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Team:NTU Taiwan/index.html
From 2013.igem.org
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| - | <title> | + | <title> iGEM-NTU-Taiwan </title> |
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| - | <h1 class=" rainbow-text header"> | + | <h1 class=" rainbow-text header">iGEM-NTU-Taiwan YeasTherm</h1> |
<p class="header container"> | <p class="header container"> | ||
<img class="spin" alt-src="images/LaboratoryLevels.png" src="/wiki/images/9/91/NTU_TAIWAN_LaboratoryLevels.png"><br/> | <img class="spin" alt-src="images/LaboratoryLevels.png" src="/wiki/images/9/91/NTU_TAIWAN_LaboratoryLevels.png"><br/> | ||
National Taiwan University<br/> | National Taiwan University<br/> | ||
| - | Working | + | Working on Thermogenic Yeast<br/> |
| - | Apps with | + | Apps with concept of iGEM competition and synthetic biology. |
</p> | </p> | ||
</section> | </section> | ||
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<h1 class="header" style="margin: 0">Basic Research</h1> | <h1 class="header" style="margin: 0">Basic Research</h1> | ||
<div class="container"> | <div class="container"> | ||
| - | <p class="header" style="margin: 0"> our | + | <p class="header" style="margin: 0"> Our final goal is to express SrUCP in <i>Rhodotorula glutinis</i>. However, hampering by its difficulties in molecular cloning, we take <i>Saccharomyces cerevisiae</i> as our first-hand research material. </p> |
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<h1 class="header">Applications</h1> | <h1 class="header">Applications</h1> | ||
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| - | + | <p class="header" style="margin: 0"> Being an special lipid productive yeast, <i>Rhodotorula glutinis</i> has strong potentiality to become an extraordinary bio-heating device. Let's find out! </p> | |
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| - | <td class="col-md-2">β<sub> | + | <td class="col-md-2">β<sub>hsp</sub>(T)<sup>a</sup></td> |
<td class="col-md-5">Maximal production rate of Hsp, a function of temperature</td> | <td class="col-md-5">Maximal production rate of Hsp, a function of temperature</td> | ||
<td class="col-md-3">Sigmoidal curve <br/>(set 37℃=1e-6, 30℃=0) </td> | <td class="col-md-3">Sigmoidal curve <br/>(set 37℃=1e-6, 30℃=0) </td> | ||
| - | <td class="col-md-2">M/s | + | <td class="col-md-2">M/s</td> |
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<div class="container"> | <div class="container"> | ||
| - | <br/><p><b>PCR</b></p | + | <br/><p><b>PCR</b></p> |
| - | + | 1: Design of appropriate forward and reverse primers<br/> | |
| - | + | 2: Prepare our template<br/> | |
| - | + | 3: Prepare the PCR mix. (Kapa Hifi PCR kit.)<br/> | |
| - | + | 4: Run PCR<br/> | |
| - | + | 5: Examine the results by electrophoresis<br/> | |
| - | Note: If the template is genomic DNA, we would adjust the annealing temperature at 45°C. It is because the copy number of target gene may be low. We use this annealing temp when perform PCR of Tir1, 26s, 5.8s ITS | + | Note: If the template is genomic DNA, we would adjust the annealing temperature at 45°C. It is because the copy number of target gene may be low. We use this annealing temp when perform PCR of Tir1, 26s, 5.8s ITS<br/><br/> |
| - | <br/><p><b>Construction of our parts</b></p>< | + | <br/><p><b>Construction of our parts</b></p><p> |
| - | + | 1: We design primers for parts with prefix and suffix.<br/> | |
| - | + | 2: Perform PCR and cleanup the PCR product<br/> | |
| - | + | 3: Before insert our parts into standard backbone, pSB1C3, we perform RE digestion to make sticky ends of both inserts and backbones.<br/> | |
| - | + | 4: Ligation of inserts and backbones<br/> | |
| - | + | 5: Transform our ligation products into DH5α and streak the transformed DH5α on LB agar plate with chloramphenicol.<br/> | |
| - | + | 6: Inoculate single colony into broth with chloramphenicol.<br/> | |
| - | + | 7: Miniprep the plasmid DNA from the overnight broth culture.<br/> | |
| - | + | 8: Confirm the products by both RE digestion and PCR sequencing<br/></p> | |
<p><b>Point mutation protocol</b></p> | <p><b>Point mutation protocol</b></p> | ||
