Team:NTU Taiwan/index.html

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     <title> Igem-Taiwan </title>
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     <title> iGEM-NTU-Taiwan </title>
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             <h1 class=" rainbow-text header">IGem-Taiwan Yeastherm</h1>
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             <h1 class=" rainbow-text header">iGEM-NTU-Taiwan YeasTherm</h1>
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                 <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/>
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                 Working with Thermogenic Yeast<br/>
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                 Working on Thermogenic Yeast<br/>
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                 Apps with knowledge of iGEM competition and synthetic biology.
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                 Apps with concept of iGEM competition and synthetic biology.
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         </section>
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                 In Taiwan, fish farmers lose a large amount of fish, because temperature falls dramatically when cold current comes in winter. Ofcourse, fish farmers try to prevent fish from death dying; however, the current methods do not work well. Moreover, they cause damage to the environment. In 2013 iGEM competition, NTU-Taiwan team tries to make a bio-heating device. We transform the SrUCP (uncoupling protein) into yeast. UCP is thermogenic protein which can produce heat by interacting with the electron transport chain. By designing the gene circuit, we want to well control the power of the bio-heating device. In addition, we want to simulate the pond environment in reality by computer and the test results after using our device in low temperature.
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                 In Taiwan, fish farmers lose a large amount of fish, because temperature falls dramatically when cold current comes in winter. Of course, fish farmers try to prevent fish from dying. However, current methods do not work well and even cause damages to the environment. In 2013 iGEM competition, NTU_Taiwan team tries to make a bio-heating device. We transform an UCP homologue from themogenic plants into yeast. UCP is a thermogenic protein which can produce heat by interacting with the electron transport chain. By designing a genetic circuit, we want to well control the power of our bio-heating device. In addition, we want to simulate the effect of our device on fish ponds in reality after testing the heating power of our device.
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             <h1 class="header" style="margin: 0">Basic Research</h1>
             <h1 class="header" style="margin: 0">Basic Research</h1>
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                 <p class="header" style="margin: 0"> our works </p>
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                 <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">Circuit</h1>
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             <h1 class="header">Applications</h1>
             <h1 class="header">Applications</h1>
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                <p class="header" style="margin: 0"></p>
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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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                 In hope of ?? the value of our biological heating device, we strive to improving the sensitivity of our sensor － the cold shock promoter. This final goal can be break down on two parts: 1. tuning the temperature-responsive range of cold shock promoter 2. amplifying its signal under low temperature. In order to understand what kind of structure of a genetic circuit and what kinds of characteristics of activator and repressor are needed for our purpose, we create several mathematical models to explore the problem. In the end, we expect to get some useful information as a guidance to screen possible biological parts when we actually start to construct the genetic circuit.
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                 In hope of putting more values in our biological heating device, we strive to improving the sensitivity of our sensor － the cold shock promoter. This final goal can be break down on two parts: 1. tuning the temperature-responsive range of cold shock promoter 2. amplifying its signal under low temperature. In order to understand what kind of structure of a genetic circuit and what kinds of characteristics of activator and repressor are needed for our purpose, we create several mathematical models to explore the problem. In the end, we expect to get some useful information as a guidance to screen possible biological parts when we actually start to construct the genetic circuit.
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                             <td class="col-md-2">β<sub>Csp</sub>(T)<sup>a</sup></td>
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                             <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>
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                             <td class="col-md-2">M/s, normalized</td>
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                             <td class="col-md-2">M/s</td>
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         <br/><p><b>PCR</b></p><br/>
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         <br/><p><b>PCR</b></p>
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        &nbsp&nbsp&nbsp&nbsp&nbspStep 1: Design of appropriate forward and reverse primers<br/>
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1: Design of appropriate forward and reverse primers<br/>
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        &nbsp&nbsp&nbsp&nbsp&nbspStep 2: Prepare our template<br/>
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2: Prepare our template<br/>
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        &nbsp&nbsp&nbsp&nbsp&nbspStep 3: Prepare the PCR mix. (Kapa Hifi PCR kit.)<br/>
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3: Prepare the PCR mix. (Kapa Hifi PCR kit.)<br/>
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        &nbsp&nbsp&nbsp&nbsp&nbspStep 4: Run PCR<br/>
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4: Run PCR<br/>
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        &nbsp&nbsp&nbsp&nbsp&nbspStep 5: Examine the results by electrophoresis<br/>
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5: Examine the results by electrophoresis<br/>
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         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
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         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/>
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<br/><p><b>Construction of our parts</b></p><br/>
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<br/><p><b>Construction of our parts</b></p><p>
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        &nbsp&nbsp&nbsp&nbsp&nbspStep 1: We design primers for parts with prefix and suffix.<br/>
