Team:NTU Taiwan/index.html

From 2013.igem.org

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                 <p>In this year, iGEM NTU_Taiwan team aim to make a <b>biological heater</b> which can produce appropriate heat in low temperature. The feature in this device is that it can responce to different temperature and produce heat in identical level.
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                <p>What a crazy project! This biological device is really charming, is't it? Let us show you our project! <br/>
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                <div class="row text-center"><h3>Let's go!</h3></div>
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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 &ldquo;Us&rdquo;, each comprising about 100 amino acids(fig.1).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.
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             <img class="tipReveal" src="http://2013.igem.org/wiki/images/2/26/NTU_TAIWAN_Image001.png" alt-src="images/image001.png"> <!--help!!!圖片放中間-->
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             <img class="tipReveal img-responsive" src="http://2013.igem.org/wiki/images/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">
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                 (fig.1 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.)
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                 <li>NTU_Taida iGEM team: <br/><br/>
                 <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>
                             <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).<br/>
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                            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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                 <li> -the environment, if released by design or by accident?</li>
                 <li> -the environment, if released by design or by accident?</li>
                 <ul></b>
                 <ul></b>
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                     <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 equipments used for bacteria cells and/or cultures are properly autoclaved. </li>
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                     <li>For the same reasons above, there would be minimum chance of the organisms causing any great harm to the environment. Our <i>E. coli</i> transformants do carry an ampicillin resistant gene, so accidental release of the bacteria might result in a spread of the antibiotic resistant phenotype. However, all our experiments are conducted under safe procedures and the equipment used for bacteria cells and/or cultures are properly autoclaved. </li>
                 </ul><b>
                 </ul><b>
                 <li> -security through malicious misuse by individuals, groups, or countries? </li>
                 <li> -security through malicious misuse by individuals, groups, or countries? </li>
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         <b>Does your project include any design features to address safety risks?</b><br/>
         <b>Does your project include any design features to address safety risks?</b><br/>
         <br/>
         <br/>
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        <div class="row pull-right" width=500>
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        <img class="pull-right img-responsive" src="http://igem.org/wiki/images/5/50/Suicide.jpg" alt-src="./images/suicide.jpg" style="display: block; margin: 10px;" width=500>
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        <div class="tip">
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        (fig.3 Overview of the HOG pathway in S. cerevisiae. Several transcriptional factors are regulated.)</div>
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        </div>
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         <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/>
         <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/>
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         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/>
+
         When faced with an increasing osmolarity of the environment, the HOG pathway is activated in yeasts and the final result is the accumulation of glycerol in yeast cells to balance the exterior osmotic pressure. Sensors of the ambient osmolarity rise activates a MAPK cascade and eventually leads to the phosphorylation and activation of the Hog1 protein. Activated Hog1 then translocates into the nucleus and activates a number of transcriptional factors via protein-protein interactions or phosphorylation.(fig.3) These transcriptional factors (mostly activators) then mediate the expression of hundreds of genes related to cell integrity and adaptation to osmostress. Among these, the GPD1 gene and the STL1 gene are the most significant targets of the HOG pathway. GPD1 encodes the sequences for NAD-dependent glycerol-3-phosphate dehydrogenase, which is the key enzyme of glycerol synthesis. Following activation of the HOG pathway, activated Hog1 binds to the transcriptional activator Hot1 and upregulates the expression of GPD1. On the other hand, STL1 codes for a glycerol/proton symporter in the plasma membrane of S. cerevisiae. Upon sensing a rise in osmolarity, STL1 is strongly and transiently induced by transcriptional activators Hot1 and Smp1, both members of the HOG pathway. Hot1 activation is as mentioned above, and Smp1 is phosphorylated and activated by the active Hog1 protein. We thus utilize, the sensing of osmolarity and the induction of GPD1 and STL1 expression in yeasts to make up the first part of our kill switch.<br/>
-
         &nbsp&nbsp&nbsp&nbsp&nbspIn order to complete our kill switch so that increasing osmolarity not only activates the HOG pathway, but also leads to cell death, we further integrate a kill gene following the promoter sequence of GPD1 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&nbspIn order to complete our kill switch so that increasing osmolarity not only activates the HOG pathway, but also leads to cell death, we further integrate a kill gene following the promoter sequence of GPD1 or STL1. The most suitable genes would be those encoding proteins that have nuclease activity. A couple of chosen examples are NUC1 (encoding endonuclease G) and YBL055C (encoding Tat-D nuclease). Endonuclease G is the major mitochondrial nuclease in S. cerevisiae, and it induces apoptosis in yeast independently of metacaspase or of apoptosis inducing factors. Tat-D is an endo-/exo-nuclease that incises the double stranded DNA without obvious specificity via its endonuclease activity and excises the DNA from 3' to 5' end by its exonuclease activity. These proteins are intrinsically expressed during apoptosis. By placing the genes downstream of the GDP1 or STL1 promoter, their expression will be induced under increasing osmolarity and cause irreversible harm to the yeasts, in the end killing them.<br/>
-
         &nbsp&nbsp&nbsp&nbsp&nbspAccording to data from current milkfish farms in Taiwan, which are saltwater farms, water osmolarity is way higher than yeast culturing environments. Therefore the HOG pathway would surely be activated once the yeasts escape from the thermogenic device, 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>
+
         &nbsp&nbsp&nbsp&nbsp&nbspAccording to data from current milkfish farms in Taiwan, which are saltwater farms, water osmolarity is way higher than yeast culturing environments. Therefore the HOG pathway would surely be activated once the yeasts escape from the thermogenic device, and with our design of kill switch, cell death follows. If the device is to be used in a fish farm with fresh water, the osmolarity would very likely be lower than the yeast culture. In light of this possibility, we are also looking into another mechanism of S. cerevisiae that is used when it is subjected to low osmolarity stress. It is called the cell integrity pathway, and is activated upon decreasing osmolarity of the environment. We hope to find similar functioning effectors downstream of the pathway like we did in the HOG pathway, and integrate the activated promoters with kill genes. If succeeded, our safety design will not be restricted to saltwater fish farms.<br/></p>
          
