Team:NTU Taiwan/index.html

     <title> Igem-Taiwan </title>

                             <li> <a href="#Background"> Background </a> </li>

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             <h1 class=" rainbow-text header">IGem-Taiwan Yeastherm</h1>

                 Working with Thermogenic Yeast<br/>

                 Apps with knowledge of iGEM competition and synthetic biology.

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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.

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

                 <div class="row text-center"><h3>Let's go!</h3></div>

                 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 pound environment in reality by computer and the test results after using our device 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&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. 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. 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.

 

             Shuttle vector is a vector that can propagate in different species, used to make gene amplifications quickly in E. coli, mutagenesis, and PCR. Generally, these plasmid vectors contain genetic material derived from the E.coli, origin of replication which enable them to be propagated in E.coli cells prior to transformation into yeast cells, and a selectable marker (mainly the ß-lactamase gene, amp) for the bacterial host.  

             The most common shuttle vector is yeast shuttle which can be propagated in yeast and E.coli.There are four types of shuttle vectors. The first one is Integrative plasmids (YIp). Foreign DNAs integrate into the host genome(YIp) by homologous recombination, then resulting in one copy of transformed DNA. The second type is Episomal plasmids (YEp). This type of vectors carry part of 2μ plasmid DNA sequence necessary for autonomous replication. Multiple copies of the transformed plasmid are propagated in the yeast cell and maintained as episomes. The next one is Autonomously replicating plasmids (YRp) which carry a yeast origin of replication (ARS sequence) that allows the transformed plasmids to be propagated several hundredfold. The last one is Cen plasmids (YCp). This type of shuttle vectors carry an ARS sequence and a centromeric sequence which normally guarantees stable mitotic segregation and reduces the copy number of self-replicated plasmid to just one. Here, we choose Episomal plasmids (YEp) as our vectors.

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

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                            <p>We help Kyoto iGEM team to charaterise their parts. They sent 13 parts to us. First, we transformed their parts into <i>E.coli</i> and do sequence. Then We keep discussing how to design the construction and which gene should we use in the characterisation.</p>

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                            <p>Because we are in the same university, we exchange a lot of information to each other. For expample, they shared the experience in iGEM compeitiotn to us and we help them conduct some experiment like preparing competent cells and transformation.</p>

                            <p>In addition, we help each other to charaterise the parts. For example, they help us to check the expression of SrUCP protein(BBa_K1125000).</p>

                            <p>We also help them in the part "BBa_K1157013". This part is very important, because this biosensor is constructed to sense C-4 AHL molecules through a method different from BBa_K1157012. This time when AHL molecules are sensed, it activates Rhl promoter, which initiates the production of CI. Therefore, CI inhibits pCI and the expression of reporter mCherry is stopped. When compared to control group, the results will be expected to express lower fluorescence level when there is existence of AHL molecules.</p>

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

             <p class="header"><i class="icon-paper-clip"> our works</i></p>

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

        <b>

            Do the biological materials used in your lab work pose any risks to…</b><br/><br/>

            <ul style="list-style:style"><b>

                <li> -the safety and health of team members or others working in the lab? <br/> -the safety and health of the general public, if released by design or by accident?</li>

                <ul></b>

                    <li> Here is a list of the chassis organisms that we use in our project : <i>Escherichia coli</i>, <i>Saccharomyces cerevisiae</i>, and <i>Rhodotorula glutinis</i>. These are all Biosafety Level 1 organisms and non-pathogenic, therefore pose no severe threat to the researchers or any healthy human. </li>

                </ul><b>

                <li> -the environment, if released by design or by accident?</li>

                <ul></b>

                    <li>For the same reasons above, there would be minimum chance of the organisms causing any great harm to the environment. Our <i>E. coli</i> transformants do carry an ampicillin resistant gene, so accidental release of the bacteria might result in a spread of the antibiotic resistant phenotype. However, all our experiments are conducted under safe procedures and the equipment used for bacteria cells and/or cultures are properly autoclaved. </li>

                </ul><b>

                <li> -security through malicious misuse by individuals, groups, or countries? </li>

                <ul></b>

                    <li> Unless high density of cell culture is spread out, no potential risk is present. </li>

