Team:NTU Taiwan/index.html

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<head>

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<title> ~~Igem~~-Taiwan </title>

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<title> iGEM-NTU-Taiwan </title>

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</div>

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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>

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.

</p>

</section>

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</a>

</div>

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</a>

</div>

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</div>

<div> Motivation </div>

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~~</a>~~

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~~</div>~~

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~~<div class="col-md-3 index-item">~~

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~~<a class="pointer-cursor" scroll-to="Result">~~

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~~<div class="row">~~

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~~<span class="icon-stack icon-4x">~~

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~~<i class="icon-circle icon-stack-base font-green"></i>~~

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~~<i class="icon-beaker icon-light"></i>~~

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~~</span>~~

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~~</div>~~

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~~<div> Result </div>~~

</a>

</div>

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</section>

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<p>In this year, iGEM NTU_Taiwan team ~~aim to make~~ a ~~<b>~~biological ~~heater</b>~~ which can produce appropriate heat in low ~~temperature~~. The feature in this device is that it can ~~responce~~ to ~~different~~ temperature and produce heat in ~~identical level~~.

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<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>

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<p>What a crazy project! This biological device is really charming, isn't it? Let us show you our project! <br/>

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<p>What a crazy project! This biological device is really charming, isn't it? Let us show you our project! <br/>

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</p>

</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. ~~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.

</p>

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<p>

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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~~</p>~~

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~~</div>~~

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~~</div>~~

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~~<div id="Result">~~

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~~<section class="green-background">~~

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~~<h1 class="header">Result</h1>~~

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~~</section>~~

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~~<div class="container divide">~~

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~~<p>~~

</p>

</div>

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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>

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<p>Following is one of the beta-version of <b>Purdue iGEM team's</b> datasheet. </p>

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<p>Following is one of the beta-version of <b>Purdue iGEM team's</b> datasheet. </p>

</li>

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<li>

<a href="http://lokeshdhakar.com/projects/lightbox2/">Lightbox</a><br />

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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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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.

</li>

<li>

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<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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<p class="header"><i>Saccharomyces cerevisiae</i></p>

</section>

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<b>What is Saccharomyces cerevisiae?</b>

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<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).

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<p>

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~~<b>As protein expression system</b>~~

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<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.

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~~<p>The yeast <i>S. cerevisiae</i> 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, <i>E~~. ~~coli</i> 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~~.

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</p>

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~~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 <i>E.coli~~.</i> ~~As a eukaryote,~~ yeast ~~is a suitable host organism for the High-level production of secreted as well as soluble cytosolic~~ proteins.</p>

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<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>

</div>

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<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 ~~<i>~~E.coli~~</i>~~ and in yeast, as well as a yeast promoter and terminator for efficient transcription of the foreign gene. ~~This plasmid contains autonomously replicating sequence~~, ~~ARS, which can help plasmids reproduction with chromosome~~ DNA. ~~Two micron plasmids also~~ have ~~FLP sequence~~, ~~which can bind with FLP protein, cause FLP-mediated recombination.</p>~~

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<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>

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~~<p>Because~~ of the ~~properties~~ of ~~two micron plasmid, such as with high copy number, stable, we choose this plasmid to transform gene into yeast~~.</p>

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<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>

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<hr>

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<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>

</div>

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</div>

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~~<div id="Circuit">~~

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~~<section class="purple-background">~~

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~~<h1 class="header">Circuit</h1>~~

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~~</section>~~

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~~<div class="essay container divide">~~

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~~</div>~~

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~~</div>~~

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<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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</legend>

<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.

</p>

</div>

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</ul>

</div>

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<p>Meanings of parameters will be explained in detail in the next paragraph.~~The~~ difference between two models are highlighted.

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<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>

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<ol>

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<li>a. All parameters except for βCsp(T) and βHsp(T) are not treated as functions of temperature.</li>

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<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>

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<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>

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<li>d. Binding of activators to the DNA results in a certain fold of increase in protein expression relative to the basal expression.</li>

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</ol>

</li>

<li><b>Parameters</b>

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~~In the~~ model~~, the parameters~~ are ~~categorized~~ into two groups~~, constant~~ and ~~varible~~. ~~The group with constant~~ parameters ~~is given~~ after ~~we references the~~ references ~~for which meets the physiological significance~~.

