Team:NTU Taiwan/index.html

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~~<!DOCTYPE html>~~

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

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

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<li> <a href="#" style="font-size: 18px"> NTU-Taiwan </a> </li>

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

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<li> <a href="#Background"><i class="icon-inbox"></i> Background </a> </li>

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<a class="dropdown-toggle pointer-cursor" data-toggle="dropdown">Project <b class="caret"></b></a>

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<a class="dropdown-toggle pointer-cursor" data-toggle="dropdown"><i class="icon-file-text"></i> Project <b class="caret"></b></a>

<li> <a href="#BasicResearch">Basic Research</a> </li>

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

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

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

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<li> <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=NTU_Taiwan">Parts</a></li>

<li> <a href="#ProjectResult">Result</a></li>

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<li><a href="#~~Modeling~~">~~Modeling~~</a></li>

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<li><a href="#~~Safety~~">~~Safety~~</a></li>

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<li><a href="#Safety"><i class="icon-warning-sign"></i> Safety</a></li>

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<li><a href="#~~humanpractice~~">~~Human practice~~</a></li>

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<li><a href="#~~teamCalendar~~"> ~~Team_Calendar~~ </a> </li>

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<a class="dropdown-toggle pointer-cursor" data-toggle="dropdown"><i class="icon-trophy"></i> Human practice <b class="caret"></b></a>

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

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

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

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<li> <a href="#Azalea_Festival">Azalea Festival</a> </li>

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<li> <a href="#Poster">Poster Presentation</a></li>

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<li> <a href="#teamMember">Team Member</a> </li>

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<li> <a href="#attribution">Attribution</a> </li>

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<li> <a href="#acknowledgement">Acknowledgement</a></li>

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<li><a href="#Calendar"> <i class="icon-calendar"></i>Calendar </a> </li>

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<li><a href="#Photos"> <i class="icon-instagram"></i>Photos </a> </li>

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<div>2013 © NTU-Taiwan.</div>

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<li><a href="https://www.facebook.com/NationalTaiwanUniversityBSTiGEM" target="_blank">Facebook</a></li>

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<li><a href="https://igem.org/Team.cgi" target="_blank">~~IGem~~ Website</a></li>

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<li><a href="https://igem.org/Team.cgi" target="_blank">iGEM Website</a></li>

<li><a href="mailto:igemntubest@gmail.com" target="_blank">Email Us</a></li>

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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.

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<div> Motivation </div>

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

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

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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.

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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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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. Of course, fish farmers try to prevent fish from dying. However, current methods do not work well and even cause damages to the environment. In 2013 iGEM competition, NTU_Taiwan team tries to make a bio-heating device. We transform an UCP homologue from themogenic plants into yeast. UCP is a thermogenic protein which can produce heat by interacting with the electron transport chain. By designing a genetic circuit, we want to well control the power of our bio-heating device. In addition, we want to simulate the effect of our device on fish ponds in reality after testing the heating power of our device.

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

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White and brown adipose tissues (WAT and BAT) serve important opposite functions in overall energy balance. While WAT is specialized in energy storage in the form of triacylglycerols, BAT functions to dissipate energy in the form of heat (thermogenesis).BAT possess abundant mitochondria with uncoupling proteins 1(UCP1). UCP1 is a 6-transmembrane protein in inner mitochondria membrane, and it has been proposed to constitute three symmetrical membrane-spanning “Us”, each comprising about 100 amino acids(fig.?).The functional UCP is believed to be dimer.

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White and brown adipose tissues (WAT and BAT) serve important opposite functions in overall energy balance. While WAT is specialized in energy storage in the form of triacylglycerols, BAT functions to dissipate energy in the form of heat (thermogenesis).BAT possess abundant mitochondria with uncoupling proteins 1(UCP1). UCP1 is a 6-transmembrane protein in inner mitochondria membrane, and it has been proposed to constitute three symmetrical membrane-spanning “Us”, each comprising about 100 amino acids(fig.1).The functional UCP is believed to be dimer.

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(fig.? The protein (33kDa,306 amino acids) contains six transmembrane alpha-helices, proposed to constitute three symmetric repeats.)

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(fig.1 The protein (33kDa,306 amino acids) contains six transmembrane alpha-helices, proposed to constitute three symmetric repeats.)

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<p>

Mitochondria uncoupling proteins 1(UCP1) are members of the family of mitochondrial anion carrier proteins (MACP). It facilitates the transfer of anions from the inner to the outer mitochondrial membrane and the return transfer of protons from the outer to the inner mitochondrial membrane. [2][3]UCPs separate oxidative phosphorylation from ATP synthesis with energy dissipated as heat, also referred to as the mitochondrial proton leak.

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UCP has been found in several species, including mammals and plants. Skunk cabbage(Symplocarpus foetidus)is a thermogenic plant, using srUCPs to resist in dramatic temperature decreases in the environment. In 1999, Dr. Kikukatsu Ito found that there are two types of SrUCPs, designated as SrUCPA and SrUCPB [4]. SrUCPA is proposed to be the functional thermogenic prtein, and SrUCPB serves as a regulator in thermogenesis in Skunk cabbage.[5][6] We use SrUCPA gene as our material to construct our plasmid.

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UCP has been found in several species, including mammals and plants. Skunk cabbage(Symplocarpus foetidus)is a thermogenic plant, using srUCPs to resist in dramatic temperature decreases in the environment. In 1999, Dr. Kikukatsu Ito found that there are two types of SrUCPs, designated as SrUCPA and SrUCPB [4]. SrUCPA is proposed to be the functional thermogenic prtein, and SrUCPB serves as a regulator in thermogenesis in Skunk cabbage.[5][6] We use SrUCPA gene as our material to construct our plasmid.<br/><br/>

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[1] Brown adipose tissue: function and physiological significance. Cannon B, Nedergaard J. Physiol Rev. 2004 Jan.

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Reference:<br/>

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[2] Uncoupling proteins: the issues from a biochemist point of view. Klingenberg M, Echtay KS. Biochim Biophys Acta. 2001 Mar 1;1504(1):128-43.

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[1] Brown adipose tissue: function and physiological significance. Cannon B, Nedergaard J. Physiol Rev. 2004 Jan.<br/>

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[3] Mitochondrial Uncoupling Proteins in Mammals and Plants. Jirı´ Borecky´, Ivan G. Maia, and Paulo Arruda. Bioscience Reports, Vol. 21, No. 2, April 2001 (2001).

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[2] Uncoupling proteins: the issues from a biochemist point of view. Klingenberg M, Echtay KS. Biochim Biophys Acta. 2001 Mar 1;1504(1):128-43.<br/>

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[4] Isolation of two distinct cold-inducible cDNAs encoding plant uncoupling proteins from the spadix of skunk cabbage (Symplocarpus foetidus). Kikukatsu Ito. Plant Science Vol. 149, Issue 2, 3 Dec. 1999, P.167–173.

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[3] Mitochondrial Uncoupling Proteins in Mammals and Plants. Jirı´ Borecky´, Ivan G. Maia, and Paulo Arruda. Bioscience Reports, Vol. 21, No. 2, April 2001 (2001).<br/>

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[5] Characterization of the plant uncoupling protein, SrUCPA, expressed in spadix mitochondria of the thermogenic skunk cabbage. Yasuko Ito-Inaba, Yamato Hida, Megumi Ichikawa, Yoshiaki Kato and Tetsuro Yamashita. Journal of Experimental Botany, Vol. 59, No. 4, pp. 995–1005, 2008.

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[4] Isolation of two distinct cold-inducible cDNAs encoding plant uncoupling proteins from the spadix of skunk cabbage (Symplocarpus foetidus). Kikukatsu Ito. Plant Science Vol. 149, Issue 2, 3 Dec. 1999, P.167–173.<br/>

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[6] Functional Coexpression of the Mitochondrial Alternative Oxidase and Uncoupling Protein Underlies Thermoregulation in the Thermogenic Florets of Skunk Cabbage. Yoshihiko Onda, Yoshiaki Kato, Yukie Abe, Takanori Ito, Miyuki Morohashi, Yuka Ito,Megumi Ichikawa4, Kazushige Matsukawa, Yusuke Kakizaki, Hiroyuki Koiwa, and Kikukatsu Ito. Plant Physiology, February 2008, Vol. 146, pp. 636–645.

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[5] Characterization of the plant uncoupling protein, SrUCPA, expressed in spadix mitochondria of the thermogenic skunk cabbage. Yasuko Ito-Inaba, Yamato Hida, Megumi Ichikawa, Yoshiaki Kato and Tetsuro Yamashita. Journal of Experimental Botany, Vol. 59, No. 4, pp. 995–1005, 2008.<br/>

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[6] Functional Coexpression of the Mitochondrial Alternative Oxidase and Uncoupling Protein Underlies Thermoregulation in the Thermogenic Florets of Skunk Cabbage. Yoshihiko Onda, Yoshiaki Kato, Yukie Abe, Takanori Ito, Miyuki Morohashi, Yuka Ito,Megumi Ichikawa4, Kazushige Matsukawa, Yusuke Kakizaki, Hiroyuki Koiwa, and Kikukatsu Ito. Plant Physiology, February 2008, Vol. 146, pp. 636–645.<br/>

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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, and a selectable marker (mainly the ß-lactamase gene, amp) for the bacterial host.

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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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[*]Thermoregulation: what role for UCPs in mammals and birds?Mozo J, Emre Y, Bouillaud F, Ricquier D, Criscuolo F. Biosci Rep. 2005 Jun-Aug;25(3-4):227-49.

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[*]Thermoregulation: what role for UCPs in mammals and birds? Mozo J, Emre Y, Bouillaud F, Ricquier D, Criscuolo F. Biosci Rep. 2005 Jun-Aug;25(3-4):227-49.

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

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

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Collaborate with Perdue iGEM team: <br/><br/>

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<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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<li>Kyoto iGEM team: <br/><br/>

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<p>We are helping <a href="https://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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<li>NTU_Taida iGEM team: <br/><br/>

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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>

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<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>

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<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>

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<h3>Let us introduce our App in this short film :)</h3><br/>

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~~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~~.

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As we known, for most of people who has never learned synthetic biology, it‘s a magical and crazy idea to take biological parts as the electronic parts. Moreover, someone might be scaried about this technique just like "Witch Hunt" in middle centry, because he don‘t know what is it.

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When we talk about synthetic biology, most of our friends will connect this technique with "Genome Modified Organism" and "Terrible Biological Weapon". They have antagonistic feeling in the first time because they don‘t know about it. However, beyond our expectation that they still feeling unbelieveble after our explaination......

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In hence, we decide to make an App to teach them the basic biotech concept and let them know the synthetic biology is not so hazardous. The main idea of this game is let players know if they don‘t care about the biosafety, they will let environment endanger. But if they are cautious in their operation, most of the results will under their control.

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

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<b><h2>Conference with UT_Tokyo Team</h2></b><br/>

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On 4th May, We held a video conference with UT_Tokyo team. It's our first time to have video conference with foreign iGEM team! We gave a briefly project introduction and discussed about experimental problems to each other. By the discussion, we found out some bugs but some new ideas in our project. On the other hand, we also gave some advices to UT_Tokyo team. That's a great chance for us to practice how can we express our project entirely and clearly.

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<b><h2>Conference with UT_Tokyo Team</h2></b><br/>

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On 4th May, We held a video conference with UT_Tokyo team. It's our first time to have video conference with foreign iGEM team! We gave a briefly project introduction and discussed about experimental problems to each other. By the discussion, we found out some bugs but some new ideas in our project. On the other hand, we also gave some advices to UT_Tokyo team. That's a great chance for us to practice how can we express our project entirely and clearly.

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<b><h2>Conference in Tokyo University</h2></b><br/>

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<b><h2>Conference in Tokyo University</h2></b><br/>

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On 14th July, Yi-Yuan Lee met UT_Tokyo iGEM team in Tokyo University. UT_Tokyo team briefly performed their currently progress and started to discuss their project. For Yi-Yuan, he learned more experience about how to well organitze an iGEM team.

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On 14th July, Yi-Yuan Lee met UT_Tokyo iGEM team in Tokyo University. UT_Tokyo team briefly performed their currently progress and started to discuss their project. For Yi-Yuan, he learned more experience about how to well organitze an iGEM team.

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<b><h2>Conference with Purdue iGEM Team</h2></b><br/>

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<b><h2>Conference with Purdue iGEM Team</h2></b><br/>

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We had a video conference with Purdue iGEM team, Manchester iGEM team, TU Eindhoven iGEM team, and Kyoto iGEM team on July 8th. This is Purdue iGEM team’s project. They want to build up a new standardized form for BioBricks register by cooperating and discussing with worldwide iGEM teams. We complete the questionnaire and gave them some advices.

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We had a video conference with Purdue iGEM team, Manchester iGEM team, TU Eindhoven iGEM team, and Kyoto iGEM team on July 8th. This is Purdue iGEM team’s project. They want to build up a new standardized form for BioBricks register by cooperating and discussing with worldwide iGEM teams. We complete the questionnaire and gave them some advices.

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<b><h2>Conference in UC Berkeley</h2></b><br/>

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<b><h2>Conference in UC Berkeley</h2></b><br/>

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YI-Yuan and Chi-Wei had a face to face conference with Berkeley iGEM team on July 10th. We presented project and experimental design to each other, and discussed about better methods to break through our problems.<br/>

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YI-Yuan and Chi-Wei had a face to face conference with Berkeley iGEM team on July 10th. We presented project and experimental design to each other, and discussed about better methods to break through our problems.<br/>

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After the conference, we had a late lunch together in fornt of the UC Berkeley with Berkeley iGEMers. We talked from our major to where did we come from. That was a great meal with them.

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After the conference, we had a late lunch together in fornt of the UC Berkeley with Berkeley iGEMers. We talked from our major to where did we come from. That was a great meal with them.

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<b><h2>Conference in Chiao Tung University</h2></b><br/>

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<h1 class="header">NTU Azalea Festival</h1>

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NTU Azalea Festival is a anneal event for introducing each department to senior high school students. By this chance, we wanted to spread the iGEM idea to high school students. We design a simple computer game which contain the concept of synthetic biology, molecular biology and bio-Technology. First, we asked them to choose a right enzyme to digest the glactose. And in second level, we asked them to make the enzyme they used in previous level by amino acids, right RNA, and right enzyme. In the last level, we asked them to build up a operon for galactase production.

</p>

+

<a class="hover-shodow hover-no-underline" href="http://ntu-taiwan-azalea.herokuapp.com"><i class="icon-gamepad icon-2x">Here’s our Little Game</i></a>

</div>

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</script>

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<h1 class="header">Poster Presentation</h1>

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<h1 class="header">Poster Presentation</h1>

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</div>

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</script>

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

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

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~~</section>~~

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-

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

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~~<div class="row text-center" style="margin-left: 20px; margin-right: 20px">~~

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~~<h3>Let us introduce our App in this short film :)</h3><br/>~~

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-

~~<iframe id="appMovie" style="width=75%;" src="http://www.youtube.com/embed/NjNNvrgOozA" frameborder="0" allowfullscreen></iframe><br/></div></div>~~

+

-

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

+

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~~<p>~~

+

-

As we known, for most of people who has never learned synthetic biology, it‘s a magical and crazy idea to take biological parts as the electronic parts. Moreover, someone might be scaried about this technique just like "Witch Hunt" in middle centry, because he don‘t know what is it.

+

-

~~</p>~~

+

-

~~<p>~~

+

-

When we talk about synthetic biology, most of our friends will connect this technique with "Genome Modified Organism" and "Terrible Biological Weapon". They have antagonistic feeling in the first time because they don‘t know about it. However, beyond our expectation that they still feeling unbelieveble after our explaination......

+

-

~~</p>~~

+

-

~~<p>~~

+

-

In hence, we decide to make an App to teach them the basic biotech concept and let them know the synthetic biology is not so hazardous. The main idea of this game is let players know if they don‘t care about the biosafety, they will let environment endanger. But if they are cautious in their operation, most of the results will under their control.

