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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>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>What a crazy project! This biological device is really charming, is'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/>
                 <div class="row text-center"><h3>Let's go!</h3></div>
                 <div class="row text-center"><h3>Let's go!</h3></div>
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                 In Taiwan, fish farmers lose a large amount of fish, because temperature falls dramatically when cold current comes in winter. 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.
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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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                 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.
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                 There are 4 farming fishing among top 15 fishing output in Taiwan. The output value of farming fish is second only to deep sea fishing. Unfortunately, in winter, we see news about large amount of fish died due to low temperature. In winter, cold current which comes from the Mongolia dramatically decreases the temperature and causes fish to die. As you know that fish is cold-blood animal, they can&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.
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             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.  
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             Shuttle vector is a vector that can propagate in different species, used to make gene amplifications quickly in <i>E. coli</i>, mutagenesis, and PCR.[1] Generally, these plasmid vectors contain genetic material derived from the <i>E.coli</i>, origin of replication which enable them to be propagated in <i>E.coli</i> cells prior to transformation into yeast cells.
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             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.
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             The most common shuttle vector is yeast shuttle which can be propagated in yeast and <i>E.coli</i>.There are four types of shuttle vectors.[2]
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            (1) Integrative plasmids (YIp) : Foreign DNAs integrate into the host genome(YIp) by homologous recombination, then resulting in one copy of transformed DNA.<br/>
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            (2) Episomal plasmids (YEp) : 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.<br/>
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            (3) Autonomously replicating plasmids (YRp) : carry a yeast origin of replication, ARS sequence, that allows the transformed plasmids to be propagated several hundredfold.<br/>
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            (4) Cen plasmids (YCp) : 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.<br/></p>
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            <p>Here, we choose Episomal plasmids (YEp) as our vectors.</p>
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            Reference:<br/>
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            [1] Transformation of yeast by a replicating hybrid plasmid. Beggs, J.D. Nature 275 (1978) 104-109.<br/>
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            [2] A System of Shuttle Vectors and Yeast Host Strains Designed for Efficient Manipulation of DNA in <i>Saccharomyces ceratisiae</i>. Robert S. Sikorski and Philip Hieter. Genetics 122: 19-27 (May, 1989).
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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>We are helping <a href="http://2013.igem.org/Team:Kyoto/Cooperation">Kyoto iGEM team</a> 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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             <h1 class="header">Attribution</h1>
             <p class="header"><i class="icon-paper-clip"> our works tools </i></p>
             <p class="header"><i class="icon-paper-clip"> our works tools </i></p>
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                 <legend><h2 style="border-bottom: 0">SrUCP</h2></legend>
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                 <legend><h2 style="border-bottom: 0">Wet Lab</h2></legend>
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                <p>All the work for this project was performed by the iGEM NTU_Taiwan 2013 team members, the lab work started from July 2013 onwards.</p>
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                <p>The original SrUCP gene was provided by Dr.Ito from Iwate University in Japan. The pRS424 and pRS423 shuttle vectors was provided by Dr. Jing-Jer Lin from Institute of Biochemistry and Molecular Biology of NTU. The pGAPZA plasmid was provided from Dr. Ching-Tsan Huang from the Department of Biochemical Science and Technology of NTU.</p>
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                <p>All of the following DNA were designed, cloned, or constructed by our wetlab team members:</p>
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                1. TAP protein tag was cloned from pRS424 shuttle vector.<br/>
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                2. Tir1 promoter sequence was amplified from genomic DNA of <i>Saccharomyces cerevisiae</i>.<br/>
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                3. 26s and 5.8s ITS rDNA was cloned from <i>Rhodotorula glutinis</i>.<br/>
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                4. All of the PCR primers of cloning were design by ourselves.<br/>
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                5. The construction of pRS424:GAL1:SrUCP:TAP, pGAPZA:26s, pGAPZA:5.8s ITS.<br/>
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                The idea of controlling the expression of UCP by cold-shock signal was developed by the team before starting our lab works. All of the experiments, the modeling, team wiki design, Android app were planned and carried out by the team.
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        <p><b>National Taiwan University</b></p>
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        <p>Our supervisor Dr. Yen-rong Chen for making our adventure in iGEM possible.</p>
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        <p>Dr. Jing-Jer Lin for providing us with the shuttle vectors, pRS424 and pRS423.</p>
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        <p>Dr. Ching-Hsuan Lin, Dr. Kai-Yin Lo and Dr. Jing-Jer Lin for their advice with the yeast molecular techniques.</p>
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        <p>Dr. Chao-Ping Hsu for her help with the modeling part of our project.</p>
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        <p>Dr. Kung-Ta Lee for his advice of how to measure the heat produce of YeasTherm.</p><br/>
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        <p><b>Institute of Molecular Biology, Academia Sinica</b></p>
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        <p>Lab N214 Dr. Michael M.C. Lai and Dr. King-Song Jeng for providing us with lab equipment and materials.</p><br/>
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        <p><b>Iwate University</b></p>
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        <p>Dr. Kikukatsu Ito for providing us the important gene, SrUCP DNA, for our project.<p><br/>
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                     <b>Does your project include any design features to address safety risks?</b><br/><br />
                     <b>Does your project include any design features to address safety risks?</b><br/><br />
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                             (fig.3 Overview of the HOG pathway in S. cerevisiae. Several transcriptional factors are regulated.)
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                             <h5>(fig.3 Overview of the HOG pathway in <i>S. cerevisiae</i>. Several transcriptional factors are regulated.)</h5>
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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>
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                <b>What is Saccharomyces cerevisiae?</b>
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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>
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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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                <b>As protein expression system</b>
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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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                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><h5>Reference: Wikipedia</h5></p>
                     <p><h5>Reference: Wikipedia</h5></p>
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                 <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>
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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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                <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>
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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>Most yeast expression vectors have been based on the multi-copy 2p plasmid and contain sequences for propagation in E.coli and in yeast, as well as a yeast promoter and terminator for efficient transcription of the foreign gene. The recent rapid expansion in yeast molecular genetics has led to a great increase in our understanding of these components, and as a result there is now a bewildering choice of promoter systems and methods for propagating foreign DNA in yeast. In many cases ingenious new approaches have been employed, for example in increasing the strength of native promoters or the stability of expression vectors. </p>
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                 <p>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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            <P><b>Cold shock promoter</b></p>
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                 <p>Cold shock promoter can overexpression protein during dramatically temperature decrease, for exsample, cspA promotor in <i>E. coli</i>, the most well-known cold shock promoter. But cold shock promotor have not been find in yeast, we choose sequence prior to one of the cold-shock protein, TIR1, as potential cold shock promoter.</p>
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            <p><b>What is <i>TIR1</i> gene ?</b></p>
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                <p>S. cerevisiae has rigid cell wall that protects the cells from mechanical injury, hypotonic lysis, and damaging extracellular enzymes. The cell well is mainly composed of P-glucan and mannoproteins. When the cell faces severe environment stresses, such as cold shock and anaerobiosis, it will express TIR gene family, producing cell wall mannoproteins.[1] Among the TIR family, TPS1, TPS2, HSP12, HSP26, HSP42, HSP104, YRO2, SSE2, Tip1, Tir1, and Tir2 genes are supposed to express after exposure to low temperature.[2] Tir1 and TPS1 are most studied gene fragments as yet.</i>
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                <p><b>Potential Cold Shock Promoter</b></p>
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                <p>Since <i>TPS1</i> is observed in cold-shock studies under near-freezing conditions, which is much lower than the low temperature ( 10℃) designed in our experiment, we choose <i>Tir1</i> promoter as our cold shock promoter.[3] <i>Tir1</i> promoter is induced by temperature decrease from 30℃to 10℃.[4] When exposing to low temperature environment (about 10~20℃), our plasmids start transcription, producing SrUCP. Once SrUCP is formed, they will generate heat to enhance environment temperature.</p>
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                <p>The most important thing is, owing to no certain cold shock promoter have been find in yeast, whether this sequence can be induce by low temperature, it have significance in yeast genetic engineering.</p>
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                Reference:<br/>
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                [1] Identification and analysis of a static culture-specific cell wall protein,Tirlp/Srplp in <i>Saccharomyces cerevisiae</i>. Hiroshi KITAGAKI, Hitoshi SHIMOI and Kiyoshi ITOH. Eur. J. Biochem. 249, 343-349 (1997).<br/>
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                [2] Acclimation of <i>Saccharomyces cerevisiae</i> to Low Temperature: A Chemostat-based Transcriptome Analysis. Siew Leng Tai, Pascale Daran-Lapujade, Michael C. Walsh,Jack T. Pronk,and Jean-Marc Daran. Molecular Biology of the Cell. Vol. 18, 5100–5112, December 2007<br/>
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                [3] Characterization and Regulation of the Trehalose Synthesis Pathway and Its Importance in the Pathogenicity of Cryptococcus neoformans. Elizabeth Wills Petzold, Uwe Himmelreich, Eleftherios Mylonakis, Thomas Rude, Dena Toffaletti, Gary M. Cox, Jackie L. Miller, and John R. Perfect. Infect Immun. 2006 October; 74(10): 5877–5887.<br/>
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                [4] Cold-shock induction of a family of TIP1-related proteins associated with the membrane in <i>Saccharomyces cerevisiae</i>. Leslie R. Z. Kowalski, Keiji Kondo^ and Masayori. Molecular Microbiology (1995) 15(2),341-353.<br/>
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                 <p>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.<p/>
                 <p>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.<p/>
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                 <p>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.<p/>
+
                <b>The HOG pathway</b>
 +
                 <p>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.</p>
 +
                <b>The GPD1 gene and the STL1 gene</b>
 +
                <p>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.<p/>
 +
                <b>Suicide mechanism</b>
                 <p>In 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.</p>
                 <p>In 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.</p>
 +
                <b>Use in milkfish farms</b>
                 <p>According 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.</p>
                 <p>According 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.</p>
              
