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2024-03-28T22:33:44Z
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2014-10-08T01:14:48Z
<p>Lemur3 93: </p>
<hr />
<div></div>
Lemur3 93
http://2013.igem.org/Template:Team:UC_Davis/Head
Template:Team:UC Davis/Head
2014-09-08T05:25:02Z
<p>Lemur3 93: </p>
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</head><br />
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<a href="/Team:UC_Davis" style="font-size:13px;">Home</a><br />
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<a href="https://2013.igem.org/Team:UC_Davis/Team_Overview" style="font-size:13px;">Team</a><br />
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<li><a href="/Team:UC_Davis/Modeling">Modeling</a></li><br />
<li><a href='/Team:UC_Davis/Assembly'>Assembly</a></li><br />
<li><a href='/Team:UC_Davis/Parts'>Parts</a></li><br />
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<a href="/Team:UC_Davis/Data" style="font-size:13px;">Data</a><br />
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<li><a href="/Team:UC_Davis/Data">Testing Constructs</a></li><br />
<li><a href="/Team:UC_Davis/AndersonPromoters">Anderson Promoters</a></li><br />
<li><a href="/Team:UC_Davis/AndersonPromoters2">Anderson II</a></li> <br />
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<li><a href="/Team:UC_Davis/Protocols">Protocols</a></li><br />
<li><a href="https://2013.igem.org/Team:UC_Davis/Gallery">Gallery</a></li><br />
<li><a href="https://2013.igem.org/Team:UC_Davis/Attributions">Attributions</a></li><br />
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<a href="/Team:UC_Davis/HumanPracticesOverview" style="font-size:13px;">Human Practices</a><br />
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<li><a href='/Team:UC_Davis/HumanPracticesOverview'>Overview</a></li><br />
<li><a href='/Team:UC_Davis/Outreach'>Outreach</a></li><br />
<li><a href='/Team:UC_Davis/Database'>The Depot</a></li><br />
</ul><br />
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Lemur3 93
http://2013.igem.org/Template:Team:UC_Davis/Head
Template:Team:UC Davis/Head
2014-09-08T05:24:13Z
<p>Lemur3 93: </p>
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</head><br />
<body><br />
<a id="top"><br />
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<a href="/Team:UC_Davis" style="font-size:13px;">Home</a><br />
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<a href="https://2013.igem.org/Team:UC_Davis/Team_Overview" style="font-size:13px;">Team</a><br />
<ul><br />
<li><a href="https://2013.igem.org/Team:UC_Davis/Team_Overview">Overview</a></li><br />
<li><a href="https://2013.igem.org/Team:UC_Davis/Bios">Bios</a></li><br />
<li><a href='/Team:UC_Davis/Contact'>Contact Us</a></li><br />
<li><a href="https://igem.org/Team.cgi?id=1212">Official Team Profile</a></li><br />
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<a href="https://2013.igem.org/Team:UC_Davis/Project_Overview" style="font-size:13px;">Project</a><br />
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<li><a href="/Team:UC_Davis/Modeling">Modeling</a></li><br />
<li><a href='/Team:UC_Davis/Assembly'>Assembly</a></li><br />
<li><a href='/Team:UC_Davis/Parts'>Parts</a></li><br />
<li><a href='/Team:UC_Davis/Safety'>Safety</a></li><br />
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<li><a href="/Team:UC_Davis/AndersonPromoters">Anderson Promoters</a></li><br />
<li><a href="/Team:UC_Davis/AndersonPromoters2">Anderson II</a></li> <br />
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<li><a href="/Team:UC_Davis/Protocols">Protocols</a></li><br />
<li><a href="https://2013.igem.org/Team:UC_Davis/Gallery">Gallery</a></li><br />
<li><a href="https://2013.igem.org/Team:UC_Davis/Attributions">Attributions</a></li><br />
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<li><a href='/Team:UC_Davis/Outreach'>Outreach</a></li><br />
<li><a href='/Team:UC_Davis/Database'>The Depot</a></li><br />
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Lemur3 93
http://2013.igem.org/Template:Team:UC_Davis/Head
Template:Team:UC Davis/Head
2014-09-08T05:20:35Z
<p>Lemur3 93: </p>
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Lemur3 93
http://2013.igem.org/Template:Team:UC_Davis/Head
Template:Team:UC Davis/Head
2014-09-08T05:20:09Z
<p>Lemur3 93: </p>
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Lemur3 93
http://2013.igem.org/Template:Team:UC_Davis/Head
Template:Team:UC Davis/Head
2014-09-08T05:19:19Z
<p>Lemur3 93: </p>
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<li><a href="https://2013.igem.org/Team:UC_Davis/Team_Overview">Overview</a></li><br />
<li><a href="https://2013.igem.org/Team:UC_Davis/Bios">Bios</a></li><br />
<li><a href='/Team:UC_Davis/Contact'>Contact Us</a></li><br />
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<li><a href="/Team:UC_Davis/Modeling">Modeling</a></li><br />
<li><a href='/Team:UC_Davis/Assembly'>Assembly</a></li><br />
<li><a href='/Team:UC_Davis/Parts'>Parts</a></li><br />
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<a href="/Team:UC_Davis/Data" style="font-size:13px;">Data</a><br />
<ul><br />
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<li><a href="/Team:UC_Davis/AndersonPromoters">Anderson Promoters</a></li><br />
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Lemur3 93
http://2013.igem.org/Team:UC_Davis/Bios
Team:UC Davis/Bios
2013-09-28T02:51:39Z
<p>Lemur3 93: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
<br />
<html><br />
<head><br />
</head><br />
<br />
<body><br />
<div><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/Teambiobanner_UCDavis.jpg" class="banner" width=967 height=226 /><br />
</div><br />
<div class="floatboxwide"><br />
<h1>2013 UC Davis iGEM Team</h1><br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/Aurabio_UCDavis.jpg" class="rightpic" width="150" height="150"><br />
<div class="teampagetext"><br />
<h3> Aura Ferreiro</h3><br />
<p><br />
I am a 5th year undergraduate majoring in Biological Systems Engineering. I enjoy playing my ukulele and coming up with ways to integrate quinoa and tofu into every meal I cook. I plan to apply for graduate programs in the same field I study now, and look forward to a career in design and modeling of industrial biological systems, or of synthetic biological devices with commercial applications.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/8/82/Ucdavis13_amy_photo.JPG" class="leftpic" width="150" height="150"><br />
<div class="teampagetext"><br />
<h3>Amy Soon</h3><br />
<p><br />
I am a senior majoring in Biomedical Engineering and specializing in Systems and Synthetic Biology. Outside of school and the lab, I enjoy fantasy and mystery stories in tv shows, games or novels. I am still in the process of deciding on my future career path and whether I will go to graduate school directly after graduating or take some time off to get work experience first. I hope to be able to continue working with synthetic biology in a career in industry.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/e/e2/Ucdavis13_alex_photo.JPG" class="rightpic" width="150" height="150"><br />
<div class="teampagetext"><br />
<h3>Alex Neckelmann</h3><br />
<p><br />
I am a Senior majoring in Biotechnology with an emphasis in microbiology and awesome. When I’m not at school, I enjoy playing tennis or reading biographies. I plan on graduating and applying to graduate school in the fall. After graduate school, I would really enjoy working on projects involved in bioremediation or optimizing industrial bioprocesses through synthetic biology.<br />
</p><br />
</div><br />
</div><br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/c/cb/Ucdavis13_david_photo.JPG" class="leftpic" width="150" height="150"><br />
<div class="teampagetext"><br />
<h3>David Hwang</h3><br />
<p><br />
Hi, I'm David Hwang, and I'm entering my fourth year as a Computer Science major this coming fall. My current professional interests are computer science applications in healthcare, education, and clearly, biology. My unprofessional interests are ultimate frisbee (I am on the school's club team where we placed in the top 8 in the nation this past year), and cooking.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d0/Lucasbio_UCDavis.jpg" class="rightpic" width="150" height="150"><br />
<div class="teampagetext"><br />
<h3>Lucas Murray</h3><br />
<p><br />
Hi, I am a third year biomedical engineering major with an emphasis in systems and synthetic biology. When I'm not going back and forth between labs, I enjoy competitive sailing, freediving, and underwater photography. If I had free time, you'd find me doing any of those three. My main background is in instrumentation and chemistry, but my academic focus is on synthetic biology, biophysics, and molecular biology. I plan to apply to graduate school next fall, where I would like to continue to study synthetic biology.<br />
</p><br />
</div><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatboxwide"><br />
<h1>Advisors:</h1><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/UCD_13_Andrew_profile.jpg" class="leftpic"><br />
<h3>Andrew Yao</h3><br />
<div class="teampagetext"><br />
<p><br />
<ul><br />
<li>Wetlab Advisor</li><br />
<li>B.S. in Biomedical Engineering</li><br />
<li>Department: Biomedical Engineering</li><br />
</ul><br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/UCD_13_Nick_profile.jpg" class="rightpic"><br />
<h3>Nick Csicsery</h3><br />
<div class="teampagetext"><br />
<p><br />
<ul><br />
<li>iGEM Veteran Advisor</li><br />
<li>B.S. in Biological Systems Engineering</li><br />
</ul><br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/5/56/UCD_13_photo_Navneet.jpg" class="leftpic"><br />
<h3>Navneet Rai</h3><br />
<div class="teampagetext"><br />
<p><br />
<ul><br />
<li>Postdoctoral Research Associate</li><br />
<li>Wetlab Advisor</li><br />
</ul><br />
</p><br />
</div><br />
</div><br />
</div><br />
<br />
<br />
<br />
<div class="floatboxwide"><br />
<h2>Instructors:</h2><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c3/UCD_2013_Marc_Bio.png" class="leftpic"><br />
<div class="teampagetext"><br />
<h3>Dr. Marc Facciotti</h3><br />
<p><br />
<ul><br />
<li>Ph.D. in Biophysics</li><br />
<li>Department: Biomedical Engineering and UC Davis Genome Center</li><br />
<li><a target="new" href=http://www.bme.ucdavis.edu/facciotti>Facciotti Lab Website</a></li><br />
</ul><br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a3/UCD_13_photo_Justin.jpg" class="rightpic"><br />
<div class="teampagetext"><br />
<h3>Dr. Justin Siegel</h3><br />
<p><br />
<ul><br />
<li>Ph.D. in BioMolecular Structure and Design</li><br />
<li>Department of Biochemistry and Molecular Medicine, School of Medicine</li><br />
<li><a target="new" href=http://sites.google.com/site/ucdsiegellab/>Siegel Lab Website</a></li><br />
</ul><br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/5/5d/UCD_13_photo_Ilias.jpg" class="leftpic"><br />
<div class="teampagetext"><br />
<h3>Dr. Ilias Tagkopoulos</h3><br />
<p><br />
<ul><br />
<li>Ph.D. in Electrical Engineering</li><br />
<li>Department: Computer Science and UC Davis Genome Center</li><br />
<li><a target="new" href=http://www.cs.ucdavis.edu/~iliast>Tagkopoulos Lab Website</a></li><br />
</ul><br />
</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="floatboxwide"><br />
<center><img src="https://static.igem.org/mediawiki/2013/e/e3/Ucdavisteampic.gif" class="genpic" width=600 height=403></center><br />
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Lemur3 93
http://2013.igem.org/Team:UC_Davis/Protocols
Team:UC Davis/Protocols
2013-09-28T02:49:41Z
<p>Lemur3 93: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
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<h1>Protocols</h1><br />
<!-- accordion starts here --><br />
<div id="firstpane" class="menu_list"><br />
<br />
<p class="menu_head"> LB Media </p><br />
<div class="menu_body"><br />
<li>950 mL dH<sub>2</sub>0</li><br />
<li>10 g Tryptone</li><br />
<li>10 g NaCl</li><br />
<li>5 g Yeast Extract</li><br />
1 L Total<br />
<li>Add 15 g Agar, if being poured into plates.</li><br />
<li>Autoclave, when cool add antibiotics if desired.</li><br />
</div><br />
<br />
<p class="menu_head"> Antibiotic Stock Solutions</p><br />
<div class="menu_body"><br />
<p>Materials</p><br />
Chloramphenicol<br />
<li>Working Concentration 12.5 μg/ml</li><br />
<li>Stock solutions can be made at 35 mg/ml in ethanol and kept at -20º C</li><br />
<br />
Kanamycin<br />
<li>Filter sterilize for kanamycin</li><br />
Working Concentration:<br />
<li>25 μg/mL for low-copy plasmids</li><br />
<li>35 µg/mL for high-copy plasmids</li><br />
<li>Stock solution is 35 mg/ml in water and kept at -20º C</li><br />
<br />
Spectinomycin <br />
<li>Filter sterilize</li><br />
<li>Working Concentration 50 μg/mL</li><br />
<li>Stock solution is 100 mg/mL in water and kept at -20º C</li><br />
</div><br />
<br />
<p class="menu_head"> Heat Shock Transformation</p><br />
<div class="menu_body"><br />
<p>Procedure</p><br />
<ol><br />
<li>Preheat water bath to 42º C.</li><br />
<li>Thaw competent cells on ice for 10 minutes.</li><br />
<li>Use 50 µL competent cells, add transforming DNA [up to 25 ng per 50 µL of cells, volume not exceeding 2.5 µL (5%)]. For control add 1 µL of control DNA (PUC19 carb resistance). Swirl to mix, store on ice 5 minutes. </li><br />
<li>Heat shock in 42º C water bath for 45 seconds.</li><br />
<li>Cool cells in ice bath for a few minutes.</li><br />
<li>Add 800 µL LB to each tube, set in shaker at 37º C for 45 minutes. </li><br />
<li>Transfer 200 µL of culture per plate (containing the appropriate antibiotic).</li><br />
<li>Spread using glass beads.</li><br />
<li>Invert plates and incubate overnight (12-16 hrs) at 37º C.</li><br />
</ol><br />
</div><br />
<br />
<p class="menu_head"> Making Chemically Competent Cells</p><br />
<div class="menu_body"><br />
<p>Procedure</p><br />
<ol><br />
<li>Prepare 0.5 M PIPES (pH 6.7) (piperazine-1,2-bis[2-ethanesulfonic acid]) by dissolving 15.1 g of PIPES in 80 ml of pure H2O (Milli-Q, or equivalent). Adjust the pH of the solution to 6.7 with 5 M KOH, and then add pure H<sub>2</sub>O to bring the final volume to 100 ml. Sterilize the solution by filtration through a disposable pre-rinsed Nalgene filter (0.45-µm pore size). Divide into aliquots and store frozen at -20°C.<br />
Prepare Inoue transformation buffer by dissolving all of the solutes listed below in 800 mL of pure H2O and then add 20 ml of 0.5 M PIPES (pH 6.7). Adjust the volume of the Inoue transformation buffer to 1 liter with pure H<sub>2</sub>O.</li><br />