+

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~~</p>~~

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~~</div>~~

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-

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-

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

+

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

+

-

~~</section>~~

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-

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-

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

+

-

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

+

-

~~<li>~~

+

-

~~Collaborate with Perdue iGEM team: <br/><br/>~~

+

-

~~<p>We collaborated with Perdue iGEM team to design a better version of datasheet when we register our parts. Following is one of the beta-version of their datasheet. </p>~~

+

-

~~<embed src="./PDF/Datasheet Composite Part Example.pdf" style="width: 100%; height: 500px"></embed>~~

+

-

~~</li>~~

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-

~~<br/>~~

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-

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~~<li>Kyoto iGEM team: <br/><br/>~~

+

-

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

+

-

~~<div class="col-md-7">~~

+

-

~~<p>We help Kyoto iGEM team to charaterise their parts. They sent 13 parts to us. We keep discussing how to design the construction and which gene should we use in the characterisation.</p>~~

+

-

~~</div>~~

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-

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-

~~<div class="col-md-5">~~

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-

~~<img class="img-responsive" src="./images/Human_practice/HUMANPRACTICE_2.jpg" style="margin-right: 30px">~~

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~~</div>~~

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~~</div>~~

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~~</li>~~

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-

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~~<li>NTU_Taida iGEM team: <br/>~~

+

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~~</ul>~~

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~~</div>~~

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</script>

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<h1 class="header" style="margin: 0;">Team Members</h1>

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</section>

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<h1 class="header">Team Members</h1>

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-

<b><u>Team Leader</u></b>

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<b><u>Team Leader & Lab Hero</u></b>

</div>

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</div>

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<b class="col-md-4"> ~~Interests~~: </b> <div class="col-md-8"> Reading, Calligraphy, Ballroom dance, Swimming, Traveling </div>

+

<b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> Reading, Calligraphy, Ballroom dance, Swimming, Traveling </div>

</div>

-

<b class="col-md-4"> Interested: </b> <div class="col-md-8"> Immunology, Bioinformatics, ~~Virology~~ </div>

+

<b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> Immunology, Bioinformatics, Programming, Asian history </div>

</div>

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</ul>

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<b><u>Team Leader</u></b>

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<b><u>Team Leader & Lab Hero</u></b>

</div>

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</div>

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<b class="col-md-4"> ~~Interests~~: </b> <div class="col-md-8"> Reading, Calligraphy, Ballroom dance, Swimming, Traveling </div>

+

<b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> Reading, Calligraphy, Ballroom dance, Swimming, Traveling </div>

</div>

-

<b class="col-md-4"> Interested: </b> <div class="col-md-8"> Immunology, Bioinformatics, ~~Virology~~ </div>

+

<b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> Immunology, Bioinformatics, Programming, Asian history </div>

</div>

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<b><u>Lily LOL Fighter</u></b>

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</div>

-

<b class="col-md-4"> ~~Interests~~: </b> <div class="col-md-8"> swim, music, stage play </div>

+

<b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> swim, music, stage play </div>

</div>

-

<b class="col-md-4"> Interested: </b> <div class="col-md-8"> Fermentology, Applied Microbiology </div>

+

<b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> Fermentology, Applied Microbiology </div>

</div>

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</div>

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</div>

</section>

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<h2>呂宗諭(Tsung-Yu Lu)</h2>

+

<h2>呂宗諭 (Tsung-Yu Lu)</h2>

<b><u>Chief of Lab Hero</u></b>

</div>

-

<b class="col-md-4"> Department: </b> <div class="col-md-8"> ~~Biochemical Science and Technology~~ </div>

+

<b class="col-md-4"> Department: </b> <div class="col-md-8"> Insititute of Biochemial Sciences </div>

</div>

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</div>

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<b class="col-md-4"> ~~Interests~~: </b> <div class="col-md-8"> swim, music, stage play </div>

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<b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> swim, music, stage play </div>

</div>

-

<b class="col-md-4"> Interested: </b> <div class="col-md-8"> protein engineering, mathematical biology </div>

+

<b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> protein engineering, mathematical biology </div>

</div>

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<h2>張宏宇(Hung-Yu Chang)</h2>

+

<h2>張宏宇 (Hung-Yu Chang)</h2>

-

<b><u>Lab Surfer</u></b>

+

<b><u>Lab Surfer & Lab Hero</u></b>

</div>

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</div>

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<b class="col-md-4"> ~~Interests~~: </b> <div class="col-md-8"> Debate, Tennis, Surfing </div>

+

<b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> Debate, Tennis, Surfing </div>

</div>

-

<b class="col-md-4"> Interested: </b> <div class="col-md-8"> Cell Biology, Tissue Engineering </div>

+

<b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> Cell Biology, Tissue Engineering </div>

</div>

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</div>

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</div>

</section>

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<b><u>Lab Physician</u></b>

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</div>

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<b class="col-md-4"> ~~Interests~~: </b> <div class="col-md-8"> reading, movie, volleyball, Japanese </div>

+

<b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> reading, movie, volleyball, Japanese </div>

</div>

-

<b class="col-md-4"> Interested: </b> <div class="col-md-8"> physiology, immunology, neurology </div>

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<b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> physiology, immunology, neurology </div>

</div>

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<b><u>Lab Anego</u></b>

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</div>

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<b class="col-md-4"> ~~Interests~~: </b> <div class="col-md-8"> traveling, reading </div>

+

<b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> traveling, reading </div>

</div>

-

<b class="col-md-4"> Interested: </b> <div class="col-md-8"> sociology, literature, biochemistry </div>

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<b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> sociology, literature, biochemistry </div>

</div>

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</div>

</section>

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<b><u>Fake Secretary</u></b>

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</div>

-

<b class="col-md-4"> ~~Interests~~: </b> <div class="col-md-8"> reading,playing,volleyball,movie </div>

+

<b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> reading,playing,volleyball,movie </div>

</div>

-

<b class="col-md-4"> Interested: </b> <div class="col-md-8"> Immunology, Nutritional </div>

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<b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> Immunology, Nutritional </div>

</div>

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</div>

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<b class="col-md-4"> ~~Interests~~: </b> <div class="col-md-8"> dance, cooking, traveling, reading </div>

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<b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> dance, cooking, traveling, reading </div>

</div>

-

<b class="col-md-4"> Interested: </b> <div class="col-md-8"> Molecular biology, cell biology </div>

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<b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> Molecular biology, cell biology </div>

</div>

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</div>

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</div>

</section>

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<h2>王柏軒(Po-Hsien Wang)</h2>

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<h2>王柏軒 (Po-Hsien Wang)</h2>

<b><u>Web Hacker</u></b>

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</div>

-

<b class="col-md-4"> ~~Interests~~: </b> <div class="col-md-8"> reading, movie </div>

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<b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> reading, movie </div>

</div>

-

<b class="col-md-4"> Interested: </b> <div class="col-md-8"> AI, Virtual Reality </div>

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<b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> AI, Virtual Reality </div>

</div>

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<b><u>Niwei</u></b>

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</div>

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<b class="col-md-4"> ~~Interests~~: </b> <div class="col-md-8"> badminton, photography, reading </div>

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<b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> badminton, photography, reading </div>

</div>

-

<b class="col-md-4"> Interested: </b> <div class="col-md-8"> Communication, AI, App & Web developing </div>

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<b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> Communication, AI, App & Web developing </div>

</div>

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</div>

</section>

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</div>

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<b class="col-md-4"> ~~Interests~~: </b> <div class="col-md-8"> baseball, girl, post rock </div>

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<b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> baseball, girl, post rock </div>

</div>

-

<b class="col-md-4"> Interested: </b> <div class="col-md-8"> ML, AI, probability graphical model </div>

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<b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> ML, AI, probability graphical model </div>

</div>

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<b><u>Secret staff</u></b>

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</div>

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<b class="col-md-4"> ~~Interests~~: </b> <div class="col-md-8"> fixed gear, table tennis, photography </div>

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<b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> fixed gear, table tennis, photography </div>

</div>

-

<b class="col-md-4"> Interested: </b> <div class="col-md-8"> Computational photography, App </div>

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<b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> Computational photography, App </div>

</div>

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</div>

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</div>

</section>

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<b><u>Lynda</u></b>

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</div>

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<b class="col-md-4"> ~~Interests~~: </b> <div class="col-md-8"> drawing, tennis, tap dance, Chinese comedy </div>

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<b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> drawing, tennis, tap dance, Chinese comedy </div>

</div>

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<b class="col-md-4"> Interested: </b> <div class="col-md-8"> UI, HCI, VR </div>

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<b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> UI, HCI, VR </div>

</div>

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</section>

</div>

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</script>

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

+

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

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</section>

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<div>

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<legend><h2 style="border-bottom: 0">Website</h2></legend>

+

<ol>

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<li>

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<a href="http://jquery.com/">jQuery</a><br />

+

a fast, small, and feature-rich JavaScript library. It makes things like HTML document traversal and manipulation, event handling, animation, and Ajax much simpler with an easy-to-use API that works across a multitude of browsers. With a combination of versatility and extensibility, jQuery has changed the way that millions of people write JavaScript.

+

</li>

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<li>

+

<a href="http://getbootstrap.com/">BootStrap</a><br />

+

Sleek, intuitive, and powerful mobile first front-end framework for faster and easier web development.

+

</li>

+

<li>

+

<a href="http://underscorejs.org/">UnderScore</a><br />

+

a utility-belt library for JavaScript that provides a lot of the functional programming support that you would expect in Prototype.js (or Ruby), but without extending any of the built-in JavaScript objects.

+

</li>

+

<li>

+

<a href="http://backbonejs.org/">BackBone</a><br />

+

BackBone gives structure to web applications by providing models with key-value binding and custom events, collections with a rich API of enumerable functions, views with declarative event handling, and connects it all to your existing API over a RESTful JSON interface.

+

</li>

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<li>

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<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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</li>

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<li>

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<a href="http://fortawesome.github.io/Font-Awesome/">FontAwesome</a><br />

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Font Awesome gives you scalable vector icons that can instantly be customized — size, color, drop shadow, and anything that can be done with the power of CSS.

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</li>

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<li>

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<a href="http://fittextjs.com/">FitText</a><br />

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FitText makes font-sizes flexible. Use this plugin on your fluid or responsive layout to achieve scalable headlines that fill the width of a parent element.

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</li>

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<li>

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<a href="http://www.dpereyra.com/scripts/dp_calendar/">SIMPLE EVENTS CALENDAR</a>

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jQuery Basic Event Calendar is a highly configurable plugin that adds calendar functionality to your pages.

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</li>

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</ol>

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</div>

+

<div>

+

+

<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>

+

<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>

+

<p>All of the following DNA were designed, cloned, or constructed by our wetlab team members:</p>

+

1. TAP protein tag was cloned from pRS424 shuttle vector.<br/>

+

2. Tir1 promoter sequence was amplified from genomic DNA of <i>Saccharomyces cerevisiae</i>.<br/>

+

3. 26s and 5.8s ITS rDNA was cloned from <i>Rhodotorula glutinis</i>.<br/>

+

4. All of the PCR primers of cloning were design by ourselves.<br/>

+

5. The construction of pRS424:GAL1:SrUCP:TAP, pGAPZA:26s, pGAPZA:5.8s ITS.<br/>

+

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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</div>

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</div>

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</script>

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

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</section>

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+

<p><b>National Taiwan University</b></p>

+

+

<p>Our supervisor Dr. Yen-rong Chen for making our adventure in iGEM possible.</p>

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+

<p>Dr. Ching-Tsan Huang for providing us with the shuttle vectors pGAPZA plasmid.</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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+

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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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+

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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/>

+

<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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</div>

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</script>

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

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Look what we have done<br/>

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</p>

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</span>

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

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</a>

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Fascinating Android App

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<ul>

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

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<li>

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

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<li> -the safety and health of team members or others working in the lab?</li>

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<ul>

-

~~<li>~~ -the safety and health of the general public, if released by design or by accident?</li>

+

<li>

-

<ul~~></b~~>

+

<b> -the safety and health of team members or others working in the lab? <br/>

-

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

+

-the safety and health of the general public, if released by design or by accident? </b>

-

</ul><b>

+

</li>

-

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

+

-

<ul~~></b~~>

+

<ul>

-

<li>For the same reasons above, there would be minimum chance of the organisms causing any great harm to the environment. Our E. coli 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 ~~equipments~~ used for bacteria cells and/or cultures are properly autoclaved. </li>

+

<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>

+

</ul>

-

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

+

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

-

<ul~~></b~~>

+

<ul>

-

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

+

<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>

+

</ul>

-

</b></ul>

+

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

-

+

<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/>

+

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

-

+

</ul>

-

<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"~~>

+

</ul>

-

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

+

</li>

-

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

+

<li>

-

+

<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/>

-

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

+

<p>Respecting our goals to prevent the damage caused by cold temperature in fish farming:</p>

-

<br/>

+

<ol>

-

<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/>

+

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

-

When faced with an increasing ~~osmaolarity~~ 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. 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 ~~sythesis~~. Following activation of the HOG pathway, activated Hog1 binds to the transcriptional ~~acitvator~~ 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. Smp1 is phosphorylated and activated by the active Hog1 protein. ~~Thus~~, the sensing of osmolarity and the induction of GPD1 and STL1 expression ~~will~~ make up ~~mainly~~ the ~~fist~~ part of our kill switch.<br/>

+

<li>Release of cell culture into the environment may cause fish infection and perturbation of the ecosystem.</li>

-

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 ~~and~~ 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, ~~and~~ their ~~induction via osmosensitive promoters would~~ cause irreversible harm to the yeasts ~~and~~ in the end ~~kill~~ them.<br/>

+

</ol>

-

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, with ~~following~~ cell death. 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>

+

</li>

-

+

<li>

-

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

+

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

-

<p ~~style="margin-left: 40px"~~>Every student who worked in the lab have had to ~~received~~ a 12-hour training. The training includes lectures on the topics "Principal of Biosafety" and "Management of Biosafety in Laboratories".</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 ~~Enviroment~~ Protection and Occupational Safety and Health Center. The assessment result was that we don't need to send the "Recombinant gene experiment" form to the Center, due to our materials all belonging to RG1.</p><br/>

+

<h5>(fig.3 Overview of the HOG pathway in <i>S. cerevisiae</i>. Several transcriptional factors are regulated.)</h5>

-

+

</div>

-

<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>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/>

-

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

+

<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/>

-

</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>

+

<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>

+

</div>

+

</div>

+

+

<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.</h5>

+

</p>

+

</li>

+

<li>

+

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

+

<p>Every student who worked in the lab have had to receive a 12-hour training. The training includes lectures on the topics "Principal of Biosafety" and "Management of Biosafety in Laboratories".</p><br/>

+

</li>

+

<li>

+

<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>We had an discussion with the National Taiwan University Environment Protection and Occupational Safety and Health Center. The assessment result was that we don't need to send the "Recombinant gene experiment" form to the Center, due to our materials all belonging to RG1.</p><br/>

+

</li>

+

<li>

+

<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/>

+

</li>

+

+

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

+

</p>

+

</ul>

</div>

</script>

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<h1 class="header" style="margin: 0">Basic Research</h1>

-

<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>

Line 1,556:

Line 1,738:

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

</section>

-

+

+

+

+

<h5>"Budding Yeast and Friend Fan Page <a href="https://www.facebook.com/yeastandfriends"> Link</a>"</h5>

+

</div>

+

+

<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). 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>

-

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Line 1,762:

-

+

-

<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>

+

<div style="margin-top: 75px"><p>The yeast <b>Saccharomyces cerevisiae</b> has several properties which have established it as an important tool in the expression of foreign protein for research, industrial or medical use. As a food organism, it is highly acceptable for the production of pharmaceutical proteins. In contrast,<b>Escherichia coli</b> have toxic cell wall pyroxenes and mammalian cells may contain oncogenic or viral DNA, so that products from these organisms must be tested hmore extensively.</p>

-

<p>Yeast can be grown rapidly on simple media and to high cell density and its genetics are more advanced than any other eukaryote, so that it can be manipulated almost as readily as ~~<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>

+

<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/>

+

<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>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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Line 1,779:

<p>UCP1 can transport protons through two different hypothesized mechanisms. In the first model, UCP1 transfers protons from intermembrane space to mitochondria matrix. Fatty acids provide essential free carboxy groups catalyzing proton (H+) translocation. In this model, Coenzyme Q(CoQ) serves as a necessary cofactor of UCP1, activating the protein functions.</p>

-

<p>In the second model, free fatty acids (FFAs) combine with protons with carboxy groups, then flipping across inner mitochondria membrane. After FFAs flipping to the matrix side, UCP1 removes protons from carboxy groups, flipping the anion form FFAs back to the innermembrane space. (fig.??)The anion form FFAs bind to protons again, transferring more protons into the matrix. </p>

+

<p>In the second model, free fatty acids (FFAs) combine with protons with carboxy groups, then flipping across inner mitochondria membrane. After FFAs flipping to the matrix side, UCP1 removes protons from carboxy groups, flipping the anion form FFAs back to the innermembrane space. (fig.2)The anion form FFAs bind to protons again, transferring more protons into the matrix. </p>

-

+

-

+

<p>Through these two div mechanisms, proton gradient on either side of inner membrane are formed. Generally, the oxidative phosphorylation of ADP into ATP takes place within the mitochondrial inner membrane respiratory chain. However, the coupling of mitochondrial respiration and ATP synthesis is not complete (Nicholls, 1974; Brand, 1977). Thus in several tissues there is a proton leak through the inner mitochondrial membrane that is not associated with ATP synthesis. And in these tissues, UCPs produce heat.

