Team:METU Turkey SoftLab

From 2011.igem.org

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<title> Methan<b>E.COLIc</b> : Decreasing the Greenhouse effects and Saving the workers life in one system</title>
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// coal mining 228,159105231 metrictone per second (cm_act)
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// livestock 467,726165724 metrictone per second (ls_act)
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    // landfills 798,430504099 metrictone per second (lf_act)
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// natural gases 456,318210462 metrictone per second (ng_act)
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<body onLoad="window.setTimeout('getSecs()',1);javascript: alert(' This website running on all versions of following browsers,better  at 1280x800 resolution,\n*Google Chrome\n*Mozilla Firefox\n*Internet Explorer\n*Opera\n*Safari')">
<div id="metutop">
<div id="metutop">
</div>
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<div id="home" class="item">
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<div class="content2">
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<embed width="480" height="360" quality="high" bgcolor="#000000" name="main" id="main" allowfullscreen="true" allowscriptaccess="always" src="http://www.dnatube.com/nvlab/player/player.swf?config=http://www.dnatube.com/nvlab/econfig.php?key=ddbdf3eff0f505975b6a" type="application/x-shockwave-flash" /></div><div class="homepage">
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<div class="text">
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<div id="homecontainer"><h3>Abstract</h3>
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<p>'''Under Construction!!!'''</p>
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        <h2><img src="https://static.igem.org/mediawiki/2011/d/df/Bannergiris2.png" height="300px" width="900px"></h2>
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<p>...</p>
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<div class="writeback">
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<h3>Methan<b>E.COLI</b>c : Decreasing the Greenhouse effects and Saving the workers life in one system</h3>
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<p>Firedamp explosions are frequently seen cases at all mines over the world. In Turkey every year, 50 miners lose their lives because of firedamp explosions. Firedamp is a flammable gas found in coal mines and it mainly contains methane. Beside its explosive property, methane is also the main contributor to global warming. However recent mine mechanisms release obtained methane into air. By offers of Synthetic biology, we aimed to design a device which will work on <i>E.coli</i> that provides solutions for side effects of methane. Device that we are planning to construct involves the genes of bacteria (<i>Methylococcus capsulatus</i>) and insect (<i>Drosophilia melanogaster</i>). Our compact system in <i>E.coli</i> is fabricated as sensation of methane, the conversion of methane to methanol and then entrapment of methanol to handle for biofuel and death of bacteria at 42 C by kill switch mechanism.</p>
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                        <p>When project is analyzed in stepwise, there are successive 4 steps of our modelled system in modified organism. As mentioned in subtitles,  methane is sensed, then converted to methanol and methanol is entrapped and to elute methanol the cells are dead. </p>
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  </div>   
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<center><div class="timer">
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<p><b><span class="num" id="cm"></span><span class="num">&nbsp;t</span></b></p>
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<br /><p><b><span class="num" id="ls"></span><span class="num">&nbsp;t</span></b></p>
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<br/><p><b><span class="num" id="lf"></span><span class="num">&nbsp;t</span></b></p>
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<br/><p><b> <span class="num" id="ng"></span><span class="num">&nbsp;t</span></b> </p>
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<br/><p><b><span class="num" id="total"></span><span class="num">&nbsp;t</span></b></p>
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<img src="https://static.igem.org/mediawiki/2011/1/1c/Projectheader.png" height="75px" width="890px">
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<h2>Introduction</h2>
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<p>Methan<b>E.COLIc</b> project is designed to solve one of the problems on worker security in mines of Turkey , by constructing the natural parts of organisms. The main reason for us to choose such a project is that in Turkey- and also in many countries- each year, huge numbers of  workers in mines lost their life due to deficiencies  and conditions in their working areas (mines). The release of Grizu gas leads to subsidence of mines and so death of workers.This project is also designed to solve one of universal problem; global warming. Methane gas is a potential greenhouse gas.This project focuses on sensing the ambient methane gas and converting to one of the bio-fuel sources, methanol. Methanol is entrapped by product of one of the constructs of project, and then cells are dead by kill switch device of project to elute methanol.</p>
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<br />
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<br />
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    <u><h3>Steps</h3></u>
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    <ul>
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      <li><a href="#project2">Methane sensing</a></li>
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      <li><a href="#project3">Conversion</a></li>
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      <li><a href="#project4">Entrapment</a></li>
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      <li><a href="#project5">Kill switch</a></li>
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</ul>
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<h2>Team</h2>
<h2>Team</h2>
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<p><b><i>Hello everyone!We are Metu Ankara IGEM team.Our team consists of high motivated,social and enjoyable  “undergraduate  and graduate scientists”  from departments of physics, chemistry ,biology, computer engineer and computer education & instructional technology. We are such a team that complete each other  so that we all experienced team  spirit in IGEM. This makes us happy  while doing experiences even in the hot summer.Our slogan is “ all for one, one for all”!!Then, who are we?</i></b></p>
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<br/>
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<p>METU Turkey Software is an interdisciplinary team of 7 students and
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<h3><u>Instructors</u></h3>
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4 advisors from various backgrounds such as Molecular Biology, Bioinformatics,
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<ul>
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Computer Engineering and Computer Education and Instructional Technology.  
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<li><a href="mailto:akkayams@metu.edu.tr">Prof. Dr. Mahinur S. Akkaya</a></li>
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We have put our knowledge and experience in our fields together to bring
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<li><a href="mailto:kursat@metu.edu.tr">Assoc. Prof. Dr.Kürşat Çağıltay</a></li>
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a much needed solution to a daily problem in field of synthetic biology
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for iGEM 2010</p>
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<h3><u>Advisors</u></h3>
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<ul>
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<li><a href="mailto:brkylmz@gmail.com">Burak Yilmaz</a></li>
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<li><a href="mailto:okdeniz@ceng.metu.edu.tr">Gökdeniz Karadağ</a></li>
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</ul>
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<h2>Team leader :<a href="mailto:ceren.seref@gmail.com">Ceren Seref</a></h2>
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<img width="130px" height="180px" src="https://static.igem.org/mediawiki/2011/2/2e/Cereh.png">
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<p>Ceren has just finished the junior year in biological sciences department in METU. It’s her second year in iGEM competition. She’s planning to study about neuroscience-especially on Alzheimer’s disease. She believes synthetic biology approaches can be a very good way of drug development in neuroscience field. She enjoys climbing, listening to music and singing.</p>
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<h2><a href="mailto:sibelataol8@gmail.com">Sibel Ataol</a></h2>
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<img width="130px" height="180px" src="https://static.igem.org/mediawiki/2011/a/a9/Sibel-01.jpg">
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<p> Sibel is first semester student in Master's program of Biomedical engineering at Middle East
 +
Technical University indeed  she is graduated from Biology Department at METU. This is the second year of her in IGEM team from METU.She is one of the experienced in team and elder sister:) As a vice team lead, planing the experiments and cloning strategies and motivating team are the responsibilities of her. Studying in bioengineering and regenerative sciences is her desire
 +
for future.</p>
</div>
</div>
</div>
</div>
<div class="smallbox">
<div class="smallbox">
-
<h2 class="black">......</h2>
+
<h2><a href="mailto:e153564@metu.edu.tr">Funda Guzey</a></h2>
-
<img src="....">
+
<img width="130px" height="180px" src="https://static.igem.org/mediawiki/2011/9/93/Funda.jpg">
<div class="smalltext">
<div class="smalltext">
-
<p>.....</p>
+
<p>She is a senior student of Biological Sciences at Middle East Technical University. This is her third year in iGEM competition. She gained  a lot of experiences  in  this summer. One of the her interests is Synthetic Biology. However, her main interest is Biomedical and Tissue Engineering. She feels lucky to have such a wonderful team and is glad to share a wonderful summer with her team.</p>
</div>
</div>
</div>
</div>
<div class="smallbox">
<div class="smallbox">
-
<h2 class="black">......</h2>
+
<h2><a href="mailto:gokcanaydogan@hotmail.com">Gökcan Aydoğan</a></h2>
-
<img src="....">
+
<img width="130px" height="180px" src="https://static.igem.org/mediawiki/2011/d/dd/Gokcan_01_%281%29.jpg">
<div class="smalltext">
<div class="smalltext">
-
<p>.....</p>
+
<p>Gökcan has just finished the third year of the B.Sc. Chemistry degree at METU. This is his first year in IGEM team. He has been a huge fan of Synthetic Biology ever since he understands nothing about the concept. Everyday, he tries hard to get the picture. In his free time, he likes gaming, drawing cartoon and eating,  eating, eating...</p>
</div>
</div>
</div>
</div>
Line 385: Line 580:
</div>
</div>
<div class="smallbox">
<div class="smallbox">
-
<h2 class="black">......</h2>
+
<h2><a href="mailto:e160446@metu.edu.tr">Filiz ORMANCI</a></h2>
-
<img src="....">
+
<img width="130px" height="180px" src="https://static.igem.org/mediawiki/2011/8/8f/Filiz.jpg">
<div class="smalltext">
<div class="smalltext">
-
<p>.....</p>
+
<p>She is a fourth year student at the Department of Physics at Middle East Technical University. This is the first year of her in iGEM team. She wants to study on the usage of optical instruments in biophysics. She likes team working and sharing the success. She is always positive and happy... ehm..also a “soothsayer”. She enjoys playing musical instruments mainly violin and learning different languages.</p>
</div>
</div>
</div>
</div>
<div class="smallbox">
<div class="smallbox">
-
<h2 class="black">......</h2>
+
<h2><a href="mailto:simgeyuz@gmail.com">G. Simge Yüz</a></h2>
-
<img src="....">
+
<img width="130px" height="180px" src="https://static.igem.org/mediawiki/2011/f/f6/Sibel_012-1.jpg">
<div class="smalltext">
<div class="smalltext">
-
<p>.....</p>
+
<p>She is a recent graduate student of Chemistry and a fourth year student of Biological Sciences. This is the first year of her in iGEM team. She wants to study on neuropharmacology in the field of neuroscience. She is glad to join to METU-Ankara-iGEM team. She enjoys dancing especially Argentine tango and swimming. She is also a member of Flashmob Ankara Society</p>
</div>
</div>
</div>
</div>
<div class="smallbox">
<div class="smallbox">
-
<h2 class="black">......</h2>
+
<h2><a href="mailto:yunusalpagu@gmail.com">Yunus Alpağu</a></h2>
-
<img src="....">
+
<img width="130px" height="180px" src="https://static.igem.org/mediawiki/2011/e/e8/Yunus_01.jpg">
<div class="smalltext">
<div class="smalltext">
-
<p>.....</p>
+
<p>Yunus is a third year student in biology department at Middle East Technical University in Turkey. This is his first year in iGEM team.He is interested in regenerative medicine, stem cells, biomaterials and tissue engineering. His favourite TV series is ‘Fringe’ and he likes playing tennis and volleyball.He is glad to join to METU-Ankara-iGEM team. </p>
</div>
</div>
</div>
</div>
<div class="smallbox">
<div class="smallbox">
-
<h2 class="black">......</h2>
+
<h2><a href="mailto:pelinispir@hotmail.com">Pelin İspir</a></h2>
-
<img src="....">
+
<img width="130px" height="180px" src="https://static.igem.org/mediawiki/2011/8/88/Pelin_01.jpg">
<div class="smalltext">
<div class="smalltext">
-
<p>.....</p>
+
<p>Pelin is currently studying the biology department of Middle East Technical University in Turkey.
 +
She is interested in stem cell, developmental biology,tissue engineering, and conservation biology.This is the first year of her in İGEM.She really enjoys with being this team and dealing with the mystery of synthetic biology.She enjoys listening latin musics and flamenco.</p>
</div>
</div>
</div>
</div>
<div class="smallbox">
<div class="smallbox">
-
<h2 class="black">......</h2>
+
<h2><a href="mailto:ibrahim.ozturk_02@metu.edu.tr">İbrahim Kutay Ozturk</a></h2>
-
<img src="....">
+
<img width="130px" height="180px"  src="https://static.igem.org/mediawiki/2011/8/87/Ibrahimkut.jpg">
<div class="smalltext">
<div class="smalltext">
-
<p>.....</p>
+
<p>Kutay is a graduate of Molecular Biology and Genetics, and now doing his M.Sc. in Biotechnology department at METU. This is his first year in IGEM team. He is interested in plant-based drugs, plant-pathogen interaction, biofuel production and bioremediation. He enjoys playing basketball, watching movies, and listening to music.</p>
</div>
</div>
</div>
</div>
-
<div class="smallbox">
+
-
<h2 class="black">......</h2>
+
                     
-
<img src="....">
+
                     
 +
                        <div class="smallbox">
 +
<h2>Advisor:<a href="mailto:brkylmz@gmail.com">Burak Yılmaz</a></h2>
 +
<img width="130px" height="180px"  src="https://static.igem.org/mediawiki/2010/a/a2/Metu-burak.jpg">
<div class="smalltext">
<div class="smalltext">
-
<p>.....</p>
+
<p>He is a recent graduate of METU Molecular Biology and Genetics department and now studying towards his masters degree on Molecular Bioengineering at METU. His interest in synthetic biology did start during his undergraduate years and after graduation he started up the Sentegen company which is the first biotechnology based company focused on synthetic biology in Turkey. He continues his research and training in synthetic biology while also contributing to the development of the field in his country. He needs new scientific revolutions to solve huge problems of life and emerging field of synthetic biology is best candidate for biotechnological revolution. He is interested in synthetic biology applications, along with Lab-on-a-Chip devices for molecular biology techniques, and he is designing gene synthesis chips to produce biobricks - raw materials of garage biology- faster and cheaper. he enjoys snowboarding, cycling and writing poems.</p>
</div>
</div>
 +
</div>
 +
               
 +
<div class="team-2">
 +
              <center><h3><u>Software Group</u></h3></center>
 +
                        <div class="smallbox">
 +
<h2><a href="mailto:agca1988@gmail.com">Muhammed Akif Ağca</a></h2>
 +
<img width="130px" height="180px" src="https://static.igem.org/mediawiki/2010/1/10/Metu-akif2.jpg">
 +
<div class="smalltext">
 +
<p>Akif is a graduate of Computer Education and Instructional Technologies department. In bachelor he has worked on Software Development, Database Management, Instructional Technologies, and Bioinformatics.<br />
 +
He was software developer and team leader of BioGuide project and Synthetic Biology Online Lecture.
 +
<br />Currently he is mastering in Software Engineering and developing scientific applications, he will continue to work on Science and Technology Management after becoming master of Software Engineering.
 +
</p>
 +
</div>
 +
</div>
 +
                        <div class="smallbox">
 +
<h2><a href="mailto:miktat_aktas@hotmail.com">Miktat Aktaş</a></h2>
 +
<img width="130px" height="180px" src="https://static.igem.org/mediawiki/2011/8/8d/Miktat.JPG">
 +
<div class="smalltext">
 +
<p>He graduated from Computer Education & Instructional Technology Department of Middle East Technical University last semester and he contributes this genius team as a coder for website. He wants to be a successful academician in his department at METU. He likes swimming and everything related to computers and automobiles.</p>
 +
</div>
 +
</div>
 +
                       
 +
                        <div class="smallbox">
 +
<h2>Ceyhun Kerimov</h2>
 +
<img width="130px" height="180px" src="https://static.igem.org/mediawiki/2011/4/4d/Ceyhunkerim.png">
 +
<div class="smalltext">
 +
<p>Ceyhun was born in Azerbaijan. He is studying in METU Computer Engineering Department. <br />
 +
He is Java and Salesforce developer now. He was in web side of the project. He tried to help the team to manupilate and store the data. <br />
 +
He is working in web and cloud computing technologies.
 +
</p>
 +
</div>
 +
</div>
 +
                      <div class="smallbox">
 +
<h2>Huseyin Lutin</h2>
 +
<img width="130px" height="180px" src="https://static.igem.org/mediawiki/2011/8/81/Husayinlut.png">
 +
<div class="smalltext">
 +
<p>Huseyin is a senior student in METU Computer Engineering Department.<br />
 +
He was helping the team during software requirements specification step of the project.<br />
 +
He is working on computer graphics in this year.
 +
</p>
 +
 +
                      </div>
 +
</div>
 +
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
-
<div id="motivation" class="item">
+
<div id="wetlab" class="item">
<div class="content2">
<div class="content2">
<div class="text">
<div class="text">
-
<h2>Motivation</h2>
+
<img src="https://static.igem.org/mediawiki/2011/2/28/Wetlabheader.png" height="75px" width="890px">
-
<p>...</p>
+
<h2>WET LAB</h2>
-
</div>
+
<p>Whole summer in lab work we aimed to design 7 new <i>E.coli</i> compatible Biobricks and  1 composite part from 2011 kit plate distributions, also we aimed to characterize each part by protein analysis and fluorescence protein measurements.</p>
 +
 
 +
<p>We achieved design and compatibility of parts in <i>E.coli</i>, however not all parts’ characterization is achieved. Only the genes of designed composite part from kit plate is characterized by GFP measurements.</p>  
 +
 
 +
<p>In this section you can find out more details on all our wet lab works. You can also find our data under results subtitle and information about design of our parts-biobricks with explanations in literature. It also contains materials and protocols, and finally there is safety questions list on our safety considerations both in the lab and on a wider environmental scale.</p>
 +
 
 +
 
 +
              </div>
</div>
</div>
</div>
</div>
-
<div id="scope" class="item">
+
 
 +
 
 +
 
 +
 
 +
<div id="humanpractice" class="item">
<div class="content2">
<div class="content2">
-
<div class="text">
+
        <div class="text">
-
<h2>Scope and Future Aspects</h2>
+
<img src="https://static.igem.org/mediawiki/2011/1/1b/Humanpractice.png" height="75px" width="890px">
-
<p>.....</p>
+
<h2>Overview</h2>
-
<p>.....</p>
+
<p>Methan<b>E.COLI</b>c projec
-
<p>....</p>
+
t arises from sustainability and conservancy of human health and safety. Therefore while we were constructing the organismic device we also wanted to inform people from all age and we wanted to increase the public awareness on our project; methane gas based (firedamp) explosions and the greenhouse effect of methane gas.</p>
 +
 
 +
<p>Our aim is to inform people in each age. After brainstorming and searching, we planned the content of information and activities according  to following subtitles.</p>
</div>
</div>
-
</div>
+
 
</div>
</div>
-
<div id="project" class="item">
 
-
<div class="content2">
 
-
<div class="text">
 
-
<h2>Project Introduction</h2>
 
-
<p>...</p>
 
-
</div>
 
-
</div>
 
</div>
</div>
-
<div id="notebook" class="item">
+
<div id="futureplan" class="item">
<div class="content2">
<div class="content2">
<div class="text">
<div class="text">
-
<h2>Notebook</h2>
+
<img src="https://static.igem.org/mediawiki/2011/5/53/Futureplanheader.png" height="75px" width="890px">
-
<div id="notebook2">
+
<h2>Future Plan</h2>
-
<div class="month">
+
-
January</div>
+
-
<div class="general">
+
-
<p>Brain Storming about the iGEM. </p>
+
-
<ul>
+
-
<li>What is iGEM.</li>
+
-
<li>Previous Wet-Lab Projects developed at METU.</li>
+
-
<li>What kind of projects can be developed as a software
+
-
team.</li>
+
-
</ul>
+
-
<p>NOTE: Still the first software team in Turkey...</p>
+
-
</div>
+
-
<div class="month">
+
-
February</div>
+
-
<div class="general">
+
-
<ul>
+
-
<li>Reading articles about iGEM WetLab and Software team
+
-
projects.</li>
+
-
<li>Looking for the members of the team.</li>
+
-
<li>Looking for the instructors who can consult the team.
+
-
</li>
+
-
</ul>
+
-
</div>
+
-
<div class="month">
+
-
March</div>
+
-
<div class="general">
+
-
<ul>
+
-
<li>Employing a member to team interested in Synthetic Biology.</li>
+
-
<li>Reading articles about Synthetic Biology, Bioinformatics
+
-
and Bio Engineering.</li>
+
-
<li>Founding the team [ An instructor, and student members
+
-
]</li>
+
-
</ul>
+
-
</div>
+
-
<div class="month">
+
-
April</div>
+
-
<div class="general">
+
-
At this month we have started regular workshop about Synthetic
+
-
Biology, Bioengineering, and Bioinformatics.
+
-
<ul>
+
-
<li>This month biologists in the team telling the needed
+
-
basics to software group.</li>
+
-
</ul>
+
-
</div>
+
-
<div class="lower">
+
-
<div class="week1" style="height: 150px;">
+
-
<div id="week">
+
-
Week 1</div>
+
-
<p>Workshop -1</p>
+
-
<p>[Biology basics, What is Synthetic Biology?, and the
+
-
works in this field ]</p>
+
-
</div>
+
-
<div class="week2" style="height: 150px;">
+
-
<div id="week">
+
-
Week 2</div>
+
-
<p>Workshop -2</p>
+
-
<p>[What is Synthetic Biology?, and the works in this field
+
-
]</p>
+
-
</div>
+
-
<div class="week3" style="height: 150px;">
+
-
<div id="week">
+
-
Week 3</div>
+
-
<p>Workshop -3</p>
+
-
<p>[iGEM, Parts, Biobricks, and Devices ]</p>
+
-
</div>
+
-
<div class="week4" style="height: 150px;">
+
-
<div id="week">
+
-
Week 4</div>
+
-
<p>Workshop – 4</p>
+
-
<p>[iGEM, Parts, Biobricks, and Devices ]</p>
+
-
</div>
+
-
</div>
+
-
<div class="month">
+
-
May</div>
+
-
<div class="general">
+
-
At this month we have completed our workshops, and as the masters
+
-
in this field started meeting with instructors. /
+
-
<ul>
+
-
<li>Meanwhile, looking for sponsors [We have designed a
+
-
document telling the iGEM, previous project and our project
+
-
generally and started to send it private companies who can
+
-
found us.] </li>
+
-
<li>This month it is turn to software group; they are transferring
+
-
the basics of software concepts to biologists in the team.
+
-
</li>
+
-
<li>Furthermore, we are discussing about how we can apply
+
-
the basics of computer engineering to synthetic biology
+
-
and iGEM parts. </li>
+
-
</ul>
+
-
</div>
+
-
<div class="lower">
+
-
<div class="week1" style="height: 180px;">
+
-
<div id="week">
+
-
Week 1</div>
+
-
<p>Meeting -1</p>
+
-
<p>[First, discussion on articles that have been selected
+
-
by consulters. Then, brain storming about the selected iGEM
+
-
projects from previous years and our project ]</p>
+
-
</div>
+
-
<div class="week2" style="height: 180px;">
+
-
<div id="week">
+
-
Week 2</div>
+
-
<p>Meeting -2[with consulters ]</p>
+
-
<p>[Tellingtheprevious projects to consulters and telling
+
-
our initial idea about project. Then, brainstorming about
+
-
our project. ]</p>
+
-
</div>
+
-
<div class="week3" style="height: 180px;">
+
-
<div id="week">
+
-
Week 3</div>
+
-
<p>Meeting -3</p>
+
-
<p>[Basic databaseconcepts and iGEM parts. ]</p>
+
-
</div>
+
-
<div class="week4" style="height: 180px;">
+
-
<div id="week">
+
-
Week 4</div>
+
-
<p>Meeting -4</p>
+
-
<p>[What is ER Diagram and How we can develop a database
+
-
model for iGEM parts with ER Model. ]</p>
+
-
</div>
+
-
</div>
+
-
<div class="month">
+
-
June</div>
+
-
<div class="general">
+
-
<p>At this month software group is going on to tell the basics
+
-
of software development and programming and computer engineering,
+
-
discussions about the computer engineering approaches are continuing.</p>
+
-
<ul>
+
-
<li>Furthermore, we have constructed the design group for
+
-
web page, poster, presentation, and an attractive animation
+
-
telling us.</li>
+
-
</ul>
+
-
</div>
+
-
<div class="lower">
+
-
<div class="week1" style="height: 250px;">
+
-
<div id="week">
+
-
Week 1</div>
+
-
<p>Meeting – 5</p>
+
-
<p>[Graph theory, Graph theoretic modeling, and graphical
+
-
modeling of iGEM parts. Using Input Output loops on iGEM
+
-
parts. ]</p>
+
-
</div>
+
-
<div class="week2" style="height: 250px;">
+
-
<div id="week">
+
-
Week 2</div>
+
-
<p>Meeting – 6 [ With Consulters ]</p>
+
-
<ul>
+
-
<li>Project Description. </li>
+
-
<li>Our tasks on holiday.</li>
+
-
<li>Telling our project and basic concepts to design
+
-
team.</li>
+
-
<li>Discussion on storyboard for animation.</li>
+
-
</ul>
+
-
</div>
+
-
<div style="height: 250px; background-color: #1e6dd4; padding: 10px;">
+
-
<div id="week">
+
-
Week 3 and Week 4</div>
+
-
<p align="center">HAVE a NICE HOLIDAY</p>
+
-
<p align="center">SEE YOU ON JULY 1 as a POWERFUL TEAM;
+
-
<em>“METU TURKEY SOFTWARE”</em>.</p>
+
-
</div>
+
-
</div>
+
-
<div class="month">
+
-
July</div>
+
-
<div class="general">
+
-
<p>At this month we have started to develop application. And
+
-
divided the team to 3 groups [ Software - Gene – Design ].</p>
+
-
<ul>
+
-
<li>Gene group is providing row data to software group by
+
-
extracting it from the part registery and other resources.</li>
+
-
<li>Software group is developing application.</li>
+
-
<li>Designers are learning new design tools, and applying
+
-
those to our project [Not all Members of the group working
+
-
actively for the team].</li>
+
-
</ul>
+
-
<p><u>NOTE: Members are not strictly assigned to a group; this
+
-
is just for organization of tasks.</u></p>
+
-
</div>
+
-
<div class="lower">
+
-
<div class="week1" style="height: 600px;">
+
-
<div id="week">
+
-
Week 1</div>
+
-
<p>Meeting-7</p>
+
-
<ul>
+
-
<li>Take stock for current situation.</li>
+
-
<li>Discussion on web, poster, animation design.</li>
+
-
<li>Discussion on storyboard for animation.</li>
+
-
<li>Task analyses for each group [ Software, Gene, Design
+
-
].</li>
+
-
</ul>
+
-
</div>
+
-
<div class="week2" style="height: 600px;">
+
-
<div id="week">
+
-
Week 2</div>
+
-
<p>Meeting – 8</p>
+
-
<p>Checking the tasks of each group. </p>
+
-
<p>· Software Group</p>
+
-
<ul>
+
-
<li>Database Design</li>
+
-
<li>Interface for DB.</li>
+
-
<li>Designing a basic SRS and SDD to be able to state
+
-
the requirements of application exactly .</li>
+
-
</ul>
+
-
<p>· Gene Group</p>
+
-
<ul>
+
-
<li>Extracting I/O information for each part in part
+
-
registry according to specified standards by Gene group.</li>
+
-
<li>Discussion about expectation from the software.
+
-
</li>
+
-
</ul>
+
-
<p>· Design Group</p>
+
-
<ul>
+
-
<li>Team Logo</li>
+
-
<li>Web site</li>
+
-
<li>Poster</li>
+
-
<li>Animation</li>
+
-
<li>Presentation</li>
+
-
</ul>
+
-
</div>
+
-
<div style="height: 600px; background-color: #1e6dd4; padding: 10px">
+
-
<div id="week">
+
-
Week 3 and Week 4
+
-
<p>DOING THE TASKS.</p>
+
-
</div>
+
-
</div>
+
-
</div>
+
-
<div class="month">
+
-
August</div>
+
-
<div class="general">
+
-
<p>At this month we have started to apply graph theory on iGEM
+
-
parts. We have specified nodes, edges, graph types. Furthermore,
+
-
we have started to develop a new “ Part Registery Form ” to
+
-
be able to more standardize the part entry to be able to apply
+
-
some algorithms on the parts more efficiently.</p>
+
-
</div>
+
-
<div class="lower">
+
-
<div class="week1" style="height: 350px;">
+
-
<div id="week">
+
-
Week 1</div>
+
-
<p>Meeting -8</p>
+
-
<p>/ </p>
+
-
<ul>
+
-
<li>Node data extraction algorithm.</li>
+
-
<li>Node description. </li>
+
-
<li>Visualization of nodes.</li>
+
-
<li>Pathway finding according to specified I/O properties.</li>
+
-
<li>Representing the nodes with original images.</li>
+
-
</ul>
+
-
</div>
+
-
<div class="week2" style="height: 350px;">
+
-
<div id="week">
+
-
Week 2</div>
+
-
<p>Meeting -9</p>
+
-
<ul>
+
-
<li>Whichone is node part or subparts or both are nodes
+
-
in different graphs?</li>
+
-
<li>Part Combination rules.</li>
+
-
<li>Web site, Poster Content</li>
+
-
<li>Animation storyboard. </li>
+
-
<li>Survey for new “Part Registery Standarts”.</li>
+
-
<li>NewPart Registery Form.</li>
+
-
</ul>
+
-
</div>
+
-
<div class="week3" style="height: 350px;">
+
-
<div id="week">
+
-
Week 3</div>
+
-
<p>Meeting -10</p>
+
-
<p>/ </p>
+
-
<ul>
+
-
<li>Extraction of Part Combination Rules </li>
+
-
<li>Web, poster, presentation contents generally.
+
-
</li>
+
-
<li></li>
+
-
</ul>
+
-
</div>
+
-
<div class="week4" style="height: 350px;">
+
-
<div id="week">
+
-
Week 4</div>
+
-
<p>Meeting -11</p>
+
-
<ul>
+
-
<li>USTC and Berkeley projects.[ https://2009.igem.org/Team:USTC_Software
+
-
and https://2009.igem.org/Team:Berkeley_Software </li>
+
-
<li>Graphical representation of node relations. </li>
+
-
<li>Part Combination Rules </li>
+
-
<li>Subpart Combinations Rules </li>
+
-
<li>Expectations from the software (SRS: Functional;
+
-
Requirements) </li>
+
-
</ul>
+
-
</div>
+
-
</div>
+
-
<div class="month">
+
-
September</div>
+
-
<div class="general">
+
-
At this time all bases for application were nearly to finish,
+
-
the software group was waiting row data from the gene group.
+
-
Meanwhile, they were working on code bases.</div>
+
-
<div class="lower">
+
-
<div class="week1" style="height: 350px;">
+
-
<div id="week">
+
-
Week 1 </div>
+
-
<p>DOING THE TASKS. </p>
+
-
</div>
+
-
<div class="week2" style="height: 350px;">
+
-
<div id="week">
+
-
Week 2</div>
+
-
<p>Meeting -12</p>
+
-
<ul>
+
-
<li>Final Database </li>
+
-
<li>Final Graphs </li>
+
-
<li>GUI </li>
+
-
<li>Expectations from the software (SRS: Functional
+
-
Requirements) (Suggestions)</li>
+
-
<li>Survey details</li>
+
-
</ul>
+
-
</div>
+
-
<div class="week3" style="height: 350px;">
+
-
<div id="week">
+
-
Week 3</div>
+
-
<p>Meeting -13</p>
+
-
<ul>
+
-
<li>Animation (Storyboard, timeline) </li>
+
-
<li>Web site (Suggestions to web site) </li>
+
-
<li>Poster (How we can use 3D stereoscopic image, How
+
-
we can tell the development progress and our concepts
+
-
by 3D effect etc...) </li>
+
-
<li>Presentation ( Suggestions about presentation )</li>
+
-
</ul>
+
-
</div>
+
-
<div class="week4" style="height: 350px;">
+
-
<div id="week">
+
-
Week 4</div>
+
-
<p>Meeting -14</p>
+
-
<ul>
+
-
<li>Final Graphs </li>
+
-
<li>GUI ( about 70 % is over ) </li>
+
-
<li>How to send the software to other teams for collaboration
+
-
( just general ideas, details will be talked later ).
+
-
</li>
+
-
<li>With survey or not , can it shade the software?
+
-
</li>
+
-
<li>What to ask to teams while sending?</li>
+
-
</ul>
+
-
</div>
+
-
</div>
+
-
<div class="month">
+
-
October</div>
+
-
<div class="general">
+
-
<p>Now everything is nearly over, it is time to put everything
+
-
together.</p>
+
-
<ul>
+
-
<li>Gene group explaining the methods that we have used
+
-
during project.</li>
+
-
<li>Software group finalizing, software, testing it importing
+
-
new function according to collaboration results with METU
+
-
TURKEY wet lab team., trying to solve infinite bugs…</li>
+
-
<li>Design group putting all together…</li>
+
-
<li>Meanwhile, all team is writing the content for web,
+
-
poster, and presentation.</li>
+
-
</ul>
+
-
</div>
+
-
<div class="lower">
+
-
<div class="week1" style="height: 170px;">
+
-
<div id="week">
+
-
Week 1</div>
+
-
<p>Meeting -15</p>
+
-
<ul>
+
-
<li>BioGuide Application, missing points.</li>
+
-
<li>Content writing</li>
+
-
<li>Web, poster, animation</li>
+
-
</ul>
+
-
</div>
+
-
<div style="float: left; width: 540px; height: 170px; font-weight: normal; background-color: #3fff72; padding: 10px;">
+
-
<div id="week">
+
-
Week 2, Week 3, &amp; Week 4
+
-
<p>GOOD NEWS Infinite meetings started :) </p>
+
-
<ul>
+
-
<li>Writing content [shared it, tasks assigned to
+
-
members according to their fields.]</li>
+
-
<li>Software; debugging, testing, adding functions…</li>
+
-
<li>Designers web, poster, presentation, animation,
+
-
importing content…</li>
+
-
</ul>
+
-
</div>
+
-
</div>
+
-
</div>
+
-
<div class="month">
+
-
November</div>
+
-
<div class="general">
+
-
<p align="center">November 1 – 12 is our ticket dates,</p>
+
-
<p align="center">See you in BOSTON …</p>
+
-
<p align="center">METU TURKEY SOFTWARE</p>
+
-
</div>
+
-
</div>
+
-
</div><h3>Follow our discussion topic at <a href="http://groups.google.com.tr/group/software_MetuTurkey/topics">our googlegroups</a></h3>
+
 +
<br />
 +
<center><iframe width="560" height="315" src="http://www.youtube.com/embed/XAkuG4Xjp2A" frameborder="0" allowfullscreen></iframe></center>
 +
<p>Methane explosions called as grisou are one of  the main problems from the beginning of the coal mines.  All countries have tried different systems to solve this methane problem. When we joined iGEM 2011 competition, our aim is to find a solution to that problem for humanity by cheaper and energy saving way. After a long literature research, we found how seriously dangerous the methane gas is and how people deal with this big problem on coal mines. Below are the countries that have faced with serious methane explosions throughout their history.</p>
 +
<ol>
 +
 +
<li><a href="http://en.wikipedia.org/wiki/Mining_accident#Australia"      target="_blank">Australia</a></li>
 +
<li><a href="http://en.wikipedia.org/wiki/Mining_accident#Belgium"        target="_blank">Belgium</a></li>
 +
<li><a href="http://en.wikipedia.org/wiki/Mining_accident#Canada"        target="_blank">Canada</a></li>
 +
<li><a href="http://en.wikipedia.org/wiki/Mining_accident#Chile"          target="_blank">Chile</a></li>
 +
<li><a href="http://en.wikipedia.org/wiki/Mining_accident#China"          target="_blank">China</a></li>
 +
<li><a href="http://en.wikipedia.org/wiki/Mining_accident#France"        target="_blank">France</a></li>
 +
<li><a href="http://en.wikipedia.org/wiki/Mining_accident#New Zealand"    target="_blank">New Zealand</a></li>
 +
<li><a href="http://en.wikipedia.org/wiki/Mining_accident#Poland"        target="_blank">Poland</a></li>
 +
<li><a href="http://en.wikipedia.org/wiki/Mining_accident#Russia"        target="_blank">Russia</a></li>
 +
<li><a href="http://en.wikipedia.org/wiki/Mining_accident#United Kingdom" target="_blank">Australia</a></li>
 +
<li><a href="http://en.wikipedia.org/wiki/Mining_accident#United States"  target="_blank">United States</a></li>
 +
 +
</ol>
 +
<p>Todays companies deal with this problem by;
 +
<br /><i>Regenerative Thermal Oxidizer from MEGTEC capable of handling 60,000 scfm of VAM. At a methane concentration of about 0.7% a unit this size generates 50,000 carbon credits per year. This unit costs about US$ 1,5 million
 +
Elizabeth Obediente from CO2IMPACT at a methane upgrading facility at a coal mine in Alabama. The gas is stripped of impurities and upgraded to 96% methane per volume and then compressed and then sold to the natural gas pipeline.</i>
 +
<b>Ventilation Air Methane (VAM):</b> VAM units cost about $20-$30 USD per scfm.</p>
 +
<p>At some mines, downtime is considerable, causing expensive losses to the mine. Ventilation cost cuts can be up to half of the original cost of the ventilation system. In one study, a mine with 400 ft3/ton of methane using vertical wells to pre-drain will result in an estimated US$ 11 million over 20 years in reduced ventilation costs, not including carbon and energy revenue[iii] (U.S. Environmental Protection Agency, 1999).
 +
Like many solution system for methane problem, the tree examples of solution of methane problem did not satisfied us, so we created our simple and very effective way to deal with the methane.( <a href="http://www.co2impact.com/cmm.html"  target="_blank">http://www.co2impact.com/cmm.html</a> )</p>
 +
<p><b>According to our methane E colic project, we aimed:</b>
 +
<ul type="disc">
 +
            <li>Save the coal workers’ life</li>
 +
            <li>Save the nature</li>
 +
            <li>Process a new energy</li>
 +
            <li>Obtain a cheap system</li>
 +
</ul></p>
 +
                                <h3><u>Save the coal workers’ life</u></h3>
 +
 +
<p>We aimed to collect the methane gas from coal mines to the tanks which are filled with water. When the methane passes the legal percent that is 1%, according to our project, the gas is dissolved in water in tanks. Then, inside the tablets containing our bacteria and that are already present on the tanks the molecular process for conversion of methane to methanol begins.
 +
Below the number of workers dead by years are given according to Prof. Dr. Mustafa ÖZTÜRK’s research,Vice President of Parliamentary Commission for the Environment in Turkey:
 +
 +
<br />
 +
<br />
 +
 +
          <center> <table border="1" cellspacing=”2″>
 +
 +
    <tr>
 +
<th width="100">Years</th>
 +
<th width="300">Death workers on Turkey</th>
 +
    </tr>
 +
    <tbody>
 +
    <tr>
 +
<th >1983</th>
 +
<th>103</th>
 +
    </tr>
 +
    <tr>
 +
<th >1990</th>
 +
<th>330</th>
 +
    </tr>
 +
    <tr>
 +
<th >1995</th>
 +
<th>40</th>
 +
    </tr>
 +
    <tr>
 +
<th >2003</th>
 +
<th>17</th>
 +
    </tr>
 +
    <tr>
 +
<th >2004</th>
 +
<th>3</th>
 +
    </tr>
 +
    <tr>
 +
<th >2005</th>
 +
<th>18</th>
 +
    </tr>
 +
    <tr>
 +
<th >2006</th>
 +
<th>21</th>
 +
    </tr>
 +
    <tr>
 +
<th >2007</th>
 +
<th>7</th>
 +
    </tr>
 +
    <tr>
 +
<th >2008</th>
 +
<th>43</th>
 +
    </tr>
 +
  </tbody>
 +
          </table></center>
 +
<br />
 +
<center><h3>Methane explosions: </h3>
 +
<iframe width="420" height="315" src="http://www.youtube.com/embed/25BE42PzZZc" frameborder="0" allowfullscreen></iframe></center>
 +
 +
 +
                                        <h3><u>Save the nature</u></h3>
 +
 +
<p>Methane is one of the greenhouse gases in the atmosphere. Methane is continuously released from coal mining, livestock, landfills and natural gases to the atmosphere. The accumulation of these greenhouse gases causes one of the greatest problems of Earth: global warming. Our project aims to prevent release of methane gas by the conversion of it to methanol in bacterial system in coal mines. </p>
 +
<center><h3>Greenhouse gases: </h3>
 +
<iframe width="420" height="315" src="http://www.youtube.com/embed/tm-zZMiL58A" frameborder="0" allowfullscreen></iframe>
 +
</center>
 +
       
