http://2009.igem.org/wiki/index.php?title=Special:Contributions/Ke_lyxaa&feed=atom&limit=50&target=Ke_lyxaa&year=&month=2009.igem.org - User contributions [en]2024-03-29T10:25:05ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:HKUST/Lab_NotebookTeam:HKUST/Lab Notebook2009-10-20T10:10:17Z<p>Ke lyxaa: </p>
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<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
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<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
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<span style="color:green"><br />
<li>Resources</li><br />
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<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
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<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Biosafety">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
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<div class="contentlab"> <h3>a</h3><br />
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<span> iGEM 2009 <br /> </span> <br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:TeamMembersTeam:TeamMembers2009-10-20T08:51:50Z<p>Ke lyxaa: </p>
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<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
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<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
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<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
</ul><br />
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<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Biosafety">BIOSAFETY</a></li><br />
<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
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<p><b>Instructor and Advisors</b><br />
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<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/King.jpeg" class="image" title="King"><img alt="King" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/King.jpeg" width="180" height="124" border="0" /><br />
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<p>Professor King-Lau CHOW, Department of Biology<br />
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<p>Guangnan Tian, Biochemistry Student<br />
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<p>Wai-Pang HO, Computer Science and Engineering Student<br />
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<p><b>Team Members</b><br />
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<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Elaine.jpg" class="image" title="Elaine"><br />
<img alt="Elaine" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Elaine.jpg" width="120" height="119" border="0" /><br />
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<p>Cai Yilei<br> Hi~ I am Elaine, a year 2 Molecular Biomedical Science student. Through this iGem project, I really learned a lot from my teammates, on both spirit and knowledge aspects.<br />
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<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><br />
<div style="margin-left: auto; margin-right: auto; width: 120px;"><br />
<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Swift.jpg" class="image" title="Swift"><br />
<img alt="Swift" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Swift.jpg" width="140" height="119" border="0" /></a></div></div><br />
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<p>My name is CHEN Xihan. You can call me Swift for short. I am an undergraduate student in HKUST studying Chemistry. I was born in Wuhan and I am now studying in Hong Kong. I am a fan of sports and soccer is my favorite.<br />
</p><br />
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<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><br />
<div style="margin-left: auto; margin-right: auto; width: 120px;"><br />
<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Emma.jpg" class="image" title="Emma"><br />
<img alt="Emma" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Emma.jpg" width="120" height="119" border="0" /><br />
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<p>I'm Chen Xinru (Emma), a year 1 student and a part-time bug buster.<br />
IGEM provided me with the great chance of learning all those fascinating stuff and I really enjoyed the past four months. Good luck to HKUST bug busters!<br />
</p><br />
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<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><br />
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<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Bonnie.jpg" class="image" title="Bonnie"><br />
<img alt="Bonnie" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Bonnie.jpg" width="120" height="119" border="0" /></a></div></div><br />
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<p>Lam Ki Yan <br><br />
Hi, I am Bonnie, a year 2 Biology students. In iGem, I have acquired many practical skills in laboratory, and most importantly, I have made a bunch of friends who worked together in the laboratory and had a lot of fun.<br />
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<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Lavinia.jpg" class="image" title="Lavinia"><br />
<img alt="Lavinia" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Lavinia.jpg" width="120" height="119" border="0" /></a></div></div><br />
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<p>Lavinia Li <br><br />
Hi, I'm Li Yunzi. I'm a sophomore in Chemical and Biomolecular Engineering Department. I really had a wonderful time in iGEM team this summer! I stepped into the fantastic world of Synthetic Biology together with all my lovely teammates! I love our team very much!!<br />
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<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><br />
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<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Anglea.jpg" class="image" title="Angela"><img alt="Angela" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Anglea.jpg" width="86" height="119" border="0" /><br />
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<p>Liu Yang, Angela<br> Hello =] I'm a freshman in Biochemistry. This summer I really had a great<br />
time massacring E.coli and cooking DNA. Thank you to those who train, advise, help, torture, scold or<br />
terrify me and who happen to read till this line!<br />
</p><br />
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<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><div style="margin-left: auto; margin-right: auto; width: 120px;"><br />
<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Kelly.jpg" class="image" title="Kelly"><br />
<img alt="Kelly" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Kelly.jpg" width="120" height="119" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p>Hi, I'm Kelly, LUO Yunqiu, a year 1 applied Physics student, responsible for Toxin<br />
Part. I have learned and experienced a lot about research in<br />
synthetic biology. What I value most for IGEM is that we<br />
are not in this game for prize, we are in to learn and experience.<br />
</p><br />
</div><br />
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<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><div style="margin-left: auto; margin-right: auto; width: 120px;"><br />
<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Kelvin.jpg" class="image" title="Kelvin"><br />
<img alt="Kelvin" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Kelvin.jpg" width="140" height="119" border="0" /></a></div></div><br />
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<p>I am Miu Kai Kei. I am an undergraduate student from Molecular Biomedical<br />
Sciences. I like science a lot, in particularly Biochemistry.<br />
</p><br />
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<p>Ng Wang Ki, Vito <br><br />
I am a year 2 student major in Molecular Biomedical Sciences. My interests are playing sports and reading. My previous research included characterizing mutants of C.elegans. I join the HKUST iGEM team and build up biological knowledge and research skills. I looked forward to 2010 HKUST iGEM project.<br />
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<div class="thumb" style="padding: 13px 0; width: 140px;"><div style="margin-left: auto; margin-right: auto; width: 120px;"><a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Aileen.jpg" class="image" title="Aileen"><img alt="Aileen" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Aileen.jpg" width="120" height="119" border="0" /></a></div></div><br />
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<p>PAN Huilin <br><br />
I'm Aileen, secretary of HKUST team, a year 2 biochemistry student. I think iGem is fairly interesting and join the team this year. I feel more comfortable communicating and making friends with other people. =) Welcome if you wanna befriend with me.<br />
</p><br />
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<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><div style="margin-left: auto; margin-right: auto; width: 120px;"><a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Alex.jpg" class="image" title="Alex"><img alt="Alex" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Alex.jpg" width="90" height="119" border="0" /></a></div></div><br />
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<p>Wong Chun Hung, Alex <br><br />
I am currently a year 3 biochemistry student, minor in chemistry and physics, responsible for the toxin part. This project gives me valuable experience to apply all my knowledges. I like running and hiking. I also play chess, read magazine, listen to music and watch movie.<br />
</p><br />
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<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><div style="margin-left: auto; margin-right: auto; width: 120px;"><a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Sandy.jpg" class="image" title="Sandy"><img alt="Sandy" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Sandy.jpg" width="120" height="119" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p>Wong Pui Shan, Sandy <br><br />
I am currently a year 2 (year 09-10) Chemical and Bioproduct Engineering student. In this project I am responsible for the design and testing of the toxin part.<br />
</p><br />
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<div class="thumb" style="padding: 13px 0; width: 140px;"><div style="margin-left: auto; margin-right: auto; width: 120px;"><a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Philip.jpg" class="image" title="Philip"><img alt="Philip" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Philip.jpg" width="82" height="119" border="0" /></a></div></div><br />
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<p>Yang Yang<br />
</p><br />
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<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><div style="margin-left: auto; margin-right: auto; width: 120px;"><a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Chuchu.jpg" class="image" title="Chuchu"><img alt="Chuchu" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Chuchu.jpg" width="120" height="119" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p>Zhang Chuchu <br><br />
Hey guys, I'm Chu Chu. I am from Wuhan - China and I have studied in Hong Kong for 2 years. I love travelling and I wish I could go to every single place in the world!<br />
</p><br />
</div><br />
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<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><div style="margin-left: auto; margin-right: auto; width: 120px;"><a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Joyce.jpg" class="image" title="Joyce"><img alt="Joyce" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Joyce.jpg" width="120" height="119" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p><br />
I am Zhang-Junyi, or you can call me Joyce as well. I am a year 1 student from HKUST Biology department. As a freshman in synthetic Biology, I really learnt a lot in this iGEM competition.<br />
</p><br />
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<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><div style="margin-left: auto; margin-right: auto; width: 120px;"><a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Helen.jpg" class="image" title="Helen"><img alt="Helen" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Helen.jpg" width="90" height="119" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p><br />
I am Helen, ZHONG Wanting, (also affectionately known as SPY), a UG Year 1 student majoring in Biochemistry at HKUST. As a member of the 2009 HKUST iGEM team, I have enjoyed a truly amazing summer on the project~<br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:TeamMembersTeam:TeamMembers2009-10-20T08:51:15Z<p>Ke lyxaa: </p>
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<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
</ul><br />
</div><br />
<div id="menubottom"><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Biosafety">BIOSAFETY</a></li><br />
<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
</div><br />
</div><br />
<div id="rightxx"><br />
<div class="contentxx"> <br />
<p><b>Instructor and Advisors</b><br />
</p><br />
<table class="gallery" cellspacing="0" cellpadding="0"><br />
<tr><br />
<td><div class="gallerybox" style="width: 195px;"><br />
<div class="thumb" style="padding: 13px 0; width: 190px;"><br />
<div style="margin-left: auto; margin-right: auto; width: 180px;"><br />
<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/King.jpeg" class="image" title="King"><img alt="King" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/King.jpeg" width="180" height="124" border="0" /><br />
</a></div></div><br />
<div class="gallerytext"><br />
<p>Professor King-Lau CHOW, Department of Biology<br />
</p><br />
</div><br />
</div></td><br />
<td><div class="gallerybox" style="width: 195px;"><br />
<div class="thumb" style="padding: 13px 0; width: 190px;"><br />
<div style="margin-left: auto; margin-right: auto; width: 180px;"><br />
<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Richard.jpg" class="image" title="Richard"><br />
<img alt="Richard" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Richard.jpg" width="190" height="120" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p>Guangnan Tian, Biochemistry Student<br />
</p><br />
</div><br />
</div></td><br />
<td><div class="gallerybox" style="width: 195px;"><br />
<div class="thumb" style="padding: 13px 0; width: 190px;"><br />
<div style="margin-left: auto; margin-right: auto; width: 180px;"><br />
<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Tony.jpg" class="image" title="Tony"><br />
<img alt="Tony" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Tony.jpg" width="190" height="120" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p>Wai-Pang HO, Computer Science and Engineering Student<br />
</p><br />
</div><br />
</div></td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<br />
<p><b>Team Members</b><br />
</p><br />
</div><br />
<table class="gallery" cellspacing="0" cellpadding="0"><br />
<tr><br />
<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><br />
<div style="margin-left: auto; margin-right: auto; width: 120px;"><br />
<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Elaine.jpg" class="image" title="Elaine"><br />
<img alt="Elaine" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Elaine.jpg" width="120" height="119" border="0" /><br />
</a></div></div><br />
<div class="gallerytext"><br />
<p>Cai Yilei<br> Hi~ I am Elaine, a year 2 Molecular Biomedical Science student. Through this iGem project, I really learned a lot from my teammates, on both spirit and knowledge aspects.<br />
</p><br />
</div><br />
</div></td><br />
<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><br />
<div style="margin-left: auto; margin-right: auto; width: 120px;"><br />
<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Swift.jpg" class="image" title="Swift"><br />