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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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~~The other group with variables~~ are ~~assigned a range~~ of values ~~after~~ we ~~considered~~ the ~~Physiological conditions~~.

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</p>

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</tr>

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<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>

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<td class="col-md-2">M/s~~, normalized~~</td>

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</tr>

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<td class="col-md-5">Fold of activation by activator</td>

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</tr>

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<h1 class="header">Result</h1>

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<h1 class="header">Result & Discussion</h1>

</section>

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</div>

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<legend><b>Fitting results of βCsp(T) and βHsp(T) show no overlapping range of promoter activity.</b></legend>

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<p>

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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.

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</p>

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<div class="tip"> Fig. 2: Fitting result of βCsp(T). </div>

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<div class="tip"> Fig. 3: Fitting result of βHsp(T) </div>

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<legend><b>Neither Hsp nor constitutive promoter suits our purpose under this circuit structure.</b></legend>

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<p>

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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!

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</p>

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<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>

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<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>

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</div>

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<h1 class="header">~~Discussion~~</h1>

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</section>

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<p>

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<legend><b>consModel</b></legend>

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由於要達到我們所預期的功能，Hsp在對溫度的表現量上必須要有一定程度與Csp重疊，才能有抑制Csp在較高溫度時表現的效果。因此以一個較符合生理情況下，Csp的溫度敏感範圍大約落在10~20℃，然而Hsp的溫度敏感範圍則大約落在30℃以上，兩者並無重疊，因此在生物學上不太容易找到一個適當的Hsp作為此基因迴路的必要元件。這個問題我們將在後續部分進行改良。

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<pre>slopeHsp = 1.75;

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</p>

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slopeCsp = 1.25;

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</div>

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n = 2;

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gamma_A = 1e-2;

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gamma_GFP = 8.2e-3;

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gamma_R = 1e-2;

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ten = 10;

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beta_A = 200*1e-6;

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[lnalpha_A, lnalpha_R, temperature] = meshgrid((-12:0.2:-3), (-12:0.2:-3), (0:0.01:40));

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beta_Csp = 1e-6 ./ (1.0+exp(slopeCsp*(temperature-15)));

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beta_Cons = 1e-6;

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Ass = beta_Csp./gamma_A;

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Rss = beta_Cons./gamma_R;

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GFP = (beta_Csp.*(1 + beta_A*(Ass.^n./(Ass.^n+(ten.^lnalpha_A).^n)))) .* ((ten.^lnalpha_R).^n./((ten.^lnalpha_R).^n+Rss.^n)) ./ gamma_GFP;

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maxGFP = max(GFP, [], 3);

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lnGFP_MAX = log10(maxGFP);

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GFP_half_temp = zeros(46, 46);

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for i = 1:46

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for j = 1:46

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value = 0;

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temp = 0;

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for t = 2:4000

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if (GFP(i, j, t)-GFP(i, j, t-1)) < value

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value = GFP(i, j, t)-GFP(i, j, t-1);

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temp = t-1;

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end

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end

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GFP_half_temp(i,j) = temp;

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end

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end

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GFP_half_temp = GFP_half_temp./100;

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[x, y] = meshgrid([-12:0.2:-3]);

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surf(x,y,lnGFP_MAX,GFP_half_temp);

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h = colorbar;

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ylabel(h, 'degree Celsius');

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xlabel('alphaA (power of 10)');

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ylabel('alphaR (power of 10)');

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zlabel('GFP [] (power of 10)');

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axis([-Inf Inf -Inf Inf -20 -2 10 20]);

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saveas(h, 'cons', 'jpg');</pre>

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<legend><b>hspModel</b></legend>

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<pre>

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slopeHsp = 1.75;

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slopeCsp = 1.25;

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n = 2;