<br />
<table> <tr><br />
<th></th> <th>Reagent</th> <th>Amount per liter</th> <th>Final Concentration</th><br />
</tr> <tr><br />
<th></th> <td>MnCl<sub>2</sub>•4H<sub>2</sub>O</td> <td>10.88 g</td> <td>55 mM</td><br />
</tr> <tr><br />
<th></th> <td>CaCl<sub>2</sub>•2H<sub>2</sub>O</td> <td>2.20 g</td> <td>15 mM</td><br />
</tr> <tr><br />
<th></th> <td>KCl</td> <td>18.65 g</td> <td>250 mM</td><br />
</tr> <tr><br />
<th></th> <td>PIPES (0.5 M, pH 6.7)</td> <td>20 ml</td> <td>10 mM</td><br />
</tr> </table><br />
Add H<sub>2</sub>O to fill solution to 1 liter. Sterilize Inoue transformation buffer by filtration through a prerinsed 0.45-µm Nalgene filter. Divide into aliquots and store at -20°C.<br />
<br />
<li>Pick a single bacterial colony (2-3 mm in diameter) from a plate that has been incubated for 16-20 hours at 37°C. Transfer the colony into 25 ml of SOB medium (LB may be used instead) in a 250-ml flask. Incubate the culture for 6-8 hours at 37°C with vigorous shaking (250-300 rpm).</li><br />
<li>At about 6 o'clock in the evening, use this starter culture to inoculate three 1-liter flasks, each containing 250 ml of SOB. The first flask receives 10 ml of starter culture, the second receives 4 ml, and the third receives 2 ml. Incubate all three flasks overnight at 18-22°C with moderate shaking.</li><br />
<li> The following morning, read the OD<sub>600</sub> of all three cultures. Continue to monitor the OD every 45 minutes.</li><br />
<li>When the OD<sub>600</sub> of one of the cultures reaches 0.55, transfer the culture vessel to an ice-water bath for 10 minutes. Discard the two other cultures.<br />
<li>Harvest the cells by centrifugation at 2500g (3900 rpm in a Sorvall GSA rotor) for 10 minutes at 4°C.</li><br />
<li>Pour off the medium and store the open centrifuge bottle on a stack of paper towels for 2 minutes. Use a vacuum aspirator to remove any drops of remaining medium adhering to walls of the centrifuge bottle or trapped in its neck.</li><br />
<li>Resuspend the cells gently in 80 ml of ice-cold Inoue transformation buffer.</li><br />
<li>Harvest the cells by centrifugation at 2500g (3900 rpm in a Sorvall GSA rotor) for 10 minutes at 4°C.</li><br />
<li>Pour off the medium and store the open centrifuge tube on a stack of paper towels for 2 minutes. Use a vacuum aspirator to remove any drops of remaining medium adhering to the walls of the centrifuge tube or trapped in its neck.</li><br />
<li>Resuspend the cells gently in 20 ml of ice-cold Inoue transformation buffer.</li><br />
<li>Add 1.5 ml of DMSO. Mix the bacterial suspension by swirling and then store it in ice for 10 minutes.</li><br />
<li>Working quickly, dispense aliquots of the suspensions into chilled, sterile microcentrifuge tubes. Immediately snap-freeze the competent cells by immersing the tightly closed tubes in a bath of liquid nitrogen. Store the tubes at -70°C until needed.<br />
</ol><br />
</div><br />
<p class="menu_head">Double Restriction (Fast) Digest</p><br />
<div class="menu_body"><br />
<p>Materials</p><br />
<li>~20 µL DNA (as much as possible)</li><br />
<li>1 µL Restriction Enzyme 1</li><br />
<li>1 µL Restriction Enzyme 2</li><br />
<li>5 µL 10X Universal Buffer (with loading dye)</li><br />
<li>Add appropriate amount of dH<sub>2</sub>0 (µL)</li><br />
<br />
50 µL Total → 37º C, 3 hrs.<br />
<br />
<p>Procedure</p><br />
<li>Treat insert with XbaI and PstI</li><br />
<li>Treat vector with SpeI and PstI</li><br />
</div><br />
<br />
<p class="menu_head"> Ligation</p><br />
<div class="menu_body"><br />
<p>Materials</p><br />
Ligation reaction:<br />
<li>__ µL vector DNA</li><br />
<li>__ µL insert DNA</li><br />
<li>2 µL T4 10x Buffer</li><br />
<li>1 µL DNA ligase</li><br />
<li>Add appropriate amount of dH<sub>2</sub>0 (µL)</li><br />
20 µL Total →Leave at room temperature for 20 minutes<br />
<br></br><br />
Vector control:<br />
<li>__ µL vector DNA (same volume as ligation reaction)</li><br />
<li>2 µL Buffer</li><br />
<li>1 µL ligase</li><br />
<li>Add appropriate amount of dH<sub>2</sub>0 (µL)</li><br />
20 µL total <br />
<br></br><br />
Insert control:<br />
<li>__ µL insert DNA (same volume as ligation reaction)</li><br />
<li>2 µL Buffer</li><br />
<li>1 µL ligase</li><br />
<li>Add appropriate amount of dH<sub>2</sub>0 (µL)</li><br />
20 uL Total<br />
<p>Procedure</p><br />
<li>Mix these materials in the amounts determined by the reaction volume calculator <a href="https://static.igem.org/mediawiki/2011/7/73/UC_Davis_Reaction_Volume_Calculator.xls">here</a>.<br />
</div><br />
<br />
<p class="menu_head">Gel Electrophoresis</p><br />
<div class="menu_body"><br />
<p>Procedure</p><br />
<ol><br />
<li>Add 0.5 grams of agarose to 50 mL of 1X TAE buffer.</li><br />
<li>Microwave agarose/TAE until agarose completely dissolved.</li><br />
<li>Cool under water, add SYBR safe dye (2.5-3µL).</li><br />
<li>Pour into mold with appropriate comb.</li><br />
<li>Wait 15 minutes for gel to solidify.</li><br />
<li>Load DNA with dye into wells while submerged in 1X TAE.</li><br />
<li>Run gel at a constant 120V.</li><br />
<li>Check gel periodically.</li><br />
</ol><br />
</div><br />
<br />
<p class="menu_head">Gel Extraction and Purification</p><br />
<div class="menu_body"><br />
<p>Procedure</p><br />
<ol><br />
<li>Prepare agarose gel and use 3 combs to make a bigger well.</li><br />
<li>Once it has run, use hand held UV lamp (in the dark, wearing goggles) to identify bands. </li><br />
<li>Cut out desired band with stamp pipette tip and transfer to a clean tube. The stamp pipette tip can be left in the tube to be cleaned out with a smaller pipette tip. </li><br />
<li>Weigh gel fragments and add 200 µL Buffer NTI for every 100 mg agarose gel.</li><br />
Incubate sample for 5-10 min at 50º C, vortexing every 2-3 min until the gel is completely dissolved.</li><br />
<li>Load sample onto column over collection tube. Centrifuge for 30 sec at 11,000 x g.</li><br />
<li>Discard flow-through and replace column on tube.</li><br />
<li>Add 700 µL Buffer NT3 and centrifuge for 30 sec. at 11,000 x g. Discard flow-through.</li><br />
<li>Centrifuge for 2 min at 11,000 x g to completely remove buffer.</li><br />
<li>Transfer column to a 1.5 mL tube and add 20 µL ddH<sub>2</sub>O.</li><br />
<li>Incubate at room temperature for 10 min.</li><br />
<li>Centrifuge for 1 min at 11,000 x g. </li><br />
</ol><br />
</div> <br />
<br />
<p class="menu_head">PCR Amplification for Golden Gate Assembly</p><br />
<div class="menu_body"><br />
<p>Materials</p><br />
<li>10 µL 5x HF Buffer</li><br />
<li>1 uL dNTPs</li><br />
<li>2.5 µL Forward Primer</li><br />
<li>2.5 µL Reverse Primer</li><br />
<li>100 ng Template DNA (2 ng/µL)</li><br />
<li>0.5 µL DNA Phusion Polymerase</li><br />
<li>Add appropriate amount of ddH<sub>2</sub>O</li><br />
50 µL Total<br />
<li>Run 1% agarose gel for verification. If the gel is good, perform PCR clean up.</li><br />
</div><br />
<p class="menu_head">Golden Gate Assembly</p><br />
<div class="menu_body"><br />
<br />
<p>Materials</p><br />
<li>100 ng for each DNA part</li><br />
<li>1 µL BsaI</li><br />
<li>1 µL T4-ligase</li><br />
<li>2 µL 10X T4 ligase buffer</li><br />
<li>Add appropriate amount of ddH<sub>2</sub>0.</li><br />
20 µL Total<br />
</div><br />
<br />
<p class="menu_head">DNA Extraction (Minipreps)</p><br />
<div class="menu_body"><br />
<br />
<p>Procedure</p><br />
<ol><br />
<li>Sediment the cells by centrifuging 1-5 mL of overnight LB-culture. Remove all medium.</li><br />
<li> Add 250 µL Resuspension Buffer (R3) with RNase A to the cell pellet and resuspend the pellet until it is homogeneous.</li><br />
<li>Add 250 µL Lysis Buffer (L7). Mix gently by inverting the capped tube five time. Do not vortex. Incubate the tube at room temperature for 5 minutes.</li><br />
<li>Add 350 µL Precipitation Buffer (N4). Mix immediately, or for large pellets, vigorously shaking the tube until the mixture is homogeneous. Do not vortex. Centrifuge the lysate at >12,000 x g for 10 minutes.</li><br />
<li>Load the supernatant from the prior step on a spin column in a 2-mL wash tube. Centrifuge at the column for 12,000 x g for 1 minute. Discard the flow-through and place the column back into the wash tube.</li><br />
<li>Add 700 µL Wash Buffer (W9) with ethanol to the column. Centrifuge the column at 12,000 x g for 1 minute. Discard the flow-through and place the column into the wash tube. Centrifuge the column at 12,000 x g for 1 minute. Discard the wash tube with the flow-through.</li><br />
<li>Place the spin column in a clean 1.5 mL recovery tube. Add 30 µL of ddH2O to the center of the column. Incubate the column for 10 minutes at room temperature.</li><br />
<li>Centrifuge the column at 12,000 x g for 2 minutes. Discard the column. Store plasmid DNA at 4º C (short-term) or store the DNA in aliquots at -20º C (long-term).</li><br />
</ol><br />
</div><br />
<br />
<p class="menu_head">Making and Reviving Glycerol Stocks</p><br />
<div class="menu_body"><br />
<br />
<p>Procedure</p><br />
<li>Add equal volumes (500-700 µL) of overnight cell culture and glycerol into a cryotube, keep sterile with a flame.</li><br />
<li>Store at -80º C.</li><br />
<li>When reviving a glycerol stock, keep the glycerol stock on dry ice. <br />
<li>Use a pipette tip to poke and/or slightly swirl glycerol stock and drop tip into 5 mL LB culture with appropriate antibiotic and shake overnight.</li><br />
</div><br />
<br />
<p class="menu_head">Sequencing Preparation</p><br />
<div class="menu_body"><br />
<br />
<p>Procedure</p><br />
<li>Primer will have [ng] content printed on label: add 10x H<sub>2</sub>0 for DNA at 100 uM.</li><br />
<li>Need 10 uM for sequencing, so dilute a portion of the hydrated primer solution 10x.</li><br />
<li>Determine DNA concentration of template DNA.</li><br />
<li>(Premixed) in 0.5 µL tube</li><br />
<li>.6 µg DNA (final concentration: 50 ng/µL)</li><br />
<li>8 pmol primer (universal primers 10 µM = .8 µL)</li><br />
<li>Add appropriate amount of H<sub>2</sub>O.</li><br />
12 µL total<br />
</div><br />
<br />
<p class="menu_head">Measuring DNA Concentration</p><br />
<div class="menu_body"><br />
<p>Procedure</p><br />
<ol><br />
<li>Log in to nanodrop program.</li><br />
<li>Moisten a Kim wipe and clean the pedestal.</li><br />
<li>Apply 2 µL H<sub>2</sub>O to pedestal and click 'OK'.</li><br />
<li>Press 'Blank' button.</li><br />
<li>Wipe blank from pedestal using Kimwipe.</li><br />
<li>Apply 2 µL of desired sample to pedestal.</li><br />
<li>Click 'Measure'.</li><br />
<li>Print results. </li><br />
</ol><br />
</div><br />
<br />
<p class="menu_head">Tecan Fluorescence Testing</p><br />
<div class="menu_body"><br />
<p>Procedure</p><br />
<ol><br />
<li>Grow cultures overnight in LB at 37 C, 150 RPM. </li><br />
<li>Measure OD<sub>600</sub> and dilute to get <0.01 OD<sub>600</sub>.</li><br />
<li>Grow until the OD<sub>600</sub> approaches 0.5.</li><br />
<li>Load 96 well plate with LB or M9, depending on the experiment, as well as the appropriate antibiotic, inducer stock solutions, and the appropriate volume of culture so as to reach an OD<sub>600</sub> of 0.1 in 200 µL. </li><br />
<li>Be sure to include the appropriate positive and negative controls.</li><br />
</ol><br />
<p>Tecan Program Parameters</p><br />
<ul><br />
<li>Temperature: 37 C</li><br />
<li>Orbital Shake Frequency: 244.5 rpm (Amplitude of 2.5 mm)</li><br />
<li>Orbital Shake Duration: 600 sec</li><br />
<li>Excitation wavelength: 485 nm</li><br />
<li>Emission wavelength: 535 nm</li><br />
<li>Absorbance wavelength (for OD readings): 595 nm</li><br />
<li>Gain: 35</li><br />
</ul><br />
</div><br />
<br />
<p class="menu_head">Primer Design for Site-Directed Mutagenesis PCR</p><br />
<div class="menu_body"><br />
<p>Procedure</p><br />
<ol><br />
<li> Identify site that needs to be mutated.</li><br />
<li>Check the amino acid sequence to create a silent mutation, generally the last base in a codon.</li><br />
<li>Check a codon usage table to help choose how the codon should be changed, try to pick a frequently used codon. </li><br />
<li>Take about 20 base pairs upstream and 20 base pairs downstream of your desired mutation site to create your primer, try to have it start and end in a G or C. The sequence should be identical to the template except for the one changed base you are trying to mutate at the center. </li><br />
<li>The reverse primer will be the reverse complement of this sequence.</li><br />
</ol><br />
<br />
</div><br />
<br />
<p class="menu_head">Site-Directed Mutagenesis PCR</p><br />
<div class="menu_body"><br />
<p>Materials</p><br />
<li>Primer will have [µg] content printed on label: add H<sub>2</sub>0 1:1 for DNA at 1 µg/µL.</li><br />
<li>Need 0.1 µg/µL for PCR reaction, so dilute a portion of the hydrated primer solution 10x.</li><br />
<li>Determine DNA concentration of template DNA.</li><br />
<br />
<li>50 ng Template DNA</li><br />
<li>5 µL 10x Turbo Buffer</li><br />
<li>1 µL Forward Primer (0.1 ug/uL)</li><br />
<li>1 µL Reverse Primer (0.1 ug/uL)</li><br />
<li>1 µL dNTPs (10 mM)</li><br />
<li>1 µL Pfu Turbo (enzyme)</li><br />
<li>Add appropriate amount of dH<sub>2</sub>O.</li><br />
50 µL Total<br />
<br></br><br />
<p>PCR program</p><br />
<ol><br />
<li>95º C 1 min</li><br />
<li>95º C 50 sec</li><br />
<li>60º C 50 sec Repeat Steps 2-4 17x (18x total) </li> <br />
<li>68º C 1 min / kb of plasmid </li><br />
<li>68º C 7 min</li><br />
<li>4º C Hold </li><br />
</ol><br />
</div><br />
<br />
<p class="menu_head">Electroporation Transformation</p><br />
<div class="menu_body"><br />
<p>Procedure</p><br />
<ol><br />
<li>Thaw electrocompetent cells on ice, keep on ice when thawed.</li><br />
<li>Aliquot 20-30µL of competent cells into separate 1.5mL microcentrifuge tubes on ice.</li><br />
<li>Pipette appropriate amount of DNA (1 µL) into tube on ice.</li><br />
<li>Transfer cell/DNA into prechilled electroporation cuvette, keep on ice for 1 minute.</li><br />
<li>Make sure the electroporator is set to:<br />
<ul>Time constant = 4.5-5.0 ms</ul><br />
<ul>Resistance = 200 W</ul><br />
<ul>Capacitance = 25 mFD</ul><br />
<ul>Volts = 1.7 kV (for 1mm gap cuvettes)</ul><br />
<li>Electrocute cells once.</li><br />
<li>Add 1mL of LB to cuvette, pipette up and down to mix, and transfer mixture to 14mL Falcon culture tube.</li><br />
<li>Incubate for 1hr at 37C shaking at 150rpm.</li><br />
<li>Plate cells on appropriate antibiotic.</li><br />
</ol><br />
</div><br />
<p class="menu_head">SOE PCR</p><br />
<div class="menu_body"><br />
<p>Materials</p><br />
<li>Primer will have [µg] content printed on label: add H<sub>2</sub>0 1:1 for DNA at 1 µg/µL.</li><br />
