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Team:NTU Taiwan/index.html
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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> |
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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/> | ||
| - | Working | + | Working on Thermogenic Yeast<br/> |
| - | Apps with | + | Apps with concept of iGEM competition and synthetic biology. |
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| - | <p>In this year, iGEM NTU_Taiwan team | + | <p>In this year, iGEM NTU_Taiwan team aims at making a biological heating device which can produce appropriate heat at low temperatures. The feature of this device is that it can respond differently to temperature and produce heat in an efficient and economical manner. |
</p> | </p> | ||
| - | <p>What a crazy project! This biological device is really charming, isn | + | <p>What a crazy project! This biological device is really charming, isn't it? Let us show you our project! <br/> |
| - | <div class="row text-center"><h3>Let | + | <div class="row text-center"><h3>Let's go!</h3></div> |
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| - | In Taiwan, fish farmers lose a large amount of fish, because temperature falls dramatically when cold current comes in winter. | + | 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> | ||
<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> | ||
<div class="container"> | <div class="container"> | ||
| - | + | <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 | + | 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> | + | <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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βCsp(T) was obtained by fitting previous studies on related cold shock promoter in human. The curve of βCsp(T) reaches a maximum at 10℃ and a minimum at 20℃ (Fig. 2). In other words, its temperature-responsive range is between 10℃ and 20℃. The value of βHsp(T) is defined by us, reaching a maximum at 37℃ and a minimum at 30℃ (Fig. 3). This setting of parameter is based on certain physiological considerations where the optimal growth temperature of Saccharomyces cerevisiae is 30℃ and where heat shock response is observed at temperatures higher than 37℃. Next, comparing Fig. 2and Fig. 3, it is obvious that the activity range of these two promoters are not overlapping, which is an critical problem to our genetic circuit. We believed that this phenomenon is going to be the flaw of our genetic circuit because two signals produced by two promoters are not able to crosstalk. Hence, the expression profile of GFP along these temperatures might not be changed. This suspect will be proved by simulation in the next paragraph. | βCsp(T) was obtained by fitting previous studies on related cold shock promoter in human. The curve of βCsp(T) reaches a maximum at 10℃ and a minimum at 20℃ (Fig. 2). In other words, its temperature-responsive range is between 10℃ and 20℃. The value of βHsp(T) is defined by us, reaching a maximum at 37℃ and a minimum at 30℃ (Fig. 3). This setting of parameter is based on certain physiological considerations where the optimal growth temperature of Saccharomyces cerevisiae is 30℃ and where heat shock response is observed at temperatures higher than 37℃. Next, comparing Fig. 2and Fig. 3, it is obvious that the activity range of these two promoters are not overlapping, which is an critical problem to our genetic circuit. We believed that this phenomenon is going to be the flaw of our genetic circuit because two signals produced by two promoters are not able to crosstalk. Hence, the expression profile of GFP along these temperatures might not be changed. This suspect will be proved by simulation in the next paragraph. | ||
</p> | </p> | ||
| - | <img class="tipReveal row" src="images/modeling/fittingresult1.jpg"> | + | <img class="tipReveal row" src="https://static.igem.org/mediawiki/2013/8/81/Fittingresult1.jpg" alt-src="images/modeling/fittingresult1.jpg"> |
<div class="tip"> Fig. 2: Fitting result of βCsp(T). </div> | <div class="tip"> Fig. 2: Fitting result of βCsp(T). </div> | ||
| - | <img class="tipReveal row" src="images/modeling/fittingresult2.jpg"> | + | <img class="tipReveal row" src="https://static.igem.org/mediawiki/2013/a/a5/Fittingresult2.jpg" alt-src="images/modeling/fittingresult2.jpg"> |
<div class="tip"> Fig. 3: Fitting result of βHsp(T) </div> | <div class="tip"> Fig. 3: Fitting result of βHsp(T) </div> | ||
<legend><b>Neither Hsp nor constitutive promoter suits our purpose under this circuit structure.</b></legend> | <legend><b>Neither Hsp nor constitutive promoter suits our purpose under this circuit structure.</b></legend> | ||
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To better understand the role of Hsp and constitutive promoter in our circuit, we analyze the expression pattern difference between repressor-regulated-Csp and Csp alone. By an steady state approach, we may validate if our genetic circuit is in effect changing the sensitivity of Csp. In order to define "markers" that help us discriminate between "bad" results and "good" results, "GFP maximal concentration" (abbreviated as <b>GFP<sub>max</sub></b>) and "temperature corresponding to half of the maximal concentration of GFP" (abbreviated as T1/2) as taken into consideration (Fig. 4). As the value of GFPmax goes up, we are more able to observe the signal under low temperatures; as T1/2 goes down, the temperature-responsive range of Csp narrows down which implies more <b>sensitive</b>. However, in our constitutive promoter model, the two markers do not become "better". The repressor suppresses the activity of Csp significantly when αR becomes small (Fig. 5). Likewise, the markers of Hsp model are "bad" too. Since the active ranges of Csp and Hsp are not overlapping, expression of GFP cannot be suppressed at all as predicted in the last paragraph (Fig. 6). We are going to solve this problem using another genetic circuit! | To better understand the role of Hsp and constitutive promoter in our circuit, we analyze the expression pattern difference between repressor-regulated-Csp and Csp alone. By an steady state approach, we may validate if our genetic circuit is in effect changing the sensitivity of Csp. In order to define "markers" that help us discriminate between "bad" results and "good" results, "GFP maximal concentration" (abbreviated as <b>GFP<sub>max</sub></b>) and "temperature corresponding to half of the maximal concentration of GFP" (abbreviated as T1/2) as taken into consideration (Fig. 4). As the value of GFPmax goes up, we are more able to observe the signal under low temperatures; as T1/2 goes down, the temperature-responsive range of Csp narrows down which implies more <b>sensitive</b>. However, in our constitutive promoter model, the two markers do not become "better". The repressor suppresses the activity of Csp significantly when αR becomes small (Fig. 5). Likewise, the markers of Hsp model are "bad" too. Since the active ranges of Csp and Hsp are not overlapping, expression of GFP cannot be suppressed at all as predicted in the last paragraph (Fig. 6). We are going to solve this problem using another genetic circuit! | ||