Line 1,600:

Line 1,795:

-

<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>

+

<P><b>Cold shock promoter</b></p>

-

</~~div~~>

+

<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>

-

</~~div~~>

+

-

+

<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>

-

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

+

<p><b>Potential Cold Shock Promoter</b></p>

-

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

+

<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>

-

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

+

<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>

-

</~~section~~>

+

-

+

-

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

+

Reference:<br/>

+

[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/>

+

[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/>

+

[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/>

+

[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/>

</div>

+

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

</section>

-

+

-

~~&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbspIn~~ 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/>

+

-

~~&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbspWhen~~ faced with an increasing ~~osmaolarity~~ 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. 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 ~~sythesis~~. Following activation of the HOG pathway, activated Hog1 binds to the transcriptional ~~acitvator~~ 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. Smp1 is phosphorylated and activated by the active Hog1 protein. ~~Thus~~, the sensing of osmolarity and the induction of GPD1 and STL1 expression ~~will~~ make up ~~mainly~~ the ~~fist~~ part of our kill switch.<br/>

+

-

~~&nbsp&nbsp&nbsp&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 ~~and~~ 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, ~~and~~ their ~~induction via osmosensitive promoters would~~ cause irreversible harm to the yeasts ~~and~~ in the end ~~kill~~ them.<br/>

+

-

~~&nbsp&nbsp&nbsp&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, with ~~following~~ cell death. 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/>

+

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

+

</div>

+

</div>

+

<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/>

+

<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>

+

+

<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>

+

<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>

+

<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>

</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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Line 1,854:

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</div>

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

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<p class="header"><i> Rhodotorula glutinis</i></p>

</section>

-

<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.<br/>

+

-

<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. <br/>

+

<p><b>What is <i>Rhodotorula glutinis</i> ?</b></p>

-

~~&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbspThe~~ 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><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>

-

~~&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbspSo we choose R.g. for the Expression vector.~~<br/>

+

<p><b>The oleaginous yeast</b></p>

-

~~&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbspThermogenic~~ 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.<br/>

+

<p>

-

~~&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbspAccording~~ 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. 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. <br/>

+

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>

-

~~&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbspAfter 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.<br/>

+

<p><b>Why <i>Rhodotorula glutinis</i> ?</b></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>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><b>Conclusion</b></p>

+

<p>

+

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>

+

Reference:<br/>

+

[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/>

+

[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/>

</div>

</script>

+

+

+

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

+

</section>

+

+

+

<h2 style="border-bottom: 0"><b>Mathematical Model－「Tuning the sensitivity of cold shock promoter」</b></h2>

+

</legend>

+

<p>

+

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>

+

+

+

<h2 style="border-bottom: 0"><b>Model A、Heat shock promoter or Constitutive promoter?</b></h2>

+

</legend>

+

+

<li><b>Purpose</b>

+

+

<li><p>Investigate on whether heat shock promoter (abbreviated as Hsp) or Constitutive promoter suit our genetic circuit as a controller of regulator.</p></li>

+

</ul>

+

</li>

+

<li><b>Circuit structure</b>

+

+

<li><p>In this model, we use GFP to report the activity of cold shock promoter (abbreviated as Csp) under different temperatures. The reporter itself is in turn regulated by an activator and a repressor through activator binding site and repressor binding site. While activators are regulated by Csp, repressors are regulated by Hsp or Constitutive promoter (Fig. 4)</p></li>

+

</ul>

+

+

<div class="tip">Fig. 4: Representative diagram of our genetic circuit</div>

+

</li>

+

<li><b>Mathematical expression</b>

+

+

<p>Gene expression can be described with certain mathematical language, with deterministic expression one of the most commonly used. Deterministic expression represent the production rate of a gene product as the difference between a fraction, determining by the promoter occupancy by activator or repressor, of maximal production rate and the removal rate. Here, we will show the mathematical expression of the model with Hsp or constitutive promoter as their circuit part, respectively.</p>

+

+

<div><b>model with Hsp as a part</b></div>

+

<ul>

+

<li>

+

+

</li>

+

<li>

+

+

</li>

+

<li>

+

+

</li>

+

</ul>

+

<div><b>model with constitutive promoter as a part:</b></div>

+

<ul>

+

<li>

+

+

</li>

+

<li>

+

+

</li>

+

<li>

+

+

</li>

+

</ul>

+

</div>

+

<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>

+

+

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).

+

</p>

+

+

+

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

+

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

+

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

+

+

</tr>

+

+

+

<td class="col-md-5">Hill’s coefficient</td>

+

+

+

</tr>

+

+

+

<td class="col-md-5">Maximal production rate of Csp, a function of temperature</td>

+

<td class="col-md-3">Sigmoidal curve <br/>(set 10℃=1e-6, 20℃=0)) </td>

+

+

</tr>

+

+

+

<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>

+

+

</tr>

+

+

+

<td class="col-md-5">Maximal production rate of constitutive promoter</td>

+

+

+

</tr>

+

+

+

<td class="col-md-5">Removal rate of activator</td>

+

+

+

</tr>

+

+

+

<td class="col-md-5">Removal rate of repressor</td>

+

+

+

</tr>

+

+

+

<td class="col-md-5">Removal rate of GFP</td>

+

+

+

</tr>

+

+

+

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

+

+

+

</tr>

+

+

+

<td class="col-md-5">Kd value of activator from DNA</td>

+

+

+

</tr>

+

+

+

<td class="col-md-5">Kd value of repressor from DNA</td>

+

+

+

</tr>

+

</table>

+

+

<ul>

+

<li>Assume their maximal activity the same as Csp</li>

+

<li>Assume only slightly higher than γGFP</li>

+

<li>Consider the action of generation time of yeast and degradation rate in E. coli</li>

+

</ul>

+

</div>

+

</li>

+

</ul>

+

</div>

+

+

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

+

</section>

+

+

<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>

+

+

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

+

+

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

+

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

+

<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>

+

+

+

<div class="tip"> Fig. 4: Expression pattern of GFP under various αA and αR－ a constitutive promoter integrated model. The X, Y axis are values of αA and αR scanned. The Z axis is the maximal GFP concentration. The color bar represents the temperature corresponding to half of the maximal GFP concentration</div>

+

+

<div class="tip"> Fig. 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>

+

+

<legend><b>consModel</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_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>

+

+

+

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

+

</section>

+

+

+

1: Design of appropriate forward and reverse primers<br/>

+

2: Prepare our template<br/>

+

3: Prepare the PCR mix. (Kapa Hifi PCR kit.)<br/>

+

4: Run PCR<br/>

+

5: Examine the results by electrophoresis<br/>

+

Note: If the template is genomic DNA, we would adjust the annealing temperature at 45°C. It is because the copy number of target gene may be low. We use this annealing temp when perform PCR of Tir1, 26s, 5.8s ITS<br/><br/>

+

<br/><p><b>Construction of our parts</b></p><p>

+

1: We design primers for parts with prefix and suffix.<br/>

+

2: Perform PCR and cleanup the PCR product<br/>

+

3: Before insert our parts into standard backbone, pSB1C3, we perform RE digestion to make sticky ends of both inserts and backbones.<br/>

+

4: Ligation of inserts and backbones<br/>

+

5: Transform our ligation products into DH5α and streak the transformed DH5α on LB agar plate with chloramphenicol.<br/>

+

6: Inoculate single colony into broth with chloramphenicol.<br/>

+

7: Miniprep the plasmid DNA from the overnight broth culture.<br/>

+

8: Confirm the products by both RE digestion and PCR sequencing<br/></p>

+

<p><b>Point mutation protocol</b></p>

+

<p>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>

+

</script>

+

+

+

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

+

</section>

+

+

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

+

<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: 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: 140px"><p> This is the pGAPZa which had been digested by <i>Bgi</i>II.</p></div>

+

</div>

+

</div>

+

</div>

+

+

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

+

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

+

+

+

<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>

+

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

+

+

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

+

<p>After both experimental group (pRS424-GAL1-SrUCP-TAP) and negative control group (pRS424-GAL1∆) induced by 2% galactose for 21 hours, we couldn’t find out statistical difference between control and experimental group by our first experimental method. Most of the heat production is come from the fermentation and shaking of the incubator.</p>

+

<p>However, we didn’t analyze the quantity of yeasts during the experiment. We consider that the experimental group (which had been transformed SrUCP) might grow slower than the control group and then cause the result t I show below:<br/>

+

Experimental: Heat(E) = Fermentation(1) + SrUCP<br/>

+

Control: Heat(C) = Fermentation(2)<br/>

+

Because the heat of fermentation(1) is lower than fermentation(2), even if SrUCP produce heat, the total Heat(E) equal to Heat(C).

+

In hence, the better method to test heat production is incubate in isothermal environment or use the isothermal titration calorimetry. We will try these more precise method.