<li>Need 0.1 µg/µL for PCR reaction, so dilute a portion of the hydrated primer solution 10x.</li><br />
<li>Determine DNA concentration of template DNA.</li><br />
<br />
<p>Amplification</p><br />
<br />
<li>100 ng Template DNA</li><br />
<li>10 µL HF 5x Buffer</li><br />
<li>2.5 µL Forward Primer (0.1 µg/µL)</li><br />
<li>2.5 µL Reverse Primer (0.1 µg/µL)</li><br />
<li>1 µL dNTPs (10 mM)</li><br />
<li>0.5 µL Phusion Polymerase (enzyme)</li><br />
<li>Add appropriate amount of dH<sub>2</sub>O.</li><br />
50 µL Total<br />
<br></br><br />
<p>PCR program</p><br />
<ol><br />
<li>98º C 30 sec</li><br />
<li>98º C 10 sec</li><br />
<li>55º C 30 sec </li> <br />
<li>72º C 1 min / kb Repeat Steps 2-4 29x (30x total) </li><br />
<li>72º C 5 min</li><br />
<li>4º C Hold </li><br />
</ol><br />
<br>Purify the PCR product and determine the resultant concentration</br><br />
<br />
<p>SOE PCR</p><br />
<br />
<li>300 ng Template DNA (larger part)</li><br />
<li>xx ng Template DNA (smaller part) keeping a 1:1 molar ratio </li><br />
<li>10 µL HF 5X Buffer</li><br />
<li>2.5 µL Forward Primer (0.1 µg/µL)</li><br />
<li>2.5 µL Reverse Primer (0.1 µg/µL)</li><br />
<li>1 µL dNTPs (10 mM) </li><br />
<li>0.5 µL Phusion Polymerase (enzyme)</li><br />
<li>Add appropriate amount of dH<sub>2</sub>O.</li><br />
50 µL Total<br />
<br />
<br />
<p>PCR program</p><br />
<br />
<li>Same as above.</li><br />
<br />
</div><br />
<br />
<p class="menu_head">Colony PCR</p><br />
<div class="menu_body"><br />
<p>When to Use</p><br />
Transformation of competent cells may yield false positives. Through colony PCR, one may screen for the presence of the assembled sequence by amplifying with the universal forward and reverse primers and checking the length of the fragments through gel electrophoresis.<br />
<br />
<p>Materials</p><br />
<br />
<li>2 µL 10x Buffer</li><br />
<li>4 µL Q solution</li><br />
<li>1 µL Universal Forward Primer (0.1 µg/µL)</li><br />
<li>1 µL Universal Reverse Primer (0.1 µg/µL)</li><br />
<li>0.5 µL dNTPs (10 mM)</li><br />
<li>1.375 µL dH<sub>2</sub>O</li><br />
<br>Pick a colony with a sterile tip and set the tip in 10 µL dH<sub>2</sub>O in a PCR tube for 10 minutes. Pipet up and down to resuspend the cell material and then add the reagent, DNA, and buffer mixture. While picking the colony, some cells should also be transferred to a new plate with the same antibiotic so that if the screen results are positive, the colony may be identified and grown up.</br><br />
<br><br />
20 µL Total</br><br />
<br />
<p>PCR program</p><br />
<ol><br />
<li>98º C 30 sec</li><br />
<li>98º C 10 sec</li><br />
<li>55º C 30 sec </li> <br />
<li>72º C 1 min / kb Repeat Steps 2-4 29x (30x total) </li><br />
<li>72º C 5 min</li><br />
<li>4º C Hold </li><br />
</ol><br />
<br />
</div><br />
<p class="menu_head">M9 Minimal Media</p><br />
<div class="menu_body"><br />
<p>Materials</p><br />
<ul><br />
<li>1x <a href="http://openwetware.org/wiki/M9_salts">M9 salts</a></hi></li><br />
<li>2 mM MgSO<sub>4</sub></li><br />
<li>0.1 mM CaCl<sub>2</sub></li><br />
<li>0.4% carbon source (eg. glucose)</li><br />
<li>Dissolve in sterile dH<sub>2</sub>0</li><br />
</div><br />
<br />
</div><br />
<br />
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Lemur3 93
http://2013.igem.org/Team:UC_Davis/Protocols
Team:UC Davis/Protocols
2013-09-28T02:49:14Z
<p>Lemur3 93: </p>
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<h1>Protocols</h1><br />
<!-- accordion starts here --><br />
<div id="firstpane" class="menu_list"><br />
<br />
<p class="menu_head"> LB Media </p><br />
<div class="menu_body"><br />
<li>950 mL dH<sub>2</sub>0</li><br />
<li>10 g Tryptone</li><br />
<li>10 g NaCl</li><br />
<li>5 g Yeast Extract</li><br />
1 L Total<br />
<li>Add 15 g Agar, if being poured into plates.</li><br />
<li>Autoclave, when cool add antibiotics if desired.</li><br />
</div><br />
<br />
<p class="menu_head"> Antibiotic Stock Solutions</p><br />
<div class="menu_body"><br />
<p>Materials</p><br />
Chloramphenicol<br />
<li>Working Concentration 12.5 μg/ml</li><br />
<li>Stock solutions can be made at 35 mg/ml in ethanol and kept at -20º C</li><br />
<br />
Kanamycin<br />
<li>Filter sterilize for kanamycin</li><br />
Working Concentration:<br />
<li>25 μg/mL for low-copy plasmids</li><br />
<li>35 µg/mL for high-copy plasmids</li><br />
<li>Stock solution is 35 mg/ml in water and kept at -20º C</li><br />
<br />
Spectinomycin <br />
<li>Filter sterilize</li><br />
<li>Working Concentration 50 μg/mL</li><br />
<li>Stock solution is 100 mg/mL in water and kept at -20º C</li><br />
</div><br />
<br />
<p class="menu_head"> Heat Shock Transformation</p><br />
<div class="menu_body"><br />
<p>Procedure</p><br />
<ol><br />
<li>Preheat water bath to 42º C.</li><br />
<li>Thaw competent cells on ice for 10 minutes.</li><br />
<li>Use 50 µL competent cells, add transforming DNA [up to 25 ng per 50 µL of cells, volume not exceeding 2.5 µL (5%)]. For control add 1 µL of control DNA (PUC19 carb resistance). Swirl to mix, store on ice 5 minutes. </li><br />
<li>Heat shock in 42º C water bath for 45 seconds.</li><br />
<li>Cool cells in ice bath for a few minutes.</li><br />
<li>Add 800 µL LB to each tube, set in shaker at 37º C for 45 minutes. </li><br />
<li>Transfer 200 µL of culture per plate (containing the appropriate antibiotic).</li><br />
<li>Spread using glass beads.</li><br />
<li>Invert plates and incubate overnight (12-16 hrs) at 37º C.</li><br />
</ol><br />
</div><br />
<br />
<p class="menu_head"> Making Chemically Competent Cells</p><br />
<div class="menu_body"><br />
<p>Procedure</p><br />
<ol><br />
<li>Prepare 0.5 M PIPES (pH 6.7) (piperazine-1,2-bis[2-ethanesulfonic acid]) by dissolving 15.1 g of PIPES in 80 ml of pure H2O (Milli-Q, or equivalent). Adjust the pH of the solution to 6.7 with 5 M KOH, and then add pure H<sub>2</sub>O to bring the final volume to 100 ml. Sterilize the solution by filtration through a disposable pre-rinsed Nalgene filter (0.45-µm pore size). Divide into aliquots and store frozen at -20°C.<br />
Prepare Inoue transformation buffer by dissolving all of the solutes listed below in 800 mL of pure H2O and then add 20 ml of 0.5 M PIPES (pH 6.7). Adjust the volume of the Inoue transformation buffer to 1 liter with pure H<sub>2</sub>O.</li><br />
<br />
<table> <tr><br />
<th></th> <th>Reagent</th> <th>Amount per liter</th> <th>Final Concentration</th><br />
</tr> <tr><br />
<th></th> <td>MnCl<sub>2</sub>•4H<sub>2</sub>O</td> <td>10.88 g</td> <td>55 mM</td><br />
</tr> <tr><br />
<th></th> <td>CaCl<sub>2</sub>•2H<sub>2</sub>O</td> <td>2.20 g</td> <td>15 mM</td><br />
</tr> <tr><br />
<th></th> <td>KCl</td> <td>18.65 g</td> <td>250 mM</td><br />
</tr> <tr><br />
<th></th> <td>PIPES (0.5 M, pH 6.7)</td> <td>20 ml</td> <td>10 mM</td><br />
</tr> </table><br />
Add H<sub>2</sub>O to fill solution to 1 liter. Sterilize Inoue transformation buffer by filtration through a prerinsed 0.45-µm Nalgene filter. Divide into aliquots and store at -20°C.<br />
<br />
<li>Pick a single bacterial colony (2-3 mm in diameter) from a plate that has been incubated for 16-20 hours at 37°C. Transfer the colony into 25 ml of SOB medium (LB may be used instead) in a 250-ml flask. Incubate the culture for 6-8 hours at 37°C with vigorous shaking (250-300 rpm).</li><br />
<li>At about 6 o'clock in the evening, use this starter culture to inoculate three 1-liter flasks, each containing 250 ml of SOB. The first flask receives 10 ml of starter culture, the second receives 4 ml, and the third receives 2 ml. Incubate all three flasks overnight at 18-22°C with moderate shaking.</li><br />
<li> The following morning, read the OD<sub>600</sub> of all three cultures. Continue to monitor the OD every 45 minutes.</li><br />
<li>When the OD<sub>600</sub> of one of the cultures reaches 0.55, transfer the culture vessel to an ice-water bath for 10 minutes. Discard the two other cultures.<br />
<li>Harvest the cells by centrifugation at 2500g (3900 rpm in a Sorvall GSA rotor) for 10 minutes at 4°C.</li><br />
<li>Pour off the medium and store the open centrifuge bottle on a stack of paper towels for 2 minutes. Use a vacuum aspirator to remove any drops of remaining medium adhering to walls of the centrifuge bottle or trapped in its neck.</li><br />
<li>Resuspend the cells gently in 80 ml of ice-cold Inoue transformation buffer.</li><br />
<li>Harvest the cells by centrifugation at 2500g (3900 rpm in a Sorvall GSA rotor) for 10 minutes at 4°C.</li><br />
<li>Pour off the medium and store the open centrifuge tube on a stack of paper towels for 2 minutes. Use a vacuum aspirator to remove any drops of remaining medium adhering to the walls of the centrifuge tube or trapped in its neck.</li><br />
<li>Resuspend the cells gently in 20 ml of ice-cold Inoue transformation buffer.</li><br />
<li>Add 1.5 ml of DMSO. Mix the bacterial suspension by swirling and then store it in ice for 10 minutes.</li><br />
<li>Working quickly, dispense aliquots of the suspensions into chilled, sterile microcentrifuge tubes. Immediately snap-freeze the competent cells by immersing the tightly closed tubes in a bath of liquid nitrogen. Store the tubes at -70°C until needed.<br />
</ol><br />
</div><br />
<p class="menu_head">Double Restriction (Fast) Digest</p><br />
<div class="menu_body"><br />
<p>Materials</p><br />
<li>~20 µL DNA (as much as possible)</li><br />
<li>1 µL Restriction Enzyme 1</li><br />
<li>1 µL Restriction Enzyme 2</li><br />
<li>5 µL 10X Universal Buffer (with loading dye)</li><br />
<li>Add appropriate amount of dH<sub>2</sub>0 (µL)</li><br />
<br />
50 µL Total → 37º C, 3 hrs.<br />
<br />
<p>Procedure</p><br />
<li>Treat insert with XbaI and PstI</li><br />
<li>Treat vector with SpeI and PstI</li><br />
</div><br />
<br />
<p class="menu_head"> Ligation</p><br />
<div class="menu_body"><br />
<p>Materials</p><br />
Ligation reaction:<br />
<li>__ µL vector DNA</li><br />
<li>__ µL insert DNA</li><br />
<li>2 µL T4 10x Buffer</li><br />
<li>1 µL DNA ligase</li><br />
<li>Add appropriate amount of dH<sub>2</sub>0 (µL)</li><br />
20 µL Total →Leave at room temperature for 20 minutes<br />
<br></br><br />
Vector control:<br />
<li>__ µL vector DNA (same volume as ligation reaction)</li><br />
<li>2 µL Buffer</li><br />
<li>1 µL ligase</li><br />
<li>Add appropriate amount of dH<sub>2</sub>0 (µL)</li><br />
20 µL total <br />
<br></br><br />
Insert control:<br />
<li>__ µL insert DNA (same volume as ligation reaction)</li><br />
<li>2 µL Buffer</li><br />
<li>1 µL ligase</li><br />
<li>Add appropriate amount of dH<sub>2</sub>0 (µL)</li><br />
20 uL Total<br />
<p>Procedure</p><br />
<li>Mix these materials in the amounts determined by the reaction volume calculator <a href="https://static.igem.org/mediawiki/2011/7/73/UC_Davis_Reaction_Volume_Calculator.xls">here</a>.<br />
</div><br />
<br />
<p class="menu_head">Gel Electrophoresis</p><br />
<div class="menu_body"><br />
<p>Procedure</p><br />
<ol><br />
<li>Add 0.5 grams of agarose to 50 mL of 1X TAE buffer.</li><br />
<li>Microwave agarose/TAE until agarose completely dissolved.</li><br />
<li>Cool under water, add SYBR safe dye (2.5-3µL).</li><br />
<li>Pour into mold with appropriate comb.</li><br />
<li>Wait 15 minutes for gel to solidify.</li><br />
<li>Load DNA with dye into wells while submerged in 1X TAE.</li><br />
<li>Run gel at a constant 120V.</li><br />
<li>Check gel periodically.</li><br />
</ol><br />
</div><br />
<br />
<p class="menu_head">Gel Extraction and Purification</p><br />
<div class="menu_body"><br />
<p>Procedure</p><br />
<ol><br />
<li>Prepare agarose gel and use 3 combs to make a bigger well.</li><br />
<li>Once it has run, use hand held UV lamp (in the dark, wearing goggles) to identify bands. </li><br />
<li>Cut out desired band with stamp pipette tip and transfer to a clean tube. The stamp pipette tip can be left in the tube to be cleaned out with a smaller pipette tip. </li><br />
<li>Weigh gel fragments and add 200 µL Buffer NTI for every 100 mg agarose gel.</li><br />
Incubate sample for 5-10 min at 50º C, vortexing every 2-3 min until the gel is completely dissolved.</li><br />
<li>Load sample onto column over collection tube. Centrifuge for 30 sec at 11,000 x g.</li><br />
<li>Discard flow-through and replace column on tube.</li><br />
<li>Add 700 µL Buffer NT3 and centrifuge for 30 sec. at 11,000 x g. Discard flow-through.</li><br />
<li>Centrifuge for 2 min at 11,000 x g to completely remove buffer.</li><br />
<li>Transfer column to a 1.5 mL tube and add 20 µL ddH<sub>2</sub>O.</li><br />
<li>Incubate at room temperature for 10 min.</li><br />
<li>Centrifuge for 1 min at 11,000 x g. </li><br />
</ol><br />
</div> <br />
<br />
<p class="menu_head">PCR Amplification for Golden Gate Assembly</p><br />
<div class="menu_body"><br />
<p>Materials</p><br />
<li>10 µL 5x HF Buffer</li><br />
<li>1 uL dNTPs</li><br />
<li>2.5 µL Forward Primer</li><br />
<li>2.5 µL Reverse Primer</li><br />
<li>100 ng Template DNA (2 ng/µL)</li><br />
<li>0.5 µL DNA Phusion Polymerase</li><br />
<li>Add appropriate amount of ddH<sub>2</sub>O</li><br />
50 µL Total<br />
<li>Run 1% agarose gel for verification. If the gel is good, perform PCR clean up.</li><br />
</div><br />
<p class="menu_head">Golden Gate Assembly</p><br />
<div class="menu_body"><br />
<br />
<p>Materials</p><br />
<li>100 ng for each DNA part</li><br />
<li>1 µL BsaI</li><br />
<li>1 µL T4-ligase</li><br />
<li>2 µL 10X T4 ligase buffer</li><br />
<li>Add appropriate amount of ddH<sub>2</sub>0.</li><br />
20 µL Total<br />
</div><br />
<br />
<p class="menu_head">DNA Extraction (Minipreps)</p><br />
<div class="menu_body"><br />
<br />
<p>Procedure</p><br />
<ol><br />
<li>Sediment the cells by centrifuging 1-5 mL of overnight LB-culture. Remove all medium.</li><br />
<li> Add 250 µL Resuspension Buffer (R3) with RNase A to the cell pellet and resuspend the pellet until it is homogeneous.</li><br />
<li>Add 250 µL Lysis Buffer (L7). Mix gently by inverting the capped tube five time. Do not vortex. Incubate the tube at room temperature for 5 minutes.</li><br />
<li>Add 350 µL Precipitation Buffer (N4). Mix immediately, or for large pellets, vigorously shaking the tube until the mixture is homogeneous. Do not vortex. Centrifuge the lysate at >12,000 x g for 10 minutes.</li><br />
<li>Load the supernatant from the prior step on a spin column in a 2-mL wash tube. Centrifuge at the column for 12,000 x g for 1 minute. Discard the flow-through and place the column back into the wash tube.</li><br />
