Team:NTU Taiwan/index.html

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

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

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

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

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

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

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

                                 <i class="icon-facebook" style="line-height: 20px;"></i>

                         <div class="col-md-7" style="margin-top: 30px">

                             <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" style="margin-right: 30px">

                         <div class="col-md-11" style="margin-top: 30px">

                            <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>

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

        <b>

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

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

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

                <ul></b>

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

                </ul><b>

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

                <ul></b>

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

                </ul><b>

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

                <ul></b>

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

                </ul>

            </b></ul>

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

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

        <p style="margin-left: 80px">

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

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

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

        <br/>

        <div class="row pull-right" width=500>

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

        <div class="tip">

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

        </div>

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

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

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

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

        <h5>Resource: <br/>

        Overview of HOG pathway<br/>

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

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

 

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

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

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

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

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

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

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

        </p>

                <div class="row" style="margin-left: 70px; margin-right: 70px; margin-top: 30px; margin-bottom: 30px">

                    <img class="pull-right 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">

 

 

                    <div class="row text-right">

                     </div>

            <div class="row pull-right" width=500>

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

            <div class="tip">

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

            </div>

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

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

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

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

            <h5>Resource: <br/>

            Overview of HOG pathway<br/>

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

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

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

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

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

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

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