+

</p>

+

+

</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>

+

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+     <title> iGEM-NTU-Taiwan </title>
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+                                </ul>
+                            </li>
+                             <li><a href="#Calendar"> <i class="icon-calendar"></i>Calendar </a> </li>
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-                             <img src="https://static.igem.org/mediawiki/2011/a/a9/DTU-Denmark2011-IGEM_logo.png" alt="">
+                             <img src="https://static.igem.org/mediawiki/2013/b/b8/NTU_TAIWAN_Igem_Logo.png" alt="">
                          </a>
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+                             <li><a href="https://igem.org/Team.cgi" target="_blank">iGEM Website</a></li>
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              </div>
-             <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>
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          <div class="container divide" id="index-icons">
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                          <div> Motivation </div>
-                    </a>
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-                            </span>
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                      </a>
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              </section>
              <div class="container divide">
-                 <p>
+                 <p>In this year, iGEM NTU_Taiwan team aims at making a biological heating device which can produce appropriate heat at low temperatures. The feature of this device is that it can respond differently to temperature and produce heat in an efficient and economical manner.
-                 </p>
+                </p>
+                <p>What a crazy project! This biological device is really charming, isn&#39;t it? Let us show you our project! <br/>
+                <div class="row text-center"><h3>Let&#39;s go!</h3></div>
+                 </p>
              </div>
          </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 pound 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. 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.
+                 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>
              </div>
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              <p>
-                 White and brown adipose tissues (WAT and BAT) serve important opposite functions in overall energy balance. While WAT is specialized in energy storage in the form of triacylglycerols, BAT functions to dissipate energy in the form of heat (thermogenesis).BAT possess abundant mitochondria with uncoupling proteins 1(UCP1). UCP1 is a 6-transmembrane protein in inner mitochondria membrane, and it has been proposed to constitute three symmetrical membrane-spanning &ldquo;Us&rdquo;, each comprising about 100 amino acids(fig.?).The functional UCP is believed to be dimer.
+                 White and brown adipose tissues (WAT and BAT) serve important opposite functions in overall energy balance. While WAT is specialized in energy storage in the form of triacylglycerols, BAT functions to dissipate energy in the form of heat (thermogenesis).BAT possess abundant mitochondria with uncoupling proteins 1(UCP1). UCP1 is a 6-transmembrane protein in inner mitochondria membrane, and it has been proposed to constitute three symmetrical membrane-spanning &ldquo;Us&rdquo;, each comprising about 100 amino acids(fig.1).The functional UCP is believed to be dimer.
              </p>
-             <img class="tipReveal img-responsive" src="images/image001.png">
+             <img class="tipReveal img-responsive" src="https://static.igem.org/mediawiki/2013/2/26/NTU_TAIWAN_Image001.png" alt-src="images/image001.png" style="display: block; margin: auto;"> <!--help!!!圖片放中間-->
              <div class="tip">
-                 (fig.? The protein (33kDa,306 amino acids) contains six transmembrane alpha-helices, proposed to constitute three symmetric repeats.)
+                 (fig.1 The protein (33kDa,306 amino acids) contains six transmembrane alpha-helices, proposed to constitute three symmetric repeats.)
              </div>
              <p>
                  Mitochondria uncoupling proteins 1(UCP1) are members of the family of mitochondrial anion carrier proteins (MACP). It facilitates the transfer of anions from the inner to the outer mitochondrial membrane and the return transfer of protons from the outer to the inner mitochondrial membrane. [2][3]UCPs separate oxidative phosphorylation from ATP synthesis with energy dissipated as heat, also referred to as the mitochondrial proton leak.
              </p><p>
-             UCP has been found in several species, including mammals and plants. Skunk cabbage(Symplocarpus foetidus)is a thermogenic plant, using srUCPs to resist in dramatic temperature decreases in the environment. In 1999, Dr. Kikukatsu Ito found that there are two types of SrUCPs, designated as SrUCPA and SrUCPB [4]. SrUCPA is proposed to be the functional thermogenic prtein, and SrUCPB serves as a regulator in thermogenesis in Skunk cabbage.[5][6] We use SrUCPA gene as our material to construct our plasmid.
+             UCP has been found in several species, including mammals and plants. Skunk cabbage(Symplocarpus foetidus)is a thermogenic plant, using srUCPs to resist in dramatic temperature decreases in the environment. In 1999, Dr. Kikukatsu Ito found that there are two types of SrUCPs, designated as SrUCPA and SrUCPB [4]. SrUCPA is proposed to be the functional thermogenic prtein, and SrUCPB serves as a regulator in thermogenesis in Skunk cabbage.[5][6] We use SrUCPA gene as our material to construct our plasmid.<br/><br/>
-             [1] Brown adipose tissue: function and physiological significance. Cannon B, Nedergaard J. Physiol Rev. 2004 Jan.
+            Reference:<br/>
-             [2] Uncoupling proteins: the issues from a biochemist point of view. Klingenberg M, Echtay KS. Biochim Biophys Acta. 2001 Mar 1;1504(1):128-43.
+             [1] Brown adipose tissue: function and physiological significance. Cannon B, Nedergaard J. Physiol Rev. 2004 Jan.<br/>
-             [3] Mitochondrial Uncoupling Proteins in Mammals and Plants. Jirı´ Borecky´, Ivan G. Maia, and Paulo Arruda. Bioscience Reports, Vol. 21, No. 2, April 2001 (2001).
+             [2] Uncoupling proteins: the issues from a biochemist point of view. Klingenberg M, Echtay KS. Biochim Biophys Acta. 2001 Mar 1;1504(1):128-43.<br/>
-             [4] Isolation of two distinct cold-inducible cDNAs encoding plant uncoupling proteins from the spadix of skunk cabbage (Symplocarpus foetidus). Kikukatsu Ito. Plant Science Vol. 149, Issue 2, 3 Dec. 1999, P.167–173.
+             [3] Mitochondrial Uncoupling Proteins in Mammals and Plants. Jirı´ Borecky´, Ivan G. Maia, and Paulo Arruda. Bioscience Reports, Vol. 21, No. 2, April 2001 (2001).<br/>
-             [5] Characterization of the plant uncoupling protein, SrUCPA, expressed in spadix mitochondria of the thermogenic skunk cabbage. Yasuko Ito-Inaba, Yamato Hida, Megumi Ichikawa, Yoshiaki Kato and Tetsuro Yamashita. Journal of Experimental Botany, Vol. 59, No. 4, pp. 995–1005, 2008.
+             [4] Isolation of two distinct cold-inducible cDNAs encoding plant uncoupling proteins from the spadix of skunk cabbage (Symplocarpus foetidus). Kikukatsu Ito. Plant Science Vol. 149, Issue 2, 3 Dec. 1999, P.167–173.<br/>
-             [6] Functional Coexpression of the Mitochondrial Alternative Oxidase and Uncoupling Protein Underlies Thermoregulation in the Thermogenic Florets of Skunk Cabbage. Yoshihiko Onda, Yoshiaki Kato, Yukie Abe, Takanori Ito, Miyuki Morohashi, Yuka Ito,Megumi Ichikawa4, Kazushige Matsukawa, Yusuke Kakizaki, Hiroyuki Koiwa, and Kikukatsu Ito. Plant Physiology, February 2008, Vol. 146, pp. 636–645.
+             [5] Characterization of the plant uncoupling protein, SrUCPA, expressed in spadix mitochondria of the thermogenic skunk cabbage. Yasuko Ito-Inaba, Yamato Hida, Megumi Ichikawa, Yoshiaki Kato and Tetsuro Yamashita. Journal of Experimental Botany, Vol. 59, No. 4, pp. 995–1005, 2008.<br/>
+             [6] Functional Coexpression of the Mitochondrial Alternative Oxidase and Uncoupling Protein Underlies Thermoregulation in the Thermogenic Florets of Skunk Cabbage. Yoshihiko Onda, Yoshiaki Kato, Yukie Abe, Takanori Ito, Miyuki Morohashi, Yuka Ito,Megumi Ichikawa4, Kazushige Matsukawa, Yusuke Kakizaki, Hiroyuki Koiwa, and Kikukatsu Ito. Plant Physiology, February 2008, Vol. 146, pp. 636–645.<br/>
              </p>
@@ Line 312: / Line 316: @@
          <div class="container divide essay">
          <p>
-             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.
+             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, and a selectable marker (mainly the ß-lactamase gene, amp) for the bacterial host.
          </p><p>
-             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.
+             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]
              </p>
+            (1)	Integrative plasmids (YIp) : Foreign DNAs integrate into the host genome(YIp) by homologous recombination, then resulting in one copy of transformed DNA.<br/>
+            (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/>
+            (3)	Autonomously replicating plasmids (YRp) : carry a yeast origin of replication, ARS sequence, that allows the transformed plasmids to be propagated several hundredfold.<br/>
+            (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>
+            <p>Here, we choose Episomal plasmids (YEp) as our vectors.</p>
+            <br/>
+            Reference:<br/>
+            [1] Transformation of yeast by a replicating hybrid plasmid. Beggs, J.D. Nature 275 (1978) 104-109.<br/>
+            [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).
          </div>
      </script>
@@ Line 332: / Line 345: @@
                  <u class='tip'>
-                     [*]Thermoregulation: what role for UCPs in mammals and birds?Mozo J, Emre Y, Bouillaud F, Ricquier D, Criscuolo F. Biosci Rep. 2005 Jun-Aug;25(3-4):227-49.
+                     [*]Thermoregulation: what role for UCPs in mammals and birds? Mozo J, Emre Y, Bouillaud F, Ricquier D, Criscuolo F. Biosci Rep. 2005 Jun-Aug;25(3-4):227-49.
                  </u>
              </p>
@@ Line 338: / Line 351: @@
      </script>
-     <script type="text/template" id="Sensor">
+     <script type="text/template" id="Cooperation">
          <section class="red-background">
-             <h1 class="header">Sensor</h1>
+             <h1 class="header">Cooperation</h1>
          </section>
+        <div class="container divide essay">
+            <ul>
+                <li>
+                    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&#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>
+                </li>
+                <br/>
+                <li>Kyoto iGEM team: <br/><br/>
+                    <div class="row">
+                        <div class="col-md-7" style="margin-top: 40px">
+                        <p>We are helping <a href="https://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>
+                        </div>
+                        <div class="col-md-5">
+                            <img class="img-responsive" src="https://static.igem.org/mediawiki/igem.org/6/6d/HUMANPRACTICE_2.jpg" alt-src="./images/Human_practice/HUMANPRACTICE_2.jpg">
+                        </div>
+                    </div>
+                </li>
+                <li>NTU_Taida iGEM team: <br/><br/>
+                    <div class="row">
+                        <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>
+                        <div class="row text-center">
+                            <img class="img-responsive" src="https://static.igem.org/mediawiki/igem.org/6/66/Cooperation_taida.png" alt-src="./images/cooperation_taida.jpg">
+                        </div>
+                </li>
+            </ul>
+        </div>
+    </script>
+    <script type="text/template" id="App">
+        <section class="yellow-background">
+            <h1 class="header">App</h1>
+        </section>
+        <div class="container divide essay">
+            <div class="row text-center" style="margin-left: 20px; margin-right: 20px">
+                <h3>Let us introduce our App in this short film :)</h3><br/>
+                <iframe id="appMovie" style="width=75%;" src="http://www.youtube.com/embed/NjNNvrgOozA" frameborder="0" allowfullscreen></iframe><br/>
+            </div>
+        </div>
          <div class="container divide essay">
              <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.
+                 As we known, for most of people who has never learned synthetic biology, it&lsquo;s a magical and crazy idea to take biological parts as the electronic parts. Moreover, someone might be scaried about this technique just like &quot;Witch Hunt&quot; in middle centry, because he don&lsquo;t know what is it.
+            </p>
+            <p>
+                When we talk about synthetic biology, most of our friends will connect this technique with &quot;Genome Modified Organism&quot; and &quot;Terrible Biological Weapon&quot;. They have antagonistic feeling in the first time because they don&lsquo;t know about it. However, beyond our expectation that they still feeling unbelieveble after our explaination......
+            </p>
+            <p>
+                In hence, we decide to make an App to teach them the basic biotech concept and let them know the synthetic biology is not so hazardous. The main idea of this game is let players know if they don&lsquo;t care about the biosafety, they will let environment endanger. But if they are cautious in their operation, most of the results will under their control.
              </p>
          </div>
      </script>
-     <script type="text/template" id="humanpractice">
+     <script type="text/template" id="Conference">
          <section class="blue-background">
              <h1 class="header">Conference</h1>
          </section>
-         <div class="row" style="margin-left: 20px; margin-right: 20px">
+         <div class="divide">
-            <img class="pull-right img-responsive" src="./images/UT_conference.jpg">
+            <div class="row" style="margin-left: 20px; margin-right: 20px">
-            <div class="row text-center" style="margin-top: 90px">
+                <img class="pull-right img-responsive" src="https://static.igem.org/mediawiki/2013/7/7b/NTU_TAIWAN_UT_conference.jpg" alt-src="./images/UT_conference.jpg">
-                <b><h2>Conference with UT_Tokyo Team</h2></b><br/>
+                <div class="row text-center" style="margin-top: 90px">
-                On 4th May, We held a video conference with UT_Tokyo team. It's our first time to have video conference with foreign iGEM team! We gave a briefly project introduction and discussed about experimental problems to each other. By the discussion, we found out some bugs but some new ideas in our project. On the other hand, we also gave some advices to UT_Tokyo team. That's a great chance for us to practice how can we express our project entirely and clearly.
+                    <b><h2>Conference with UT_Tokyo Team</h2></b><br/>
+                    On 4th May, We held a video conference with UT_Tokyo team. It's our first time to have video conference with foreign iGEM team! We gave a briefly project introduction and discussed about experimental problems to each other. By the discussion, we found out some bugs but some new ideas in our project. On the other hand, we also gave some advices to UT_Tokyo team. That's a great chance for us to practice how can we express our project entirely and clearly.
+                </div>
              </div>
-        </div>
-        <div class="row" style="margin-left: 20px; margin-right: 20px">
+            <div class="row" style="margin-left: 20px; margin-right: 20px">
-            <img class="pull-left img-responsive" src="./images/UT_conference in Tokyo.jpg">
+                <img class="pull-left img-responsive" src="https://static.igem.org/mediawiki/2013/2/27/NTU_TAIWAN_UT_conference_in_Tokyo.jpg" alt-src="./images/UT_conference in Tokyo.jpg">
-            <div class="row text-center" style="margin-top: 150px">
+                <div class="row text-center" style="margin-top: 150px">
-                <b><h2>Conference in Tokyo University</h2></b><br/>
+                    <b><h2>Conference in Tokyo University</h2></b><br/>
-                On 14th July, Yi-Yuan Lee met UT_Tokyo iGEM team in Tokyo University. UT_Tokyo team briefly performed their currently progress and started to discuss their project. For Yi-Yuan, he learned more experience about how to well organitze an iGEM team.
+                    On 14th July, Yi-Yuan Lee met UT_Tokyo iGEM team in Tokyo University. UT_Tokyo team briefly performed their currently progress and started to discuss their project. For Yi-Yuan, he learned more experience about how to well organitze an iGEM team.
+                </div>
              </div>
-        </div>
-        <div class="row" style="margin-left: 20px; margin-right: 20px">
+            <div class="row" style="margin-left: 20px; margin-right: 20px">
-            <img class="pull-right img-responsive" src="./images/Purdue_conference.jpg">
+                <img class="pull-right img-responsive" src="https://static.igem.org/mediawiki/2013/8/80/NTU_TAIWAN_Purdue_conference.jpg" alt-src="./images/Purdue_conference.jpg">
-            <div class="row text-center" style="margin-top: 150px">
+                <div class="row text-center" style="margin-top: 150px">
-                <b><h2>Conference with Purdue iGEM Team</h2></b><br/>
+                    <b><h2>Conference with Purdue iGEM Team</h2></b><br/>
-                We had a video conference with Purdue iGEM team, Manchester iGEM team, TU Eindhoven iGEM team, and Kyoto iGEM team on July 8th. This is Purdue iGEM team&rsquo;s project. They want to build up a new standardized form for BioBricks register by cooperating and discussing with worldwide iGEM teams. We complete the questionnaire and gave them some advices.
+                    We had a video conference with Purdue iGEM team, Manchester iGEM team, TU Eindhoven iGEM team, and Kyoto iGEM team on July 8th. This is Purdue iGEM team&rsquo;s project. They want to build up a new standardized form for BioBricks register by cooperating and discussing with worldwide iGEM teams. We complete the questionnaire and gave them some advices.
+                </div>
              </div>
-        </div>
-        <div class="row" style="margin-left: 20px; margin-right: 20px">
+            <div class="row" style="margin-left: 20px; margin-right: 20px">
-            <img class="pull-left img-responsive" src="./images/Berkeley_conference.jpg" width=700 height=525>
+                <img class="pull-left img-responsive" src="https://static.igem.org/mediawiki/2013/b/bc/NTU_TAIWAN_Berkeley_conference.jpg" alt-src="./images/Berkeley_conference.jpg">
-            <div class="row text-center" style="margin-top: 150px">
+                <div class="row text-center" style="margin-top: 150px">
-                <b><h2>Conference in UC Berkeley</h2></b><br/>
+                    <b><h2>Conference in UC Berkeley</h2></b><br/>
-                YI-Yuan and Chi-Wei had a face to face conference with Berkeley iGEM team on July 10th. We presented project and experimental design to each other, and discussed about better methods to break through our problems.<br/>
+                    YI-Yuan and Chi-Wei had a face to face conference with Berkeley iGEM team on July 10th. We presented project and experimental design to each other, and discussed about better methods to break through our problems.<br/>
-                After the conference, we had a late lunch together in fornt of the UC Berkeley with Berkeley iGEMers. We talked from our major to where did we come from. That was a great meal with them.
+                    After the conference, we had a late lunch together in fornt of the UC Berkeley with Berkeley iGEMers. We talked from our major to where did we come from. That was a great meal with them.
+                </div>
              </div>
-        </div>
-        <div class="row" style="margin-left: 20px; margin-right: 20px">
+            <div class="row" style="margin-left: 20px; margin-right: 20px">
-             <img class="pull-right img-responsive" src="./images/Chiao_Tung_Conference/(27).jpg" width=700>
+             <img class="pull-right img-responsive" src="https://static.igem.org/mediawiki/2013/5/55/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2827%29.jpg" alt-src="./images/Chiao_Tung_Conference/(27).jpg" width=700>
              <div class="row text-center" style="margin-top: 100px">
                  <b><h2>Conference in Chiao Tung University</h2></b><br/>
@@ Line 394: / Line 461: @@
              </div>
          </div>
+        </div>
+    </script>
+    <script type="text/template" id="Azalea_Festival">
          <section class="purple-background">
              <h1 class="header">NTU Azalea Festival</h1>
          </section>
          <div class="container divide essay">
-             <div class="row divide" style="margin-left: 20px; margin-right: 20px">
+             <div class="row divide">
                  <div class="col-md-6">
-                     <img class="pull-left img-responsive" src="./images/AZALEA/(1).jpg">
+                     <img class="pull-left img-responsive" src="https://static.igem.org/mediawiki/2013/7/7a/NTU_TAIWAN_AZALEA%281%29.jpg" alt-src="./images/AZALEA/(1).jpg">
                  </div>
                  <div class="col-md-6">
-                     <img class="pull-right img-responsive" src="./images/AZALEA/(10).jpg">
+                     <img class="pull-right img-responsive" src="https://static.igem.org/mediawiki/2013/1/14/NTU_TAIWAN_AZALEA%2810%29.jpg" alt-src="./images/AZALEA/(10).jpg">
                  </div>
              </div>
@@ Line 411: / Line 481: @@
                  NTU Azalea Festival is a anneal event for introducing each department to senior high school students. By this chance, we wanted to spread the iGEM idea to high school students. We design a simple computer game which contain the concept of synthetic biology, molecular biology and bio-Technology. First, we asked them to choose a right enzyme to digest the glactose. And in second level, we asked them to make the enzyme they used in previous level by amino acids, right RNA, and right enzyme. In the last level, we asked them to build up a operon for galactase production.
              </p>
+            <a class="hover-shodow hover-no-underline" href="http://ntu-taiwan-azalea.herokuapp.com"><i class="icon-gamepad icon-2x">Here’s our Little Game</i></a>
          </div>
+    </script>
+    <script type="text/template" id="Poster">
