Difference between revisions of "Team:NYMU-Taipei/Notebook"

(Undo revision 124204 by Pinyuchen (talk))
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{{NYMU-Taipei-css}}
 
{{NYMU-Taipei-css}}
  
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<style>
 
body{
 
body{
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.Nitrogen_Starvation{
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/* Content of Notebook */
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#Protocol{
 
width: 81%;
 
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float:right;  
 
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font-size:20px;
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background-color:#cee1ea;
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font-family:'Fresca',sans-serif;
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font-size:18px;
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}
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#Labnote{
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padding:2%;
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font-family:'Fresca',sans-serif;
 
font-family:'Fresca',sans-serif;
 
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padding-bottom:250px;
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.Nitrogen_Starvation h4{
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#Protocol h3 {
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font-size: 28px;
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font-family: 'Francois One', sans-serif;
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font-size: 40px;
 
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font-family: 'Acme', sans-serif;
 
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.Nitrogen_Starvation p {
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#Labnote h1{
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#Protocol {width:96%; margin-left:0px;}
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#Protocol {width:96%; margin-left:50px;}
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#Timeline {width:96%; margin-left:0px;}
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#Labnote {width:96%; margin-left:50px;}
 
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function toggleHeight1(e, maxHeight) {
 
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</script>
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</head>
 
</head>
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<body>
 
<body>
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<div class="Nitrogen_Starvation_top">
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<div id="Protocol" >
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<img src="https://static.igem.org/mediawiki/2017/2/20/T--NYMU-Taipei--nitrogen_starvation.jpg" width="100%">
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<center>
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<h3></h3>
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<img src="https://static.igem.org/mediawiki/2017/f/f9/T--NYMU-Taipei--notebook.jpg"
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style="width:50%;border-radius:50%;border:5px solid #f6ffb2;">
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</center>
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<h3></h3>
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<h3></h3>
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<h1>Protocol</h1>
 +
<h3></h3>
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<h3>NrtA expression</h3>
 +
<p><a herf="">Download the file</a></p>
 +
<h3>Suicide gene expression</h3>
 +
<p><a herf="">Download the file</a></p>
 +
<h3>OD measurement</h3>
 +
<p><a herf="">Download the file</a></p>
 
</div>
 
</div>
 
 
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<div class="Nitrogen_Starvation">
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<div id="Timeline" >
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<h1>Timeline</h1>
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<p></p>
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<div class='panel'>
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<div id="s1" class="expandable" style='height: 24px;'>
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<a href="#!" onclick="toggleHeight1(this, 440); return false"  
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<a href="#!" onclick="toggleHeight1(this, 220); return false"  
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style="font-family:'Acme', sans-serif;font-size:30px;color:#205e1a;height: 30px;">
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style="font-family: 'Acme', sans-serif;font-size:28px;color: #479fcc;height: 20px;">
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background
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June, 2017
 
</a>
 
</a>
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<p>　　With the development of global economy in the latest demands of energy in world, Taiwanese person produce 2.58 billion tons of carbon emission a year. The carbon emission of each person in Taiwan is far ahead China, Japan and South Korea. So in our project, we want to use biofuel as an alternative to fossil fuel in our project. We choose Microalgae as biofuel because it will produce more oil during the period of nitrogen starvation. </p>
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<p>
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<p>　　One research  indicates nitrogen starvation promotion fuel accumulation in microalgae. Under nitrogen starvation, de novo synthesis of triacylglycerol from acyl-CoA increases. Then acyl moieties from the degradation of membrane lipids recycle into triacylglycerol and finally increase carbon flux towards glycerol-3 -phosphate and acyl-CoA for fatty acid synthesis. Therefore, the oil accumulation under nitrogen starvation will increase.</p>
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<b>2017-06-19</b>&nbsp;&nbsp; prepare stock1 and stock5<br>
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<p>　　How to reach nitrogen starvation? In the past, people cultivate microalgae in closed pond. Give microalgae no nitrogen mediate and extract nitrogen.  But this method is consumes lots of energy like closed pond cultivation need electricity to maintain the temperature, nutrition, light etc. So, we want to develop a new method to make microalgae reach nitrogen starvation and evaluate oil production in open pond.</p>
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<b>2017-06-20</b>&nbsp;&nbsp; (1) reprepare stock5<br>
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　　　　　(2) prepare BG11 medium<br>
 +
　　　　　(3) unfreeze <i>Synechocystis</i> sp. PCC6803 and start to cultivate in BG11<br>
 +
<b>2017-06-29</b>&nbsp;&nbsp; (1) prepare Pseudomonas aeruginosa growing medium<br>
 +
　　　　　(2) activate Pseudomonas aeruginosa and start to cultivate in Petri dish<br>
 +
　　　　　(3) subculture <i>Synechocystis</i> sp. PCC6803
 +
</p>
 