<img alt="Swift" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Swift.jpg" width="140" height="119" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p>My name is CHEN Xihan. You can call me Swift for short. I am an undergraduate student in HKUST studying Chemistry. I was born in Wuhan and I am now studying in Hong Kong. I am a fan of sports and soccer is my favorite.<br />
</p><br />
</div><br />
</div></td><br />
<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><br />
<div style="margin-left: auto; margin-right: auto; width: 120px;"><br />
<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Emma.jpg" class="image" title="Emma"><br />
<img alt="Emma" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Emma.jpg" width="120" height="119" border="0" /><br />
</a></div></div><br />
<div class="gallerytext"><br />
<p>I'm Chen Xinru (Emma), a year 1 student and a part-time bug buster.<br />
IGEM provided me with the great chance of learning all those fascinating stuff and I really enjoyed the past four months. Good luck to HKUST bug busters!<br />
</p><br />
</div><br />
</div></td><br />
<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><br />
<div style="margin-left: auto; margin-right: auto; width: 120px;"><br />
<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Bonnie.jpg" class="image" title="Bonnie"><br />
<img alt="Bonnie" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Bonnie.jpg" width="120" height="119" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p>Lam Ki Yan <br><br />
Hi, I am Bonnie, a year 2 Biology students. In iGem, I have acquired many practical skills in laboratory, and most importantly, I have made a bunch of friends who worked together in the laboratory and had a lot of fun.<br />
</p><br />
</div><br />
</div></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><br />
<div style="margin-left: auto; margin-right: auto; width: 120px;"><br />
<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Lavinia.jpg" class="image" title="Lavinia"><br />
<img alt="Lavinia" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Lavinia.jpg" width="120" height="119" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p>Lavinia Li <br><br />
Hi, I'm Li Yunzi, Lavinia. I'm a sophomore in Chemical and Biomolecular Engineering Department. I really had a wonderful time in iGEM team this summer! I stepped into the fantastic world of Synthetic Biology together with all my lovely teammates! I love our team very much!!<br />
</p><br />
</div><br />
</div></td><br />
<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><br />
<div style="margin-left: auto; margin-right: auto; width: 120px;"><br />
<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Anglea.jpg" class="image" title="Angela"><img alt="Angela" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Anglea.jpg" width="86" height="119" border="0" /><br />
</a></div></div><br />
<div class="gallerytext"><br />
<p>Liu Yang, Angela<br> Hello =] I'm a freshman in Biochemistry. This summer I really had a great<br />
time massacring E.coli and cooking DNA. Thank you to those who train, advise, help, torture, scold or<br />
terrify me and who happen to read till this line!<br />
</p><br />
</div><br />
</div></td><br />
<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><div style="margin-left: auto; margin-right: auto; width: 120px;"><br />
<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Kelly.jpg" class="image" title="Kelly"><br />
<img alt="Kelly" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Kelly.jpg" width="120" height="119" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p>Hi, I'm Kelly, LUO Yunqiu, a year 1 applied Physics student, responsible for Toxin<br />
Part. I have learned and experienced a lot about research in<br />
synthetic biology. What I value most for IGEM is that we<br />
are not in this game for prize, we are in to learn and experience.<br />
</p><br />
</div><br />
</div></td><br />
<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><div style="margin-left: auto; margin-right: auto; width: 120px;"><br />
<a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Kelvin.jpg" class="image" title="Kelvin"><br />
<img alt="Kelvin" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Kelvin.jpg" width="140" height="119" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p>I am Miu Kai Kei. I am an undergraduate student from Molecular Biomedical<br />
Sciences. I like science a lot, in particularly Biochemistry.<br />
</p><br />
</div><br />
</div></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><br />
<div style="margin-left: auto; margin-right: auto; width: 120px;"><a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Vito.jpg" class="image" title="Vito"><br />
<img alt="Vito" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Vito.jpg" width="120" height="119" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p>Ng Wang Ki, Vito <br><br />
I am a year 2 student major in Molecular Biomedical Sciences. My interests are playing sports and reading. My previous research included characterizing mutants of C.elegans. I join the HKUST iGEM team and build up biological knowledge and research skills. I looked forward to 2010 HKUST iGEM project.<br />
</p><br />
</div><br />
</div></td><br />
<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><div style="margin-left: auto; margin-right: auto; width: 120px;"><a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Aileen.jpg" class="image" title="Aileen"><img alt="Aileen" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Aileen.jpg" width="120" height="119" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p>PAN Huilin <br><br />
I'm Aileen, secretary of HKUST team, a year 2 biochemistry student. I think iGem is fairly interesting and join the team this year. I feel more comfortable communicating and making friends with other people. =) Welcome if you wanna befriend with me.<br />
</p><br />
</div><br />
</div></td><br />
<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><div style="margin-left: auto; margin-right: auto; width: 120px;"><a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Alex.jpg" class="image" title="Alex"><img alt="Alex" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Alex.jpg" width="90" height="119" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p>Wong Chun Hung, Alex <br><br />
I am currently a year 3 biochemistry student, minor in chemistry and physics, responsible for the toxin part. This project gives me valuable experience to apply all my knowledges. I like running and hiking. I also play chess, read magazine, listen to music and watch movie.<br />
</p><br />
</div><br />
</div></td><br />
<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><div style="margin-left: auto; margin-right: auto; width: 120px;"><a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Sandy.jpg" class="image" title="Sandy"><img alt="Sandy" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Sandy.jpg" width="120" height="119" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p>Wong Pui Shan, Sandy <br><br />
I am currently a year 2 (year 09-10) Chemical and Bioproduct Engineering student. In this project I am responsible for the design and testing of the toxin part.<br />
</p><br />
</div><br />
</div></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><div style="margin-left: auto; margin-right: auto; width: 120px;"><a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Philip.jpg" class="image" title="Philip"><img alt="Philip" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Philip.jpg" width="82" height="119" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p>Yang Yang<br />
</p><br />
</div><br />
</div></td><br />
<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><div style="margin-left: auto; margin-right: auto; width: 120px;"><a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Chuchu.jpg" class="image" title="Chuchu"><img alt="Chuchu" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Chuchu.jpg" width="120" height="119" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p>Zhang Chuchu <br><br />
Hey guys, I'm Chu Chu. I am from Wuhan - China and I have studied in Hong Kong for 2 years. I love travelling and I wish I could go to every single place in the world!<br />
</p><br />
</div><br />
</div></td><br />
<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><div style="margin-left: auto; margin-right: auto; width: 120px;"><a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Joyce.jpg" class="image" title="Joyce"><img alt="Joyce" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Joyce.jpg" width="120" height="119" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p><br />
I am Zhang-Junyi, or you can call me Joyce as well. I am a year 1 student from HKUST Biology department. As a freshman in synthetic Biology, I really learnt a lot in this iGEM competition.<br />
</p><br />
</div><br />
</div></td><br />
<td><div class="gallerybox" style="width: 145px;"><br />
<div class="thumb" style="padding: 13px 0; width: 140px;"><div style="margin-left: auto; margin-right: auto; width: 120px;"><a href="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Helen.jpg" class="image" title="Helen"><img alt="Helen" src="http://igem2009hkust.fileave.com/wiki/template/selfPhoto/Helen.jpg" width="90" height="119" border="0" /></a></div></div><br />
<div class="gallerytext"><br />
<p><br />
I am Helen, ZHONG Wanting, (also affectionately known as SPY), a UG Year 1 student majoring in Biochemistry at HKUST. As a member of the 2009 HKUST iGEM team, I have enjoyed a truly amazing summer on the project~<br />
</p><br />
</div><br />
</div></td><br />
<br />
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</table><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Lab_NotebookTeam:HKUST/Lab Notebook2009-10-19T16:14:36Z<p>Ke lyxaa: </p>
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<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li><br />
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
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<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
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<span style="color:green"><br />
<li>Resources</li><br />
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<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Lab_NotebookTeam:HKUST/Lab Notebook2009-10-19T16:12:00Z<p>Ke lyxaa: </p>
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<div id="mainxx"><br />
<div id="leftxx"><br />
<div id="menuxx"><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/GFP_testingTeam:HKUST/Protocols/GFP testing2009-10-19T16:03:28Z<p>Ke lyxaa: </p>
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<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Fluorescence microscopy visualization</p><br />
<br />
<br />
<p>Procedure: </p><br />
1. Grow yeast transformed cells containing appropriate plasmids in 5 ml SCM minus His & glucose at 30ºC overnight.<br><br />
2. Add galactose (to a conc.of 2%) at a cell density corresponding to an OD600 of a 10-fold dilution reaching 0.8.<br><br />
3. Continue growing cells at 30ºC for 30min to 1.5hr.<br><br />
4. At time 30min, 60min, 90min, use 1ml of cells, spin down and resuspend cells in 10μl ddH2O.<br><br />
5. Use 2μl cells and visualize the cells in a fluorescency microscopy.<br />
<br> <br><br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/Western_blottingTeam:HKUST/Protocols/Western blotting2009-10-19T16:02:09Z<p>Ke lyxaa: </p>
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<li>Main Parts</li><br />
</span><br />
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<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
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<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Western Blotting</p><br />
<br />
<p>Procedure: </p><br />
1. Yeast transformed cells containing appropriate plasmids were grown in SCM with appropriate amino acids deleted and harvested at a cell density corresponding to an OD600 of a 10-fold dilution reaching 0.2. <br><br />
2. 2× Laemmli’s buffer (20% v/v glycerol, 0.1M Tris-HCl pH 6.9, 4% SDS, 0.2% bromophenol blue, 10% mercaptoethanol) was added and yeast crude extracts were prepared by glass bead lysis and separated on 10% SDS-PAGE.<br><br />
3. The 10% resolving gel and the 5% stacking gel were cast according to the method described in Molecular Cloning (Sambrook et al., 1989).Separation was performed on a Mini Protean 3 gel and transblot system (Biorad). Proteins were stacked at 60-80 volts in the stacking gel and resolved at 120-150 volts in the resolving gel. <br><br />
4. The proteins were then transferred onto a nitrocellulose membrane (pore size 0.45 μm, Schleicher &Schuell) in electrophoretic blotting system (C.B.S. Scientific) by applying a constant current of 0.5 mA for 1.5 hours.<br><br />
5. After transferring, the membrane was taken out and socked in Ponceau S solution (0.5% Ponceau S in 1% HAc) for 1 min. After rinsed with ddH2O and labeled with protein marker bands.<br><br />
6. The membrane was destained with TBST buffer (10 mM Tris-HCl pH8.0, 150 mM NaCl, 0.05% v/v Tween-20) and blocked with 5% milk in TBST for 30 min. <br><br />
7. Then the membrane was incubated with the primary antibody (mouse monoclonal antibody against Flag) for 1 hour at room temperature followed by 3 × 15 min washing with TBST. <br><br />
8. The membrane was then incubated with secondary antibody (goat anti-mouse antibody conjugated with horseradish peroxidase) for 1 hour at room temperature followed by 3 × 15 min washing with TBST. <br><br />
9. After that, the membrane was incubated in SuperSignal chemiluminescence substrate (Pierce) for 5 min and exposed to light-sensitive films (Fuji). In the end, the film was developed in a film-processing machine (Eastman Kodak).<br> <br><br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/Western_blottingTeam:HKUST/Protocols/Western blotting2009-10-19T16:01:26Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
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<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
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<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
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<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
</div><br />
</div><br />
<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Western Blotting</p><br />
<br />
<p>Procedure: </p><br />
1. Yeast transformed cells containing appropriate plasmids were grown in SCM with appropriate amino acids deleted and harvested at a cell density corresponding to an OD600 of a 10-fold dilution reaching 0.2. <br />
2. 2× Laemmli’s buffer (20% v/v glycerol, 0.1M Tris-HCl pH 6.9, 4% SDS, 0.2% bromophenol blue, 10% mercaptoethanol) was added and yeast crude extracts were prepared by glass bead lysis and separated on 10% SDS-PAGE.<br />
3. The 10% resolving gel and the 5% stacking gel were cast according to the method described in Molecular Cloning (Sambrook et al., 1989).Separation was performed on a Mini Protean 3 gel and transblot system (Biorad). Proteins were stacked at 60-80 volts in the stacking gel and resolved at 120-150 volts in the resolving gel. <br />
4. The proteins were then transferred onto a nitrocellulose membrane (pore size 0.45 μm, Schleicher &Schuell) in electrophoretic blotting system (C.B.S. Scientific) by applying a constant current of 0.5 mA for 1.5 hours.<br />
5. After transferring, the membrane was taken out and socked in Ponceau S solution (0.5% Ponceau S in 1% HAc) for 1 min. After rinsed with ddH2O and labeled with protein marker bands.<br />
6. The membrane was destained with TBST buffer (10 mM Tris-HCl pH8.0, 150 mM NaCl, 0.05% v/v Tween-20) and blocked with 5% milk in TBST for 30 min. <br />
7. Then the membrane was incubated with the primary antibody (mouse monoclonal antibody against Flag) for 1 hour at room temperature followed by 3 × 15 min washing with TBST. <br />
8. The membrane was then incubated with secondary antibody (goat anti-mouse antibody conjugated with horseradish peroxidase) for 1 hour at room temperature followed by 3 × 15 min washing with TBST. <br />
9. After that, the membrane was incubated in SuperSignal chemiluminescence substrate (Pierce) for 5 min and exposed to light-sensitive films (Fuji). In the end, the film was developed in a film-processing machine (Eastman Kodak).<br><br><br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