          <section class="green-background">
-        <h1 class="header">Poster Presentation</h1>
+            <h1 class="header">Poster Presentation</h1>
          </section>
          <div class="container divide essay">
              <div class="row" style="margin-left: 20px; margin-right: 20px">
                  <div class="col-md-4">
-                     <img class="img-responsive" src="./images/MEETING/Meeting-(10).jpg" width=400>
+                     <img class="img-responsive" src="https://static.igem.org/mediawiki/igem.org/1/13/Meeting-%2810%29.jpg" alt-src="./images/MEETING/Meeting-(10).jpg" width=400>
                  </div>
@@ Line 430: / Line 503: @@
              </div>
          </div>
+    </script>
-        <section class="yellow-background">
-        <h1 class="header">App</h1>
-        </section>
-        <div class="container divide essay">
-        <div class="row text-center" style="margin-left: 20px; margin-right: 20px">
-        <h3>Let us introduce our App in this short film :)</h3><br/>
-        <iframe id="appMovie" style="width=75%;" src="http://www.youtube.com/embed/NjNNvrgOozA" frameborder="0" allowfullscreen></iframe><br/></div></div>
-        <div class="container divide essay">
-            <p>
-                As we known, for most of people who has never learned synthetic biology, it&lsquo;s a magical and crazy idea to take biological parts as the electronic parts. Moreover, someone might be scaried about this technique just like &quot;Witch Hunt&quot; in middle centry, because he don&lsquo;t know what is it.
-            </p>
-            <p>
-                When we talk about synthetic biology, most of our friends will connect this technique with &quot;Genome Modified Organism&quot; and &quot;Terrible Biological Weapon&quot;. They have antagonistic feeling in the first time because they don&lsquo;t know about it. However, beyond our expectation that they still feeling unbelieveble after our explaination......
-            </p>
-            <p>
-                In hence, we decide to make an App to teach them the basic biotech concept and let them know the synthetic biology is not so hazardous. The main idea of this game is let players know if they don&lsquo;t care about the biosafety, they will let environment endanger. But if they are cautious in their operation, most of the results will under their control.
-            </p>
-        </div>
-        <section class="red-background">
-            <h1 class="header">Cooperation</h1>
-        </section>
-        <div class="container divide essay">
-            <ul style="list-style:style">
-                <li>
-                    Collaborate with Perdue iGEM team: <br/><br/>
-                    <p>We collaborated with Perdue iGEM team to design a better version of datasheet when we register our parts. Following is one of the beta-version of their datasheet. </p>
-                    <embed src="./PDF/Datasheet Composite Part Example.pdf" style="width: 100%; height: 500px"></embed>
-                </li>
-                <br/>
-                <li>Kyoto iGEM team: <br/><br/>
-                    <div class="row">
-                        <div class="col-md-7">
-                            <p>We help Kyoto iGEM team to charaterise their parts. They sent 13 parts to us. We keep discussing how to design the construction and which gene should we use in the characterisation.</p>
-                        </div>
-                        <div class="col-md-5">
-                            <img class="img-responsive" src="./images/Human_practice/HUMANPRACTICE_2.jpg" style="margin-right: 30px">
-                        </div>
-                    </div>
-                </li>
-                <li>NTU_Taida iGEM team: <br/>
-            </ul>
-        </div>
-        </script>
-     <script type="text/template" id="teamCalendar">
+     <script type="text/template" id="teamMember">
          <section class="purple-background" style="padding-bottom: 0;">
              <h1 class="header" style="margin: 0;">Team Members</h1>
@@ Line 758: / Line 783: @@
              </div>
          </section>
          <div class="teamMember divide">
-             <section id="teamContainer" class="blue-background" cur-color="blue-background">
+             <section id="teamContainer" class="blue-background">
+                <h1 class="header">Team Members</h1>
                  <div class="row">
-                     <div class="col-md-5"><img class="img-responsive" src="./images/people/李易遠.jpg"></div>
+                     <div class="col-md-4"><img class="img-responsive" src="https://static.igem.org/mediawiki/2013/3/38/李易遠.jpg" alt-src="./images/people/李易遠.jpg"></div>
-                     <div class="col-md-7">
+                     <div class="col-md-8">
-                         <h2>李易遠(Yi-Yuan Lee)</h2>
+                         <h2>李易遠 (Yi-Yuan Lee)</h2>
                          <div class="row">
-                             <b><u>Team Leader</u></b>
+                             <b><u>Team Leader & Lab Hero</u></b>
                          </div>
                          <div class="row">
@@ Line 775: / Line 800: @@
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interests: </b> <div class="col-md-8"> Reading, Calligraphy, Ballroom dance, Swimming, Traveling </div>
+                             <b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> Reading, Calligraphy, Ballroom dance, Swimming, Traveling </div>
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interested: </b> <div class="col-md-8"> Immunology, Bioinformatics, Virology </div>
+                             <b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> Immunology, Bioinformatics, Programming, Asian history </div>
                          </div>
                          <div class="row">
@@ Line 788: / Line 813: @@
              <div class="row red-background">
                  <ul class="img-gallery">
-                     <li class="active" data-toggle="tooltip" title="Team Leader"><img src="./images/people/李易遠.jpg" toggle="李易遠" ></li>
+                     <li class="active" data-toggle="tooltip" title="Team Leader"><img src="https://static.igem.org/mediawiki/2013/3/38/李易遠.jpg" alt-src="./images/people/李易遠.jpg" toggle="李易遠" ></li>
-                     <li data-toggle="tooltip" title="Chief of Lab Hero"><img src="./images/people/呂宗諭.jpg" toggle="呂宗諭"></li>
+                     <li data-toggle="tooltip" title="Chief of Lab Hero"><img src="https://static.igem.org/mediawiki/2013/6/67/呂宗諭.jpg" alt-src="./images/people/呂宗諭.jpg" toggle="呂宗諭"></li>
-                     <li data-toggle="tooltip" title="Lab Surfer"><img src="./images/people/張宏宇.jpg" toggle="張宏宇"></li>
+                     <li data-toggle="tooltip" title="Lab Surfer"><img src="https://static.igem.org/mediawiki/2013/9/99/張宏宇.jpg" alt-src="./images/people/張宏宇.jpg" toggle="張宏宇"></li>
-                     <li data-toggle="tooltip" title="Lab Anego"><img src="./images/people/馬蓁華.jpg" toggle="馬蓁華"></li>
+                     <li data-toggle="tooltip" title="Lab Anego"><img src="https://static.igem.org/mediawiki/2013/0/0e/馬蓁華.jpg" alt-src="./images/people/馬蓁華.jpg" toggle="馬蓁華"></li>
-                     <li data-toggle="tooltip" title="Fake Secretary"><img src="./images/people/陳俋秀.jpg" toggle="陳俋秀"></li>
+                     <li data-toggle="tooltip" title="Fake Secretary"><img src="https://static.igem.org/mediawiki/2013/5/5e/陳俋秀.jpg" alt-src="./images/people/陳俋秀.jpg" toggle="陳俋秀"></li>
-                     <li data-toggle="tooltip" title="LuLu"><img src="./images/people/盧彥云.jpg" toggle="盧彥云"></li>
+                     <li data-toggle="tooltip" title="LuLu"><img src="https://static.igem.org/mediawiki/2013/8/86/盧彥云.jpg" alt-src="./images/people/盧彥云.jpg" toggle="盧彥云"></li>
-                     <li data-toggle="tooltip" title="Lily LOL Fighter"><img src="./images/people/吳泰億.jpg" toggle="吳泰億"></li>
+                     <li data-toggle="tooltip" title="Lily LOL Fighter"><img src="https://static.igem.org/mediawiki/2013/a/a6/吳泰億.jpg" alt-src="./images/people/吳泰億.jpg" toggle="吳泰億"></li>
-                     <li data-toggle="tooltip" title="Lab Physician"><img src="./images/people/王德緯.jpg" toggle="王德緯"></li>
+                     <li data-toggle="tooltip" title="Lab Physician"><img src="https://static.igem.org/mediawiki/2013/4/46/王德緯.jpg" alt-src="./images/people/王德緯.jpg" toggle="王德緯"></li>
-                     <li data-toggle="tooltip" title="Web Hacker"><img src="./images/people/王柏軒.jpg" toggle="王柏軒"></li>
+                     <li data-toggle="tooltip" title="Web Hacker"><img src="https://static.igem.org/mediawiki/2013/6/68/王柏軒.jpg" alt-src="./images/people/王柏軒.jpg" toggle="王柏軒"></li>
-                     <li data-toggle="tooltip" title="Niwei"><img src="./images/people/李威.jpg" toggle="李威"></li>
+                     <li data-toggle="tooltip" title="Niwei"><img src="https://static.igem.org/mediawiki/2013/1/1b/李威.jpg" alt-src="./images/people/李威.jpg" toggle="李威"></li>
-                     <li data-toggle="tooltip" title="Y-way"><img src="./images/people/李啟為.jpg" toggle="李啟為"></li>
+                     <li data-toggle="tooltip" title="Y-way"><img src="https://static.igem.org/mediawiki/2013/3/33/李啟為.jpg" alt-src="./images/people/李啟為.jpg" toggle="李啟為"></li>
-                     <li data-toggle="tooltip" title="Secret staff"><img src="./images/people/王凡.jpg" toggle="王凡"></li>
+                     <li data-toggle="tooltip" title="Secret staff"><img src="https://static.igem.org/mediawiki/2013/e/e4/王凡.jpg" alt-src="./images/people/王凡.jpg" toggle="王凡"></li>
-                     <li data-toggle="tooltip" title="Lynda"><img src="./images/people/林湧達.jpg" toggle="林湧達"></li>
+                     <li data-toggle="tooltip" title="Lynda"><img src="https://static.igem.org/mediawiki/2013/f/fa/林湧達.jpg" alt-src="./images/people/林湧達.jpg" toggle="林湧達"></li>
                  </ul>
              </div>
@@ Line 808: / Line 833: @@
              <section id="李易遠" color="blue-background">
                  <div class="row">
-                     <div class="col-md-4"><img class="img-responsive" src="./images/people/李易遠.jpg"></div>
+                     <div class="col-md-4"><img class="img-responsive" src="https://static.igem.org/mediawiki/2013/3/38/李易遠.jpg" alt-src="./images/people/李易遠.jpg"></div>
                      <div class="col-md-8">
-                         <h2>李易遠(Yi-Yuan Lee)</h2>
+                         <h2>李易遠 (Yi-Yuan Lee)</h2>
                          <div class="row">
-                             <b><u>Team Leader</u></b>
+                             <b><u>Team Leader & Lab Hero</u></b>
                          </div>
                          <div class="row">
@@ Line 821: / Line 846: @@
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interests: </b> <div class="col-md-8"> Reading, Calligraphy, Ballroom dance, Swimming, Traveling </div>
+                             <b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> Reading, Calligraphy, Ballroom dance, Swimming, Traveling </div>
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interested: </b> <div class="col-md-8"> Immunology, Bioinformatics, Virology </div>
+                             <b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> Immunology, Bioinformatics, Programming, Asian history </div>
                          </div>
                          <div class="row">
@@ Line 836: / Line 861: @@
                  <div class="row">
                      <div class="col-md-8">
-                         <h2>吳泰億(Tai-Yi Wu)</h2>
+                         <h2>吳泰億 (Tai-Yi Wu)</h2>
                          <div class="row">
                              <b><u>Lily LOL Fighter</u></b>
@@ Line 847: / Line 872: @@
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interests: </b> <div class="col-md-8"> swim, music, stage play </div>
+                             <b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> swim, music, stage play </div>
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interested: </b> <div class="col-md-8"> Fermentology, Applied Microbiology  </div>
+                             <b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> Fermentology, Applied Microbiology  </div>
                          </div>
                          <div class="row">
@@ Line 856: / Line 881: @@
                          </div>
                      </div>
-                     <div class="col-md-4"><img class="img-responsive" src="./images/people/吳泰億.jpg"></div>
+                     <div class="col-md-4"><img class="img-responsive" src="https://static.igem.org/mediawiki/2013/a/a6/吳泰億.jpg" alt-src="./images/people/吳泰億.jpg"></div>
                  </div>
              </section>
@@ Line 862: / Line 887: @@
              <section id="呂宗諭" color="yellow-background">
                  <div class="row">
-                     <div class="col-md-4"><img class="img-responsive" src="./images/people/呂宗諭.jpg"></div>
+                     <div class="col-md-4"><img class="img-responsive" src="https://static.igem.org/mediawiki/2013/6/67/呂宗諭.jpg" alt-src="./images/people/呂宗諭.jpg"></div>
                      <div class="col-md-8">
-                         <h2>呂宗諭(Tsung-Yu Lu)</h2>
+                         <h2>呂宗諭 (Tsung-Yu Lu)</h2>
                          <div class="row">
                              <b><u>Chief of Lab Hero</u></b>
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Department: </b> <div class="col-md-8"> Biochemical Science and Technology </div>
+                             <b class="col-md-4"> Department: </b> <div class="col-md-8"> Insititute of Biochemial Sciences </div>
                          </div>
                          <div class="row">
@@ Line 875: / Line 900: @@
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interests: </b> <div class="col-md-8"> swim, music, stage play </div>
+                             <b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> swim, music, stage play </div>
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interested: </b> <div class="col-md-8"> protein engineering, mathematical biology </div>
+                             <b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> protein engineering, mathematical biology </div>
                          </div>
                          <div class="row">
@@ Line 890: / Line 915: @@
                  <div class="row">
                      <div class="col-md-8">
-                         <h2>張宏宇(Hung-Yu Chang)</h2>
+                         <h2>張宏宇 (Hung-Yu Chang)</h2>
                          <div class="row">
-                             <b><u>Lab Surfer</u></b>
+                             <b><u>Lab Surfer & Lab Hero</u></b>
                          </div>
                          <div class="row">
@@ Line 901: / Line 926: @@
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interests: </b> <div class="col-md-8"> Debate, Tennis, Surfing </div>
+                             <b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> Debate, Tennis, Surfing </div>
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interested: </b> <div class="col-md-8"> Cell Biology, Tissue Engineering </div>
+                             <b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> Cell Biology, Tissue Engineering </div>
                          </div>
                          <div class="row">
@@ Line 910: / Line 935: @@
                          </div>
                      </div>
-                     <div class="col-md-4"><img class="img-responsive" src="./images/people/張宏宇.jpg"></div>
+                     <div class="col-md-4"><img class="img-responsive" src="https://static.igem.org/mediawiki/2013/9/99/張宏宇.jpg" alt-src="./images/people/張宏宇.jpg"></div>
                  </div>
              </section>
@@ Line 916: / Line 941: @@
              <section id="王德緯" color="green-background">
                  <div class="row">
-                     <div class="col-md-4"><img class="img-responsive" src="./images/people/王德緯.jpg"></div>
+                     <div class="col-md-4"><img class="img-responsive" src="https://static.igem.org/mediawiki/2013/4/46/王德緯.jpg" alt-src="./images/people/王德緯.jpg"></div>
                      <div class="col-md-8">
-                         <h2>王德緯(Te-Wei Wang)</h2>
+                         <h2>王德緯 (Te-Wei Wang)</h2>
                          <div class="row">
                              <b><u>Lab Physician</u></b>
@@ Line 929: / Line 954: @@
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interests: </b> <div class="col-md-8"> reading, movie, volleyball, Japanese </div>
+                             <b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> reading, movie, volleyball, Japanese </div>
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interested: </b> <div class="col-md-8"> physiology, immunology, neurology </div>
+                             <b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> physiology, immunology, neurology </div>
                          </div>
                          <div class="row">
@@ Line 945: / Line 970: @@
                  <div class="row">
                      <div class="col-md-8">
-                         <h2>馬蓁華(Chen-Hwa Ma)</h2>
+                         <h2>馬蓁華 (Chen-Hwa Ma)</h2>
                          <div class="row">
                              <b><u>Lab Anego</u></b>
@@ Line 956: / Line 981: @@
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interests: </b> <div class="col-md-8"> traveling, reading </div>
+                             <b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> traveling, reading </div>
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interested: </b> <div class="col-md-8"> sociology, literature, biochemistry </div>
+                             <b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> sociology, literature, biochemistry </div>
                          </div>
                          <div class="row">
@@ Line 965: / Line 990: @@
                          </div>
                      </div>
-                     <div class="col-md-4"><img class="img-responsive" src="./images/people/馬蓁華.jpg"></div>
+                     <div class="col-md-4"><img class="img-responsive" src="https://static.igem.org/mediawiki/2013/0/0e/馬蓁華.jpg" alt-src="./images/people/馬蓁華.jpg"></div>
                  </div>
              </section>
@@ Line 971: / Line 996: @@
              <section id="陳俋秀" color="pink-background">
                  <div class="row">
-                     <div class="col-md-4"><img class="img-responsive" src="./images/people/陳俋秀.jpg"></div>
+                     <div class="col-md-4"><img class="img-responsive" src="https://static.igem.org/mediawiki/2013/5/5e/陳俋秀.jpg" alt-src="./images/people/陳俋秀.jpg"></div>
                      <div class="col-md-8">
-                         <h2>陳俋秀(Yi-Hsiu Chen)</h2>
+                         <h2>陳俋秀 (Yi-Hsiu Chen)</h2>
                          <div class="row">
                              <b><u>Fake Secretary</u></b>
@@ Line 984: / Line 1,009: @@
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interests: </b> <div class="col-md-8"> reading,playing,volleyball,movie </div>
+                             <b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> reading,playing,volleyball,movie </div>
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interested: </b> <div class="col-md-8"> Immunology, Nutritional </div>
+                             <b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> Immunology, Nutritional </div>
                          </div>
                          <div class="row">
@@ Line 999: / Line 1,024: @@
                  <div class="row">
                      <div class="col-md-8">
-                         <h2>盧彥云(Yen-Yun Lu)</h2>
+                         <h2>盧彥云 (Yen-Yun Lu)</h2>
                          <div class="row">
                              <b><u>LuLu</u></b>
@@ Line 1,010: / Line 1,035: @@
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interests: </b> <div class="col-md-8"> dance, cooking, traveling, reading </div>
+                             <b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> dance, cooking, traveling, reading </div>
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interested: </b> <div class="col-md-8"> Molecular biology, cell biology </div>
+                             <b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> Molecular biology, cell biology </div>
                          </div>
                          <div class="row">
@@ Line 1,019: / Line 1,044: @@
                          </div>
                      </div>
-                     <div class="col-md-4"><img class="img-responsive" src="./images/people/盧彥云.jpg"></div>
+                     <div class="col-md-4"><img class="img-responsive" src="https://static.igem.org/mediawiki/2013/8/86/盧彥云.jpg" alt-src="./images/people/盧彥云.jpg"></div>
                  </div>
              </section>
@@ Line 1,025: / Line 1,050: @@
              <section id="王柏軒" color="brown-background">
                  <div class="row">
-                     <div class="col-md-4"><img class="img-responsive" src="./images/people/王柏軒.jpg"></div>
+                     <div class="col-md-4"><img class="img-responsive" src="https://static.igem.org/mediawiki/2013/6/68/王柏軒.jpg" alt-src="./images/people/王柏軒.jpg"></div>
                      <div class="col-md-8">
-                         <h2>王柏軒(Po-Hsien Wang)</h2>
+                         <h2>王柏軒 (Po-Hsien Wang)</h2>
                          <div class="row">
                              <b><u>Web Hacker</u></b>
@@ Line 1,038: / Line 1,063: @@
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interests: </b> <div class="col-md-8"> reading, movie </div>
+                             <b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> reading, movie </div>
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interested: </b> <div class="col-md-8"> AI, Virtual Reality </div>
+                             <b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> AI, Virtual Reality </div>
                          </div>
                          <div class="row">
@@ Line 1,053: / Line 1,078: @@
                  <div class="row">
                      <div class="col-md-8">
-                         <h2>李威(Wei Lee)</h2>
+                         <h2>李威 (Wei Lee)</h2>
                          <div class="row">
                              <b><u>Niwei</u></b>
@@ Line 1,064: / Line 1,089: @@
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interests: </b> <div class="col-md-8"> badminton, photography, reading </div>
+                             <b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> badminton, photography, reading </div>
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interested: </b> <div class="col-md-8"> Communication, AI, App & Web developing </div>