</div>
 
</div>
 
</div>
 
</div>
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<div class='panel'>
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<div id="s2" class="expandable" style='height: 24px;'>
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<a href="#!" onclick="toggleHeight2(this, 1470); return false"  
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<a href="#!" onclick="toggleHeight2(this, 1440); return false"  
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style="font-family:'Acme', sans-serif;font-size:30px;color:#205e1a;height: 30px;">
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style="font-family: 'Acme', sans-serif;font-size:28px;color: #479fcc;height: 20px;">
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design
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July, 2017
 
</a>
 
</a>
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<p>　　NrtA protein sticks to the periplasmic membrane by a flexible linker and it can capture nitrite or nitrate in the periplasm. Then delivery to the transmembrane complexed that made by NrtB. In our project, we try to transform NrtA gene from cyanobacteria Synechosistis PCC 6803 to E.coli. Engineering E.coli will be capable of clutching nitrite or nitrate in the environment. They will not intake nitrate or nitrite since the gas accumulation may be lethal to cells. But the amount of cells that contain nitrite will decrease. Therefore, the microalgae will undergo nitrogen Starvation and produce oil more efficient.</p>
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<p>
−
<p>　　After building up the nitrogen starvation and extract the oil from microalgae, we need to kill E.coli to prevent contamination. So we plan to use endolysin and holin for cell lysis, which is similar to the mechanism used by team Pecking  (2014iGEM Beijing). Holin can trigger the formation of holes on cell membrane. When the holin successfully triggers holes on cell membrane, endolysin can pass the membrane through holes and decompose peptidoglycan. E.coli is lysed after the cell membrane and cell wall are destroyed. To control the suicide timing, we design an inducible promoter for holin, so that we can induce E.coli suicide at the exactly time we want.</p>
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<b>2017-07-02</b>&nbsp;&nbsp; extract <i>Pseudomonas aeruginosa</i> plasmid and freeze into refrigerator<br>
−
 