<br />
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<span> iGEM 2009 <br /> </span> <br />
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<div id="payment"><img src="http://igem2009hkust.fileave.com/wiki/template/12092009/images/HKUSTLogo.jpg" alt="HKUST" /></div><br />
</div> <br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extractionTeam:HKUST/Protocols/Yeast genomic DNA extraction2009-10-19T16:00:02Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
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<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
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<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
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<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Yeast genomic DNA extraction</p><br />
<br />
<p> Purpose: To extract yeast genomic DNA from yeast strain (strain YPH501, in our case).</p><br />
Materials: yeast strain, lysis buffer, phenol, chloroform, 3M NaAc, 100% ethanol, 70% ethanol, ddH2O, glass beads. <br> <br><br />
<br />
<p>Procedure: </p><br />
a.Add 200μL lysis buffer.<br><br />
b.Pick a whole patch of colony and add it in the tube.<br><br />
c.Add 100μL phenol, and mix it well.<br><br />
d.Add 100μL chloroform, and a few glass beads.<br><br />
e.Vortex for 4-5mins and centrifuge for 5mins (13,000 rpm). <br><br />
f.Put the supernatant (~160μL) to another tube. <br><br />
g.Add 200μL phenol/chloroform (1:1 in volume)<br><br />
h.Vortex for 4-5mins and centrifuge for 5mins (13,000 rpm). <br><br />
i.Put the supernatant (~120μL) to another tube. <br><br />
j.Add 0.1 volume NaAc(pH5.2), approximately 12μL.<br><br />
k.Add 2 volume of 100% ethanol, approximately 240μL.<br><br />
l.Store it at -20 °C for 20mins.<br><br />
m.Centrifuge for 10mins (13,000 rpm) and remove the supernatant.<br><br />
n.Add 2 volume of 70% ethanol.<br><br />
o.Centrifuge for 5mins (13,000 rpm) and remove the supernatant. <br><br />
p.Stand the tube without closing it for a while to let the ethanol evaporate.<br><br />
q.Add 20μL ddH2O to resuspend it.<br><br />
r.Store it at -20 °C.<br><br><br />
<br />
<p> Safety tips: </p><br />
Phenol is a strong toxin, make sure to wear gloves and be careful with this chemical. <br><br><br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
<br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/Cell_lysisTeam:HKUST/Protocols/Cell lysis2009-10-19T15:58:16Z<p>Ke lyxaa: </p>
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<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Cell lysis</p><br />
<br />
<p> Purpose: to lyse E.coli cell to get linearized ligated plasmid.</p><br />
Materials: cultured transformed E.coli colony, phenol, chloroform, ddH2O <br> <br><br />
<br />
<p>Procedure: </p><br />
a.Add 40μL ddH2O in a 1.5mL microcentrigfuge tube. <br><br />
b.Use a toothpick to pick a colony and dissolve it in the water. <br><br />
c.Add 20μL phenol and 20μL chloroform. <br><br />
d.Scrape the tube to mix the solution. A well mixed white solution can be seen after this step. <br><br />
e.Centrifuge it for 10mins (13,000 rpm). <br><br />
f.Place the supernatant in another tube. <br><br />
g.Load 10μL to test the result by gel electrophoresis. <br> <br><br />
<p> Tips: </p><br />
Ignite the fire for sterile environment when transforming the colony.<br><br />
<p> Safety tips: </p><br />
1. Phenol is a strong toxin, make sure to wear gloves and be careful with this chemical.<br><br />
2. Be careful with the fire, and extinguish it after use. <br><br><br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeastTeam:HKUST/Protocols/Transformation to yeast2009-10-19T15:56:06Z<p>Ke lyxaa: </p>
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<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Transformation to E.coli</p><br />
<br />
<p> Purpose: To transform desired plasmid DNA in to E.coli in order to amplify it.</p><br />
Materials: E.coli competent cell, desired plasmid DNA, LB media, agar plate with desired selection antibiotic (ampicillin) <br> <br><br />
<br />
<p>Procedure: </p><br />
a.Perform ligation or any other manipulation of DNA to yield circularized plasmids.<br><br />
b.Remove bacterial aliquots (usually the DH5 strain of E.coli) from -70°C freezer and thaw on ice for 10-15 min; briefly flick the tube to resuspend the bacterial cells, but minimize any time the cells are not on ice.<br><br />
c.Add desired amount of plasmid DNA (usually 1-5μL of a standard ligation or 1.0ng of purified plasmid) to the bacteria at the bottom of the tube, mix it with pipette and incubate 5min on ice.<br><br />
d.Remove the tube from ice and immediately begin heat shock in a water bath at 42°C for 45 sec.<br><br />
e.After heat shock, immediately add 500μL of room temperature LB media to the bacterial cells, then incubate in a 37 °C shaker incubator for 45mins.<br><br />
f.Plate 50μL of the bacterial culture to a LB/agar plate with desired selection antibiotic (ampicillin) using a sterile bacterial streaking rod, and allow the plate to absorb the extra liquid for 15 min on the benchtop at room temperature.<br><br />
g.Invert the plates and place in the 37°C bacterial incubator overnight.<br><br><br />
<p> Tips: </p><br />
A. The competent cell is quite fragile, so make sure that it does not leave the ice before heat shocking.<br><br />
B. LB is easily contaminated, make sure to sterile it with fire each time when opening or closing it. Add it quickly to minimize the time of leave it open.<br><br />
C. Ignite the fire for sterile environment when adding LB.<br><br />
<p> Safety tips: </p><br />
Be careful with the fire, and extinguish it after use. <br><br><br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/Transformation_to_EcoliTeam:HKUST/Protocols/Transformation to Ecoli2009-10-19T15:53:35Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Transformation to yeast</p><br />
<br />
<p> Purpose: To transform desired constructed plamid DNA to yeast to test its effect. </p><br />
Materials: LiOAc solution, PEG4000 solution, DMSO (dimethyl sulfoxide), yeast strain, desired plasmid, YEPD, SD media with specific selection marker absent (-Leu, -His, or -Ura), ddH2O <br><br><br />
<br />
<p>Procedure: </p><br />
a.A stationary culture of yeast grown in YEPD is used to inoculate 10mL of YEPD in a 100mL Pyrex flask.<br><br />
b.Cells are grown at 30 °C with shaking (200 rpm) until a density of 1-4x107 is reached (OD660=0.4).<br><br />
c.Yeast is transformed into 4 sterile 1.5mL tubes and are centrifuged for 2mins (4,000 rpm).<br><br />
d.Damp off media and wash the pellet with 100μL ddH2O (to culture 1:1). Resuspend the cells by gently shake it or flip it.<br><br />
e.Centrifuge it again for 2mins (4,000 rpm). Remove the supernatant.<br><br />
f.Wash the pellet with 1mL LiOAc solution and pour it into a single tube. Resuspend the cell by gently shake it or flip it. <br><br />
g.Repeat step e and f once.<br><br />
h.Add 100μL of yeast suspension to a 1.5mL microcentrifuge tube, and add 10μL of DNA to be transformed. Mix them gently and stand the tube at room temperature for 5mins.<br><br />
i.Add 280μL PEG4000 solution. Mix it gently by inverting 4-6 times, then the tube is stored at 30 °C for 45mins without shaking.<br><br />
j.Add 43μL DMSO to give an approximate 10% (volume percentage) DMSO solution. Mix the solution gently by inverting.<br><br />
k.Heat shock at 42°C for 5mins.<br><br />
l.Centrifuge it for time long enough to get pellet (usually 5 sec) at 12,000rpm.<br><br />
m.Remove the supernatant and wash it with ddH2O.<br><br />
n.Centrifuge it again for 50sec (13,000 rpm). Remove the supernatant.<br><br />
o.Resuspend the cell with 1mL ddH2O.<br><br />
p.Plate the solution on SD media with specific selection marker absent.<br><br />
<p> Tips: </p><br />
Ignite the fire for sterile environment when transforming the yeast cell, since there is no any antibiotic in the culture.br><br />
<p> Safety tips: </p><br />
Be careful with the fire, and extinguish it after use. <br> <br><br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/LigationTeam:HKUST/Protocols/Ligation2009-10-19T15:51:21Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
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<span style="color:green"><br />
<li>Resources</li><br />
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<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
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<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
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<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
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<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Ligation</p><br />
<br />
<p> Purpose: To ligate desired insert DNA fragment into vector with specific restriction site.</p><br />
Materials: digested insert DNA and plasmid DNA with the same restriction site, T4 DNA ligase, 10X T4 Ligation Buffer, CIP (Calf Intestinal Alkaline Phosphatase), ddH2O <br><br><br />
<br />
<p>Procedure: </p><br />
a.Add sufficient water to bring to a total volume of 10 μL or 20 μL, depending on the volume of the two DNA pieces.<br><br />
b.Add 50ng of vector DNA.<br><br />
c.Add enough amount of insert DNA, which can be calculated by ligation calculator online.<br><br />
d.Add 2μL ligation buffer and 0.5μL ligase.<br><br />
e.Incubate at 16 °C overnight or at room temperature for 8 hours.</p><br />
<p> Tips: </p><br />
A. Ligase blank control group is recommended.<br><br />
B. The plasmid DNA and insert DNA for ligation must be added with CIP when enzyme digestion. <br><br><br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/gel_extractionTeam:HKUST/Protocols/gel extraction2009-10-19T15:49:33Z<p>Ke lyxaa: </p>
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<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
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<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Gel extraction</p><br />
<br />
<p> Purpose: To clean up the gel after band cutting from the gel and leave only the desired DNA fragment.</p><br />
Materials: GEX Buffer, WN Buffer, WS Buffer, Elution Buffer (all provided in FavorPrepTM Gel/PCR DNA Clean-Up Kit) <br><br><br />
<br />
<p>Procedure: </p><br />
a.Add 0.5ml GEX buffer into the tube with gel fragment in it. <br><br />
b.Incubate at 60°C for 5-10mins until the gel is completely dissolved. Invert the tube every 2 mins during the incubation. Stop incubation after the gel is completely dissolved. Let the gel mixture to cool down to room temperature.<br><br />
c.Place a GP column onto a collection tube. Load no more than 0.7ml gel mixture into each column. Centrifuge for 30s-60s (9000rpm). Discard the flow through.<br><br />
d.Repeat step c for the excessive gel mixture.<br><br />
e.Wash the column once with 0.5ml WN buffer by centrifuging for 30s-60s (9000rpm). Discard the flow through.<br><br />
f.Wash the column once with 0.5ml WS buffer by centrifuging for 30s-60s (9000rpm).<br><br />
g.Centrifuge for another 3 mins (13200rpm) to remove all the ethanol<br><br />
h.Place the column onto a new 1.5ml centrifuge tube. Add 15μL -30μL elution buffer onto the center of the membrane. (Preheated the Elution buffer to 60°C).<br><br />
i.Stand the column for another 3mins and then centrifuge at full speed for 1-2 mins to elute DNA.<br><br><br />
<p> Tips: </p><br />
The gel extraction has a low recovery rate of about 10%. Stand the column longer in step i may give relatively higher recovery rate.<br />
</p><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/plasmid_extractionTeam:HKUST/Protocols/plasmid extraction2009-10-19T15:49:07Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
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<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Consolidation">Consolidation</a></li><br />
<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
</div><br />
</div><br />
<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Plamid DNA extraction</p><br />
<br />
<p> Purpose: To extract plasmid DNA from E.coli.</p><br />
Materials: MX1 Buffer, MX2 Buffer, MX3 Buffer, WN Buffer, WS Buffer, Elution Buffer (all provided in Mini Plus Plasmid DNA Extraction System)<br> <br><br />
<br />
<p>Procedure: </p><br />
a.Grow 1 to 5 ml plasmid-containing bacterial cells in LB medium with appropriate anlibiotic overnight (12-16 hours) with vigorous agitation.<br><br />
b. Pellet the cells by centrifuging for 1-2 minutes. Decant the supernatant and remove all medium residues by pipette.<br><br />
c.Add 200 μL of MX1 Buffer 10 the pellet, and resuspend the cells completely by vortexing or pipetting.<br><br />
d.Add 250 μL of MX2 Buffer and gently mix well (invert the tube 6-10 times) to lyse the cells until the lysate becomes d ear. Incubate at room temperature for 2-5 minutes.<br><br />
e.Add 350 μL of MX3 Buffer to neutralize the lysate, then immediately and gently mix the solution well. A white precipitate should be formed.<br><br />
f.Centrifuge at 10,000 x g (13,000rpm) for 5-10 minutes, meanwhile place a Mini Plus™ Column onto a Collection Tube. Transfer the supernatant carefully into the column.<br><br />
g.Centrifuge at 7,000x g (9,000rpm) for 30-60 seconds. Discard the flow-through.<br><br />
h.Wash the column once with 0.5 ml of WN Buffer by centrifuging at 7,000 x g (9,000rpm) for 30-60 seconds. Discard the flow-through.<br><br />
i.Wash the column once with 0.7 ml of WS Buffer by centrifuging at 7,000 x g (9,000rpm) for 30-60 seconds. Discard the flow-through.<br><br />
j.Centrifuge the column at 10,000 x g (13,000rpm) for another 3 minutes to remove residual ethanol.<br><br />
k.Place the column into a new 1.5-ml centrifuge tube. Add 50 μL of Elution Buffer onto the center of the membrane.<br><br />
l.Stand the column for 2-3 minutes, and centrifuge at 10,000 x g (13,000rpm) for 2-3 minutes to elute DNA. Store plasmid DNA at 4 °C or -20 °C.</p><br />
<p> Tips: </p><br />
A. In step c, no clump should be visible after resuspension. Clumped cells lead to bad plasmid yield and quality.<br><br />
B. Do not vortex in step d, otherwise genomic DNA will be sheared and contaminate the sample, which can be observed as serious foaming.<br><br />
C. In step d, the lysate should become clear and viscous. Insufficient cell-lysis leads to low plasmid yield and quality.<br><br />
D. MX1 Buffer must be stored at 4 °C.<br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestionTeam:HKUST/Protocols/Enzyme digestion2009-10-19T15:48:39Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
</ul><br />
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<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Consolidation">Consolidation</a></li><br />
<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
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<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Enzyme digestion (vector, insert) </p><br />
<br />
<p> Purpose: To cut the sequence with specific restriction enzyme</p><br />
Materials: substrate DNA, restriction enzymes, corresponding 10X buffer, ddH2O, CIP (Calf Intestinal Alkaline Phosphatase, for vector digestion and DNA to be ligated)<br> <br><br />
<br />
<p>Procedure: </p><br />
a.Add sufficient water to make it a 20 μL reaction. <br><br />
b.Add 1 μg DNA, 2 μL buffer in total, 0.5 μL each restriction enzyme.<br><br />
c.Vortex and then spin down.<br><br />
d.Keep it in 37 °C incubator for one hour. (If needed, incubate for 1.5-2hours)<br><br />
e.If the substrate DNA is vector or pieces to be ligated, add 0.5 ul (for 20ul reaction) CIP after incubated for 1.5-2 hours, and then incubate at 37 °C for another 30mins.<br><br />
f.Check the result by gel electrophoresis.<br><br />
<p> Tips: </p><br />
A. Restriction enzymes are easy to be denatured, so do not leave it at room temperature.<br><br />
B. If two restriction enzymes must be added with different buffer, digest the DNA with respective restriction enzyme sequentially. Incubate for one hour after each enzyme added.</p><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/PCR_cleanupTeam:HKUST/Protocols/PCR cleanup2009-10-19T15:48:21Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>PCR product clean-up</p><br />
<br />
<p> Purpose: To clean up primers, dNTPs, enzymes, short-failed PCR products in the PCR reaction.</p><br />