          
 +
        <h5>Resource: <br/>
 +
        Overview of HOG pathway<br/>
 +
        Microbiol Mol Biol Rev. 2002 Jun;66(2):300-72.<br/>
 +
        Osmotic stress signaling and osmoadaptation in yeasts. by Hohmann S.</p></h5>
 +
       
 +
         <b>What safety training have you received?</b><br/><br/>
         <b>What safety training have you received?</b><br/><br/>
-
         <p style="margin-left: 40px">Every student who worked in the lab have had to 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/>
+
         <p style="margin-left: 40px">Every student who worked in the lab have had to receive a 12-hour training. The training includes lectures on the topics &#34;Principal of Biosafety&#34; and &#34;Management of Biosafety in Laboratories&#34;.</p><br/>
          
          
         <b>Does your institution have an Institutional Biosafety Committee, or an equivalent group? If yes, have you discussed your project with them? Describe any concerns they raised with your project, and any changes you made to your project plan based on their review.</b><br/><br/>
         <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&#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/>
+
         <p style="margin-left: 40px">We had an discussion with the National Taiwan University Environment Protection and Occupational Safety and Health Center. The assessment result was that we don&#39;t need to send the &#34;Recombinant gene experiment&#34; form to the Center, due to our materials all belonging to RG1.</p><br/>
          
          
         <b>Does your country have national biosafety regulations or guidelines? If so, please provide a link to these regulations or guidelines if possible.</b><br/><br/>
         <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/>
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             </section>
             <div class="essay container divide">
             <div class="essay container divide">
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                <p>In case of an accidental release of the thermogenic yeasts from our device to the environment, we have designed a kill switch that would ideologically lead the yeasts to death under such circumstance.</p>
+
            <div class="row pull-right" width=500>
-
                <p>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.</p>
+
            <img class="pull-right img-responsive" src="http://igem.org/wiki/images/5/50/Suicide.jpg" alt-src="./images/suicide.jpg" style="display: block; margin: 10px;" width=500>
-
                <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 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.</p>
+
            <div class="tip">
-
                <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, 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.</p>
+
            (fig.3 Overview of the HOG pathway in S. cerevisiae. Several transcriptional factors are regulated.)</div>
 +
            </div>
 +
           