              
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             <div class="container divide essay">
             <div class="container divide essay">
                 <img class="pull-right" src="http://igem.org/wiki/images/e/e2/Meeting-%2827%29.jpg" alt-src="./images/MEETING/Meeting-(27).jpg" width=500>
                 <img class="pull-right" src="http://igem.org/wiki/images/e/e2/Meeting-%2827%29.jpg" alt-src="./images/MEETING/Meeting-(27).jpg" width=500>
 +
                <p><b>What is <i>Rhodotorula glutinis</i> ?</b></p>
 +
                    <p><i><u>Rhodotorula</u></i> is an oleaginous yeast which is able to activate non-esterified fatty acids for the synthesis of triacylglycerol. <i>Rhodotorula</i> also a common environmental inhabitant. It can be cultured from soil, water, and air samples. It is able to scavenge nitrogenous compounds from its environment remarkably well, growing even in air which has been carefully cleaned of any fixed nitrogen contaminants. In such conditions, the nitrogen content of the dry weight of <i>Rhodotorula</i> can drop as low as 1%, compared to around 14% for most bacteria growing in normal conditions.</p>
 +
                <p><b>The oleaginous yeast</b></p>
                 <p>
                 <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.
+
                     The increasing cost of vegetable oils is turning the use of microbial lipids into a competitive alternative for the production of biodiesel fuel. The oleaginous yeast <i>R. glutinis</i> is able to use a broad range of carbon sources for lipid production, and is able to resist some of the inhibitors commonly released during hydrolysis of lignocellulose materials.</p>
-
                 </p>
+
                 <p><b>Why <i>Rhodotorula glutinis</i> ?</b></p>
                 <p>
                 <p>
-
                    <i><u>Rhodotorula</u></i> is a common environmental inhabitant. It can be cultured from soil, water, and air samples. It is able to scavenge nitrogenous compounds from its environment remarkably well, growing even in air which has been carefully cleaned of any fixed nitrogen contaminants. In such conditions, the nitrogen content of the dry weight of <i><u>Rhodotorula</u></i> can drop as low as 1%, compared to around 14% for most bacteria growing in normal conditions.
+
                 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>
+
                 <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>R. glutinis</i>’s 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>
-
                <p>
+
                  