 +
 +
                                        <h3><u>Process a new energy</u></h3>
 +
 +
<p>After the conversion of methane to methanol, methanol can be used as a precursor of bio-fuel so that accumulated methanol can be recovered. Ethanol is used as biofuel, also; however, methanol is more cheaper to obtain and use as a biofuel. According to Dr. Robert Zubrin, Methanol is cheaper than ethanol. It can also be made from a broader variety of biomass material, as well as from coal and natural gas. And methanol is the safest motor fuel.(American enterprise, February 13th, 2006).</p>
 +
 +
 +
                                   
 +
                                  <h3><u>Cheap system</u></h3>
 +
 +
<p>There are many systems used to prevent or minimize methane explosions. However, they are so expensive to use and none of them are based on a biological system. The biological system that we build contains a bacterial cell having a molecular process of conversion of methane to methanol is completely harmless and cheaper since expensive equipments are not needed.</p>
 +
       
 +
 +
 +
 +
</div>
</div>
</div>
</div>
</div>
-
<div id="download" class="item">
+
<div id="partnership" class="item">
<div class="content2">
<div class="content2">
<div class="text">
<div class="text">
-
<h2>Download Executable and Code</h2>
+
<img src="https://static.igem.org/mediawiki/2011/f/f8/Partnershipheader.png" height="75px" width="890px">
-
<p>You can download BioGUIDE at <a href="http://sourceforge.net/projects/bioguide">http://sourceforge.net/projects/bioguide</a></p>
+
<h2>PartnerShip</h2>
-
<p>Download & install it and feel it's power!!</p>
+
<ul>                       
 +
                        <li><a href="#partnership2">Collaboration</a></li>
 +
                        <li><a href="#partnership3">Sponsors</a></li>
 +
                        <li><a href="#partnership4">METU</a></li>
 +
                        <li><a href="#partnership5">Acknowledgements</a></li>
 +
                        <ul>                        
</div>
</div>
</div>
</div>
</div>
</div>
-
<div id="miscellaneous" class="item">
+
<div id="extras" class="item">
<div class="content2">
<div class="content2">
<div class="text">
<div class="text">
-
<h2>Collaboration</h2>
+
<img src="https://static.igem.org/mediawiki/2011/f/f4/Extraheader.png" height="75px" width="890px">
-
<h3>Part Registry Survey</h3>
+
                <h2>Extras</h2>
-
<p>
+
-
<a href="http://www.kwiksurveys.com/online-survey.php?surveyID=HIMIGG_469f28c4&amp;ooo=HKDDJHO_95dcbe36">
+
<br />
-
Click here to go to survey page..</a></p>
+
<br />
-
<br>
+
<center><p><img src="https://static.igem.org/mediawiki/2011/7/70/COLLY.jpg"></p><center>
-
<h3>Results</h3>
+
<br />
-
<p>(including responses between 10th -22nd of Oct 2010)</p>
+
<br />
-
<h4>General Profile of Participants</h4>
+
<div id="slider2">
-
<ul>
+
<img src="https://static.igem.org/mediawiki/2011/d/d7/IMG_0788.JPG">  
-
<li>The following teams are dedicated as collaborators with more
+
<img src="https://static.igem.org/mediawiki/2011/c/c9/IMG_0793.JPG">  
-
than 60% team participation are </li>
+
<img src="https://static.igem.org/mediawiki/2011/f/fd/DSCN2037.JPG">  
-
<ul>
+
<img src="https://static.igem.org/mediawiki/2011/4/45/DSCN1102.JPG">  
-
<li>INSA-Lyon</li>
+
<img src="https://static.igem.org/mediawiki/2011/1/1a/IMG_0794.JPG">  
-
<li>Lethbridge</li>
+
<img src="https://static.igem.org/mediawiki/2011/9/9f/IMG_0148.JPG">  
-
<li>WashU</li>
+
<img src="https://static.igem.org/mediawiki/2011/2/2a/IMG_0796.JPG">
-
</ul>
+
<img src="https://static.igem.org/mediawiki/2011/d/d0/DSCN1905.JPG">
-
<li>Out of 244 participants between 10 to 22.10.2010, 57% of the
+
<img src="https://static.igem.org/mediawiki/2011/8/84/DSCN1786.JPG">
-
participant had scientific degrees from B.Sc to Professor and 18%
+
<img src="https://static.igem.org/mediawiki/2011/1/12/DSCN1797.JPG">
-
had graduate degrees. 18% of participants are enrolled in their
+
<img src="https://static.igem.org/mediawiki/2011/a/af/DSCN1812.JPG">
-
teams as either Instructors or Advisors. </li>
+
<img src="https://static.igem.org/mediawiki/2011/3/31/DSCN1895.JPG">
-
<li>95 teams have responded to the survey while we are still waiting
+
<img src="https://static.igem.org/mediawiki/2011/9/90/DSCN1917.JPG">
-
to hear from 33 teams. 74% teams participated with one or more members
+
<img src="https://static.igem.org/mediawiki/2011/d/d4/IMG_0210.JPG">
-
to the survey. </li>
+
<img src="https://static.igem.org/mediawiki/2011/3/34/DSCN1818.JPG">
-
<li>75% of participants were interested with synthetic biology field
+
</div>
-
for academic purposes.</li>
+
<br />
-
</ul>
+
<br />
-
<h4>Browsing the Registry of Standard Parts </h4>
+
<center><h3>Hands on show!</h3></center>
-
<ul>
+
<center><iframe width="560" height="315" src="http://www.youtube.com/embed/_b50ti5L1_M" frameborder="0" allowfullscreen></iframe></center>
-
<li><strong>56% of participants think that it is not easy to search
+
<br />
-
for the parts in Registry of Standard Parts.</strong> Many comments
+
<br />
-
indicate a need for a better search engine and more flexible keyword
+
<p><div class="extra"><iframe width="646px" height="350px" src="http://www.metu.edu.tr/~e172018/metu-bin/self-evaluation.html"></iframe></div></p>
-
search options, especially excepting aliases. Also many are longing
+
<br />
-
for recognizable parts names, which will ease searching with keyword.
+
<br />
-
</li>
+
<center><h3>Photos</h3>
-
</ul>
+
<br />
-
<p><i>Partnership with Google and enforcing standardized parts names
+
<br />
-
are suggested </i></p>
+
<p><img src="https://static.igem.org/mediawiki/2011/2/26/Background.png"></p>
-
<p><i>As a global organization iGEM can offer the Parts Registry in
+
<p><img  src="https://static.igem.org/mediawiki/2011/d/df/Bannergiris2.png"></p>
-
different languages and more illustrations describing how the system
+
<p><img src="https://static.igem.org/mediawiki/2011/d/d1/Bannergiris.png"></p>
-
works.</i></p>
+
<p><img src="https://static.igem.org/mediawiki/2011/3/3f/Bioguide.png"></p>
-
<h4>Content of Registry of Standard Parts </h4>
+
<p><img src="https://static.igem.org/mediawiki/2011/4/4a/Labguardrulez_copy.jpg"></p>
-
<ul>
+
<p><img src="https://static.igem.org/mediawiki/2011/4/45/Methanecanavar%C4%B1.jpg"></p>
-
<li><strong>57% of participants agree that the number of parts registered
+
-
in the Registry of Standard Parts is not enough for their projects.</strong>
+
-
</li>
+
-
<li><strong>55% of participants think that there are enough and
+
-
useful parts distributed in iGEM Plates that we can use in our projects.
+
-
</strong></li>
+
-
</ul>
+
-
<p>Even though most agree the number of parts in the registry is impressive,
+
-
still they find it limited when it comes to design different devices
+
-
for diverse applications especially in different species other than
+
-
E. Coli. Participants believe that if there are more functional standardized
+
-
parts, especially protein coding sequences and promoter-RBS , they can
+
-
design devices according to the needs of the community instead of designing
+
-
what can simply be assembled into a device. </p>
+
-
<p><i>Encouraging development of vectors and standards for new species
+
-
and new standardized parts in different research areas is suggested.
+
-
</i></p>
+
-
<p><i>Enforcing submission of right DNA sequences and working conditions
+
-
for each part is suggested.</i> </p>
+
-
<p><i>Few recommend expanding iGEM into a collaborative effort rather
+
-
than an undergraduate tournament, which will increase the number and
+
-
the diversity of the parts designed and submitted all throughout the
+
-
year. </i></p>
+
-
<h4>Submission to the Parts Registry </h4>
+
-
<ul>
+
-
<li><strong>52% of participants said that they have not encountered
+
-
difficulties during submitting parts.</strong> Even though participants
+
-
are satisfied with the web interface of the registry, most complains
+
-
about the pSB1C3 as the new standard plasmid to submit DNA. </li>
+
-
<li><strong>71% of participants are like minded with our team&#39;s
+
-
opinion, which is that The nomenclature of part IDs such as construct,
+
-
device, composite parts, protein generator, is confusing as there
+
-
is no consensus on how to use them correctly.</strong> </li>
+
-
</ul>
+
-
<p><i>Terminology and categorization used on iGEM’s Parts Registry should
+
-
be re-described and correct use of terminology should be enforced during
+
-
the submission process.</i> </p>
+
-
<ul>
+
-
<li><strong>75% of participants agree that different, specified
+
-
submission interfaces should be designed for contructs, promoter,
+
-
RBS, CDS and terminals is needed during Registry of Standard Parts.</strong>  
+
-
But, there are very strong and valid arguments against it such as,
+
-
losing the flexibility of the registry will not allow future submission
+
-
of unclassified parts. </li>
+
-
</ul>
+
-
<p><i>We suggest keeping the parts submission interface as is, until
+
-
these concerns are addressed.</i> </p>
+
-
<ul>
+
-
<li><strong>75% of participants agree that Out-dated, un-available
+
-
and not-characterized parts in the Registry of Standard Parts should
+
-
be removed to an archive after the consent of the designer.</strong>
+
-
</li>
+
-
</ul>
+
-
<p>“It would be great to see some sort of organization like this! I
+
-
agree that unavailable parts should be followed up on and removed if
+
-
necessary. I also think that parts which are not sufficiently documented
+
-
should be highlighted in some way. Once these parts are identified,
+
-
teams can actively characterize them as part of their projects or as
+
-
side projects.” </p>
+
-
<p>“Think about these things: (i) who decides when a part is out-dated,
+
-
and how can that person know that an old part cannot have a novel use
+
-
in the future? (ii) likewise, an uncharacterized part may be both characterized
+
-
and used in the future” </p>
+
-
<p><i>We suggest building a backup system, such as an archive, to sort
+
-
out the rarely used, un-available and un-categorized parts until they
+
-
are in line with the enforced standards.</i> </p>
+
-
<ul>
+
-
<li><strong>91% of participants have same opinion with us, which
+
-
is that standardization of the nomenclatures used for each different
+
-
composition of parts is necessary.</strong></li>
+
-
</ul>
+
-
<h4>Standards that should be enforced and Additional New Standards
+
-
</h4>
+
-
<p>According to our survey, from high rated to low, these standards
+
-
have been rated which has been used while assigning a name to parts
+
-
</p>
+
-
<ul>
+
-
<li><strong>33% Type of part</strong> </li>
+
-
<li><strong>17% Input</strong> </li>
+
-
<li><strong>17% Output</strong> </li>
+
-
<li><strong>14% Version</strong> </li>
+
-
<li><strong>10% Year</strong> </li>
+
-
<li><strong>9% Group</strong> </li>
+
-
</ul>
+
-
<p>Along with above, having short recognizable part names along with
+
-
function and performance , Genbank/EMBL link and organism information
+
-
is important. </p>
+
-
<ul>
+
-
<li><strong>93% of participants have said that for the parts that
+
-
are marked as “WORKS” distinguishing the parts with quantitative
+
-
experimental validation vs parts without this information is important.</strong>
+
-
Most participants have encountered with similar problems about parts
+
-
that don’t work under their lab conditions or works but not they
+
-
were claimed for. </li>
+
-
<li><strong>89% of participants have same opinion with us, which
+
-
is that iGEM should sub-categorize the “WORKS” comment into 1) “Quantitative”
+
-
for parts which are characterized with experiments and 2) “Qualitative”
+
-
for parts which are not characterized will be an appropriate measure
+
-
for standardization of Biobrick database.</strong> </li>
+
-
</ul>
+
-
<p><i>In order to overcome these problems we suggest enforcing the working
+
-
conditions title for the registry entrance, in order to collect quantitative
+
-
experimental details on submitted parts, which might slow down the registration
+
-
process but will definitely increase the quality of the database.</i>
+
-
</p>
+
-
<ul>
+
-
<li><strong>61% of participants agree that POPS (Polymerase Per
+
-
Second) should be assigned to every part or biobricks with a promoter,
+
-
where appropriate. - 57% of participants have been agree that RIPS
+
-
(Ribosome per Second) should be assigned to every part or biobricks
+
-
with a RBS brick.</strong> </li>
+
-
</ul>
+
-
<p>Though most participants agree the need for POPS and RBS information
+
-
, they are concerned about the workload it would bring to individual
+
-
labs. </p>
+
-
<p>“To do this, the Registry need to define a reliable and easy method
+
-
of determining the PoPS for teams to use. However, I would say that
+
-
there are better systems for quantifying promoter output than PoPS,
+
-
and they should be used instead, if possible”. </p>
+
-
<ul>
+
-
<li><strong>67% of participants have thought that entering POPS
+
-
information should not be mandatory while submitting new parts.  
+
-
Similarly, 65% of participants disagree that entering RIBS information
+
-
should be mandatory while submitting new parts </strong></li>
+
-
</ul>
+
-
<p>Even though the researchers feeling the need for this information
+
-
they are shying away from requesting it as a mandatory title for parts
+
-
registry as it would be difficult for underfunded and inexperienced
+
-
groups to perform these measurements. </p>
+
-
<p><i>We strongly suggest starting a forum on how to quantify the performance
+
-
of promoters and genes to bring an easy to measure standard for the
+
-
efficiency of the parts. Additionally iGEM should the responsibility
+
-
and provide the measurements for the each promoter and gene included
+
-
in the distributions. The second choice would be even better in terms
+
-
of standardization as all the measurement will be performed by one center
+
-
under similar conditions and with experienced researchers, which will
+
-
allow user to compare and contrast the efficiencies of the parts more
+
-
accurately. </i></p>
+
-
<ul>
+
-
<li><strong>82% of participants have thought that information on
+
-
working conditions of the parts should be mandatory while submitting
+
-
new parts.</strong> Most find submiting the detailed experimental
+
-
information and working conditions is crucial and even easier than
+
-
submitting measurements of POPS or RBS. </li>
+
-
</ul>
+
-
<h4>Definitions you would like to see at the Registry of Standard Parts
+
-
</h4>
+
-
<ul>
+
-
<li><strong>Transcriptional efficiency 13%</strong> </li>
+
-
<li><strong>Protein lifetime 10%</strong> </li>
+
-
<li><strong>Ribosome binding efficiency 10%</strong> </li>
+
-
<li><strong>mRNA lifetime 9%</strong> </li>
+
-
<li><strong>Translation initiation and efficiency 9%</strong>
+
-
</li>
+
-
<li><strong>Protein concentration 9%</strong> </li>
+
-
<li><strong>Cooperative effects with other molecules 9%</strong>
+
-
</li>
+
-
<li><strong>Protein-DNA binding rates and efficiencies 8%</strong>
+
-
</li>
+
-
<li><strong>RNA polymerase affects 8% </strong></li>
+
-
<li><strong>System copy count 8%</strong> </li>
+
-
<li><strong>Protein multimerization 6%</strong> </li>
+
-
</ul>
+
-
<p>Additional titles includes: Catalytic rates and affinities for substrates,
+
-
leakiness of promoter in lack of stimulus, POPS at various inducer/repressor
+
-
concentrations. </p>
+
-
<h4>Efficiency of the Database Entries </h4>
+
-
<ul>
+
-
<li><strong>86% of participants would like to see a ranking/rating
+
-
system for the parts by the other iGEM users which will be one indication
+
-
of if a part is working and how well in different laboratories.</strong>
+
-
Few had concerns about how well the rating system will work for
+
-
rarely used parts while the widely used parts would even more popular
+
-
due the the rating system. Still many believes this would be one
+
-
futher towards a peer-reviewed quality control system for the parts.
+
-
</li>
+
-
<li><strong>61% of participants agreed that parts should be updated
+
-
regularly by the designers, where most agreed at least when there
+
-
is new information on the parts.</strong> It has also been suggested
+
-
to give permission to all the users of that part for updating information.
+
-
</li>
+
-
<li><strong>73% of participants have been agree with us that excluding
+
-
the low ranking parts or the parts with negative feedback from the
+
-
future plates will increase efficiency of the system.</strong> The
+
-
major concern about excluding any part is losing the variety of
+
-
parts in the database. Few recommends excluding only the parts that
+
-
are not working. </li>
+
-
</ul>
+
-
<p>“Efficiency shouldn&#39;t be top priority in a database. First and foremost,
+
-
data is the top priority. Excluding those parts would make the system
+
-
more efficient” </p>
+
-
<p>“Some parts may be rare or new and have low efficiency, but can be
+
-
very important! Getting rid of them would eliminate any chance of improvement
+
-
to these parts, which not only a qualifier for an iGEM gold medal, but
+
-
also one of the focuses of biobricks.” </p>
+
-
<p><i>We suggest excluding the parts not-working, low rated or with
+
-
negative feedbacks from the annual distribution plates but still archive
+
-
them and make their data available through the parts registry. So the
+
-
while the individuals labs are receiving plates with higher rated, fully
+
-
working parts for their projects, anyone who wants to work on a more
+
-
exotic part can search through the achieves and re-vitalize the parts
+
-
stored there. The challenge of re-vitalization of parts can be encouraged
+
-
as an collaborative effort.</i> </p>
+
-
<h4>New Options for the Parts Registry Database </h4>
+
-
<ul>
+
-
<li><strong>96% of participants are like minded with us that it
+
-
will be useful to have a link out to the gene/protein information
+
-
of the parts and - %97 of participants have been agree that they
+
-
would like to know if a part is also involved in known biological
+
-
pathways.</strong> </li>
+
-
</ul>
+
-
<p><strong>For receiving pathway information more participants have
+
-
voted for NCBI Cog (59%) than KEGG pathways (38%) when the responses
+
-
for both has been distributed among the choices according to response
+
-
rates.</strong> Adding the blast option to the parts registry has also
+
-
been suggested to locate parts of interest. We are sure all of us would
+
-
like to see gene-protein and pathway information if these information
+
-
was integrated into the database and offered automatically for each
+
-
entry in the database.</p>
+
-
<p><i>We are planning to provide this information about the parts to
+
-
all parts registry users as a build-in option in the next version of
+
-
BioGuide in iGEM 2011. </i></p>
+
-
<br>
+
-
<h2>New Parts Registry Form Suggested for The New Standards</h2>
+
-
<img src="https://static.igem.org/mediawiki/2010/1/17/Metu-form1.png">
+
-
<h3>Description</h3>
+
-
<p>Warning Boxes: </p>
+
-
<ul>
+
-
<li>If Out-dated, un-available and not-characterized parts exist
+
-
in the Registry of Standard Parts, bring to an archive after the
+
-
consent of the designer. Divide archive into three title: Out-dated,
+
-
un-available and not-characterized parts </li>
+
-
<li>Besides shown as “works”, in the works box there should be explanation
+
-
whether the part is characterized or non-characterized. </li>
+
-
<li>Parts should be updated regularly by the designers </li>
+
-
<li>Excluding the low ranking parts or the parts with negative feedback
+
-
from the future plates </li>
+
-
</ul>
+
-
<p>Characterization Boxes: </p>
+
-
<ul>
+
-
<li>transcriptional efficiency </li>
+
-
<li>mRNA lifetime </li>
+
-
<li>ribosome binding efficiency </li>
+
-
<li>translation initiation and efficiency </li>
+
-
<li>protein lifetime </li>
+
-
<li>protein concentration </li>
+
-
<li>protein multimerization </li>
+
-
<li>protein-DNA binding rates and efficiencies </li>
+
-
<li>cooperative effects with other molecules </li>
+
-
<li>RNA polymerase effects </li>
+
-
<li>system copy count </li>
+
-
</ul>
+
-
<img src="https://static.igem.org/mediawiki/2010/9/91/Metu-form2.png">
+
-
<h3>Desription</h3>
+
-
<p>Search box </p>
+
-
<ul>
+
-
<li>with click options </li>
+
-
<li>options: searched parts are:
+
-
<ul>
+
-
<li>Available </li>
+
-
<li>Length OK </li>
+
-
<li>Building </li>
+
-
<li>Planning </li>
+
-
<li>Missing </li>
+
-
<li>Unavailable </li>
+
-
</ul>
+
-
</li>
+
-
</ul>
+
-
<p>according to the clicks of above options, search is modified</p>
+
-
<img src="https://static.igem.org/mediawiki/2010/5/54/Metu-form3.png">
+
-
<img src="https://static.igem.org/mediawiki/2010/1/1d/Metu-form4.png">
+
-
<h3>Description</h3>
+
-
<p>Assume on the part image; </p>
+
-
<ul>
+
-
<li>part DNA sequence is not confirmed, then tag with &quot;non-confirmed
+
-
DNA sequence&quot; </li>
+
-
<li>non-characterized parts in the Parts Registry are not characterized
+
-
further, then it will be tagged as &quot;deprecated&quot; </li>
+
-
</ul>
+
-
<p>also: </p>
+
-
<ul>
+
-
<li>comment box stated that any team can make comment about experiences
+
-
with the part is opened </li>
+
-
<li>boxes which had been not filled with the data are highlighted;
+
-
<ul>
+
-
<li>transcriptional efficiency </li>
+
-
<li>mRNA lifetime </li>
+
-
<li>ribosome binding efficiency </li>
+
-
<li>translation initiation and efficiency </li>
+
-
<li>protein lifetime </li>
+
-
<li>protein concentration </li>
+
-
<li>protein multimerization </li>
+
-
<li>protein-DNA binding rates and efficiencies </li>
+
-
<li>cooperative effects with other molecules </li>
+
-
<li>RNA polymerase effects </li>
+
-
<li>system copy count </li>
+
-
</ul>
+
-
</li>
+
-
<li>if the part is not characterized but &quot;works&quot; then a &quot;Qualitative
+
-
part&quot; tag is added </li>
+
-
<li>besides &quot;works&quot;, &quot;Characterized&quot; or &quot;non-characterized&quot; box
+
-
is added </li>
+
-
<li>ranking/rating stars for the parts voted by the other iGEM users
+
-
which indicate how well the parts perform in different laboratories
+
-
is added. For example 4.5 star voted by 27 teams (number of stars
+
-
and number of votes) </li>
+
-
</ul>
+
</div>
</div>
</div>
</div>
</div>
</div>
 +
 +
 +
<div class="clear">
<div class="clear">
</div>
</div>
Line 1,227: Line 895:
<div class="item">
<div class="item">
</div>
</div>
-
<div class="item">
+
<div id="project2" class="item">
 +
<div class="content2">
 +
<div class="text">
 +
<img src="https://static.igem.org/mediawiki/2011/1/1c/Projectheader.png" height="75px" width="890px">
 +
<h2>Methane sensing</h2>
 +
<center><p><img src="https://static.igem.org/mediawiki/2011/1/10/Sensingesas.gif" height="454px" width="640px"></center></p>
 +
                          <h3>i) Introduction </h3>
 +
        <p>In our methanE.COLIc project, the first critical point is to sense the ambient methane gas. While in literature search, we have focused on the methane and DNA or methane and  regulator protein  interaction which binds to DNA after binding to methane. In structure, methane consists of one carbon atom  and four hydrogen atom which means it is smaller in size and weight. Therefore it is hard to find any study based on methane interaction with DNA or protein to regulate transcription. This made us search for carbon hydrogen bond interaction with any oligo or protein to trigger transcription of any metabolite product. So that we also searched the literature for any organism which utilizes  any alkane chain and alkane which regulates the transcription of organismal metabolites by  interaction with DNA or protein. Since bacteria are environmental adapting organisms, it is possible to find any alkane to regulate the transcription of degradation metabolism. Then we have found the organism, <i>Pseudomonas oleovorans</i> to analyze the alkane degradation  of organism for its carbon source.</p>
 +
                         
 +
                            <h3>ii) Background </h3>
 +
<p> Many microorganisms live in the environments where the conditions changing frequently and so the evolution is inevitable for mechanisms to withstand unfavorable situations.  Therefore microorganisms can use their specific and sensitive mechanisms for sensing the required nutrients for them or any pollution to affect their sustainability. Otherwise they can expose to mutations which change the gene expression and gain new functionalities.  In case they have ability to survive in such conditions. </p>
 +
<p>While we were scanning the literature based on methane and alkane degrading organisms we have found some organisms that sensitive to methane presence and have mechanisms to activate transcription of related gene clusters.</p>
 +
<p>We analyzed the soil bacteria, <i>Pseudomonas oleovorans</i>. This strain can assimilate the alkane for its carbon source and one of the microbial whole cell –biosensor.  They have a gene cluster which codes for degradative pathway and includes the activator which interacts especially with linear alkanes. This activator protein, AlkS, in the presence of alkanes, induces the transcription from PalkB promoter which initiates the expression of genes code for assimilation of alkanes. We have analyzed the related articles in which the promoter region  is studied and showed that it is expressed in <i>E.coli</i> correctly because of the corresponding RNA polymerase binding regions.</p>
 +
 
 +
<b><p>REFERENCE:</p></b>
 +
<ol>
 +
<li>Canosa, I. & Yuste, L. (2000). A positive feedback mechanism controls expression of AlkS, the transcriptional regulator of the Pseudomonas oleovorans alkane degradation pathway.<i> Molecular Microbiology</i>, 35 (4), 791-799.</li>
 +
 
 +
</ol>
 +
 
 +
 +
                          <h3>iii) Modelling </h3>
 +
<p> Since the parts that we have found belongs to yeast organism, it was hard to us to manipulate it in bacteria <i>E.coli</i>. We designed the tests of this part by characterizing one of the subunits of methane monooxgenase (MMo). For the functional activity of monooxygenase enzyme, there are required 3 subunits as A,B and C. The A subunit of methane monooxygenase is 210 kDa protein that is supposed as the methane binding active site of monoxygenase complex. This protein consists of non-haem iron and contains p-hydroxo bridge structure. We planned the experiments of this step as characterization of protein A by PCR amplification with specific primers of full construct device to obtained the sequence of protein A. The methane gas is applied to cell culture with our modelled device then the cells were centrifuged and the gas concentration is measured from supernatant.</p>
 +
 
 +
 
 +
 
 +
 
 +
           
 +
 
 +
              </div>
 +
</div>
</div>
</div>
<div class="item">
<div class="item">
</div>
</div>
-
<div class="item">
+
<div id="wetlab2" class="item">
 +
<div class="content2">
 +
<div class="text">
 +
<img src="https://static.igem.org/mediawiki/2011/2/28/Wetlabheader.png" height="75px" width="890px">
 +
 +
 
 +
<h3>Clonning procedures</h3>
 +
<ol type="1">
 +
      <li><strong>Getting the DNA parts</strong><br />
 +
        <br />
 +
      </li>
 +
              <ol type="a">
 +
              <li><strong>For parts in kit plates</strong></li>
 +
  </b>
 +
                    <ol>
 +
                    <li>With a pipette tip, punch a hole through the foil cover into the corresponding well of the Biobrick<sup>TM</sup>-standard part that you want.</li>
 +
                    <li>Pipette 10 uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully re-suspended.</li>
 +
                    <li><a href="http://partsregistry.org/Help:Transformation_Protocol" target="_blank">Transform</a>
 +
1 or 2 uL of the re-suspended DNA into desired competent cells, plate your transformation with the appropriate antibiotic and grow overnight.</li>
 +
                    <li>Pick a single colony and inoculate LB medium (again, with the correct antibiotic) and grow for 16 hours.</li>
 +
                    <li>Use the resulting culture to <a href="http://openwetware.org/wiki/Miniprep/Kit-free_high-throughput_protocol" target="_blank">miniprep</a> the DNA and make your own glycerol stock.</li>
 +
                    </ol>
 +
 +
              <li><strong>For new synthesized DNA parts</strong></li>
 +
                   