Materials: FAPC Buffer,Wash Buffer, Elution Buffer, PCR Product (all provided in FavorPrepTM PCR Clean-Up Kit)<br> <br><br />
<br />
<p>Procedure: </p><br />
a.Transfer 30μL of PCR product and add 5 volumes of FAPC buffer to a 1.5mL microcentrifuge tube, then mix well by vortexing.<br><br />
b.Place a FAPC Column into a Collection Tube and transfer the sample mixture to FAPC Column.<br><br />
c.Centrifuge at 6,000 rpm for1 min then discard the flow-through.<br><br />
d.Add 750 μl of Wash Buffer (ethanol added) to FAPC Column. Centrifuge at 6,000 rpm for 1 min, then discard the flow-through.<br><br />
e.Centrifuge at 14,000 rpm for an additional 3 min to dry the column.<br><br />
f.Place FAPC Column into an Elution Tube.<br><br />
g.Add 40 μl of Elution Buffer or ddH2O (pH 7.0~8.5) to the membrane center of FAPC Column. Stand FAPC Column for 2 minutes.<br><br />
h.Centrifuge at 14,000 rpm for 1 min to elute the DNA.<br><br />
<p> Tips: </p><br />
In step f, for effective elution, make sure that the elution solution is dispensed onto the membrane center and is absorbed completely.</p><br />
<br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/PCRTeam:HKUST/Protocols/PCR2009-10-19T15:47:58Z<p>Ke lyxaa: </p>
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</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
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<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
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<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>PCR</p><br />
<br />
<p> Purpose: To amplify a specific piece of DNA out from the whole. </p><br />
Materials: ddH2O, 10X ThermoPol Buffer, 1mM dNTP, Forward primer, Reverse Primer, Taq or Vent DNA polymerase, DNA template<br />
Choice of DNA polymerase: Taq gives higher rate of mutation compared with Vent.<br />
Taq gives sticky ends after PCR, while Vent gives blunt ends. <br><br><br />
<br />
<p>Procedure: </p><br />
1. Add 10 μL water to make it a 20 μL reaction. <br><br />
2. Add 2 μL buffer, 4 μL dNTP, 1 μL forward primer (5 μM), 1 μL reverse primer (5 μM), 1 μL DNA template, 1 μL DNA polymerase <br><br />
3. Vortex and then spin down.<br><br />
4. Put it into the PCR machine and set the program.<br><br />
&nbsp; Program<br><br />
&nbsp; &nbsp; (1) Initial denaturation 95 °C 4 mins<br><br />
&nbsp; &nbsp; (2) Run 25-30 cycles of:<br><br />
&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Denaturation &nbsp; 95 °C 30 secs<br><br />
&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Annealing &nbsp; &nbsp; 30 secs <br><br />
&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Temperature is depended on melting temperature of primer. <br><br />
&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Lower Tm-5°C, for Taq; Lower Tm +3°C for Vent<br><br />
&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Extension 72 °C 30 secs per 500bp PCR product length<br><br />
&nbsp; &nbsp; (3) Final extension &nbsp; 72 °C 3~5 mins<br><br />
<p> Tips: </p><br />
The DNA polymerase is easy to be denatured, so do not leave it at room temperature. It is highly recommended to use an ice holder or do the adding in the fridge.</p><br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitationTeam:HKUST/Protocols/Ethanol precipitation2009-10-19T15:47:09Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Ethanol precipitation</p><br />
<br />
<p> Purpose: To concentrate DNA product.</p><br />
Materials: 100% ethanol, 75% ethanol, 3M NaAc, ddH2O, DNA product<br> <br><br />
<br />
<p>Procedure: </p><br />
a.Add 2 volume of 100% ethanol.<br><br />
b.Add 0.1 volume of 3M NaAc.<br><br />
c.Put the tube in fridge at -20 °C for 10 minutes.<br><br />
d.Put it out and centrifuge it for 10-15 minutes (13,000 rpm). Remove the supernatant.<br><br />
e.Wash with 100μL 75% ethanol.<br><br />
f.Centrifuge for 5 minutes (13,000 rpm). Remove all the supernatant.<br><br />
g.Add 10μL ddH2O to resuspend DNA.<br><br />
<p> Tips: </p><br />
A.If the volume of DNA product is too low, make it to higher volume with ddH2O. The recommended lowest volume is 50μL. Combine several tubes of DNA product is also suggested for higher DNA product concentration.<br><br />
B. In step f, make sure to remove all the supernatant without touching the pellet. If it is too hard to do so, open the tube and leave it at room temperature for a while to make the ethanol evaporate. <br><br><br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/gel_extractionTeam:HKUST/Protocols/gel extraction2009-10-19T15:45:20Z<p>Ke lyxaa: </p>
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<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Gel extraction</p><br />
<br />
<p> Purpose: To clean up the gel after band cutting from the gel and leave only the desired DNA fragment.</p><br />
Materials: GEX Buffer, WN Buffer, WS Buffer, Elution Buffer (all provided in FavorPrepTM Gel/PCR DNA Clean-Up Kit) <br><br><br />
<br />
<p>Procedure: </p><br />
a.Add 0.5ml GEX buffer into the tube with gel fragment in it. <br><br />
b.Incubate at 60°C for 5-10mins until the gel is completely dissolved. Invert the tube every 2 mins during the incubation. Stop incubation after the gel is completely dissolved. Let the gel mixture to cool down to room temperature.<br><br />
c.Place a GP column onto a collection tube. Load no more than 0.7ml gel mixture into each column. Centrifuge for 30s-60s (9000rpm). Discard the flow through.<br><br />
d.Repeat step c for the excessive gel mixture.<br><br />
e.Wash the column once with 0.5ml WN buffer by centrifuging for 30s-60s (9000rpm). Discard the flow through.<br><br />
f.Wash the column once with 0.5ml WS buffer by centrifuging for 30s-60s (9000rpm).<br><br />
g.Centrifuge for another 3 mins (13200rpm) to remove all the ethanol<br><br />
h.Place the column onto a new 1.5ml centrifuge tube. Add 15μL -30μL elution buffer onto the center of the membrane. (Preheated the Elution buffer to 60°C).<br><br />
i.Stand the column for another 3mins and then centrifuge at full speed for 1-2 mins to elute DNA.<br><br><br />
<p> Tips: </p><br />
The gel extraction has a low recovery rate of about 10%. Stand the column longer in step i may give relatively higher recovery rate.<br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/gel_cutTeam:HKUST/Protocols/gel cut2009-10-19T15:43:13Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Cutting bands on the gel</p><br />
<br />
<p> Purpose: To extract specific DNA fragment.</p><br />
<br />
<p>Procedure: </p><br />
a.Visualize the bands under UV light. Use long-wavelength UV to minimize damage to the DNA. <br><br />
b.Cut the band with a clean razor blade.<br><br />
c.Turn the gel slice on its side to trim off extra agarose. Place the gel in a microcentrifuge tube.</p><br />
<p> Tips: </p><br />
EB staining is needed for higher resolution under UV light.<br />
<br><br><br />
<p> Safety tips: </p><br />
1. ermission is needed from technician before doing this! Follow the safety instruction in the room.<br><br><br />
2.When using UV light, protect your skin by wearing safety goggles or a face shield, gloves, and a lab coat. <br><br><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestionTeam:HKUST/Protocols/Enzyme digestion2009-10-19T15:40:13Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
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<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Enzyme digestion (vector, insert) </p><br />
<br />
<p> Purpose: To cut the sequence with specific restriction enzyme</p><br />
Materials: substrate DNA, restriction enzymes, corresponding 10X buffer, ddH2O, CIP (Calf Intestinal Alkaline Phosphatase, for vector digestion and DNA to be ligated)<br> <br><br />
<br />
<p>Procedure: </p><br />
a.Add sufficient water to make it a 20 μL reaction. <br><br />
b.Add 1 μg DNA, 2 μL buffer in total, 0.5 μL each restriction enzyme.<br><br />
c.Vortex and then spin down.<br><br />
d.Keep it in 37 °C incubator for one hour. (If needed, incubate for 1.5-2hours)<br><br />
e.If the substrate DNA is vector or pieces to be ligated, add 0.5 ul (for 20ul reaction) CIP after incubated for 1.5-2 hours, and then incubate at 37 °C for another 30mins.<br><br />
f.Check the result by gel electrophoresis.<br><br />
<p> Tips: </p><br />
A. Restriction enzymes are easy to be denatured, so do not leave it at room temperature.<br><br />
B. If two restriction enzymes must be added with different buffer, digest the DNA with respective restriction enzyme sequentially. Incubate for one hour after each enzyme added.<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/plasmid_extractionTeam:HKUST/Protocols/plasmid extraction2009-10-19T15:39:38Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
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<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Consolidation">Consolidation</a></li><br />
<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
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</div><br />
<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Plamid DNA extraction</p><br />
<br />
<p> Purpose: To extract plasmid DNA from E.coli.</p><br />
Materials: MX1 Buffer, MX2 Buffer, MX3 Buffer, WN Buffer, WS Buffer, Elution Buffer (all provided in Mini Plus Plasmid DNA Extraction System)<br> <br><br />
<br />
<p>Procedure: </p><br />
a.Grow 1 to 5 ml plasmid-containing bacterial cells in LB medium with appropriate anlibiotic overnight (12-16 hours) with vigorous agitation.<br><br />
b. Pellet the cells by centrifuging for 1-2 minutes. Decant the supernatant and remove all medium residues by pipette.<br><br />
c.Add 200 μL of MX1 Buffer 10 the pellet, and resuspend the cells completely by vortexing or pipetting.<br><br />
d.Add 250 μL of MX2 Buffer and gently mix well (invert the tube 6-10 times) to lyse the cells until the lysate becomes d ear. Incubate at room temperature for 2-5 minutes.<br><br />
e.Add 350 μL of MX3 Buffer to neutralize the lysate, then immediately and gently mix the solution well. A white precipitate should be formed.<br><br />
f.Centrifuge at 10,000 x g (13,000rpm) for 5-10 minutes, meanwhile place a Mini Plus™ Column onto a Collection Tube. Transfer the supernatant carefully into the column.<br><br />
g.Centrifuge at 7,000x g (9,000rpm) for 30-60 seconds. Discard the flow-through.<br><br />
h.Wash the column once with 0.5 ml of WN Buffer by centrifuging at 7,000 x g (9,000rpm) for 30-60 seconds. Discard the flow-through.<br><br />
i.Wash the column once with 0.7 ml of WS Buffer by centrifuging at 7,000 x g (9,000rpm) for 30-60 seconds. Discard the flow-through.<br><br />
j.Centrifuge the column at 10,000 x g (13,000rpm) for another 3 minutes to remove residual ethanol.<br><br />
k.Place the column into a new 1.5-ml centrifuge tube. Add 50 μL of Elution Buffer onto the center of the membrane.<br><br />
l.Stand the column for 2-3 minutes, and centrifuge at 10,000 x g (13,000rpm) for 2-3 minutes to elute DNA. Store plasmid DNA at 4 °C or -20 °C.<br><br />
<p> Tips: </p><br />
A. In step c, no clump should be visible after resuspension. Clumped cells lead to bad plasmid yield and quality.<br><br />
B. Do not vortex in step d, otherwise genomic DNA will be sheared and contaminate the sample, which can be observed as serious foaming.<br><br />
C. In step d, the lysate should become clear and viscous. Insufficient cell-lysis leads to low plasmid yield and quality.<br><br />
D. MX1 Buffer must be stored at 4 °C.<br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestionTeam:HKUST/Protocols/Enzyme digestion2009-10-19T15:36:23Z<p>Ke lyxaa: </p>
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<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Agarose gel preparation and gel electrophoresis</p><br />
<br />
<p> Purpose: To cut the sequence with specific restriction enzyme</p><br />
Materials: substrate DNA, restriction enzymes, corresponding 10X buffer, ddH2O, CIP (Calf Intestinal Alkaline Phosphatase, for vector digestion and DNA to be ligated)<br> <br><br />
<br />
<p>Procedure: </p><br />
a.Add sufficient water to make it a 20 μL reaction. <br><br />
b.Add 1 μg DNA, 2 μL buffer in total, 0.5 μL each restriction enzyme.<br><br />
c.Vortex and then spin down.<br><br />
d.Keep it in 37 °C incubator for one hour. (If needed, incubate for 1.5-2hours)<br><br />
e.If the substrate DNA is vector or pieces to be ligated, add 0.5 ul (for 20ul reaction) CIP after incubated for 1.5-2 hours, and then incubate at 37 °C for another 30mins.<br><br />
f.Check the result by gel electrophoresis.<br><br />
<p> Tips: </p><br />
A. Restriction enzymes are easy to be denatured, so do not leave it at room temperature.<br><br />
B. If two restriction enzymes must be added with different buffer, digest the DNA with respective restriction enzyme sequentially. Incubate for one hour after each enzyme added.<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/PCRTeam:HKUST/Protocols/PCR2009-10-19T15:34:19Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
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<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>PCR</p><br />
<br />
<p> Purpose: To amplify a specific piece of DNA out from the whole. </p><br />
Materials: ddH2O, 10X ThermoPol Buffer, 1mM dNTP, Forward primer, Reverse Primer, Taq or Vent DNA polymerase, DNA template<br />
Choice of DNA polymerase: Taq gives higher rate of mutation compared with Vent.<br />
Taq gives sticky ends after PCR, while Vent gives blunt ends. <br><br><br />
<br />
<p>Procedure: </p><br />
1. Add 10 μL water to make it a 20 μL reaction. <br><br />
2. Add 2 μL buffer, 4 μL dNTP, 1 μL forward primer (5 μM), 1 μL reverse primer (5 μM), 1 μL DNA template, 1 μL DNA polymerase <br><br />
3. Vortex and then spin down.<br><br />
4. Put it into the PCR machine and set the program.<br><br />
&nbsp; Program<br><br />
&nbsp; &nbsp; (1) Initial denaturation 95 °C 4 mins<br><br />
&nbsp; &nbsp; (2) Run 25-30 cycles of:<br><br />
&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Denaturation &nbsp; 95 °C 30 secs<br><br />
&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Annealing &nbsp; &nbsp; 30 secs <br><br />
&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Temperature is depended on melting temperature of primer. <br><br />
&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Lower Tm-5°C, for Taq; Lower Tm +3°C for Vent<br><br />
&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Extension 72 °C 30 secs per 500bp PCR product length<br><br />
&nbsp; &nbsp; (3) Final extension &nbsp; 72 °C 3~5 mins<br><br />
<p> Tips: </p><br />
The DNA polymerase is easy to be denatured, so do not leave it at room temperature. It is highly recommended to use an ice holder or do the adding in the fridge.<br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/PCRTeam:HKUST/Protocols/PCR2009-10-19T15:32:30Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
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<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>PCR</p><br />
<br />
<p> Purpose: To amplify a specific piece of DNA out from the whole. </p><br />
Materials: ddH2O, 10X ThermoPol Buffer, 1mM dNTP, Forward primer, Reverse Primer, Taq or Vent DNA polymerase, DNA template<br />
Choice of DNA polymerase: Taq gives higher rate of mutation compared with Vent.<br />
Taq gives sticky ends after PCR, while Vent gives blunt ends. <br><br><br />
<br />
<p>Procedure: </p><br />
1. Add 10 μL water to make it a 20 μL reaction. <br><br />
2. Add 2 μL buffer, 4 μL dNTP, 1 μL forward primer (5 μM), 1 μL reverse primer (5 μM), 1 μL DNA template, 1 μL DNA polymerase <br><br />
3. Vortex and then spin down.<br><br />
4. Put it into the PCR machine and set the program.<br><br />
&nbsp; Program<br><br />
&nbsp; &nbsp; (1) Initial denaturation 95 °C 4 mins<br><br />
&nbsp; &nbsp; (2) Run 25-30 cycles of:<br><br />
&nbsp; &nbsp; Denaturation &nbsp; 95 °C 30 secs<br><br />
&nbsp; &nbsp; Annealing &nbsp; &nbsp; 30 secs <br><br />
&nbsp; &nbsp; &nbsp; Temperature is depended on melting temperature of primer. <br><br />
&nbsp; &nbsp; &nbsp; Lower Tm-5°C, for Taq; Lower Tm +3°C for Vent<br><br />
&nbsp; &nbsp; Extension 72 °C 30 secs per 500bp PCR product length<br><br />
(3) Final extension &nbsp; 72 °C 3~5 mins<br><br />
<p> Tips: </p><br />