</p> | </p> | ||
| - | <img class="img-responsive" src="images/modeling/modeling3.jpg"> | + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2013/a/af/Modeling3.JPG" alt-src="images/modeling/modeling3.jpg"> |
| - | <img class="tipReveal img-responsive" src="images/modeling/modelresult1.jpg"> | + | <img class="tipReveal img-responsive" src="https://static.igem.org/mediawiki/2013/4/42/Modelresult1.jpg" alt-src="images/modeling/modelresult1.jpg"> |
<div class="tip"> Fig. 4: Expression pattern of GFP under various αA and αR－ a constitutive promoter integrated model. The X, Y axis are values of αA and αR scanned. The Z axis is the maximal GFP concentration. The color bar represents the temperature corresponding to half of the maximal GFP concentration</div> | <div class="tip"> Fig. 4: Expression pattern of GFP under various αA and αR－ a constitutive promoter integrated model. The X, Y axis are values of αA and αR scanned. The Z axis is the maximal GFP concentration. The color bar represents the temperature corresponding to half of the maximal GFP concentration</div> | ||
| - | <img class="tipReveal img-responsive" src="images/modeling/modelresult2.jpg"> | + | <img class="tipReveal img-responsive" src="https://static.igem.org/mediawiki/2013/b/bf/Modelresult2.jpg" alt-src="images/modeling/modelresult2.jpg"> |
<div class="tip"> Fig. 5: Expression pattern of GFP under various αA and αR－ an Hsp integrated model. The X, Y axis are values of αA and αR scanned. The Z axis is the maximal GFP concentration. The color bar represents the temperature corresponding to half of the maximal GFP concentration</div> | <div class="tip"> Fig. 5: Expression pattern of GFP under various αA and αR－ an Hsp integrated model. The X, Y axis are values of αA and αR scanned. The Z axis is the maximal GFP concentration. The color bar represents the temperature corresponding to half of the maximal GFP concentration</div> | ||
</div> | </div> | ||
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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="red-background row"> | <div class="red-background row"> | ||
| + | <h1 class="header"> Plasmid Construction</h1> | ||
| + | <p class="header">For characterization </h1> | ||
<div class="container essay"> | <div class="container essay"> | ||
| - | <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> | + | <div class="container essay"> |
| - | + | <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> | |
| - | </div> | + | <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> |
| - | + | </div> | |
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| + | <div class="container"> | ||
| + | <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> | ||
| + | <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> | ||
| + | </div> | ||
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| + | <div class="container"> | ||
| + | <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> | ||
| + | <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> | ||
| + | </div> | ||
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| + | <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=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> | ||
| + | <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> | ||
| - | + | <div class="container" style="margin-top: 20px"> | |
| - | + | <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> | |
| - | + | <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"> | ||
| + | <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> | ||
| + | <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> | ||
| + | </div> | ||
| + | </div> | ||
</div> | </div> | ||
| - | + | <div class="yellow-background row"> | |
| - | <div class=" | + | <h1 class="header"> Characterize the biological part </h1> |
| - | + | <p class="header">Test the expression of SrUCP by Western blotting.</p> | |
| - | + | <div class="container" style="margin-top: 20px"> | |
| - | + | <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> | |
| - | + | <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> | </div> | ||
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| - | + | <p class="header"> Analyze the heat-production ability of transformant</p> | |
| - | + | <div class="container essay"> | |
| - | + | <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>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<br/> | |
| - | + | 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). | |
| - | + | 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. | |
| + | </p> | ||
| + | <img src="https://static.igem.org/mediawiki/2013/3/37/NTU_TAIWAN_Capture.JPG" alt-src="images/Capture.jpg"> | ||
| + | |||
| + | </div> | ||
| + | <p class="header"> Rhodotorula glutinis Growth curve</p> | ||
| + | |||
| + | <div class="container essay"> | ||
| + | <img src="https://static.igem.org/mediawiki/2013/2/2f/NTU_TAIWAN_Capture2.JPG" alt-src="images/Capture2.jpg"> | ||
| + | <img src="https://static.igem.org/mediawiki/2013/5/52/NTU_TAIWAN_Capture3.JPG" alt-src="images/Capture3.jpg"> | ||
| + | <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> | ||
| + | Results: <br/> | ||
| + | <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> | ||
| + | <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> | ||
| + | </div> | ||
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</div> | </div> | ||
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</script> | </script> | ||
Latest revision as of 04:22, 28 September 2013