 +
                    <ol>
 +
                    <li>The DNA parts are sent in nmol values in each. These data are determined for all of them individually</li>
 +
                    <li>As a stock solution preparation, these DNA parts are pipetted with PCR water or TE buffer which is 100 times of DNA amount. The final solution is prepared as 100uM.</li>
 +
                    <li>For each transformation procedures, the 10uM of stock DNA part in water is transformed. 1uL is taken from stock DNA solution and is added to 9uL PCR water.<br />
 +
                      <br />
 +
                    </li>
 +
                    </ol>
 +
              </ol>
 +
              <li><strong>Preparation  of Competent Cells</strong>
 +
                <br />
 +
                <br />
 +
                <ol>
 +
                  <ol>
 +
                    <li>Inoculate a single colony of E. Coli cells into 50 ml  LB medium. Grow overnight at 37 C with moderate shaking (250rpm)</li>
 +
                    <li>Inoculate  4 mL of the culture into 400 mL LB medium in a sterile 2-liter flask. Grow at  37 C shaking at 250 rpm to an OD590 of 0.375.
 +
                      <p><em>This  procedure requires that cells be growing rapidly (early- or mid-log phase).  Accordingly, it is very important that the growing cells have sufficient air.  Overgrowth of culture (beyond OD590 of 0.4) decreases the efficiency of  transformation.</em>                        </p>
 +
                    </li>
 +
                    <li>Aliquot culture into eight 50 mL prechilled, sterile polypropylene tubes and leave the tubes on ice 5 to 10 min.
 +
                      <p><em>Cells should be kept cold for all subsequent steps.<br />
 +
                      Larger tubes or bottles can be used to centrifuge cells if volumes of all subsequent solutions are increased in direct proportion. </em>                        </p>
 +
                    </li>
 +
                    <li>Centrifuge  cells 7 min at 1600 g,   4 C.</li>
 +
                    <p>Allow  centrifuge to decelerate without breake.<br />
 +
                      <em>We have not  attempted to determine whether deceleration without braking is critical to this  procedure. However, we do not use the breake for this step or for the  subsequent centrifugation steps. </em>                    </p>
 +
                    <li>Pour off subernant and resuspend each pellet in 10 ml ice-cold CaCl2 solution.Re-suspension should be performed very gently and all cells kept on ice.</li>
 +
                    <li>Cetrifuge cells 5 min at 1100  g, 4 C. Discard supernatant and re-suspend each pellet in 10 mL ice-cold CaCl2 solution. Keep re-suspend cells on ice for 30 min.</li>
 +
                    <li>Centrifuge cells 5 min at 1100 g, 4 C. Discard supernatant and re-suspend each pellet in 2 mL ice-cold CaCl2 solution.
 +
                      <p><em>It is important to re-suspend this final pellet well. The suspension may be left on ice for several days.</em>                        </p>
 +
                    </li>
 +
                    <li>Dispense cells into prechilled, sterile polypropylene tubes (250 ml aliquots are convenient). Freeze immediately at -70 C.</li>
 +
                  </ol>
 +
                </ol>
 +
              </li>
 +
            <li><strong>Transformation</strong>              <br />
 +
                <br />
 +
              </li>
 +
 
 +
  <ol>
 +
                <ol>
 +
                  <li>Aliquot  10 ng of DNA in a volume 10 to 25  mL  into a sterile 15 mL round bottom test tube and place on ice.</li>
 +
                  <p><em>Plasmid DNA  can be used directly from ligation reactions. When this is done , more DNA is  usually used. However, if there is &lt;1 mg of DNA in the ligation reaction, or  if the ligation reaction is from low gelling/melting temperature agarose, it is  wise to dilute the ligation mix.</em></p>
 +
                  <li>Rapidly  thaw competent cells by warming between hands and dispense 100 mL immediately  into test tubes containing DNA. Gently swirl tubes to mix, then place on ice  for 10 min.</li>
 +
                  <p><em>Competent  cells should be used immediately after thawing. Remaining cells should be  discarded rather than refrozen</em></p>
 +
                  <li>Heat  shock cells by placing tubes into a 42 C water bath for 2 min</li>
 +
                  <p><em>Alternatively  incubate at 37 C for 5 min.</em></p>
 +
                  <li>Add 1 ml  LB medium to each tube. Place each tube on a roler drum at 250 rpm for 1 hr at  37 C.</li>
 +
                  <li>Plate  aliquots of transformatin culture on LB /ampicillin or other approprate  antibiotic- containing plates.</li><p><em>It is  convenient to plate several different dilutions of each transformation culture.  The remainder of the mixture can be stored at 4 C for subsequent platings.</em></p>
 +
                  <li>When  plates are dry, incubate 12 to 16 hr at 37 C.</li>
 +
                </ol>
 +
  </ol>
 +
  <li><strong>Plasmid isolation</strong></li>
 +
  <p>This procedure is performed with Fermentas GeneJET™ Plasmid Miniprep Kit</p>
 +
  <ol>
 +
    <ol>
 +
      <ol>
 +
      </ol>
 +
                <li>Pick a single  colony</li>
 +
                <li> Inoculate  in 5 mL LB medium for high-copy or 10 mL for low-copy of LB medium supplemented  with the appropriate selection antibiotic.</li>
 +
                <li> Incubate at  225 rpm not longer than 12-14 h at 37 C .</li>
 +
      <li> Centrifuge  at 4000 rpm for 5 min at 4 C .</li>
 +
                <li> Discard the  supernatant and keep pellet.</li>
 +
                <li>  Resuspend  the pellet with 250 ul  Resuspension solution. (Bacteria should be resuspended completely by vortexing  until no cell clumps remain)</li>
 +
                <li>Transfer the  cell suspension to eppendorf.</li>
 +
         
 +
                <li> Add 250 uL Lysis Solution and mix gently by  inverting the tube 4-6 times until the solution becomes slightly clearPlate  aliquots of transformatin culture on LB /ampicillin or other approprate  antibiotic- containing plates.</li>
 +
        <p><em>Do not  vortex!</em><br />
 +
        <em>Do not incubate for more than 5 min. (To avoid denaturation of  supercoiled plasmid DNA!)</em></p>
 +
        <li>Add 350  uL Neutralization solution and mix immediately and thoroughly by inverting the  tube 4-6 times.</li>
 +
        <p><em>(The neutralized bacterial lysate is cloudy and viscous)  (&quot;thoroughly&quot; to avoid localized precipitation of bacterial cell  debris)</em></p>
 +
        <li>Centrifuge  at 13000 rpm for 5 min to precipitate cell debris and chromosomal DNA.</li>
 +
        <li>Transfer  the supernatant to spin column (about 600 uL).</li>
 +
        <p><em>(Avoid disturbing or transferring the white precipitate)</em></p>
 +
        <li>Centrifuge  at 13000 rpm for 1 min.        </li>
 +
        <li>Discard  the flow-through.        </li>
 +
      <li> Place the  column back into same collection tube.</li>
 +
        <li> Add 500 uL  Wash solution to spin column.</li>
 +
        <li> Centrifuge  at 13000 for 1 min</li>
 +
        <li> Discard  the flow-through.</li>
 +
        <li> Repeat  this step with using 500 uL Wash solution.</li>
 +
        <li> Discard  the flow-through</li>
 +
        <li> Centrifuge  at 13000 rpm for an additional 1 min to remove residual<strong> </strong>Wash solution.<br />
 +
          (This step is  essential to avoid residual ethanol in plasmid preps)</li>
 +
        <li> Transfer spin column into a fresh 1.5 mL epp.</li>
 +
        <li> Add 50 uL Elution buffer into center of the  spin column membrane to elute the plasmid DNA</li>
 +
        <p><em>Do not contact the membrane with pipette tip!</em></p>
 +
        <li>Incubate  for 2 min at 25 C.  </li>
 +
        <p><em>To increase  yield incubation is done for 2 min at heat block at 42 C</em></p>
 +
        <li>Centrifuge  at 13000 for 2 min.</li>
 +
        <li>Discard  the column and store the purified plasmid DNA at -20 C.</li>
 +
        <p><em>OPTIONAL= additional elution step with elution buffer or water. This  step increases the yield by 10-20 %.</em><br />
 +
          <em>NOTE:  For elution of plasmids ≥ 20 kb, prewarm  Elution buffer to 70 C before applying.</em></p>
 +
    </ol>
 +
  </ol>
 +
  <li><strong>Restriction  digestion</strong></li>
 +
 
 +
  <p>This procedure  is performed with NEB  BioBrick™ Assembly  Kit</p>
 +
 
 +
<ul>
 +
  <li>Digest  Upstream Part with EcoRI-HF and Spel<br />
 +
    500 ng  Upstream Part Plasmid<br />
 +
    1 uL EcorI-HF<br />
 +
    1 uL Spe I<br />
 +
    5 uL  10XNE  Buffer 2<br />
 +
    0.5 uL 100X  BSA<br />
 +
    To 50 uL add  dH2O<br />
 +
    <br />
 +
  </li>
 +
  <li>Digest  downstream part will  Xbal and PstI<br />
 +
    500 ng  Downstream Part Plasmid<br />
 +
    1 ul XbaI<br />
 +
    5 uL 10X NE  Buffer 2<br />
 +
    0.5 uL 100X BSA<br />
 +
  To 50 uL add  dH2O<br />
 +
  <br />
 +
  </li>
 +
  <li>Digest  the destination plasmid with EcorI-HF and Pst I:</li>
 +
 
 +
<p>The destination plasmid DNA should either be prepared with PCR or  contain the toxic gene (e.g, ccdB, sacB) in the cloning site to avoid the need  for gene purification.<br />
 +
The Destination Plasmid should also have a different antibiotic  resistance marker from both the plasmid containing the Upstream Part and the  plasmid containing the Downstream Part to avoid the need to purify the Upstream  and Downstream Parts.<br />
 +
500 ng  Destination Plasmid DNA<br />
 +
1 uL EcorI- HF<br />
 +
1 uL PstI<br />
 +
15 uL 10X NE  Buffer 2<br />
 +
0.5 uL 100X BSA<br />
 +
To 50 uL add  dH2O<br />
 +
 <br />
 +
Incubate all 3  restriction digest reactions at 37 C for 10 min and then heat inactivate at 80  C for 20 min.</p>
 +
</ul>
 +
<li><strong>AGE (Confirmation)</strong></li>
 +
<h3>Background</h3>
 +
<ul><li>EtBr stains only dsDNA. You cannot see ssDNA on gel</li>
 +
<li>Minimum amount of DNA load:</li>
 +
<p>300 ng <br />
 +
  •  Each band of 1kb ladder is approximately 125 ng (2.5 ul loaded) and can be seen on gel</p>
 +
</ul>
 +
 
 +
<h3>Preparation</h3>
 +
<ul><li>50X TAE (Tris-Acetate) Buffer<br />
 +
 
 +
121 g/0.5 L Tris<br />
 +
28.6 g/0.5 L Glacial Acetic Acid
 +
<br />
 +
7.31 g/0.5 L EDTA
 +
<br />
 +
pH 8.0<br />
 +
 
 +
</li>
 +
<li>Sterile 1.5 mL eppendorf tubes</li>
 +
<li>Sterile long pipette tips</li>
 +
<li>DNA standards
 +
  <ul>
 +
    <li>  Promega 1.0 kb (1.0-10Kbp), 100 bp (0.1-1.0 Kbp)</li>
 +
  </ul>
 +
</li>
 +
<li>TAE Buffer</li>
 +
<li>EtBr sln (10 mg/mL). Purchase as solution ! Store at RT. <strong>Light-sensitive and carcinogenic !</strong></li>
 +
<li>Sample loading buffer. Store at 4 C.</li>
 +
</ul>
 +
<h3>Protocol</h3>
 +
<ul><li>Prepare 360 ml, 1X TAE running buffer</li>
 +
 
 +
 
 +
  <li>Prepare  gel compartment with tape (do not use parafilm)</li> 
 +
  <li>Prepare  60 ml, 1% agarose gel (0.6 g agarose <u>in 60 ml</u> <u>1X TAE buffer)</u>
 +
    <ul>
 +
      <li>1-2 min  in microwave. Check and swirl after each 30 sec. It must be dissolved completely.</li>
 +
      </ul>
 +
  </li>
 +
  <li>Add 1.0  ul EtBr (10 mg/ml) per 10 ml gel solution, swirl to mix</li>
 +
  <li>Add add 4  ul EtBr (10 mg/ml) per 100 ml running buffer.</li>
 +
  <li>Pour gel  into gel compartment. Put comb.</li>
 +
  <li> After gel solidifies (30 min), take  out the comb and load buffer to one well to measure its capacity.</li>
 +
  <li>Position the  gel compartment and fill gel chamber with running buffer.</li>
 +
  <li> Prepare samples and standards (ladder)</li>
 +
  <li> Prepare samples by mixing 4 ul 6X  Loading Buffer + 20 ul sample</li>
 +
  <li> Load the samples (for 24 ul sample, set  pipette 24 ul).</li>
 +
 
 +
  <p><em>Do not push the tip to the bottom of the wells. Make a 45 degree angle and support the pipette tip on the edge of the well. Don’t worry, gel will not brake when you take support. Slowly release the sample. Do not use second pipet push not to risk air injection.</em></p>
 +
  <li>Make the  connections: Black (-) on sample side, Red (+) opposite side</li>
 +
  <li>Run at 60V<br />
 +
      5-10V/cm (distance between anode and cathode) is recommended.<br />
 +
      90V causes high heat generation for our unit. Use 60V maximum.<br />
 +
      For overnight runs: 2V is too low, causes diffusion<br />
 +
    35V / 4hrs / ¾ progress</li>
 +
  <li>Gel can  be stored in 4 C until next day after completion</li>
 +
</ul>
 +
<h3>Gel Photo</h3>
 +
<ul><li>Adjust zoom and position using visible light</li>
 +
 
 +
 
 +
 
 +
 
 +
  <li>Before  turning on UV load your settings file which has the following parameters:
 +
    <ul>
 +
      <li>Preview tab,  all three options checked</li>
 +
      <li> Active  image</li>
 +
      <li> Dynamic  integration, auto exposure, 10 frames</li>
 +
      <li> 50/50  brigthness/contrast</li>
 +
     
 +
      </ul>
 +
  </li> 
 +
  <li> Maximize brigthness with camera knob  (counterclockwise)</li>
 +
  <li> Turn on UV light</li>
 +
  <li>  Lower brightness from camera knob if necessary</li>
 +
  <p>&nbsp;</p>
 +
</ul>
 +
<li><strong>Gel  extraction (For  Fermentas Gel Extraction Kit)</strong>
 +
  <br />
 +
  <br />
 +
  <ol>
 +
    <ol>
 +
      <li>Excise  gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut  as close to the DNA as possible to minimize the gel volume. Place the gel slice  into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel slice.</li>
 +
    <p><em>Note: If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a glass or plastic plate during UV illumination.</em></p>
 +
    <li>Add 1:1 volume of Binding Buffer to the gel slice (volume: weight) (e.g., add 100 µl of Binding Buffer for every 100 mg of agarose gel)</li>
 +
    p><em>Note. For gels with an agarose content greater than 2%, add 2:1 volumes of Binding Buffer to the gel slice.</em></p>
 +
    <li>Incubate the gel mixture at 50-60°C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Check the color of the solution. A yellow color indicates an optimal pH for DNA  binding. If the color of the solution is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow</li>
 +
   
 +
    <li><em>Optional:  use this step only when DNA fragment is &lt;500 bp or &gt;10 kb long</em>.
 +
      <ul><li>If the DNA fragment is &lt;500 bp, add a 1:2 volume of 100% isopropanol to the solubilized gel solution (e.g. 100 µl of isopropanol should be added to 100 mg gel slice solubilized in 100 µl of Binding Buffer). Mix thoroughly</li>
 +
        <li>- If the  DNA fragment is &gt;10 kb, add a 1:2 volume of water to the solubilized gel solution  (e.g. 100 µl of water should be added to 100 mg gel slice solubilized in 100 µl  of Binding Buffer). Mix thoroughly.</li>
 +
        </ul>
 +
    </li>
 +
    <li>Transfer  up to 800 µl of the solubilized gel solution (from step 3 or 4) to the GeneJET™  purification column. Centrifuge for 1 min. Discard the flow-through and place  the column back into the same collection tube. </li>
 +
    <p><em>Note. If  the total volume exceeds 800 µl, the solution can be added to the column in  stages. After each application, centrifuge the column for 30-60 s and discard  the flow-through after each spin. Repeat until the entire volume has been  applied to the column membrane. Do not   exceed 1 g of total agarose gel per column.</em></p>
 +
    <li>
 +
      <p><em>Optional:  use this additional binding step only if the purified DNA will be used for sequencing</em>
 +
      <p>Add 100 µl of Binding Buffer to the GeneJET™ purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.</p>
 +
    </li> 
 +
    <li>Add 700  µl of Wash Buffer (diluted with ethanol as described on p. 3) to the GeneJET™  purification column. Centrifuge for 1 min. Discard the flow-through and place  the column back into the same collection tube.</li>
 +
    <li>Centrifuge  the empty GeneJET™ purification column for an additional 1 min to completely  remove residual wash buffer. </li>
 +
    <p><em>Note. This  step is essential to avoid residual ethanol in the purified DNA solution. The  presence of ethanol in the DNA sample may inhibit downstream enzymatic  reactions.</em></p>
 +
    <li>Transfer  the  purification column into a clean 1.5  ml microcentrifuge tube (not included). Add 50 µl of Elution Buffer to the  center of the purification column membrane. Centrifuge for 1 min.</li>
 +
   
 +
    <p><em>Note:</em></p>
 +
      <em><ul><li>For low  DNA amounts the elution volumes can be reduced to increase DNA concentration.  An elution volume between 20-50 µl does not significantly reduce the DNA yield.  However, elution volumes less than 10 µl are not recommended.</li>
 +
      <li>If DNA  fragment is &gt;10 kb, prewarm Elution Buffer to 65°C before applying to column</li>
 +
    <li>If the  elution volume is 10 µl and DNA amount is ≤5 µg, incubate column for 1 min at  room temperature before centrifugation</li></ul></em>
 +
    <li>Discard  the GeneJET™ purification column and store the purified DNA at -20°C</li>
 +
    </ol> 
 +
    </ol>
 +
</li>
 +
<li><strong>Ligation</strong></li>
 +
 
 +
 
 +
<p>This procedure  is performed with  NEB BioBrick™ Assembly  Kit</p>
 +
<ol><li>Ligate  the Upstream and Downstream Parts into digested Destination Plasmid.
 +
  <ul>
 +
    <li> 2 uL Upstream  Part Digestion</li>
 +
    <li>      2 uL  Downstream Part Digestion</li>
 +
    <li>      2 uL  Desination Plasmid Digestion</li>
 +
    <li>      2 uL 10X T4  DNA ligase bufferl</li>
 +
    <li>      2 uL T4 DNA  ligase buffer</li>
 +
    <li>11 uL dH2O</li>
 +
    </ul>
 +
</li>
 +
 +
  <li>Incubate at RT  for 10 min and then heat inactivate at 80 C for 20 min.</li>
 +
  <li>    Transform 2 uL  of the ligation product into 50 uL of competent E.Coli cells ( or other  suitable host strain). </li>
 +
  <li>Select using the antibiotic corresponding to the  Destination Plasmid.<br />
 +
    <br />
 +
  </li>
 +
</ol>
 +
<li><strong>Glycerol Stock Preparation</strong> <br />
 +
    <br />
 +
      <ol>
 +
    <li>Centrifuge  5 mL overnight cells at 5000 rpm for 5 min.</li>
 +
    <li>Discard 4  mL supernatant, remain 1 mL with pellet.</li>
 +
    <li>Add 176 uL 15% glycerol  and resuspend the pellet.</li>
 +
    <li>Aliquot  100 uL and immediately freeze at -70 C.<br />
 +
      <br />
 +
    </li>
 +
    </ol>
 +
</li>
 +
<li><strong>Cell  imaging</strong></li>
 +
<h3>Checklist Procedure</h3>
 +
<ol>
 +
  <li>Suspension culture preparation  </li>
 +
 
 +
<ul>
 +
  <li>
 +
    Dissolve a single colony in 10 mL  LB+Amp  </li>
 +
    <li>Incubate 14-15 hrs, shaking at  225 rpm<br />
 +
      <br />
 +
    </li>
 +
  </ul>
 +
 
 +
 
 +
  <li>Scale-up of the suspension culture</li>
 +
    <ul>
 +
      <li> Add 2-3 mL of the suspension culture to 100 mL LB+Amp </li>
 +
 
 +
  <li>Incubate 5-6 hrs, shaking at 225 rpm<br />
 +
    <br />
 +
  </li>
 +
  </ul>
 +
<li>Equalizing OD readings</li>
 +
    <ul>
 +
      <li>Take 10 mL samples from GFP/RFP  &amp; NC cultures</li>
 +
      <li>Centrifuge at 10000 rpm for 5  mins</li>
 +
      <li> Discard supernatant</li>
 +
      <li>Resuspend pellet in 500 uL 1X PBS</li>
 +
      <li>Take 250 uL &amp; dilute to 3 mL  with 1X PBS</li>
 +
      <li> Read in UV-Vis at 600 nm</li>
 +
      <li> Multiply the OD readings by 12<br />
 +
      </li>
 +
      <li>Dilute remaining 250 uL samples as  required to make all samples have the same ODvalue, final volume should also be 250 uL<br />
 +
        <br />
 +
      </li>
 +
    </ul>
 +
    <li>Fluorescent  signal reading</li>
 +
    <ul>
 +
      <li>Set  parameters<br />
 +
      [GFP]</li>
 +
      <li> Mode:  Emission scan</li>
 +
      <li> Config:  Digital</li>
 +
      <li> Slit: 1 turn  / 2 nm</li>
 +
      <li> Blaze: 750  nm</li>
 +
      <li> Excitation:  395 nm</li>
 +
      <li> Emission  range: 475-600 nm</li>
 +
      <li> Average: 3</li>
 +
      <li> Step size:  5 nm<br />
 +
        [RFP]</li>
 +
      <li> Mode:  Emission scan</li>
 +
      <li> Config:  Digital</li>
 +
      <li> Slit: 1  turn / 2 nm</li>
 +
      <li> Blaze: 750  nm</li>
 +
      <li> Excitation:  570 nm</li>
 +
      <li> Emission  range: 585-650 nm</li>
 +
      <li> Average: 3</li>
 +
      <li> Step size:  5 nm</li>
 +
      <li> Transfer  samples to small 150 uL cuvette and scan</li>
 +
    </ul>
 +
    <li>Data Analysis</li>
 +
    <ul>
 +
      <li>Emission  maxima: <br />
 +
        GFP 515 nm<br />
 +
      RFP 605 nm</li>
 +
      <li> Record  intensity values at 509 nm for GFP &amp; 606 nm for RFP</li>
 +
      <li> Subtract NC  intensity value from GFP/RFP value</li>
 +
      <li> Result is  the fluorescent signal of the protein in the bacteria<br />
 +
        <br />
 +
      </li>
 +
    </ul>
 +
  </ol>
 +
<li><strong>Protein expression</strong></li>
 +
<p> <strong>MAIN STEPS/TIME TABLE</strong><br />
 +
  - Pre-culturing/5 hours<br />
 +
  - Culturing/5 hours <br />
 +
  - IPTG induction/15 hours<br />
 +
  <br />
 +
  <strong>Materials</strong><br />
 +
  - LB medium with a suitable antibiotic<br />
 +
  - TB medium with a suitable antibiotic<br />
 +
  - IPTG<br />
 +
  - 2-liter flask<br />
 +
  - Incubator with shaker<br />
 +
  - Centrifuge<br />
 +
  <br />
 +
  <strong>Check List Procedure</strong><br />
 +
  - Culture transformed E.coli BL21 for 5 hrs at 37 C in 10 LB medium containing antibiotic<br />
 +
  - Inoculate 4 ml pre-cultured cells into 400 ml of TB medium containing antibiotic in a 2-liter cultivation flask<br />
 +
  - Culture it for 5 hrs at 37 C with a rotary shaker at 180 rpm!<br />
 +
  - Add 0.5 mM IPTG in a final concentration<br />
 +
  - Continue the cultivation for 15 hrs at 22 C.<br />
 +
  - Harvest the cells with centrifuge.<br />
 +
  <br />
 +
  <strong>Alternative Check List Procedure</strong><br />
 +
  - Inoculate single colony for 5 hrs at 37 C in 10 mL LB medium containing antibiotic<br />
 +
  - Inoculate 4 ml pre-cultured cells into 400 ml of TB medium containing antibiotic in a 2-liter cultivation flask<br />
 +
  - Culture it for 5 hrs at 37 C (OD600 to 0.5 - 0.6) with a rotary shaker at 180 rpm<br />
 +
  - Add 1 mM IPTG in a final concentration ( 0.5- 2.0 mM recomended for pT7)<br />
 +
  - Continue the cultivation for 3-4 h at 37 C (15 hrs at 22 C.)<br />
 +
  - Harvest the cells with centrifuge.<br />
 +
  <br />
 +
  <strong>Solutions</strong><br />
 +
  - TB (Terrific Broth)<br />
 +
  <br />
 +
  Reference:<a href="http://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/" target="_blank"> http://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/</a><br />
 +
  <br />
 +
  <br />
 +
  <br />
 +
  <br />
 +
  <strong>SDS-PAGE
 +
  <br />
 +
  MATERIALS </strong>  
 +
  <br />
 +
  - Vertical Electrophoresis apparatus
 +
  <br />
 +
  - Power supply
 +
  <br />
 +
  - pH meter
 +
  <br />
 +
  - Balance
 +
  <br />
 +
  - Silver staining shaker platform
 +
  <br />
 +
  - Transilluminator
 +
  <br />
 +
  - Filter paper
 +
  <br />
 +
  <br />
 +
  <strong>For Gel preparation  </strong><br />
 +
  - Acrylamide
 +
  <br />
 +
  - Bisacrylamide
 +
  <br />
 +
  - Deionized Water
 +
  <br />
 +
  - Tris base
 +
  <br />
 +
  - SDS
 +
  <br />
 +
  - APS
 +
  <br />
 +
  - TEMED
 +
  <br />
 +
  <br />
 +
  <strong>For Silver Stanning  </strong><br />
 +
  - Fixer
 +
  <br />
 +
  - %50 ethanol
 +
  <br />
 +
  - Pretreatment solution
 +
  <br />
 +
  - Silver nitrate
 +
  <br />
 +
  - Devoloping solution
 +
  <br />
 +
  - Stop solution
 +
  <br />
 +
  <br />
 +
  - Marker: Fermentas / Page Ruler Protein Ladder SM0661 (10-200kDa)
 +
  <br />
 +
  <br />
 +
  <strong>SOLUTIONS</strong>
 +
  <br />
 +
  <strong>Running Buffer (10L, 1X)</strong><br />
 +
  <br />
 +
  - 25mM Tris, pH 8.3
 +
  <br />
 +
  - 250mM Glycine
 +
  <br />
 +
  - 0.1% SDS
 +
<br />
 +
  or
 +
 
 +
    <br />
 +
  - 60 ml 10X stock + 6 ml 10% SDS + 534 ml dH2O
 +
 +
  <br />
 +
  -----------------------
 +
 
 +
  <br />
 +
  - 30.3g Tris
 +
 
 +
  <br />
 +
  - 187.7g Glycine
 +
 
 +
  <br />
 +
  - 10g SDS
 +
 +
  <br />
 +
  - Final volume 10 L in demijon
 +
 
 +
  <br />
 +
 
 +
  <br />
 +
  <strong>Staining Solution (Coomassie Blue) ( 1 L, 1 X)</strong>
 +
 
 +
  <br />
 +
  - 2 g brilliant blue (R-250)
 +
 
 +
  <br />
 +
  - 450 ml methanol
 +
 
 +
  <br />
 +
  - 450 ml dH2O
 +
 
 +
  <br />
 +
  - 100 ml acetic acid
 +
 
 +
  <br />
 +
  - store at 24 C
 +
 +
  <br />
 +
 
 +
  <br />
 +
  <strong>Destaining Solution ( 4 L, 1 X)  </strong><br />
 +
  - Methanol:Acetic Acid:dH2O 40:10:50
 +
 
 +
  <br />
 +
  - Prepare 4.0 L (1.6 L MeOH, 0.4 L AcA, 2.0 L dH2O) in 1 gallon amber bottle, cap tightly
 +
 
 +
  <br />
 +
  - store at room temp.
 +
 
 +
  <br />
 +
 
 +
  <br />
 +
  <strong>4X Sample Loading Buffer  </strong>  <br />
 +
  - 400 mM DTT
 +
 
 +
  <br />
 +
  - 40 mM Tris
 +
 
 +
  <br />
 +
  - 10% Glycerol
 +
 +
  <br />
 +
  - 4% SDS
 +
 +
  <br />
 +
  - 0.4% Bromophenol blue
 +
 
 +
  <br />
 +
  <br />
 +
  - prepare 15 ml
 +
 
 +
  <br />
 +
  - 925.2 mg DTT
 +
 
 +
  <br />
 +
  - 72.6 mg Tris
 +
 +
  <br />
 +
  - adjust pH to 6.8
 +
 
 +
  <br />
 +
  - 1500 ul Glycerol
 +
 
 +
  <br />
 +
  - 600 mg SDS
 +
 
 +
  <br />
 +
  - 6 mg Bromophenol blue
 +
 
 +
  <br />
 +
  - aliquot 50 x 600 ul
 +
 
 +
  <br />
 +
  - store at -20 C
 +
 
 +
   
 +
  <br />
 +
 
 +
  <br />
 +
  <strong>30% Acrylamide 1% Bis-acrylimide  </strong>  <br />
 +
  - prepare in laminar flow
 +
 
 +
  <br />
 +
  - 30 g acrylimide and 1 g bis-acrylimide in 100 ml
 +
 
 +
  <br />
 +
  - 75 mL acrylimide solution ( 40% stock, Apllichem) and 25 mL bis-acrylimide solution  ( 2% stock, Apllichem)to final volume 100 ml
 +
 
 +
  <br />
 +
  - store at 4 C, stable for 1 month
 +
 
 +
  <br />
 +
 
 +
  <br />
 +
  <strong>Seperating Gel Buffer</strong>
 +
 
 +
  <br />
 +
  - 1.5 M Tris pH 8.8
 +
 
 +
  <br />
 +
  ------------------------------------------
 +
 
 +
  <br />
 +
  - for 100 mL Buffer solution
 +
 
 +
  <br />
 +
  - 18,15 g Tris pH 8.8
 +
 
 +
  <br />
 +
  - store at room temp.
 +
 
 +
  <br />
 +
 
 +
  <br />
 +
  <strong>Stacking Gel Buffer</strong>
 +
 
 +
  <br />
 +
  - 0.5 M Tris
 +
 
 +
  <br />
 +
  ------------------------------------------
 +
 
 +
  <br />
 +
  - for 50 mL Buffer solution
 +
 
 +
  <br />
 +
  - 3 g Tris pH 6.8
 +
 
 +
  <br />
 +
  - store at room temp.
 +
 
 +
  <br />
 +
 
 +
  <br />
 +
  <strong>APS (Ammonium Persulfate)  Stocks (100 mg/ml)  </strong>  <br />
 +
  - dissolve 0.6 g in 6 ml dH2O
 +
 
 +
  <br />
 +
  - aliquot 10 x 600 ul and store at -20 C
 +
 
 +
  <br />
 +
 
 +
  <br />
 +
  <strong>1% Bromophenol Blue  </strong>  <br />
 +
  - 0.01 g in 1 ml 1M Tris, pH 7.0
 +
 
 +
  <br />
 +
  - store at room temp. in amber bottle<br />
 +
  <br />
 +
 
 +
  <strong>CASTING 13% GEL</strong><br />
 +
  - set heater to 100 C for sample prep step<br />
 +
  <br />
 +
 
 +
  <br />  
 +
  <strong>  Seperating Gel (30 ml)</strong>
 +
 
 +
  <br />  
 +
  - 13 ml 30% Acrylimide 1% Bis-acrylimide
 +
 
 +
  <br />
 +
  - 7.5 ml seperating gel buffer
 +
 
 +
  <br />
 +
  - 8,45 ml dH2O
 +
 
 +
  <br />
 +
  - 500 ul %10 SDS
 +
 
 +
  <br />
 +
  - 250 ul APS (initiator of polymerization)
 +
 
 +
  <br />
 +
  - 25 ul TEMED (catalyst of polymerization)
 +
 
 +
  <br />
 +
 
 +
  <br />
 +
  - Load 5.4 ml separating gel between glasses
 +
 
 +
  <br />
 +
 
 +
  <br />  
 +
  <strong>Stacking Gel (10 ml)  </strong>  <br />  
 +
  - 1.6 ml 30% Acrylimide 1% Bis-acrylimide
 +
 
 +
  <br />
 +
  - 2.5 ml stacking gel buffer
 +
 
 +
  <br />
 +
  - 5.85 ml dH2O
 +
 
 +
  <br />
 +
  - 100 ul %10 SDS
 +
 
 +
  <br />
 +
  - 15 ul 1% Bromophenol Blue
 +
 
 +
  <br />
 +
  - 50 ul APS (initiator of polymerization) [add after resolving gel is casted]
 +
 
 +
  <br />
 +
  - 10 ul TEMED (catalyst of polymerization) [add after resolving gel is casted]
 +
 
 +
  <br />
 +
 
 +
  <br />
 +
  - Load 1.7 ml stacking gel between glasses
 +
 
 +
  <br />
 +
  - Inset the comb
 +
 
 +
  - make sure the comb has not been inserted in a tilted way. check from behind the apparatus<br />
 +
  <br />
 +
  - load seperating gel
 +
 