The DNA polymerase is easy to be denatured, so do not leave it at room temperature. It is highly recommended to use an ice holder or do the adding in the fridge.<br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/PCR_cleanupTeam:HKUST/Protocols/PCR cleanup2009-10-19T15:30:55Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
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<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>PCR product clean-up</p><br />
<br />
<p> Purpose: To clean up primers, dNTPs, enzymes, short-failed PCR products in the PCR reaction.</p><br />
Materials: FAPC Buffer,Wash Buffer, Elution Buffer, PCR Product (all provided in FavorPrepTM PCR Clean-Up Kit)<br> <br><br />
<br />
<p>Procedure: </p><br />
a.Transfer 30μL of PCR product and add 5 volumes of FAPC buffer to a 1.5mL microcentrifuge tube, then mix well by vortexing.<br><br />
b.Place a FAPC Column into a Collection Tube and transfer the sample mixture to FAPC Column.<br><br />
c.Centrifuge at 6,000 rpm for1 min then discard the flow-through.<br><br />
d.Add 750 μl of Wash Buffer (ethanol added) to FAPC Column. Centrifuge at 6,000 rpm for 1 min, then discard the flow-through.<br><br />
e.Centrifuge at 14,000 rpm for an additional 3 min to dry the column.<br><br />
f.Place FAPC Column into an Elution Tube.<br><br />
g.Add 40 μl of Elution Buffer or ddH2O (pH 7.0~8.5) to the membrane center of FAPC Column. Stand FAPC Column for 2 minutes.<br><br />
h.Centrifuge at 14,000 rpm for 1 min to elute the DNA.<br><br />
<p> Tips: </p><br />
In step f, for effective elution, make sure that the elution solution is dispensed onto the membrane center and is absorbed completely.<br />
<br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/PCRTeam:HKUST/Protocols/PCR2009-10-19T15:28:07Z<p>Ke lyxaa: </p>
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</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>PCR</p><br />
<br />
<p> Purpose: To amplify a specific piece of DNA out from the whole. </p><br />
Materials: ddH2O, 10X ThermoPol Buffer, 1mM dNTP, Forward primer, Reverse Primer, Taq or Vent DNA polymerase, DNA template<br />
Choice of DNA polymerase: Taq gives higher rate of mutation compared with Vent.<br />
Taq gives sticky ends after PCR, while Vent gives blunt ends. <br><br><br />
<br />
<p>Procedure: </p><br />
1. Add 10 μL water to make it a 20 μL reaction. <br><br />
2. Add 2 μL buffer, 4 μL dNTP, 1 μL forward primer (5 μM), 1 μL reverse primer (5 μM), 1 μL DNA template, 1 μL DNA polymerase <br><br />
3. Vortex and then spin down.<br><br />
4. Put it into the PCR machine and set the program.<br><br />
Program<br><br />
(1) Initial denaturation 95 °C 4 mins<br><br />
(2) Run 25-30 cycles of:<br><br />
Denaturation 95 °C 30 secs<br><br />
Annealing 30 secs <br><br />
Temperature is depended on melting temperature of primer. <br><br />
Lower Tm-5°C, for Taq; Lower Tm +3°C for Vent<br><br />
Extension 72 °C 30 secs per 500bp PCR product length<br><br />
(3) Final extension 72 °C 3~5 mins<br><br />
<p> Tips: </p><br />
The DNA polymerase is easy to be denatured, so do not leave it at room temperature. It is highly recommended to use an ice holder or do the adding in the fridge.<br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Protocols/Agarose_gelTeam:HKUST/Protocols/Agarose gel2009-10-19T15:22:47Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
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<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
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<span style="color:green"><br />
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<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Agarose gel preparation and gel electrophoresis</p><br />
<br />
<p> Purpose: To check the result </p><br />
Materials: Agarose, 0.5X TBE, SYBR safe DNA gel stain, DNA loading dye, DNA ladder/marker <br><br><br />
Choice of ladders: VC100bp Plus DNA ladder (highest band 3000bp); Lamda DNA BstEII marker (highest band 8400bp, 1% argarose) <br><br><br />
<br />
<p>Procedure: </p><br />
1. To make a 0.8% agarose gel, use 0.16 g agarose per 20 mL 0.5x TBE. <br><br />
2. Cover the flask with plastic wrap to prevent boiling over, then microwave the solution for 1-2 minutes to dissolve the agarose. <br><br />
3. Let agarose solution cool for 5 minutes, then add SYBR safe DNA gel stain to a final concentration of 0.5 μg/mL. <br><br />
4. Pour the agarose solution into a casting tray with well comb in place. Allow 20-30 minutes to completely solidify.<br><br />
5. Place the agarose gel into the gel box and fill the box with 0.5X TBE until the gel is immersed.<br><br />
6. Load an appropriate molecular weight ladder into the first lane of the gel. If needed, an additional ladder with different molecular weight could be loaded into the last lane.<br><br />
7. Add 1 μL loading dye per 5 μL of sample.<br><br />
8. Load the samples from left to right.<br><br />
9. Cover the gel box and plug in the electrodes in a way that the samples will run towards the positive, red electrode.<br><br />
10. Run the gel at 100V until the dye line is approximately 50-75% of the way down the gel. Approximately 20-30 minutes.<br><br />
11. Carefully remove the gel from the gel box and check the result under UV exposure.<br><br />
<p> Tips: </p><br />
A. Higher concentration of agarose solution makes better resolution for less molecular weight expected band.<br><br />
B. Let bottom of the flask be immersed in a cup of cold water for faster cooling.<br><br />
C. In order for clearer band to cut specific band on the gel, immerse the gel in Ethidium bromide for 10-15 minutes after step k.<br><br />
<p> Safety tips: </p><br />
1. Be sure to wear a glove before treating the hot flask.<br><br><br />
2. Ethidium bromide is a strong mutagen. Wear a lab coat, eye protection, and gloves when working with this chemical. <br><br><br />
<br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
</ul><br />
<br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/ProtocolsTeam:HKUST/Protocols2009-10-19T15:22:05Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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<li><a href="https://2009.igem.org/Team:Consolidation">Consolidation</a></li><br />
<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
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<div id="rightxx"> <br />
<div class="contentlist"> <h3>a</h3><br />
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<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel <br />
electrophoresis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to <br />
yeast</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA <br />
extraction</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li><br />
<br />
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<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin production</a></li><br />
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<h3>Welcome</h3><br />
<br />
<iframe src="http://www.google.com/calendar/embed?src=hkust2009igem%40gmail.com&ctz=Asia/Hong_Kong" style="border: 0" width="500" height="460" frameborder="0" scrolling="no"></iframe> <br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Group4Team:HKUST/Group42009-10-17T13:41:07Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol list</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other resources</a></li> <br />
</ul><br />
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<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Consolidation">Consolidation</a></li><br />
<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
</div><br />
</div><br />
<div id="rightxx"><br />
<div class="contentxx"><br />
<h3>Welcome</h3><br />
<br />
<p>Protein Expression</p><br />
We have designed several constructs to test its toxicity in to insects as well as to yeast:<br><br />
(1) BinA and BinB genes each fused with GST, cloned into the multi-cloning sites of pESC-Leu expression vector under the GAL10 and GAL1 promoter, respectively.</p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group4/figure1.jpg " width=450; height=90 /></a><br><br />
<br />
(2) Only BinA gene is fused with GST, cloned into the same multi-cloning site of pESC-Leu expression vector under the Gal 10 promoter.</p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group4/figure2.jpg " width=450; height=90 /></a><br><br />
(3) Only BinB gene is fused with GST, cloned into the same multi-cloning site of pESC-Leu expression vector under the Gal 1 promoter.</p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group4/figure3.jpg " width=450; height=90 /></a><br><br />
These constructs will be transformed into the yeast and induced by galactose. Then we will use western blot to test the expression of toxin by anti-GST antibody. We can estimate the protein level as well as the yeast phenotype: whether they are resistant to the toxin.</p><br />
<br><br />
<p>Drosophila Larvicidal Essay</p><br />
Next we will test the biological function of toxin produced in yeast to kill Drosophila larvae. The Drosophila larvicidal activity is tested by diluting yeast cells expressing the toxin in water and adding them to water containing 10 larvae in each well of a 24-well tissue culture plate (diameter of the well is 1.5 cm)(Figure 1). Mortality is recorded after incubation at 30°C for 48 h. LC50 is determined by Probit analysis[6].</p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group4/figure4.jpg " width=450; height=700 /></a><br><br />
Fig 1. Drosophila Larvicidal assay. Diluted yeast cells expressing the toxin and 10 larvae are incubated in a 24-well tissue culture plate. Mortality is recorded after incubation is measured. LC50 is determined by Probit analysis.</p> <br><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Group4Team:HKUST/Group42009-10-17T13:40:02Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol list</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other resources</a></li> <br />
</ul><br />
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<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
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<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
</div><br />
</div><br />
<div id="rightxx"><br />
<div class="contentxx"><br />
<h3>Welcome</h3><br />
<br />
<p>Protein Expression</p><br />
We have designed several constructs to test its toxicity in to insects as well as to yeast:<br><br />
(1) BinA and BinB genes each fused with GST, cloned into the multi-cloning sites of pESC-Leu expression vector under the GAL10 and GAL1 promoter, respectively.</p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group4/figure1.jpg " width=450; height=200 /></a><br><br />
<br />
<br />
(2) Only BinA gene is fused with GST, cloned into the same multi-cloning site of pESC-Leu expression vector under the Gal 10 promoter.</p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group4/figure2.jpg " width=450; height=200 /></a><br><br />
(3) Only BinB gene is fused with GST, cloned into the same multi-cloning site of pESC-Leu expression vector under the Gal 1 promoter.</p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group4/figure3.jpg " width=450; height=200 /></a><br><br />
These constructs will be transformed into the yeast and induced by galactose. Then we will use western blot to test the expression of toxin by anti-GST antibody. We can estimate the protein level as well as the yeast phenotype: whether they are resistant to the toxin.</p><br />
<br><br />
<p>Drosophila Larvicidal Essay</p><br />
Next we will test the biological function of toxin produced in yeast to kill Drosophila larvae. The Drosophila larvicidal activity is tested by diluting yeast cells expressing the toxin in water and adding them to water containing 10 larvae in each well of a 24-well tissue culture plate (diameter of the well is 1.5 cm)(Figure 1). Mortality is recorded after incubation at 30°C for 48 h. LC50 is determined by Probit analysis[6].</p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group4/figure4.jpg " width=450; height=700 /></a><br><br />
Fig 1. Drosophila Larvicidal assay. Diluted yeast cells expressing the toxin and 10 larvae are incubated in a 24-well tissue culture plate. Mortality is recorded after incubation is measured. LC50 is determined by Probit analysis.</p> <br><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Group4Team:HKUST/Group42009-10-17T13:38:27Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol list</a></li> <br />
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</ul><br />
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</div><br />
<div id="rightxx"><br />
<div class="contentxx"><br />
<h3>Welcome</h3><br />
<br />
<p>Protein Expression</p><br />
We have designed several constructs to test its toxicity in to insects as well as to yeast:<br><br />
(1) BinA and BinB genes each fused with GST, cloned into the multi-cloning sites of pESC-Leu expression vector under the GAL10 and GAL1 promoter, respectively.</p><br />
<br />
<br />
(2) Only BinA gene is fused with GST, cloned into the same multi-cloning site of pESC-Leu expression vector under the Gal 10 promoter.</p><br />
<br />
(3) Only BinB gene is fused with GST, cloned into the same multi-cloning site of pESC-Leu expression vector under the Gal 1 promoter.</p><br />
<br />
These constructs will be transformed into the yeast and induced by galactose. Then we will use western blot to test the expression of toxin by anti-GST antibody. We can estimate the protein level as well as the yeast phenotype: whether they are resistant to the toxin.</p><br />
<br><br />
<p>Drosophila Larvicidal Essay</p><br />
Next we will test the biological function of toxin produced in yeast to kill Drosophila larvae. The Drosophila larvicidal activity is tested by diluting yeast cells expressing the toxin in water and adding them to water containing 10 larvae in each well of a 24-well tissue culture plate (diameter of the well is 1.5 cm)(Figure 1). Mortality is recorded after incubation at 30°C for 48 h. LC50 is determined by Probit analysis[6].</p><br />
<br />
Fig 1. Drosophila Larvicidal assay. Diluted yeast cells expressing the toxin and 10 larvae are incubated in a 24-well tissue culture plate. Mortality is recorded after incubation is measured. LC50 is determined by Probit analysis.</p> <br><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Back4Team:HKUST/Back42009-10-17T13:34:41Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant sensoring</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol list</a></li> <br />
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<h3>Welcome</h3><br />
</p> <br />
The binary toxin BinA and BinB, which is produced in Bacillus sphaericus, is a mosquito-larvicidal crystal protein. It has its maximum activity when both components are present in equimolar ratio[1]. It could kill larvae by forming pores once binA binds to and binB inserts to membrane lipid bilayer, leading to swelling and lysis of the cell[2]. </p><br />
Attempts have been made to produce the binary toxin at a high level in many organisms including Escherichia coli[3]. To obtain a high expression level and a high-potency toxin, one approach is to improving solubility by tagging T7 tag [4] or glutathione S-transferase (GST), between which GST seems to work better. Not only did GST increases the expression level; it also improves the solubility of those proteins[2].</p><br />
Yeast has also been a good tool for bacterial toxin studies. It can functionally express many bacterial toxins[5]. Since the binary toxin Bin A and Bin B are very specific to insects, we propose that yeast can express this toxin without damaging itself; at the same time, when insects eat the yeast the insect will die. This is why we choose this binary toxin as our “insect killer”.<br><br />
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<br />
[1] Charles JF, Nielson-LeRoux C, Delecluse A (1996) Bacillus sphaericus toxins: Molecular biology and mode of action. Annu Rev Entomol 41:451–472<br><br />
[2] El-Bendary,Magda A (2005) Bacillus thuringiensis and Bacillus sphaericus biopesticides production. J. Basic Microbiol. 46, 2, 158–170 <br><br />
[3] Promdonkoy B, Promdonkoy P, Panyim S (2008) High-level expression in Escherichia coli, purification and mosquito-larvicidal activity of the binary toxin from Bacillus sphaericus. Curr Microbiol 57:626–630<br><br />