-
                    The increasing cost of vegetable oils is turning the use of microbial lipids into a competitive alternative for the production of biodiesel fuel. The oleaginous yeast <i><u>Rhodotorula glutinis</u></i> is able to use a broad range of carbon sources for lipid production, and is able to resist some of the inhibitors commonly released during hydrolysis of lignocellulose materials. <br/>
+
                <p><b>Conclusion</b></p>
-
                </p>
+
-
                <p>
+
-
                    So we choose <i>R.g.</i> for the Expression system.
+
-
                </p>
+
-
                <p>
+
-
                    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>
+
                 <p>
                 <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/>
+
                     Because of the ability of <i>R. glutinis</i> to synthesis  triacylglycerol, we choose <i>R. glutinis</i> as one ofour expression system. After expressing SrUCP in <i>R. glutinis</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]
                 </p>
                 </p>
                     Reference:<br/>
                     Reference:<br/>
                     [1]Biodiesel generation from oleaginous yeast Rhodotorula glutinis with xylose assimilating capacity
                     [1]Biodiesel generation from oleaginous yeast Rhodotorula glutinis with xylose assimilating capacity
                     African Journal of Biotechnology Vol. 6 (18), pp. 2130-2134, 19 September 2007<br/>
                     African Journal of Biotechnology Vol. 6 (18), pp. 2130-2134, 19 September 2007<br/>
-
                    Available online at <a href="http://www.academicjournals.org/AJB">http://www.academicjournals.org/AJB</a><br/>
 