 +
            <p style="margin-left: 40 px">In case of an accidental release of the thermogenic yeasts from our device to the environment, we have designed a kill switch that would ideologically lead the yeasts to death under such circumstance.<br/>
 +
            When faced with an increasing osmolarity of the environment, the HOG pathway is activated in yeasts and the final result is the accumulation of glycerol in yeast cells to balance the exterior osmotic pressure. Sensors of the ambient osmolarity rise activates a MAPK cascade and eventually leads to the phosphorylation and activation of the Hog1 protein. Activated Hog1 then translocates into the nucleus and activates a number of transcriptional factors via protein-protein interactions or phosphorylation.(fig.3) These transcriptional factors (mostly activators) then mediate the expression of hundreds of genes related to cell integrity and adaptation to osmostress. Among these, the GPD1 gene and the STL1 gene are the most significant targets of the HOG pathway. GPD1 encodes the sequences for NAD-dependent glycerol-3-phosphate dehydrogenase, which is the key enzyme of glycerol synthesis. Following activation of the HOG pathway, activated Hog1 binds to the transcriptional activator Hot1 and upregulates the expression of GPD1. On the other hand, STL1 codes for a glycerol/proton symporter in the plasma membrane of S. cerevisiae. Upon sensing a rise in osmolarity, STL1 is strongly and transiently induced by transcriptional activators Hot1 and Smp1, both members of the HOG pathway. Hot1 activation is as mentioned above, and Smp1 is phosphorylated and activated by the active Hog1 protein. We thus utilize, the sensing of osmolarity and the induction of GPD1 and STL1 expression in yeasts to make up the first part of our kill switch.<br/>
 +
            &nbsp&nbsp&nbsp&nbsp&nbspIn order to complete our kill switch so that increasing osmolarity not only activates the HOG pathway, but also leads to cell death, we further integrate a kill gene following the promoter sequence of GPD1 or STL1. The most suitable genes would be those encoding proteins that have nuclease activity. A couple of chosen examples are NUC1 (encoding endonuclease G) and YBL055C (encoding Tat-D nuclease). Endonuclease G is the major mitochondrial nuclease in S. cerevisiae, and it induces apoptosis in yeast independently of metacaspase or of apoptosis inducing factors. Tat-D is an endo-/exo-nuclease that incises the double stranded DNA without obvious specificity via its endonuclease activity and excises the DNA from 3' to 5' end by its exonuclease activity. These proteins are intrinsically expressed during apoptosis. By placing the genes downstream of the GDP1 or STL1 promoter, their expression will be induced under increasing osmolarity and cause irreversible harm to the yeasts, in the end killing them.<br/>
 +
            &nbsp&nbsp&nbsp&nbsp&nbspAccording to data from current milkfish farms in Taiwan, which are saltwater farms, water osmolarity is way higher than yeast culturing environments. Therefore the HOG pathway would surely be activated once the yeasts escape from the thermogenic device, and with our design of kill switch, cell death follows. If the device is to be used in a fish farm with fresh water, the osmolarity would very likely be lower than the yeast culture. In light of this possibility, we are also looking into another mechanism of S. cerevisiae that is used when it is subjected to low osmolarity stress. It is called the cell integrity pathway, and is activated upon decreasing osmolarity of the environment. We hope to find similar functioning effectors downstream of the pathway like we did in the HOG pathway, and integrate the activated promoters with kill genes. If succeeded, our safety design will not be restricted to saltwater fish farms.<br/></p>
 +
           
 +
            <h5>Resource: <br/>
 +
            Overview of HOG pathway<br/>
 +
            Microbiol Mol Biol Rev. 2002 Jun;66(2):300-72.<br/>
 +
            Osmotic stress signaling and osmoadaptation in yeasts. by Hohmann S.</p></h5>
             </div>
             </div>
         </div>
         </div>

Revision as of 13:04, 27 September 2013

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