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gamma_A = 1e-2;

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gamma_GFP = 8.2e-3;

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gamma_R = 1e-2;

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ten = 10;

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beta_A = 200*1e-6;

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[lnalpha_A, lnalpha_R, temperature] = meshgrid((-12:0.2:-3), (-12:0.2:-3), (0:0.01:40));

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beta_Csp = 1e-6 ./ (1.0+exp(slopeCsp*(temperature-15)));

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beta_Hsp = 1e-6 ./ (1.0+exp(-slopeHsp*(temperature-33.5)));

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Ass = beta_Csp./gamma_A;

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Rss = beta_Hsp./gamma_R;

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GFP = (beta_Csp.*(1 + beta_A*(Ass.^n./(Ass.^n+(ten.^lnalpha_A).^n)))) .* ((ten.^lnalpha_R).^n./((ten.^lnalpha_R).^n+Rss.^n)) ./ gamma_GFP;

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maxGFP = max(GFP, [], 3);

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lnGFP_MAX = log10(maxGFP);

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GFP_half_temp = zeros(46, 46);

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for i = 1:46

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for j = 1:46

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value = 0;

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temp = 0;

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for t = 2:4000

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if (GFP(i, j, t)-GFP(i, j, t-1)) < value

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value = GFP(i, j, t)-GFP(i, j, t-1);

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temp = t-1;

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end

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end

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GFP_half_temp(i,j) = temp;

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end

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end

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GFP_half_temp = GFP_half_temp./100;

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[x, y] = meshgrid([-12:0.2:-3]);

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surf(x,y,lnGFP_MAX,GFP_half_temp);

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h = colorbar;

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ylabel(h, 'degree Celsius');

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xlabel('alphaA (power of 10)');

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ylabel('alphaR (power of 10)');

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zlabel('GFP [] (power of 10)');

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axis([-Inf Inf -Inf Inf -20 -2 10 20]);

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saveas(h, 'latest', 'jpg');</pre>

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</div>

</script>

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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>

Line 2,143:

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<h1 class="header">Project Result</h1>

</section>

-

+

-

+

-

<div class="col-md-4" style="margin-top: ~~200px~~"><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>

+

<h1 class="header"> Plasmid Construction</h1>

-

</div>

+

<p class="header">For characterization </h1>

+

+

+

+

<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>

+

+

+

<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>

+

+

+

<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>

+

+

+

+

<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="col-md-4" style="margin-top: ~~190px~~"><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 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>

+

</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>

+

-

~~</div>~~

+

-

+

-

~~<div class="container" style="margin-top: 20px">~~

+

-

~~<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 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>

-

+

-

+

<h1 class="header"> Characterize the biological part </h1>

-

<~~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~~>

+

<p class="header">Test the expression of SrUCP by Western blotting.</p>

-

<~~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>~~

+

-

+

<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>

-

+

-

<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>

+

</div>

-

+

-

<~~div~~ class="~~container~~" ~~style="margin~~-~~top: 20px"~~>

+

<p class="header"> Analyze the heat-production ability of transformant</p>

-

<~~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~~>

+

<p>For estimate the heat-production ability of the SrUCP in yeast. We built up a straight way to analyze it. </p>

-

~~</div>~~

+

<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).