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1: We design primers for parts with prefix and suffix.<br/>
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        &nbsp&nbsp&nbsp&nbsp&nbspStep 2: Perform PCR and cleanup the PCR product<br/>
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2: Perform PCR and cleanup the PCR product<br/>
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        &nbsp&nbsp&nbsp&nbsp&nbspStep 3: Before insert our parts into standard backbone, pSB1C3, we perform RE digestion to make sticky ends of both inserts and backbones.<br/>
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3: Before insert our parts into standard backbone, pSB1C3, we perform RE digestion to make sticky ends of both inserts and backbones.<br/>
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        &nbsp&nbsp&nbsp&nbsp&nbspStep 4: Ligation of inserts and backbones<br/>
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4: Ligation of inserts and backbones<br/>
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        &nbsp&nbsp&nbsp&nbsp&nbspStep 5: Transform our ligation products into DH5α and streak the transformed DH5α on LB agar plate with chloramphenicol.<br/>
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5: Transform our ligation products into DH5α and streak the transformed DH5α on LB agar plate with chloramphenicol.<br/>
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        &nbsp&nbsp&nbsp&nbsp&nbspStep 6: Inoculate single colony into broth with chloramphenicol.<br/>
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6: Inoculate single colony into broth with chloramphenicol.<br/>
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        &nbsp&nbsp&nbsp&nbsp&nbspStep 7: Miniprep the plasmid DNA from the overnight broth culture.<br/>
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7: Miniprep the plasmid DNA from the overnight broth culture.<br/>
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        &nbsp&nbsp&nbsp&nbsp&nbspStep 8: Confirm the products by both RE digestion and PCR sequencing<br/>
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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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            <h1 class="header"> Plasmid Construction</h1>
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            <p class="header">For characterization </h1>
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                 <img class="pull-right img-responsive" src="https://static.igem.org/mediawiki/2013/9/91/Ucp.png" alt-src="./images/result/ucp_1.png" width=400>
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                <div class="col-md-4" style="margin-top: 100px"><p>After we got the SrUCP cDNA fro Dr.Ito, we did restrict enzyme analysis and sequencing to make sure the sequence is right.</p></div>
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                    <img class="pull-right img-responsive" src="https://static.igem.org/mediawiki/2013/9/91/Ucp.png" alt-src="./images/result/ucp_1.png" width=400>
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                    <div class="col-md-4" style="margin-top: 100px"><p>After we got the SrUCP cDNA fro Dr.Ito, we did restrict enzyme analysis and sequencing to make sure the sequence is right.</p></div>
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                    <img class="pull-left img-responsive" src="https://static.igem.org/mediawiki/2013/9/99/Backbone.png" alt-src="./images/result/backbone.png" width=400>
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                    <p class="col-md-4 pull-right" style="margin-top: 40px">  We also check the shuttle vector before the experiment and find out some problem on it. Because we had to insert our SrUCP gene into pRS424 by NcoI and SpeI, we use these two enzymes to check the restrict enzyme sites on it. However we found out there was only one NcoI site on pRS424, it was different to the map.</p>
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                    <img class="pull-right img-responsive" src="https://static.igem.org/mediawiki/2013/d/d4/Prs424.png" alt-src="./images/result/prs424.png" width=700>
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                    <div class="col-md-4" style="margin-top: 140px"><p>Because the size of shuttle vector is too large to transform by heat shock method. We got only one successful construction in 22 samples. But it’s great enough!</p></div>
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                    <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>
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                    <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>
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                    <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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                    <img class="pull-right img-responsive" src="https://static.igem.org/mediawiki/2013/d/d4/Prs424.png" alt-src="./images/result/prs424.png" width=700>
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                <img class="pull-left img-responsive" src="https://static.igem.org/mediawiki/2013/9/99/Backbone.png" alt-src="./images/result/backbone.png" width=400>
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                    <div class="col-md-4" style="margin-top: 140px"><p>Because the size of shuttle vector is too large to transform by heat shock method. We got only one successful construction in 22 samples. But it’s great enough!</p></div>
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                <div class="col-md-4" style="margin-top: 60px"><p> We also check the shuttle vector before the experiment and find out some problem on it. Because we had to insert our SrUCP gene into pRS424 by NcoI and SpeI, we use these two enzymes to check the restrict enzyme sites on it. However we found out there was only one NcoI site on pRS424, it was different to the map.</p></div>
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        <img class="pull-right img-responsive" src="https://static.igem.org/mediawiki/2013/d/d4/Prs424.png" alt-src="./images/result/prs424.png" width=700>
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        <div class="col-md-4" style="margin-top: 140px"><p>Because the size of shuttle vector is too large to transform by heat shock method. We got only one successful construction in 22 samples. But it’s great enough!</p></div>
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        <div class="container" style="margin-top: 20px">
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        <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>
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        <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>
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                 <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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                <div class="container" style="margin-top: 20px">
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                    <img class="pull-right img-responsive" src="https://static.igem.org/mediawiki/2013/1/1d/Pgapza.png" alt-src="./images/result/pgapza.png" width=550>