 +
  <br />
 +
  - add some butanol or isopropanol before resolving gel solidifies
 +
 
 +
  <br />
 +
  - make sure gel stays on flat surface while solidifies to prevent tilted surface
 +
 
 +
  <br />
 +
  - load stacking gel
 +
 
 +
  <br />
 +
 
 +
  <br />
 +
 
 +
  <br />
 +
  - if bubbles form in the stacking gel after polymerization, press the plates between hands to push them out
 +
 
 +
  <br />
 +
 
 +
  <br />
 +
  <strong>SAMPLE PREPARATION AND LOADING  </strong><br />
 +
 
 +
  <br />
 +
  - Do not overload the the samples, purity check is difficult with overloaded samples.
 +
 
 +
  <br />
 +
  - Sample volume: 5 ul sample+ 5 ul loading buffer + 10 ul dH2O
 +
 
 +
  <br />
 +
  - vortex loading buffer before use
 +
 
 +
  <br />
 +
  - put samples in heating block (100 C) for 5 min
 +
 
 +
  <br />
 +
  - if possible, do not load into the first and last lanes
 +
 
 +
  <br />
 +
  - load 5 ul marker
 +
 
 +
  <br />
 +
  - load 17 ul samples
 +
 
 +
  <br />
 +
  <br />
 +
 
 +
  <br />
 +
  <strong>PREPERATION  </strong>
 +
 
 +
  <br />
 +
  - check the wire on running apparatus, clean and test
 +
 
 +
  <br />
 +
 
 +
  <br />
 +
  <strong>RUNNING</strong>
 +
 
 +
  <br />
 +
  - never terminate the run early, lighter bands dont separate
 +
 
 +
  <br />
 +
  - 600 ml running buffer is required for each run
 +
 
 +
  <br />
 +
 
 +
  <br />
 +
  <strong>Running Standards  </strong><br />
 +
  - 5 mA &gt; 3/4 of gel &gt; 25hrs &gt;&gt;&gt; overnight running
 +
 
 +
  <br />
 +
  - max: 80 mA for both old and new gel systems
 +
 
 +
  <br />
 +
  - For Lab 103 Tankı
 +
 
 +
  <br />   
 +
  - for 145 min at 30 mA 120 V through seperating gel
 +
 
 +
  <br />   
 +
  - amper constant
 +
 
 +
  <br />
 +
 
 +
  <br />
 +
  <strong>After Run  </strong>
 +
 
 +
  <br />
 +
  - wash electrophoresis unit after each use
 +
 
 +
  <br />
 +
  - weekly cleaning of power connections recommended to prevent oxidation<<br /><br />
 +
    <strong>STAINING    </strong>  <br />
 +
  - Stain the gel for 30 min<br /><br />
 +
  <strong>DESTAINING  </strong>  <br />
 +
  - load the tray fully with destaining buffer <br />
 +
  - do not put too many (over-destaining) or too less (under-staining) paper sheets <br />
 +
  - destaining takes 2-3 hrs
 +
<li><strong>Device Experiments</strong></li>
 +
<p>  <strong>MAIN STEPS/TIME TABLE</strong><br />
 +
    - Pre-culturing/7 hours<br />
 +
    - Culturing to 250ml flasks/3 hours<br />
 +
    <br />
 +
    <strong>Materials</strong><br />
 +
    - LB medium with a suitable antibiotic (Ampicillin)<br />
 +
    - 250 ml flasks<br />
 +
    - Incubator with shaker<br />
 +
    - Centrifuge<br />
 +
    <br />
 +
    <strong>Check List Procedure</strong><br />
 +
    - Cell culture (with ROSE regulated kill switch) transformed E.coli BL21 for 7 hrs at 37 C in 10 ml LB medium containing antibiotic<br />
 +
    - Inoculate 100ul pre-cultured cells into 100 ml of LB medium containing antibiotic in a 250 ml cultivation flask<br />
 +
    - Culture it for 3 hrs at 37 C with a rotary shaker at 220 rpm up to OD reading reaches to 0.3<br />
 +
    - Continue taking OD readings up to 0.7 at 37C.<br />
 +
    - Harvest the 5 ml of cells with centrifuge for 5 min at 5000rpm.<br />
 +
    - Resuspend in 5ml PBS and take fluorescence readings.
 +
</p>
 +
</ol>
 +
</div>
 +
</div>
</div>
</div>
-
<div id="project2" class="item">
+
<div id="humanpractice2" class="item">
<div class="content2">
<div class="content2">
<div class="text">
<div class="text">
-
<h2>Design</h2>
+
<img src="https://static.igem.org/mediawiki/2011/1/1b/Humanpractice.png" height="75px" width="890px">
-
<a href="https://static.igem.org/mediawiki/2010/6/6a/Metu_Turkey_Software_ER3.jpg"><img src="http://ung.igem.org/wiki/images/7/71/Metu_Software_ER3.png" /></a>
+
<h2>Online Lecture</h2>
 +
<p><b><i><u>Anybody interested and novice in Synthetic Biology can use the lecture</b></i></u>;</p>
 +
<h3>Here is our animation; Industrial Revolution 2 After Synthetic Biology,  to introduce the basics of the field; </h3>
 +
<center><p><iframe width="420" height="315" src="http://www.youtube.com/embed/g6RICJsKQAI" frameborder="0" allowfullscreen></iframe></p></center>
 +
<h3>Here is our Online Lecture, Click the picture to go;</h3>
 +
<center><p><a href="http://ocw.metu.edu.tr/course/view.php?id=137" target="_blank"  alink="red" vlink="red" ><img src="https://static.igem.org/mediawiki/2011/5/50/Syntheticbiol.png" height="115px" width="632px" alt="Click picture to reach Online Lectures" ></a></center></p>
 +
</div>
</div>
</div>
</div>
Line 1,244: Line 1,773:
<div class="item">
<div class="item">
</div>
</div>
-
<div id="download2" class="item">
+
<div id="partnership2" class="item">
<div class="content2">
<div class="content2">
<div class="text">
<div class="text">
-
<h2>User Guide</h2>
+
<img src="https://static.igem.org/mediawiki/2011/f/f8/Partnershipheader.png" height="75px" width="890px">
-
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/Znb1o7cr5dc?hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/Znb1o7cr5dc?hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/Fk7eJ-Ig_IQ?hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/Fk7eJ-Ig_IQ?hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><p>For new users whom want to interest with synthetic biology and for experienced scientists, in our program you have two options for Input Output properties ;</p>
+
<h2>Collaboration</h2>
-
<p>To find input-output related biobrick parts or larger constructs! If you want to provide specific chemical or physical inputs from external environment, you may choose these inputs from the list. if you want other specific inputs which will be expressed from a coding sequence, the list can provide these specified protein names.</p>
+
<h3>Collaboration with Turkish teams in iGEM 2011 </h3><br />
-
<img src="https://static.igem.org/mediawiki/2010/6/60/Metu-guide1.jpg">
+
    <br />
-
<p>Wanted features of outputs can be selected by either from list or entering individually into text boxes.</p>
+
  Since iGEM 2007, there is a team to research and prepare a project for iGEM competition from Middle East Technical University(METU). On behalf, there are advisors and instructors who are familiar with competition and content of it. In this year there are 4 registered teams from Turkey and 3 of them are new participants. Therefore in order to come together and criticize our projects and any deficiencies or ambugity related with project. So we organized and arranged a meeting on Synthetic Biology and iGEM competition which is the first in our country.The participants of this meeting were teams;<b>Fatih Turkey, Bilkent_UNAM, METU-BIN</b> and researchers who related with Synthetic Biology and company owners in Biotechnology field also there was special guests via online talk, Drew Endy, he is an assistant Professor of Bioengineering, Stanford University and Scott Mohr Professor of Biological Chemistry, Boston University This meeting was held in September 10, that was close to Regional Jamboree in order to also check the presentation preparations of teams. The news agencies were also invited and the interviews were done with all teams publicity of teams were enhanced. <br />
-
<img src="https://static.igem.org/mediawiki/2010/c/c0/Metu-guide2.jpg">
+
    <br />
-
<p>Then click on the “Show Parts ” button.</p>
+
  Except from this, we established a collaboration with <b>METU-BIN Software iGEM 2011</b> team  on their software program. This year their project, <a href="http://dayhoff.ii.metu.edu.tr:8080/m4b/" target="_blank">M4B: Mining for BioBricks</a> is on enhancement and simplification of parts registry and gene library usage to ease the wet lab researchers job. They requested us to use and try their software program. One of our parts in this year, kill switch is composite of this year distributions. By the way we tried their program to search and try on this composite. we gave them the following as feedback and also according to our comments on visual of program, we had collaborated this year.<br />
-
<img src="https://static.igem.org/mediawiki/2010/9/95/Metu-guide3.jpg">
+
    <br />
-
<p>Then the program will show you the parts have specified I/O properties</p>
+
  “We are constructing a device that is induced by IPTG and that gives lysis enzymes and GFP as the outputs because, in order to elute methanol we need to kill our modified cells that convert ambient methane to methanol and the reason why we want GFP as one output is to be sure that it works. Our modelled device has promoter induced by IPTG, RBS, gene coding GFP and lysis cassette ending with cell death and we are glad to have it and it's working how we expected. So then, we use M4B to see that if it finds the exact device or the device that can do the same job. Since the software has only one place for output, we queried IPTG as an input and GFP as an output on the software and got corresponding 117 results. These results contain devices exactly what we have one for the same operation. However, we couldn't find the lysozyme output for the IPTG input in the software.”   <br />
-
<img src="https://static.igem.org/mediawiki/2010/7/7f/Metu-guide4.jpg">
+
    <br />
-
<p>To see the properties of a part just click on it.</p>
+
  We had requested them to support us on wiki page design and same tricks on loading the links on wiki page. We had prepared the self evaluating lab safety questionnaire and they made them manipulable on wiki page  and the methane release meter on home page is their work.<br />
-
<img src="https://static.igem.org/mediawiki/2010/3/33/Metu-guide5.jpg">
+
  <br />
-
<img src="https://static.igem.org/mediawiki/2010/2/27/Metu-guide6.jpg">
+
  <br />
-
<p>After clicking, you will see a highlighted pathway on the 2nd graph. Which are the subparts of the clicked part. Showing the sub graph in network.</p>
+
  <br />
 +
<h3>Helping to other new Turkish teams in the future</h3> <br />
 +
  <br />
 +
  Our university, METU has an online lecture application; METU OpenCourseware to support the open information source not only members of METU  and to reach people to inform in all ages. In this online application, For Synthetic Biology field, last year the sessions were loaded and this year it is upgraded. The protocols for Synthetic biology methods were downloaded and supported with tutorials of procedures that taken during experiments. We established this page and enhanced for Synthetic Biology to reach more students to meet with Synthetic Biology in any region of Turkey. We had requested  from President of METU made people to reach this webpage from universities with related departments in an official way. By the way, we believe to reach more students or researchers to iGEM competition and Synthetic Biology. <br />
 +
  <br />
 +
  <h3>Collaboration with 2011 Uppsala Sweeden iGEM Team </h3><br />
 +
  <br />
 +
  <p>We asked them to use and give feedback to our software tool on our wiki. This tool is named as BioGuide which was first studied on last year’s competition iGEM 2010 and gained silver medal. This year this team joined with wet lab team. While they were progressing the BioGuide project, this software tool also supported the web page and experiments of METU_Ankara team. We publish this software program on our wiki page to made other iGEM teams to reach and benefit from program on part  and path choices. Uppsala-Sweeden team was one of the teams that use and try the BioGuide program. They tried to reach their used parts in kit distributions and then gave us feedback on them. </p><br />
 +
  They tried this software tool according to parts of their project As an input parameters they tried to type<strong> red light 'cph8',  blue light 'LovTAP', green light 'Ccas', IPTG(PT5-Lac), YF1 </strong>and as output parameters they typed <strong>amil GFP, amil CP and mCHERRY</strong>.  However they found the library of program restricted and they could not reach all parts  because BioGuide software tool library is designed and programmed on parts that are in 2010 and 2011kit plate distributions. By the way, they found this software program managable. Given part names or input and output data results with Biobrick codes of parts in parts registry library. Therefore, it made easier their job on longstanding assembly plans. For mutual collaboration, we read and evaluated their Biosafety web page and gave feedback below<br />
 +
  <br />
 +
  “You replied the first question having main title “materials used in your project” as bacteria strain used in your project. We suggest that you should focus on chemical or any other material to answer this question.However we also used the E.coli Top 10 as host strain and found that these strains are classified as hazard group 2 pathogen by the UK Advisory Committee on the Dangerous Pathogens (ACDP).<br />
 +
  In the question 3 that you explained “Institutional Biosafety Committee”, you described the threats of Synthetic biology in 3 main items based on your interview, we think you should expand content of items.<br />
 +
  <br />
 +
  And lastly, it would be better if you give more detail on your last question to be more clear for future iGEM teams.”
 +
<p><a href="https://2011.igem.org/Team:Uppsala-Sweden/Feedback" target="_blank">https://2011.igem.org/Team:Uppsala-Sweden/Feedback</a></p>
 +
</div>
</div>
</div>
</div>
</div>
</div>
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<div id="miscellaneous2" class="item">
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<div class="item">
 +
</div>
 +
<div id="software" class="item">
<div class="content2">
<div class="content2">
<div class="text">
<div class="text">
-
<h2>Human Practices</h2>
+
<img src="https://static.igem.org/mediawiki/2011/4/4c/Softwaretoolheader.png" height="75px" width="890px">
-
<p>iGEM’s parts registry is the only current database that holds information
+
<h2>Software Tool</h2>
-
and DNA for over 3000 standard parts for the use of synthetic biology
+
<p>BioGuide is a Biological System Design tool. It has been developed in 2010 by the team METU TURKEY SOFTWARE. You can check the page for detailed information about the project :<br /> <a href="https://2010.igem.org/Team:METU_Turkey_Software" target="_blank">https://2010.igem.org/Team:METU_Turkey_Software</a></p>
-
community. BioGUIDE is the first designed software that organizes the
+
                     
-
parts in iGEM spring 2010 distribution as possible atomics parts to
+
 
-
build new biological device and systems for specific input and outputs
+
<h3>Here is rough draft and setup of BioGuide Paper you can read for technical details and download the software;</h3>
-
based on graph theory. We are the first group who has applied a novel
+
 
-
algorithm to search for input/output relations between the parts to
+
<p><a href="https://static.igem.org/mediawiki/2011/d/db/BioGuide.pdf" target="_blank">BioGuide.pdf</a>
-
reveal possible construct assemblies. This new approach will chance
+
<p><a href="http://sourceforge.net/projects/bioguidesetup/files/BioGuideSetup.rar/download" target="_blank">Download the Software</a>
-
how the parts registry is used by the scientific community. And the
+
 
-
availability of software implementing our algorithm with a very user
+
                      <h3>This year;</h3>
-
friendly graphical user interface will allow all the users of the parts
+
<p>We have completed biological system design part of BioGuide and enabled wet lab teams to use it.</p>
-
registry to explore new and novel constructs according to their parameter
+
 
-
with ease. Additionally as the BioGUIDE software is an open source ware
+
<h3>You can watch the "how to" video of BioGuide to see what and how you can do with it;</h3>
-
, any user can contribute to the development of the application. So,
+
 
-
BioGUIDE will be improving with a collaborative effort, which will make
+
<center><iframe width="425" height="349" src="http://www.youtube.com/embed/P4pMri0bAMc?hl=en&fs=1" frameborder="0" allowfullscreen></iframe></center>
-
it even more widely used among the synthetic biologists. </p>
+
<br/>
-
<p>We also had many collabrations with the teams such as, INSA-Lyon,
+
<br/>
-
Lethbridge, WashU through our survey and WARSAW as we have participated
+
<h3>Here is how METU ANKARA wet lab team is using BioGuide;</h3>
-
in their survey. Also our sister team METU_TURKEY is our main collaborator.
+
<p><i>This year we are using it while building one of our devices, kill switch composite. Kill switch composite consists of 4 main parts which are in 2011 kit plate distributions; T7 promoter, RNA thermometer, GFP and lysis casette. The upstream part of this composite is T7 promoter  (BBa_I712074) which is strong promoter from T7 bacteriophage mostly used expression system and so for strong transcription T7 promoter expression system is chosen.</p>
-
They have provided feedback BioGUIDE at different stages of the development.  
+
<p>We also tried the inducible promoters (pLac-IPTG inducer) to design and express the lysis casette. We desired to use a promoter that could be induced and enhance the expression to control and manipulate the translation of device. When we typed IPTG on input box of Bioguide we had reached the possible promoters that could be induced by IPTG. This program gave their standard biobrick codes and finally we got these parts by knowing their location in kit plate.</p>
-
And they had the chance to analyze their constructs input/output relations
+
<p>In downstream of promoter there is RNA thermometer(ROSE) coding region which determines the transcription of our lysis composite part. We had reached this part by coding the temperature 42 C in input box of BioGuide. RNA thermometer (BBa_K115001) is temperature sensitive at which 42 C translation initiates. </p>
-
throughout this collaboration. </p>
+
<p>In downstream of RNA thermometer, green fluorescence protein(BBa_E0040) is ligated. This ligation is done to measure expression level and to comment on RNA thermometer efficiency. We typed GFP in output box since we wanted to measure fluorescence.</p>  
-
<p><strong>ALL TEAMS</strong>: The Parts Registry Survey that has been
+
<p>We had designed this composite and it's parts mostly by processing the BioGuide program that made our job easier in intense lab works. We reached the shortest path with our input and output parameters for this device to construct by BioGuide.</i></p>  
-
developed by METU_TURKEY_SOFTWARE got response from 253 of participant
+
-
from 94 teams. Analysis of the survey results we have received so far
+
-
indicates that we were able outreach to the iGEM community and help
+
-
them verbalize their concerns and suggestions for the parts registry
+
-
standards and the maintenance of the database.</p>
+
-
<p><strong>WARSAW</strong>: Our team members have participated in their
+
-
survey abour iGEM participants profile. </p>
+
-
<p><strong>METU_TURKEY</strong>: Beta testing of the algorithm developed
+
-
and the BioGUIDE software has been performed by METU_TURKEY on few case
+
-
studies. Additionally they have tested their construct for 2010 competition
+
-
with our algorithm. </p>
+
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<div class="content2">
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<div class="text">
<div class="text">
-
<h2>Material</h2>
+
<img src="https://static.igem.org/mediawiki/2011/1/1c/Projectheader.png" height="75px" width="890px">
-
<p>Our main data source for BIO Guide Software Program was the available
+
                          <h2>Conversion</h3>
-
background information of parts distributed in 2010 iGEM plates (Total
+
-
of three384-well plates of dried DNA) to the wetlab teams. This data
+
 
-
was available through both the parts registry main website (<a href="http://partsregistry.org/Main_Page">http://partsregistry.org/Main_Page</a>)  
+
<center><p><img src="https://static.igem.org/mediawiki/2011/7/71/Conversion.gif" height="454px" width="640px"></center></p>
-
in XML format and parts registry libraries (http://partsregistry.org/assembly/libraries.cgi?id=31)  
+
                          <h3>i) Introduction </h3>
-
in Excel format. Data from parts with specific part IDs have been parsed
+
 
-
with a custom code developed to modify SAX Parser. Then, the rest of  
+
        <p>When examined in order, conversion is the second step of device system. While in literature searches, it is found that methanotrophs which live in either extreme conditions or in deep oceans, the process is the same, methane is converted to methanol which is the oxygenated form of hydrocarbon group. The searches directed us to organism <i>Methylococcus capsulatus</i> which is one of the mostly studied organisms among methanotrophs. We designed the conversion part of this organism and modified coding operon according to <i>E.coli</i> strain to enhance the expression. The monoxygenase coding part with  protein A, B and C products are designed for conversion step of  this project.</p>
-
the data which needs to be standardized according to biological importance
+
                         
-
have been extracted from the Registry of Parts Page manually. The chemical
+
                            <h3>ii) Background </h3>
-
(IPTG, galactose etc.) or physical (UV irradiation, temperature etc)  
+
<p> Methanotrophs - methane oxidizing bacteria- are ubiquitous in the environment and play an important role in mitigating global warming because they are a unique group of gram-negative bacteria that grow aerobically on methane as sole source of carbon and energy (Hanson and Hanson 1996). They are also potentially interesting for industrial applications such as production of bulk chemicals or bioremediation. Methanotrophs converts methane to methanol and subsequently to formaldehyde which is assimilated or further oxidized to CO2 for biosynthesis of cellular forms.In this biosynthesis pathway the conversion of methane to methanol is catalysed  by  methane monooxygenase (MMO) which is a non-specific enzyme complex that also catalyse the oxidation of a wide range of aliphatic,  alicyclic  and aromatic compounds  (Colby  et a l . , 1977; Higgins et a l . ,1979). The first step in the oxidation of methane to precursors of organic compounds is the conversion to methanol by methane monooxygenase, the key enzyme, which exists in two forms: the cytoplasmic, soluble methane monooxygenase (sMMO) and the membrane-bound, particulate methane monooxygenase (pMMO).  sMMO components have been expressed in heterologous and homologous hosts. Therefore we searched more on soluble form of monooxygenase enzyme. When analyzed in detail, pMMo differs from sMMo by copper ion concentration. Copper ions have been shown to play a key role in regulating the expression of both MMO enzyme complexes. When its concentration increases the differentiation goes through particulate form. Here we analyzed the soluble form of methane monooxygenase to express in heterologous host.</p>
-
external inputs and proteins synthesized from a biobrick coding sequence
+
 
-
can affect promoters on the parts. These effectors are identified under  
+
-
the title “Input”. And the “Output” s of these effectors are classified
+
                          <h4>a) Soluble Methane Monooxygenase </h4>
-
as inducers (a molecule that starts gene expression), repressors (blocker
+
<p> In contrast to pMMO, sMMO has extremely broad substrate specificity and can oxidise a wide range of non-growth substrates such as alkanes, alkenes and aromatic compounds thus making it the more attractive enzyme for co-oxidation reactions. sMMO is expressed only under conditions in which the copper-to-biomass ratio is low, i.e. under “low-copper” growth conditions, when copper ions are omitted from the trace elements solution of a standard mineral salts medium or cells are grown in a fermentor to high cell densities.There is also some evidence that copper ions inhibit the activity of sMMO (Jahng and Wood 1996). Like many other multi-component oxygenase systems, sMMO contains a component of approximately 16 kDa, Protein B, which serves an “effector” or regulatory role. The activity of Protein B may be regulated by proteolysis at its amino terminus (Lloyd et al. 1997). At low concentrations, Protein B converts the hydroxylase from an oxidase and stabilizes intermediates necessary for oxygen activation. Saturating amounts of Protein B dramatically increase the rates of formation of intermediates and accelerate catalysis of methane to methanol by sMMO (Lee and Lipscomb 1999). When analyzed, the most extensively characterised sMMO enzymes are those from <i>Methylococcus capsulatus</i> (Bath) and <i>Methylosinus trichosporium</i> OB3b (reviewed in Lipscomb 1994; Deeth and Dalton 1998). Therefore we preferred to study on <i>Methylococcus capsulatus</i> (Bath) for monooxygenase.The sMMO is a non-haem iron-containing enzyme complex consisting of three components. The hydroxylase consists of three subunits of 60, 45 and 20 kDa arranged in an α2 β2 γ2 configuration. sMMO genes are clustered on the chromosome of <i>Methylococcus capsulatus</i> (Bath) and <i>Methylosinus trichosporium</i> OB3b. mmoX, mmoY and mmoZ encode the α-,  β- and  γ-subunits respectively of the hydroxylase. mmoB and mmoC code for Protein B and the reductase component. Interestingly, mmoB lies between mmoY and mmoZ;  an ORF of unknown function, designated orfY, with a coding capacity of 12 kDa, lies between mmoZ and mmoC in all genes clusters analysed to date (McDonald et al. 1997).</p>
-
of attachment of RNA polymerase to promoter), activators (increasing
+
 
-
the rate of transcription) and inhibitor s(decreasing the rate of transcription).  
+
                      <p> <b>REFERENCE:</b></p>
-
These standardizations on the database helped us to build the algorithm
+
<ol>
-
based on input/output relationships. MySQL Server is used for Database
+
<li>J. Colin Murrell · Bettina Gilbert · Ian R. McDonald, (2000), Molecular biology and regulation of methane monooxygenase Arch <i>Microbiology</i>(2000) 173 :325–33</li>
-
development and organization. All of our illustrations for ER and algorithm
+
<li>Colby, J . &  Dalton, H.  (1978).  Resolution  of  the methane monooxygenase  of Methylococcus capsulatus  (Bath)  into  three components. Biochemical Journal 171,461-468</li>
-
is created in SmartDraw (trial version). Java Programming Language,  
+
<li>Hanson Te&Hanson RS(1996) Methanotrophic bacteria.<i>American Society forMicrobiology</i>.Microbiol. Rev., Jun 1996, 439-471, Vol 60, No. 2</li>
-
and NetBeans Development environment is used or for software development.  
+
<li>Charlotte, A., West, G. & Horward, D. (1992). Functional expression in Escherichia coli of proteins B and C from soluble methane monooxygenase of Methylococcus capsulatus (Bath. <i>Journal of General Microbiology,</i> (138), 1301-1307.</li>
-
The graphical visualization of the software is done with Cyctoscape
+
</ol>
-
and yfiles libraries (trial version) are used for the presentation of  
+
 
-
graphical events. We have utilized css Javascripts for our webdesign.  
+
 
-
Autodesk Maya 2011 with Academic Licence, Adobe Creative Suite 5 Master
+
                          <h3>iii) Modelling </h3>
-
Collection (Trial Version) have been used for animations and illustrations.  
+
 
-
Video tutorial for the BioGUIDE has been created by camstudio and trial
+
<p>For this step of MethanE.COLIc project, the experiments were planned and modelled as protein characterizations. The protein B and C were previously characterized and analyzed proteins in literature. Based on this publications we planned the experiments as purifying protein from cell extract then SDS-PAGE analysis. </p>
-
versions of Flash and After Effects are also used for the videos.
+
-
</p>
+
</div>
</div>
</div>
</div>
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<div class="item">
<div class="item">
</div>
</div>
-
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+
<div id="wetlab3" class="item">
<div class="content2">
<div class="content2">
<div class="text">
<div class="text">
-
<h2>Supporting Tools</h2>
+
<img src="https://static.igem.org/mediawiki/2011/2/28/Wetlabheader.png" height="75px" width="890px">
-
<ul>
+
<h2>Biobricks</h2>
-
<li>SAX Parser ( modified )to parse XML files</li>
+
<div class="biobir"><img src="https://static.igem.org/mediawiki/2011/f/f1/Lush.PNG">&nbsp;&nbsp;&nbsp;<strong>LUSH  protein coding device / </strong><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K593002" target="_blank">BBa_K593002</a></div>
-
<li>Java ProramingLanguage, NetBeans Developement environment for
+
 
-
software developement</li>
+
<div class="biobir"><img  src="https://static.igem.org/mediawiki/2011/d/d8/Composite.PNG" height="75px" >&nbsp;&nbsp;&nbsp;<strong>ROSE  regulated kill-switch / </strong><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K593000" target="_blank">BBa_K593000</a></div>
-
<li>MySQL Server for DataBase</li>
+
<br />
-
<li>cystoscape for graph visualization</li>
+
<br />
-
<li>yfiles library for graph events</li>
+
<br />
-
<li>SmartDraw for illutration of ER and Algorithm</li>
+
<br />
-
<li>maya &amp; Cinema 4D for 3D animation, Adobe Master Collection and
+
<br />
-
Microsoft Expression Studio for design</li>
+
<br />
-
<li>CSS, Java Script for web</li>
+
<br />
-
</ul>
+
<br />
 +
<div class="biobir"><img src="https://static.igem.org/mediawiki/2011/3/3a/Untitled1.PNG">&nbsp;&nbsp;&nbsp;<strong>Lush  protein / </strong><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K593003" target="_blank">BBa_K593003</a></div>
 +
 
 +
<div class="biobir"><img src="https://static.igem.org/mediawiki/2011/3/3a/Untitled1.PNG">&nbsp;&nbsp;&nbsp;<strong>mmo  X / </strong><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K593004" target="_blank">BBa_K593004</a></div>
 +
 
 +
<div class="biobir"><img src="https://static.igem.org/mediawiki/2011/3/3a/Untitled1.PNG">&nbsp;&nbsp;&nbsp;<strong>mmo  Y / </strong><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K593005" target="_blank">BBa_K593005</a></div>
 +
 
 +
<div class="biobir"><img src="https://static.igem.org/mediawiki/2011/3/3a/Untitled1.PNG">&nbsp;&nbsp;&nbsp;<strong>mmo  Z / </strong><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K593006" target="_blank">BBa_K593006</a></div>
 +
 
 +
<div class="biobir"><img src="https://static.igem.org/mediawiki/2011/3/3a/Untitled1.PNG">&nbsp;&nbsp;&nbsp;<strong>mmo  B / </strong><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K593007" target="_blank">BBa_K593007</a></div>
 +
 
 +
<div class="biobir"><img src="https://static.igem.org/mediawiki/2011/3/3a/Untitled1.PNG">&nbsp;&nbsp;&nbsp;<strong>mmo  C / </strong><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K593008" target="_blank">BBa_K593008</a></div>
 +
 
 +
<div class="biobir"><img src="https://static.igem.org/mediawiki/2011/3/3a/Untitled1.PNG">&nbsp;&nbsp;&nbsp;<strong>mmo  D / </strong><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K593010" target="_blank">BBa_K593010</a></div>
 +
 
 +
<div class="biobir"><img src="https://static.igem.org/mediawiki/2011/f/fa/Untitled12.PNG">&nbsp;&nbsp;&nbsp;<strong>ROSE  regulated kill switch / </strong><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K593009" target="_blank">BBa_K593009</a></div>
 +
 
 +
<div class="biobir"><img src="https://static.igem.org/mediawiki/2011/c/c0/Rosebr.png">&nbsp;&nbsp;&nbsp;<strong>ROSE  regulated  GFP generator/ </strong><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K593011" target="_blank">BBa_K593011</a></div>
</div>
</div>
</div>
</div>
</div>
</div>
-
<div id="miscellaneous3" class="item">
+
<div id="humanpractice3" class="item">
<div class="content2">
<div class="content2">
<div class="text">
<div class="text">
-
<h2>Safety</h2>
+
<img src="https://static.igem.org/mediawiki/2011/1/1b/Humanpractice.png" height="75px" width="890px">
-
<p>Synthetic biology has the potential to impact many areas of society.  
+
<h2>Public Awareness</h2>
-
Synthetic biologists may use artificial molecules to reproduce emergent
+
                      <h3>5-10 year old group</h3>
-
behavior from natural biology, with the goal of creating artificial
+
<center><iframe width="560" height="315" src="http://www.youtube.com/embed/DemmlqdfPnk" frameborder="0" allowfullscreen></iframe></center>
-
life or seeking interchangeable biological parts to assemble them into
+
<p>For this age group, we prepared and modified one of the national child game. In this game there is a circle of group of child, one is not included in that circle. He/she runs around the circle and labels two of the participants in the circle. When the labelled kids realized that, they leave the circle and new kids join the circle. The video below shows our modified game in which four kids that out of circle are our restriction enzymes wearing white caps. Moreover, we have three insert groups which wear red, green and yellow caps. The blue capped ones are our backbone.<br /> During the game we explained kids what DNA, enzyme and insert are, and what we have done as a Synthetic Biology research group.</p>
-
devices and systems that function in a manner not found in nature (Benner
+
                      <br />
-
and Sismour 2005, Endy 2005, Heinemann and Panke 2006, Luisi 2007, Serrano
+
                      <div id="slider4">
-
2007).There is possibility of causing intentional or accidental harm
+
-
to humans, agriculture or the environment. While deliberate damage is
+
<img src="https://static.igem.org/mediawiki/2011/4/4e/DSCN1645.JPG">
-
dealt with under the heading biosecurity, the potential unintended consequences
+
<img src="https://static.igem.org/mediawiki/2011/d/d6/DSCN1651.JPG">
-
have to be considered under the term biosafety. As a software developer
+
<img src="https://static.igem.org/mediawiki/2011/d/d2/1682.JPG">
-
, we have to consider all possible maliciously use of synthetic biology  
+
<img src="https://static.igem.org/mediawiki/2011/9/9b/IMG_0084.JPG">
-
tools. However, it is diffucult to understand for which purposesour
+
<img src="https://static.igem.org/mediawiki/2011/0/04/IMG_0087.JPG">
-
tools will be used, bestway avoiding garage bioterrorism is all parts
+
<img src="https://static.igem.org/mediawiki/2011/c/cb/IMG_0092.JPG">
-
before adding to partsregistry must be checked , looked for toxic affect
+
<img src="https://static.igem.org/mediawiki/2011/6/65/IMG_0106.JPG">
-
and any environmental or human harmness. After scanning possible candidate
+
<img src="https://static.igem.org/mediawiki/2011/f/f2/IMG_0122.JPG">
-
parts, a committe should decide whether novel part can be added parts
+
<img src="https://static.igem.org/mediawiki/2011/8/81/IMG_0134.JPG">
-
registry or not.And we can only warn the user about our intention while
+
<img src="https://static.igem.org/mediawiki/2011/1/17/IMG_0136.JPG">
-
building the application: </p>
+
 
-
<p>“BioGUIDE v1.0 software is <strong>FOR RESEARCH USE ONLY</strong>,
+
</div>
-
no medical or diagnostic use for applications of the novel Biobrick
+
 
-
constructs generated through our software has been described “</p>
+
                      <h3>10-17 year old group</h3>
-
<p>“No military (defense or combat) applications will be allowed in  
+
<p>This group consists of high school aged children groups.Through out the workshop,  we have paired them in doubles. Firstly, we have explained  Synthetic Biolgy  and what Synthetic Biology is able to do. After these training, we explained them our project and taught them basic molecular biology procedures and what are the requirements of basic synthetic biology based experiments. We have done the experiments (cloning procedures) together and also they did individually. We also encouraged them to join iGEM High School division.</p>
-
future”</p>
+
 