-

<~~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>

+

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>

+

+

</div>

+

<p class="header"> Rhodotorula glutinis Growth curve</p>

+

+

+

+

<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>

+

</div>

+

</script>

@@ Line 1: / Line 1: @@
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-     <title> Igem-Taiwan </title>
+     <title> iGEM-NTU-Taiwan </title>
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-             <h1 class=" rainbow-text header">IGem-Taiwan Yeastherm</h1>
+             <h1 class=" rainbow-text header">iGEM-NTU-Taiwan YeasTherm</h1>
              <p class="header container">
                  <img class="spin" alt-src="images/LaboratoryLevels.png" src="/wiki/images/9/91/NTU_TAIWAN_LaboratoryLevels.png"><br/>
                  National Taiwan University<br/>
-                 Working with Thermogenic Yeast<br/>
+                 Working on Thermogenic Yeast<br/>
-                 Apps with knowledge of iGEM competition and synthetic biology.
+                 Apps with concept of iGEM competition and synthetic biology.
              </p>
          </section>
          <div class="container divide" id="index-icons">
              <div class="row text-center">
-                 <div class="col-md-3 index-item">
+                 <div class="col-md-4 index-item">
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+                 <div class="col-md-4 index-item">
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-                 <div class="col-md-3 index-item">
+                 <div class="col-md-4 index-item">
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                          </div>
                          <div> Motivation </div>
-                    </a>
-                </div>
-                <div class="col-md-3 index-item">
-                    <a class="pointer-cursor" scroll-to="Result">
-                        <div class="row">
-                            <span class="icon-stack icon-4x">
-                              <i class="icon-circle icon-stack-base font-green"></i>
-                              <i class="icon-beaker icon-light"></i>
-                            </span>
-                        </div>
-                        <div> Result </div>
                      </a>
                  </div>
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              </section>
              <div class="container divide">
-                 <p>In this year, iGEM NTU_Taiwan team aim to make a <b>biological heater</b> which can produce appropriate heat in low temperature. The feature in this device is that it can responce to different temperature and produce heat in identical level.
+                 <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>What a crazy project! This biological device is really charming, isn't it? Let us show you our project! <br/>
+                 <p>What a crazy project! This biological device is really charming, isn&#39;t it? Let us show you our project! <br/>
-                 <div class="row text-center"><h3>Let's go!</h3></div>
+                 <div class="row text-center"><h3>Let&#39;s go!</h3></div>
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              </div>
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              <div class="container divide">
                  <p class="col-md-8">
-                 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.
+                 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.
                  </p>
                  <div class="col-md-4">
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                  <p>
                  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&rsquo;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&lsquo;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.
-                </p>
-            </div>
-        </div>
-        <div id="Result">
-            <section class="green-background">
-                <h1 class="header">Result</h1>
-            </section>
-            <div class="container divide">
-                <p>
                  </p>
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                      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>Following is one of the beta-version of <b>Purdue iGEM team's</b> datasheet. </p>
+                     <p>Following is one of the beta-version of <b>Purdue iGEM team&#39;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>
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                      <li>
                          <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's a snap to setup and works on all modern browsers.
+                         Lightbox is small javascript library used to overlay images on top of the current page. It&#39;s a snap to setup and works on all modern browsers.
