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Team:NTU Taiwan/index.html
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There are 4 farming fishing among top 15 fishing output in Taiwan. The output value of farming fish is second only to deep sea fishing. Unfortunately, in winter, we see news about large amount of fish died due to low temperature. In winter, cold current which comes from the Mongolia dramatically decreases the temperature and causes fish to die. As you know that fish is cold-blood animal, they can’t get with the rapid temperature change. For example, milkfish (Chanos chanos) dies for two major reasons. The first one is dramatical temperature decrease. The second one is vibrios infection. If the temperature stays low in about 10 degree, the mucosa on the fish body will peel off and cause milkfish to die for vibrios infection. </p><p>Fish farmers currently pump the groundwater to warm up the pound but it will damage the stratum. On the other hand, they build up wind shields and dig deeper pounds to resist the cold wind, but it can only increase about 2-3 degree. In addition, some engineers try to heat up the water by electricity, however, fish farmers can‘t afford the expenses, The method is not realistic. Fish farmers are in passive position because no one knows whether the fish can survive in this time or not. It just likes a gambling, they can only fish the fish before the coming of cold current. Besides Taiwan, Japanese fish farmers also have this problem. The farming fishers in Japan heat up the water by hot water from nuclear power plant. Lack of this heating source brought huge loss in Japanese farming fish business. In May, 2012, they lost 47% output of white trevally and 35% output of shellfish in Fukui Prefecture.</p><p> To sum up, we want to solve this problem by using a brand new method called synthetic biology. We want to make a device to slow down the decreasing of temperature and keep water in a specific temperature. It will be helpful in lessening the death of fish. Our goal is to make a device which can heat up the water in low temperature. | There are 4 farming fishing among top 15 fishing output in Taiwan. The output value of farming fish is second only to deep sea fishing. Unfortunately, in winter, we see news about large amount of fish died due to low temperature. In winter, cold current which comes from the Mongolia dramatically decreases the temperature and causes fish to die. As you know that fish is cold-blood animal, they can’t get with the rapid temperature change. For example, milkfish (Chanos chanos) dies for two major reasons. The first one is dramatical temperature decrease. The second one is vibrios infection. If the temperature stays low in about 10 degree, the mucosa on the fish body will peel off and cause milkfish to die for vibrios infection. </p><p>Fish farmers currently pump the groundwater to warm up the pound but it will damage the stratum. On the other hand, they build up wind shields and dig deeper pounds to resist the cold wind, but it can only increase about 2-3 degree. In addition, some engineers try to heat up the water by electricity, however, fish farmers can‘t afford the expenses, The method is not realistic. Fish farmers are in passive position because no one knows whether the fish can survive in this time or not. It just likes a gambling, they can only fish the fish before the coming of cold current. Besides Taiwan, Japanese fish farmers also have this problem. The farming fishers in Japan heat up the water by hot water from nuclear power plant. Lack of this heating source brought huge loss in Japanese farming fish business. In May, 2012, they lost 47% output of white trevally and 35% output of shellfish in Fukui Prefecture.</p><p> To sum up, we want to solve this problem by using a brand new method called synthetic biology. We want to make a device to slow down the decreasing of temperature and keep water in a specific temperature. It will be helpful in lessening the death of fish. Our goal is to make a device which can heat up the water in low temperature. | ||
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Collaborate with Perdue iGEM team: <br/><br/> | Collaborate with Perdue iGEM team: <br/><br/> | ||
<p>We collaborated with Perdue iGEM team to design a better version of datasheet with them. We complete their questionnaire and provide some idea.</p> | <p>We collaborated with Perdue iGEM team to design a better version of datasheet with them. We complete their questionnaire and provide some idea.</p> | ||