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<b>2017-07-03</b>&nbsp;&nbsp; proliferate <i>Pseudomonas aeruginosa</i> and freeze into refrigerator<br>
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<center>
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<b>2017-07-04</b>&nbsp;&nbsp; (1) prepare BG11 medium<br>
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<img src="https://static.igem.org/mediawiki/2017/8/83/T--NYMU-Taipei--nitrogen_starvation_design.png" width="100%">
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　　　　　(2) proliferate <i>Chlorella vulgaris</i> in Petri dish and centrifuge tube<br>
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</center>
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<b>2017-07-05 to 07-10</b>&nbsp;&nbsp; cultivate <i>Synechocystis</i> sp. PCC6803 and <i>Chlorella vulgaris</i> in incubator<br>
 +
<b>2017-07-11</b>&nbsp;&nbsp;&nbsp;&nbsp; (1) extract <i>Synechocystis</i> sp. PCC6803 plasmid<br>
 +
　　　　　(2) prepare LB containing ampicillin and chloramphenicol and prepare Petri dish<br>
 +
<b>2017-07-12</b>&nbsp;&nbsp; transform BBa_K112806, BBa_K737007, BBa_K112000, BBa_J04450, BBa_K1356003, BBa_K1356004, and BBa_K356005 into competent cell<br>
 +
<b>2017-07-13</b>&nbsp;&nbsp;&nbsp; (1) proliferate 07-12 transformed E.coli<br>
 +
　　　　　(2) proliferate <i>Chlorella vulgaris</i><br>
 +
　　　　　(3) proliferate <i>Synechocystis</i> sp. PCC6803<br>
 +
　　　　　(4) activate <i>Synechococcus elongatus</i> PCC7942<br>
 +
<b>2017-07-14</b>&nbsp;&nbsp;&nbsp; (1)extract 07-13 proliferated E.coli plasmid<br>
 +
　　　　　(2) digestion: BBa_K112806, BBa_K737007, BBa_K112000, BBa_J04450, BBa_K1356003, BBa_K1356004, and BBa_K1356005<br>
 +
　　　　　　(check the correction of transformed E.coli)<br>
 +
<b>2017-07-17</b>&nbsp;&nbsp;&nbsp; (1) digestion again: BBa_K1356005, BBa_K112806, and BBa_K112000<br>
 +
　　　　　(2) retransform BBa_K112806 and BBa_K112000<br>
 +
<b>2017-07-18</b>&nbsp;&nbsp;&nbsp; (1) proliferate BBa_K112806 transformed E.coli (BBa_K112000 failed)<br>
 +
　　　　　(2) subculture <i>Synechococcus elongatus</i> PCC7942<br>
 +
　　　　　(3) cultivate <i>Synechocystis</i> sp. PCC6803 in BG11<br>
 +
<b>2017-07-19</b>&nbsp;&nbsp;&nbsp; (1) extract 07-18 BBa_K112806 transformed E.coli plasmid<br>
 +
　　　　　(2) digestion check<br>
 +
　　　　　(3) save BBa_K112806 transformed E.coli<br>
 +
　　　　　(4) save 07-18 <i>Synechocystis</i> sp. PCC6803<br>
 +
<b>2017-07-20</b>&nbsp;&nbsp; (1) activate Rhizobium etli in PY medium<br>
 +
　　　　　(2) dilute primer 001~008<br>
 +
　　　　　(3) run NrtA and terminator PCR (failed)<br>
 +
<b>2017-07-21 to 07-23</b>&nbsp;&nbsp; cultivate <i>Chlorella vulgaris</i> again for measuring growth curve<br>
 +
<b>2017-07-24</b>&nbsp;&nbsp; (1) prepare <i>Rhizobium etli</i> liquid culture<br>
 +
　　　　　(2) activate <i>Synechococcus elongatus</i> PCC7942<br>
 +
<b>2017-07-25</b>&nbsp;&nbsp; (1) extract <i>Synechocystis</i> sp. PCC6803 genomic DNA<br>
 +
　　　　　(2) run NrtA PCR<br>
 +
<b>2017-07-26</b>&nbsp;&nbsp; (1) extract <i>Rhizobium etli</i> genomic DNA<br>
 +
　　　　　(2) run NrtA and terminator PCR<br>
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　　　　　(3) run NrtA+ terminator fusion PCR (failed)<br>
 +
<b>2017-07-27</b>&nbsp;&nbsp; (1) run NrtA+ terminator fusion PCR again<br>
 +
　　　　　(2) purify the successful fusion product <br>
 +
　　　　　(3) digestion check<br>
 +
<b>2017-07-28</b>&nbsp;&nbsp; run the whole NrtA cloning procedure again, using KOD polymerase<br>
 +
　　　　　(1) run NrtA and terminator PCR<br>
 +
　　　　　(2) digestion check NrtA and terminator and purify the PCR product<br>
 +
　　　　　(3) run NrtA and terminator fusion PCR<br>
 +
　　　　　(4) digestion check and purify the fusion PCR product (failed)<br>
 +
<b>2017-07-29</b>&nbsp;&nbsp; (1) run NrtA and terminator fusion PCR again<br>
 +
　　　　　(2) digestion check and purify the fusion PCR product<br>
 +
　　　　　(3) ligation: backbone(psb1C3) and promoter, RBS, NrtA, and terminator<br>
 +
　　　　　(4) transformation in to E.coli<br>
 +
<b>2017-07-30</b>&nbsp;&nbsp; cultivate the transformed E.coli in liquid culture<br>
 +
<b>2017-07-31</b>&nbsp;&nbsp;&nbsp; (1) extract 07-30 transformed E.coli plasmid<br>
 +
　　　　　(2) restriction enzyme check (failed)<br>
 +
</p>
 