                      </li>
                      <li>
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              <h1 class="header" style="margin: 0">Basic Research</h1>
              <div class="container">
-                 <p class="header" style="margin: 0"> our works </p>
+                 <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>
                  <div id="beaker">
                      <span class="bubble"><span class="glow">&nbsp;</span></span>
@@ Line 1,760: / Line 1,738: @@
                  <p class="header"><i>Saccharomyces cerevisiae</i></p>
              </section>
-             <div class="row" style="margin-left: 70px; margin-right: 70px; margin-top: 30px; margin-bottom: 30px">
+             <div class="container essay divide">
                  <div class="pull-right col-md-5">
                      <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">
@@ Line 1,768: / Line 1,746: @@
                  <div class="row text-letf" style="margin-top: 20px">
                  <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>
-                <b>As protein expression system</b>
+                        <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>The yeast <i>S. cerevisiae</i> 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, <i>E. coli</i>  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>
-                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 <i>E.coli.</i> As a eukaryote, yeast is a suitable host organism for the High-level production of secreted as well as soluble cytosolic proteins.</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>
                  </div>
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              <div class="container divide">
                  <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 <i>E.coli</i> and in yeast, as well as a yeast promoter and terminator for efficient transcription of the foreign gene. This plasmid contains autonomously replicating sequence, ARS, which can help plasmids reproduction with chromosome DNA. Two micron plasmids also have FLP sequence, which can bind with FLP protein, cause FLP-mediated recombination.</p>
+                 <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>Because of the properties of two micron plasmid, such as with high copy number, stable, we choose this plasmid to transform gene into yeast.</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>
                  </div>
              </div>
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          </div>
-        <div id="Circuit">
-            <section class="purple-background">
-                <h1 class="header">Circuit</h1>
-            </section>
-            <div class="essay container divide">
-            </div>
-        </div>
          <div id="Suicide">
@@ Line 1,876: / Line 1,848: @@
              <h1 class="header">Applications</h1>
              <div class="container">
-                <p class="header" style="margin: 0"></p>
+               <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>
                  <div id="beaker">
                      <span class="bubble"><span class="glow">&nbsp;</span></span>
@@ Line 1,927: / Line 1,899: @@
              </legend>
              <p>
-                 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.
+                 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.
              </p>
          </div>
@@ Line 1,977: / Line 1,949: @@
                              </ul>
                          </div>
-                         <p>Meanings of parameters will be explained in detail in the next paragraph.The difference between two models are highlighted.
+                         <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>
+                        <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><b>Parameters</b>
                      <p class="container">
-                         In the model, the parameters are categorized into two groups, constant and varible. The group with constant parameters is given after we references the references for which meets the physiological significance.
+                         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).
-                        The other group with variables are assigned a range of values after we considered the Physiological conditions.
                      </p>
                      <table class="row tipReveal">
@@ Line 2,005: / Line 1,982: @@
                          </tr>
                          <tr class="row">
-                             <td class="col-md-2">β<sub>Csp</sub>(T)<sup>a</sup></td>
+                             <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-3">Sigmoidal curve <br/>(set 37℃=1e-6, 30℃=0) </td>
-                             <td class="col-md-2">M/s, normalized</td>
+                             <td class="col-md-2">M/s</td>