-
</div>
+
<h3> Official Announcement To All Universities For Introduction Of Synthetic Biology and iGEM Competetion</h3>
-
</div>
+
                   
 +
<p>For the last human practice approach, we believed that we  should inform especially students from other universities about synthetic  biology as students in universities are the ones who can improve this field for  further generations. For that purpose, we published an official message from  our instructor, Prof. Dr. Mahinur Akkaya, with this message, students from  other universities –regardless of their department that they’re studying in-  will be aware of the field synthetic biology and may not only consider about  learning more but also consider about creating an iGEM team for coming years.  Here is the original official message and its brief translation for you to read:<br />
 +
  Dean Office of Arts and Science Department<br />
 +
  Synthetic biology is a field where  natural sciences and engineering sciences go  together hand in hand. It is an emerging science that requires the literature  search, knowledge  of natural sciences  and methods of engineering sciences. Synthetic biology is the engineering of organisms to give them desired properties, except their regular metabolic  activities. These desired properties are entegrated into genome of the organism  with molecular cloning procedures.<br />
 +
  There are lots of studies going on to introduce this newly  emerging science field to people from all age groups and producing an  information repository and improving it at the same time. One of this studies  is the iGEM (International Genetically Engineered Machines) competition.<br />
 +
  We, as METU iGEM teams, are attending this competition  for four years. For this newly emerging science field to improve more, we believe that it should be spread out to other universities and more and more  people should attend this competiton. We are sending you our study about the  videos of lab protocols that are mainly used in synthetic biology and  references that you can search for more information.  We feel honored to tell that these sources  are available for everyone who would like to reach them. Links for the sources  are as follows:<br />
 +
  <a href="http://ocw.metu.edu.tr/course/view.php?id=137">http://ocw.metu.edu.tr/course/view.php?id=137</a> <br />
 +
  <a href="http://www.youtube.com/user/Metuankara">http://www.youtube.com/user/Metuankara</a>
 +
<br />
 +
<br />
 +
<p><img width="900px" src="https://static.igem.org/mediawiki/2011/b/bc/Imagetif.png"></p>
 +
<br />
 +
<br />
 +
 
 +
                        <h3>18-24 year old group</h3>
 +
<p>This group consists of METU students. We arranged the Physics lectures in order to reach students from several departments. We explained the miracle of Synthetic Biology and our project. At the end of the presentation, we gained many volunteers to join METU-iGEM team for the following years. Also, we have an online lecture application as METU open course ware. For  this web page, we have prepare the course notes and they were uploaded to reach many students from all around  to inform and contact for further relations about Synthetic Biology.
 +
<br />
 +
In addition, we distributed hand outs about our project and Synthetic Biology at METU campus, when the registration of new students were done. We aimed to  inform students about our project and about Synthetic Biology to attract their attention on the issue.</p>
 +
<p>Online lecture link:&nbsp: <a href="http://ocw.metu.edu.tr/course/view.php?id=137" target="_blank">http://ocw.metu.edu.tr/course/view.php?id=137</a>
 +
 
 +
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<img src="https://static.igem.org/mediawiki/2011/6/60/DSCN2099.JPG">
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<img src="https://static.igem.org/mediawiki/2011/1/11/111.JPG">  
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</div>
 +
                       
 +
 
 +
                                <h3>Above 24 year old</h3>
 +
 
 +
 
 +
<p>For this group of people we met with group of engineers who work in mining fields in Turkey. We explained our project in details and consulted them on background of our project. What the conditions in mines are, what the general regulations for worker safety are and what the rooted controls for any gas release are the main points which we discussed on.</p>
 +
<h3>List of Engineers:</h3>
 +
<h3>Turkish Architects and Engineers Association members:</h3>
 +
 
 +
<center> <table align="center" border="1" cellspacing=”2″>
 +
<tr>
 +
<b><td width="50">NO</td>
 +
<td width="200">NAME & SURNAME</td>
 +
<td width="250">INSTITUTION</td>
 +
<td width="100">CITY</td></b>
 +
    </tr>
 +
    <tr>
 +
<td width="50">1</td>
 +
<td width="200">Başak GÜLEKEN</td>
 +
<td width="250">ERKUNT DÖKÜM A.Ş.</td>
 +
<td width="100">ANKARA</td>
 +
    </tr>
 +
    <tr>
 +
<td width="50">2</td>
 +
<td width="200">Saliha SİVRİ</td>
 +
<td width="250">ETİ MADEN İŞLETMELERİ</td>
 +
<td width="100">ANKARA</td>
 +
    </tr>
 +
    <tr>
 +
<td width="50">3</td>
 +
<td width="200">Aynur ODAMAN</td>
 +
<td width="250">KOSGEB</td>
 +
<td width="100">ANKARA</td>
 +
    </tr>
 +
    <tr>
 +
<td width="50">4</td>
 +
<td width="200">Mehmet Şevki TÜRÜDÜ</td>
 +
<td width="250">MADEN TETKİK ARAMA KURUMU</td>
 +
<td width="100">ANKARA</td>
 +
    </tr>
 +
    <tr>
 +
<td width="50">5</td>
 +
<td width="200">Galip Şevket YİNSEL</td>
 +
<td width="250">MİTAŞ A.Ş</td>
 +
<td width="100">ANKARA</td>
 +
    </tr>
 +
    <tr>
 +
<td width="50">6</td>
 +
<td width="200">Cemil Hakan GÜR</td>
 +
<td width="250">ORTA DOĞU TEKNİK ÜNİVERSİTESİ</td>
 +
<td width="100">ANKARA</td>
 +
    </tr>
 +
 +
          </table></center>
 +
<br />
 +
<br />
 +
 
 +
                           
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<div class="text">
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<img src="https://static.igem.org/mediawiki/2011/f/f8/Partnershipheader.png" height="75px" width="890px">
 +
<h2>Sponsors</h2>
 +
<p>As METU Ankara iGEM 2011 team we would like to thank to our partners and sponsors in listed below for their supports and for their encouragement.</p>
 +
<center>
 +
<div class="sponsors"><center><h3>Cargill Comart</h3>
 +
<p><img src="https://static.igem.org/mediawiki/2011/d/d6/Logo_cargill_reg.jpg" height="100px" width="200px"></p></center></div>
 +
<div class="sponsors"><center><h3>Sentegen</h3>
 +
<p><img src="https://static.igem.org/mediawiki/2011/d/d8/Sentegen.jpg" height="100px" width="200px"></p></center></div>
 +
<div class="sponsors"><center><h3>MetuTech</h3>
 +
<p><img src="https://static.igem.org/mediawiki/2011/9/9d/Metutech.jpg" height="100px" width="200px"></p></center></div>
 +
<div class="sponsors"><center><h3>Onkogen</h3>
 +
<p><img src="https://static.igem.org/mediawiki/2011/a/a7/Onkogen.jpg" height="100px" width="200px"></p></center></div>
 +
<div class="sponsors"><center><h3>Sacem</h3>
 +
<p><img src="https://static.igem.org/mediawiki/2011/8/8c/Sacem.png" height="100px" width="200px"></p></center></div>
 +
<div class="sponsors"><center><h3>Metu</h3>
 +
<p><img src="https://static.igem.org/mediawiki/2011/8/8a/Metud.png" height="100px" width="200px"></p></center></div>
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-
<h2>Methods</h2>
+
<img src="https://static.igem.org/mediawiki/2011/1/1c/Projectheader.png" height="75px" width="890px">
-
<h3>Part Extraction Standards</h3>
+
<h2>Entrapment</h2>
-
<p>All information about the parts that are essential in experimental
+
<center><p><img src="https://static.igem.org/mediawiki/2011/c/c2/Entrapment.gif" height="454px" width="640px"></center></p>
-
setup of iGEM projects has been utilized. The information for the parts
+
<h3>i) Introduction</h3>
-
available provided with all three 384 well plates in Spring 2010 distribution
+
<p>After conversion of methane to methanol, the successive step is the entrapment of methanol. Methanol is an alcohol form of hydrocarbon methane and includes hydroxyl group. Hydroxyl radical groups are highly reactive and, and so short-lived; however, they form an important part of radical chemistry. Hydroxyl groups are especially important in biological systems and their chemistry because free radicals tend to form hydrogen bonds both as donor and acceptor. This property is also related to their ability to increase hydrophilicity and water solubility. Therefore hydroxyl free radicals cause damage to oxidative cells and cellular membranes. In order to produce the methanol for further industrial manufacturing in cellular organism, the organismic conditions should be adjusted. In the light of this, for our bacterial system we transferred the protein coding sequence(named as LUSH) of <i>Drosophilia melanogaster</i> to attack to free methanols and hydroxyl group. Here is some structural information about protein LUSH.</p>
-
have been standardized. Our standardization criteria have been discussed
+
-
in detail under Database Standardization. ER diagram has been generated
+
<h3>ii) Background</h3>
-
which simply describes the organization of the data. Around 70% of the
+
<p>LUSH is an alcohol-sensitive odorant binding protein expressed in the olfactory organs of <i>Drosophila melanogaster</i>, and it is used as a model system to investigate the biophysical nature of
-
parts information has been fetched by the custom parsing code from XML
+
alcohol-protein interactions at alcohol concentrations that produce intoxication in humans. In this study, by using NMR spectroscopy, they have identified the regions of  LUSH that show increased conformational stability on binding alcohols. These residues primarily line the alcohol-binding pocket. A direct measure of the degree of stability that alcohol imparts on LUSH has been provided. LUSH was originally identified as responsible for mediating an avoidance response to short-chain n-alcohols.</p>
-
and Excel files provided by iGEM. Rest of the data had to be collected
+
<p>The general structure of odorant binding proteins consists of six α-helices surrounding a hydrophobic ligand- binding pocket which differs in size and shape between each protein. All these odorant binding proteins have a set of six cysteines that form three conserved disulfide bonds. In the study, by observing the X-ray crystal structures of LUSH-alcohol complexes, it was found that alcohol binds to a single site in the protein formed by a network of concerted hydrogen-binding residues located at one end of hyrophobic pocket. This binding site has some sequence and/or structural similarities to regions of several ligand gated ion channels (LGICs) that have previously been implicated in inferring sensitivity to alcohol. It is hypothesized in the study that the alcohol-binding site in LUSH may represent a more general structural motif for functionally relevant alcohol-binding sites in proteins. The characterization of the effects of n-alcohols on the structure and stability of LUSH is presented. Also, in the absence of ligand, LUSH exists in vitro in a partially unstructured state and binding of alcohols shifts the solution conformation to a more compact folded state which is accompanied by an increase in the overall protein stability. Those regions of the protein that show the largest changes in local dynamics on binding alcohol have been identified and it have been shown that these are predominantly associated with the residues that line the alcohol-binding pocket. The results provide a quantitative measure of the ability of short-chain alcohols to stabilize protein structure at physiological relevant concentrations. </p>
-
and organized manually as the organization of these data cannot be standardized
+
 
-
to generate an algorithm. This step was one of the most time consuming
+
                    <p> <b>REFERENCE:</b></p>
-
steps in our project. For each construct and Biobrick the information
+
<ol>
-
collected was; Activity, Inducer, Activator, Repressor and Inhibitor
+
<li>Bucci, B. K., Kruse, S. W. & Thode, A. B. (2006). Effect of n-Alcohols on the Structure and Stability of the <i>Drosophila</i> Odorant Binding Protein LUSH.<i> Biochemistry,</i> 45 1693-1701</li>
-
for promoters and Inducer, Activator, Repressor and Inhibitor information
+
 
-
valid for synthesized molecules (mostly proteins and RNA fragments etc.)</p>
+
<li>Thode, A. B., Kruse, S. W. & Nix, J. C. (2008). The role of multiple hydrogen bonding groups in specific alcohol binding sites in proteins: Insights from structural studies of LUSH.<i> J.Mol Biol,</i> 376 (5), 1360-1376</li>
-
<h3>Combination</h3>
+
</ol>
-
<p>Rules (Image Combinations) In order to build our input/output relations
+
 
-
graphs first we run our algorithm on the real combination dataset which
+
-
contains all few thousand different possible combinations of the biobricks.  
+
                        <h3>iii) Modelling </h3>
-
But after performing all combinations for the first few hundred biobricks
+
<p>In Methan<b>E.COLI</b>c project, LUSH protein is designed to hold methanol; the conversion product of methane. Methanol is bounded to LUSH protein through its hydroxyl group. In device experiments of this part, we planned to isolate and denaturate protein and then refold in presence of methanol and we planned to analyze the alcohol bounded protein in X-ray diffraction crystallogrophy.</p>
-
application’s rate slowed downed tremendously, which also become very
+
 
-
time consuming for displaying biobricks graphs. To overcome this bottleneck
+
-
we have developed a new strategy, where we have only used the construct
+
-
combinations of the biobricks distributed within the plates. Moreover,  
+
-
according to information gathered from the subparts of the constructs
+
-
distrubuted, we also collected the subpart assembly order, such as 1st:  
+
-
promoter, 2nd:rbs, 3rd:coding seq, any internal parts and the Last:
+
-
terminator. Each specific Biobrick type has been assigned a number as
+
-
a unique image ID from 1 to 19. Gathering the information on subparts
+
-
was not a direct forward process. ImageID assembly orders for each construct
+
-
has been used to extract the type information for each subpart with
+
-
that construct. This innovative approach helped us to reveal 400 possible
+
-
brick combinations present within the 3x384 well plates distributed
+
-
by iGEM in Spring 2010.</p>
+
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+
<div id="wetlab4" class="item">
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<div class="text">
 +
<img src="https://static.igem.org/mediawiki/2011/2/28/Wetlabheader.png" height="75px" width="890px">
 +
<h2>Notebook</h2>
 +
 
 +
<div id="slider6">
 +
 +
<img src="https://static.igem.org/mediawiki/2011/8/87/Notebook-1.png">
 +
<img src="https://static.igem.org/mediawiki/2011/c/c5/Week2.png">
 +
<img src="https://static.igem.org/mediawiki/2011/8/84/Week3.png">
 +
<img src="https://static.igem.org/mediawiki/2011/4/48/Week4.png">
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<img src="https://static.igem.org/mediawiki/2011/5/5d/Week5.png">
 +
<img src="https://static.igem.org/mediawiki/2011/8/8d/Week6.png">
 +
<img src="https://static.igem.org/mediawiki/2011/0/00/Week7.png">
 +
<img src="https://static.igem.org/mediawiki/2011/7/72/Week8.png">
 +
<img src="https://static.igem.org/mediawiki/2011/a/ab/Week9.png">
 +
<img src="https://static.igem.org/mediawiki/2011/1/12/Week10.png">
 +
<img src="https://static.igem.org/mediawiki/2011/8/8e/Week11.png">
 +
<img src="https://static.igem.org/mediawiki/2011/a/a4/Week13.png">
 +
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 +
 
 +
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 +
<div id="humanpractice4" class="item">
<div class="content2">
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<div class="text">
-
<h2>Contact</h2>
+
<img src="https://static.igem.org/mediawiki/2011/1/1b/Humanpractice.png" height="75px" width="890px">
-
<p>For critics, suggestion, or appraisal, you can contact us on
+
<h2>Media Press</h2>
-
<a href="mailto:software_metuturkey@googlegroups.com">software_metuturkey@googlegroups.com</a></p>
+
 
 +
<center><p><iframe width="420" height="315" src="http://www.youtube.com/embed/Femv5f6V8UM" frameborder="0" allowfullscreen></iframe>
 +
<p><img src="https://static.igem.org/mediawiki/2011/2/26/Untitled-ntvmsnbc.png"></p></center>
</div>
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<h2>Future Plan</h2>
+
<img src="https://static.igem.org/mediawiki/2011/f/f8/Partnershipheader.png" height="75px" width="890px">
-
<p>The application we have developed can be used by all iGEM members.
+
<h2>Middle East Technical University</h2>
-
As the iGEMs database expands and the recognition of the field of Synthetic
+
<p><img src="https://static.igem.org/mediawiki/2011/8/8a/Metud.png" height="100px" width="200px"></p>
-
Biology increases, data resources from other biological databases such
+
<p>Middle East Technical University is a pioneering university in education, research and community service in the country. In addition to being highly preferred by students, it is internationally recognized.Our country is successfully represented by METU academic staff in numerous scientific and professional institutions and committees.The wide variety of cultural, artistic and intellectual activities, and sports opportunities that are offered by our campuses are a part of the "education environment" of international quality. METU has been defined in national rankings as the university which has the "Richest Social Environment".Whatever your reason to visit METU is, you will find here an innovative, dynamic and exciting environment which offers diversity and variety of choices. Come and join us at METU where the most successful students of the country, the most outstanding teaching and research staff, and the most distinguished administrative-technical staff live, learn, and produce hand in hand...</p>
-
as NCBI might be needed to be integrated to the application. In such
+
 
-
a situation, extendibility of the application is vital. New data resources
+
<h3> For more information, visit following link;</h3>
-
and new functions should be added easily. </p>
+
 
-
<p>Before planning ahead, feedback from other teams and iGEM headquarters
+
<a href="http://metu.edu.tr" target="_blank">Middle East Technical University</a>
-
about the BioGuide 1.0 will be collected. That will help us to fill
+
-
in the missing features of the application and check the theories which
+
-
are basis of our algorithms. </p>
+
-
<p>As our application is not geared towards any commercial use and will
+
-
stay as an academic application, keeping track of the weekly developmental
+
-
process on the wiki notebook environment was satisfactory. If the need
+
-
for a commercial application emerges, we should be utilizing professional
+
-
software development approaches to determine the exact requirements
+
-
and to facilitate the use of a common language between interdiciplinary
+
-
members in the team. </p>
+
-
<p>For constructing BioGuide 2.0 we have some plans. </p>
+
-
<h3>Short Term plan:</h3>
+
-
<p>Next year we are planning to generate BioGuide 2.0 by using all parts
+
-
data but inorder to do this we will update our part database but easyway
+
-
is standardization and reorganizing all parts in partsregistrty.org
+
-
according to our suggestion because re-organizing and normalization
+
-
are crucial. We are planning to add new tools to improve graphs.Our  
+
-
ultimate aim is finding best pathway based on automated construction
+
-
and input-output relation. BioGuide 2.0 will be more faster because
+
-
we are planning to use OODBMS and all all platform will support BioGuide
+
-
2.0. </p>
+
-
<h3>Long term plan:</h3>
+
-
<p>We want to improve our algoritm and add more parameter to make graphs
+
-
more effective. Our dream is embeding our software into partsregistry.org
+
-
so no mere iGEMers will choose parts in real time by using our software.
+
-
</p>
+
-
<h3>Suggestions based on PartsRegistry Survey Results</h3>
+
-
<p>First suggesiton is offering fartnership with Google for easy search
+
-
and founding a committe to enforce a standardized nomenclature for terminology
+
-
and parts registry entries. </p>
+
-
<p>We strongly suggest starting a forum on how to quantify the performance
+
-
of promoters and genes to bring an easy to measure standard for the  
+
-
efficiency of the parts. Additionally iGEM should assume the responsibility
+
-
and provide the measurements for the each promoter and gene included
+
-
in the distributions. The second choice would beeven better in terms
+
-
of standardization as all the measurement will be performed by one center
+
-
under similar conditions and with experienced researchers, which will
+
-
allow user to compare and contrast the efficiencies of the parts more
+
-
accurately. We suggest excluding the parts not-working, low rated or
+
-
with negative feedbacks from the annual distribution plates but still
+
-
archive them and make their data available through the parts registry.
+
-
So the while the individuals labs are receiving plates with higher rated,  
+
-
fully working parts for their projects, anyone who wants to work on
+
-
a more exotic part can search through the achieves and re-vitalize the
+
-
parts stored there. The challenge of re-vitalization of parts can be
+
-
encouraged as an collaborative effort. We are sure, all of us would
+
-
like to see gene-protein and pathway information if these information  
+
-
was integrated into the database and offered automatically for each
+
-
entry in the database. We are planning to provide this information about
+
-
the parts to all parts registry users as a build-in option in the next
+
-
version of BioGuide in iGEM 2011. </p>
+
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<div class="text">
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<h2>Database Standardization</h2>
+
<img src="https://static.igem.org/mediawiki/2011/1/1c/Projectheader.png" height="75px" width="890px">
-
<p>Two main focuses of our project was the organization of the available
+
<h2>Kill switch</h2>
-
information about Biobricks on iGEM’s website and development of a software
+
<center><p><img src="https://static.igem.org/mediawiki/2011/6/66/Killswitch.gif" height="454px" width="640px"></center></p>
-
application to help synthetic biologists at the experimental set-up
+
<h3>i) Introduction </h3>
-
level by providing all available construct combinations for any given
+
<p>In this project the last step is the  death of our bacteria to elute the protein(LUSH) bounded product methanol which is one of the precursors of biofuel. In our modified bacteria, the death of cells occurs by temperature dependent cell lysis device. The expression of this device takes place at 42 C since there is an RNA thermometer part in upstream of this device. At the downstream of this device there is an lysis casette. When transcription initiated at 42C holin and endolysin is expressed from this device and make cell dead.</p>
-
input and output relations ,which they can utilize for their own project.</p>
+
                        <h3>ii) Background </h3>
-
<p>Normalization and re-organization of the part information at iGEM’s
+
<p>This composite part consists of promoter, RNA thermometer, GFP and lysis casette. The upstream part of this composite is T7 promoter  (BBa_I712074) which is strong promoter from T7 bacteriophage. For strong transcription T7 promoter expression system is chosen. In downstream of promoter there is RNA thermometer(ROSE) coding region which determines the transcription of our lysis composite part. RNA thermometer (BBa_K115001) is temperature sensitive that at 42C translation initiates. That means, up to 42 C RNA thermometer is in dimer form and at 42C it becomes linear. In downstream of RNA thermometer, green fluorescence protein(BBa_E0040) is ligated. This ligation is done to measure expression level and to comment on RNA thermometer efficiency. The last ligate to this composite is bacteriophage 21 lysis casette (BBa_K124003). This lysis casette induces lysis in E.coli. This gene cassette consists of the S protein (Holin), R protein (Endolysin), and Rz protein. When translation initiates at 42 C then the following coding gene begins to produce the proteins which lyse the cell.</p>
-
web site was needed in order to develop our application, which will
+
                          <h3>iii) Modelling </h3>
-
automatically search the possible construct combinations. For the organization
+
<p>In Methan<b>E.COLI</b>c project, this composite is modelled to lyse and kill the bacteria when switched on at 42C. In experimental design, we ligated this composite without lysis casette (with promoter, ROSE and GFP) We planned two control groups. One is T7 promoter and GFP ligate, this is designed to measure expression level of T7 promoter by reading the GFP expression. The second control group is T7 promoter, ROSE and GFP ligate, this is designed to measure the expression level of T7 promoter with ROSE ligate at 37 C. The device experiment is done with T7 promoter ROSE and GFP ligate at 42 C in which  the GFP expression is expected.</p>
-
and analysis of the Biobricks, we used part info for Spring 2010 distribution.  
+
-
The information on all three 384 well plates distributed by iGEM scrutinized
+
-
and checked individually to specify the standards available and needed.
+
-
iGEM is providing so many parts within a hierarchical way, but there
+
-
is no order in the information flow and no common standards. Furthermore,
+
-
the information bulk is being used in an ineffective manner. Some of
+
-
the parts distributed are known to be nonfunctional. Web pages for parts
+
-
contain lots of information, but majority of them, are again not ordered.
+
-
Moreover, some additional information had to be removed or replaced
+
-
in such a way that the information for parts can be used effectively.
+
-
And removal of the redundant bulk information related with parts at
+
-
iGEM’s web site had been recommended for future. </p>
+
-
<p>Although, the final standardization, which we have suggested is not
+
-
for general public use and it was urgently needed in order to satisfy
+
-
the needs of our algorithm. But, still it will be a valuable resource,
+
-
since it summarizes the basic information about the parts.</p>
+
-
<p>As the first step to build the proposed standardization template,
+
-
the headings selected related to parts are listed on Table 1. Submission
+
-
of part IDs for individual parts is an accepted and quite valuable way
+
-
of tracking information. Although, every part has unique partID, for
+
-
every part there is a need to assign unique part names as official iGEM
+
-
names. Part names will have an important role as they will be providing
+
-
the short description about the part, which synthetic biologists can
+
-
immediately recognize and utilize during the construction of unique
+
-
Biobricks. Additionally unique part names will be helpful to identify
+
-
the devices with more than one Biobrick in their constructs. Assignment
+
-
of unique and distinct names for parts describing their nature and content
+
-
will be helpful to researchers for the recognition of and search for
+
-
the parts.</p>
+
-
<br>
+
-
<h3>Headings Selected From Previous Entry Forms for Indication of Standardized
+
-
Information</h3>
+
-
<p>=========================================</p>
+
-
<p>PartID:</p>
+
-
<p>PartName:</p>
+
-
<p>Bricks:</p>
+
-
<p>BrickIDs:</p>
+
-
<p>ImageIDs:</p>
+
-
<p>RFC10:</p>
+
-
<p>RFC21:</p>
+
-
<p>RFC23:</p>
+
-
<p>RFC25:</p>
+
-
<p>=========================================</p>
+
-
<p><span>Table 1: The table above basically describes and designates
+
-
qualities of parts which identifies their compositions and demonstrates
+
-
the status of previously assigned standards. PartID refers to the unique
+
-
ID number for parts including atomic parts and assemblies. PartName
+
-
refers to the given unique names to parts. Bricks, refers to the shortcut
+
-
names which specifies atomic parts. ImageIDs, refers to individual or
+
-
combination of numbers that are assigned by us. RFCs refers to the states
+
-
of parts based on RFC standards.</span></p>
+
-
<p>iGEM both provides individual, atomic parts and pre-combined constructs
+
-
such as devices and systems. Availability of combined constructs is
+
-
important to the researchers as combining individual bio-bricks one
+
-
at a time will be very time consuming. These previously merged constructs,
+
-
serve as the repository for puzzle and they can be used for different
+
-
purposes. Up to date the largest and most trustworthy source, for synthetic
+
-
biology and its components, is iGEM’s parts registry. In 2010, iGEM
+
-
provided over 1000 parts that have initiated many projects. Having more
+
-
atomic parts available in the iGEM’s repository, will lead to the design
+
-
of more complex and robust constructs, and we would have a better chance
+
-
to design different constructs for unique purposes. Also, for the parts
+
-
that are already available, extra steps needs to be taken for the quality
+
-
control and surveillance of these products. The quality control of the
+
-
information for the parts is essential for the future of iGEM and synthetic
+
-
biology. Even though we have found pre-determined RFC standards useful
+
-
and included those to our standardized template, some individual parts
+
-
still requires re-organization of the information as RFC standards alone
+
-
for the functionality of parts, does not satisfy the needs for wet lab
+
-
biologists.</p>
+
-
<p>Without a question there is an urgent need to build a distinct and  
+
-
specific database well organized with its own standards for synthetic
+
-
biology; however, development of such a database is not an easy task.</p>
+
-
<br>
+
-
<h3>Contact Information of Part Owners and Qualitative Group Comments
+
-
about Parts</h3>
+
-
<p>=========================================</p>
+
-
<p>Designers: Mail:</p>
+
-
<p>GroupFavorite:</p>
+
-
<p>StarRating:</p>
+
-
<p>Parameters:</p>
+
-
<p>=========================================</p>
+
-
<p><span>Table 2: The above table simply depicts information about possessors
+
-
of parts and their contact information and the popularity of the parts
+
-
for groups. Parameters heading, refers distinctive experimental details
+
-
unique to the usage of parts which should be decided by groups.</span></p>
+
-
<p>Second step for building the standardized template was to get the
+
-
phylogenic information about the parts development process which includes
+
-
the name of the group, designer and contact information, along with
+
-
the comments from the group on the parts they have submitted. Contact
+
-
information is especially important for iGEM as other groups who need
+
-
extra information about the available part can reach to the required
+
-
information. Even though contacting with the designers of the individual
+
-
parts which are available is highly encouraged by iGEM, unavailability
+
-
of contact information points at out the fact that iGEM’s parts registry
+
-
needs strong re-organization in order to serve to the synthetic biology
+
-
community properly.</p>
+
-
<p>Additionally, the “group favorite” and “starRating” fields are also
+
-
important for individual evaluation of the parts, which doesn’t get
+
-
the deserved attention from the iGEM groups. “Group Favorite” defines
+
-
the confidence on the part by the designer group. “StarRating” defines
+
-
the related part in terms of popularity and usage efficiency among the
+
-
groups. According to our observations, most groups are not aware of
+
-
either of the fields or they are used incorrectly or ineffectively.
+
-
For example for a part with a full reporter which is known to be functional
+
-
and gives precise and expected results the StarRating should be at least
+
-
2 stars, but for most of the parts in 2010 distribution, it is very
+
-
difficult to observe a part whose “StarRating” is above one. For quick
+
-
determination of functionality of the parts these two evaluations are
+
-
important so they have been included in the proposed standardization
+
-
template. But, as they were not properly used up to now for the re-organization
+
-
of the parts information during the development of our software application
+
-
we had to include all parts to our queries regardless of their evaluations
+
-
based on “Group Favorites” and “ StarRatings”</p>
+
-
<p>Second step for building the standardized template was to get the
+
-
phylogenic information about the parts development process which includes
+
-
the name of the group, designer and contact information, along with
+
-
the comments from the group on the parts they have submitted. Contact
+
-
information is especially important for iGEM as other groups who need
+
-
extra information about the available part can reach to the required
+
-
information. Even though contacting with the designers of the individual
+
-
parts which are available is highly encouraged by iGEM, unavailability
+
-
of contact information points at out the fact that iGEM’s parts registry
+
-
needs strong re-organization in order to serve to the synthetic biology
+
-
community properly.</p>
+
-
<p>Additionally, the “group favorite” and “starRating” fields are also
+
-
important for individual evaluation of the parts, which doesn’t get
+
-
the deserved attention from the iGEM groups. “Group Favorite” defines
+
-
the confidence on the part by the designer group. “StarRating” defines
+
-
the related part in terms of popularity and usage efficiency among the
+
-
groups. According to our observations, most groups are not aware of
+
-
either of the fields or they are used incorrectly or ineffectively.
+
-
For example for a part with a full reporter which is known to be functional
+
-
and gives precise and expected results the StarRating should be at least
+
-
2 stars, but for most of the parts in 2010 distribution, it is very
+
-
difficult to observe a part whose “StarRating” is above one. For quick
+
-
determination of functionality of the parts these two evaluations are
+
-
important so they have been included in the proposed standardization
+
-
template. But, as they were not properly used up to now for the re-organization
+
-
of the parts information during the development of our software application
+
-
we had to include all parts to our queries regardless of their evaluations
+
-
based on “Group Favorites” and “ StarRatings”</p>
+
-
<br>
+
-
<h3>Input and Output Characteristics of Parts</h3>
+
-
<p>=========================================</p>
+
-
<p>Parameters:</p>
+
-
<p>-Input:</p>
+
-
<p>• Promoter:</p>
+
-
<p>• Activity:</p>
+
-
<p>• Inducer:</p>
+
-
<p>• Activator:</p>
+
-
<p>• Repressor:</p>
+
-
<p>• Inhibitor:</p>
+
-
<p>• Promoter2:</p>
+
-
<p>• Activity:</p>
+
-
<p>• Inducer:</p>
+
-
<p>• Activator:</p>
+
-
<p>• Repressor:</p>
+
-
<p>• Inhibitor:</p>
+
-
<p>-Output:</p>
+
-
<p>• Reporter:</p>
+
-
<p>• Reporter2:</p>
+
-
<p>• Regulator:</p>
+
-
<p>• Inducer:</p>
+
-
<p>• Activator:</p>
+
-
<p>• Repressor:</p>
+
-
<p>• Inhibitor:</p>
+
-
<p>• Regulator2:</p>
+
-
<p>• Inducer:</p>
+
-
<p>• Activator:</p>
+
-
<p>• Repressor:</p>
+
-
<p>• Inhibitor:</p>
+
-
<p>-Working Condition:</p>
+
-
<p>=========================================</p>
+
-
<p><span>Table 3: The table above elaborately describes the input relations
+
-
based on promoters and the output products based on the functional genes
+
-
and RNAs which are included within the parts. Working condition simply
+
-
describes any influencing factor or circumstance which is directly related
+
-
with the functional properties of parts.</span></p>
+
-
<p>Third part of our standardization template includes parameters of
+
-
contingent input and output elements. These parameters are classified
+
-
into two groups for simplicity as presented on Table 3. This final part
+
-
of the standardization template includes the upmost important information
+
-
about the Biobricks that are required for the BioGuide Software to run
+
-
its searching algorithm.</p>
+
-
<p>Briefly, BioGuide application is designed to catch the input and  
+
-
output relations of individual parts to examine possible Biobricks pathways
+
-
for specific input and output queries. In other words, at pre-experimental
+
-
stage, it helps wet lab biologists to design their unique constructs
+
-
by revealing possible alternative options for pre-determined purposes,
+
-
along with the primary paths. Our ultimate goal is to improve the algorithm
+
-
designed for iGEM 2010 and present a new version of the BioGuide in
+
-
iGEM 2011, which will provide optimum design of constructs for predetermined
+
-
parameters.</p>
+
-
<p>Most of the parts are composed of functional and nonfunctional constructs
+
-
which are formed by atomic parts. And every part should carry the information
+
-
for all of its atomic parts within itself. The “input” heading actually
+
-
stands for promoters. Parts with one or more promoters can be found
+
-
at iGEM’s Parts Registry. Along with the information on which and how
+
-
many promoters a part might have, the activity level of promoters are
+
-
also important to distinguish between a constitutively active promoter
+
-
or a promoter activated by specific physiological processes or states
+
-
etc. This information was crucial for us to dissect in order to run
+
-
our algorithm as it directly affects which inputs can activate the devices
+
-
or the systems.</p>
+
-
<p>Throughout our investigations on the Parts Registry, we found out
+
-
that much of the terminology was being used ambiguously. Although this
+
-
might not be vital for synthetic biologists, it is still endeavoring
+
-
to understand the function of certain regulatory elements which also
+
-
becomes a time consuming task for the researcher. Thus, we recommend
+
-
that the explanations of certain regulatory elements should be redefined
+
-
and fixed especially for synthetic biology for easy communication, sharing
+
-
and searching of information.</p>
+
-
<p>Common misuses of the terminology can guide us to figure out how
+
-
to construct a standard nomenclature for synthetic biology. We claim
+
-
that a standard nomenclature is urgently needed for synthetic biology
+
-
for the following reasons. First of all, synthetic biology is an emerging
+
-
research discipline and an industrial application area which is highly
+
-
promising. Secondly, redefinition of the terminology to build a standard
+
-
nomenclature is needed as some of the terms are prone to be used instead
+
-
of another causing problems related to misuse for the global communication
+
-
about synthetic biology. Lastly, the nomenclature has major importance
+
-
for the construction of a persistent and trustworthy database for synthetic
+
-
biology which serves for the information exhibition and exchange globally.
+
-
For instance, there are obvious misunderstandings about the words which
+
-
are predominantly used for regulation process. We have noticed that,
+
-
the terms “inhibitor” and “repressor” are being used as equivocally
+
-
in the part information pages. Like the lactose inhibitor protein, a
+
-
widely used DNA-binding transcriptional repressor, that have been labeled
+
-
both as “inhibitor” and “repressor” at iGEM’s Parts Registry. Similar
+
-
problems resulting from ambiguous use of terminology also observed with
+
-
regulatory elements. To sum up, we investigated all input elements for
+
-
promoters and classify these elements in terms of their function, affect
+
-
and required input element for them. So, we suggest that terminology
+
-
used for regulation of transcription should be defined clearly on iGEM’s
+
-
website and correct use of terminology should be enforced.</p>
+
-
<p>The second group of parameters was collected under the title “Output”,
+
-
which refers to products of functional genes. In contradiction, the
+
-
term “reporter” has also been described within the same list. Reporters
+
-
are also genes whose products, can be used for screening as an output.
+
-
According to our group, the usage of the term “reporter” for genes is
+
-
unnecessary and cause extra complexity for information distribution
+
-
and gives rise to discrepancies. Instead of using the term “reporter”,
+
-
predefined “gene” description should be used for genes, which can function
+
-
as reporters. The special information which is related with the characteristic
+
-
of that gene should also be presented on part info web page.</p>
+
-
<p>Furthermore, the same terminology “reporter” was used for both atomic
+
-
parts and composite bio-bricks. Also the overall image descriptions
+
-
for these were defined as “reporters”. We want to point out that using
+
-
same nomenclature for both atomic genes and for whole functional constructs
+
-
contributes to the complexity and makes specific explorations difficult
+
-
through the Parts Registry. So, assigning “reporter” for both atomic
+
-
parts and for whole constructs is not a good practice. Instead, we are
+
-
suggesting the usage of other available terminology for the parts listed
+
-
as reporters, which most of the constructs, now known as reporters,
+
-
can be grouped into, such as “protein generators”, “composite parts”
+
-
or “inverters”.</p>
+
-
<p>Devices are whole constructs which are functional and have specific
+
-
and distinct functions. But, as we have observed, unfortunately, the
+
-
term “device” is also being used for parts which are not functional
+
-
and do not have specific functional at all. Moreover, within the classification
+
-
of devices, we argue that some terms are also being used unnecessarily
+
-
and ambiguously. Devices are classified into five types which are protein
+
-
generators, reporters, inverters, receivers and senders, measurement
+
-
devices. For example iGEM defines protein generators as:</p>
+
-
<p>Protein generator = promoter + rbs +gene + terminator</p>
+
-
<p>Though we accept the definition for protein generators, we observed
+
-
that there exist numerous parts which are defined as protein generators
+
-
but actually most of them do not fit to the definition provided above.
+
-
Although some parts are not functional and do not generate proteins
+
-
at all, they are classified as protein generators, which makes searching
+
-
for the parts difficult in the registry. Furthermore, there are also
+
-
numerous parts which are defined as “composite parts” but actually they
+
-
fit to the same definition with protein generators. In order to overcome
+
-
the problem of misuse of device type we have extracted related image
+
-
ID information for the composite parts. Image ID information helped
+
-
us to correctly categorize composite parts depending on its individual
+
-
atomic parts and identify the ones with more than one function, such
+
-
as being both inhibitor and activator. In other words, we used image
+
-
and part IDs in order to merge an input for its outputs.</p>
+
-
<p>Subtitle working conditions, includes all the detailed information
+
-
about the experimental properties of parts, and the details about the
+
-
working process of individual parts and complete devices. Additionally,
+
-
we marked the subtitle “Working Condition” in our standardization template
+
-
as potentially the most important title that helps synthetic biologist
+
-
to better understand the parts functions at iGEM’s part registry database.
+
-
The main problem we have encounter with the subtitle “working condition”
+
-
is within most of the parts the details about working process is not
+
-
enough and not provided regularly. </p>
+
-
<br>
+
-
<h3>Examples of Misuse of Terminology:</h3>
+
-
<h4>For Composite Parts:</h4>
+
-
<p>PartID: BBa_S04055</p>
+
-
<p>PartName: Synthetic lacYZ operon</p>
+
-
<img src="https://static.igem.org/mediawiki/2010/a/ac/Metu-database1.png" />
+
-
<p>This part is functional and responsible for the production of LacY
+
-
and LacZ proteins. This part partially fits the definition for “composite
+
-
part” but actually should be a protein generator as it fits fully to
+
-
the definition of “protein generators”.</p>
+
-
<h4>For Protein Generators:</h4>
+
-
<p>PartID: BBa_J45299</p>
+
-
<p>PartName: PchA &amp; PchB enzyme generator</p>
+
-
<img src="https://static.igem.org/mediawiki/2010/2/2c/Metu-database2.png" />
+
-
<p>The part which is illustrated above actually fits the definition
+
-
for “composite part” but in part registry it is classified as protein
+
-
generator. This part can be functional but it needs a promoter. Even
+
-
though this part is not functional and is not capable of producing protein,  
+
-
part registry assigns this product as protein generator. We suggest
+
-
that all parts in the registry, which are composed of more than one
+
-
atomic part and which are not functional on their own but can be functional,
+
-
should be classified as “composite parts”.</p>
+
-
<h4>For Reporters:</h4>
+
-
<p>PartID: BBa_J04451</p>
+
-
<p>PartName: RFP Coding Device with an LVA tag</p>
+
-
<img src="https://static.igem.org/mediawiki/2010/0/0a/Metu-database3.png" />
+
-
<p>This functional part is classified as “Reporter” in the parts registry
+
-
database. It is very clear that this part fits the same description
+
-
as Protein Generator in Biobrick part registry standards. Although,  
+
-
this part has specific and known functional role, characterizing this  
+
-
part as a reporter is unnecessary and contributes to the level of complexity
+
-
of information provided. Instead, we suggest that this part should be
+
-
classified as “protein generator” and related detailed information about
+
-
the specific function of this part, should be provided in the part information
+
-
page.</p>
+
-
<p>In conclusion, as mentioned above we tried to reorganize and normalize
+
-
the information about parts which is provided in part registry for 2010
+
-
in order to develop our algorithm for the BioGuide application. During
+
-
this process, we encountered some inconsistencies and misuses of the
+
-
terminology being used and also inadequacies about the information provided
+
-
about parts. First of all, we claim that a standard nomenclature should
+
-
be constituted for future use in the field of synthetic biology. Based
+
-
on the information gathered according to new nomenclature a professional
+
-
database should be constructed to address the needs of synthetic biology.
+
-
This will enable easy information exchange and exhibition globally.
+
-
Secondly, although there are enough information about parts exists on
+
-
parts registry database, the information which is provided for parts
+
-
need to be ordered urgently. Furthermore, there should be new experimental
+
-
standards which must be introduced to groups in the part submission
+
-
process for the subtitle “working condition”. These experimental standards
+
-
will be important because the experimental details about parts are not
+
-
satisfying the needs of wet-lab biologists for the design and the construction
+
-
of new Biobricks.</p>
+
</div>
</div>
</div>
</div>
</div>
</div>
<div class="item">
<div class="item">
 +
</div>
 +
<div id="wetlab5" class="item">
 +
<div class="content2">
 +
<div class="text">
 +
<img src="https://static.igem.org/mediawiki/2011/2/28/Wetlabheader.png" height="75px" width="890px">
 +
<h2>Contact</h2>
 +
<strong>Device Experiment Results</strong><br />
 +
    <br />
 +
  -<strong>Flask experiments</strong><br />
 +
    <br />
 +
  In characterization experiments, we choosed temperature sensitive RNA thermometer (BBa_K115001) and modelled the experiments on GFP measurements. RNA thermometer is a temperature sensitive DNA part that up to 42 C it forms a dimer. This dimer formation prevents polymerase readings that the translation is obstructed. At 42 C the linear form of part forms and translation initiates. This part is submitted to parts registry by iGEM 2008 TUDelft team and the experience of part is represented as none. We modelled the characterization of this part by planing two apart control groups.<br />
 +
    <br />
 +
  One pair group for characterization was T7 promoter,RNA thermometer and GFP from upstream to downstream at 37C and the same device at 42C. The aim of this control group was to control any expression of green fluorescence protein at 37 C to check RNA thermometer dimer formation. We prepared a flask experiments and from protein formation to folding range we measured the spectrophotometer and fluoresence spectrometer readings. It is showed the dimer formation of RNA thermometer at lower than 42 C caused inefficient binding of RBS to DNA. That means we measured the GFP readings at two temperatures however at 42 C the expression was observed in higher level. As seen at OD:0.6 reading there is a dramatic difference in expressions.<br />
 +
  <br />
 +
  Other pair group for characterization was T7 promoter with GFP at 37 C  and  T7 promoter,RNA thermometer and GFP from upstream to downstream at 42C. This control group was modelled in order to check and compare the expression levels of two devices. The expected result was to observe the similar readings. Because without RNA thermometer temperature switch off device express similar readings at 37C with RNA thermometer device at 42 C. In the data analysis we observed that there is deviation in readings between control T7 with GFP at 37C and to construct, T7 RNA thermometer and GFP at 42 C.<br />
 +
  <br />
 +
  <br />
 +
<center><h3>GFP Reading Data</h3>
 +
<p><img  src="https://static.igem.org/mediawiki/2011/d/db/Datapage.png"></p>
 +
<br />
 +
<br/>
 +
<p><img width="900px" src="https://static.igem.org/mediawiki/2011/3/34/Ans_paper.jpg"></center></p>
 +
<br />
 +
<br />
 +
<p><strong>Assembly Results</strong><br />
 +
    <br />
 +
  According our clonning plan, we planned to ligate the coding sequences of methane monooxygenase as subunits and to express the functional monooxygenase enzyme. Since we synthesized the long coding sequence we did PCR experiments with specific primers we extracted the parts in each the gel electrophoresis results gave validation for successfully synthesied DNA fragments, then we digested and ligated each part with related promoters and vector (pSB1C3) again gel electrophoresis data gave us validation to correctly digest and ligated form. <br />
 +
    <br />
 +
    <br />
 +
    <br />
 +
<center><p><img src="https://static.igem.org/mediawiki/2011/2/28/1KB-Ladder-X-Lush.png">
 +
<br />
 +
    <br />
 +
    <br />
 +
<img src="https://static.igem.org/mediawiki/2011/e/ec/09%2C16-1kb-control-mmoB-D-Z-C.png">
 +
<br />
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    <br />
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    <br />
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<img src="https://static.igem.org/mediawiki/2011/6/6c/Z-B-c3-c.png">
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    <br />
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    <br />
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<img src="https://static.igem.org/mediawiki/2011/a/a5/09%2C19-a3-d-lush.png">
 +
<br />
 +
    <br />
 +
    <br />
 +
<img src="https://static.igem.org/mediawiki/2011/e/e4/09%2C18-1k%C4%B1b-D-control-C-Z.png"></p></center>
 +
<br />
 +
    <br />
 +
    <br />
 +
   