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<div class="container"> | <div class="container"> | ||
| - | <img class="pull-left img-responsive" src="https://static.igem.org/mediawiki/2013/d/d4/Tir1-1-1.png" alt-src="./images/result/tir1-1.png" style="margin-top: 50px" width= | + | <img class="pull-left img-responsive" src="https://static.igem.org/mediawiki/2013/d/d4/Tir1-1-1.png" alt-src="./images/result/tir1-1.png" style="margin-top: 50px"width=600> |
<img class="pull-right img-responsive" src="https://static.igem.org/mediawiki/2013/b/b1/Tir1-2.png" alt-src="./images/result/tir1-2.png" width= 500> | <img class="pull-right img-responsive" src="https://static.igem.org/mediawiki/2013/b/b1/Tir1-2.png" alt-src="./images/result/tir1-2.png" width= 500> | ||
<div class="col-md-11" style="margin-top: 10px"><p>We predicted the Tir-1 promoter should be at about 1000 base pairs upstream, so we tried to amplified the Tir-1 promoter sequence from Saccharomyces cerevisiae by PCR. We design the primer with expanded restriction enzyme sites and about 30 base pairs complementary to the S.c. genome sequence, preventing from non-specific product. However, it’s harder to PCR a sequence from genomic DNA than plasmid. In hence, we tried different annealing temperature to make sure we have target product and decrease non-specific band.</p></div> | <div class="col-md-11" style="margin-top: 10px"><p>We predicted the Tir-1 promoter should be at about 1000 base pairs upstream, so we tried to amplified the Tir-1 promoter sequence from Saccharomyces cerevisiae by PCR. We design the primer with expanded restriction enzyme sites and about 30 base pairs complementary to the S.c. genome sequence, preventing from non-specific product. However, it’s harder to PCR a sequence from genomic DNA than plasmid. In hence, we tried different annealing temperature to make sure we have target product and decrease non-specific band.</p></div> | ||
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<p>For estimate the heat-production ability of the SrUCP in yeast. We built up a straight way to analyze it. </p> | <p>For estimate the heat-production ability of the SrUCP in yeast. We built up a straight way to analyze it. </p> | ||
<p>After both experimental group (pRS424-GAL1-SrUCP-TAP) and negative control group (pRS424-GAL1∆) induced by 2% galactose for 21 hours, we couldn’t find out statistical difference between control and experimental group by our first experimental method. Most of the heat production is come from the fermentation and shaking of the incubator.</p> | <p>After both experimental group (pRS424-GAL1-SrUCP-TAP) and negative control group (pRS424-GAL1∆) induced by 2% galactose for 21 hours, we couldn’t find out statistical difference between control and experimental group by our first experimental method. Most of the heat production is come from the fermentation and shaking of the incubator.</p> | ||
| - | <p>However, we didn’t analyze the quantity of yeasts during the experiment. We consider that the experimental group (which had been transformed SrUCP) might grow slower than the control group and then cause the result t I show below: | + | <p>However, we didn’t analyze the quantity of yeasts during the experiment. We consider that the experimental group (which had been transformed SrUCP) might grow slower than the control group and then cause the result t I show below:<br/> |
| - | Experimental: Heat(E) = Fermentation(1) + SrUCP | + | Experimental: Heat(E) = Fermentation(1) + SrUCP<br/> |
| - | Control: Heat(C) = Fermentation(2) | + | Control: Heat(C) = Fermentation(2)<br/> |
Because the heat of fermentation(1) is lower than fermentation(2), even if SrUCP produce heat, the total Heat(E) equal to Heat(C). | Because the heat of fermentation(1) is lower than fermentation(2), even if SrUCP produce heat, the total Heat(E) equal to Heat(C). | ||
In hence, the better method to test heat production is incubate in isothermal environment or use the isothermal titration calorimetry. We will try these more precise method. | In hence, the better method to test heat production is incubate in isothermal environment or use the isothermal titration calorimetry. We will try these more precise method. | ||
Latest revision as of 04:22, 28 September 2013