[4] Promdonkoy B, Promdonkoy P, Audtho M, Tanapongpipat S, Chewawiwat N, Luxananil P, Panyim S (2003) Efficient expression of the mosquito larvicidal binary toxin gene from Bacillus sphaericus in Escherichia coli. Curr Microbiol 47:383–387<br><br />
[5] Turgeon Z. et al.(2009) Yeast as a tool for characterizing mono-ADP-ribosyltransferase toxins. FEMS Microbiology Letters Volume 300 Issue 1, Pages 97 - 106<br><br />
[6] Finney D (1971) Probit analysis, 3rd edn. Cambridge University<br />
Press, London, UK<br />
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<h3>Welcome</h3><br />
[1]Promdonkoy B, Promdonkoy P, Panyim S (2008) High-level expression in Escherichia coli, purification and mosquito-larvicidal activity of the binary toxin from Bacillus sphaericus. Curr Microbiol 57:626–630<br><br />
[2] Turgeon Z. et al.(2009) Yeast as a tool for characterizing mono-ADP-ribosyltransferase toxins. FEMS Microbiology Letters Volume 300 Issue 1, Pages 97 - 106<br><br />
[3] Finney D (1971) Probit analysis, 3rd edn. Cambridge University Press, London, UK<br><br />
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<h3>Welcome</h3><br />
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The binary toxin BinA and BinB, which is produced in Bacillus sphaericus, is a mosquito-larvicidal crystal protein. It has its maximum activity when both components are present in equimolar ratio[1]. It could kill larvae by forming pores once binA binds to and binB inserts to membrane lipid bilayer, leading to swelling and lysis of the cell[1]. </p><br />
Attempts have been made to produce the binary toxin at a high level in many organisms including Escherichia coli[1]. To obtain a high expression level and a high-potency toxin, one approach is to improving solubility by tagging T7 tag [2] or glutathione S-transferase (GST), between which GST seems to work better. Not only did GST increases the expression level; it also improves the solubility of those proteins[2].</p><br />
Yeast has also been a good tool for bacterial toxin studies. It can functionally express many bacterial toxins[2]. Since the binary toxin Bin A and Bin B are very specific to insects, we propose that yeast can express this toxin without damaging itself; at the same time, when insects eat the yeast the insect will die. This is why we choose this binary toxin as our “insect killer”.<br><br />
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1. Finish constructing expression vectors of our design.<br><br />
2. Carry out protein expression test and Drosophila larvicidal test.<br><br />
3. Testing the toxin production optimization conditions.<br><br />
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<h3>Welcome</h3><br />
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<p>Protein Expression</p><br />
We have designed several constructs to test its toxicity in to insects as well as to yeast:<br><br />
(1) BinA and BinB genes each fused with GST, cloned into the multi-cloning sites of pESC-Leu expression vector under the GAL10 and GAL1 promoter, respectively.</p><br />
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(2) Only BinA gene is fused with GST, cloned into the same multi-cloning site of pESC-Leu expression vector under the Gal 10 promoter.</p><br />
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(3) Only BinB gene is fused with GST, cloned into the same multi-cloning site of pESC-Leu expression vector under the Gal 1 promoter.</p><br />
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These constructs will be transformed into the yeast and induced by galactose. Then we will use western blot to test the expression of toxin by anti-GST antibody. We can estimate the protein level as well as the yeast phenotype: whether they are resistant to the toxin.</p><br />
<br><br />
<p>Drosophila Larvicidal Essay</p><br />
Next we will test the biological function of toxin produced in yeast to kill Drosophila larvae. The Drosophila larvicidal activity is tested by diluting yeast cells expressing the toxin in water and adding them to water containing 10 larvae in each well of a 24-well tissue culture plate (diameter of the well is 1.5 cm)(Figure 1). Mortality is recorded after incubation at 30°C for 48 h. LC50 is determined by Probit analysis[3].</p><br />
<br />
Fig 1. Drosophila Larvicidal assay. Diluted yeast cells expressing the toxin and 10 larvae are incubated in a 24-well tissue culture plate. Mortality is recorded after incubation is measured. LC50 is determined by Probit analysis.</p> <br><br />
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<h3>Welcome</h3><br />
</p> <br />
The binary toxin BinA and BinB, which is produced in Bacillus sphaericus, is a mosquito-larvicidal crystal protein. It has its maximum activity when both components are present in equimolar ratio[1]. It could kill larvae by forming pores once binA binds to and binB inserts to membrane lipid bilayer, leading to swelling and lysis of the cell[1]. </p><br />
Attempts have been made to produce the binary toxin at a high level in many organisms including Escherichia coli .To obtain a high expression level and a high-potency toxin, one approach is to improving solubility by tagging T7 tag [2] or glutathione S-transferase (GST), between which GST seems to work better. Not only did GST increases the expression level; it also improves the solubility of those proteins[2].</p><br />
Yeast has also been a good tool for bacterial toxin studies. It can functionally express many bacterial toxins . Since the binary toxin Bin A and Bin B are very specific to insects, we propose that yeast can express this toxin without damaging itself; at the same time, when insects eat the yeast the insect will die. This is why we choose this binary toxin as our “insect killer”.<br><br />
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<h3>Welcome</h3><br />
</p> <br />
The binary toxin BinA and BinB, which is produced in Bacillus sphaericus, is a mosquito-larvicidal crystal protein. It has its maximum activity when both components are present in equimolar ratio[1]. It could kill larvae by forming pores once binA binds to and binB inserts to membrane lipid bilayer, leading to swelling and lysis of the cell[1]. </r><br />
Attempts have been made to produce the binary toxin at a high level in many organisms including Escherichia coli .To obtain a high expression level and a high-potency toxin, one approach is to improving solubility by tagging T7 tag [2] or glutathione S-transferase (GST), between which GST seems to work better. Not only did GST increases the expression level; it also improves the solubility of those proteins[2].</p><br />
Yeast has also been a good tool for bacterial toxin studies. It can functionally express many bacterial toxins . Since the binary toxin Bin A and Bin B are very specific to insects, we propose that yeast can express this toxin without damaging itself; at the same time, when insects eat the yeast the insect will die. This is why we choose this binary toxin as our “insect killer”.<br><br />
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<h3>Welcome</h3><br />
<br />
The binary toxin BinA and BinB, which is produced in Bacillus sphaericus, is a mosquito-larvicidal crystal protein. It has its maximum activity when both components are present in equimolar ratio[1]. It could kill larvae by forming pores once binA binds to and binB inserts to membrane lipid bilayer, leading to swelling and lysis of the cell.[1] <br><br />
Attempts have been made to produce the binary toxin at a high level in many organisms including Escherichia coli .To obtain a high expression level and a high-potency toxin, one approach is to improving solubility by tagging T7 tag [2] or glutathione S-transferase (GST), between which GST seems to work better. Not only did GST increases the expression level; it also improves the solubility of those proteins[2].<br><br />
Yeast has also been a good tool for bacterial toxin studies. It can functionally express many bacterial toxins . Since the binary toxin Bin A and Bin B are very specific to insects, we propose that yeast can express this toxin without damaging itself; at the same time, when insects eat the yeast the insect will die. This is why we choose this binary toxin as our “insect killer”.<br><br />
<br />
<br />
<br />
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project description</a></li><br />
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<li>Main Parts</li><br />
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<h3>Welcome</h3><br />
<br />
In this sub-project, we are going to engineer a yeast strain able to produce a kind of toxin. We have designed to clone the mosquito-larvicidal binary toxin genes in the expression vector pESC-Leu into the yeast. In that case, it can continuously express binary toxin proteins under the promoter GAL10 and GAL1. Later we could potentially use this strain to kill some disease vectors, such as mosquitoes and fleas, or as a pesticide in agriculture.<br />
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<br />
<li> Odorant sensoring </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/View1">Overview</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back1">Background</a></li><br />
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<li><a href="https://2009.igem.org/Team:HKUST/Ref1">Reference</a></li><br />
<br />
<li> Attranctant production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/View3">Overview</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back3">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group3">Experimental design</a></li> <br />
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<li><a href="https://2009.igem.org/Team:HKUST/Ref3">Reference</a></li><br />
<br />
<li>Toxin production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/View4">Overview</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back4">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group4">Experimental design</a></li> <br />
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<br />
<li>Resources</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol list</a></li> <br />
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<h3>Welcome</h3><br />
<br />
<br />
<p>Construction of Receptor Expression Cassette</p><br />
To functionally express C. elegans Odr10 receptor, we have designed an expression cassette. Based on the fact that rat I7 (RI7) receptor has been successfully expressed in the yeast system and it couples well to Gαolf subunit, we have designed such a chimeric receptor with the RI7 N (amino acids 1-61 of RI7) and C termini (animo acids 295-327 of RI7) fused with Odr10 transmambrane domains two to seven (TM2 – 7, amino acids 48-305 of Odr10, starting from the fourth amino acid of Odr10 TM2) [i] (figure 3). At the N termini side, they are fused at the junctions PMYFF (RI7) and YLMAFF (Odr10) because these two sequences are conserved in both receptor junction sites (Figure 4). They are cloned into the multiple cloning site of pESC-HIS expression vector under the Gal1 promoter. They are also tagged with FLAG or GFP at the C termini for protein expression detection or localization test. Given that we have retained the N and C termini of RI7, localization and subsequent coupling should not be perturbed.</p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/receptor expression strategy.jpg " width=310; height=270 /></a><br><br />
Fig3. Schemetic illustration of receptor expression strategy. The receptor expression cassette of the pESC-HIS vector was constructed to contain an insert that encodes the N and C termini of the RI7 receptor flanking an intervening sequence containing multiple cloning sites. The ligand-binding pocket of Odr10 protein is inserted between N and C termini. Adopted from Venkat,B.,el al, 2007.</p><br />
<br />
Fig 4. Sequence of the chimeric receptor. Transmambrane domains are highlighted in red;sequences from RI7 are highlight in yellow; the fusion site is separated by a green line. </p><br />
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<br />
<p>Test of Chimeric Protein Localization</p><br />
To test the localization of chimeric proteins, we have made the construct pESC-Fusion-GFP, which is the chimeric protein with the GFP tag at the C termini in the expression vector pESC-HIS, as well as the negative control pESC-GFP, which is only the GFP at the same site in the expression vector pESC-HIS (Figure 5). We choose to tag GFP at the C termini because N termini of the fusion protein is important for membrane localization while C termini is important for coupling. Since we want to ensure its proper localization, it is better to fuse GFP at the C termini. </p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/Sequence of the chimeric receptor.jpg " width=450; height=350 /></a><br><br />
Fig 5. Constructs for chimeric receptor fluorescence miscroscopy test, in order to confirm its localization to the yeast membrane. (a) is pESC-Fusion-GFP, which is the chimeric protein with the GFP tag at the C termini in the expression vector pESC-HIS; (b) is the negative control pESC-GFP, which is only the GFP at the same site in the expression vector pESC-HIS. </p><br />
After transformation and selection, we would induce the transformants with galactose at the exponential stage of yeast growth. After some time, we would harvest cells for the fluorescence microscopy test. We would expect to see the cells transformed with pESC-Fusion-GFP to have strong green fluorescence surrounding the cell, forming a green circle; while the negative control does not. In that case, we could say that the fusion protein is able to localize in the yeast membrane. </p><br />
<br />
<p>Test of Chimeric Protein Function</p><br />
To test the functional sensing and coupling to Gαolf or Gpa1, we have made several constructs: <br><br />
(1) pESC-Fusion-FLAG, which is the chimeric protein with the FLAG tag at the C termini in the expression vector pESC-HIS under the Gal1 promotor. <br><br />
(2) pESC-RI7-FLAG, which is the rat RI7 receptor protein with the FLAG tag at the C termini in the expression vector pESC-HIS under the Gal1 promotor, serving as the positive control. <br><br />
(3) pESC-Gαolf-FLAG , which is rat Gαolf in the pESC-HIS under the Gal10 promotor. <br><br />
(4) pRS426-FUS1P-GFP-FUS1T, which is the FUS1 promotor, GFP and FUS1 terminator in the expression vector pRS426. <br><br />
(5) pESC empty plasmid, serving as the negative control. </p><br />
<br />
There are two parts of the fuctional coupling test:</p><br />
Part 1<br><br />
First we test whether our chimeric protein could couple to Gpa1. <br><br />
The yeasts would be transformed with construct (1)+(4) or (2)+(4) or (5)+(4), respectively. After induction with galactose to express the receptors, we would add in the ligands diacetyl (for Odr10) and hexanal (for RI7). After some time of ligands binding, we would expect to see that the functional receptors could couple to Gpa1 and then trigger downstream FUS1-promoter-driven expression of GFP, together with cell cycle arrest at G1 phase (figure 6). The expression of GFP could be viewed by fluorescence microscopy; the cell cycle arrest could be confirmed by fluorescence-activated cell sorting (FACS). We might expect to see the fusion receptor and RI7 response to diacetyl and hexanal, respectively, to have GFP and cell cycle arrest. In that case we could say that fusion receptor could functionally couple to Gpa1 and start downstream signalling. <br><br />
<br />
Fig 6 Ligands sensing functional assay showing control experiments. (a) is yeast transformed with pESC-Fusion-FLAG & pRS426-FUS1P-GFP-FUS1T, and we would expect to see GFP and cell cycle arrest only when both galactose and diacetyl are added; (b) is yeast transformed with pESC-RI7-FLAG & pRS426-FUS1P-GFP-FUS1T, and we would expect to see GFP and cell cycle arrest only when both galactose and hexanal are added; (c) is yeast transformed with pESC empty plasmid and pRS426-FUS1P-GFP-FUS1T, and we would expect to see no GFP and cell cycle arrest. These cells are analyzed with fluorescence microscopy and FACS. </p><br />
<br />
Part 2<br><br />
Second, we test whether our chimeric receptor could couple to Gαolf for optimized signalling transduction as well as constructing a testable yeast system for odorant sensing. <br><br />