 +
  <br />
 +
  <br />
 +
  <strong>-The device parts control experiments</strong></p>
 +
<p>The synthesized methane monoxygenase construct was so long part that we had problems in synthesizing that we got genes so late. By the way the long sequence was divided into two sequences that one of the regions were splitted into two parts therefore we could not ligated the two parts of coding sequence due to unidentified restriction sites, and full construct unfortunately did not reach to us. The main methane interacting region of monooxygenase could not be expressed functionally.
 +
  We expressed the protein B and C of methane monoxygenase encoeded from mmo B and mmo C genes in protein expression host E.coli BL21 strain. We planned to characterize the proteins in their theoretical molecular weights by SDS-PAGE analysis. However due to technical problems in gel formation we lost samples that we could not reached the data.
 +
  We planned another part on kit plate distributions to check works or not. The bacteriophage 21 lysis casette S, R, and Rz (PVJ4) (BBa_K124003). This part was designed by 2008 iGEM Brown team which induces lysis in E.coli bacteria. We ligated this part with ROSE regulated GFP generator  to induce the lysis of bacteria at 42C. Apart from this ligate, we ligated lysis casette with plac promoter and lacZ expressing gene to observe the blue colonies on plates. However we could not observe any blue colony on plates.
 +
 
 +
  </p>
 +
 +
 +
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<img src="https://static.igem.org/mediawiki/2011/1/1b/Humanpractice.png" height="75px" width="890px">
 +
<h2>Fun & Learn</h2>
 +
                        <h3>Flashmob!</h3>
 +
   
 +
<br />
 +
<center><p><iframe width="560" height="315" src="http://www.youtube.com/embed/gdRz8IICg5E" frameborder="0" allowfullscreen></iframe></p></center>
 +
<br />
 +
<p>In order to reach more people from several age groups, we have organized a flashmob activity which is related with our project -in molecular level-. (A flashmob is an activity done by a group of people who assemble suddenly in a public <a href="http://en.wikipedia.org/wiki/Public_place " target="_blank">place</a>, perform an unusual and sometimes seemingly pointless act for a brief time, then disperse, often for the purposes of entertainment, satire, artistic expression. Flashmob activities are organized via <a href="http://en.wikipedia.org/wiki/Telecommunications" target="_blank">telecommunications</a>, <a href="http://en.wikipedia.org/wiki/Social_media" target="_blank">social media</a>, or <a href="http://en.wikipedia.org/wiki/Viral_email " target="_blank">viral emails</a> ).One of our group member who is also a member of Flashmob society in Ankara helped us for arrangements. We use flashmob activity in order to explain what our project is and  to increase the public awareness about Synthetic Biology and especially about our project. Flashmob event took place at  one of the biggest shopping malls in Ankara where people often strolled around.  Afterwards, we had interviewed with people who watched  our sketch and informed people about nearly limitless capabilities of synthetic biology.  Indeed we prepared handouts that contains interesting statistical data about methane and its side effects, also information about iGEM and about our project.</p>
 +
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<h2>Sponsors</h2>
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<img src="https://static.igem.org/mediawiki/2011/f/f8/Partnershipheader.png" height="75px" width="890px">
-
<ul id="sponsorlist">
+
<h2>Acknowledgements</h2>
-
<li>...</li>
+
We are grateful to;</p>
-
<li>...</li>
+
 
-
<li>...</li>
+
<i><p>Gönenç Gürsoy, for helping in modelling, being our travel agent and ordering the best potato chips for us (with beer of course)...</p>
-
<li>...</li>
+
 
-
</ul>
+
<p>Duygu Yıldız, for being a mother and bringing delicious food for our breaks, being our light graffiti maker...</p>
 +
 
 +
<p>Gence Bektaş for being a methane molecule...</p>
 +
 
 +
<p>Önder Alparslan, for helping in modelling and ending our desperate search for a graphic tablet :)...</p>
 +
 
 +
<p>Mustafa Türkkan for being the best lipid bilayer member ever :) even though being a civil engineer...</p>
 +
 
 +
<p>Efe Köksal for being our moviemaker...</p>
 +
 
 +
<p>Demir Berkay Yılmaz for our second moviemaker and being in lab with us most of the time... </p>
 +
 
 +
<p>Ankara Flashmob society for being with us in our presentations and making it real fun...</p>
 +
 
 +
<p>Tufan Öz, Burcu Tefon, Aslıhan Kurt and Çiğdem Yılmaz for their incredible patience to our questions and supplying chemicals at speed of light!</p>
 +
 
 +
<p>As a team, we’d like to thank our instructor Professor Dr. Mahinur S. Akkaya for her patience, love, support and all of her solutions to our unending problems. :)</p></i>
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<h2>Algorithm</h2>
+
<img src="https://static.igem.org/mediawiki/2011/1/1c/Projectheader.png" height="75px" width="890px">
-
<p>In this section, the step by step functioning of our application,
+
<h2>Safety</h2>
-
along with the encapsulation of the algorithmic concepts of ‘standardization’
+
<h3>Safety Questions</h3>
-
of functional iGEM devices are depicted in pictorial forms called flowcharts.  
+
<p><b>1.  Would the materials used in your project and/or your final product pose:</b><br />
-
Rectangular boxes represent the encapsulation of implementations of
+
    <b>a.  Risks to the safety and health of team members or others in the lab?</b></p>
-
the computer programs to perform the particular tasks stated in that
+
<ol>
-
box on the flowcharts. These boxes are sometimes called subprograms,  
+
  <li>While in cloning steps the most  dangerous material that we used is <b>ethidium bromide</b> which is used in  agarose gel electrophoresis. Ethidium bromide is a potential mutagen because it  works by inserting itself between the two strands of double-stranded DNA. Since  the amount of ethidium bromide kept in our lab is relatively small, it does not  pose devastating effects.However it is a toxic chemical, when got in contact,  it causes eye and skin irritation. To prevent the damages that we would be face  with, we wraped it with aluminium and put in brown bottle and wore gloves while  using.</li>
-
objects or packages in Object Oriented software Engineering context.
+
  <li>While device training experiments we  planned to use <b>methane gas CH4</b> and <b>methanol CH3OH</b>. Methane  is  inactive biologically and essentially  nontoxic.Methane is not listed in the IARC, NTP or by OSHA as a carcinogen or  potential carcinogen. When inhaled in high concentrations,  so as to exclude an adequate supply of  oxygen to the lungs causes dizziness, deeper  breathing due to air hunger, possible   nausea and eventual unconsciousness. In case of protection, we adjusted  the progressing the steps for methane in glove boxes in order to provide the  respiratory protection. The ventilation is   supported by hood. However due to methane gas is potent for greenhouse  effect for doing experiments, special hood system is designed that could be  used which has its own gas container for storage of gases is used. Thus,  emission of methane gas is controlled. Later on, filled gas container is taken  by department of chemistry to empty with certain procedures. We arranged the conditions in department for possible experiments with methane gas. And  protective gloves and goggles are equiped for personal safety.Out of the  experiments for storage the gas tanks were saved in area where is cool enough  and never used open flames.</li>
-
The diamonds represent decision branching and they are found between
+
</ol>
-
two rectangular boxes. The arrows show the direction in which subprograms
+
<p>REFERENCE:<a href="http://www.isocinfo.com/DocumentRoot/13/Methane.pdf" target="_blank">http://www.isocinfo.com/DocumentRoot/13/Methane.pdf</a> <br />
-
work and communicate. The subprogram at the head of the arrow starts
+
  For the steps with <b>methanol t</b>he  characterization tests for LUSH protein, methanol is        used. Methanol is hazardous in case of  skin contact, eye contact, ingestion and inhalation. It is flammable liquid.The  safety for this chemical is provided by experimenting in hood with high  ventilation and and so much exposed because it pose a health risk to anyone in  lab. <br />
-
executing after the termination of the subprogram at the tail of the  
+
  REFERENCE :<a href="http://www.sciencelab.com/msds.php?msdsId=9927227" target="_blank">http://www.sciencelab.com/msds.php?msdsId=9927227</a> </p>
-
arrow. Following flowcharts are the high level representations of our
+
<p>We had studied with ANS; 1-Anilino-8-Naphthalene  Sulfonate as protein conformational tightening agent for LUSH protein folding  experiments. We searched on safety of this chemical and we found that it is non  hazardous chemical according to Directive 67/548/EE Following reference belongs  to Sigma Aldrich MSDS report.<br />
-
algorithms developed for the BioGuide software.</p>
+
    <a href="http://www.sigmaaldrich.com/catalog/DisplayMSDSContent.do" target="_blank">http://www.sigmaaldrich.com/catalog/DisplayMSDSContent.do</a></p>
-
<br>
+
<p><b>b. Risks to the safety and health of the general public if  released by design or accident?</b></p>
-
<h3>1</h3>
+
<ol>
-
<img src="https://static.igem.org/mediawiki/2010/9/95/Metu-algorithm1.png" />
+
  <li>The materials  mentioned in part a. could potentially be  dangerous to the general public. We use just enough concentration for our experiments. We always check the amount both for methane and methanol and EtBr,  because if accidentally released, they could be dangerous to us while  experiments and to lab security.</li>
-
<p><span>Diagram 1. Flowchart of collection, formatting and storage
+
</ol>
-
of devices data algorithm</span></p>
+
<p><b>c. Risks to environmental quality if released by design or  accident?</b></p>
-
<p>Information about the iGEM parts had to be collected in a standardized
+
<ol>
-
format for our application to function properly. Following data collection
+
  <li>Since methane is a gas at normal temperature  and pressure, the inhalation of this gas pose a risk for human health and so  for environmental quality. Methane is potent greenhouse gas and compared to carbon dioxide it has greater potential. This makes methane gas dangerous for  environmental quality in case of release by accident or design.However, as  mentioned in part a, we controlled the conditions for gas and examined in air  ventilated glove boxes. Another material which could pose risk for  environmental quality is methanol. This chemical is volatile and flammable  liquid. When this chemical release near open flame it could result with harmful  effects both for health and environment conditions.</li>
-
custom subprograms is needed to parse and forward the data the application’s
+
</ol>
-
database. In order to achieve this we have designed and implemented
+
<p><b> d. Risks to security  through malicious misuse by individuals, groups or states?</b><br />
-
the algorithm shown in diagram 1. In this algorithm, the first stage
+
    <b><br />
-
was to find the list of part IDs of devices which were supplied by iGEM
+
    </b>Only the methane gas has risks for  security if reaches to malicious someone. Our group members have experience and  training on gas used experiments and there is no possibility on misuse indeed  malicious misuse.<b></b><br />
-
in Spring 2010 distribution. This information has been collected from
+
  e.<b>Please explain your responses  (whether yes or no) to these questions.<br />
-
two sources 1) plate files in excel format which was available online
+
    Specifically, are any parts or devices in your project associated with (or  known to cause):</b><br />
-
2) device data provided in xml format, both provided by iGEM. The last
+
  <b>- pathogenicity, infectivity, or  toxicity? </b> No<b></b><br />
-
step in the algorithm was to send the collected partID data to the application’s
+
  <b>- threats to environmental quality? </b>None of our designed parts pose a risk to environment<b></b><br />
-
database.</p>
+
  <b>- security concerns? </b>No<b></b></p>
-
<br>
+
<p>     There are no part on our experiments and our project that could be use  as treat to environment or infect to human life as individually. Only one of  the parts, subunit A of methane monooxygenase may pose a health risk while  studying since it has the region where methane interacts and the conversion  steps initialize. As host cell we used E.coli BL21 (DE3) and Top10 strains, these strains are classified as hazard group 2 pathogen by the UK  Advisory Committee on the Dangerous Pathogens (ACDP). <br />
-
<h3>2.</h3>
+
    <b>2. If your response to any of the questions above is yes:</b><br />
-
<img src="https://static.igem.org/mediawiki/2010/e/e6/Metu-algorithm2.png" />
+
    <b>a. Explain how you addressed these  issues in project design and while conducting laboratory work. </b>Explained in each question<b></b><br />
-
<p><span>Diagram 2. Flowchart for BioGuide execution before and during
+
    <b>b. Describe and document safety,  security, health and/or environmental issues as you submit your parts to the Registry. </b>Explained in each question<b></b><br />
-
user interaction</span></p>
+
    <b><br />
-
<p>Diagram 2 presents the main algorithm, which shows how BioGuide application
+
    </b><br />
-
works. In BioGuide the major components are device and Biobrick graphs.  
+
    <b>3. Under what biosafety provisions will / do you operate?</b><br />
-
While the device graph represents input-output (promoter-regulator)
+
    <b>a. Does your institution have its  own biosafety rules and if so what are they? Provide a link to them online if  possible.</b><br />
-
compatibility combination of iGEM devices, the Biobrick graph represents
+
  Yes, there is general laboratory biosafety  rules for lab security and researcher security obeyed in Biology and Chemistry  departments. In research laboratories the students are provided with general  lab security and rules which are compilation of general and international  procedures for individual safety. These procedures are the compilation of  several links as<br />
-
combinations of atomic parts assembled in a device or system. The flowchart
+
  <a href="http://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Gdlnes_lnk_2002z.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Gdlnes_lnk_2002z.pdf</a></p>
-
shows how these graphs are created and embedded into the program, which
+
<p>&nbsp;</p>
-
displays both of the graphs to the user when launched. Application presents
+
<p><b>b. Does your institution have an  Institutional Biosafety Committee or equivalent group? If yes, have you  discussed your project with them? Describe any concerns or changes that were  made based on this review.</b></p>
-
few interactive options to the user when started, which were shown on  
+
<p>Yes there is biosafety and ethical  research center in METU until 2000. This committee helds  many conferences about ethics, environmental  ethics in studies and the medicinal ethics based on human researchs. The below  link belongs to this committee however in Turkish language.<br />
-
the flowchart under the horizontal, bolded line. As shown on the diagram
+
    <a href="http://www.ueam.metu.edu.tr/" target="_blank">http://www.ueam.metu.edu.tr</a></p>
-
2, there are four interactive tasks BioGuide can do, where the device
+
<p>      Indeed in Biology department at METU one of our professors, Prof.Dr  Huseyin Avni Oktem was one of the members of National Biosafety Coordinating  Committee. He is our pioneer for biosafety and security issues. In the case of any safety issues, he is the one to consult   the critical safety points in our project and also discussed the project  based on safety. </p>
-
and Biobricks graphs are utilized. Upon clicking a node on a devices
+
<p><b>      c. Will / did you receive any biosafety and/or lab training before  beginning your project? If so, describe this training.</b></p>
-
or Biobricks graph, that node changes in size and color and the various
+
<p>Before we begin to experiments, we  firstly were taught on lab security and personal safety for cases of individual  injure, indeed in emergency situations. This interval was training for us. Also  team leaders, the experienced ones in team, prepared an exam for new team  members as a part of training for general lab regulations and project details.</p>
-
functions shown on the flowchart can be performed then after.</p>
+
<ol>
 +
  <li><b>Does your country have national  biosafety regulations or guidelines? If so, provide a link to them online if  possible.</b></li>
 +
</ol>
 +
<p><b>&nbsp;</b></p>
 +
<p>In Turkey, biosafety regulations are  organized by the Biosafety Information Exchange  Mechanism of Turkey, facility of Ministry  of Agriculture and Rural Affair.<br />
 +
    <a href="http://www.tbbdm.gov.tr/en/Home.aspx" target="_blank">http://www.tbbdm.gov.tr/en/Home.aspx</a></p>
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+
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<h3>Graphical Modeling for Bio-Guide</h3>
+
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<h4>Introduction</h4>
+
-
<p>Graphical Modeling Theory has been applied to construct four different
+
-
graphs where relations of atomic parts, devices and systems and the
+
-
functional combinations that can build new constructs are presented
+
-
for the iGEMs parts registry database. Three graphs are composed of
+
-
iGEM devices and one graph is based on Biobricks. Each graph comprises
+
-
a set of vertices or nodes and a set of edges. In the set of nodes each
+
-
node represents a device, while in the set of edges each edge represents
+
-
the input-output combination of the nodes. These graphs are directed
+
-
graphs as the edges are created according to input-output combination.
+
-
All compatibilities between a regulator and a promoter of an edge is
+
-
created, where the source of this edge is the device with the corresponding
+
-
regulator and target of the edge is the device with the promoter in
+
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concern.</p>
+
-
<img src="https://static.igem.org/mediawiki/2010/3/3f/Metu-node1.png" />
+
-
<p><span>Fig. 1: A node representing a device</span></p>
+
-
<img src="https://static.igem.org/mediawiki/2010/1/14/Metu-node2.png" />
+
-
<p><span>Fig. 2: Arrow representing an edge between two nodes</span></p>
+
-
<p>The atomic structures used in our graphical model have been represented
+
-
in Figures 1 and 2. A node is represented with a solid circle where
+
-
the label, the part/device ID according to iGEM standards, of the device
+
-
is marked on the foreground. The blue arrows between nodes connect the
+
-
related devices, representing the input-output connectivity. End style
+
-
of the arrow helps us to determine the direction of the node, like in
+
-
Figure 2 where the node labeled BBa_S03520 is the source and BBa_JO9250
+
-
is the target.</p>
+
-
<br>
+
-
<h3>Directivity</h3>
+
-
<p>All the four constructed graphs build for BioGuide are directed graphs.
+
-
So that, for every edge there must be a single source and a target.
+
-
There is no single edge which is bidirectional. In mathematical form
+
-
this can be represented as:</p>
+
-
<p>If an edge e has node v as source and node w as target then the edge
+
-
can be expressed as</p>
+
-
<img src="https://static.igem.org/mediawiki/2010/6/6c/Metu-equation1.png" />
+
-
<p>For a directed graph the combination (v, w) is totally different
+
-
from (w, v). Therefore,</p>
+
-
<img src="https://static.igem.org/mediawiki/2010/a/ac/Metu-equation2.png" />
+
-
<p>The direction of the edges has been represented with the arrows,
+
-
as explained in Figure 2.</p>
+
-
<br>
+
-
<h3>Connectivity</h3>
+
-
<p>The nodes forming their own sub-graphs disconnected from the rest
+
-
of the nodes have been recognized, which showed us the presence of incompatibility
+
-
between few regulators and promoters of the devices. We have observed
+
-
this disconnection in all four of our graphs. The basis of the disconnection
+
-
has been shown in Figure 3, where the two sub-graphs without any edge
+
-
that connects them to the main graph has been presented on the right
+
-
hand side of the diagram. These features classify our graphs as disconnected
+
-
graphs [1].</p>
+
-
<img src="https://static.igem.org/mediawiki/2010/8/8b/Metu-node3.png" />
+
-
<p><span>Fig. 3: A zoomed in screenshot showing two sub-graphs within
+
-
the disconnected graph.</span></p>
+
-
<br>
+
-
<h3>&quot;Semi-Simplicity&quot;</h3>
+
-
<p>A simple graph is a graph in which no more than one edge contains
+
-
the same set of nodes. So, in a simple graph it is not possible to find
+
-
more than one edge with the same source and the same target. Additionally,
+
-
an edge with the same source and target, forming a loop is not allowed.
+
-
But, in synthetic biology it is possible to construct a device consisting
+
-
of devices or bio bricks of the same species or type. Accordingly, our
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Revision as of 17:27, 13 October 2011

Methan<b>E.COLIc</b> : Decreasing the Greenhouse effects and Saving the workers life in one system

MethanE.COLIc : Decreasing the Greenhouse effects and Saving the workers life in one system

Firedamp explosions are frequently seen cases at all mines over the world. In Turkey every year, 50 miners lose their lives because of firedamp explosions. Firedamp is a flammable gas found in coal mines and it mainly contains methane. Beside its explosive property, methane is also the main contributor to global warming. However recent mine mechanisms release obtained methane into air. By offers of Synthetic biology, we aimed to design a device which will work on E.coli that provides solutions for side effects of methane. Device that we are planning to construct involves the genes of bacteria (Methylococcus capsulatus) and insect (Drosophilia melanogaster). Our compact system in E.coli is fabricated as sensation of methane, the conversion of methane to methanol and then entrapment of methanol to handle for biofuel and death of bacteria at 42 C by kill switch mechanism.

When project is analyzed in stepwise, there are successive 4 steps of our modelled system in modified organism. As mentioned in subtitles, methane is sensed, then converted to methanol and methanol is entrapped and to elute methanol the cells are dead.

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Introduction

MethanE.COLIc project is designed to solve one of the problems on worker security in mines of Turkey , by constructing the natural parts of organisms. The main reason for us to choose such a project is that in Turkey- and also in many countries- each year, huge numbers of workers in mines lost their life due to deficiencies and conditions in their working areas (mines). The release of Grizu gas leads to subsidence of mines and so death of workers.This project is also designed to solve one of universal problem; global warming. Methane gas is a potential greenhouse gas.This project focuses on sensing the ambient methane gas and converting to one of the bio-fuel sources, methanol. Methanol is entrapped by product of one of the constructs of project, and then cells are dead by kill switch device of project to elute methanol.



Steps

Team

Hello everyone!We are Metu Ankara IGEM team.Our team consists of high motivated,social and enjoyable “undergraduate and graduate scientists” from departments of physics, chemistry ,biology, computer engineer and computer education & instructional technology. We are such a team that complete each other so that we all experienced team spirit in IGEM. This makes us happy while doing experiences even in the hot summer.Our slogan is “ all for one, one for all”!!Then, who are we?


Instructors

Advisors

Team leader :Ceren Seref

Ceren has just finished the junior year in biological sciences department in METU. It’s her second year in iGEM competition. She’s planning to study about neuroscience-especially on Alzheimer’s disease. She believes synthetic biology approaches can be a very good way of drug development in neuroscience field. She enjoys climbing, listening to music and singing.

Sibel Ataol

Sibel is first semester student in Master's program of Biomedical engineering at Middle East Technical University indeed she is graduated from Biology Department at METU. This is the second year of her in IGEM team from METU.She is one of the experienced in team and elder sister:) As a vice team lead, planing the experiments and cloning strategies and motivating team are the responsibilities of her. Studying in bioengineering and regenerative sciences is her desire for future.

Funda Guzey

She is a senior student of Biological Sciences at Middle East Technical University. This is her third year in iGEM competition. She gained a lot of experiences in this summer. One of the her interests is Synthetic Biology. However, her main interest is Biomedical and Tissue Engineering. She feels lucky to have such a wonderful team and is glad to share a wonderful summer with her team.

