wildtype plants (B), which had been grown separately in sealed boxes, were exposed to formaldehyde evaporated from a micro tube (0.5ml) containing formaldehyde solution (37%, 10μl)
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<div align="center"><img style="width:50% ;" src="https://static.igem.org/mediawiki/2014/b/bb/Result1.png">
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<div align="center"><img style="width: 54%;
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position: relative;
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left: 20px;
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" src="https://static.igem.org/mediawiki/2014/9/95/Uestcresult9.png">
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<strong>Fig.9</strong>
<strong>Fig.9</strong>
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Phenotype testing of transgenetic seedlings and wildtype. A: Before exposure to HCHO. B: Exposure to HCHO for one week. The transgenetic seedling is stronger than wildtype after formaldehyde exposure. 20ul 37% HCHO, one week.
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Phenotype testing of transgenic plants and wildtype. A: Before exposure to HCHO. B: Exposure to HCHO for one week. The transgenic plant is stronger than wildtype after formaldehyde exposure. 10μl 37% HCHO, two weeks.
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<h1 class="SectionTitles" style="width:900px; ">Enhanced HCHO Absorbing</h1><br/>
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<h1 class="SectionTitles" style="width:1100px; ">Enhanced formaldehyde absorbance</h1><br/>
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<p style="color:#1b1b1b;">We detected the concentration of gaseous formaldehyde evaporated from a micro tube (0.5ml) containing HCHO solution (37%,10ul) and made standard curve (Fig.10). And we saw a linear relationship between HCHO concentration and time before formaldehyde is saturated. For quantity result, we used a HCHO detector to detect the concentration of  gaseous HCHO (Fig.11). The transgenic plants and wildtype, which had been grown separately in sealed boxes, were exposed to HCHO evaporated from a micro tube (0.5ml) containing HCHO solution (37%, 50ul) for about 2 weeks. Two weeks later, the covers of the plant boxes were removed and quickly replaced with covers equipped with HCHO dose-monitoring tubes in order to determine roughly the gaseous HCHO levels remaining in the boxes. We have not got the precise data results now, and this work is to be continued.</p>
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<p style="color:#1b1b1b;">We detected the concentration of gaseous formaldehyde evaporated from a micro tube (0.5ml) containing formaldehyde solution (37%, 10μl) and made a curve (Fig.10) about relationship between formaldehyde concentration and time. And we saw a linear relationship between formaldehyde concentration and time before formaldehyde is saturated. For quantity result, we used a formaldehyde detector to detect the concentration of  gaseous formaldehyde (Fig.11). The transgenic and wildtype plants, which had been grown separately in sealed boxes, were exposed to formaldehyde evaporated from a micro tube (0.5ml) containing formaldehyde solution (37%, 10μl) for about 3 weeks. Three weeks later, the covers of the plant boxes were removed and quickly replaced with covers equipped with formaldehyde dose-monitoring tubes in order to determine roughly the gaseous formaldehyde levels remaining in the boxes. We have not got the precise data results now, and this work is to be continued.</p>
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<div align="center"><img style="width:50% ;" src="https://static.igem.org/mediawiki/2014/3/3d/Result_10.png">
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<div align="center"><img style="width:50% ;" src="https://static.igem.org/mediawiki/2014/3/3c/Graph1.png">
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<p style="position:relative; left:0px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:650px; color:#1b1b1b;"><strong>Fig.10</strong>
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<p style="position:relative; left:50px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:650px; color:#1b1b1b;"><strong>Fig.10</strong>
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Standard Curve of Formaldehyde.
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The relationship between formaldehyde concentration and time
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<div align="center"><img style="width:40% ;" src="https://static.igem.org/mediawiki/2014/2/24/Result_10.jpg">
<div align="center"><img style="width:40% ;" src="https://static.igem.org/mediawiki/2014/2/24/Result_10.jpg">
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<p style="position:relative; left:0px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:650px; color:#1b1b1b;"><strong>Fig.11</strong>
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<p style="position:relative; left:50px; padding:15 5px; font-size:20px; font-family: calibri, arial, helvetica, sans-serif; font-style: calibri; text-align:justify; width:500px; color:#1b1b1b;"><strong>Fig.11</strong>
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folmaldehyde Concentration Detection.