                </ul>

            </b></ul>

        <b>If your project moved from a small-scale lab study to become widely used as a commercial/industrial product, what new risks might arise?</b><br/><br/>

        <p style="margin-left: 40px">Respecting our goals to prevent the damage caused by cold temperature in fish farming:</p>

        <p style="margin-left: 80px">

        1.  Contact with high density of cells may cause harmful effects on people.<br/>

        &nbsp &nbsp2.  Release of cell culture into the environment may cause fish infection and perturbation of the ecosystem.<br/></p>

        <b>Does your project include any design features to address safety risks?</b><br/>

        <br/>

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        <img class="pull-right img-responsive" src="https://static.igem.org/mediawiki/igem.org/5/50/Suicide.jpg" alt-src="./images/suicide.jpg" style="display: block; margin: 10px;" width=500>

        <div class="tip">

        (fig.3 Overview of the HOG pathway in S. cerevisiae. Several transcriptional factors are regulated.)</div>

        </div>

        <p style="margin-left: 40 px">In case of an accidental release of the thermogenic yeasts from our device to the environment, we have designed a kill switch that would ideologically lead the yeasts to death under such circumstance.<br/>

        When faced with an increasing osmolarity of the environment, the HOG pathway is activated in yeasts and the final result is the accumulation of glycerol in yeast cells to balance the exterior osmotic pressure. Sensors of the ambient osmolarity rise activates a MAPK cascade and eventually leads to the phosphorylation and activation of the Hog1 protein. Activated Hog1 then translocates into the nucleus and activates a number of transcriptional factors via protein-protein interactions or phosphorylation.(fig.3) These transcriptional factors (mostly activators) then mediate the expression of hundreds of genes related to cell integrity and adaptation to osmostress. Among these, the GPD1 gene and the STL1 gene are the most significant targets of the HOG pathway. GPD1 encodes the sequences for NAD-dependent glycerol-3-phosphate dehydrogenase, which is the key enzyme of glycerol synthesis. Following activation of the HOG pathway, activated Hog1 binds to the transcriptional activator Hot1 and upregulates the expression of GPD1. On the other hand, STL1 codes for a glycerol/proton symporter in the plasma membrane of S. cerevisiae. Upon sensing a rise in osmolarity, STL1 is strongly and transiently induced by transcriptional activators Hot1 and Smp1, both members of the HOG pathway. Hot1 activation is as mentioned above, and Smp1 is phosphorylated and activated by the active Hog1 protein. We thus utilize, the sensing of osmolarity and the induction of GPD1 and STL1 expression in yeasts to make up the first part of our kill switch.<br/>

        &nbsp&nbsp&nbsp&nbsp&nbspIn order to complete our kill switch so that increasing osmolarity not only activates the HOG pathway, but also leads to cell death, we further integrate a kill gene following the promoter sequence of GPD1 or STL1. The most suitable genes would be those encoding proteins that have nuclease activity. A couple of chosen examples are NUC1 (encoding endonuclease G) and YBL055C (encoding Tat-D nuclease). Endonuclease G is the major mitochondrial nuclease in S. cerevisiae, and it induces apoptosis in yeast independently of metacaspase or of apoptosis inducing factors. Tat-D is an endo-/exo-nuclease that incises the double stranded DNA without obvious specificity via its endonuclease activity and excises the DNA from 3' to 5' end by its exonuclease activity. These proteins are intrinsically expressed during apoptosis. By placing the genes downstream of the GDP1 or STL1 promoter, their expression will be induced under increasing osmolarity and cause irreversible harm to the yeasts, in the end killing them.<br/>

        &nbsp&nbsp&nbsp&nbsp&nbspAccording to data from current milkfish farms in Taiwan, which are saltwater farms, water osmolarity is way higher than yeast culturing environments. Therefore the HOG pathway would surely be activated once the yeasts escape from the thermogenic device, and with our design of kill switch, cell death follows. If the device is to be used in a fish farm with fresh water, the osmolarity would very likely be lower than the yeast culture. In light of this possibility, we are also looking into another mechanism of S. cerevisiae that is used when it is subjected to low osmolarity stress. It is called the cell integrity pathway, and is activated upon decreasing osmolarity of the environment. We hope to find similar functioning effectors downstream of the pathway like we did in the HOG pathway, and integrate the activated promoters with kill genes. If succeeded, our safety design will not be restricted to saltwater fish farms.<br/></p>