<li>Add 700 µL Wash Buffer (W9) with ethanol to the column. Centrifuge the column at 12,000 x g for 1 minute. Discard the flow-through and place the column into the wash tube. Centrifuge the column at 12,000 x g for 1 minute. Discard the wash tube with the flow-through.</li><br />
<li>Place the spin column in a clean 1.5 mL recovery tube. Add 30 µL of ddH2O to the center of the column. Incubate the column for 10 minutes at room temperature.</li><br />
<li>Centrifuge the column at 12,000 x g for 2 minutes. Discard the column. Store plasmid DNA at 4º C (short-term) or store the DNA in aliquots at -20º C (long-term).</li><br />
</ol><br />
</div><br />
<br />
<p class="menu_head">Making and Reviving Glycerol Stocks</p><br />
<div class="menu_body"><br />
<br />
<p>Procedure</p><br />
<li>Add equal volumes (500-700 µL) of overnight cell culture and glycerol into a cryotube, keep sterile with a flame.</li><br />
<li>Store at -80º C.</li><br />
<li>When reviving a glycerol stock, keep the glycerol stock on dry ice. <br />
<li>Use a pipette tip to poke and/or slightly swirl glycerol stock and drop tip into 5 mL LB culture with appropriate antibiotic and shake overnight.</li><br />
</div><br />
<br />
<p class="menu_head">Sequencing Preparation</p><br />
<div class="menu_body"><br />
<br />
<p>Procedure</p><br />
<li>Primer will have [ng] content printed on label: add 10x H<sub>2</sub>0 for DNA at 100 uM.</li><br />
<li>Need 10 uM for sequencing, so dilute a portion of the hydrated primer solution 10x.</li><br />
<li>Determine DNA concentration of template DNA.</li><br />
<li>(Premixed) in 0.5 µL tube</li><br />
<li>.6 µg DNA (final concentration: 50 ng/µL)</li><br />
<li>8 pmol primer (universal primers 10 µM = .8 µL)</li><br />
<li>Add appropriate amount of H<sub>2</sub>O.</li><br />
12 µL total<br />
</div><br />
<br />
<p class="menu_head">Measuring DNA Concentration</p><br />
<div class="menu_body"><br />
<p>Procedure</p><br />
<ol><br />
<li>Log in to nanodrop program.</li><br />
<li>Moisten a Kim wipe and clean the pedestal.</li><br />
<li>Apply 2 µL H<sub>2</sub>O to pedestal and click 'OK'.</li><br />
<li>Press 'Blank' button.</li><br />
<li>Wipe blank from pedestal using Kimwipe.</li><br />
<li>Apply 2 µL of desired sample to pedestal.</li><br />
<li>Click 'Measure'.</li><br />
<li>Print results. </li><br />
</ol><br />
</div><br />
<br />
<p class="menu_head">Tecan Fluorescence Testing</p><br />
<div class="menu_body"><br />
<p>Procedure</p><br />
<ol><br />
<li>Grow cultures overnight in LB at 37 C, 150 RPM. </li><br />
<li>Measure OD<sub>600</sub> and dilute to get <0.01 OD<sub>600</sub>.</li><br />
<li>Grow until the OD<sub>600</sub> approaches 0.5.</li><br />
<li>Load 96 well plate with LB or M9, depending on the experiment, as well as the appropriate antibiotic, inducer stock solutions, and the appropriate volume of culture so as to reach an OD<sub>600</sub> of 0.1 in 200 µL. </li><br />
<li>Be sure to include the appropriate positive and negative controls.</li><br />
</ol><br />
<p>Tecan Program Parameters</p><br />
<ul><br />
<li>Temperature: 37 C</li><br />
<li>Orbital Shake Frequency: 244.5 rpm (Amplitude of 2.5 mm)</li><br />
<li>Orbital Shake Duration: 600 sec</li><br />
<li>Excitation wavelength: 485 nm</li><br />
<li>Emission wavelength: 535 nm</li><br />
<li>Absorbance wavelength (for OD readings): 595 nm</li><br />
<li>Gain: 35</li><br />
</ul><br />
</div><br />
<br />
<p class="menu_head">Primer Design for Site-Directed Mutagenesis PCR</p><br />
<div class="menu_body"><br />
<p>Procedure</p><br />
<ol><br />
<li> Identify site that needs to be mutated.</li><br />
<li>Check the amino acid sequence to create a silent mutation, generally the last base in a codon.</li><br />
<li>Check a codon usage table to help choose how the codon should be changed, try to pick a frequently used codon. </li><br />
<li>Take about 20 base pairs upstream and 20 base pairs downstream of your desired mutation site to create your primer, try to have it start and end in a G or C. The sequence should be identical to the template except for the one changed base you are trying to mutate at the center. </li><br />
<li>The reverse primer will be the reverse complement of this sequence.</li><br />
</ol><br />
<br />
</div><br />
<br />
<p class="menu_head">Site-Directed Mutagenesis PCR</p><br />
<div class="menu_body"><br />
<p>Materials</p><br />
<li>Primer will have [µg] content printed on label: add H<sub>2</sub>0 1:1 for DNA at 1 µg/µL.</li><br />
<li>Need 0.1 µg/µL for PCR reaction, so dilute a portion of the hydrated primer solution 10x.</li><br />
<li>Determine DNA concentration of template DNA.</li><br />
<br />
<li>50 ng Template DNA</li><br />
<li>5 µL 10x Turbo Buffer</li><br />
<li>1 µL Forward Primer (0.1 ug/uL)</li><br />
<li>1 µL Reverse Primer (0.1 ug/uL)</li><br />
<li>1 µL dNTPs (10 mM)</li><br />
<li>1 µL Pfu Turbo (enzyme)</li><br />
<li>Add appropriate amount of dH<sub>2</sub>O.</li><br />
50 µL Total<br />
<br></br><br />
<p>PCR program</p><br />
<ol><br />
<li>95º C 1 min</li><br />
<li>95º C 50 sec</li><br />
<li>60º C 50 sec Repeat Steps 2-4 17x (18x total) </li> <br />
<li>68º C 1 min / kb of plasmid </li><br />
<li>68º C 7 min</li><br />
<li>4º C Hold </li><br />
</ol><br />
</div><br />
<br />
<p class="menu_head">Electroporation Transformation</p><br />
<div class="menu_body"><br />
<p>Procedure</p><br />
<ol><br />
<li>Thaw electrocompetent cells on ice, keep on ice when thawed.</li><br />
<li>Aliquot 20-30µL of competent cells into separate 1.5mL microcentrifuge tubes on ice.</li><br />
<li>Pipette appropriate amount of DNA (1 µL) into tube on ice.</li><br />
<li>Transfer cell/DNA into prechilled electroporation cuvette, keep on ice for 1 minute.</li><br />
<li>Make sure the electroporator is set to:<br />
<ul>Time constant = 4.5-5.0 ms</ul><br />
<ul>Resistance = 200 W</ul><br />
<ul>Capacitance = 25 mFD</ul><br />
<ul>Volts = 1.7 kV (for 1mm gap cuvettes)</ul><br />
<li>Electrocute cells once.</li><br />
<li>Add 1mL of LB to cuvette, pipette up and down to mix, and transfer mixture to 14mL Falcon culture tube.</li><br />
<li>Incubate for 1hr at 37C shaking at 150rpm.</li><br />
<li>Plate cells on appropriate antibiotic.</li><br />
</ol><br />
</div><br />
<p class="menu_head">SOE PCR</p><br />
<div class="menu_body"><br />
<p>Materials</p><br />
<li>Primer will have [µg] content printed on label: add H<sub>2</sub>0 1:1 for DNA at 1 µg/µL.</li><br />
<li>Need 0.1 µg/µL for PCR reaction, so dilute a portion of the hydrated primer solution 10x.</li><br />
<li>Determine DNA concentration of template DNA.</li><br />
<br />
<p>Amplification</p><br />
<br />
<li>100 ng Template DNA</li><br />
<li>10 µL HF 5x Buffer</li><br />
<li>2.5 µL Forward Primer (0.1 µg/µL)</li><br />
<li>2.5 µL Reverse Primer (0.1 µg/µL)</li><br />
<li>1 µL dNTPs (10 mM)</li><br />
<li>0.5 µL Phusion Polymerase (enzyme)</li><br />
<li>Add appropriate amount of dH<sub>2</sub>O.</li><br />
50 µL Total<br />
<br></br><br />
<p>PCR program</p><br />
<ol><br />
<li>98º C 30 sec</li><br />
<li>98º C 10 sec</li><br />
<li>55º C 30 sec </li> <br />
<li>72º C 1 min / kb Repeat Steps 2-4 29x (30x total) </li><br />
<li>72º C 5 min</li><br />
<li>4º C Hold </li><br />
</ol><br />
<br>Purify the PCR product and determine the resultant concentration</br><br />
<br />
<p>SOE PCR</p><br />
<br />
<li>300 ng Template DNA (larger part)</li><br />
<li>xx ng Template DNA (smaller part) keeping a 1:1 molar ratio </li><br />
<li>10 µL HF 5X Buffer</li><br />
<li>2.5 µL Forward Primer (0.1 µg/µL)</li><br />
<li>2.5 µL Reverse Primer (0.1 µg/µL)</li><br />
<li>1 µL dNTPs (10 mM) </li><br />
<li>0.5 µL Phusion Polymerase (enzyme)</li><br />
<li>Add appropriate amount of dH<sub>2</sub>O.</li><br />
50 µL Total<br />
<br />
<br />
<p>PCR program</p><br />
<br />
<li>Same as above.</li><br />
<br />
</div><br />
<br />
<p class="menu_head">Colony PCR</p><br />
<div class="menu_body"><br />
<p>When to Use</p><br />
Transformation of competent cells may yield false positives. Through colony PCR, one may screen for the presence of the assembled sequence by amplifying with the universal forward and reverse primers and checking the length of the fragments through gel electrophoresis.<br />
<br />
<p>Materials</p><br />
<br />
<li>2 µL 10x Buffer</li><br />
<li>4 µL Q solution</li><br />
<li>1 µL Universal Forward Primer (0.1 µg/µL)</li><br />
<li>1 µL Universal Reverse Primer (0.1 µg/µL)</li><br />
<li>0.5 µL dNTPs (10 mM)</li><br />
<li>1.375 µL dH<sub>2</sub>O</li><br />
<br>Pick a colony with a sterile tip and set the tip in 10 µL dH<sub>2</sub>O in a PCR tube for 10 minutes. Pipet up and down to resuspend the cell material and then add the reagent, DNA, and buffer mixture. While picking the colony, some cells should also be transferred to a new plate with the same antibiotic so that if the screen results are positive, the colony may be identified and grown up.</br><br />
<br><br />
20 µL Total</br><br />
<br />
<p>PCR program</p><br />
<ol><br />
<li>98º C 30 sec</li><br />
<li>98º C 10 sec</li><br />
<li>55º C 30 sec </li> <br />
<li>72º C 1 min / kb Repeat Steps 2-4 29x (30x total) </li><br />
<li>72º C 5 min</li><br />
<li>4º C Hold </li><br />
</ol><br />
<br />
</div><br />
<p class="menu_head">M9 Minimal Media</p><br />
<div class="menu_body"><br />
<p>Materials</p><br />
<ul><br />
<li>1x <a href="http://openwetware.org/wiki/M9_salts">M9 salts</a></hi></li><br />
<li>2 mM MgSO<sub>4</sub></li><br />
<li>0.1 mM CaCl<sub>2</sub></li><br />
<li>0.4% carbon source (eg. glucose)</li><br />
<li>Dissolve in sterile dH<sub>2</sub>0</li><br />
</div><br />
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Lemur3 93
http://2013.igem.org/Team:UC_Davis/Modeling
Team:UC Davis/Modeling
2013-09-28T02:40:16Z
<p>Lemur3 93: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
<br />
<html><br />
<head><br />
</head><br />
<body><br />
<br />
<div><br />
<img src="https://static.igem.org/mediawiki/2013/c/cf/Modelingbanner_UCDavis.jpg" class="banner" width="967" height="226"><br />
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</div><br />
<br />
<div class="floatboxwide"><br />
<h1>Equations</h1><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/e/ef/Ucdavismodelingscheme.png" width= 630 height 195"></center><br />
<br></br><br />
<br><br />
The equations below model the concentrations of bound transcription factors. That is, they serve to model the concentration of araC bound to pBAD and tetR bound to pTET given the concentrations of the ligands, arabinose and aTc.</br><br>The subsequent equations model the probability of active complex for each element in our circuit. P<sub>BAD</sub> represents the probability that the pBAD promoter will be unbound by araC and thus active. P<sub>TET</sub> represents the probability that the pTET promoter will be unbound by tetR and thus active. P<sub>Riboswitch</sub> expresses the probability that the riboswitch is bound by theophylline, and thus active. For simplicity, it has been modeled here as an activator-controlled promoter. P<sub>Tale Binding Site</sub>, which may be abbreviated to P<sub>TBS</sub> expresses the probability that the TALe binding site is unbound by the TAL repressor, and thus active.</br><br />
<br>The third set of equations are ordinary differential equations modeling the change in concentration over time of the riboswitch-TALe transcript, TAL repressor, GFP mRNA, GFP protein intermediate, and GFP protein. In this model we have taken into account the maturation time of GFP.</br> <br />
<br></br><br />
<img src="https://static.igem.org/mediawiki/2013/9/96/Ucdavismodeling1n.png" width=1019 height=299><br />
<br></br><br />
<img src="https://static.igem.org/mediawiki/2013/8/8d/Ucdavismodeling2.png" width=1019 height=410><br />
<br></br><br />
<img src="https://static.igem.org/mediawiki/2013/9/90/Ucdavismodeling3nb.png" width=938 height=544><br />
<br></br><br />
<h1>Parameters</h1><br />
Included here are the parameters used in this model. Please refer to the References section of this page for the source of each parameter value. <br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4e/Ucdavisparameters1.jpg" class="genpic"></center><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/d/de/Ucdavisparameters2.jpg" class="genpic"></center><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/a/aa/Ucdavisparameters3.jpg" class="genpic"></center><br />
<br></br><br />
<h1>MATLAB Simulation</h1><br />
<br></br><br />
<h3>TALe Binding Site K<sub>D</sub> as a source of tunability</h3><br />
<br>Each state variable in the system of ODEs was given an initial condition of 0. The dynamic response of the system was calculated and plotted over a time span of 10 hours. The results of the model support our data in that the RiboTALe with the larger dissociation constant (RiboTALe 1) is less effective at repressing GFP than RiboTALe 8 under the same induction conditions. The peak seen in the dynamic response of both simulations is a result of the kinematics of the system; there is lag between the initiation of GFP production and when the concentration of active TAL repressors is enough to tip the system.</br><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/0/0a/Ucdavismodel2.png" class="genpic"></center><br />
<br></br><br />
<h3>RiboTALe Modulation Through Theophylline Induction Levels</h3><br />
<br>This simulation was carried out under the same conditions defined above, but interrogated only one RiboTALe, RiboTALe 1 with a K<sub>D</sub> of 240 nM. The concentration of theophylline, however, was varied over a range of 1 mM to 10 mM and the results plotted. This simulation also supports our data in that it is clear that the riboswitch is in fact responsive to theophylline and that final GFP counts are inversely proportional to the amount of theophylline added.</br><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/2c/Ucdavismodel3.png"class="genpic"></center><br />
<br></br><br />
<h3>Amplifying System Response Through Transcript Induction</h3> <br />
</div><br />
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Lemur3 93
http://2013.igem.org/Team:UC_Davis/Modeling
Team:UC Davis/Modeling
2013-09-28T02:39:41Z
<p>Lemur3 93: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
<br />
<html><br />
<head><br />
</head><br />
<body><br />
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<div><br />
<img src="https://static.igem.org/mediawiki/2013/c/cf/Modelingbanner_UCDavis.jpg" class="banner" width="967" height="226"><br />
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</div><br />
<br />
<div class="floatboxwide"><br />