Gökcan Aydoğan

Gökcan has just finished the third year of the B.Sc. Chemistry degree at METU. This is his first year in IGEM team. He has been a huge fan of Synthetic Biology ever since he understands nothing about the concept. Everyday, he tries hard to get the picture. In his free time, he likes gaming, drawing cartoon and eating, eating, eating...

Filiz ORMANCI

She is a fourth year student at the Department of Physics at Middle East Technical University. This is the first year of her in iGEM team. She wants to study on the usage of optical instruments in biophysics. She likes team working and sharing the success. She is always positive and happy... ehm..also a “soothsayer”. She enjoys playing musical instruments mainly violin and learning different languages.

G. Simge Yüz

She is a recent graduate student of Chemistry and a fourth year student of Biological Sciences. This is the first year of her in iGEM team. She wants to study on neuropharmacology in the field of neuroscience. She is glad to join to METU-Ankara-iGEM team. She enjoys dancing especially Argentine tango and swimming. She is also a member of Flashmob Ankara Society

Yunus Alpağu

Yunus is a third year student in biology department at Middle East Technical University in Turkey. This is his first year in iGEM team.He is interested in regenerative medicine, stem cells, biomaterials and tissue engineering. His favourite TV series is ‘Fringe’ and he likes playing tennis and volleyball.He is glad to join to METU-Ankara-iGEM team.

Pelin İspir

Pelin is currently studying the biology department of Middle East Technical University in Turkey. She is interested in stem cell, developmental biology,tissue engineering, and conservation biology.This is the first year of her in İGEM.She really enjoys with being this team and dealing with the mystery of synthetic biology.She enjoys listening latin musics and flamenco.

İbrahim Kutay Ozturk

Kutay is a graduate of Molecular Biology and Genetics, and now doing his M.Sc. in Biotechnology department at METU. This is his first year in IGEM team. He is interested in plant-based drugs, plant-pathogen interaction, biofuel production and bioremediation. He enjoys playing basketball, watching movies, and listening to music.

Advisor:Burak Yılmaz

He is a recent graduate of METU Molecular Biology and Genetics department and now studying towards his masters degree on Molecular Bioengineering at METU. His interest in synthetic biology did start during his undergraduate years and after graduation he started up the Sentegen company which is the first biotechnology based company focused on synthetic biology in Turkey. He continues his research and training in synthetic biology while also contributing to the development of the field in his country. He needs new scientific revolutions to solve huge problems of life and emerging field of synthetic biology is best candidate for biotechnological revolution. He is interested in synthetic biology applications, along with Lab-on-a-Chip devices for molecular biology techniques, and he is designing gene synthesis chips to produce biobricks - raw materials of garage biology- faster and cheaper. he enjoys snowboarding, cycling and writing poems.

Software Group

Muhammed Akif Ağca

Akif is a graduate of Computer Education and Instructional Technologies department. In bachelor he has worked on Software Development, Database Management, Instructional Technologies, and Bioinformatics.
He was software developer and team leader of BioGuide project and Synthetic Biology Online Lecture.
Currently he is mastering in Software Engineering and developing scientific applications, he will continue to work on Science and Technology Management after becoming master of Software Engineering.

Miktat Aktaş

He graduated from Computer Education & Instructional Technology Department of Middle East Technical University last semester and he contributes this genius team as a coder for website. He wants to be a successful academician in his department at METU. He likes swimming and everything related to computers and automobiles.

Ceyhun Kerimov

Ceyhun was born in Azerbaijan. He is studying in METU Computer Engineering Department.
He is Java and Salesforce developer now. He was in web side of the project. He tried to help the team to manupilate and store the data.
He is working in web and cloud computing technologies.

Huseyin Lutin

Huseyin is a senior student in METU Computer Engineering Department.
He was helping the team during software requirements specification step of the project.
He is working on computer graphics in this year.

WET LAB

Whole summer in lab work we aimed to design 7 new E.coli compatible Biobricks and 1 composite part from 2011 kit plate distributions, also we aimed to characterize each part by protein analysis and fluorescence protein measurements.

We achieved design and compatibility of parts in E.coli, however not all parts’ characterization is achieved. Only the genes of designed composite part from kit plate is characterized by GFP measurements.

In this section you can find out more details on all our wet lab works. You can also find our data under results subtitle and information about design of our parts-biobricks with explanations in literature. It also contains materials and protocols, and finally there is safety questions list on our safety considerations both in the lab and on a wider environmental scale.

Overview

MethanE.COLIc projec t arises from sustainability and conservancy of human health and safety. Therefore while we were constructing the organismic device we also wanted to inform people from all age and we wanted to increase the public awareness on our project; methane gas based (firedamp) explosions and the greenhouse effect of methane gas.

Our aim is to inform people in each age. After brainstorming and searching, we planned the content of information and activities according to following subtitles.

Future Plan


Methane explosions called as grisou are one of the main problems from the beginning of the coal mines. All countries have tried different systems to solve this methane problem. When we joined iGEM 2011 competition, our aim is to find a solution to that problem for humanity by cheaper and energy saving way. After a long literature research, we found how seriously dangerous the methane gas is and how people deal with this big problem on coal mines. Below are the countries that have faced with serious methane explosions throughout their history.

  1. Australia
  2. Belgium
  3. Canada
  4. Chile
  5. China
  6. France
  7. New Zealand
  8. Poland
  9. Russia
  10. Australia
  11. United States

Todays companies deal with this problem by;
Regenerative Thermal Oxidizer from MEGTEC capable of handling 60,000 scfm of VAM. At a methane concentration of about 0.7% a unit this size generates 50,000 carbon credits per year. This unit costs about US$ 1,5 million Elizabeth Obediente from CO2IMPACT at a methane upgrading facility at a coal mine in Alabama. The gas is stripped of impurities and upgraded to 96% methane per volume and then compressed and then sold to the natural gas pipeline. Ventilation Air Methane (VAM): VAM units cost about $20-$30 USD per scfm.

At some mines, downtime is considerable, causing expensive losses to the mine. Ventilation cost cuts can be up to half of the original cost of the ventilation system. In one study, a mine with 400 ft3/ton of methane using vertical wells to pre-drain will result in an estimated US$ 11 million over 20 years in reduced ventilation costs, not including carbon and energy revenue[iii] (U.S. Environmental Protection Agency, 1999). Like many solution system for methane problem, the tree examples of solution of methane problem did not satisfied us, so we created our simple and very effective way to deal with the methane.( http://www.co2impact.com/cmm.html )

According to our methane E colic project, we aimed:

  • Save the coal workers’ life
  • Save the nature
  • Process a new energy
  • Obtain a cheap system

Save the coal workers’ life

We aimed to collect the methane gas from coal mines to the tanks which are filled with water. When the methane passes the legal percent that is 1%, according to our project, the gas is dissolved in water in tanks. Then, inside the tablets containing our bacteria and that are already present on the tanks the molecular process for conversion of methane to methanol begins. Below the number of workers dead by years are given according to Prof. Dr. Mustafa ÖZTÜRK’s research,Vice President of Parliamentary Commission for the Environment in Turkey:

Years Death workers on Turkey
1983 103
1990 330
1995 40
2003 17
2004 3
2005 18
2006 21
2007 7
2008 43

Methane explosions:

Save the nature

Methane is one of the greenhouse gases in the atmosphere. Methane is continuously released from coal mining, livestock, landfills and natural gases to the atmosphere. The accumulation of these greenhouse gases causes one of the greatest problems of Earth: global warming. Our project aims to prevent release of methane gas by the conversion of it to methanol in bacterial system in coal mines.

Greenhouse gases:

Process a new energy

After the conversion of methane to methanol, methanol can be used as a precursor of bio-fuel so that accumulated methanol can be recovered. Ethanol is used as biofuel, also; however, methanol is more cheaper to obtain and use as a biofuel. According to Dr. Robert Zubrin, Methanol is cheaper than ethanol. It can also be made from a broader variety of biomass material, as well as from coal and natural gas. And methanol is the safest motor fuel.(American enterprise, February 13th, 2006).

Cheap system

There are many systems used to prevent or minimize methane explosions. However, they are so expensive to use and none of them are based on a biological system. The biological system that we build contains a bacterial cell having a molecular process of conversion of methane to methanol is completely harmless and cheaper since expensive equipments are not needed.

Extras







Hands on show!





Photos



Methane sensing

i) Introduction

In our methanE.COLIc project, the first critical point is to sense the ambient methane gas. While in literature search, we have focused on the methane and DNA or methane and regulator protein interaction which binds to DNA after binding to methane. In structure, methane consists of one carbon atom and four hydrogen atom which means it is smaller in size and weight. Therefore it is hard to find any study based on methane interaction with DNA or protein to regulate transcription. This made us search for carbon hydrogen bond interaction with any oligo or protein to trigger transcription of any metabolite product. So that we also searched the literature for any organism which utilizes any alkane chain and alkane which regulates the transcription of organismal metabolites by interaction with DNA or protein. Since bacteria are environmental adapting organisms, it is possible to find any alkane to regulate the transcription of degradation metabolism. Then we have found the organism, Pseudomonas oleovorans to analyze the alkane degradation of organism for its carbon source.

ii) Background

Many microorganisms live in the environments where the conditions changing frequently and so the evolution is inevitable for mechanisms to withstand unfavorable situations. Therefore microorganisms can use their specific and sensitive mechanisms for sensing the required nutrients for them or any pollution to affect their sustainability. Otherwise they can expose to mutations which change the gene expression and gain new functionalities. In case they have ability to survive in such conditions.

While we were scanning the literature based on methane and alkane degrading organisms we have found some organisms that sensitive to methane presence and have mechanisms to activate transcription of related gene clusters.

We analyzed the soil bacteria, Pseudomonas oleovorans. This strain can assimilate the alkane for its carbon source and one of the microbial whole cell –biosensor. They have a gene cluster which codes for degradative pathway and includes the activator which interacts especially with linear alkanes. This activator protein, AlkS, in the presence of alkanes, induces the transcription from PalkB promoter which initiates the expression of genes code for assimilation of alkanes. We have analyzed the related articles in which the promoter region is studied and showed that it is expressed in E.coli correctly because of the corresponding RNA polymerase binding regions.

REFERENCE:

  1. Canosa, I. & Yuste, L. (2000). A positive feedback mechanism controls expression of AlkS, the transcriptional regulator of the Pseudomonas oleovorans alkane degradation pathway. Molecular Microbiology, 35 (4), 791-799.

iii) Modelling

Since the parts that we have found belongs to yeast organism, it was hard to us to manipulate it in bacteria E.coli. We designed the tests of this part by characterizing one of the subunits of methane monooxgenase (MMo). For the functional activity of monooxygenase enzyme, there are required 3 subunits as A,B and C. The A subunit of methane monooxygenase is 210 kDa protein that is supposed as the methane binding active site of monoxygenase complex. This protein consists of non-haem iron and contains p-hydroxo bridge structure. We planned the experiments of this step as characterization of protein A by PCR amplification with specific primers of full construct device to obtained the sequence of protein A. The methane gas is applied to cell culture with our modelled device then the cells were centrifuged and the gas concentration is measured from supernatant.

Clonning procedures

  1. Getting the DNA parts

    1. For parts in kit plates
      1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the BiobrickTM-standard part that you want.
      2. Pipette 10 uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully re-suspended.
      3. Transform 1 or 2 uL of the re-suspended DNA into desired competent cells, plate your transformation with the appropriate antibiotic and grow overnight.
      4. Pick a single colony and inoculate LB medium (again, with the correct antibiotic) and grow for 16 hours.
      5. Use the resulting culture to miniprep the DNA and make your own glycerol stock.
    2. For new synthesized DNA parts
      1. The DNA parts are sent in nmol values in each. These data are determined for all of them individually
      2. As a stock solution preparation, these DNA parts are pipetted with PCR water or TE buffer which is 100 times of DNA amount. The final solution is prepared as 100uM.
      3. For each transformation procedures, the 10uM of stock DNA part in water is transformed. 1uL is taken from stock DNA solution and is added to 9uL PCR water.

  2. Preparation of Competent Cells

      1. Inoculate a single colony of E. Coli cells into 50 ml LB medium. Grow overnight at 37 C with moderate shaking (250rpm)
      2. Inoculate 4 mL of the culture into 400 mL LB medium in a sterile 2-liter flask. Grow at 37 C shaking at 250 rpm to an OD590 of 0.375.

        This procedure requires that cells be growing rapidly (early- or mid-log phase). Accordingly, it is very important that the growing cells have sufficient air. Overgrowth of culture (beyond OD590 of 0.4) decreases the efficiency of transformation.

      3. Aliquot culture into eight 50 mL prechilled, sterile polypropylene tubes and leave the tubes on ice 5 to 10 min.

        Cells should be kept cold for all subsequent steps.
        Larger tubes or bottles can be used to centrifuge cells if volumes of all subsequent solutions are increased in direct proportion.

      4. Centrifuge cells 7 min at 1600 g,  4 C.
      5. Allow centrifuge to decelerate without breake.
        We have not attempted to determine whether deceleration without braking is critical to this procedure. However, we do not use the breake for this step or for the subsequent centrifugation steps.

      6. Pour off subernant and resuspend each pellet in 10 ml ice-cold CaCl2 solution.Re-suspension should be performed very gently and all cells kept on ice.
      7. Cetrifuge cells 5 min at 1100 g, 4 C. Discard supernatant and re-suspend each pellet in 10 mL ice-cold CaCl2 solution. Keep re-suspend cells on ice for 30 min.
      8. Centrifuge cells 5 min at 1100 g, 4 C. Discard supernatant and re-suspend each pellet in 2 mL ice-cold CaCl2 solution.

        It is important to re-suspend this final pellet well. The suspension may be left on ice for several days.

      9. Dispense cells into prechilled, sterile polypropylene tubes (250 ml aliquots are convenient). Freeze immediately at -70 C.
  3. Transformation

      1. Aliquot 10 ng of DNA in a volume 10 to 25  mL into a sterile 15 mL round bottom test tube and place on ice.
      2. Plasmid DNA can be used directly from ligation reactions. When this is done , more DNA is usually used. However, if there is <1 mg of DNA in the ligation reaction, or if the ligation reaction is from low gelling/melting temperature agarose, it is wise to dilute the ligation mix.

      3. Rapidly thaw competent cells by warming between hands and dispense 100 mL immediately into test tubes containing DNA. Gently swirl tubes to mix, then place on ice for 10 min.
      4. Competent cells should be used immediately after thawing. Remaining cells should be discarded rather than refrozen

      5. Heat shock cells by placing tubes into a 42 C water bath for 2 min
      6. Alternatively incubate at 37 C for 5 min.

      7. Add 1 ml LB medium to each tube. Place each tube on a roler drum at 250 rpm for 1 hr at 37 C.
      8. Plate aliquots of transformatin culture on LB /ampicillin or other approprate antibiotic- containing plates.
      9. It is convenient to plate several different dilutions of each transformation culture. The remainder of the mixture can be stored at 4 C for subsequent platings.

      10. When plates are dry, incubate 12 to 16 hr at 37 C.
  4. Plasmid isolation
  5. This procedure is performed with Fermentas GeneJET™ Plasmid Miniprep Kit

      1. Pick a single colony
      2. Inoculate in 5 mL LB medium for high-copy or 10 mL for low-copy of LB medium supplemented with the appropriate selection antibiotic.
      3. Incubate at 225 rpm not longer than 12-14 h at 37 C .
      4. Centrifuge at 4000 rpm for 5 min at 4 C .
      5. Discard the supernatant and keep pellet.
      6. Resuspend  the pellet with 250 ul Resuspension solution. (Bacteria should be resuspended completely by vortexing until no cell clumps remain)
      7. Transfer the cell suspension to eppendorf.
      8. Add 250 uL Lysis Solution and mix gently by inverting the tube 4-6 times until the solution becomes slightly clearPlate aliquots of transformatin culture on LB /ampicillin or other approprate antibiotic- containing plates.
      9. Do not vortex!
        Do not incubate for more than 5 min. (To avoid denaturation of supercoiled plasmid DNA!)

      10. Add 350 uL Neutralization solution and mix immediately and thoroughly by inverting the tube 4-6 times.
      11. (The neutralized bacterial lysate is cloudy and viscous) ("thoroughly" to avoid localized precipitation of bacterial cell debris)

      12. Centrifuge at 13000 rpm for 5 min to precipitate cell debris and chromosomal DNA.
      13. Transfer the supernatant to spin column (about 600 uL).
      14. (Avoid disturbing or transferring the white precipitate)

      15. Centrifuge at 13000 rpm for 1 min.
      16. Discard the flow-through.
      17. Place the column back into same collection tube.
      18. Add 500 uL Wash solution to spin column.
      19. Centrifuge at 13000 for 1 min
      20. Discard the flow-through.
      21. Repeat this step with using 500 uL Wash solution.
      22. Discard the flow-through
      23. Centrifuge at 13000 rpm for an additional 1 min to remove residual Wash solution.
        (This step is essential to avoid residual ethanol in plasmid preps)
      24. Transfer spin column into a fresh 1.5 mL epp.
      25. Add 50 uL Elution buffer into center of the spin column membrane to elute the plasmid DNA
      26. Do not contact the membrane with pipette tip!

      27. Incubate for 2 min at 25 C. 
      28. To increase yield incubation is done for 2 min at heat block at 42 C

      29. Centrifuge at 13000 for 2 min.
      30. Discard the column and store the purified plasmid DNA at -20 C.
      31. OPTIONAL= additional elution step with elution buffer or water. This step increases the yield by 10-20 %.
        NOTE:  For elution of plasmids ≥ 20 kb, prewarm Elution buffer to 70 C before applying.

  6. Restriction digestion
  7. This procedure is performed with NEB  BioBrick™ Assembly Kit

    • Digest Upstream Part with EcoRI-HF and Spel
      500 ng Upstream Part Plasmid
      1 uL EcorI-HF
      1 uL Spe I
      5 uL 10XNE  Buffer 2
      0.5 uL 100X BSA
      To 50 uL add dH2O

    • Digest downstream part will  Xbal and PstI
      500 ng Downstream Part Plasmid
      1 ul XbaI
      5 uL 10X NE Buffer 2
      0.5 uL 100X BSA
      To 50 uL add dH2O

    • Digest the destination plasmid with EcorI-HF and Pst I:
    • The destination plasmid DNA should either be prepared with PCR or contain the toxic gene (e.g, ccdB, sacB) in the cloning site to avoid the need for gene purification.
      The Destination Plasmid should also have a different antibiotic resistance marker from both the plasmid containing the Upstream Part and the plasmid containing the Downstream Part to avoid the need to purify the Upstream and Downstream Parts.
      500 ng Destination Plasmid DNA
      1 uL EcorI- HF
      1 uL PstI
      15 uL 10X NE Buffer 2
      0.5 uL 100X BSA
      To 50 uL add dH2O
       
      Incubate all 3 restriction digest reactions at 37 C for 10 min and then heat inactivate at 80 C for 20 min.

  8. AGE (Confirmation)
  9. Background

    • EtBr stains only dsDNA. You cannot see ssDNA on gel
    • Minimum amount of DNA load:
    • 300 ng
      • Each band of 1kb ladder is approximately 125 ng (2.5 ul loaded) and can be seen on gel

    Preparation

    • 50X TAE (Tris-Acetate) Buffer
      121 g/0.5 L Tris
      28.6 g/0.5 L Glacial Acetic Acid
      7.31 g/0.5 L EDTA
      pH 8.0
    • Sterile 1.5 mL eppendorf tubes
    • Sterile long pipette tips
    • DNA standards
      • Promega 1.0 kb (1.0-10Kbp), 100 bp (0.1-1.0 Kbp)
    • TAE Buffer
    • EtBr sln (10 mg/mL). Purchase as solution ! Store at RT. Light-sensitive and carcinogenic !
    • Sample loading buffer. Store at 4 C.

    Protocol

    • Prepare 360 ml, 1X TAE running buffer
    • Prepare gel compartment with tape (do not use parafilm)
    • Prepare 60 ml, 1% agarose gel (0.6 g agarose in 60 ml 1X TAE buffer)
      • 1-2 min in microwave. Check and swirl after each 30 sec. It must be dissolved completely.
    • Add 1.0 ul EtBr (10 mg/ml) per 10 ml gel solution, swirl to mix
    • Add add 4 ul EtBr (10 mg/ml) per 100 ml running buffer.
    • Pour gel into gel compartment. Put comb.
    • After gel solidifies (30 min), take out the comb and load buffer to one well to measure its capacity.
    • Position the gel compartment and fill gel chamber with running buffer.
    • Prepare samples and standards (ladder)
    • Prepare samples by mixing 4 ul 6X Loading Buffer + 20 ul sample
    • Load the samples (for 24 ul sample, set pipette 24 ul).
    • Do not push the tip to the bottom of the wells. Make a 45 degree angle and support the pipette tip on the edge of the well. Don’t worry, gel will not brake when you take support. Slowly release the sample. Do not use second pipet push not to risk air injection.

    • Make the connections: Black (-) on sample side, Red (+) opposite side
    • Run at 60V
      5-10V/cm (distance between anode and cathode) is recommended.
      90V causes high heat generation for our unit. Use 60V maximum.
      For overnight runs: 2V is too low, causes diffusion
      35V / 4hrs / ¾ progress
    • Gel can be stored in 4 C until next day after completion

    Gel Photo

    • Adjust zoom and position using visible light
    • Before turning on UV load your settings file which has the following parameters:
      • Preview tab, all three options checked
      • Active image
      • Dynamic integration, auto exposure, 10 frames
      • 50/50 brigthness/contrast
    • Maximize brigthness with camera knob (counterclockwise)
    • Turn on UV light
    • Lower brightness from camera knob if necessary
    •  

  10. Gel extraction (For Fermentas Gel Extraction Kit)

      1. Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel slice.
      2. Note: If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a glass or plastic plate during UV illumination.

      3. Add 1:1 volume of Binding Buffer to the gel slice (volume: weight) (e.g., add 100 µl of Binding Buffer for every 100 mg of agarose gel)
      4. p>Note. For gels with an agarose content greater than 2%, add 2:1 volumes of Binding Buffer to the gel slice.

      5. Incubate the gel mixture at 50-60°C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow
      6. Optional: use this step only when DNA fragment is <500 bp or >10 kb long.
        • If the DNA fragment is <500 bp, add a 1:2 volume of 100% isopropanol to the solubilized gel solution (e.g. 100 µl of isopropanol should be added to 100 mg gel slice solubilized in 100 µl of Binding Buffer). Mix thoroughly
        • - If the DNA fragment is >10 kb, add a 1:2 volume of water to the solubilized gel solution (e.g. 100 µl of water should be added to 100 mg gel slice solubilized in 100 µl of Binding Buffer). Mix thoroughly.
      7. Transfer up to 800 µl of the solubilized gel solution (from step 3 or 4) to the GeneJET™ purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube. 
      8. Note. If the total volume exceeds 800 µl, the solution can be added to the column in stages. After each application, centrifuge the column for 30-60 s and discard the flow-through after each spin. Repeat until the entire volume has been applied to the column membrane. Do not  exceed 1 g of total agarose gel per column.

      9. Optional: use this additional binding step only if the purified DNA will be used for sequencing

        Add 100 µl of Binding Buffer to the GeneJET™ purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.

      10.  
      11. Add 700 µl of Wash Buffer (diluted with ethanol as described on p. 3) to the GeneJET™ purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
      12. Centrifuge the empty GeneJET™ purification column for an additional 1 min to completely remove residual wash buffer.
      13. Note. This step is essential to avoid residual ethanol in the purified DNA solution. The presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions.

      14. Transfer the  purification column into a clean 1.5 ml microcentrifuge tube (not included). Add 50 µl of Elution Buffer to the center of the purification column membrane. Centrifuge for 1 min.
      15. Note:

        • For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 µl does not significantly reduce the DNA yield. However, elution volumes less than 10 µl are not recommended.
        • If DNA fragment is >10 kb, prewarm Elution Buffer to 65°C before applying to column
        • If the elution volume is 10 µl and DNA amount is ≤5 µg, incubate column for 1 min at room temperature before centrifugation
      16. Discard the GeneJET™ purification column and store the purified DNA at -20°C
  11. Ligation
  12. This procedure is performed with  NEB BioBrick™ Assembly Kit

    1. Ligate the Upstream and Downstream Parts into digested Destination Plasmid.
      • 2 uL Upstream Part Digestion
      • 2 uL Downstream Part Digestion
      • 2 uL Desination Plasmid Digestion
      • 2 uL 10X T4 DNA ligase bufferl
      • 2 uL T4 DNA ligase buffer
      • 11 uL dH2O
    2. Incubate at RT for 10 min and then heat inactivate at 80 C for 20 min.
    3. Transform 2 uL of the ligation product into 50 uL of competent E.Coli cells ( or other suitable host strain).
    4. Select using the antibiotic corresponding to the Destination Plasmid.

  13. Glycerol Stock Preparation

    1. Centrifuge 5 mL overnight cells at 5000 rpm for 5 min.
    2. Discard 4 mL supernatant, remain 1 mL with pellet.
    3. Add 176 uL 15% glycerol  and resuspend the pellet.
    4. Aliquot 100 uL and immediately freeze at -70 C.

  14. Cell imaging
  15. Checklist Procedure

    1. Suspension culture preparation
      • Dissolve a single colony in 10 mL LB+Amp
      • Incubate 14-15 hrs, shaking at 225 rpm

    2. Scale-up of the suspension culture
      • Add 2-3 mL of the suspension culture to 100 mL LB+Amp
      • Incubate 5-6 hrs, shaking at 225 rpm

    3. Equalizing OD readings
      • Take 10 mL samples from GFP/RFP & NC cultures
      • Centrifuge at 10000 rpm for 5 mins
      • Discard supernatant
      • Resuspend pellet in 500 uL 1X PBS
      • Take 250 uL & dilute to 3 mL with 1X PBS
      • Read in UV-Vis at 600 nm
      • Multiply the OD readings by 12
      • Dilute remaining 250 uL samples as required to make all samples have the same ODvalue, final volume should also be 250 uL

    4. Fluorescent signal reading
      • Set parameters
        [GFP]
      • Mode: Emission scan
      • Config: Digital
      • Slit: 1 turn / 2 nm
      • Blaze: 750 nm
      • Excitation: 395 nm
      • Emission range: 475-600 nm
      • Average: 3
      • Step size: 5 nm
        [RFP]
      • Mode: Emission scan
      • Config: Digital
      • Slit: 1 turn / 2 nm
      • Blaze: 750 nm
      • Excitation: 570 nm
      • Emission range: 585-650 nm
      • Average: 3
      • Step size: 5 nm
      • Transfer samples to small 150 uL cuvette and scan
    5. Data Analysis
      • Emission maxima:
        GFP 515 nm
        RFP 605 nm
      • Record intensity values at 509 nm for GFP & 606 nm for RFP
      • Subtract NC intensity value from GFP/RFP value
      • Result is the fluorescent signal of the protein in the bacteria

  16. Protein expression
  17. MAIN STEPS/TIME TABLE
    - Pre-culturing/5 hours
    - Culturing/5 hours
    - IPTG induction/15 hours

    Materials
    - LB medium with a suitable antibiotic
    - TB medium with a suitable antibiotic
    - IPTG
    - 2-liter flask
    - Incubator with shaker
    - Centrifuge

    Check List Procedure
    - Culture transformed E.coli BL21 for 5 hrs at 37 C in 10 LB medium containing antibiotic
    - Inoculate 4 ml pre-cultured cells into 400 ml of TB medium containing antibiotic in a 2-liter cultivation flask
    - Culture it for 5 hrs at 37 C with a rotary shaker at 180 rpm!
    - Add 0.5 mM IPTG in a final concentration
    - Continue the cultivation for 15 hrs at 22 C.
    - Harvest the cells with centrifuge.

    Alternative Check List Procedure
    - Inoculate single colony for 5 hrs at 37 C in 10 mL LB medium containing antibiotic
    - Inoculate 4 ml pre-cultured cells into 400 ml of TB medium containing antibiotic in a 2-liter cultivation flask
    - Culture it for 5 hrs at 37 C (OD600 to 0.5 - 0.6) with a rotary shaker at 180 rpm
    - Add 1 mM IPTG in a final concentration ( 0.5- 2.0 mM recomended for pT7)
    - Continue the cultivation for 3-4 h at 37 C (15 hrs at 22 C.)
    - Harvest the cells with centrifuge.

    Solutions
    - TB (Terrific Broth)

    Reference: http://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/




    SDS-PAGE
    MATERIALS
      
    - Vertical Electrophoresis apparatus
    - Power supply
    - pH meter
    - Balance
    - Silver staining shaker platform
    - Transilluminator
    - Filter paper

    For Gel preparation
    - Acrylamide
    - Bisacrylamide
    - Deionized Water
    - Tris base
    - SDS
    - APS
    - TEMED

    For Silver Stanning
    - Fixer
    - %50 ethanol
    - Pretreatment solution
    - Silver nitrate
    - Devoloping solution
    - Stop solution

    - Marker: Fermentas / Page Ruler Protein Ladder SM0661 (10-200kDa)

    SOLUTIONS
    Running Buffer (10L, 1X)

    - 25mM Tris, pH 8.3
    - 250mM Glycine
    - 0.1% SDS
    or
    - 60 ml 10X stock + 6 ml 10% SDS + 534 ml dH2O
    -----------------------
    - 30.3g Tris
    - 187.7g Glycine
    - 10g SDS
    - Final volume 10 L in demijon

    Staining Solution (Coomassie Blue) ( 1 L, 1 X)
    - 2 g brilliant blue (R-250)
    - 450 ml methanol
    - 450 ml dH2O
    - 100 ml acetic acid
    - store at 24 C

    Destaining Solution ( 4 L, 1 X)
    - Methanol:Acetic Acid:dH2O 40:10:50
    - Prepare 4.0 L (1.6 L MeOH, 0.4 L AcA, 2.0 L dH2O) in 1 gallon amber bottle, cap tightly
    - store at room temp.

    4X Sample Loading Buffer
    - 400 mM DTT
    - 40 mM Tris
    - 10% Glycerol
    - 4% SDS
    - 0.4% Bromophenol blue

    - prepare 15 ml
    - 925.2 mg DTT
    - 72.6 mg Tris
    - adjust pH to 6.8
    - 1500 ul Glycerol
    - 600 mg SDS
    - 6 mg Bromophenol blue
    - aliquot 50 x 600 ul
    - store at -20 C

    30% Acrylamide 1% Bis-acrylimide
    - prepare in laminar flow
    - 30 g acrylimide and 1 g bis-acrylimide in 100 ml
    - 75 mL acrylimide solution ( 40% stock, Apllichem) and 25 mL bis-acrylimide solution  ( 2% stock, Apllichem)to final volume 100 ml
    - store at 4 C, stable for 1 month

    Seperating Gel Buffer
    - 1.5 M Tris pH 8.8
    ------------------------------------------
    - for 100 mL Buffer solution
    - 18,15 g Tris pH 8.8
    - store at room temp.

    Stacking Gel Buffer
    - 0.5 M Tris
    ------------------------------------------
    - for 50 mL Buffer solution
    - 3 g Tris pH 6.8
    - store at room temp.

    APS (Ammonium Persulfate)  Stocks (100 mg/ml)
    - dissolve 0.6 g in 6 ml dH2O
    - aliquot 10 x 600 ul and store at -20 C

    1% Bromophenol Blue
    - 0.01 g in 1 ml 1M Tris, pH 7.0
    - store at room temp. in amber bottle

    CASTING 13% GEL
    - set heater to 100 C for sample prep step


      Seperating Gel (30 ml)
      - 13 ml 30% Acrylimide 1% Bis-acrylimide
    - 7.5 ml seperating gel buffer
    - 8,45 ml dH2O
    - 500 ul %10 SDS
    - 250 ul APS (initiator of polymerization)
    - 25 ul TEMED (catalyst of polymerization)

    - Load 5.4 ml separating gel between glasses

      Stacking Gel (10 ml)
      - 1.6 ml 30% Acrylimide 1% Bis-acrylimide
    - 2.5 ml stacking gel buffer
    - 5.85 ml dH2O
    - 100 ul %10 SDS
    - 15 ul 1% Bromophenol Blue
    - 50 ul APS (initiator of polymerization) [add after resolving gel is casted]
    - 10 ul TEMED (catalyst of polymerization) [add after resolving gel is casted]

    - Load 1.7 ml stacking gel between glasses
    - Inset the comb - make sure the comb has not been inserted in a tilted way. check from behind the apparatus

    - load seperating gel
    - add some butanol or isopropanol before resolving gel solidifies
    - make sure gel stays on flat surface while solidifies to prevent tilted surface
    - load stacking gel


    - if bubbles form in the stacking gel after polymerization, press the plates between hands to push them out

    SAMPLE PREPARATION AND LOADING

    - Do not overload the the samples, purity check is difficult with overloaded samples.
    - Sample volume: 5 ul sample+ 5 ul loading buffer + 10 ul dH2O
    - vortex loading buffer before use
    - put samples in heating block (100 C) for 5 min
    - if possible, do not load into the first and last lanes
    - load 5 ul marker
    - load 17 ul samples


    PREPERATION
    - check the wire on running apparatus, clean and test

    RUNNING
    - never terminate the run early, lighter bands dont separate
    - 600 ml running buffer is required for each run

    Running Standards
    - 5 mA > 3/4 of gel > 25hrs >>> overnight running
    - max: 80 mA for both old and new gel systems
    - For Lab 103 Tankı
       - for 145 min at 30 mA 120 V through seperating gel
       - amper constant

    After Run
    - wash electrophoresis unit after each use
    - weekly cleaning of power connections recommended to prevent oxidation<

    STAINING
    - Stain the gel for 30 min

    DESTAINING
    - load the tray fully with destaining buffer
    - do not put too many (over-destaining) or too less (under-staining) paper sheets
    - destaining takes 2-3 hrs

  18. Device Experiments
  19. MAIN STEPS/TIME TABLE
    - Pre-culturing/7 hours
    - Culturing to 250ml flasks/3 hours

    Materials
    - LB medium with a suitable antibiotic (Ampicillin)
    - 250 ml flasks
    - Incubator with shaker
    - Centrifuge

    Check List Procedure
    - Cell culture (with ROSE regulated kill switch) transformed E.coli BL21 for 7 hrs at 37 C in 10 ml LB medium containing antibiotic
    - Inoculate 100ul pre-cultured cells into 100 ml of LB medium containing antibiotic in a 250 ml cultivation flask
    - Culture it for 3 hrs at 37 C with a rotary shaker at 220 rpm up to OD reading reaches to 0.3
    - Continue taking OD readings up to 0.7 at 37C.
    - Harvest the 5 ml of cells with centrifuge for 5 min at 5000rpm.
    - Resuspend in 5ml PBS and take fluorescence readings.