                          </tr>
                          <tr class="row">
@@ Line 2,037: / Line 2,014: @@
                              <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-3">2e-6 ~ 2e-4</td>
+                             <td class="col-md-3">2~200</td>
                              <td class="col-md-2">M/s</td>
                          </tr>
@@ Line 2,065: / Line 2,042: @@
          <section class="brown-background">
-             <h1 class="header">Result</h1>
+             <h1 class="header">Result & Discussion</h1>
          </section>
          <div class="container essay divide">
-         </div>
+            <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="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>
+            <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>
+            <legend><b>Neither Hsp nor constitutive promoter suits our purpose under this circuit structure.</b></legend>
+            <p>
+                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>
+            <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="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>
+            <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>
-         <section class="yellow-background">
+         <section class="brown-background">
-             <h1 class="header">Discussion</h1>
+             <h1 class="header">Code</h1>
          </section>
          <div class="container essay divide">
-             <p>
+             <legend><b>consModel</b></legend>
-                 由於要達到我們所預期的功能，Hsp在對溫度的表現量上必須要有一定程度與Csp重疊，才能有抑制Csp在較高溫度時表現的效果。因此以一個較符合生理情況下，Csp的溫度敏感範圍大約落在10~20℃，然而Hsp的溫度敏感範圍則大約落在30℃以上，兩者並無重疊，因此在生物學上不太容易找到一個適當的Hsp作為此基因迴路的必要元件。這個問題我們將在後續部分進行改良。
+            <pre>slopeHsp = 1.75;
-             </p>
+slopeCsp = 1.25;
-         </div>
+n = 2;
+gamma_A = 1e-2;
+gamma_GFP = 8.2e-3;
+gamma_R = 1e-2;
+ten = 10;
+beta_A = 200*1e-6;
+[lnalpha_A, lnalpha_R, temperature] = meshgrid((-12:0.2:-3), (-12:0.2:-3), (0:0.01:40));
+beta_Csp = 1e-6 ./ (1.0+exp(slopeCsp*(temperature-15)));
+beta_Cons = 1e-6;
+Ass = beta_Csp./gamma_A;
+Rss = beta_Cons./gamma_R;
+GFP = (beta_Csp.*(1 + beta_A*(Ass.^n./(Ass.^n+(ten.^lnalpha_A).^n)))) .* ((ten.^lnalpha_R).^n./((ten.^lnalpha_R).^n+Rss.^n)) ./ gamma_GFP;
+maxGFP = max(GFP, [], 3);
+lnGFP_MAX = log10(maxGFP);
+GFP_half_temp = zeros(46, 46);
+for i = 1:46
+    for j = 1:46
+        value = 0;
+        temp = 0;
+        for t = 2:4000
+            if (GFP(i, j, t)-GFP(i, j, t-1)) < value
+                 value = GFP(i, j, t)-GFP(i, j, t-1);
+                temp = t-1;
+             end
+        end
+        GFP_half_temp(i,j) = temp;
+    end
+end
+GFP_half_temp = GFP_half_temp./100;
+[x, y] = meshgrid([-12:0.2:-3]);
+surf(x,y,lnGFP_MAX,GFP_half_temp);
+h = colorbar;
+ylabel(h, 'degree Celsius');
+xlabel('alphaA (power of 10)');
+ylabel('alphaR (power of 10)');
+zlabel('GFP [] (power of 10)');
+axis([-Inf Inf -Inf Inf -20 -2 10 20]);
+saveas(h, 'cons', 'jpg');</pre>
+            <legend><b>hspModel</b></legend>
+            <pre>
+slopeHsp = 1.75;
+slopeCsp = 1.25;
+n = 2;
+gamma_A = 1e-2;
+gamma_GFP = 8.2e-3;
+gamma_R = 1e-2;
+ten = 10;
+beta_A = 200*1e-6;
+[lnalpha_A, lnalpha_R, temperature] = meshgrid((-12:0.2:-3), (-12:0.2:-3), (0:0.01:40));
+beta_Csp = 1e-6 ./ (1.0+exp(slopeCsp*(temperature-15)));
+beta_Hsp = 1e-6 ./ (1.0+exp(-slopeHsp*(temperature-33.5)));
+Ass = beta_Csp./gamma_A;
+Rss = beta_Hsp./gamma_R;
+GFP = (beta_Csp.*(1 + beta_A*(Ass.^n./(Ass.^n+(ten.^lnalpha_A).^n)))) .* ((ten.^lnalpha_R).^n./((ten.^lnalpha_R).^n+Rss.^n)) ./ gamma_GFP;
+maxGFP = max(GFP, [], 3);
+lnGFP_MAX = log10(maxGFP);
+GFP_half_temp = zeros(46, 46);
+for i = 1:46
+    for j = 1:46
+        value = 0;
+        temp = 0;
+        for t = 2:4000
+            if (GFP(i, j, t)-GFP(i, j, t-1)) < value
+                value = GFP(i, j, t)-GFP(i, j, t-1);
+                temp = t-1;
+            end
+        end
+        GFP_half_temp(i,j) = temp;
+    end
+end
+GFP_half_temp = GFP_half_temp./100;
+[x, y] = meshgrid([-12:0.2:-3]);
+surf(x,y,lnGFP_MAX,GFP_half_temp);
+h = colorbar;
+ylabel(h, 'degree Celsius');
+xlabel('alphaA (power of 10)');
+ylabel('alphaR (power of 10)');
+zlabel('GFP [] (power of 10)');
+axis([-Inf Inf -Inf Inf -20 -2 10 20]);
+saveas(h, 'latest', 'jpg');</pre>
+         </div>
      </script>
@@ Line 2,086: / Line 2,162: @@
          <div class="container">
-         <br/><p><b>PCR</b></p><br/>
+         <br/><p><b>PCR</b></p>
-        &nbsp&nbsp&nbsp&nbsp&nbspStep 1: Design of appropriate forward and reverse primers<br/>
+: Design of appropriate forward and reverse primers<br/>
-        &nbsp&nbsp&nbsp&nbsp&nbspStep 2: Prepare our template<br/>
+: Prepare our template<br/>
-        &nbsp&nbsp&nbsp&nbsp&nbspStep 3: Prepare the PCR mix. (Kapa Hifi PCR kit.)<br/>
+: Prepare the PCR mix. (Kapa Hifi PCR kit.)<br/>
-        &nbsp&nbsp&nbsp&nbsp&nbspStep 4: Run PCR<br/>
+: Run PCR<br/>