<h1>Equations</h1><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/e/ef/Ucdavismodelingscheme.png" width= 630 height 195"></center><br />
<br></br><br />
<br><br />
The equations below model the concentrations of bound transcription factors. That is, they serve to model the concentration of araC bound to pBAD and tetR bound to pTET given the concentrations of the ligands, arabinose and aTc.</br><br>The subsequent equations model the probability of active complex for each element in our circuit. P<sub>BAD</sub> represents the probability that the pBAD promoter will be unbound by araC and thus active. P<sub>TET</sub> represents the probability that the pTET promoter will be unbound by tetR and thus active. P<sub>Riboswitch</sub> expresses the probability that the riboswitch is bound by theophylline, and thus active. For simplicity, it has been modeled here as an activator-controlled promoter. P<sub>Tale Binding Site</sub>, which may be abbreviated to P<sub>TBS</sub> expresses the probability that the TALe binding site is unbound by the TAL repressor, and thus active.</br><br />
<br>The third set of equations are ordinary differential equations modeling the change in concentration over time of the riboswitch-TALe transcript, TAL repressor, GFP mRNA, GFP protein intermediate, and GFP protein. In this model we have taken into account the maturation time of GFP.</br> <br />
<br></br><br />
<img src="https://static.igem.org/mediawiki/2013/9/96/Ucdavismodeling1n.png" width=1019 height=299><br />
<br></br><br />
<img src="https://static.igem.org/mediawiki/2013/8/8d/Ucdavismodeling2.png" width=1019 height=410><br />
<br></br><br />
<img src="https://static.igem.org/mediawiki/2013/9/90/Ucdavismodeling3nb.png" width=938 height=544><br />
<br></br><br />
<h1>Parameters</h1><br />
Included here are the parameters used in this model. Please refer to the References section of this page for the source of each parameter value. <br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4e/Ucdavisparameters1.jpg" class="genpic"></center><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/d/de/Ucdavisparameters2.jpg" class="genpic"></center><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/a/aa/Ucdavisparameters3.jpg" class="genpic"></center><br />
<br></br><br />
<h1>MATLAB Simulation</h1><br />
<br></br><br />
<h3>TALe Binding Site K<sub>D</sub> as a source of tunability</h3><br />
<br>Each state variable in the system of ODEs was given an initial condition of 0. The dynamic response of the system was calculated and plotted over a time span of 10 hours. The results of the model support our data in that the RiboTALe with the larger dissociation constant (RiboTALe 1) is less effective at repressing GFP than RiboTALe 8 under the same induction conditions. The peak seen in the dynamic response of both simulations is a result of the kinematics of the system; there is lag between the initiation of GFP production and when the concentration of active TAL repressors is enough to tip the system.</br><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/0/0a/Ucdavismodel2.png"></center><br />
<br></br><br />
<h3>RiboTALe Modulation Through Theophylline Induction Levels</h3><br />
<br>This simulation was carried out under the same conditions defined above, but interrogated only one RiboTALe, RiboTALe 1 with a K<sub>D</sub> of 240 nM. The concentration of theophylline, however, was varied over a range of 1 mM to 10 mM and the results plotted. This simulation also supports our data in that it is clear that the riboswitch is in fact responsive to theophylline and that final GFP counts are inversely proportional to the amount of theophylline added.</br><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/2c/Ucdavismodel3.png"></center><br />
<br></br><br />
<h3>Amplifying System Response Through Transcript Induction</h3> <br />
</div><br />
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Lemur3 93
http://2013.igem.org/Team:UC_Davis/HumanPracticesOverview
Team:UC Davis/HumanPracticesOverview
2013-09-28T02:37:00Z
<p>Lemur3 93: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
<html><br />
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<div class="floatboxwide"><br />
<br />
<h1>Human Practices</h1><br />
<br />
<br />
<div class="floatbox1"><br />
<a href="https://2013.igem.org/Team:UC_Davis/Database"><img src="https://static.igem.org/mediawiki/2013/4/4e/Depotbutton_UCDavis.jpg" class="leftpic" width=150 height=150 /></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Database" class="white"><h3>BioBrick Characterization Data Depot</h3></a><br />
<p>Sharing is what makes us human, but as synthetic biologists, how can we improve the ways we share with each other? Find out more at the Data Depot<br /><br /><br /></p><br />
</div><br />
<br />
<div class=floatbox1><br />
<a href="https://2013.igem.org/Team:UC_Davis/Outreach"><img src="https://static.igem.org/mediawiki/2013/a/a5/UCDavis_outreachbutton.jpg" class="leftpic" width=150 /></a><br />
<div class="teampagetext"><br />
<a href="https://2013.igem.org/Team:UC_Davis/Outreach" class="white"><h3>Outreach</h3></a><br />
<p><br />
<ul><br />
<li>Provided DNA extractions for E.coli expressing RFP for the biotechnology class at Davis Senior High School</li><br />
<li>Hosted the NorCal iGEM team meet-up at UC Davis with the UC Berkeley, UCSF and Stanford-Brown teams attending</li><br />
<li>Met with a fellow student to discuss the creation of a Synthetic Biology Club at UC Davis</li><br />
</ul><br />
<br /><br />
</p><br />
</div><br />
</div><br />
</div><br />
<div class="floatboxwide"><br />
<br />
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<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project"><img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project"><h3>Project Background</h3></a><br />
<p>Learn about how we combine riboswitches and TAL's into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br />
</p><br />
</td><br />
<br />
<br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><img src="https://static.igem.org/mediawiki/igem.org/0/00/UCD_Outreach_2013.png" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we designed a new database for better raw data characterization of Biobricks. <br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
</p><br />
</td><br />
<br />
<br />
<br />
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Lemur3 93
http://2013.igem.org/Team:UC_Davis/Attributions
Team:UC Davis/Attributions
2013-09-28T02:32:51Z
<p>Lemur3 93: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
<html><br />
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</head><br />
<br />
<body><br />
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<img src="https://static.igem.org/mediawiki/2013/9/90/Attribanner_UCDavis.jpg" class="banner" width=967 height=226 /><br />
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<div class="floatboxwide"><br />
<h1>Attributions</h1><br />
<div class="floatbox1"><br />
<p>What we did:<br />
<ul><br />
<li>Wet lab work</li> <br />
<li>Conceptual and experimental design</li><br />
<li>Wiki and graphic design</li><br />
<li>Photography, figures and modeling</li><br />
<li>The Depot development</li><br />
</ul><br />
and analysis of all of the data we generated with some help from our advisors.<br />
</p><br />
</div><br />
<div class="floatbox1"><br />
<p>Special thanks to:<br />
<ul><br />
<li><a href="http://www.gallivanlab.org/Justin.html">Dr. Justin Gallivan</a> and <a href="http://www.bme.ucdavis.edu/people/departmental-faculty/profiles2/yohei-yokobayashi/">Dr. Yohei Yokobayashi</a> for advice on riboswitches</li><br />
<li>The Tagkopoulos Lab and the <a href="http://www.genomecenter.ucdavis.edu/segallab">Segal Lab</a> for providing us with the DNA for our TAL repressors</li><br />
<li><a href="http://www.genomecenter.ucdavis.edu/people/ludaesch">Dr. Bertram Ludaescher</a>, Sven Koehler, Anandarup Sarkar for advising in the design and implementation process of The Depot.</li><br />
<li><a href="http://www.jbei.org/people/directors/hector-garcia-martin/"> Hector Garcia Martin</a> for insight and direction from his work with JBEI's experimental data. </li><br />
<li><a href="http://www.tagkopouloslab.ucdavis.edu/people.html#">Linh Viet Huynh</a> of the Tagkopoulos Lab for looking over our modeling</li><br />
<li>Keegan Owsley, a past UC Davis iGEMer, for allowing us to use his 3D plotting software KO3D seen on our <a href="https://2013.igem.org/Team:UC_Davis/Data#widget">data page</a>.</li> <br />
<br />
<li><br />
And all of our sponsors:</li><br />
</ul><br /><br /><br />
<center><table class="genpic"><br />
<tr><br />
<td><br />
<a href="http://engineering.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2011/f/f6/UCD_CoE.png" width="200" height="40"></a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="http://biosci.ucdavis.edu/index_js.html" target="_blank"><img src="https://static.igem.org/mediawiki/2011/b/b1/UCD_biosci_sponsor.jpg" width="200" height="90"></a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="http://www.genomecenter.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2011/1/1b/UCD_Genome_center_sponsor.jpg" width="200" height="60"></a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="http://www.idtdna.com/site" target="_blank"><img src="https://static.igem.org/mediawiki/2012/2/22/IDT-Logo.jpg" width="200"></a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="http://www.bme.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2011/4/40/UCD_BME_logo_minimal_copy.png" width="200 height="70"></a><br />
</td><br />
</tr><br />
</table></center><br />
<br /><br />
We could not have done it without all of you. Thank you!<br />
</p><br />
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Lemur3 93
http://2013.igem.org/Team:UC_Davis/Notebook
Team:UC Davis/Notebook
2013-09-28T02:30:45Z
<p>Lemur3 93: </p>
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<h1 class="title">Week 0</h1><br />
<p><br />
<br />
<br />6/19/13<br />
<br />Part of the group met for the first day to discuss iGEM. In spite of some of the technical difficulties with Skype and Google Hangout, we learned about some of the principles to consider when choosing an iGEM project and began brainstorming. With consideration of time, we decided to limit our chassis to Escherichia coli. We also believed that providing a foundational advance for the field of synthetic biology would be an advantageous project to pursue. After our discussions, we had a small list of ideas. We created a Google Doc for further collaboration and created a list consisting of all of our contact information.<br /><br />
<br />
<br />6/20/13<br />
<br />We continued brainstorming ideas about different problems that we could address through synthetic biology. We came up with the following list of ideas for potential projects:<br />
<ul><br />
<li>Tal Repressors + riboswitches </li><br />
<li>Red, green, blue color output in response to light </li><br />
<li>Cas9 kill switch </li> <br />
<li>RNA Devices - develop some sort of assembly method protocol </li><br />
<li>Integrase switch</li><br />
<li>Faster reporting system - RNA fluorophore + repressing RNA binder</li><br />
<li>Optimization of taxol biosynthesis through augmentation of mevalonate and isoprenoid pathway</li><br />
<li>Shewanella RFID tag used in detection of volatile compounds present in produce<br />
<li>Applications of expansin proteins for control of plant growth</li><br />
<li>Method to suppress infectivity of the soybean cyst nematode</li><br />
<li>Assessment of noise production in different promoters via production of ribosome competing RNAs</li><br />
<li>Use of Integrase and excisionase for memory storage, lysis device, binary counter<br />
<li>Oscillator with different RNA fluorophores</li><br />
<li>Cas9 inserting a fluorophore for further expression</li><br />
<li>Vitamin B12 biosynthesis</li><br />
<li>Engineering E. coli for production of wine ingredients</li><br />
<li>Detection of harmful UV light levels</li><br />
<li>Detection of plant pathogen small molecule</li><br />
<li>Induced self cleaving of ribozymes with homologous RNA ends for the intracellular production of dsRNA to stimulate antiviral interferon response</li><br />
</ul><br />
<br />6/21/13<br />
<br />We talked extensively about the potential projects for the summer with our advisers. The advisers gave ratings for each of the possible projects and helped in identifying pros and cons for each of them. With consideration of the adviser’s input, the group decided to meet over the weekend to narrow the down the list to four ideas.<br />
</p><br />
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</body><br />
<script type="text/javascript"><br />
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Lemur3 93
http://2013.igem.org/Template:Team:UC_Davis/NotebookTemplate
Template:Team:UC Davis/NotebookTemplate
2013-09-28T02:25:41Z
<p>Lemur3 93: </p>
<hr />
<div><html><br />
<head><br />
<br />
<style><br />
<br />
/* Smaller floatbox for the Notebook START*/<br />
.floatnotebook<br />
{<br />
width:635px;<br />
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/* Smaller floatbox for the Notebook END*/<br />
<br />
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<body><br />
<div><img src="https://static.igem.org/mediawiki/2013/7/7d/Notebkbanner_UCDavis.jpg" class="banner" width=967px height=226/><br />
</div><br />
<table class="linktable"><br />
<tr><br />
<td><span class="weeklink" id="zero">June 19-21</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook">0</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="one">June 24-28</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_1">1</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="two">July 1-5</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_2">2</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="three">July 8-12</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_3">3</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="four">July 15-19</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_4">4</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="five">July 22-26</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_5">5</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="six">July 29-August 2</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_6">6</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="seven">August 5-9</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_7">7</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="eight">August 12-16</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_8">8</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="nine">August 19-24</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_9">9</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="ten">August 26-31</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_10">10</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="eleven">September 1-7</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_11">11</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="twelve">September 8-14</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_12">12</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="thirteen">September 15-21</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_13">13</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="fourteen">September 22-28</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_14">14</a></td><br />