        <h5>Resource: <br/>

        Overview of HOG pathway<br/>

        Microbiol Mol Biol Rev. 2002 Jun;66(2):300-72.<br/>

        Osmotic stress signaling and osmoadaptation in yeasts. by Hohmann S.</p></h5>

 

        <b>What safety training have you received?</b><br/><br/>

        <p style="margin-left: 40px">Every student who worked in the lab have had to receive a 12-hour training. The training includes lectures on the topics &#34;Principal of Biosafety&#34; and &#34;Management of Biosafety in Laboratories&#34;.</p><br/>

        <b>Does your institution have an Institutional Biosafety Committee, or an equivalent group? If yes, have you discussed your project with them? Describe any concerns they raised with your project, and any changes you made to your project plan based on their review.</b><br/><br/>

        <p style="margin-left: 40px">We had an discussion with the National Taiwan University Environment Protection and Occupational Safety and Health Center. The assessment result was that we don&#39;t need to send the &#34;Recombinant gene experiment&#34; form to the Center, due to our materials all belonging to RG1.</p><br/>

        <b>Does your country have national biosafety regulations or guidelines? If so, please provide a link to these regulations or guidelines if possible.</b><br/><br/>

        <p style="margin-left: 40px">&#34; http://esh.ntu.edu.tw/epc/e-home.php &#34;<br/>

        This is also the biosafety guidelines that our institution follows.<br/>

        </p>

                 <p class="header" style="margin: 0"> our works </p>

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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). 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>

                    </div>

                    </div>

                 <p>The yeast <b><i>Saccharomyces cerevisiae</i></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><i>Escherichia coli</i></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 <b><i>E.coli</i></b>. 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>

                 <p>We use temperature changes as the main sensors. With a cold shock promoter prior to UCP genes, our constructed plasmid will produces heat when temperature decreasing dramatically.</p>

 

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            <img class="pull-right img-responsive" src="https://static.igem.org/mediawiki/igem.org/5/50/Suicide.jpg" alt-src="./images/suicide.jpg" style="display: block; margin: 10px;" width=500>

            <div class="tip">

            (fig.3 Overview of the HOG pathway in S. cerevisiae. Several transcriptional factors are regulated.)</div>

            </div>

            <p style="margin-left: 40 px">In case of an accidental release of the thermogenic yeasts from our device to the environment, we have designed a kill switch that would ideologically lead the yeasts to death under such circumstance.<br/>

            When faced with an increasing osmolarity of the environment, the HOG pathway is activated in yeasts and the final result is the accumulation of glycerol in yeast cells to balance the exterior osmotic pressure. Sensors of the ambient osmolarity rise activates a MAPK cascade and eventually leads to the phosphorylation and activation of the Hog1 protein. Activated Hog1 then translocates into the nucleus and activates a number of transcriptional factors via protein-protein interactions or phosphorylation.(fig.3) These transcriptional factors (mostly activators) then mediate the expression of hundreds of genes related to cell integrity and adaptation to osmostress. Among these, the GPD1 gene and the STL1 gene are the most significant targets of the HOG pathway. GPD1 encodes the sequences for NAD-dependent glycerol-3-phosphate dehydrogenase, which is the key enzyme of glycerol synthesis. Following activation of the HOG pathway, activated Hog1 binds to the transcriptional activator Hot1 and upregulates the expression of GPD1. On the other hand, STL1 codes for a glycerol/proton symporter in the plasma membrane of S. cerevisiae. Upon sensing a rise in osmolarity, STL1 is strongly and transiently induced by transcriptional activators Hot1 and Smp1, both members of the HOG pathway. Hot1 activation is as mentioned above, and Smp1 is phosphorylated and activated by the active Hog1 protein. We thus utilize, the sensing of osmolarity and the induction of GPD1 and STL1 expression in yeasts to make up the first part of our kill switch.<br/>