Online Lecture

Anybody interested and novice in Synthetic Biology can use the lecture;

Here is our animation; Industrial Revolution 2 After Synthetic Biology, to introduce the basics of the field;

Here is our Online Lecture, Click the picture to go;

Click picture to reach Online Lectures

Collaboration

Collaboration with Turkish teams in iGEM 2011



Since iGEM 2007, there is a team to research and prepare a project for iGEM competition from Middle East Technical University(METU). On behalf, there are advisors and instructors who are familiar with competition and content of it. In this year there are 4 registered teams from Turkey and 3 of them are new participants. Therefore in order to come together and criticize our projects and any deficiencies or ambugity related with project. So we organized and arranged a meeting on Synthetic Biology and iGEM competition which is the first in our country.The participants of this meeting were teams;Fatih Turkey, Bilkent_UNAM, METU-BIN and researchers who related with Synthetic Biology and company owners in Biotechnology field also there was special guests via online talk, Drew Endy, he is an assistant Professor of Bioengineering, Stanford University and Scott Mohr Professor of Biological Chemistry, Boston University This meeting was held in September 10, that was close to Regional Jamboree in order to also check the presentation preparations of teams. The news agencies were also invited and the interviews were done with all teams publicity of teams were enhanced.

Except from this, we established a collaboration with METU-BIN Software iGEM 2011 team  on their software program. This year their project, M4B: Mining for BioBricks is on enhancement and simplification of parts registry and gene library usage to ease the wet lab researchers job. They requested us to use and try their software program. One of our parts in this year, kill switch is composite of this year distributions. By the way we tried their program to search and try on this composite. we gave them the following as feedback and also according to our comments on visual of program, we had collaborated this year.

“We are constructing a device that is induced by IPTG and that gives lysis enzymes and GFP as the outputs because, in order to elute methanol we need to kill our modified cells that convert ambient methane to methanol and the reason why we want GFP as one output is to be sure that it works. Our modelled device has promoter induced by IPTG, RBS, gene coding GFP and lysis cassette ending with cell death and we are glad to have it and it's working how we expected. So then, we use M4B to see that if it finds the exact device or the device that can do the same job. Since the software has only one place for output, we queried IPTG as an input and GFP as an output on the software and got corresponding 117 results. These results contain devices exactly what we have one for the same operation. However, we couldn't find the lysozyme output for the IPTG input in the software.”  

We had requested them to support us on wiki page design and same tricks on loading the links on wiki page. We had prepared the self evaluating lab safety questionnaire and they made them manipulable on wiki page  and the methane release meter on home page is their work.



Helping to other new Turkish teams in the future



Our university, METU has an online lecture application; METU OpenCourseware to support the open information source not only members of METU  and to reach people to inform in all ages. In this online application, For Synthetic Biology field, last year the sessions were loaded and this year it is upgraded. The protocols for Synthetic biology methods were downloaded and supported with tutorials of procedures that taken during experiments. We established this page and enhanced for Synthetic Biology to reach more students to meet with Synthetic Biology in any region of Turkey. We had requested  from President of METU made people to reach this webpage from universities with related departments in an official way. By the way, we believe to reach more students or researchers to iGEM competition and Synthetic Biology.

Collaboration with 2011 Uppsala Sweeden iGEM Team



We asked them to use and give feedback to our software tool on our wiki. This tool is named as BioGuide which was first studied on last year’s competition iGEM 2010 and gained silver medal. This year this team joined with wet lab team. While they were progressing the BioGuide project, this software tool also supported the web page and experiments of METU_Ankara team. We publish this software program on our wiki page to made other iGEM teams to reach and benefit from program on part  and path choices. Uppsala-Sweeden team was one of the teams that use and try the BioGuide program. They tried to reach their used parts in kit distributions and then gave us feedback on them.


They tried this software tool according to parts of their project As an input parameters they tried to type red light 'cph8',  blue light 'LovTAP', green light 'Ccas', IPTG(PT5-Lac), YF1 and as output parameters they typed amil GFP, amil CP and mCHERRY.  However they found the library of program restricted and they could not reach all parts  because BioGuide software tool library is designed and programmed on parts that are in 2010 and 2011kit plate distributions. By the way, they found this software program managable. Given part names or input and output data results with Biobrick codes of parts in parts registry library. Therefore, it made easier their job on longstanding assembly plans. For mutual collaboration, we read and evaluated their Biosafety web page and gave feedback below

“You replied the first question having main title “materials used in your project” as bacteria strain used in your project. We suggest that you should focus on chemical or any other material to answer this question.However we also used the E.coli Top 10 as host strain and found that these strains are classified as hazard group 2 pathogen by the UK Advisory Committee on the Dangerous Pathogens (ACDP).
In the question 3 that you explained “Institutional Biosafety Committee”, you described the threats of Synthetic biology in 3 main items based on your interview, we think you should expand content of items.

And lastly, it would be better if you give more detail on your last question to be more clear for future iGEM teams.”

https://2011.igem.org/Team:Uppsala-Sweden/Feedback

Software Tool

BioGuide is a Biological System Design tool. It has been developed in 2010 by the team METU TURKEY SOFTWARE. You can check the page for detailed information about the project :
https://2010.igem.org/Team:METU_Turkey_Software

Here is rough draft and setup of BioGuide Paper you can read for technical details and download the software;

BioGuide.pdf

Download the Software

This year;

We have completed biological system design part of BioGuide and enabled wet lab teams to use it.

You can watch the "how to" video of BioGuide to see what and how you can do with it;



Here is how METU ANKARA wet lab team is using BioGuide;

This year we are using it while building one of our devices, kill switch composite. Kill switch composite consists of 4 main parts which are in 2011 kit plate distributions; T7 promoter, RNA thermometer, GFP and lysis casette. The upstream part of this composite is T7 promoter (BBa_I712074) which is strong promoter from T7 bacteriophage mostly used expression system and so for strong transcription T7 promoter expression system is chosen.

We also tried the inducible promoters (pLac-IPTG inducer) to design and express the lysis casette. We desired to use a promoter that could be induced and enhance the expression to control and manipulate the translation of device. When we typed IPTG on input box of Bioguide we had reached the possible promoters that could be induced by IPTG. This program gave their standard biobrick codes and finally we got these parts by knowing their location in kit plate.

In downstream of promoter there is RNA thermometer(ROSE) coding region which determines the transcription of our lysis composite part. We had reached this part by coding the temperature 42 C in input box of BioGuide. RNA thermometer (BBa_K115001) is temperature sensitive at which 42 C translation initiates.

In downstream of RNA thermometer, green fluorescence protein(BBa_E0040) is ligated. This ligation is done to measure expression level and to comment on RNA thermometer efficiency. We typed GFP in output box since we wanted to measure fluorescence.

We had designed this composite and it's parts mostly by processing the BioGuide program that made our job easier in intense lab works. We reached the shortest path with our input and output parameters for this device to construct by BioGuide.

Conversion

i) Introduction

When examined in order, conversion is the second step of device system. While in literature searches, it is found that methanotrophs which live in either extreme conditions or in deep oceans, the process is the same, methane is converted to methanol which is the oxygenated form of hydrocarbon group. The searches directed us to organism Methylococcus capsulatus which is one of the mostly studied organisms among methanotrophs. We designed the conversion part of this organism and modified coding operon according to E.coli strain to enhance the expression. The monoxygenase coding part with protein A, B and C products are designed for conversion step of this project.

ii) Background

Methanotrophs - methane oxidizing bacteria- are ubiquitous in the environment and play an important role in mitigating global warming because they are a unique group of gram-negative bacteria that grow aerobically on methane as sole source of carbon and energy (Hanson and Hanson 1996). They are also potentially interesting for industrial applications such as production of bulk chemicals or bioremediation. Methanotrophs converts methane to methanol and subsequently to formaldehyde which is assimilated or further oxidized to CO2 for biosynthesis of cellular forms.In this biosynthesis pathway the conversion of methane to methanol is catalysed by methane monooxygenase (MMO) which is a non-specific enzyme complex that also catalyse the oxidation of a wide range of aliphatic, alicyclic and aromatic compounds (Colby et a l . , 1977; Higgins et a l . ,1979). The first step in the oxidation of methane to precursors of organic compounds is the conversion to methanol by methane monooxygenase, the key enzyme, which exists in two forms: the cytoplasmic, soluble methane monooxygenase (sMMO) and the membrane-bound, particulate methane monooxygenase (pMMO). sMMO components have been expressed in heterologous and homologous hosts. Therefore we searched more on soluble form of monooxygenase enzyme. When analyzed in detail, pMMo differs from sMMo by copper ion concentration. Copper ions have been shown to play a key role in regulating the expression of both MMO enzyme complexes. When its concentration increases the differentiation goes through particulate form. Here we analyzed the soluble form of methane monooxygenase to express in heterologous host.

a) Soluble Methane Monooxygenase

In contrast to pMMO, sMMO has extremely broad substrate specificity and can oxidise a wide range of non-growth substrates such as alkanes, alkenes and aromatic compounds thus making it the more attractive enzyme for co-oxidation reactions. sMMO is expressed only under conditions in which the copper-to-biomass ratio is low, i.e. under “low-copper” growth conditions, when copper ions are omitted from the trace elements solution of a standard mineral salts medium or cells are grown in a fermentor to high cell densities.There is also some evidence that copper ions inhibit the activity of sMMO (Jahng and Wood 1996). Like many other multi-component oxygenase systems, sMMO contains a component of approximately 16 kDa, Protein B, which serves an “effector” or regulatory role. The activity of Protein B may be regulated by proteolysis at its amino terminus (Lloyd et al. 1997). At low concentrations, Protein B converts the hydroxylase from an oxidase and stabilizes intermediates necessary for oxygen activation. Saturating amounts of Protein B dramatically increase the rates of formation of intermediates and accelerate catalysis of methane to methanol by sMMO (Lee and Lipscomb 1999). When analyzed, the most extensively characterised sMMO enzymes are those from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b (reviewed in Lipscomb 1994; Deeth and Dalton 1998). Therefore we preferred to study on Methylococcus capsulatus (Bath) for monooxygenase.The sMMO is a non-haem iron-containing enzyme complex consisting of three components. The hydroxylase consists of three subunits of 60, 45 and 20 kDa arranged in an α2 β2 γ2 configuration. sMMO genes are clustered on the chromosome of Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. mmoX, mmoY and mmoZ encode the α-, β- and γ-subunits respectively of the hydroxylase. mmoB and mmoC code for Protein B and the reductase component. Interestingly, mmoB lies between mmoY and mmoZ; an ORF of unknown function, designated orfY, with a coding capacity of 12 kDa, lies between mmoZ and mmoC in all genes clusters analysed to date (McDonald et al. 1997).

REFERENCE:

  1. J. Colin Murrell · Bettina Gilbert · Ian R. McDonald, (2000), Molecular biology and regulation of methane monooxygenase Arch Microbiology(2000) 173 :325–33
  2. Colby, J . & Dalton, H. (1978). Resolution of the methane monooxygenase of Methylococcus capsulatus (Bath) into three components. Biochemical Journal 171,461-468
  3. Hanson Te&Hanson RS(1996) Methanotrophic bacteria.American Society forMicrobiology.Microbiol. Rev., Jun 1996, 439-471, Vol 60, No. 2
  4. Charlotte, A., West, G. & Horward, D. (1992). Functional expression in Escherichia coli of proteins B and C from soluble methane monooxygenase of Methylococcus capsulatus (Bath. Journal of General Microbiology, (138), 1301-1307.

iii) Modelling

For this step of MethanE.COLIc project, the experiments were planned and modelled as protein characterizations. The protein B and C were previously characterized and analyzed proteins in literature. Based on this publications we planned the experiments as purifying protein from cell extract then SDS-PAGE analysis.

Biobricks

   LUSH protein coding device / BBa_K593002
   ROSE regulated kill-switch / BBa_K593000








   Lush protein / BBa_K593003
   mmo X / BBa_K593004
   mmo Y / BBa_K593005
   mmo Z / BBa_K593006
   mmo B / BBa_K593007
   mmo C / BBa_K593008
   mmo D / BBa_K593010
   ROSE regulated kill switch / BBa_K593009
   ROSE regulated GFP generator/ BBa_K593011

Public Awareness

5-10 year old group

For this age group, we prepared and modified one of the national child game. In this game there is a circle of group of child, one is not included in that circle. He/she runs around the circle and labels two of the participants in the circle. When the labelled kids realized that, they leave the circle and new kids join the circle. The video below shows our modified game in which four kids that out of circle are our restriction enzymes wearing white caps. Moreover, we have three insert groups which wear red, green and yellow caps. The blue capped ones are our backbone.
During the game we explained kids what DNA, enzyme and insert are, and what we have done as a Synthetic Biology research group.


10-17 year old group

This group consists of high school aged children groups.Through out the workshop, we have paired them in doubles. Firstly, we have explained Synthetic Biolgy and what Synthetic Biology is able to do. After these training, we explained them our project and taught them basic molecular biology procedures and what are the requirements of basic synthetic biology based experiments. We have done the experiments (cloning procedures) together and also they did individually. We also encouraged them to join iGEM High School division.

Official Announcement To All Universities For Introduction Of Synthetic Biology and iGEM Competetion

For the last human practice approach, we believed that we should inform especially students from other universities about synthetic biology as students in universities are the ones who can improve this field for further generations. For that purpose, we published an official message from our instructor, Prof. Dr. Mahinur Akkaya, with this message, students from other universities –regardless of their department that they’re studying in- will be aware of the field synthetic biology and may not only consider about learning more but also consider about creating an iGEM team for coming years. Here is the original official message and its brief translation for you to read:
Dean Office of Arts and Science Department
Synthetic biology is a field where  natural sciences and engineering sciences go together hand in hand. It is an emerging science that requires the literature search, knowledge  of natural sciences and methods of engineering sciences. Synthetic biology is the engineering of organisms to give them desired properties, except their regular metabolic activities. These desired properties are entegrated into genome of the organism with molecular cloning procedures.
There are lots of studies going on to introduce this newly emerging science field to people from all age groups and producing an information repository and improving it at the same time. One of this studies is the iGEM (International Genetically Engineered Machines) competition.
We, as METU iGEM teams, are attending this competition for four years. For this newly emerging science field to improve more, we believe that it should be spread out to other universities and more and more people should attend this competiton. We are sending you our study about the videos of lab protocols that are mainly used in synthetic biology and references that you can search for more information.  We feel honored to tell that these sources are available for everyone who would like to reach them. Links for the sources are as follows:
http://ocw.metu.edu.tr/course/view.php?id=137
http://www.youtube.com/user/Metuankara



18-24 year old group

This group consists of METU students. We arranged the Physics lectures in order to reach students from several departments. We explained the miracle of Synthetic Biology and our project. At the end of the presentation, we gained many volunteers to join METU-iGEM team for the following years. Also, we have an online lecture application as METU open course ware. For this web page, we have prepare the course notes and they were uploaded to reach many students from all around to inform and contact for further relations about Synthetic Biology.
In addition, we distributed hand outs about our project and Synthetic Biology at METU campus, when the registration of new students were done. We aimed to inform students about our project and about Synthetic Biology to attract their attention on the issue.

Online lecture link:&nbsp: http://ocw.metu.edu.tr/course/view.php?id=137

Above 24 year old

For this group of people we met with group of engineers who work in mining fields in Turkey. We explained our project in details and consulted them on background of our project. What the conditions in mines are, what the general regulations for worker safety are and what the rooted controls for any gas release are the main points which we discussed on.

List of Engineers:

Turkish Architects and Engineers Association members:

NO NAME & SURNAME INSTITUTION CITY
1 Başak GÜLEKEN ERKUNT DÖKÜM A.Ş. ANKARA
2 Saliha SİVRİ ETİ MADEN İŞLETMELERİ ANKARA
3 Aynur ODAMAN KOSGEB ANKARA
4 Mehmet Şevki TÜRÜDÜ MADEN TETKİK ARAMA KURUMU ANKARA
5 Galip Şevket YİNSEL MİTAŞ A.Ş ANKARA
6 Cemil Hakan GÜR ORTA DOĞU TEKNİK ÜNİVERSİTESİ ANKARA


Sponsors

As METU Ankara iGEM 2011 team we would like to thank to our partners and sponsors in listed below for their supports and for their encouragement.

Cargill Comart

Sentegen

MetuTech

Onkogen

Sacem

Metu

Entrapment

i) Introduction

After conversion of methane to methanol, the successive step is the entrapment of methanol. Methanol is an alcohol form of hydrocarbon methane and includes hydroxyl group. Hydroxyl radical groups are highly reactive and, and so short-lived; however, they form an important part of radical chemistry. Hydroxyl groups are especially important in biological systems and their chemistry because free radicals tend to form hydrogen bonds both as donor and acceptor. This property is also related to their ability to increase hydrophilicity and water solubility. Therefore hydroxyl free radicals cause damage to oxidative cells and cellular membranes. In order to produce the methanol for further industrial manufacturing in cellular organism, the organismic conditions should be adjusted. In the light of this, for our bacterial system we transferred the protein coding sequence(named as LUSH) of Drosophilia melanogaster to attack to free methanols and hydroxyl group. Here is some structural information about protein LUSH.

ii) Background

LUSH is an alcohol-sensitive odorant binding protein expressed in the olfactory organs of Drosophila melanogaster, and it is used as a model system to investigate the biophysical nature of alcohol-protein interactions at alcohol concentrations that produce intoxication in humans. In this study, by using NMR spectroscopy, they have identified the regions of LUSH that show increased conformational stability on binding alcohols. These residues primarily line the alcohol-binding pocket. A direct measure of the degree of stability that alcohol imparts on LUSH has been provided. LUSH was originally identified as responsible for mediating an avoidance response to short-chain n-alcohols.

The general structure of odorant binding proteins consists of six α-helices surrounding a hydrophobic ligand- binding pocket which differs in size and shape between each protein. All these odorant binding proteins have a set of six cysteines that form three conserved disulfide bonds. In the study, by observing the X-ray crystal structures of LUSH-alcohol complexes, it was found that alcohol binds to a single site in the protein formed by a network of concerted hydrogen-binding residues located at one end of hyrophobic pocket. This binding site has some sequence and/or structural similarities to regions of several ligand gated ion channels (LGICs) that have previously been implicated in inferring sensitivity to alcohol. It is hypothesized in the study that the alcohol-binding site in LUSH may represent a more general structural motif for functionally relevant alcohol-binding sites in proteins. The characterization of the effects of n-alcohols on the structure and stability of LUSH is presented. Also, in the absence of ligand, LUSH exists in vitro in a partially unstructured state and binding of alcohols shifts the solution conformation to a more compact folded state which is accompanied by an increase in the overall protein stability. Those regions of the protein that show the largest changes in local dynamics on binding alcohol have been identified and it have been shown that these are predominantly associated with the residues that line the alcohol-binding pocket. The results provide a quantitative measure of the ability of short-chain alcohols to stabilize protein structure at physiological relevant concentrations.

REFERENCE:

  1. Bucci, B. K., Kruse, S. W. & Thode, A. B. (2006). Effect of n-Alcohols on the Structure and Stability of the Drosophila Odorant Binding Protein LUSH. Biochemistry, 45 1693-1701
  2. Thode, A. B., Kruse, S. W. & Nix, J. C. (2008). The role of multiple hydrogen bonding groups in specific alcohol binding sites in proteins: Insights from structural studies of LUSH. J.Mol Biol, 376 (5), 1360-1376

iii) Modelling

In MethanE.COLIc project, LUSH protein is designed to hold methanol; the conversion product of methane. Methanol is bounded to LUSH protein through its hydroxyl group. In device experiments of this part, we planned to isolate and denaturate protein and then refold in presence of methanol and we planned to analyze the alcohol bounded protein in X-ray diffraction crystallogrophy.

Notebook

Media Press

Middle East Technical University

Middle East Technical University is a pioneering university in education, research and community service in the country. In addition to being highly preferred by students, it is internationally recognized.Our country is successfully represented by METU academic staff in numerous scientific and professional institutions and committees.The wide variety of cultural, artistic and intellectual activities, and sports opportunities that are offered by our campuses are a part of the "education environment" of international quality. METU has been defined in national rankings as the university which has the "Richest Social Environment".Whatever your reason to visit METU is, you will find here an innovative, dynamic and exciting environment which offers diversity and variety of choices. Come and join us at METU where the most successful students of the country, the most outstanding teaching and research staff, and the most distinguished administrative-technical staff live, learn, and produce hand in hand...

For more information, visit following link;

Middle East Technical University

Kill switch

i) Introduction

In this project the last step is the death of our bacteria to elute the protein(LUSH) bounded product methanol which is one of the precursors of biofuel. In our modified bacteria, the death of cells occurs by temperature dependent cell lysis device. The expression of this device takes place at 42 C since there is an RNA thermometer part in upstream of this device. At the downstream of this device there is an lysis casette. When transcription initiated at 42C holin and endolysin is expressed from this device and make cell dead.

ii) Background

This composite part consists of promoter, RNA thermometer, GFP and lysis casette. The upstream part of this composite is T7 promoter (BBa_I712074) which is strong promoter from T7 bacteriophage. For strong transcription T7 promoter expression system is chosen. In downstream of promoter there is RNA thermometer(ROSE) coding region which determines the transcription of our lysis composite part. RNA thermometer (BBa_K115001) is temperature sensitive that at 42C translation initiates. That means, up to 42 C RNA thermometer is in dimer form and at 42C it becomes linear. In downstream of RNA thermometer, green fluorescence protein(BBa_E0040) is ligated. This ligation is done to measure expression level and to comment on RNA thermometer efficiency. The last ligate to this composite is bacteriophage 21 lysis casette (BBa_K124003). This lysis casette induces lysis in E.coli. This gene cassette consists of the S protein (Holin), R protein (Endolysin), and Rz protein. When translation initiates at 42 C then the following coding gene begins to produce the proteins which lyse the cell.

iii) Modelling

In MethanE.COLIc project, this composite is modelled to lyse and kill the bacteria when switched on at 42C. In experimental design, we ligated this composite without lysis casette (with promoter, ROSE and GFP) We planned two control groups. One is T7 promoter and GFP ligate, this is designed to measure expression level of T7 promoter by reading the GFP expression. The second control group is T7 promoter, ROSE and GFP ligate, this is designed to measure the expression level of T7 promoter with ROSE ligate at 37 C. The device experiment is done with T7 promoter ROSE and GFP ligate at 42 C in which the GFP expression is expected.

Contact

Device Experiment Results

-Flask experiments

In characterization experiments, we choosed temperature sensitive RNA thermometer (BBa_K115001) and modelled the experiments on GFP measurements. RNA thermometer is a temperature sensitive DNA part that up to 42 C it forms a dimer. This dimer formation prevents polymerase readings that the translation is obstructed. At 42 C the linear form of part forms and translation initiates. This part is submitted to parts registry by iGEM 2008 TUDelft team and the experience of part is represented as none. We modelled the characterization of this part by planing two apart control groups.

One pair group for characterization was T7 promoter,RNA thermometer and GFP from upstream to downstream at 37C and the same device at 42C. The aim of this control group was to control any expression of green fluorescence protein at 37 C to check RNA thermometer dimer formation. We prepared a flask experiments and from protein formation to folding range we measured the spectrophotometer and fluoresence spectrometer readings. It is showed the dimer formation of RNA thermometer at lower than 42 C caused inefficient binding of RBS to DNA. That means we measured the GFP readings at two temperatures however at 42 C the expression was observed in higher level. As seen at OD:0.6 reading there is a dramatic difference in expressions.

Other pair group for characterization was T7 promoter with GFP at 37 C  and  T7 promoter,RNA thermometer and GFP from upstream to downstream at 42C. This control group was modelled in order to check and compare the expression levels of two devices. The expected result was to observe the similar readings. Because without RNA thermometer temperature switch off device express similar readings at 37C with RNA thermometer device at 42 C. In the data analysis we observed that there is deviation in readings between control T7 with GFP at 37C and to construct, T7 RNA thermometer and GFP at 42 C.


GFP Reading Data





Assembly Results

According our clonning plan, we planned to ligate the coding sequences of methane monooxygenase as subunits and to express the functional monooxygenase enzyme. Since we synthesized the long coding sequence we did PCR experiments with specific primers we extracted the parts in each the gel electrophoresis results gave validation for successfully synthesied DNA fragments, then we digested and ligated each part with related promoters and vector (pSB1C3) again gel electrophoresis data gave us validation to correctly digest and ligated form.




















-The device parts control experiments

The synthesized methane monoxygenase construct was so long part that we had problems in synthesizing that we got genes so late. By the way the long sequence was divided into two sequences that one of the regions were splitted into two parts therefore we could not ligated the two parts of coding sequence due to unidentified restriction sites, and full construct unfortunately did not reach to us. The main methane interacting region of monooxygenase could not be expressed functionally. We expressed the protein B and C of methane monoxygenase encoeded from mmo B and mmo C genes in protein expression host E.coli BL21 strain. We planned to characterize the proteins in their theoretical molecular weights by SDS-PAGE analysis. However due to technical problems in gel formation we lost samples that we could not reached the data. We planned another part on kit plate distributions to check works or not. The bacteriophage 21 lysis casette S, R, and Rz (PVJ4) (BBa_K124003). This part was designed by 2008 iGEM Brown team which induces lysis in E.coli bacteria. We ligated this part with ROSE regulated GFP generator  to induce the lysis of bacteria at 42C. Apart from this ligate, we ligated lysis casette with plac promoter and lacZ expressing gene to observe the blue colonies on plates. However we could not observe any blue colony on plates.

Fun & Learn

Flashmob!



In order to reach more people from several age groups, we have organized a flashmob activity which is related with our project -in molecular level-. (A flashmob is an activity done by a group of people who assemble suddenly in a public place, perform an unusual and sometimes seemingly pointless act for a brief time, then disperse, often for the purposes of entertainment, satire, artistic expression. Flashmob activities are organized via telecommunications, social media, or viral emails ).One of our group member who is also a member of Flashmob society in Ankara helped us for arrangements. We use flashmob activity in order to explain what our project is and to increase the public awareness about Synthetic Biology and especially about our project. Flashmob event took place at one of the biggest shopping malls in Ankara where people often strolled around. Afterwards, we had interviewed with people who watched our sketch and informed people about nearly limitless capabilities of synthetic biology. Indeed we prepared handouts that contains interesting statistical data about methane and its side effects, also information about iGEM and about our project.

Acknowledgements

We are grateful to;

Gönenç Gürsoy, for helping in modelling, being our travel agent and ordering the best potato chips for us (with beer of course)...

Duygu Yıldız, for being a mother and bringing delicious food for our breaks, being our light graffiti maker...

Gence Bektaş for being a methane molecule...

Önder Alparslan, for helping in modelling and ending our desperate search for a graphic tablet :)...

Mustafa Türkkan for being the best lipid bilayer member ever :) even though being a civil engineer...

Efe Köksal for being our moviemaker...

Demir Berkay Yılmaz for our second moviemaker and being in lab with us most of the time...

Ankara Flashmob society for being with us in our presentations and making it real fun...

Tufan Öz, Burcu Tefon, Aslıhan Kurt and Çiğdem Yılmaz for their incredible patience to our questions and supplying chemicals at speed of light!

As a team, we’d like to thank our instructor Professor Dr. Mahinur S. Akkaya for her patience, love, support and all of her solutions to our unending problems. :)

Safety

Safety Questions

1. Would the materials used in your project and/or your final product pose:
a. Risks to the safety and health of team members or others in the lab?

  1. While in cloning steps the most dangerous material that we used is ethidium bromide which is used in agarose gel electrophoresis. Ethidium bromide is a potential mutagen because it works by inserting itself between the two strands of double-stranded DNA. Since the amount of ethidium bromide kept in our lab is relatively small, it does not pose devastating effects.However it is a toxic chemical, when got in contact, it causes eye and skin irritation. To prevent the damages that we would be face with, we wraped it with aluminium and put in brown bottle and wore gloves while using.
  2. While device training experiments we planned to use methane gas CH4 and methanol CH3OH. Methane is  inactive biologically and essentially nontoxic.Methane is not listed in the IARC, NTP or by OSHA as a carcinogen or potential carcinogen. When inhaled in high concentrations,  so as to exclude an adequate supply of  oxygen to the lungs causes dizziness, deeper breathing due to air hunger, possible  nausea and eventual unconsciousness. In case of protection, we adjusted the progressing the steps for methane in glove boxes in order to provide the respiratory protection. The ventilation is  supported by hood. However due to methane gas is potent for greenhouse effect for doing experiments, special hood system is designed that could be used which has its own gas container for storage of gases is used. Thus, emission of methane gas is controlled. Later on, filled gas container is taken by department of chemistry to empty with certain procedures. We arranged the conditions in department for possible experiments with methane gas. And protective gloves and goggles are equiped for personal safety.Out of the experiments for storage the gas tanks were saved in area where is cool enough and never used open flames.

REFERENCE:http://www.isocinfo.com/DocumentRoot/13/Methane.pdf
For the steps with methanol the characterization tests for LUSH protein, methanol is        used. Methanol is hazardous in case of skin contact, eye contact, ingestion and inhalation. It is flammable liquid.The safety for this chemical is provided by experimenting in hood with high ventilation and and so much exposed because it pose a health risk to anyone in lab.
REFERENCE :http://www.sciencelab.com/msds.php?msdsId=9927227

We had studied with ANS; 1-Anilino-8-Naphthalene Sulfonate as protein conformational tightening agent for LUSH protein folding experiments. We searched on safety of this chemical and we found that it is non hazardous chemical according to Directive 67/548/EE Following reference belongs to Sigma Aldrich MSDS report.
http://www.sigmaaldrich.com/catalog/DisplayMSDSContent.do

b. Risks to the safety and health of the general public if released by design or accident?

  1. The materials  mentioned in part a. could potentially be dangerous to the general public. We use just enough concentration for our experiments. We always check the amount both for methane and methanol and EtBr, because if accidentally released, they could be dangerous to us while experiments and to lab security.

c. Risks to environmental quality if released by design or accident?

  1. Since methane is a gas at normal temperature and pressure, the inhalation of this gas pose a risk for human health and so for environmental quality. Methane is potent greenhouse gas and compared to carbon dioxide it has greater potential. This makes methane gas dangerous for environmental quality in case of release by accident or design.However, as mentioned in part a, we controlled the conditions for gas and examined in air ventilated glove boxes. Another material which could pose risk for environmental quality is methanol. This chemical is volatile and flammable liquid. When this chemical release near open flame it could result with harmful effects both for health and environment conditions.

 d. Risks to security through malicious misuse by individuals, groups or states?

Only the methane gas has risks for security if reaches to malicious someone. Our group members have experience and training on gas used experiments and there is no possibility on misuse indeed malicious misuse.
e.Please explain your responses (whether yes or no) to these questions.
Specifically, are any parts or devices in your project associated with (or known to cause):

- pathogenicity, infectivity, or toxicity?  No
- threats to environmental quality? None of our designed parts pose a risk to environment
- security concerns? No

    There are no part on our experiments and our project that could be use as treat to environment or infect to human life as individually. Only one of the parts, subunit A of methane monooxygenase may pose a health risk while studying since it has the region where methane interacts and the conversion steps initialize. As host cell we used E.coli BL21 (DE3) and Top10 strains, these strains are classified as hazard group 2 pathogen by the UK Advisory Committee on the Dangerous Pathogens (ACDP).
2. If your response to any of the questions above is yes:
a. Explain how you addressed these issues in project design and while conducting laboratory work. Explained in each question
b. Describe and document safety, security, health and/or environmental issues as you submit your parts to the Registry. Explained in each question


3. Under what biosafety provisions will / do you operate?
a. Does your institution have its own biosafety rules and if so what are they? Provide a link to them online if possible.
Yes, there is general laboratory biosafety rules for lab security and researcher security obeyed in Biology and Chemistry departments. In research laboratories the students are provided with general lab security and rules which are compilation of general and international procedures for individual safety. These procedures are the compilation of several links as
http://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Gdlnes_lnk_2002z.pdf

 

b. Does your institution have an Institutional Biosafety Committee or equivalent group? If yes, have you discussed your project with them? Describe any concerns or changes that were made based on this review.

Yes there is biosafety and ethical research center in METU until 2000. This committee helds  many conferences about ethics, environmental ethics in studies and the medicinal ethics based on human researchs. The below link belongs to this committee however in Turkish language.
http://www.ueam.metu.edu.tr

     Indeed in Biology department at METU one of our professors, Prof.Dr Huseyin Avni Oktem was one of the members of National Biosafety Coordinating Committee. He is our pioneer for biosafety and security issues. In the case of any safety issues, he is the one to consult  the critical safety points in our project and also discussed the project based on safety.

     c. Will / did you receive any biosafety and/or lab training before beginning your project? If so, describe this training.

Before we begin to experiments, we firstly were taught on lab security and personal safety for cases of individual injure, indeed in emergency situations. This interval was training for us. Also team leaders, the experienced ones in team, prepared an exam for new team members as a part of training for general lab regulations and project details.

  1. Does your country have national biosafety regulations or guidelines? If so, provide a link to them online if possible.

 

In Turkey, biosafety regulations are organized by the Biosafety Information Exchange Mechanism of Turkey, facility of Ministry of Agriculture and Rural Affair.
http://www.tbbdm.gov.tr/en/Home.aspx