                     [2]Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid<br/>
                     [2]Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid<br/>
                     Bioresource Technology Volume 102, Issue 10, May 2011, Pages 6134–6140<br/>
                     Bioresource Technology Volume 102, Issue 10, May 2011, Pages 6134–6140<br/>
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         </section>
         <div class="container">
         <div class="container">
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+
       
 +
        <br/><p><b>PCR</b></p><br/>
 +
        &nbsp&nbsp&nbsp&nbsp&nbspStep 1: Design of appropriate forward and reverse primers<br/>
 +
        &nbsp&nbsp&nbsp&nbsp&nbspStep 2: Prepare our template<br/>
 +
        &nbsp&nbsp&nbsp&nbsp&nbspStep 3: Prepare the PCR mix. (Kapa Hifi PCR kit.)<br/>
 +
        &nbsp&nbsp&nbsp&nbsp&nbspStep 4: Run PCR<br/>
 +
        &nbsp&nbsp&nbsp&nbsp&nbspStep 5: Examine the results by electrophoresis<br/>
 +
        Note: If the template is genomic DNA, we would adjust the annealing temperature at 45°C. It is because the copy number of target gene may be low. We use this annealing temp when perform PCR of Tir1, 26s, 5.8s ITS
 +
 
 +
 
 +
<br/><p><b>Construction of our parts</b></p><br/>
 +
        &nbsp&nbsp&nbsp&nbsp&nbspStep 1: We design primers for parts with prefix and suffix.<br/>
 +
        &nbsp&nbsp&nbsp&nbsp&nbspStep 2: 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/>
 +
        &nbsp&nbsp&nbsp&nbsp&nbspStep 4: 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/>
 +
        &nbsp&nbsp&nbsp&nbsp&nbspStep 6: Inoculate single colony into broth with chloramphenicol.<br/>
 +
        &nbsp&nbsp&nbsp&nbsp&nbspStep 7: 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/>
 +
 