-        &nbsp&nbsp&nbsp&nbsp&nbspStep 5: Examine the results by electrophoresis<br/>
+: 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/>
+<br/><p><b>Construction of our parts</b></p><p>
-        &nbsp&nbsp&nbsp&nbsp&nbspStep 1: We design primers for parts with prefix and suffix.<br/>
+: We design primers for parts with prefix and suffix.<br/>
-        &nbsp&nbsp&nbsp&nbsp&nbspStep 2: Perform PCR and cleanup the PCR product<br/>
+: Perform PCR and cleanup the PCR product<br/>
-        &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/>
+: Before insert our parts into standard backbone, pSB1C3, we perform RE digestion to make sticky ends of both inserts and backbones.<br/>
-        &nbsp&nbsp&nbsp&nbsp&nbspStep 4: Ligation of inserts and backbones<br/>
+: Ligation of inserts and backbones<br/>
-        &nbsp&nbsp&nbsp&nbsp&nbspStep 5: Transform our ligation products into DH5α and streak the transformed DH5α on LB agar plate with chloramphenicol.<br/>
+: Transform our ligation products into DH5α and streak the transformed DH5α on LB agar plate with chloramphenicol.<br/>
-        &nbsp&nbsp&nbsp&nbsp&nbspStep 6: Inoculate single colony into broth with chloramphenicol.<br/>
+: Inoculate single colony into broth with chloramphenicol.<br/>
-        &nbsp&nbsp&nbsp&nbsp&nbspStep 7: Miniprep the plasmid DNA from the overnight broth culture.<br/>
+: Miniprep the plasmid DNA from the overnight broth culture.<br/>
-        &nbsp&nbsp&nbsp&nbsp&nbspStep 8: Confirm the products by both RE digestion and PCR sequencing<br/>
+: Confirm the products by both RE digestion and PCR sequencing<br/></p>
          <p><b>Point mutation protocol</b></p>
@@ Line 2,143: / Line 2,219: @@
              <h1 class="header">Project Result</h1>
          </section>
-         <div class="container" style="margin-top: 20px">
-             <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=700>
+         <div class="red-background row">
-            <div class="col-md-4" style="margin-top: 200px"><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>
+            <h1 class="header"> Plasmid Construction</h1>
-        </div>
+             <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="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 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>
+                <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>
+                <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">
+                <div class="container" style="margin-top: 20px">
-        <img class="pull-left img-responsive" src="https://static.igem.org/mediawiki/2013/9/99/Backbone.png" alt-src="./images/result/backbone.png" width=700>
+                    <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: 190px"><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 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>
+                </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>
-        </div>
-        <div class="container" style="margin-top: 20px">
-        <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 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 class="yellow-background row">
-         <div class="container" style="margin-top: 20px">
+            <h1 class="header"> Characterize the biological part  </h1>
-        <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>
+            <p class="header">Test the expression of SrUCP by Western blotting.</p>
-        <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 class="container" style="margin-top: 20px">
-        </div>
+                <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 class="container" style="margin-top: 20px">
+                </div>
-            <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 class="container" style="margin-top: 20px">
+            <p class="header"> Analyze the heat-production ability of transformant</p>
-        <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="container essay">
-        <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>
+                <p>For estimate the heat-production ability of the SrUCP in yeast. We built up a straight way to analyze it. </p>
-        </div>
+                <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/>
-        <div class="container" style="margin-top: 20px">
+                    Experimental: Heat(E) = Fermentation(1) + SrUCP<br/>
-        <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>
+                    Control: Heat(C) = Fermentation(2)<br/>
-        <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>
+                    Because the heat of fermentation(1) is lower than fermentation(2), even if SrUCP produce heat, the total Heat(E) equal to Heat(C).
-        <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>
+                    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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