Team:UESTC-China/result

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<h1 class="SectionTitles" style="width:1100px; ">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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<h1 class="SectionTitles" style="width:1100px; ">Vectors construction</h1><br/>
<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>
<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 ><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;">
<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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<strong>A.</strong> Digestion the plasmid piGEM001 with <i>Hpa</i>I and <i>Spe</i>I.  
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A: Digestion the plasmid piGEM001 with <i>Hpa</i>I and <i>Spe</i>I.  
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 <i>Hpa</i>I and <i>Dra</i>III.  
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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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<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;">
<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 <i>Xba</i>I and <i>Sal</i>I.  
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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:
Digestion the plasmid piGEM010 with <i>Hind</i>III and <i>Sac</i>I.  
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:
Digestion of the plasmid piGEM011 with <i>EcoR</i>I and <i>Sac</i>I.  
Digestion of the plasmid piGEM011 with <i>EcoR</i>I and <i>Sac</i>I.  
M: DNA marker;
M: DNA marker;
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<h1 class="SectionTitles" style="width:1100px; ">Plant transformation</h1><br/>
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<h1 class="SectionTitles" style="width:1100px; ">Tobacco transformation</h1><br/>
<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>
<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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<strong>Fig.5</strong>
<strong>Fig.5</strong>
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positive tobacco plantlets of each transgenic line.
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Positive tobacco plantlets of each transgenic line.
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<h1 class="SectionTitles" style="width:1100px; ">Enhanced formaldehyde 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 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%, 10ul) (Fig.8). One week later we observed the phenotype of transgeneic  and widetype plants (Fig.9). We found that the transgenetic 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 plants.</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">
<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;">
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<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 formaldehyde evaporated from a micro tube (0.5ml) containing formaldehyde solution (37%, 10μl)
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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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position: relative;
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left: 20px;
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" src="https://static.igem.org/mediawiki/2014/b/bb/Result1.png">
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" src="https://static.igem.org/mediawiki/2014/9/95/Uestcresult9.png">
<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;">
<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.9</strong>
<strong>Fig.9</strong>
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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. 10ul 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:1100px; ">Enhanced formaldehyde Absorbance</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 formaldehyde solution (37%, 10ul) 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 plants and wildtype, which had been grown separately in sealed boxes, were exposed to formaldehyde evaporated from a micro tube (0.5ml) containing formaldehyde 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 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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<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/2014/3/3c/Graph1.png">
<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:600px; 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>
The relationship between formaldehyde concentration and time
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:400px; 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>
Folmaldehyde concentration detection.
Folmaldehyde concentration detection.
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<h1 class="SectionTitles" style="width:1100px; ">Transit Peptides Affect Formaldehyde 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;"><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%, 10ul). 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:1100px; ">Different Genes Affect Formaldehyde 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 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%, 10ul). 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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Latest revision as of 02:29, 18 October 2014

UESTC-China