</div>
 
</div>
 
</div>
 
</div>
 
 
 
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<div class='panel'>
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<div id="s3" class="expandable" style='height: 30px;'>
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<div id="s3" class="expandable" style='height: 24px;'>
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<a href="#!" onclick="toggleHeight3(this, 440); return false"  
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<a href="#!" onclick="toggleHeight3(this, 240); return false"  
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style="font-family:'Acme', sans-serif;font-size:30px;color:#205e1a;height: 30px;">
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style="font-family: 'Acme', sans-serif;font-size:28px;color: #479fcc;height: 20px;">
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experiments
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Augest, 2017
 
</a>
 
</a>
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<h4>PCC 6803 gDNA extraction</h4>
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<p>
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<h4>NrtA expression</h4>
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<b>2017-06-19</b>&nbsp;&nbsp; prepare stock1 and stock5<br>
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<h4>endolysin construct</h4>
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</p>
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<h4>holin construct</h4>
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−
<h4>endolysin-holin construct</h4>
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<h4>endolysin-holin-NrtA construct</h4>
+
−
<p>*See our parts <a href="https://2017.igem.org/Team:NYMU-Taipei/Parts">click</a></p>
+
−
<p>*See our experiments protocols <a href="https://2017.igem.org/Team:NYMU-Taipei/Notebook">click</a></p>
+
 
</div>
 
</div>
 
</div>
 
</div>
 
 
 
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<div class='panel'>
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<a href="#!" onclick="toggleHeight4(this, 240); return false"  
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style="font-family: 'Acme', sans-serif;font-size:28px;color: #479fcc;height: 20px;">
−
functional test
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September, 2017
 
</a>
 
</a>
−
<h4>PCC 6803 gDNA extraction</h4>
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<p>
−
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<b>2017-06-19</b>&nbsp;&nbsp; prepare stock1 and stock5<br>
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</p>
 
</div>
 
</div>
 
</div>
 
</div>
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 +
<div class='panel'>
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<div id="s5" class="expandable" style='height: 24px;'>
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<a href="#!" onclick="toggleHeight5(this, 240); return false"
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style="font-family: 'Acme', sans-serif;font-size:28px;color: #479fcc;height: 20px;">
 +
October, 2017
 +
</a>
 +
<p>
 +
<b>2017-06-19</b>&nbsp;&nbsp; prepare stock1 and stock5<br>
 +
</p>
 +
</div>
 +
</div>
 +
 +
</div>
 
 
 +
<div id="Labnote">
 +
<h1>Labnote</h1>
 +
<p></p>
 +
<a herf="https://static.igem.org/mediawiki/2017/7/7d/T--NYMU-Taipei--Labnote001.pdf">Download the file</a>
 +
<p></p>
 +
<embed src="https://static.igem.org/mediawiki/2017/7/7d/T--NYMU-Taipei--Labnote001.pdf" style="width:80%;height:500px;padding-left:10%">
 
</div>
 
</div>
 
</body>
 
</body>
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</html>
 
</html>

Revision as of 06:15, 17 September 2017

HOME
PROJECT
Overview
Pigments
Nitrogen starvation
Modeling
Hardware
Contribution
Improvement
Demonstration
LABWORK
Parts
Safety
Notebook
HUMAN PRACTICES
Silver HP
Gold & Integrated HP
Collaborations
ABOUT US
AWARDS
Applied Design
Modeling
Measurement
Plant
Basic Parts
Composite Parts
Part Collection
Public Engagement
JUDGING FORM

CONTACT US

Email us: 2017igem.nymutaipei@gmail.com
Call us: 886-2-28267316
Facebook: NYMU iGEM Team

AFFILIATIONS & ACKNOWLEDGMENT

IGEM 2017
National Yang-Ming University
Special Thanks

Protocol

NrtA expression

Download the file

Suicide gene expression

Download the file

OD measurement

Download the file

Timeline