| - | <p>Following is one of the beta-version of <b>Purdue iGEM team | + | <p>Following is one of the beta-version of <b>Purdue iGEM team's</b> datasheet. </p> |
<embed src="https://static.igem.org/mediawiki/igem.org/c/c7/Datasheet_Composite_Part_Example.pdf" style="width: 100%; height: 500px"></embed> | <embed src="https://static.igem.org/mediawiki/igem.org/c/c7/Datasheet_Composite_Part_Example.pdf" style="width: 100%; height: 500px"></embed> | ||
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<a href="http://lokeshdhakar.com/projects/lightbox2/">Lightbox</a><br /> | <a href="http://lokeshdhakar.com/projects/lightbox2/">Lightbox</a><br /> | ||
| - | Lightbox is small javascript library used to overlay images on top of the current page. It | + | Lightbox is small javascript library used to overlay images on top of the current page. It's a snap to setup and works on all modern browsers. |
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<p class="header"><i>Saccharomyces cerevisiae</i></p> | <p class="header"><i>Saccharomyces cerevisiae</i></p> | ||
</section> | </section> | ||
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<img class="img-responsive" src="https://static.igem.org/mediawiki/igem.org/e/ec/Sc_pic.jpg" width=500 alt-src="./images/LabTime_2/Sc_pic.jpg"> | <img class="img-responsive" src="https://static.igem.org/mediawiki/igem.org/e/ec/Sc_pic.jpg" width=500 alt-src="./images/LabTime_2/Sc_pic.jpg"> | ||
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<b>What is Saccharomyces cerevisiae?</b> | <b>What is Saccharomyces cerevisiae?</b> | ||
| - | <p><i><b>Saccharomyces cerevisiae</b></i> is a species of yeast. It is perhaps the most useful yeast, having been instrumental to winemaking, baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes (one can see the yeast as a component of the thin white film on the skins of some dark-colored fruits such as plums; it exists among the waxes of the cuticle). | + | <p> |
| - | + | <i><b>Saccharomyces cerevisiae</b></i> is a species of yeast. It is perhaps the most useful yeast, having been instrumental to winemaking, baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes (one can see the yeast as a component of the thin white film on the skins of some dark-colored fruits such as plums; it exists among the waxes of the cuticle). It is one of the most intensively studied eukaryotic model organisms in molecular and cell biology, much like Escherichia coli as the model bacterium. It is the microorganism behind the most common type of fermentation. S. cerevisiae cells are round to ovoid, 5–10 micrometres in diameter. It reproduces by a division process known as budding. | |
| - | + | </p> | |
| - | + | <p>Many proteins important in human biology were first discovered by studying their homologs in yeast; these proteins include cell cycle proteins, signaling proteins, and protein-processing enzymes.</p> | |
<p><h5>Reference: Wikipedia</h5></p> | <p><h5>Reference: Wikipedia</h5></p> | ||
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<img src="https://static.igem.org/mediawiki/2013/1/1e/NTU_TAIWAN_E_coli.jpg" alt-src="images/e_coli.jpg" class="pull-right img-responsive"> | <img src="https://static.igem.org/mediawiki/2013/1/1e/NTU_TAIWAN_E_coli.jpg" alt-src="images/e_coli.jpg" class="pull-right img-responsive"> | ||
| - | <div style="margin-top: 75px"><p>Most yeast expression vectors have been based on the multi-copy 2p plasmid and contain sequences for propagation in | + | <div style="margin-top: 75px"><p>The yeast <b>Saccharomyces cerevisiae</b> has several properties which have established it as an important tool in the expression of foreign protein for research, industrial or medical use. As a food organism, it is highly acceptable for the production of pharmaceutical proteins. In contrast,<b>Escherichia coli</b> have toxic cell wall pyroxenes and mammalian cells may contain oncogenic or viral DNA, so that products from these organisms must be tested hmore extensively.</p> |
| - | + | <p>Yeast can be grown rapidly on simple media and to high cell density and its genetics are more advanced than any other eukaryote, so that it can be manipulated almost as readily as E.coli. As a eukaryote, yeast is a suitable host organism for the High-level production of secreted as well as soluble cytosolic proteins.</p> | |
| + | <hr> | ||