</tr><br />
</table> <br />
</body><br />
</html></div>
Lemur3 93
http://2013.igem.org/Template:Team:UC_Davis/NotebookTemplate
Template:Team:UC Davis/NotebookTemplate
2013-09-28T02:24:23Z
<p>Lemur3 93: </p>
<hr />
<div><html><br />
<head><br />
<br />
<style><br />
<br />
/* Smaller floatbox for the Notebook START*/<br />
.floatnotebook<br />
{<br />
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<body><br />
<div><img src="https://static.igem.org/mediawiki/2013/7/7d/Notebkbanner_UCDavis.jpg" class="banner" width=967px height=226/><br />
</div><br />
<table class="linktable"><br />
<tr><br />
<td><span class="weeklink" id="zero">June 19-21</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook">0</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="one">June 24-28</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_1">1</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="two">July 1-5</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_2">2</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="three">July 8-12</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_3">3</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="four">July 15-19</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_4">4</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="five">July 22-26</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_5">5</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="six">July 29-August 2</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_6">6</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="seven">August 5-9</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_7">7</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="eight">August 12-16</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_8">8</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="nine">August 19-24</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_9">9</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="ten">August 26-31</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_10">10</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="eleven">September 1-7</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_11">11</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="twelve">September 8-14</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_12">12</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="thirteen">September 15-21</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_13">13</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="fourteen">September 22-28</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_14">14</a></td><br />
</tr><br />
</table> <br />
</body><br />
</html></div>
Lemur3 93
http://2013.igem.org/Template:Team:UC_Davis/NotebookTemplate
Template:Team:UC Davis/NotebookTemplate
2013-09-28T02:22:06Z
<p>Lemur3 93: </p>
<hr />
<div><html><br />
<head><br />
<br />
<style><br />
<br />
/* Smaller floatbox for the Notebook START*/<br />
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<br />
<body><br />
<div><img src="https://static.igem.org/mediawiki/2013/7/7d/Notebkbanner_UCDavis.jpg" class="banner" width=967px height=226/><br />
</div><br />
<table class="linktable"><br />
<tr><br />
<td><span class="weeklink" id="zero">June 19-21</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook">0</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="one">June 24-28</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_1">1</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="two">July 1-5</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_2">2</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="three">July 8-12</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_3">3</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="four">July 15-19</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_4">4</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="five">July 22-26</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_5">5</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="six">July 29-August 2</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_6">6</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="seven">August 5-9</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_7">7</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="eight">August 12-16</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_8">8</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="nine">August 19-24</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_9">9</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="ten">August 26-31</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_10">10</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="eleven">September 1-7</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_11">11</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="twelve">September 8-14</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_12">12</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="thirteen">September 15-21</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_13">13</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="fourteen">September 22-28</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_14">14</a></td><br />
</tr><br />
</table> <br />
</body><br />
</html></div>
Lemur3 93
http://2013.igem.org/Template:Team:UC_Davis/NotebookTemplate
Template:Team:UC Davis/NotebookTemplate
2013-09-28T02:20:00Z
<p>Lemur3 93: </p>
<hr />
<div><html><br />
<head><br />
<br />
<style><br />
<br />
/* Smaller floatbox for the Notebook START*/<br />
.floatnotebook<br />
{<br />
width:635px;<br />
float:left;<br />
background-color: rgba(0,0,0,.65);<br />
margin-top: -1px;<br />
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padding: 15px 15px 15px 15px;<br />
font: sans-serif;<br />
font-size: 13px;<br />
color: rgba(225,225,225,.9);<br />
}<br />
/* Smaller floatbox for the Notebook END*/<br />
<br />
/* Link bar for each week in the Notebook START*/<br />
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float:left;<br />
background-color: rgba(0,0,0,.65);<br />
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border-radius: 4px;<br />
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font: sans-serif;<br />
font-size: 13px;<br />
}<br />
.linktable td>a {<br />
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margin: 2px;<br />
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border-radius: 4px;<br />
color: rgba(0,0,0,.9);<br />
width: 20px;<br />
text-align: center;<br />
text-shadow: 1px #2a2a2a;<br />
cursor: pointer;<br />
}<br />
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text-decoration:none;<br />
}<br />
/* Link bar for each week in the Notebook END*/<br />
<br />
/*Show dates when hovering over a link for a week START*/<br />
span.weeklink { <br />
position:absolute; <br />
left:-9999px; <br />
border-radius: 4px;<br />
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font-size: 13px;<br />
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td:hover>#eleven { left:-76px }<br />
td:hover>#twelve{ left:-83px }<br />
td:hover>#thirteen { left:-90px }<br />
td:hover>#fourteen { left:-90px }<br />
/*Show dates when hovering over a link for a week END*/<br />
<br />
</style><br />
</head><br />
<br />
<body><br />
<div><img src="https://static.igem.org/mediawiki/2013/7/7d/Notebkbanner_UCDavis.jpg" class="banner" width=967px height=226/><br />
</div><br />
<table class="linktable"><br />
<tr><br />
<td><span class="weeklink" id="zero">June 19-21</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook">0</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="one">June 24-28</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_1">1</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="two">July 1-5</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_2">2</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="three">July 8-12</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_3">3</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="four">July 15-19</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_4">4</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="five">July 22-26</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_5">5</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="six">July 29-August 2</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_6">6</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="seven">August 5-9</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_7">7</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="eight">August 12-16</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_8">8</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="nine">August 19-24</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_9">9</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="ten">August 26-31</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_10">10</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="eleven">September 1-7</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_11">11</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="twelve">September 8-14</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_12">12</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="thirteen">September 15-21</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_13">13</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="fourteen">September 22-28</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_14">14</a></td><br />
</tr><br />
</table> <br />
</body><br />
</html></div>
Lemur3 93
http://2013.igem.org/Template:Team:UC_Davis/NotebookTemplate
Template:Team:UC Davis/NotebookTemplate
2013-09-28T02:12:35Z
<p>Lemur3 93: </p>
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<td><span class="weeklink" id="zero">June 19-21</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook">0</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="one">June 24-28</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_1">1</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="two">July 1-5</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_2">2</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="three">July 8-12</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_3">3</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="four">July 15-19</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_4">4</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="five">July 22-26</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_5">5</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="six">July 29-August 2</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_6">6</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="seven">August 5-9</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_7">7</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="eight">August 12-16</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_8">8</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="nine">August 19-24</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_9">9</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="ten">August 26-31</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_10">10</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="eleven">September 1-7</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_11">11</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="twelve">September 8-14</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_12">12</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="thirteen">September 15-21</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_13">13</a></td><br />
</tr><br />
<tr><br />
<td><span class="weeklink" id="fourteen">September 22-28</span><a href="https://2013.igem.org/Team:UC_Davis/Notebook/Week_14">14</a></td><br />
</tr><br />
</table> <br />
</body><br />
</html></div>
Lemur3 93
http://2013.igem.org/Team:UC_Davis/Contact
Team:UC Davis/Contact
2013-09-28T02:01:53Z
<p>Lemur3 93: </p>
<hr />
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<div class="floatbox"><br />
<h1>Contact Us</h1><br />
<br />
<div class="floatbox1"><br />
<img class="contactimage" src="https://static.igem.org/mediawiki/2013/9/9a/UC_Davis_E-mail.png"></img><br />
<p><br />
<h3>E-mail us at: ucdigem@gmail.com</h3><br />
</p><br />
</div><br />
<br />
<div class="floatbox1"><br />
<img class="contactimage" src="https://static.igem.org/mediawiki/2013/1/10/UC_Davis_Twitter_logo.png" width=100px height=100px></img><br />
<p><br />
<h3>Follow us on twitter at: <a href="https://twitter.com/UCDavisiGEM">@UCDavisiGEM</a></hi></h3><br />
</p><br />
</div><br />
<br />
<div class="floatbox1"><br />
<img class="contactimage" src="https://static.igem.org/mediawiki/2013/3/37/UC_Davis_13_phone.jpg" width="150px"></img><br />
<p><br />
<h3>Give us a call at (530)-752-3781</h3><br />
</p><br />
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Lemur3 93
http://2013.igem.org/Team:UC_Davis/Project_Overview
Team:UC Davis/Project Overview
2013-09-28T01:59:05Z
<p>Lemur3 93: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
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<br />
<br />
<br />
<div class="floatbox2"> <br />
<h2>Project Links<h2><br />
<ul><br />
<li><a href="#motivation">Project Motivation</a></li><br />
<li><a href="#solution">The Solution</a></li><br />
<li><a href="#projbackground">Parts Details</a></li><br />
</ul><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="motivation"><br />
<h1 class="title">Project Motivation</h1><br />
</a><br />
<p><br />
In synthetic biology, every circuit or device contains, at its core, at least one promoter and one protein coding region. While there are countless usefully proteins we could want to create, circuit design is limited by the small number of well characterized inducible promoters at our disposal, and their respective transcription factors. TetR, LacI, AraC, LuxR, and cI...do these sound familiar? <br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/ac/ProjMotive_UCDavis.png" class="centerimg"/><br></br><br />