 +
        <p><b>Point mutation protocol</b></p>
 +
        <p>For a standard 100 @L reaction:<br/>
 +
        1, 10 pL of 10X reaction buffer, prepared as follows:<br/>
 +
        For 10 mL of 10 X PCR reaction buffer:<br/>
 +
        &nbsp&nbsp&nbsp&nbsp&nbspa. 0.5 mL of 1M KCl, final concentration of 50 mM.<br/>
 +
        &nbsp&nbsp&nbsp&nbsp&nbspb. 0.1 mL of 1M Tris-HCI, pH 8.4, final concentration of 10 mM.<br/>
 +
        &nbsp&nbsp&nbsp&nbsp&nbspc. 15 pL of 1M MgCl,, final concentration of 1.5 mM.<br/>
 +
        &nbsp&nbsp&nbsp&nbsp&nbspd. 100 pL of a 1% solution of gelatin (heated and dissolved in water), final concentratton 100 clg/mL.<br/>
 +
        2. 100-500 ng of each primer (0.2-l .O pL of a 500 pg/rnL stock).<br/><br/>
 +
       
 +
       
 +
        1. Whenever possible, design primers such that the G + C composition IS 50-60%. If the target DNA sequence is A + T rich, choose a primer with one or more G or C nucleotldes at or near the 3’ termim. This will stabilize the primer-template annealing at the end of the primer that will be extended.<br/>
 +
        2. Primer pairs should have approximately the same temperature of denaturation. The approximate temperature of denaturation (TJ for a primer may be calculated as follows: Td = 2[A + T] + 4[G + C] (20).<br/>
 +
3. Primers 17-30 nucleotldes in length work well. Since shorter primers cost less to synthesize, start with pnmers m the range of 18-22 nucleotldes.<br/>
 +
4. Determine that there is not extensive complementarity at the 3’ ends of a prrmer pair. Complementarrty ~111 cause the generatton of prtmerdrmer artifacts that ~111 reduce the yield of the desired product.<br/>
 +
5. An annealing temperature of 5 degrees below the Td will generally work well. Higher temperatures produce more strmgent annealing conditions and will decrease primer bmdmg to mismatched sequences.<br/>
 +
6. For most reactions the standard magnesium ion concentration of 1.5 mM works well. The magnesium concentration can affect both the product yield and the specificity of the reaction; the optimum will generally be in the range of 1.0-3.5 mM.<br/>
 +
7. Primer design is still an imperfect science. If after optrmizing the reaction conditions the product yield or reaction speclftcity remams poor, try a different primer pair.<br/>
 +
 
 +
        <p><b>Western blot analysis of SrUCP</b></p>
 +
        <p>Mutiple colonies of <i>Saccharomyces cerevisiae</i> strain BJ2168 carrying either pRS424-GAL1-SrUCP-TAP or pRS424-GAL1∆ were suspended in 2 mL SD+DO(Trp-) medium supplemented with 2% raffinose until O.D.600 = 3~5. Transfer 1 mL of suspended colonies into 50 mL SD+DO(Trp-) medium supplemented with 2% raffinose and incubate at 30℃ overnight. Dilute the overnight culture to O.D.600 = 0.3~0.8 with fresh SD+DO(Trp-) medium supplemented with 2% raffinose and grow for an additional 2 hr if O.D.600 is higher than 0.8. Before adding galactose and adjusting to 2% for induction, collect 2.5 O.D.600 of yeast cells. After induction, also collect 2.5 O.D.600 of yeast cells at certain time point. After all samples collected, total protein extraction was performed using post-alkaline extraction (Kushnirov, V.V., 2000). Treated samples were run on 10% SDS-PAGE. Transfer and Western blot were performed with normal procedures. SrUCP-TAP fusion protein was detected with primary anti-TAP antibody (Thermo #CAB1001) and secondary anti-rabbit IgG antibody.</p>
 +
       