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                    <div class="col-md-4" style="margin-top: 140px"><p> This is the pGAPZa which had been digested by <i>Bgi</i>II.</p></div>
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            <h1 class="header"> Characterize the biological part  </h1>
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        <img class="pull-right img-responsive" src="https://static.igem.org/mediawiki/2013/d/d4/Prs424.png" alt-src="./images/result/prs424.png" width=700>
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            <p class="header">Test the expression of SrUCP by Western blotting.</p>
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        <div class="col-md-4" style="margin-top: 140px"><p>Because the size of shuttle vector is too large to transform by heat shock method. We got only one successful construction in 22 samples. But it’s great enough!</p></div>
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            <div class="container" style="margin-top: 20px">
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                <img class="pull-left img-responsive" src="https://static.igem.org/mediawiki/2013/9/95/Western.png" alt-src="./images/result/western.png" width=700>
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                <div class="col-md-4" style="margin-top: 190px"><p>  Based on the sequence analysis, we predict the protein size of SrUCP(with TAP tag) is about 53 kDa. We did the Western blotting and confirmed our SrUCP gene have expressed in Saccharomyces cerevisiae.</p>
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        <div class="container" style="margin-top: 20px">
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            <img class="pull-left img-responsive" src="https://static.igem.org/mediawiki/2013/9/95/Western.png" alt-src="./images/result/western.png" width=700>
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            <div class="col-md-4" style="margin-top: 190px"><p>  Based on the sequence analysis, we predict the protein size of SrUCP(with TAP tag) is about 53 kDa. We did the Western blotting and confirmed our SrUCP gene have expressed in Saccharomyces cerevisiae.</p></div>
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        <div class="container" style="margin-top: 20px">
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            <p class="header"> Analyze the heat-production ability of transformant</p>
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        <img class="pull-right img-responsive" src="https://static.igem.org/mediawiki/2013/1/1d/Pgapza.png" alt-src="./images/result/pgapza.png" width=550>
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            <div class="container essay">
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        <div class="col-md-4" style="margin-top: 140px"><p> This is the pGAPZa which had been digested by <i>Bgi</i>II.</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>
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        </div>
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                <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>
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                <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/>
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        <div class="container" style="margin-top: 20px">
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                    Experimental: Heat(E) = Fermentation(1) + SrUCP<br/>
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        <img class="pull-left img-responsive" src="https://static.igem.org/mediawiki/2013/a/a9/25.png" alt-src="./images/result/25.png" style="margin-top: 0px"width=530>
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                    Control: Heat(C) = Fermentation(2)<br/>
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        <img class="pull-right img-responsive" src="https://static.igem.org/mediawiki/2013/d/d6/15.png" alt-src="./images/result/15.png" width=570>
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                    Because the heat of fermentation(1) is lower than fermentation(2), even if SrUCP produce heat, the total Heat(E) equal to Heat(C).
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        <div class="col-md-10" style="margin-top: 10px"><p> For understanding the physical function of both strains in normal temperature and low temperature. We built up four growth curve.</p></div>
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                    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.
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                </p>
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                <img src="https://static.igem.org/mediawiki/2013/3/37/NTU_TAIWAN_Capture.JPG" alt-src="images/Capture.jpg">
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            </div>
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            <p class="header"> Rhodotorula glutinis Growth curve</p>
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            <div class="container essay">
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                <img src="https://static.igem.org/mediawiki/2013/2/2f/NTU_TAIWAN_Capture2.JPG" alt-src="images/Capture2.jpg">
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                <img src="https://static.igem.org/mediawiki/2013/5/52/NTU_TAIWAN_Capture3.JPG" alt-src="images/Capture3.jpg">
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                <p>To realize our ultimate goal, that is, to express SrUCP in Rhodotorula glutinis, we analyze this organism’s growth property. This information is useful for use to prepare the competent cell of Rhodotorula glutinis. This is the fundamental and important step to express exogenous gene in this species.</p>
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                Results: <br/>
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                <p>At 25℃, R.glutinis has the optimal growth curve, it’s lower than the S. cerevisiae. Also, no matter under 25 or 15℃, the growth rates are both slower than S. cerevisiae. However, the lag phase of these two curves are close to each other.(fig2, fig3). Interestingly, this strain has a faster growth rate at 4℃ relatively. This phenomenon is easily to be observed when inoculating on agar plate.(The result is not shown) Therefore, R. glutinis maybe a better chassis than S. cerevisiae to produce heat in low temperature.</p>
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                <p>According to the growth curve, we suppose that between 6 to 8 hours (at early log phase) would be the best time for making competent cell of R.glutinis, but we still need more experiments for further characterization.</p>
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            </div>
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         </div>
         </div>
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     </script>
     </script>

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iGEM-NTU-Taiwan

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