<center><h3>We need more available inputs for transcriptional and translational control.</h3></center><br />
<p>What if ... <br />
<ul><br />
<li>we had transcriptional regulators that could be used in any strain or any chassis?</li> <br />
<li>we could directly engineer repressors for target sequences, instead of having to assemble parts to place them under the control of an inducible promoter? </li><br />
<li>we could control repression with our molecule of choice?</li><br />
<li>we could increase the degrees of freedom that we as researchers have over the control of gene expression?</li><br />
</ul><br />
We would have the ability to host multiple, orthogonal systems within the same chassis. A large part of synthetic biology is, ultimately, designing constructs that generate a response to an input stimulus. A construct that is entirely flexible both at its inputs and outputs is the ideal tool to facilitate the engineering of synthetic biology devices. If we decoupled transcription and translation of a repressor device, maintaining fine-tuned control of both processes, and characterized the behavior of all the parts involved, the dynamic range achievable would be stupendous.<br />
</p><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="solution"><br />
<h1>The Solution</h1><br />
</a><br />
<img src="https://static.igem.org/mediawiki/2013/9/94/Ucdavisoverview2.gif" width=600 height=245 class="centerimg genpic" /><br />
</div><br />
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<a id="projbackground"><br />
<h1 class="title">Parts Details</h1><br />
</a><br />
<p>Transcription activator-like effectors (TALEs) are proteins secreted by the bacterial plant pathogen <i>Xanthomonas</i>. TALEs contain sequence specific DNA binding domains and can act as transcriptional repressors or activators <a href="#ref">[1]</a>. This binding occurs through hydrogen bonds and van der Waals interactions and is stabilized by the protein's secondary structure. The DNA binding domains are sequence specific due to consecutive protein repeats, which are composed to correspond to a certain base preference <a href="#ref">[2]</a>. TAL repressors can therefore be engineered to bind to any DNA sequence of interest, following now well-understood rules for TAL-DNA binding <a href="#ref">[3,4]</a>. TALEs are thus a powerful and modular tool for the control of gene expression in genetic circuits. Current efforts to quantify and predict TALE binding affinities and functionalities are being made in order to create libraries of TALE systems that will serve to streamline research and the development of genetic devices <a href="#ref">[2]</a>.</p><br />
<br />
<p><br />Riboswitches, on the other hand, are regulatory structures in the 5’-UTR of mRNA that undergo a conformational change in the presence of a specific ligand that binds to the aptamer domain of the structure <a href="#ref">[5]</a>. This conformational change can regulate the initiation of translation by sequestering the ribosome binding site of the mRNA sequence, making it unavailable for binding <a href="#ref">[5]</a>. Riboswitches have been shown to work in diverse bacterial species and many natural examples have been found for riboswitches that turn off translation in the presence of the target ligand as well <a href="#ref">[6,7]</a>. Riboswitches have also been well characterized, are dose-dependent and have been engineered to respond to non-natural ligands thus providing an orthogonal control system <a href="#ref">[8]</a>. These RNA-based devices, like the TALE proteins, are also modular and powerful tools for the control of gene expression.</p><br />
<br />
<p><br />The fusion of these two devices--placing the TAL repressors under the control of riboswitches--offers a means by which to control the expression of any gene of interest, using wider variety of inducers than there are currently available. As our understanding of riboswitches and our ability to engineer aptamer binding domains develop, it will be possible to develop a fully orthogonal, highly versatile systems for control of gene expression. There is a potential for multiplexing, as riboswitches designed to respond to different molecules and fused to different TAL repressors can be used in parallel within a single chassis, or can be induced in a temporally sequential manner for applications such as developmental research. We have to demonstrated that RiboTALes function as synthetic transcription factors that are orthogonal to the natural biochemistry of the cell and increase the degrees of freedom available to us in the control of genetic circuits.</p><br />
</div><br />
<br />
<div class="floatbox2"><br />
<h2>TALE binding to DNA <a href="#ref">[1]</a></h2><br />
<img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" width=195 height=195 class="genpic"></img><br />
<h2>Riboswitch with inducer bound <a href="#ref">[9]</a></h2><br />
<img src="https://static.igem.org/mediawiki/2013/7/7e/Riboswitchpic_UCDavis.jpg" width=195 height=195 class="genpic"><img/><br />
</div><br />
<br />
<br />
<div class="floatbox"><br />
<table class="showbox"><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project"><img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project"><h3>Project Background</h3></a><br />
<p>Learn about how we combine riboswitches and TAL's into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br />
</p><br />
</td><br />
<br />
<br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><img src="https://static.igem.org/mediawiki/igem.org/0/00/UCD_Outreach_2013.png" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we designed a new database for better raw data characterization of Biobricks. <br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
</p><br />
</td><br />
<br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="ref"><h3>References</h3></a><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Structural+Basis+for+Sequence-Specific+Recognition+of+DNA+by+TAL+Effectors">[1] D. Dong, Y. Chuangye, P. Xiaojing, M. Mahfouz, W. Jiawei, Z. Jian-Kang, et al., "Structural Basis for Sequence-Specific Recognition of DNA by TAL Effectors," Science, vol. 335, pp. 720-723, 10 2012.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Quantitative+analysis+of+TALE-DNA+interactions+suggests+polarity+effects">[2] J. F. Meckler, M. S. Bhakta, M. S. Kim, R. Ovadia, C. H. Habrian, A. Zykovich, et al., "Quantitative analysis of TALE-DNA interactions suggests polarity effects," Nucleic Acids Res, vol. 41, pp. 4118-28, Apr 2013.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Breaking+the+Code+of+DNA+Binding+Specificity+of+TAL-Type+III+Effectors">[3] J. Boch, H. Scholze, S. Schornack, A. Landgraf, S. Hahn, S. Kay, et al., "Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors," Science, vol. 326, pp. 1509-1512, Dec 2009.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+Simple+Cipher+Governs+DNA+Recognition+by+TAL+Effectors">[4] M. J. Moscou and A. J. Bogdanove, "A Simple Cipher Governs DNA Recognition by TAL Effectors," Science, vol. 326, pp. 1501-1501, Dec 11 2009.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Dual-acting+riboswitch+control+of+translation+initiation+and+mRNA+decay">[5] M. P. Caron, L. Bastet, A. Lussier, M. Simoneau-Roy, E. Masse, and D. A. Lafontaine, "Dual-acting riboswitch control of translation initiation and mRNA decay," Proceedings of the National Academy of Sciences of the United States of America, vol. 109, pp. E3444-E3453, Dec 2012.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Synthetic+Riboswitches+That+Induce+Gene+Expression+in+Diverse+Bacterial+Species">[6] S. Topp, C. M. K. Reynoso, J. C. Seeliger, I. S. Goldlust, S. K. Desai, D. Murat, et al., "Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species (vol 76, pg 7881, 2010)," Applied and Environmental Microbiology, vol. 77, pp. 2199-2199, Mar 2011.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Mechanism-Guided+Library+Design+and+Dual+Genetic+Selection+of+Synthetic+OFF+Riboswitches">[7] N. Muranaka, K. Abe, and Y. Yokobayashi, "Mechanism-Guided Library Design and Dual Genetic Selection of Synthetic OFF Riboswitches," Chembiochem, vol. 10, pp. 2375-2381, Sep 2009.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Reengineering+orthogonally+selective+riboswitches">[8] N. Dixon, J. N. Duncan, T. Geerlings, M. S. Dunstan, J. E. G. McCarthy, D. Leys, et al., "Reengineering orthogonally selective riboswitches," Proceedings of the National Academy of Sciences of the United States of America, vol. 107, pp. 2830-2835, Feb 16 2010.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[9] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
</p><br />
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Lemur3 93
http://2013.igem.org/Team:UC_Davis/Project_Overview
Team:UC Davis/Project Overview
2013-09-28T01:58:39Z
<p>Lemur3 93: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
{{Team:UC_Davis/Slideshow}}<br />
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<img src="https://static.igem.org/mediawiki/2013/4/4b/UCD_2013_ProjectOverview_Bannerv3.PNG" class="banner" width="967" height="226"><br />
</div><br />
<br />
<br />
<br />
<br />
<div class="floatbox2"> <br />
<h2>Project Links<h2><br />
<ul><br />
<li><a href="#motivation">Project Motivation</a></li><br />
<li><a href="#solution">The Solution</a></li><br />
<li><a href="#projbackground">Parts Details</a></li><br />
</ul><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="motivation"><br />
<h1 class="title">Project Motivation</h1><br />
</a><br />
<p><br />
In synthetic biology, every circuit or device contains, at its core, at least one promoter and one protein coding region. While there are countless usefully proteins we could want to create, circuit design is limited by the small number of well characterized inducible promoters at our disposal, and their respective transcription factors. TetR, LacI, AraC, LuxR, and cI...do these sound familiar? <br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/ac/ProjMotive_UCDavis.png" class="centerimg"/><br></br><br />
<center><h3>We need more available inputs for transcriptional and translational control.</h3></center><br />
<p>What if ... <br />
<ul><br />
<li>we had transcriptional regulators that could be used in any strain or any chassis?</li> <br />
<li>we could directly engineer repressors for target sequences, instead of having to assemble parts to place them under the control of an inducible promoter? </li><br />
<li>we could control repression with our molecule of choice?</li><br />
<li>we could increase the degrees of freedom that we as researchers have over the control of gene expression?</li><br />
</ul><br />
We would have the ability to host multiple, orthogonal systems within the same chassis. A large part of synthetic biology is, ultimately, designing constructs that generate a response to an input stimulus. A construct that is entirely flexible both at its inputs and outputs is the ideal tool to facilitate the engineering of synthetic biology devices. If we decoupled transcription and translation of a repressor device, maintaining fine-tuned control of both processes, and characterized the behavior of all the parts involved, the dynamic range achievable would be stupendous.<br />
</p><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="solution"><br />
<h1>The Solution</h1><br />
</a><br />
<img src="https://static.igem.org/mediawiki/2013/9/94/Ucdavisoverview2.gif" width=600 height=245 class="centerimg genpic" /><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="projbackground"><br />
<h1 class="title">Parts Details</h1><br />
</a><br />
<p>Transcription activator-like effectors (TALEs) are proteins secreted by the bacterial plant pathogen <i>Xanthomonas</i>. TALEs contain sequence specific DNA binding domains and can act as transcriptional repressors or activators <a href="#ref">[1]</a>. This binding occurs through hydrogen bonds and van der Waals interactions and is stabilized by the protein's secondary structure. The DNA binding domains are sequence specific due to consecutive protein repeats, which are composed to correspond to a certain base preference <a href="#ref">[2]</a>. TAL repressors can therefore be engineered to bind to any DNA sequence of interest, following now well-understood rules for TAL-DNA binding <a href="#ref">[3,4]</a>. TALEs are thus a powerful and modular tool for the control of gene expression in genetic circuits. Current efforts to quantify and predict TALE binding affinities and functionalities are being made in order to create libraries of TALE systems that will serve to streamline research and the development of genetic devices <a href="#ref">[2]</a>.</p><br />
<br />
<p><br />Riboswitches, on the other hand, are regulatory structures in the 5’-UTR of mRNA that undergo a conformational change in the presence of a specific ligand that binds to the aptamer domain of the structure <a href="#ref">[5]</a>. This conformational change can regulate the initiation of translation by sequestering the ribosome binding site of the mRNA sequence, making it unavailable for binding <a href="#ref">[5]</a>. Riboswitches have been shown to work in diverse bacterial species and many natural examples have been found for riboswitches that turn off translation in the presence of the target ligand as well <a href="#ref">[6,7]</a>. Riboswitches have also been well characterized, are dose-dependent and have been engineered to respond to non-natural ligands thus providing an orthogonal control system <a href="#ref">[8]</a>. These RNA-based devices, like the TALE proteins, are also modular and powerful tools for the control of gene expression.</p><br />
<br />
<p><br />The fusion of these two devices--placing the TAL repressors under the control of riboswitches--offers a means by which to control the expression of any gene of interest, using wider variety of inducers than there are currently available. As our understanding of riboswitches and our ability to engineer aptamer binding domains develop, it will be possible to develop a fully orthogonal, highly versatile systems for control of gene expression. There is a potential for multiplexing, as riboswitches designed to respond to different molecules and fused to different TAL repressors can be used in parallel within a single chassis, or can be induced in a temporally sequential manner for applications such as developmental research. We have to demonstrated that RiboTALes function as synthetic transcription factors that are orthogonal to the natural biochemistry of the cell and increase the degrees of freedom available to us in the control of genetic circuits.</p><br />