+                             <b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> Communication, AI, App & Web developing </div>
                          </div>
                          <div class="row">
@@ Line 1,073: / Line 1,098: @@
                          </div>
                      </div>
-                     <div class="col-md-4"><img class="img-responsive" src="./images/people/李威.jpg"></div>
+                     <div class="col-md-4"><img class="img-responsive" src="https://static.igem.org/mediawiki/2013/1/1b/李威.jpg" alt-src="./images/people/李威.jpg"></div>
                  </div>
              </section>
@@ Line 1,079: / Line 1,104: @@
              <section id="李啟為" color="green-background">
                  <div class="row">
-                     <div class="col-md-4"><img class="img-responsive" src="./images/people/李啟為.jpg"></div>
+                     <div class="col-md-4"><img class="img-responsive" src="https://static.igem.org/mediawiki/2013/3/33/李啟為.jpg" alt-src="./images/people/李啟為.jpg"></div>
                      <div class="col-md-8">
-                         <h2>李啟為(Chi-Wei Lee)</h2>
+                         <h2>李啟為 (Chi-Wei Lee)</h2>
                          <div class="row">
                              <b><u>Y-way</u></b>
@@ Line 1,092: / Line 1,117: @@
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interests: </b> <div class="col-md-8"> baseball, girl, post rock </div>
+                             <b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> baseball, girl, post rock </div>
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interested: </b> <div class="col-md-8"> ML, AI, probability graphical model </div>
+                             <b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> ML, AI, probability graphical model </div>
                          </div>
                          <div class="row">
@@ Line 1,107: / Line 1,132: @@
                  <div class="row">
                      <div class="col-md-8">
-                         <h2>王凡(Fang Wang)</h2>
+                         <h2>王凡 (Fang Wang)</h2>
                          <div class="row">
                              <b><u>Secret staff</u></b>
@@ Line 1,118: / Line 1,143: @@
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interests: </b> <div class="col-md-8"> fixed gear, table tennis, photography </div>
+                             <b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> fixed gear, table tennis, photography </div>
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interested: </b> <div class="col-md-8"> Computational photography, App </div>
+                             <b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> Computational photography, App </div>
                          </div>
                          <div class="row">
@@ Line 1,127: / Line 1,152: @@
                          </div>
                      </div>
-                     <div class="col-md-4"><img class="img-responsive" src="./images/people/王凡.jpg"></div>
+                     <div class="col-md-4"><img class="img-responsive" src="https://static.igem.org/mediawiki/2013/e/e4/王凡.jpg" alt-src="./images/people/王凡.jpg"></div>
                  </div>
              </section>
@@ Line 1,133: / Line 1,158: @@
              <section id="林湧達" color="lightblue-background">
                  <div class="row">
-                     <div class="col-md-4"><img class="img-responsive" src="./images/people/林湧達.jpg"></div>
+                     <div class="col-md-4"><img class="img-responsive" src="https://static.igem.org/mediawiki/2013/f/fa/林湧達.jpg" alt-src="./images/people/林湧達.jpg"></div>
                      <div class="col-md-8">
-                         <h2>林湧達(Yung-Ta Lin)</h2>
+                         <h2>林湧達 (Yung-Ta Lin)</h2>
                          <div class="row">
                              <b><u>Lynda</u></b>
@@ Line 1,146: / Line 1,171: @@
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interests: </b> <div class="col-md-8"> drawing, tennis, tap dance, Chinese comedy </div>
+                             <b class="col-md-4"> Hobbies: </b> <div class="col-md-8"> drawing, tennis, tap dance, Chinese comedy </div>
                          </div>
                          <div class="row">
-                             <b class="col-md-4"> Interested: </b> <div class="col-md-8"> UI, HCI, VR </div>
+                             <b class="col-md-4"> Interested fields: </b> <div class="col-md-8"> UI, HCI, VR </div>
                          </div>
                          <div class="row">
@@ Line 1,158: / Line 1,183: @@
              </section>
          </div>
+    </script>
+    <script type="text/template" id="attribution">
+        <section class="yellow-background">
+            <h1 class="header">Attribution</h1>
+            <p class="header"><i class="icon-paper-clip"> our works tools </i></p>
+        </section>
+        <div class="divide essay container">
+            <div>
+                <legend><h2 style="border-bottom: 0">Website</h2></legend>
+                <ol>
+                    <li>
+                        <a href="http://jquery.com/">jQuery</a><br />
+                        a fast, small, and feature-rich JavaScript library. It makes things like HTML document traversal and manipulation, event handling, animation, and Ajax much simpler with an easy-to-use API that works across a multitude of browsers. With a combination of versatility and extensibility, jQuery has changed the way that millions of people write JavaScript.
+                    </li>
+                    <li>
+                        <a href="http://getbootstrap.com/">BootStrap</a><br />
+                        Sleek, intuitive, and powerful mobile first front-end framework for faster and easier web development.
+                    </li>
+                    <li>
+                        <a href="http://underscorejs.org/">UnderScore</a><br />
+                        a utility-belt library for JavaScript that provides a lot of the functional programming support that you would expect in Prototype.js (or Ruby), but without extending any of the built-in JavaScript objects.
+                    </li>
+                    <li>
+                        <a href="http://backbonejs.org/">BackBone</a><br />
+                        BackBone gives structure to web applications by providing models with key-value binding and custom events, collections with a rich API of enumerable functions, views with declarative event handling, and connects it all to your existing API over a RESTful JSON interface.
+                    </li>
+                    <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&#39;s a snap to setup and works on all modern browsers.
+                    </li>
+                    <li>
+                        <a href="http://fortawesome.github.io/Font-Awesome/">FontAwesome</a><br />
+                        Font Awesome gives you scalable vector icons that can instantly be customized — size, color, drop shadow, and anything that can be done with the power of CSS.
+                    </li>
+                    <li>
+                        <a href="http://fittextjs.com/">FitText</a><br />
+                        FitText makes font-sizes flexible. Use this plugin on your fluid or responsive layout to achieve scalable headlines that fill the width of a parent element.
+                    </li>
+                    <li>
+                        <a href="http://www.dpereyra.com/scripts/dp_calendar/">SIMPLE EVENTS CALENDAR</a>
+                        jQuery Basic Event Calendar is a highly configurable plugin that adds calendar functionality to your pages.
+                    </li>
+                </ol>
+            </div>
+            <div>
+                <legend><h2 style="border-bottom: 0">Wet Lab</h2></legend>
+                <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>
+                <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>
+                <p>All of the following DNA were designed, cloned, or constructed by our wetlab team members:</p>
+.	TAP protein tag was cloned from pRS424 shuttle vector.<br/>
+.	Tir1 promoter sequence was amplified from genomic DNA of <i>Saccharomyces cerevisiae</i>.<br/>
+.	26s and 5.8s ITS rDNA was cloned from <i>Rhodotorula glutinis</i>.<br/>
+.	All of the PCR primers of cloning were design by ourselves.<br/>
+.	The construction of pRS424:GAL1:SrUCP:TAP, pGAPZA:26s, pGAPZA:5.8s ITS.<br/>
+                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.
+            </div>
+        </div>
+    </script>
+    <script type="text/template" id="acknowledgement">
+        <section class="red-background">
+            <h1 class="header">Acknowledgement</h1>
+        </section>
+        <div class="divide essay container">
+        <p><b>National Taiwan University</b></p>
+        <img src="http://www.bst.ntu.edu.tw/faculty/ChenYR/ChenYR1.files/image009.png" width=300>
+        <p>Our supervisor Dr. Yen-rong Chen for making our adventure in iGEM possible.</p>
+                <img src="http://www.bst.ntu.edu.tw/faculty/HuangCT/HuangCT1.files/image010.png" width=300>
+        <p>Dr. Ching-Tsan Huang for providing us with the shuttle vectors pGAPZA plasmid.</p>
+                <img src="http://www.ym.edu.tw/bps/linjj.files/image001.jpg" width=300>
+        <p>Dr. Jing-Jer Lin for providing us with the shuttle vectors, pRS424 and pRS423.</p>
+                <img src="http://www.bst.ntu.edu.tw/faculty/LinCH/LinCH1.files/image010.jpg" width=300>
+                <img src="http://www.ac.ntu.edu.tw/images/people/Kaiyin%20Lo.jpg" width=300>
+        <p>Dr. Ching-Hsuan Lin, Dr. Kai-Yin Lo and Dr. Jing-Jer Lin for their advice with the yeast molecular techniques.</p>
+                <img src="http://www.chem.sinica.edu.tw/faculty/photo/B/cherri.jpg" width=300>
+        <p>Dr. Chao-Ping Hsu for her help with the modeling part of our project.</p>
+                <img src="http://www.bst.ntu.edu.tw/faculty/LeeKT/LeeKT1.files/image010.png" width=300>
+        <p>Dr. Kung-Ta Lee for his advice of how to measure the heat produce of YeasTherm.</p><br/>
+        <p><b>Institute of Molecular Biology, Academia Sinica</b></p>
+                <img src="http://www.cctmf.org.tw/campcurie/2011/images/speakers/1-2.jpg" width=300>
+                <img src="http://www.imb.sinica.edu.tw/ch/adm-photo/jeng.jpg" width=200>
+        <p>Lab N214 Dr. Michael M.C. Lai and Dr. King-Song Jeng for providing us with lab equipment and materials.</p><br/>
+        <p><b>Iwate University</b></p>
+                <img src="http://www.iwate-u.ac.jp/coe/en/images/kikuito.jpg" width=300>
+        <p>Dr. Kikukatsu Ito for providing us the important gene, SrUCP DNA, for our project.<p><br/>
+        </div>
+    </script>
+    <script type="text/template" id="Calendar">
          <section class="brown-background">
              <h1 class="header">Calendar</h1>
@@ Line 1,177: / Line 1,297: @@
                  <p class="header container">
                      Look what we have done<br/>
-                     <img class="nohover" id="TaiwanLogo" src="images/TaiwanLogo.png">
+                     <img class="nohover" id="TaiwanLogo" src="https://static.igem.org/mediawiki/2013/f/f3/NTU_TAIWAN_TaiwanLogo.png" alt-src="images/TaiwanLogo.png">
                  </p>
              </div>
@@ Line 1,240: / Line 1,360: @@
                  </div>
                  <div class="col-md-2 photo-item">
-                     <a class="pointer-cursor" scroll-to="Result">
+                     <a class="pointer-cursor" scroll-to="App">
                          <div class="row">
                              <span class="icon-stack icon-4x">
                                <i class="icon-stop icon-stack-base font-pink"></i>
-                               <i class="icon-beaker icon-light"></i>
+                               <i class="icon-gamepad icon-light"></i>
                              </span>
                          </div>
-                         <div> Result </div>
+                         <div> App </div>
                      </a>
                  </div>
@@ Line 1,253: / Line 1,373: @@
          </div>
          <section class="red-background divide" id="Meeting">
@@ Line 1,262: / Line 1,383: @@
                  <div class="row">
                      <ul class="tilt-gallery img-2x" lightbox-id="meeting">
-                         <li><img src="./images/MEETING/Meeting-(1).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/8/8f/Meeting-%281%29.jpg" alt-src="./images/MEETING/Meeting-(1).jpg"  ></li>
-                         <li><img src="./images/MEETING/Meeting-(2).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/7/70/Meeting-%282%29.jpg" alt-src="./images/MEETING/Meeting-(2).jpg"  ></li>
-                         <li><img src="./images/MEETING/Meeting-(3).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/a/a7/Meeting-%283%29.jpg" alt-src="./images/MEETING/Meeting-(3).jpg"  ></li>
-                         <li><img src="./images/MEETING/Meeting-(4).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/4/44/Meeting-%284%29.jpg" alt-src="./images/MEETING/Meeting-(4).jpg"  ></li>
-                         <li><img src="./images/MEETING/Meeting-(5).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/6/66/Meeting-%285%29.jpg" alt-src="./images/MEETING/Meeting-(5).jpg"  ></li>
-                         <li><img src="./images/MEETING/Meeting-(6).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/d/d4/Meeting-%286%29.jpg" alt-src="./images/MEETING/Meeting-(6).jpg"  ></li>
-                         <li><img src="./images/MEETING/Meeting-(7).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/a/a1/Meeting-%287%29.jpg" alt-src="./images/MEETING/Meeting-(7).jpg"  ></li>
-                         <li><img src="./images/MEETING/Meeting-(8).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/3/3c/Meeting-%288%29.jpg" alt-src="./images/MEETING/Meeting-(8).jpg"  ></li>
-                         <li><img src="./images/MEETING/Meeting-(9).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/e/e5/Meeting-%289%29.jpg" alt-src="./images/MEETING/Meeting-(9).jpg"  ></li>
-                         <li><img src="./images/MEETING/Meeting-(10).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/1/13/Meeting-%2810%29.jpg" alt-src="./images/MEETING/Meeting-(10).jpg" ></li>
-                         <li><img src="./images/MEETING/Meeting-(11).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/0/0e/Meeting-%2811%29.jpg" alt-src="./images/MEETING/Meeting-(11).jpg" ></li>
-                         <li><img src="./images/MEETING/Meeting-(12).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/6/67/Meeting-%2812%29.jpg" alt-src="./images/MEETING/Meeting-(12).jpg" ></li>
-                         <li><img src="./images/MEETING/Meeting-(13).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/1/1c/Meeting-%2813%29.jpg" alt-src="./images/MEETING/Meeting-(13).jpg" ></li>
-                         <li><img src="./images/MEETING/Meeting-(14).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/0/01/Meeting-%2814%29.jpg" alt-src="./images/MEETING/Meeting-(14).jpg" ></li>
-                         <li><img src="./images/MEETING/Meeting-(15).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/a/a8/Meeting-%2815%29.jpg" alt-src="./images/MEETING/Meeting-(15).jpg" ></li>
-                         <li><img src="./images/MEETING/Meeting-(16).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/f/fd/Meeting-%2816%29.jpg" alt-src="./images/MEETING/Meeting-(16).jpg" ></li>
-                         <li><img src="./images/MEETING/Meeting-(17).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/6/65/Meeting-%2817%29.jpg" alt-src="./images/MEETING/Meeting-(17).jpg" ></li>
-                         <li><img src="./images/MEETING/Meeting-(18).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/a/a2/Meeting-%2818%29.jpg" alt-src="./images/MEETING/Meeting-(18).jpg" ></li>
-                         <li><img src="./images/MEETING/Meeting-(19).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/e/e1/Meeting-%2819%29.jpg" alt-src="./images/MEETING/Meeting-(19).jpg" ></li>
-                         <li><img src="./images/MEETING/Meeting-(20).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/b/bd/Meeting-%2820%29.jpg" alt-src="./images/MEETING/Meeting-(20).jpg" ></li>
-                         <li><img src="./images/MEETING/Meeting-(21).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/e/e1/Meeting-%2821%29.jpg" alt-src="./images/MEETING/Meeting-(21).jpg" ></li>
-                         <li><img src="./images/MEETING/Meeting-(22).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/6/63/Meeting-%2822%29.jpg" alt-src="./images/MEETING/Meeting-(22).jpg" ></li>
-                         <li><img src="./images/MEETING/Meeting-(23).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/0/05/Meeting-%2823%29.jpg" alt-src="./images/MEETING/Meeting-(23).jpg" ></li>
-                         <li><img src="./images/MEETING/Meeting-(24).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/b/bb/Meeting-%2824%29.jpg" alt-src="./images/MEETING/Meeting-(24).jpg" ></li>
-                         <li><img src="./images/MEETING/Meeting-(25).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/0/09/Meeting-%2825%29.jpg" alt-src="./images/MEETING/Meeting-(25).jpg" ></li>
-                         <li><img src="./images/MEETING/Meeting-(26).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/a/a8/Meeting-%2826%29.jpg" alt-src="./images/MEETING/Meeting-(26).jpg" ></li>
-                         <li><img src="./images/MEETING/Meeting-(27).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/e/e2/Meeting-%2827%29.jpg" alt-src="./images/MEETING/Meeting-(27).jpg" ></li>
-                         <li><img src="./images/MEETING/Meeting-(28).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/1/1f/Meeting-%2828%29.jpg" alt-src="./images/MEETING/Meeting-(28).jpg" ></li>
                      </ul>
                  </div>
@@ Line 1,303: / Line 1,424: @@
                  <div class="row">
                      <ul class="tilt-gallery img-2x" lightbox-id="meeting2">
-                         <li><img src="./images/MEETING_2/MEETING_1.jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/3/3d/MEETING_1.jpg" alt-src="./images/MEETING_2/MEETING_1.jpg"  ></li>
-                         <li><img src="./images/MEETING_2/MEETING_2.jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/c/c6/MEETING_2.jpg" alt-src="./images/MEETING_2/MEETING_2.jpg"  ></li>
-                         <li><img src="./images/MEETING_2/MEETING_3.jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/a/ad/MEETING_3.jpg" alt-src="./images/MEETING_2/MEETING_3.jpg"  ></li>
-                         <li><img src="./images/MEETING_2/MEETING_4.jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/7/75/MEETING_4.jpg" alt-src="./images/MEETING_2/MEETING_4.jpg"  ></li>
-                         <li><img src="./images/MEETING_2/MEETING_5.jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/8/82/MEETING_5.jpg" alt-src="./images/MEETING_2/MEETING_5.jpg"  ></li>
-                         <li><img src="./images/MEETING_2/MEETING_6.jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/7/7b/MEETING_6.jpg" alt-src="./images/MEETING_2/MEETING_6.jpg"  ></li>
-                         <li><img src="./images/MEETING_2/MEETING_7.jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/igem.org/3/3c/MEETING_7.jpg" alt-src="./images/MEETING_2/MEETING_7.jpg"  ></li>
+                        <li><img src="https://static.igem.org/mediawiki/igem.org/d/d1/LABTIME_1.jpg" alt-src="./images/LabTime_2/LABTIME_1.jpg"</li>
+                        <li><img src="https://static.igem.org/mediawiki/igem.org/b/b7/LABTIME_2.jpg" alt-src="./images/LabTime_2/LABTIME_2.jpg"</li>
+                        <li><img src="https://static.igem.org/mediawiki/igem.org/7/7b/LABTIME_3.jpg" alt-src="./images/LabTime_2/LABTIME_3.jpg"</li>
+                        <li><img src="https://static.igem.org/mediawiki/igem.org/e/e4/LABTIME_4.jpg" alt-src="./images/LabTime_2/LABTIME_4.jpg"</li>
+                        <li><img src="https://static.igem.org/mediawiki/igem.org/6/62/LABTIME_5.jpg" alt-src="./images/LabTime_2/LABTIME_5.jpg"</li>
+                        <li><img src="https://static.igem.org/mediawiki/igem.org/d/d8/LABTIME_6.jpg" alt-src="./images/LabTime_2/LABTIME_6.jpg"</li>
+                        <li><img src="https://static.igem.org/mediawiki/igem.org/1/12/LABTIME_7.jpg" alt-src="./images/LabTime_2/LABTIME_7.jpg"</li>
+                        <li><img src="https://static.igem.org/mediawiki/igem.org/f/f9/LABTIME_8.jpg" alt-src="./images/LabTime_2/LABTIME_8.jpg"</li>
+                        <li><img src="https://static.igem.org/mediawiki/igem.org/d/d6/LABTIME_9.jpg" alt-src="./images/LabTime_2/LABTIME_9.jpg"</li>
+                        <li><img src="https://static.igem.org/mediawiki/igem.org/d/d7/LABTIME_10.jpg" alt-src="./images/LabTime_2/LABTIME_10.jpg"</li>
+                        <li><img src="https://static.igem.org/mediawiki/igem.org/9/91/LABTIME_11.jpg" alt-src="./images/LabTime_2/LABTIME_11.jpg"</li>
+                        <li><img src="https://static.igem.org/mediawiki/igem.org/8/8b/LABTIME_12.jpg" alt-src="./images/LabTime_2/LABTIME_12.jpg"</li>
+                        <li><img src="https://static.igem.org/mediawiki/igem.org/7/78/LABTIME_13.jpg" alt-src="./images/LabTime_2/LABTIME_13.jpg"</li>
+                        <li><img src="https://static.igem.org/mediawiki/igem.org/6/63/LABTIME_14.jpg" alt-src="./images/LabTime_2/LABTIME_14.jpg"</li>
+                        <li><img src="https://static.igem.org/mediawiki/igem.org/0/0c/LABTIME_15.jpg" alt-src="./images/LabTime_2/LABTIME_15.jpg"</li>
+                        <li><img src="https://static.igem.org/mediawiki/igem.org/e/ec/Sc_pic.jpg" alt-src="./images/LabTime_2/Sc_pic.jpg"</li>
                      </ul>
                  </div>
@@ Line 1,322: / Line 1,459: @@
                  <div class="row">
                      <ul class="tilt-gallery img-2x" lightbox-id="Azalea">
-                         <li><img src="./images/AZALEA/(1).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/7/7a/NTU_TAIWAN_AZALEA%281%29.jpg" alt-src="./images/AZALEA/(1).jpg"  ></li>
-                         <li><img src="./images/AZALEA/(2).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/4/41/%282%29.jpg" alt-src="./images/AZALEA/(2).jpg"  ></li>
-                         <li><img src="./images/AZALEA/(3).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/9/96/NTU_TAIWAN_AZALEA%283%29.jpg" alt-src="./images/AZALEA/(3).jpg"  ></li>
-                         <li><img src="./images/AZALEA/(4).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/0/01/NTU_TAIWAN_AZALEA%284%29.jpg" alt-src="./images/AZALEA/(4).jpg"  ></li>
-                         <li><img src="./images/AZALEA/(5).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/5/51/NTU_TAIWAN_AZALEA%285%29.jpg" alt-src="./images/AZALEA/(5).jpg"  ></li>