Gene deletion<br><br />
Before testing, yeast strain needs to be manipulated: we need to knock out the FAR1 gene, which encodes a protein controlling cell cycle arrest upon activation of MAPK pathway, so that the yeasts will still be viable after ligand binding; and also endogenous GPA1 gene, so that we could replace it with Gαolf. Due to the second messenger activity of the free G protein βγ subunit, haploid S. cerevisiae strains containing a null mutation of GPA1 undergo constitutive pheromone response of G1 arrest resulting in non-viability[ii]. So, we need to first knock out cell cycle regulatory gene FAR1 and afterwards, GPA1.To knock out these two genes, we have adopted the PCR-based tagging of yeast genes method, introduced by Janke C., el al, 2004[iii]. <br><br />
To test the successful deletion, we will use both PCR confirmation (figure 7) and also phenotype observation after adding α-factor (no mating phenotype). </p><br />
<br />
<br />
<br />
Figure 7. Primers designed for colony PCR to confirm successful deletion of yeast ORF. The correct replacement of the gene with hphNT1 or natNT2 is verified in the mutants by the appearance of PCR products of the expected size using primers that span the left and right junctions of the deletion module within the genome. </p><br />
Next, we need to transform Gαolf into the double knock-out strain to check for positive transformants. It has been reported that Gαolf could not only complement for a GPA1 deficiency but also can functionally couple to the pheromone receptor STE211. Thus we have developed assays to select functional Gαolf (figure8). </p><br />
<br />
Fig 8. Assay testing the Gαolf functional expression in yeasts that have been knocked out with FAR1 and GPA1. In (a), yeasts are transformed with pESC-Gαolf & pRS426-FUS1P-GFP-FUS1T; in (b), yeasts are transformed with pESC empty vector & pRS426-FUS1P-GFP-FUS1T. Both are induced with galactose and then α factor, and afterwards cells are viewed with fluorescence microscopy. In (a) Gαolf could couple with STE2 and Gβγ subunits and hence triggers GFP production; negative transformants and control in (b) will not trigger GFP production. </p><br />
<br />
After confirmation of functional Gαolf, we would use this strain to test its ligand binding using the same method shown in figure 6. However, this time we would expect to see no cell cycle arrest in any case since FAR1 has been deleted. We could expect the ligand binding assay this time could give an optimized coupling and signalling transduction. </p><br />
<br />
<p>Further Characterization of the Chimeric Protein Function</p><br />
To further characterize how this chimeric receptor function, we can do the following assays: <br><br />
First, we can test the how it responses to different ligand concentrations. According to literature, C. elegans Odr10 responses strongest to 100μM diacetyl[i], and we can then compare the intensity of GFP production under a series of diacetyl concentrations. <br><br />
Second, we can test the ligand specificity of the chimeric protein. According to literature, Odr10 responses to volatile molecule diacetyl, as well as non-volatile molecule pyruvic acid and citric acid13. We can also test whether it can response to other volatile molecules by setting up similar assays shown in figure 6. <br><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Group1Team:HKUST/Group12009-10-11T07:13:33Z<p>Ke lyxaa: </p>
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<h3>Welcome</h3><br />
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<p>Construction of Receptor Expression Cassette</p><br />
To functionally express C. elegans Odr10 receptor, we have designed an expression cassette. Based on the fact that rat I7 (RI7) receptor has been successfully expressed in the yeast system and it couples well to Gαolf subunit, we have designed such a chimeric receptor with the RI7 N (amino acids 1-61 of RI7) and C termini (animo acids 295-327 of RI7) fused with Odr10 transmambrane domains two to seven (TM2 – 7, amino acids 48-305 of Odr10, starting from the fourth amino acid of Odr10 TM2) [i] (figure 3). At the N termini side, they are fused at the junctions PMYFF (RI7) and YLMAFF (Odr10) because these two sequences are conserved in both receptor junction sites (Figure 4). They are cloned into the multiple cloning site of pESC-HIS expression vector under the Gal1 promoter. They are also tagged with FLAG or GFP at the C termini for protein expression detection or localization test. Given that we have retained the N and C termini of RI7, localization and subsequent coupling should not be perturbed.</p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/receptor expression strategy.jpg " width=400; height=300 /></a><br><br />
Fig3. Schemetic illustration of receptor expression strategy. The receptor expression cassette of the pESC-HIS vector was constructed to contain an insert that encodes the N and C termini of the RI7 receptor flanking an intervening sequence containing multiple cloning sites. The ligand-binding pocket of Odr10 protein is inserted between N and C termini. Adopted from Venkat,B.,el al, 2007.</p><br />
<br />
Fig 4. Sequence of the chimeric receptor. Transmambrane domains are highlighted in red;sequences from RI7 are highlight in yellow; the fusion site is separated by a green line. </p><br />
<br />
<br />
<p>Test of Chimeric Protein Localization</p><br />
To test the localization of chimeric proteins, we have made the construct pESC-Fusion-GFP, which is the chimeric protein with the GFP tag at the C termini in the expression vector pESC-HIS, as well as the negative control pESC-GFP, which is only the GFP at the same site in the expression vector pESC-HIS (Figure 5). We choose to tag GFP at the C termini because N termini of the fusion protein is important for membrane localization while C termini is important for coupling. Since we want to ensure its proper localization, it is better to fuse GFP at the C termini. </p><br />
<br />
Fig 5. Constructs for chimeric receptor fluorescence miscroscopy test, in order to confirm its localization to the yeast membrane. (a) is pESC-Fusion-GFP, which is the chimeric protein with the GFP tag at the C termini in the expression vector pESC-HIS; (b) is the negative control pESC-GFP, which is only the GFP at the same site in the expression vector pESC-HIS. </p><br />
After transformation and selection, we would induce the transformants with galactose at the exponential stage of yeast growth. After some time, we would harvest cells for the fluorescence microscopy test. We would expect to see the cells transformed with pESC-Fusion-GFP to have strong green fluorescence surrounding the cell, forming a green circle; while the negative control does not. In that case, we could say that the fusion protein is able to localize in the yeast membrane. </p><br />
<br />
<p>Test of Chimeric Protein Function</p><br />
To test the functional sensing and coupling to Gαolf or Gpa1, we have made several constructs: <br><br />
(1) pESC-Fusion-FLAG, which is the chimeric protein with the FLAG tag at the C termini in the expression vector pESC-HIS under the Gal1 promotor. <br><br />
(2) pESC-RI7-FLAG, which is the rat RI7 receptor protein with the FLAG tag at the C termini in the expression vector pESC-HIS under the Gal1 promotor, serving as the positive control. <br><br />
(3) pESC-Gαolf-FLAG , which is rat Gαolf in the pESC-HIS under the Gal10 promotor. <br><br />
(4) pRS426-FUS1P-GFP-FUS1T, which is the FUS1 promotor, GFP and FUS1 terminator in the expression vector pRS426. <br><br />
(5) pESC empty plasmid, serving as the negative control. </p><br />
<br />
There are two parts of the fuctional coupling test:</p><br />
Part 1<br><br />
First we test whether our chimeric protein could couple to Gpa1. <br><br />
The yeasts would be transformed with construct (1)+(4) or (2)+(4) or (5)+(4), respectively. After induction with galactose to express the receptors, we would add in the ligands diacetyl (for Odr10) and hexanal (for RI7). After some time of ligands binding, we would expect to see that the functional receptors could couple to Gpa1 and then trigger downstream FUS1-promoter-driven expression of GFP, together with cell cycle arrest at G1 phase (figure 6). The expression of GFP could be viewed by fluorescence microscopy; the cell cycle arrest could be confirmed by fluorescence-activated cell sorting (FACS). We might expect to see the fusion receptor and RI7 response to diacetyl and hexanal, respectively, to have GFP and cell cycle arrest. In that case we could say that fusion receptor could functionally couple to Gpa1 and start downstream signalling. <br><br />
<br />
Fig 6 Ligands sensing functional assay showing control experiments. (a) is yeast transformed with pESC-Fusion-FLAG & pRS426-FUS1P-GFP-FUS1T, and we would expect to see GFP and cell cycle arrest only when both galactose and diacetyl are added; (b) is yeast transformed with pESC-RI7-FLAG & pRS426-FUS1P-GFP-FUS1T, and we would expect to see GFP and cell cycle arrest only when both galactose and hexanal are added; (c) is yeast transformed with pESC empty plasmid and pRS426-FUS1P-GFP-FUS1T, and we would expect to see no GFP and cell cycle arrest. These cells are analyzed with fluorescence microscopy and FACS. </p><br />
<br />
Part 2<br><br />
Second, we test whether our chimeric receptor could couple to Gαolf for optimized signalling transduction as well as constructing a testable yeast system for odorant sensing. <br><br />
Gene deletion<br><br />
Before testing, yeast strain needs to be manipulated: we need to knock out the FAR1 gene, which encodes a protein controlling cell cycle arrest upon activation of MAPK pathway, so that the yeasts will still be viable after ligand binding; and also endogenous GPA1 gene, so that we could replace it with Gαolf. Due to the second messenger activity of the free G protein βγ subunit, haploid S. cerevisiae strains containing a null mutation of GPA1 undergo constitutive pheromone response of G1 arrest resulting in non-viability[ii]. So, we need to first knock out cell cycle regulatory gene FAR1 and afterwards, GPA1.To knock out these two genes, we have adopted the PCR-based tagging of yeast genes method, introduced by Janke C., el al, 2004[iii]. <br><br />
To test the successful deletion, we will use both PCR confirmation (figure 7) and also phenotype observation after adding α-factor (no mating phenotype). </p><br />
<br />
<br />
<br />
Figure 7. Primers designed for colony PCR to confirm successful deletion of yeast ORF. The correct replacement of the gene with hphNT1 or natNT2 is verified in the mutants by the appearance of PCR products of the expected size using primers that span the left and right junctions of the deletion module within the genome. </p><br />
Next, we need to transform Gαolf into the double knock-out strain to check for positive transformants. It has been reported that Gαolf could not only complement for a GPA1 deficiency but also can functionally couple to the pheromone receptor STE211. Thus we have developed assays to select functional Gαolf (figure8). </p><br />
<br />
Fig 8. Assay testing the Gαolf functional expression in yeasts that have been knocked out with FAR1 and GPA1. In (a), yeasts are transformed with pESC-Gαolf & pRS426-FUS1P-GFP-FUS1T; in (b), yeasts are transformed with pESC empty vector & pRS426-FUS1P-GFP-FUS1T. Both are induced with galactose and then α factor, and afterwards cells are viewed with fluorescence microscopy. In (a) Gαolf could couple with STE2 and Gβγ subunits and hence triggers GFP production; negative transformants and control in (b) will not trigger GFP production. </p><br />
<br />
After confirmation of functional Gαolf, we would use this strain to test its ligand binding using the same method shown in figure 6. However, this time we would expect to see no cell cycle arrest in any case since FAR1 has been deleted. We could expect the ligand binding assay this time could give an optimized coupling and signalling transduction. </p><br />
<br />
<p>Further Characterization of the Chimeric Protein Function</p><br />
To further characterize how this chimeric receptor function, we can do the following assays: <br><br />
First, we can test the how it responses to different ligand concentrations. According to literature, C. elegans Odr10 responses strongest to 100μM diacetyl[i], and we can then compare the intensity of GFP production under a series of diacetyl concentrations. <br><br />
Second, we can test the ligand specificity of the chimeric protein. According to literature, Odr10 responses to volatile molecule diacetyl, as well as non-volatile molecule pyruvic acid and citric acid13. We can also test whether it can response to other volatile molecules by setting up similar assays shown in figure 6. <br><br />
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<h3>Welcome</h3><br />
<br />
<p>GPCRs</p><br />
G protein-coupled receptors (GPCRs) are the main sensors of cells. They respond to ligands such as most hormones, neurotransmitters, light, odorants, taste substances, pheromones, and a variety of medicines. GPCRs comprise one of the largest protein superfamilies: it is estimated that there are 1050 and 160 GPCR genes in the genomes of Caenorhabditis elegans and Drosophila melanogaster, corresponding to 5.5% and 1% of their total genes, respectively[1]. Identifying GPCR the ligands may lead to the discovery of novel hormones, neurotransmitters etc., and thus contribute to the development of new medicines. In fact, GPCRs are known to target 30-60% of present medicines[2]. <br><br />
GPCRs are seven transmembrane proteins (Fig1). Stimulated by extracellular ligands, the receptors act through heterotrimeric G proteins to regulate intracellular effector proteins. The G proteins are composed of Gα,Gβ and Gγ subunits. In the inactive state, the Gα-subunit is bound to GDP and associated with the Gβ-and Gγ-dimer. Upon ligand binding, the receptor stimulates the release of GDP from Gα, allowing the binding of GTP. Gα-GTP is released from the Gβ-and Gγ-dimer, and the dissociated subunits regulate the activity of effector proteins such as adenylate cyclase, phospholipase C, mitogen-activated protein kinase (MAP kinase) cascades and ion channels. </p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/Model of GPCRs.jpg " width=450; height=300 /></a><br><br />
Fig.1 Model of GPCRs. Adopted from Hinako Suga, Tatsuya Haga, 2007.[3] </p><br />
Olfactory receptors (OR) are also seven transmembrane proteins. Insect OR gene families have been identified in the fruit fly Drosophila melanogaster, the malaria vector mosquito Anopheles gambiae, the yellow fever vector mosquito Aedes aegypti, the honeybee Apis mellifera, the silkmoth Bombyx mori, and the flour beetle Tribolium castaneum.[4] Though in vertebrates ORs consist largely of GPCRs, in insects there remains doubt whether ORs belong to GPCRs. However, various studies have been carried out on Drosophila and C. elegans G-protein coupled olfactory receptors, which have uncovered much of their structure and functions. <br><br />
Based on these facts, in this iGEM project we choose to study C.elegans Odr10 GPCR and rat RI7 GPCR to demonstrate the working principle in our design. Odr10 protein is an odorant receptor in C. elegans. It responses to a volatile odorant diacetyl and then triggers chemotaxis of C.elegans towards the molecule. RI7 is a rat odorant receptor that has been functionally expressed in yeast. We will show that C.elegans odorant receptor could also be functionally expressed in yeast and later we could potentially extend this study method to other insects ORs or other GPCRs.</p><br />
<p>Yeast Pheromone Sensing Pathway</p><br />
Saccharomyces cerevisiae contains two GPCR-based signalling pathways, one that mediates the pheromone response during mating and a second that senses glucose to regulate filamentous differentiation in diploid cells and invasive growth in haploid cells[5], between which there appears to be little crosstalk. By providing a null background, the yeast pheromone response pathway provides a robust and experimentally tractable system to study heterologous GPCRs. <br><br />