            &nbsp&nbsp&nbsp&nbsp&nbspIn order to complete our kill switch so that increasing osmolarity not only activates the HOG pathway, but also leads to cell death, we further integrate a kill gene following the promoter sequence of GPD1 or STL1. The most suitable genes would be those encoding proteins that have nuclease activity. A couple of chosen examples are NUC1 (encoding endonuclease G) and YBL055C (encoding Tat-D nuclease). Endonuclease G is the major mitochondrial nuclease in S. cerevisiae, and it induces apoptosis in yeast independently of metacaspase or of apoptosis inducing factors. Tat-D is an endo-/exo-nuclease that incises the double stranded DNA without obvious specificity via its endonuclease activity and excises the DNA from 3' to 5' end by its exonuclease activity. These proteins are intrinsically expressed during apoptosis. By placing the genes downstream of the GDP1 or STL1 promoter, their expression will be induced under increasing osmolarity and cause irreversible harm to the yeasts, in the end killing them.<br/>

            &nbsp&nbsp&nbsp&nbsp&nbspAccording to data from current milkfish farms in Taiwan, which are saltwater farms, water osmolarity is way higher than yeast culturing environments. Therefore the HOG pathway would surely be activated once the yeasts escape from the thermogenic device, and with our design of kill switch, cell death follows. If the device is to be used in a fish farm with fresh water, the osmolarity would very likely be lower than the yeast culture. In light of this possibility, we are also looking into another mechanism of S. cerevisiae that is used when it is subjected to low osmolarity stress. It is called the cell integrity pathway, and is activated upon decreasing osmolarity of the environment. We hope to find similar functioning effectors downstream of the pathway like we did in the HOG pathway, and integrate the activated promoters with kill genes. If succeeded, our safety design will not be restricted to saltwater fish farms.<br/></p>

            <h5>Resource: <br/>

            Overview of HOG pathway<br/>

            Microbiol Mol Biol Rev. 2002 Jun;66(2):300-72.<br/>

            Osmotic stress signaling and osmoadaptation in yeasts. by Hohmann S.</p></h5>

                <p class="header" style="margin: 0"></p>

                     <i><u>Rhodotorula</u></i> is pigment produce yeast quite easily identifiable by distinctive orange/red colonies when grown on SDA. This distinctive color is the result of pigments that the yeast creates to block out certain wavelengths of light that would otherwise be damaging to the cell. Colony color can vary from being cream colored to orange/red/pink or yellow.

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                    Thermogenic yeast relies on cultural media to grow and to generate heat. This way, the Yeastherm is kind of like burning media as fuel, which is expensive compared with other heating methods such as gas, coal, or electricity. To make our project a more competitive choice when considering large-scale heat production such as heating up a pound in the winter, it is necessary to reduce the cost of culturing yeast. Thanks to previous study in the field of biofuel, we have several solutions of cheaper substitutions of cultural medium contents.

                </p>

                 <p>

                    According to Jie Tao and his colleague’s study, agricultural and forestry residues can be used as an alternative of carbon source, taking advantage of <i><u>Rhodotorula glutinis’s</u></i> ability to assimilate xylose.[1] Agricultural residues such as rice straw and corn stalk are usually burned after harvest. Using these materials not only lower the expense but also benefits the environment. Raw materials like rice or wheat straw are first cut into pieces of appropriate size. They are then hydrolyzed using sulfuric acid with boiling water bath, turninig into hemicellulosic hydrolyzate. Saccharides can be obtained in supernatant after centrifugation and washings of the residue with hot water.

                 </p>

                     After expressing SrUCP in <i><u>Rhodotorula glutinis</u></i>, we will test for appropriate concentration for Agricultural residue extraction considering growth condition and heating power. The optimized heat power will then be used in large scale simulation as well as calculation of cost reduced. If time permitted, we will dig further into the component of the hemicellulosic hydrolyzate, as it is reviewed that there might be inhibiting compound.[2][3]<br/>

                 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.

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

                         在模型中，參數分為給定或是可變動的兩種。給定的參數是參考文獻資料後而給定符合生理意義的值；可變動的參數，則是考慮生理情況所可能出現的參數的範圍而設定可被模型掃描的數值範圍，並將討論適合我們選用的參數範圍

                             <td class="col-md-2">給定參數</td>

                             <td class="col-md-5">描述</td>

                             <td class="col-md-3">數值</td>

                             <td class="col-md-2">單位</td>

                             <td class="col-md-2">β_Csp(T)^a</td>

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

                             <td class="col-md-3">2e-6 ~ 2e-4</td>

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

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

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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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