-                         <li><img src="./images/AZALEA/(6).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/6/60/NTU_TAIWAN_AZALEA%286%29.jpg" alt-src="./images/AZALEA/(6).jpg"  ></li>
-                         <li><img src="./images/AZALEA/(7).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/c/c5/NTU_TAIWAN_AZALEA%287%29.jpg" alt-src="./images/AZALEA/(7).jpg"  ></li>
-                         <li><img src="./images/AZALEA/(8).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/0/05/NTU_TAIWAN_AZALEA%288%29.jpg" alt-src="./images/AZALEA/(8).jpg"  ></li>
-                         <li><img src="./images/AZALEA/(9).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/6/60/NTU_TAIWAN_AZALEA%286%29.jpg" alt-src="./images/AZALEA/(9).jpg"  ></li>
-                         <li><img src="./images/AZALEA/(10).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/1/14/NTU_TAIWAN_AZALEA%2810%29.jpg" alt-src="./images/AZALEA/(10).jpg" ></li>
-                         <li><img src="./images/AZALEA/(11).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/3/32/NTU_TAIWAN_AZALEA%2811%29.jpg" alt-src="./images/AZALEA/(11).jpg" ></li>
-                         <li><img src="./images/AZALEA/(12).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/4/4a/NTU_TAIWAN_AZALEA%2812%29.jpg" alt-src="./images/AZALEA/(12).jpg" ></li>
-                         <li><img src="./images/AZALEA/(13).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/c/ce/NTU_TAIWAN_AZALEA%2813%29.jpg" alt-src="./images/AZALEA/(13).jpg" ></li>
-                         <li><img src="./images/AZALEA/(14).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/4/44/NTU_TAIWAN_AZALEA%2814%29.jpg" alt-src="./images/AZALEA/(14).jpg" ></li>
                      </ul>
@@ Line 1,349: / Line 1,486: @@
                  <div class="row">
                      <ul class="tilt-gallery img-2x" lightbox-id="Chiao_Tung_Conference">
-                         <li><img src="./images/Chiao_Tung_Conference/(1).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/0/03/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%281%29.jpg" alt-src="./images/Chiao_Tung_Conference/(1).jpg"  ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(2).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/6/62/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%282%29.jpg" alt-src="./images/Chiao_Tung_Conference/(2).jpg"  ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(3).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/c/c6/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%283%29.jpg" alt-src="./images/Chiao_Tung_Conference/(3).jpg"  ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(4).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/8/87/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%284%29.jpg" alt-src="./images/Chiao_Tung_Conference/(4).jpg"  ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(5).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/7/74/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%285%29.jpg" alt-src="./images/Chiao_Tung_Conference/(5).jpg"  ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(6).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/1/1b/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%286%29.jpg" alt-src="./images/Chiao_Tung_Conference/(6).jpg"  ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(7).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/1/1e/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%287%29.jpg" alt-src="./images/Chiao_Tung_Conference/(7).jpg"  ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(8).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/6/62/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%288%29.jpg" alt-src="./images/Chiao_Tung_Conference/(8).jpg"  ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(9).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/c/c0/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%289%29.jpg" alt-src="./images/Chiao_Tung_Conference/(9).jpg"  ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(10).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/f/ff/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2810%29.jpg" alt-src="./images/Chiao_Tung_Conference/(10).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(11).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/9/92/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2811%29.jpg" alt-src="./images/Chiao_Tung_Conference/(11).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(12).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/b/b6/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2812%29.jpg" alt-src="./images/Chiao_Tung_Conference/(12).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(13).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/6/63/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2813%29.jpg" alt-src="./images/Chiao_Tung_Conference/(13).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(14).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/1/13/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2814%29.jpg" alt-src="./images/Chiao_Tung_Conference/(14).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(15).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/f/f3/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2815%29.jpg" alt-src="./images/Chiao_Tung_Conference/(15).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(16).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/8/89/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2816%29.jpg" alt-src="./images/Chiao_Tung_Conference/(16).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(17).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/a/a0/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2817%29.jpg" alt-src="./images/Chiao_Tung_Conference/(17).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(18).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/0/0f/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2818%29.jpg" alt-src="./images/Chiao_Tung_Conference/(18).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(19).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/5/57/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2819%29.jpg" alt-src="./images/Chiao_Tung_Conference/(19).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(20).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/8/84/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2820%29.jpg" alt-src="./images/Chiao_Tung_Conference/(20).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(21).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/9/9e/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2821%29.jpg" alt-src="./images/Chiao_Tung_Conference/(21).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(22).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/4/49/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2822%29.jpg" alt-src="./images/Chiao_Tung_Conference/(22).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(23).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/d/d8/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2823%29.jpg" alt-src="./images/Chiao_Tung_Conference/(23).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(24).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/5/59/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2824%29.jpg" alt-src="./images/Chiao_Tung_Conference/(24).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(25).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/c/ca/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2825%29.jpg" alt-src="./images/Chiao_Tung_Conference/(25).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(26).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/4/49/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2826%29.jpg" alt-src="./images/Chiao_Tung_Conference/(26).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(27).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/5/55/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2827%29.jpg" alt-src="./images/Chiao_Tung_Conference/(27).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(28).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/2/22/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2828%29.jpg" alt-src="./images/Chiao_Tung_Conference/(28).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(29).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/e/e5/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2829%29.jpg" alt-src="./images/Chiao_Tung_Conference/(29).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(30).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/f/f8/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2830%29.jpg" alt-src="./images/Chiao_Tung_Conference/(30).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(31).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/4/48/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2831%29.jpg" alt-src="./images/Chiao_Tung_Conference/(31).jpg" ></li>
-                         <li><img src="./images/Chiao_Tung_Conference/(32).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/6/66/NTU_TAIWAN_%E4%BA%A4%E5%A4%A7%2832%29.jpg" alt-src="./images/Chiao_Tung_Conference/(32).jpg" ></li>
                      </ul>
                  </div>
@@ Line 1,393: / Line 1,530: @@
                  <div class="row">
                      <ul class="tilt-gallery img-2x" lightbox-id="LabTime">
-                         <li><img src="./images/LabTime/(1).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/b/b3/NTU_TAIWAN_LABTIME_%281%29.JPG" alt-src="./images/LabTime/(1).jpg"  ></li>
-                         <li><img src="./images/LabTime/(2).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/6/6f/NTU_TAIWAN_LABTIME_%282%29.jpg" alt-src="./images/LabTime/(2).jpg"  ></li>
-                         <li><img src="./images/LabTime/(3).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/f/fc/NTU_TAIWAN_LABTIME_%283%29.JPG" alt-src="./images/LabTime/(3).jpg"  ></li>
-                         <li><img src="./images/LabTime/(4).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/b/b1/NTU_TAIWAN_LABTIME_%284%29.jpg" alt-src="./images/LabTime/(4).jpg"  ></li>
-                         <li><img src="./images/LabTime/(5).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/a/a2/NTU_TAIWAN_LABTIME_%285%29.jpg" alt-src="./images/LabTime/(5).jpg"  ></li>
-                         <li><img src="./images/LabTime/(6).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/4/41/NTU_TAIWAN_LABTIME_%286%29.jpg" alt-src="./images/LabTime/(6).jpg"  ></li>
-                         <li><img src="./images/LabTime/(7).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/9/97/NTU_TAIWAN_LABTIME_%287%29.jpg" alt-src="./images/LabTime/(7).jpg"  ></li>
-                         <li><img src="./images/LabTime/(8).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/2/29/NTU_TAIWAN_LABTIME_%288%29.jpg" alt-src="./images/LabTime/(8).jpg"  ></li>
-                         <li><img src="./images/LabTime/(9).jpg"  ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/0/0e/NTU_TAIWAN_LABTIME_%289%29.jpg" alt-src="./images/LabTime/(9).jpg"  ></li>
-                         <li><img src="./images/LabTime/(10).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/e/e7/NTU_TAIWAN_LABTIME_%2810%29.jpg" alt-src="./images/LabTime/(10).jpg" ></li>
-                         <li><img src="./images/LabTime/(11).jpg" ></li>
+                         <li><img src="https://static.igem.org/mediawiki/2013/b/b1/NTU_TAIWAN_LABTIME_%2811%29.jpg" alt-src="./images/LabTime/(11).jpg" ></li>
+                    </ul>
+                </div>
+            </div>
+        </section>
+        <section class="pink-background divide" id="App">
+            <h1 class="header">App</h1>
+            <p class="header container">
+                 Fascinating Android App
+            </p>
+            <div class="divide">
+                <div class="row">
+                    <ul class="tilt-gallery img-2x" lightbox-id="meeting">
+                        <li><img src="https://static.igem.org/mediawiki/2013/b/ba/NTU_TAIWAN_APP%281%29.png" alt-src="./images/App/(1).png"  ></li>
+                        <li><img src="https://static.igem.org/mediawiki/2013/1/10/NTU_TAIWAN_APP%282%29.png" alt-src="./images/App/(2).png"  ></li>
+                        <li><img src="https://static.igem.org/mediawiki/2013/a/ab/NTU_TAIWAN_APP%283%29.png" alt-src="./images/App/(3).png"  ></li>
+                        <li><img src="https://static.igem.org/mediawiki/2013/b/b4/NTU_TAIWAN_APP%284%29.png" alt-src="./images/App/(4).png"  ></li>
+                        <li><img src="https://static.igem.org/mediawiki/2013/3/39/NTU_TAIWAN_APP%285%29.png" alt-src="./images/App/(5).png"  ></li>
+                        <li><img src="https://static.igem.org/mediawiki/2013/e/e6/NTU_TAIWAN_APP%286%29.png" alt-src="./images/App/(6).png"  ></li>
                      </ul>
                  </div>
              </div>
          </section>
      </script>
@@ Line 1,417: / Line 1,573: @@
          <div class="container divide essay">
-        <b>
+            <ul>
-            Do the biological materials used in your lab work pose any risks to…</b><br/><br/>
+                <li>
-            <ul style="list-style:style"><b>
+                    <b>Do the biological materials used in your lab work pose any risks to…</b><br/><br/>
-                <li> -the safety and health of team members or others working in the lab?</li>
+                    <ul>
-                <li> -the safety and health of the general public, if released by design or by accident?</li>
+                        <li>
-                <ul></b>
+                            <b> -the safety and health of team members or others working in the lab? <br/>
-                    <li> Here is a list of the chassis organisms that we use in our project : Escherichia coli, Saccharomyces cerevisiae, and Rhodotorula glutinis. These are all Biosafety Level 1 organisms and non-pathogenic, therefore pose no severe threat to the researchers or any healthy human. </li>
+                                -the safety and health of the general public, if released by design or by accident? </b>
-                </ul><b>
+                        </li>
-                <li> -the environment, if released by design or by accident?</li>
-                <ul></b>
+                        <ul>
-                    <li>For the same reasons above, there would be minimum chance of the organisms causing any great harm to the environment. Our E. coli 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 equipments used for bacteria cells and/or cultures are properly autoclaved. </li>
+                            <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>
+                        </ul>
-                <li> -security through malicious misuse by individuals, groups, or countries? </li>
+                        <li> <b>-the environment, if released by design or by accident?</b></li>
-                <ul></b>
+                        <ul>
-                    <li> Unless high density of cell culture is spread out, no potential risk is present. </li>
+                            <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>
+                        </ul>
-            </b></ul>
+                        <li> <b>-security through malicious misuse by individuals, groups, or countries? </b></li>
+                        <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/>
+                            <li> Unless high density of cell culture is spread out, no potential risk is present. </li>
+                        </ul>
-        <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">
+                    </ul>
-.  Contact with high density of cells may cause harmful effects on people.<br/>
+                </li>
-.  Release of cell culture into the environment may cause fish infection and perturbation of the ecosystem.<br/></p>
+                <li>
+                    <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/>
-        <b>Does your project include any design features to address safety risks?</b><br/>
+                    <p>Respecting our goals to prevent the damage caused by cold temperature in fish farming:</p>
-        <br/>
+                    <ol>
-        <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/>
+                        <li>Contact with high density of cells may cause harmful effects on people.</li>
-        When faced with an increasing osmaolarity 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. 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 sythesis. Following activation of the HOG pathway, activated Hog1 binds to the transcriptional acitvator 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. Smp1 is phosphorylated and activated by the active Hog1 protein. Thus, the sensing of osmolarity and the induction of GPD1 and STL1 expression will make up mainly the fist part of our kill switch.<br/>
+                        <li>Release of cell culture into the environment may cause fish infection and perturbation of the ecosystem.</li>
-        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 and 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, and their induction via osmosensitive promoters would cause irreversible harm to the yeasts and in the end kill them.<br/>
+                    </ol>
-        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, with following cell death. 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>
+                </li>
+                <li>
-        <b>What safety training have you received?</b><br/><br/>
+                    <b>Does your project include any design features to address safety risks?</b><br/><br />
-        <p style="margin-left: 40px">Every student who worked in the lab have had to received 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/>
+                    <div class="row">
+                        <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="500px">
-        <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/>
+                        <div class="pull-right">
-        <p style="margin-left: 40px">We had an discussion with the National Taiwan University Enviroment 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/>
+                            <h5>(fig.3 Overview of the HOG pathway in <i>S. cerevisiae</i>. Several transcriptional factors are regulated.)</h5>
+                        </div>
-        <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/>
+                        <div class="margin-left: 20px;">
-        <p style="margin-left: 40px">&#34; http://esh.ntu.edu.tw/epc/e-home.php &#34;<br/>
+                        <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/>
-        This is also the biosafety guidelines that our institution follows.<br/>
+                        <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/>
-        </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>
+                        <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>
+                        </div>
+                    </div>
+                    <p style="margin-left: 40 px">
+                        <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.</h5>
+                    </p>
+                </li>
+                <li>
+                    <b>What safety training have you received?</b><br/><br/>
+                    <p>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/>
+                </li>
+                <li>
+                    <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>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/>
+                </li>
+                <li>
+                    <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/>
+                </li>
+                <p>&#34; http://esh.ntu.edu.tw/epc/e-home.php &#34;<br/>
+                    This is also the biosafety guidelines that our institution follows.<br/>
+                </p>
+            </ul>
          </div>
      </script>
@@ Line 1,466: / Line 1,648: @@
              <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 1,556: / Line 1,738: @@
                  <p class="header"><i>Saccharomyces cerevisiae</i></p>
              </section>
-             <div class="container divide essay">
+             <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">
+                    <h5>"Budding Yeast and Friend Fan Page <a href="https://www.facebook.com/yeastandfriends"> Link</a>"</h5>
+                </div>
+                <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). 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>
          </div>
          <div id="Expression">
@@ Line 1,568: / Line 1,762: @@
              <div class="container divide">
-                 <img src="images/e_coli.jpg" class="pull-right img-responsive">
+                 <img src="https://static.igem.org/mediawiki/2013/1/1e/NTU_TAIWAN_E_coli.jpg" alt-src="images/e_coli.jpg" class="pull-right img-responsive">
-                 <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>
+                 <div style="margin-top: 75px"><p>The yeast <b>Saccharomyces cerevisiae</b> has several properties which have established it as an important tool in the expression of foreign protein for research, industrial or medical use. As a food organism, it is highly acceptable for the production of pharmaceutical proteins. In contrast,<b>Escherichia coli</b> have toxic cell wall pyroxenes and mammalian cells may contain oncogenic or viral DNA, so that products from these organisms must be tested hmore extensively.</p>
-                 <p>Yeast can be grown rapidly on simple media and to high cell density and its genetics are more advanced than any other eukaryote, so that it can be manipulated almost as readily as <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>
+                 <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/>
+                 <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>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>
          </div>
@@ Line 1,584: / Line 1,779: @@
              <div class="container essay divide">
                  <p>UCP1 can transport protons through two different hypothesized mechanisms. In the first model, UCP1 transfers protons from intermembrane space to mitochondria matrix. Fatty acids provide essential free carboxy groups catalyzing proton (H+) translocation. In this model, Coenzyme Q(CoQ) serves as a necessary cofactor of UCP1, activating the protein functions.</p>
-                 <p>In the second model, free fatty acids (FFAs) combine with protons with carboxy groups, then flipping across inner mitochondria membrane. After FFAs flipping to the matrix side, UCP1 removes protons from carboxy groups, flipping the anion form FFAs back to the innermembrane space. (fig.??)The anion form FFAs bind to protons again, transferring more protons into the matrix. </p>