Haploid S. cerevisiae exists in one of two mating types(a and α), and mating occurs between cells of opposite types and is controlled by the exchange of mating pheromones; a-cells secrete a-factor and express the α-factor receptor (Ste2) whereas α-cells secrete α-factor and express the a-factor receptor (Ste3). Both receptors couple to the same heterotrimeric G protein composed of Gα(Gpa1), Gβ(Ste4) and Gγ (Ste18) subunits. Binding of the mating pheromone to the receptor results in the release of the Gβγ dimer, which then interacts with effectors to bring about conjugation (Fig.2A). A key role of Gβγ is to activate a MAP kinase cascade in which sequential activity of Ste11 (MEK kinase), Ste7 (MAP kinase kinase or MEK) and Fus3 (MAP kinase) leads to activation of a transcription factor (Ste12) that induces gene expression in preparation for mating. Fus3 also activates the cyclindependent kinase inhibitor Far1 to bring about cell cycle arrest[6].<br><br />
<br />
The Expression of Heterologous GPCRs in S. cerevisiae</p><br />
S. cerevisiae is chosen in this project for several reasons as the ideal system to study heterologous GPCR (Box 1). </p><br />
Box1. Reasons for choosing yeast to study heterologous GPCRs<br><br />
To functionally express heterologous GPCRs, we need to do some modifications to the strain to improve the localization of the receptor to membrane and the coupling of the of the heterologous receptor to the Gα subunit (Fig.2B). </p><br />
<p>Modifying GPCRs</p><br />
Though some GPCRs may be degraded or retained within intracellular compartments, most GPCRs can be expressed unmodified in yeast. Rat I7 GPCR is one of them, which has been shown to be functionally expressed in the yeast system8. Sequence analyses of ORs and GPCRs have shown that the N termini of these receptors are involved in plasma membrane localization, whereas the C termini define the specificity for G protein interaction; the spanning transmembrane domains two (TMII) and seven (TMVII) are the ligand-binding region . It is possible that a chimeric OR can be expressed using N termini and C termini of known GPCRs that can be functional expressed, for example, RI7, and the ligand binding region of our desired ORs.(For more details, see Experiment Design). </p><br />
<p>Modifying the G protein</p><br />
The yeast Gα (Gpa1) has been shown to interact with several non-yeast GPCRs. This functional coupling can be improved by replacing Gpa1 with mammalian Gα to the related GPCR, since yeast Gpa1, Ste4 and Ste18 are structurally and functionally similar to mammalian Gα. For example, rat Gαolf and RI7 are both expressed in yeast and RI7 is effectively coupled to Gαolf[8]. <br />
Developing Reporter Genes <br />
Most yeast GPCR assays use reporter genes under the control of the Ste12-inducible FUS1 promoter. We can either introduce β-galactosidase to provide a quantitative assay suitable for high-throughput analysis; or we can incorporate GFP or luciferase under the FUS1 promoter to fast detect the signal transduction. This method has also been verified in previous studies[8].<br><br />
Fig.2 Overview of genetic modifications of the pheromone signaling pathway to allow functional characterization of heterologously expressed GPCRs in S. cerevisiae. (A) Pheromone sensing pathway in budding yeast (B) Yeast can be engineered to express heterogenous GPCRs using the pheromone response pathway. Adopted from J Minic,et al,2005.[9]<br><br />
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<h3>Welcome</h3><br />
<br />
<p>Attractant production and reporter Construct </p><br />
The attractant production pathway contains three parts, which are FUS1 promoter, ARO9 gene and FUS1 terminator. These three genes are first derived through PCR from yeast genome and then cloned into PRS426 yeast expression vector for further functional assay. <br><br />
The reporter construct also contains three parts, which are FUS1 promoter, EGFP gene and FUS1 terminator. These three genes are first derived through PCR and then cloned into PRS426 yeast expression vector for further functional assay. </p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/Attractant production pathway and Reporter detailed design.jpg " width=550; height=320 /></a><br><br />
<br />
Figure 5. Attractant production pathway and Reporter detailed design</p><br />
<br />
<br />
<p> I. Primer Design</p><br />
We have designed several sets of primers for our construction, which are listed in Table 1. These mainly consist of primers for FUS1 promoter amplification, EGFP reporter amplification, ARO9 gene amplification and FUS1 terminator amplification. Primer statistics are calculated using NetPrimer. </p><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/Gp3 tablePrimer.jpg " width=550; height=650 /></a><br><br />
Table 4 Primer sequences designed for the constructions<br><br />
<br />
*Note: Restriction sites are highlighted in yellow. </p><br />
<br />
<p> II. PCR</p><br />
Three out of four genes, FUS1 promoter, FUS1 terminator and ARO9 gene are directly amplified out from yeast genomic DNA (strain YPH501). The other gene, EGFP gene, is amplified from plasmid YTK-6/Tpi/EGFP. <br><br />
For optimized PCR efficiency and accuracy, Taq polymerase is chosen for amplification PCR reactions. Gradient PCR is carried out for each step to optimize the reaction. </p><br />
<br />
<p> III. Cloning</p><br />
After the all the fragments are successfully derived through PCR, standard cloning procedures are followed to construct an integrated expression vector. <br><br />
The PRS426 yeast expression vector is used in the expression of our construction. The FUS1 promoter is cloned into the multicloning site via Sac I and Not I digest. The ARO9 gene and the EGFP gene are cloned via blunt-end Sma I digest. FUS1 terminator is cloned via Hind III and Xho I digest. </p><br />
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<h3>Welcome</h3><br />
<br />
<p>2-Phenylethanol</p><br />
Among the various attractant compounds available, there is one compound called 2-Phenylethanol (PEA). 2-Phenylethanol (PEA) is an aromatic alcohol with a rose-like odor and occurs in many essential oils and fermented foods. An annual production of 7000 tons is generated by chemical processes, mainly by the Friedel–Craft reaction of ethylene oxide with benzene, or by hydrogenation of styrene oxide with Raney nickel as a catalyst. It can be also biologically produced through the Ehrlich pathway, which originally exists in Saccharomyces cerevisiae. Through this pathway, ‘natural’ PEA can be obtained at high purity by an environmentally friendly process. 2-Phenylethanol can work very well as an attractant towards fruit flies, and it demonstrates higher attraction when mixed with some other compounds (figure 1). </p> <br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/Mean catches of Drosophila melanogaster in a trap assay.jpg" width=500; height=330 /></a><br />
FIG.1. Mean catches of Drosophila melanogaster in a trap assay. This shows different chemicals have attraction towards insects. Adopted from J. Zhu, et al, 2003.</p><br />
Therefore, based on the above information, we choose 2-phenylethanol as our target attractant molecule, which may also be potentially applied to attract other pests in a biological pesticide system. </p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/2-Phenylethanol.jpg " width=120; height=80 /></a><br />
<p>Yeast Ehrlich pathway</p><br />
Saccharomyces cerevisiae has been used for at least 8 millennia in the production of alcoholic beverages. Along with ethanol and carbon dioxide, fermenting cultures of this yeast produces many low-molecular-weight flavor compounds, including fusel alcohols. Fusel alcohols are derived from amino acid catabolism via an yeast endogenous pathway that was first proposed a century ago by Ehrlich. <br><br />
The Ehrlich pathway contains three steps, transamination, decarbonxylation and reduction. The first step, transamination, is mainly catalyzed by Aro8p and Aro9p, which are initially characterized as the aromatic amino acid aminotransferases I and II. 2-Phenylethanol production is mainly catalyzed by ARO9p. The second step, decarbonxylation, can be catalyzed by no fewer than 5 genes that show sequence similarity with thiamine diphosphate dependent decarboxylase genes. Three of these (PDC1, PDC5 and PDC6) encode pyruvate decarboxylases, while ARO10 and THI3 represent alternative candidates for Ehrlich-pathway decarboxylases. The third step, reduction, is catalyzed by dehydrogenase to yield the final product, 2-phenylethanol. <br><br />
In our project, we plan to adopt this yeast endogenous pathway to produce 2-phenylethanol from 2-phenylalanine, as well as control this pathway for our future application. </p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/2-Phenylethanol biosynthetic pathway in Saccharomyces cerevisiae.jpg " width=550; height=420 /></a><br />
Figure 2. 2-Phenylethanol biosynthetic pathway in Saccharomyces cerevisiae</p><br />
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<h3>Welcome</h3><br />
<br />
<p>Attractant production and reporter Construct </p><br />
The attractant production pathway contains three parts, which are FUS1 promoter, ARO9 gene and FUS1 terminator. These three genes are first derived through PCR from yeast genome and then cloned into PRS426 yeast expression vector for further functional assay. <br><br />
The reporter construct also contains three parts, which are FUS1 promoter, EGFP gene and FUS1 terminator. These three genes are first derived through PCR and then cloned into PRS426 yeast expression vector for further functional assay. </p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/Attractant production pathway and Reporter detailed design.jpg " width=550; height=380 /></a><br><br />
<br />
Figure 5. Attractant production pathway and Reporter detailed design</p><br />
<br />
<br />
<p> I. Primer Design</p><br />
We have designed several sets of primers for our construction, which are listed in Table 1. These mainly consist of primers for FUS1 promoter amplification, EGFP reporter amplification, ARO9 gene amplification and FUS1 terminator amplification. Primer statistics are calculated using NetPrimer. </p><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/Gp3 tablePrimer.jpg " width=550; height=650 /></a><br><br />
Table 4 Primer sequences designed for the constructions<br><br />
<br />
*Note: Restriction sites are highlighted in yellow. </p><br />
<br />
<p> II. PCR</p><br />
Three out of four genes, FUS1 promoter, FUS1 terminator and ARO9 gene are directly amplified out from yeast genomic DNA (strain YPH501). The other gene, EGFP gene, is amplified from plasmid YTK-6/Tpi/EGFP. <br><br />
For optimized PCR efficiency and accuracy, Taq polymerase is chosen for amplification PCR reactions. Gradient PCR is carried out for each step to optimize the reaction. </p><br />
<br />
<p> III. Cloning</p><br />
After the all the fragments are successfully derived through PCR, standard cloning procedures are followed to construct an integrated expression vector. <br><br />
The PRS426 yeast expression vector is used in the expression of our construction. The FUS1 promoter is cloned into the multicloning site via Sac I and Not I digest. The ARO9 gene and the EGFP gene are cloned via blunt-end Sma I digest. FUS1 terminator is cloned via Hind III and Xho I digest. </p><br />
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</html></div>Ke lyxaahttp://2009.igem.org/Team:HKUST/Group3Team:HKUST/Group32009-10-11T07:05:13Z<p>Ke lyxaa: </p>
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<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project description</a></li><br />
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<li> Odorant sensoring </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/View1">Overview</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back1">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group1">Experimental design</a></li> <br />
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<li> Attranctant production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/View3">Overview</a></li><br />
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<li>Toxin production</a></li><br />
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<h3>Welcome</h3><br />
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<p>Construction of Attractant production and Reporter pathway</p><br />
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To functionally express the yeast ARO9 gene and EGFP gene, we have designed a reporter and an attractant production constructs. Based on the fact that yeast endogenous FUS1 promoter can sense the signal generated by the receptor, and the yeast endogenous FUS1 terminator can stop the transcription activated by the promoter, we have designed such a pathway that puts ARO9 gene (with the original promoter deleted) and EGFP gene under the control of the FUS1 promoter. We have also have control constructs for later experiments. The whole system is cloned into the multiple cloning site of the pRS426 yeast expression vector. </p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/Gp3 design1.jpg " width=180; height=80 /></a><br><br />
&nbsp; (1) pRS426-FUS1P-ARO9-FUS1T, which is ARO9 gene under the control of FUS1 promoter, cloned into the plasmid pRS426.</p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/Gp3 design2.jpg " width=180; height=80 /></a><br><br />
&nbsp; (2) pRS426-FUS1P-EGFP-FUS1T, which is EGFP gene under the control of FUS1 promoter, cloned into the plasmid pRS426.</p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/pRS426-EGFP.jpg" width=150; height=80 /></a><br><br />
&nbsp; (3) pRS426-EGFP, which is EGFP gene without the FUS1 promoter, cloned into the plasmid pRS406. </p><br />
Figure 3. Attractant, reporter and control constructs</p><br />
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<p>Test of the Reporter System </p><br />
The yeast strain YPH501 with ARO9 gene deleted would be transformed with pRS426-FUS1P-EGFP-FUS1T, pRS426-EGFP, respectively. After α factor binding, we would expect to see that the MAPK pathway is activated and then the downstream FUS1-promotor-driven expression of GFP. The expression of GFP could be viewed by fluorescence microscopy. </p><br />
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<img src="http://igem2009hkust.fileave.com/wiki/Group3/Table 1 Reporter functional assay and expected results.jpg " width=550; height=250 /></a><br><br />
Table 1 Reporter functional assay and expected results. (a) is the YPH501 strain deleted with ARO9 gene and transformed with pRS426-FUS1P-EGFP-FUS1T; (b) is the same strain transformed with pRS426-EGFP. </p><br />
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<p>Attractant production test</p><br />
Second we will test our attractant production pathway and the attractiveness towards Drosophilae using the Cage Bioassays method given by J. Zhu, et al, 2003.(Figure 4) </p><br />
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<img src="http://igem2009hkust.fileave.com/wiki/Group3/test.jpg " width=400; height=230 /></a><br><br />
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Figure 4, The cage bioassay to show the attractant effect. </p><br />
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By comparing these six conditions, we can decide whether the attractant pathway under the FUS1 promoter can response to signals generated by the receptor and can effectively produce attractant molecules (table 2). </p><br />
<a href="http://www.freewebsitetemplates.com"><img src="http://igem2009hkust.fileave.com/wiki/Group3/Different traps and expected results.jpg " width=550; height=350 /></a><br><br />
Table 2. Different traps and expected results</p><br />
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<p>Further characterization of the Attractant production pathway function</p><br />
To further characterize the attractant production pathway function, we can do the following assays. <br><br />
First, we can use the HPLC or some other analytical chemical methods to analyze the attractant concentration in the yeast extract and find the best attractant concentration towards Drosophilae. <br><br />
Second, we can change the strain of the yeast to test the attractiveness differences between strains and find the best strain for attractant production. </p><br />
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