| + | <p>Most yeast expression vectors have been based on the multi-copy 2p plasmid and contain sequences for propagation in E.coli and in yeast, as well as a yeast promoter and terminator for efficient transcription of the foreign gene. The recent rapid expansion in yeast molecular genetics has led to a great increase in our understanding of these components, and as a result there is now a bewildering choice of promoter systems and methods for propagating foreign DNA in yeast. In many cases ingenious new approaches have been employed, for example in increasing the strength of native promoters or the stability of expression vectors.</p> | ||
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| - | <p>Meanings of parameters will be explained in detail in the next paragraph. | + | <p>Meanings of parameters will be explained in detail in the next paragraph. To make it clearer, the difference between two models are highlighted. Another things to be careful is that some <b>assumptions</b> are made, as listed below, when formulating these mathematical equations |
</p> | </p> | ||
| + | <ol> | ||
| + | <li>a. All parameters except for βCsp(T) and βHsp(T) are not treated as functions of temperature.</li> | ||
| + | <li>b. Production of GFP occurs only when the repressor is not binding to the promoter region. At the same time, production of GFP is contributed from two factors: the basal expression from Csp and the transcription activation by an activator.</li> | ||
| + | <li>c. The repressor has the same effect on basal expression and transcription activation. That is to say, it knock down their activities by an equal ratio.</li> | ||
| + | <li>d. Binding of activators to the DNA results in a certain fold of increase in protein expression relative to the basal expression.</li> | ||
| + | </ol> | ||
</li> | </li> | ||
<li><b>Parameters</b> | <li><b>Parameters</b> | ||
<p class="container"> | <p class="container"> | ||
| - | + | Parameters in our model are separated into two groups: fixed parameters and adjustable parameters. Fixed parameters are assigned values that are reasonable in biological context after considering related parameters in references. Adjustable parameters are parameters whose ranges of values with application potentiality are to be determined by simulation where we will scan all the possible values in biological contexts (Table 1). | |
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<table class="row tipReveal"> | <table class="row tipReveal"> | ||
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<td class="col-md-2">β<sub>A</sub></td> | <td class="col-md-2">β<sub>A</sub></td> | ||
<td class="col-md-5">Fold of activation by activator</td> | <td class="col-md-5">Fold of activation by activator</td> | ||
| - | <td class="col-md-3"> | + | <td class="col-md-3">2~200</td> |
<td class="col-md-2">M/s</td> | <td class="col-md-2">M/s</td> | ||
</tr> | </tr> | ||
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<section class="brown-background"> | <section class="brown-background"> | ||
| - | <h1 class="header">Result | + | <h1 class="header">Result & Discussion</h1> |
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| + | <legend><b>Fitting results of βCsp(T) and βHsp(T) show no overlapping range of promoter activity.</b></legend> | ||
| + | <p> | ||
| + | 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> | ||
| + | <img class="tipReveal row" src="images/modeling/fittingresult1.jpg"> | ||
| + | <div class="tip"> Fig. 2: Fitting result of βCsp(T). </div> | ||
| + | <img class="tipReveal row" src="images/modeling/fittingresult2.jpg"> | ||
| + | <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> | ||
<p> | <p> | ||
| - | + | 為了實際了解Hsp或cons在circuit中扮演的功能，我們利用求解穩定態的方式來分析GFP在被Hsp 或cons驅動的repressor調控下，會 與單純由Csp所驅動表現會有什麼差異，進而借此了解是否能透過迴圈設計來達到一個形同改變csp靈敏度的效應。在結果分析上，我們選用GFP maximal conc和T 1/2 GFP max作為評估CSP靈敏度的指標，由於GFP max越大，表示csp在低溫時的訊號越能被我們所觀察到；另一方面，T 1/2 GFP max越低時，代表csp對溫度敏感的範圍變得更小更靈敏。結果顯示，在cons的model中，兩個指標不僅沒有改善的狀況，反而在aR變小時，大幅壓制Csp的活性。另外在Hsp model中，由於Hsp所能表現的溫度範圍和Csp無法重疊，因此如我們上一節所推測的，Hsp完全無法壓低GFP的表現。 | |
</p> | </p> | ||
| - | </div> | + | <img class="tipReveal" 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> | ||
</script> | </script> | ||
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<h1 class="header">Project Result</h1> | <h1 class="header">Project Result</h1> | ||
</section> | </section> | ||
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| - | + | <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="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> | ||
</div> | </div> | ||
| - | <div class=" | + | <div class="green-background row"> |
| - | + | <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> | |
| + | <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> | ||
| + | </div> | ||
</div> | </div> | ||
Revision as of 03:05, 28 September 2013