+                 <p>In the second model, free fatty acids (FFAs) combine with protons with carboxy groups, then flipping across inner mitochondria membrane. After FFAs flipping to the matrix side, UCP1 removes protons from carboxy groups, flipping the anion form FFAs back to the innermembrane space. (fig.2)The anion form FFAs bind to protons again, transferring more protons into the matrix. </p>
-                 <img class="tipReveal img-responsive" src="images/image002.png"></img>
+                 <img class="tipReveal img-responsive" src="https://static.igem.org/mediawiki/2013/8/8f/NTU_TAIWAN_Image002.png" alt-src="images/image002.png"></img>
-                 <div class="tip">(fig.??)</div>
+                 <div class="tip">(fig.2)</div>
                  <p>Through these two div mechanisms, proton gradient on either side of inner membrane are formed. Generally, the oxidative phosphorylation of ADP into ATP takes place within the mitochondrial inner membrane respiratory chain. However, the coupling of mitochondrial respiration and ATP synthesis is not complete (Nicholls, 1974; Brand, 1977). Thus in several tissues there is a proton leak through the inner mitochondrial membrane that is not associated with ATP synthesis. And in these tissues, UCPs produce heat.
                      <span class="tipReveal">[*]</span><br/>
@@ Line 1,600: / Line 1,795: @@
              <div class="essay container divide">
-                 <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>
+            <P><b>Cold shock promoter</b></p>
-             </div>
+                 <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>
-        </div>
+             <p><b>What is <i>TIR1</i> gene ?</b></p>
+                <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>
-        <div id="Circuit">
+                 <p><b>Potential Cold Shock Promoter</b></p>
-            <section class="purple-background">
+                <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>
-                 <h1 class="header">Circuit</h1>
+                <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>
-            </section>
-            <div class="essay container divide">
+                Reference:<br/>
+                [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/>
+                [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/>
+                [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/>
+                [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/>
              </div>
          </div>
          <div id="Suicide">
@@ Line 1,618: / Line 1,817: @@
                  <h1 class="header">Suicide</h1>
              </section>
-             <div class="essay container divide" style="margin-left: 40 px">
+             <div class="essay container divide">
-             &nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbspIn 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/>
+                <div class="pull-right col-md-6">
-            &nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbspWhen faced with an increasing osmaolarity 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. 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 sythesis. Following activation of the HOG pathway, activated Hog1 binds to the transcriptional acitvator 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. Smp1 is phosphorylated and activated by the active Hog1 protein. Thus, the sensing of osmolarity and the induction of GPD1 and STL1 expression will make up mainly the fist part of our kill switch.<br/>
+                    <img class="tipReveal 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>
-            &nbsp&nbsp&nbsp&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 and 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, and their induction via osmosensitive promoters would cause irreversible harm to the yeasts and in the end kill them.<br/>
+                    <div class="tip">
-            &nbsp&nbsp&nbsp&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, with following cell death. 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/>
+                        (fig.3 Overview of the HOG pathway in S. cerevisiae. Several transcriptional factors are regulated.)
+                    </div>
+                </div>
+                <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/>
+                <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>
+                <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>
+                <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>
              </div>
          </div>
@@ Line 1,632: / 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 1,638: / Line 1,854: @@
                      <span class="bubble3"><span class="glow">&nbsp;</span></span>
                      <span class="bubble4"><span class="glow">&nbsp;</span></span>
-                     <img class="nohover" id="redBeaker" src="images/beaker.png" alt="the beaker" >
+                     <img class="nohover" id="redBeaker" src="https://static.igem.org/mediawiki/2013/6/6f/NTU_TAIWAN_Beaker.png" alt-src="images/beaker.png" alt="the beaker" >
                  </div>
              </div>
@@ Line 1,646: / Line 1,862: @@
              <section class="brown-background">
                  <h1 class="header">Chassis</h1>
-                 <p class="header">R.g.</p>
+                 <p class="header"><i> Rhodotorula glutinis</i></p>
              </section>
              <div class="container divide essay">
-                 <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.<br/>
+                <img class="pull-right" src="https://static.igem.org/mediawiki/igem.org/e/e2/Meeting-%2827%29.jpg" alt-src="./images/MEETING/Meeting-(27).jpg" width=500>
-                     <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. <br/>
+                 <p><b>What is <i>Rhodotorula glutinis</i> ?</b></p>
-                     &nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbspThe 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><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>
-                    &nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbspSo we choose R.g. for the Expression vector.<br/>
+                <p><b>The oleaginous yeast</b></p>
-                    &nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbspThermogenic 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.<br/>
+                <p>
-                    &nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbspAccording 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.  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. <br/>
+                     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>
-                     &nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbspAfter 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.<br/>
+                <p><b>Why <i>Rhodotorula glutinis</i> ?</b></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>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><b>Conclusion</b></p>
+                <p>
+                     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>
+                    Reference:<br/>
+                    [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/>
+                    [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/>
              </div>
          </div>
      </script>
+    <script type="text/template" id="Modeling">
+        <section class="red-background">
+            <h1 class="header">Modeling</h1>
+        </section>
+        <div class="container divide">
+            <legend>
+                <h2 style="border-bottom: 0"><b>Mathematical Model－「Tuning the sensitivity of cold shock promoter」</b></h2>
+            </legend>
+            <p>
+                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>
+        <div class="container divide">
+            <legend>
+                <h2 style="border-bottom: 0"><b>Model A、Heat shock promoter or Constitutive promoter?</b></h2>
+            </legend>
+            <ul class="essay">
+                <li><b>Purpose</b>
+                    <ul class="nav">
+                        <li><p>Investigate on whether heat shock promoter (abbreviated as Hsp) or Constitutive promoter suit our genetic circuit as a controller of regulator.</p></li>
+                    </ul>
+                </li>
+                <li><b>Circuit structure</b>
+                    <ul class="nav">
+                        <li><p>In this model, we use GFP to report the activity of cold shock promoter (abbreviated as Csp) under different temperatures. The reporter itself is in turn regulated by an activator and a repressor through activator binding site and repressor binding site. While activators are regulated by Csp, repressors are regulated by Hsp or Constitutive promoter (Fig. 4)</p></li>
+                    </ul>
+                    <img class="tipReveal img-responsive" src="https://static.igem.org/mediawiki/2013/e/ee/NTU_TAIWAN_Model_Image001.jpg" alt-src="images/modeling/image001.jpg">
+                    <div class="tip">Fig. 4: Representative diagram of our genetic circuit</div>
+                </li>
+                <li><b>Mathematical expression</b>
+                    <div class="container">
+                        <p>Gene expression can be described with certain mathematical language, with deterministic expression one of the most commonly used. Deterministic expression represent the production rate of a gene product as the difference between a fraction, determining by the promoter occupancy by activator or repressor, of maximal production rate and the removal rate. Here, we will show the mathematical expression of the model with Hsp or constitutive promoter as their circuit part, respectively.</p>
+                        <div class="container">
+                            <div><b>model with Hsp as a part</b></div>
+                            <ul>
+                                <li>
+                                    <img style="display:inline" class="img-responsive"  src="https://static.igem.org/mediawiki/2013/2/20/NTU_TAIWAN_Model_Image002.gif" alt-src="images/modeling/image002.gif">
+                                </li>
+                                <li>
+                                    <img style="display:inline" class="img-responsive"  src="https://static.igem.org/mediawiki/2013/1/1f/NTU_TAIWAN_Model_Image003.gif" alt-src="images/modeling/image003.gif">
+                                </li>
+                                <li>
+                                    <img style="display:inline" class="img-responsive"  src="https://static.igem.org/mediawiki/2013/1/15/NTU_TAIWAN_Model_Image004.gif" alt-src="images/modeling/image004.gif">
+                                </li>
+                            </ul>
+                            <div><b>model with constitutive promoter as a part:</b></div>
+                            <ul>
+                                <li>
+                                    <img style="display:inline" class="img-responsive" src="https://static.igem.org/mediawiki/2013/2/20/NTU_TAIWAN_Model_Image002.gif" alt-src="images/modeling/image002.gif">
+                                </li>
+                                <li>
+                                    <img style="display:inline" class="img-responsive" src="https://static.igem.org/mediawiki/2013/a/ad/NTU_TAIWAN_Model_Image005.gif" alt-src="images/modeling/image005.gif">
+                                </li>
+                                <li>
+                                    <img style="display:inline" class="img-responsive" src="https://static.igem.org/mediawiki/2013/1/15/NTU_TAIWAN_Model_Image004.gif" alt-src="images/modeling/image004.gif">
+                                </li>
+                            </ul>
+                        </div>
+                        <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">
+                        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).
+                    </p>
+                    <table class="row tipReveal">
+                        <tr class="row pink-background">
+                            <td class="col-md-2">Parameters</td>
+                            <td class="col-md-5">Discription</td>
+                            <td class="col-md-3">Value</td>
+                            <td class="col-md-2">Unit</td>
+                        </tr>
+                        <tr class="row">
+                            <td class="col-md-2">n</td>
+                            <td class="col-md-5">Hill’s coefficient</td>
+                            <td class="col-md-3">2</td>
+                            <td class="col-md-2">-</td>
+                        </tr>
+                        <tr class="row">
+                            <td class="col-md-2">β<sub>Csp</sub>(T)</td>
+                            <td class="col-md-5">Maximal production rate of Csp, a function of temperature</td>
+                            <td class="col-md-3">Sigmoidal curve <br/>(set 10℃=1e-6, 20℃=0)) </td>
+                            <td class="col-md-2">M/s</td>
+                        </tr>
+                        <tr class="row">
+                            <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</td>
+                        </tr>
+                        <tr class="row">
+                            <td class="col-md-2">β<sub>cons</sub><sup>a</sup></td>
+                            <td class="col-md-5">Maximal production rate of constitutive promoter</td>
+                            <td class="col-md-3">1e-6</td>
+                            <td class="col-md-2">-</td>
+                        </tr>
+                        <tr class="row">
+                            <td class="col-md-2">γ<sub>A</sub><sup>b</sup></td>
+                            <td class="col-md-5">Removal rate of activator</td>
+                            <td class="col-md-3">1e-2</td>
+                            <td class="col-md-2">s<sup>-1</sup></td>
+                        </tr>
+                        <tr class="row">
+                            <td class="col-md-2">γ<sub>R</sub><sup>b</sup></td>
+                            <td class="col-md-5">Removal rate of repressor</td>
+                            <td class="col-md-3">1e-2</td>
+                            <td class="col-md-2">s<sup>-1</sup></td>
+                        </tr>
+                        <tr class="row">
+                            <td class="col-md-2">γ<sub>G</sub><sup>P</sup> c</td>
+                            <td class="col-md-5">Removal rate of GFP</td>
+                            <td class="col-md-3">8.2e-3</td>
+                            <td class="col-md-2">s<sup>-1</sup></td>
+                        </tr>
+                        <tr class="row">
+                            <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">2~200</td>
+                            <td class="col-md-2">M/s</td>
+                        </tr>
+                        <tr class="row">
+                            <td class="col-md-2">α<sub>A</sub><sup>d</sup></td>
+                            <td class="col-md-5">Kd value of activator from DNA</td>
+                            <td class="col-md-3">1e+3 ~ 1e-6</td>
+                            <td class="col-md-2">M</td>
+                        </tr>
+                        <tr class="row">
+                            <td class="col-md-2">α<sub>R</sub><sup>d</sup></td>
+                            <td class="col-md-5">Kd value of repressor from DNA</td>
+                            <td class="col-md-3">1e+3 ~ 1e-6</td>
+                            <td class="col-md-2">M</td>
+                        </tr>
+                    </table>
+                    <div class="tip">
+                    <ul>
+                        <li>Assume their maximal activity the same as Csp</li>
+                        <li>Assume only slightly higher than γGFP</li>
+                        <li>Consider the action of generation time of yeast and degradation rate in E. coli</li>
+                    </ul>
+                    </div>
+                </li>
+            </ul>
+        </div>
+        <section class="brown-background">
+            <h1 class="header">Result & Discussion</h1>
+        </section>
+        <div class="container essay divide">
+            <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="brown-background">
+            <h1 class="header">Code</h1>
+        </section>
+        <div class="container essay divide">
+            <legend><b>consModel</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_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>
+    <script type="text/template" id="Protocol">
+        <section class="lightblue-background">
+            <h1 class="header">Protocol</h1>
+        </section>
+        <div class="container">
+        <br/><p><b>PCR</b></p>
+: Design of appropriate forward and reverse primers<br/>
+: Prepare our template<br/>
+: Prepare the PCR mix. (Kapa Hifi PCR kit.)<br/>
+: Run PCR<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<br/><br/>
+<br/><p><b>Construction of our parts</b></p><p>
+: We design primers for parts with prefix and suffix.<br/>
+: Perform PCR and cleanup the PCR product<br/>
+: Before insert our parts into standard backbone, pSB1C3, we perform RE digestion to make sticky ends of both inserts and backbones.<br/>
+: Ligation of inserts and backbones<br/>
+: Transform our ligation products into DH5α and streak the transformed DH5α on LB agar plate with chloramphenicol.<br/>
+: Inoculate single colony into broth with chloramphenicol.<br/>
+: Miniprep the plasmid DNA from the overnight broth culture.<br/>
+: Confirm the products by both RE digestion and PCR sequencing<br/></p>
+        <p><b>Point mutation protocol</b></p>
+        <p>For a standard 100 @L reaction:<br/>
+, 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/>
+. 100-500 ng of each primer (0.2-l .O pL of a 500 pg/rnL stock).<br/><br/>
+. 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/>
+. 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/>
+. 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/>
+. 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/>
+. 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/>
+. 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/>
+. 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>
+    </script>
+    <script type="text/template" id="ProjectResult">
+        <section class="yellow-background">
+            <h1 class="header">Project Result</h1>
+        </section>
+        <div class="red-background row">
+            <h1 class="header"> Plasmid Construction</h1>
+            <p class="header">For characterization </h1>
+            <div class="container essay">
+                <div class="container essay">
+                    <img class="pull-right img-responsive" src="https://static.igem.org/mediawiki/2013/9/91/Ucp.png" alt-src="./images/result/ucp_1.png" width=400>
+                    <div class="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">
+                    <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-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">
+            <h1 class="header"> Characterize the biological part  </h1>
+            <p class="header">Test the expression of SrUCP by Western blotting.</p>
+            <div class="container" style="margin-top: 20px">
+                <img class="pull-left img-responsive" src="https://static.igem.org/mediawiki/2013/9/95/Western.png" alt-src="./images/result/western.png" width=700>
+                <div class="col-md-4" style="margin-top: 190px"><p>  Based on the sequence analysis, we predict the protein size of SrUCP(with TAP tag) is about 53 kDa. We did the Western blotting and confirmed our SrUCP gene have expressed in Saccharomyces cerevisiae.</p>
+                </div>
+            </div>
+            <p class="header"> Analyze the heat-production ability of transformant</p>
+            <div class="container essay">
+                <p>For estimate the heat-production ability of the SrUCP in yeast. We built up a straight way to analyze it. </p>
+                <p>After both experimental group (pRS424-GAL1-SrUCP-TAP) and negative control group (pRS424-GAL1∆) induced by 2% galactose for 21 hours, we couldn’t find out statistical difference between control and experimental group by our first experimental method. Most of the heat production is come from the fermentation and shaking of the incubator.</p>
+                <p>However, we didn’t analyze the quantity of yeasts during the experiment. We consider that the experimental group (which had been transformed SrUCP) might grow slower than the control group and then cause the result t I show below:<br/>
+                    Experimental: Heat(E) = Fermentation(1) + SrUCP<br/>
+                    Control: Heat(C) = Fermentation(2)<br/>
+                    Because the heat of fermentation(1) is lower than fermentation(2), even if SrUCP produce heat, the total Heat(E) equal to Heat(C).
+                    In hence, the better method to test heat production is incubate in isothermal environment or use the isothermal titration calorimetry. We will try these more precise method.
+                </p>
+                <img src="https://static.igem.org/mediawiki/2013/3/37/NTU_TAIWAN_Capture.JPG" alt-src="images/Capture.jpg">
+            </div>
+            <p class="header"> Rhodotorula glutinis Growth curve</p>
+            <div class="container essay">
+                <img src="https://static.igem.org/mediawiki/2013/2/2f/NTU_TAIWAN_Capture2.JPG" alt-src="images/Capture2.jpg">
+                <img src="https://static.igem.org/mediawiki/2013/5/52/NTU_TAIWAN_Capture3.JPG" alt-src="images/Capture3.jpg">
+                <p>To realize our ultimate goal, that is, to express SrUCP in Rhodotorula glutinis, we analyze this organism’s growth property. This information is useful for use to prepare the competent cell of Rhodotorula glutinis. This is the fundamental and important step to express exogenous gene in this species.</p>
+                Results: <br/>
+                <p>At 25℃, R.glutinis has the optimal growth curve, it’s lower than the S. cerevisiae. Also, no matter under 25 or 15℃, the growth rates are both slower than S. cerevisiae. However, the lag phase of these two curves are close to each other.(fig2, fig3). Interestingly, this strain has a faster growth rate at 4℃ relatively. This phenomenon is easily to be observed when inoculating on agar plate.(The result is not shown) Therefore, R. glutinis maybe a better chassis than S. cerevisiae to produce heat in low temperature.</p>
+                <p>According to the growth curve, we suppose that between 6 to 8 hours (at early log phase) would be the best time for making competent cell of R.glutinis, but we still need more experiments for further characterization.</p>
+            </div>
+        </div>
+    </script>
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