 +
        <p><b>Functional analysis of SrUCP - Thermometry method</b></p>
 +
        <p>Single colony of Saccharomyces cerevisiae strain BJ2168 carrying either pRS424-GAL1-SrUCP-TAP or pRS424-GAL1∆ was inoculated into 2 mL SD+DO(Trp-) medium supplemented with 2% glucose and grown at 30℃ overnight. Overnight yeast culture was then inoculate into 10 mL same fresh medium and grown at 30℃ overnight. O.D.600 of overnight yeast culture was measured and calculated the cell amount needed for inoculation into 10 mL fresh SD+DO(Trp-) medium to O.D.600 = 2. The yeast cells needed is then collected, washed twice with 1mL distilled H2O, inoculated into medium and subject to 2% galactose induction. Meanwhile, a calibrated thermometer was placed into the yeast culture to collect its temperature profile along fermentation process until 24 hr.</p>
 +
       
 +
        <p><b>Functional analysis of SrUCP - Isothermal Titration Calorimetry method</b></p>
 +
        <p>Yeast culture was obtained as described in Western blot analysis of SrUCP. 2 mL of yeast culture was collected at each time point after induction. Yeast culture was then immediately analyzed by isothermal titration calorimetry (ITC), where yeast culture was placed in injection tube and sterile medium in the two cells. Heating power (mcal/s/O.D.600 of yeasts) of the yeast transformant is calculated from integration of the titration curve and normalized by time and O.D.600. SrUCP-responsible heat production was calculated from subtracting the heating power of the control group from that of the experimental group. Baseline of the titration curve was obtained from another control group where supernatant of yeast culture was injected into the same cell.</p><br/>
 +
       
 +
        Reference:<br/>
 +
        Kushnirov, V.V. Rapid and reliable protein extraction from yeast. Yeast 16, 857–860 (2000).
 +
 
         </div>
         </div>
     </script>
     </script>
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             <h1 class="header">Project Result</h1>
             <h1 class="header">Project Result</h1>
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         </section>
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         <div class="container" style="margin-top: 20px">
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             <img class="img-responsive" src="http://2013.igem.org/wiki/images/e/e2/NTU_TAIWAN_Website_Under_Construction.gif" alt-src="images/Website_Under_Construction.gif">
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             <img class="pull-right img-responsive" src="http://2013.igem.org/wiki/images/9/91/Ucp.png" alt-src="./images/result/ucp_1.png" width=700>
 +
            <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>
         </div>
         </div>
 +
       
 +
        <div class="container" style="margin-top: 20px">
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        <img class="pull-left img-responsive" src="http://2013.igem.org/wiki/images/9/99/Backbone.png" alt-src="./images/result/backbone.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>
 +
       
 +
        <div class="container" style="margin-top: 20px">
 +
        <img class="pull-right img-responsive" src="http://2013.igem.org/wiki/images/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="http://2013.igem.org/wiki/images/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="http://2013.igem.org/wiki/images/b/b1/Tir1-2.png" alt-src="./images/result/tir1-2.png" width= 500>
 +
                <div class="col-md-11" style="margin-top: 10px"><p>We predicted the Tir-1 promoter should be at about 1000 base pairs upstream, so we tried to amplified the Tir-1 promoter sequence from Saccharomyces cerevisiae by PCR. We design the primer with expanded restriction enzyme sites and about 30 base pairs complementary to the S.c. genome sequence, preventing from non-specific product. However, it’s harder to PCR a sequence from genomic DNA than plasmid. In hence, we tried different annealing temperature to make sure we have target product and decrease non-specific band.</p></div>
 +
       
 +
        </div>
 +
   
 +
        <div class="container" style="margin-top: 20px">
 +
        <img class="pull-right img-responsive" src="http://2013.igem.org/wiki/images/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="http://2013.igem.org/wiki/images/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>
 +
            </div>
 +
   
 +
        <div class="container" style="margin-top: 20px">
 +
        <img class="pull-right img-responsive" src="http://2013.igem.org/wiki/images/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??</p></div>
 +
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        <img class="pull-left img-responsive" src="http://2013.igem.org/wiki/images/a/a9/25.png" alt-src="./images/result/25.png" style="margin-top: 0px"width=530>
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        <div class="col-md-10" style="margin-top: 10px"><p>  For understanding the physical function of both strains in normal temperature and low temperature. We built up four growth curve.</p></div>
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Revision as of 23:24, 27 September 2013

Igem-Taiwan