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Folmaldehyde concentration detection.
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<h1 class="SectionTitles" style="width:900px; ">Transit Peptides Affect HCHO Degrading Efficiency </h1>
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<h1 class="SectionTitles" style="width:1100px; ">Transit peptides affect formaldehyde degrading efficiency </h1>
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<p style="color:#1b1b1b;">HPS, PHI, and FDH are located in chloroplast, while FALDH plays a role in cytoplasm. So we used transit peptides to locate the productions of these genes. We hope to know the effects of transit peptide on degrading HCHO. So we exposed transgenic tobaccos with and without transit peptides to HCHO (37%, 50ul). However, there are no obvious phenotype difference compairing different transgenic lines because time limited.</p>
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<p style="color:#1b1b1b;"><i>HPS</i>, <i>PHI</i>, and <i>FDH</i> are located in chloroplast, while <i>FALDH</i> plays a role in cytoplasm. So we used transit peptides to locate the productions of these genes. We hope to know the effects of transit peptide on degrading formaldehyde. So we exposed transgenic tobaccos with and without transit peptides to formaldehyde (37%, 10μl). However, there are no obvious phenotype difference compairing different transgenic lines because time limited.</p>
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<h1 class="SectionTitles" style="width:900px; ">Different Genes Affect HCHO Degrading Efficiency </h1>
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<h1 class="SectionTitles" style="width:1100px; ">Different genes affect formaldehyde degrading efficiency </h1>
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<p style="color:#1b1b1b;">To test which enzymes play the most important role in pathway of metabolizing HCHO, we constructed mono-gene expression vectors to express each enzyme individually. We also constructed multi-gene expression vectors to test whether the ability of metabolizing HCHO of transgenic tobacco enhanced. Then these transgenic tobaccos were exposed to HCHO (37%, 50ul). However, we have not got obvious phenotype difference among transgenic lines, because there is not enough time</p>
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<p style="color:#1b1b1b;">To test which enzymes play the most important role in pathway of metabolizing formaldehyde, we constructed mono-gene expression vectors to express each enzyme individually. We also constructed multi-gene expression vectors to test whether the ability of metabolizing formaldehyde of transgenic tobacco enhanced. Then these transgenic tobaccos were exposed to formaldehyde (37%, 10μl). However, we have not got obvious phenotype difference among transgenic lines, because there is not enough time</p>
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<h1 class="SectionTitles" style="width:900px; ">Tapetal expression of AdCP results in male sterility in high expression plants</h1><br/>
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<h1 class="SectionTitles" style="width:1100px; ">Tapetal expression of <i>AdCP</i> results in male sterility in high expression plants</h1><br/>
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<p style="color:#1b1b1b;">Considering the problem of environment and safety, we use male sterility system which prevents the horizontal transgene flow. Morphological and histological analysis of AdCP transgenic plants showed ablated tapetum and complete pollen abortion. However, there is not enough time to wait until transgenic tobacco flowers.</p>
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<p style="color:#1b1b1b;">Considering the problem of environment and safety, we use male sterility system which prevents the horizontal transgene flow. Morphological and histological analysis of <i>AdCP</i> transgenic plants showed ablated tapetum and complete pollen abortion. However, there is not enough time to wait for transgenic tobacco flowering.</p>
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<div><img style="width:20% ;" src="https://static.igem.org/mediawiki/2014/2/2e/Tobecontinued.png"></div>
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<div><img style="width:40% ;" src="https://static.igem.org/mediawiki/2014/2/2e/Tobecontinued.png"></div>
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Latest revision as of 02:29, 18 October 2014

UESTC-China