</div><br />
<br />
<div class="floatbox2"><br />
<h2>TALE binding to DNA <a href="#ref">[1]</a></h2><br />
<img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" width=195 height=195 class="genpic"></img><br />
<h2>Riboswitch with inducer bound <a href="#ref">[9]</a></h2><br />
<img src="https://static.igem.org/mediawiki/2013/7/7e/Riboswitchpic_UCDavis.jpg" width=195 height=195 /><br />
</div><br />
<br />
<br />
<div class="floatbox"><br />
<table class="showbox"><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project"><img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project"><h3>Project Background</h3></a><br />
<p>Learn about how we combine riboswitches and TAL's into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br />
</p><br />
</td><br />
<br />
<br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><img src="https://static.igem.org/mediawiki/igem.org/0/00/UCD_Outreach_2013.png" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we designed a new database for better raw data characterization of Biobricks. <br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
</p><br />
</td><br />
<br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="ref"><h3>References</h3></a><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Structural+Basis+for+Sequence-Specific+Recognition+of+DNA+by+TAL+Effectors">[1] D. Dong, Y. Chuangye, P. Xiaojing, M. Mahfouz, W. Jiawei, Z. Jian-Kang, et al., "Structural Basis for Sequence-Specific Recognition of DNA by TAL Effectors," Science, vol. 335, pp. 720-723, 10 2012.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Quantitative+analysis+of+TALE-DNA+interactions+suggests+polarity+effects">[2] J. F. Meckler, M. S. Bhakta, M. S. Kim, R. Ovadia, C. H. Habrian, A. Zykovich, et al., "Quantitative analysis of TALE-DNA interactions suggests polarity effects," Nucleic Acids Res, vol. 41, pp. 4118-28, Apr 2013.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Breaking+the+Code+of+DNA+Binding+Specificity+of+TAL-Type+III+Effectors">[3] J. Boch, H. Scholze, S. Schornack, A. Landgraf, S. Hahn, S. Kay, et al., "Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors," Science, vol. 326, pp. 1509-1512, Dec 2009.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+Simple+Cipher+Governs+DNA+Recognition+by+TAL+Effectors">[4] M. J. Moscou and A. J. Bogdanove, "A Simple Cipher Governs DNA Recognition by TAL Effectors," Science, vol. 326, pp. 1501-1501, Dec 11 2009.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Dual-acting+riboswitch+control+of+translation+initiation+and+mRNA+decay">[5] M. P. Caron, L. Bastet, A. Lussier, M. Simoneau-Roy, E. Masse, and D. A. Lafontaine, "Dual-acting riboswitch control of translation initiation and mRNA decay," Proceedings of the National Academy of Sciences of the United States of America, vol. 109, pp. E3444-E3453, Dec 2012.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Synthetic+Riboswitches+That+Induce+Gene+Expression+in+Diverse+Bacterial+Species">[6] S. Topp, C. M. K. Reynoso, J. C. Seeliger, I. S. Goldlust, S. K. Desai, D. Murat, et al., "Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species (vol 76, pg 7881, 2010)," Applied and Environmental Microbiology, vol. 77, pp. 2199-2199, Mar 2011.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Mechanism-Guided+Library+Design+and+Dual+Genetic+Selection+of+Synthetic+OFF+Riboswitches">[7] N. Muranaka, K. Abe, and Y. Yokobayashi, "Mechanism-Guided Library Design and Dual Genetic Selection of Synthetic OFF Riboswitches," Chembiochem, vol. 10, pp. 2375-2381, Sep 2009.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Reengineering+orthogonally+selective+riboswitches">[8] N. Dixon, J. N. Duncan, T. Geerlings, M. S. Dunstan, J. E. G. McCarthy, D. Leys, et al., "Reengineering orthogonally selective riboswitches," Proceedings of the National Academy of Sciences of the United States of America, vol. 107, pp. 2830-2835, Feb 16 2010.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[9] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
</p><br />
</div><br />
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Lemur3 93
http://2013.igem.org/Team:UC_Davis/Project_Overview
Team:UC Davis/Project Overview
2013-09-28T01:57:48Z
<p>Lemur3 93: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
{{Team:UC_Davis/Slideshow}}<br />
<br />
<html><br />
<head><br />
<style type="text/css"><br />
img.centerimg{<br />
display: block;<br />
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margin-right:auto;<br />
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<img src="https://static.igem.org/mediawiki/2013/4/4b/UCD_2013_ProjectOverview_Bannerv3.PNG" class="banner" width="967" height="226"><br />
</div><br />
<br />
<br />
<br />
<br />
<div class="floatbox2"> <br />
<h2>Project Links<h2><br />
<ul><br />
<li><a href="#motivation">Project Motivation</a></li><br />
<li><a href="#solution">The Solution</a></li><br />
<li><a href="#projbackground">Parts Details</a></li><br />
</ul><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="motivation"><br />
<h1 class="title">Project Motivation</h1><br />
</a><br />
<p><br />
In synthetic biology, every circuit or device contains, at its core, at least one promoter and one protein coding region. While there are countless usefully proteins we could want to create, circuit design is limited by the small number of well characterized inducible promoters at our disposal, and their respective transcription factors. TetR, LacI, AraC, LuxR, and cI...do these sound familiar? <br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/ac/ProjMotive_UCDavis.png" class="centerimg"/><br></br><br />
<center><h3>We need more available inputs for transcriptional and translational control.</h3></center><br />
<p>What if ... <br />
<ul><br />
<li>we had transcriptional regulators that could be used in any strain or any chassis?</li> <br />
<li>we could directly engineer repressors for target sequences, instead of having to assemble parts to place them under the control of an inducible promoter? </li><br />
<li>we could control repression with our molecule of choice?</li><br />
<li>we could increase the degrees of freedom that we as researchers have over the control of gene expression?</li><br />
</ul><br />
We would have the ability to host multiple, orthogonal systems within the same chassis. A large part of synthetic biology is, ultimately, designing constructs that generate a response to an input stimulus. A construct that is entirely flexible both at its inputs and outputs is the ideal tool to facilitate the engineering of synthetic biology devices. If we decoupled transcription and translation of a repressor device, maintaining fine-tuned control of both processes, and characterized the behavior of all the parts involved, the dynamic range achievable would be stupendous.<br />
</p><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="solution"><br />
<h1>The Solution</h1><br />
</a><br />
<img src="https://static.igem.org/mediawiki/2013/9/94/Ucdavisoverview2.gif" width=600 height=245 class="centerimg genpic" /><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="projbackground"><br />
<h1 class="title">Parts Details</h1><br />
</a><br />
<p>Transcription activator-like effectors (TALEs) are proteins secreted by the bacterial plant pathogen <i>Xanthomonas</i>. TALEs contain sequence specific DNA binding domains and can act as transcriptional repressors or activators <a href="#ref">[1]</a>. This binding occurs through hydrogen bonds and van der Waals interactions and is stabilized by the protein's secondary structure. The DNA binding domains are sequence specific due to consecutive protein repeats, which are composed to correspond to a certain base preference <a href="#ref">[2]</a>. TAL repressors can therefore be engineered to bind to any DNA sequence of interest, following now well-understood rules for TAL-DNA binding <a href="#ref">[3,4]</a>. TALEs are thus a powerful and modular tool for the control of gene expression in genetic circuits. Current efforts to quantify and predict TALE binding affinities and functionalities are being made in order to create libraries of TALE systems that will serve to streamline research and the development of genetic devices <a href="#ref">[2]</a>.</p><br />
<br />
<p><br />Riboswitches, on the other hand, are regulatory structures in the 5’-UTR of mRNA that undergo a conformational change in the presence of a specific ligand that binds to the aptamer domain of the structure <a href="#ref">[5]</a>. This conformational change can regulate the initiation of translation by sequestering the ribosome binding site of the mRNA sequence, making it unavailable for binding <a href="#ref">[5]</a>. Riboswitches have been shown to work in diverse bacterial species and many natural examples have been found for riboswitches that turn off translation in the presence of the target ligand as well <a href="#ref">[6,7]</a>. Riboswitches have also been well characterized, are dose-dependent and have been engineered to respond to non-natural ligands thus providing an orthogonal control system <a href="#ref">[8]</a>. These RNA-based devices, like the TALE proteins, are also modular and powerful tools for the control of gene expression.</p><br />
<br />
<p><br />The fusion of these two devices--placing the TAL repressors under the control of riboswitches--offers a means by which to control the expression of any gene of interest, using wider variety of inducers than there are currently available. As our understanding of riboswitches and our ability to engineer aptamer binding domains develop, it will be possible to develop a fully orthogonal, highly versatile systems for control of gene expression. There is a potential for multiplexing, as riboswitches designed to respond to different molecules and fused to different TAL repressors can be used in parallel within a single chassis, or can be induced in a temporally sequential manner for applications such as developmental research. We have to demonstrated that RiboTALes function as synthetic transcription factors that are orthogonal to the natural biochemistry of the cell and increase the degrees of freedom available to us in the control of genetic circuits.</p><br />
</div><br />
<br />
<div class="floatbox2"><br />
<h2>TALE binding to DNA <a href="#ref">[1]</a></h2><br />
<img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" width=195 height=195 /><br />
<h2>Riboswitch with inducer bound <a href="#ref">[9]</a></h2><br />
<img src="https://static.igem.org/mediawiki/2013/7/7e/Riboswitchpic_UCDavis.jpg" width=195 height=195 /><br />
</div><br />
<br />
<br />
<div class="floatbox"><br />
<table class="showbox"><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project"><img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project"><h3>Project Background</h3></a><br />
<p>Learn about how we combine riboswitches and TAL's into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br />
</p><br />
</td><br />
<br />
<br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><img src="https://static.igem.org/mediawiki/igem.org/0/00/UCD_Outreach_2013.png" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we designed a new database for better raw data characterization of Biobricks. <br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
</p><br />
</td><br />
<br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="ref"><h3>References</h3></a><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Structural+Basis+for+Sequence-Specific+Recognition+of+DNA+by+TAL+Effectors">[1] D. Dong, Y. Chuangye, P. Xiaojing, M. Mahfouz, W. Jiawei, Z. Jian-Kang, et al., "Structural Basis for Sequence-Specific Recognition of DNA by TAL Effectors," Science, vol. 335, pp. 720-723, 10 2012.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Quantitative+analysis+of+TALE-DNA+interactions+suggests+polarity+effects">[2] J. F. Meckler, M. S. Bhakta, M. S. Kim, R. Ovadia, C. H. Habrian, A. Zykovich, et al., "Quantitative analysis of TALE-DNA interactions suggests polarity effects," Nucleic Acids Res, vol. 41, pp. 4118-28, Apr 2013.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Breaking+the+Code+of+DNA+Binding+Specificity+of+TAL-Type+III+Effectors">[3] J. Boch, H. Scholze, S. Schornack, A. Landgraf, S. Hahn, S. Kay, et al., "Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors," Science, vol. 326, pp. 1509-1512, Dec 2009.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+Simple+Cipher+Governs+DNA+Recognition+by+TAL+Effectors">[4] M. J. Moscou and A. J. Bogdanove, "A Simple Cipher Governs DNA Recognition by TAL Effectors," Science, vol. 326, pp. 1501-1501, Dec 11 2009.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Dual-acting+riboswitch+control+of+translation+initiation+and+mRNA+decay">[5] M. P. Caron, L. Bastet, A. Lussier, M. Simoneau-Roy, E. Masse, and D. A. Lafontaine, "Dual-acting riboswitch control of translation initiation and mRNA decay," Proceedings of the National Academy of Sciences of the United States of America, vol. 109, pp. E3444-E3453, Dec 2012.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Synthetic+Riboswitches+That+Induce+Gene+Expression+in+Diverse+Bacterial+Species">[6] S. Topp, C. M. K. Reynoso, J. C. Seeliger, I. S. Goldlust, S. K. Desai, D. Murat, et al., "Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species (vol 76, pg 7881, 2010)," Applied and Environmental Microbiology, vol. 77, pp. 2199-2199, Mar 2011.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Mechanism-Guided+Library+Design+and+Dual+Genetic+Selection+of+Synthetic+OFF+Riboswitches">[7] N. Muranaka, K. Abe, and Y. Yokobayashi, "Mechanism-Guided Library Design and Dual Genetic Selection of Synthetic OFF Riboswitches," Chembiochem, vol. 10, pp. 2375-2381, Sep 2009.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Reengineering+orthogonally+selective+riboswitches">[8] N. Dixon, J. N. Duncan, T. Geerlings, M. S. Dunstan, J. E. G. McCarthy, D. Leys, et al., "Reengineering orthogonally selective riboswitches," Proceedings of the National Academy of Sciences of the United States of America, vol. 107, pp. 2830-2835, Feb 16 2010.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[9] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
</p><br />
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Lemur3 93
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File:Humanoutbanner UCDavis.jpg
2013-09-28T01:53:20Z
<p>Lemur3 93: uploaded a new version of &quot;File:Humanoutbanner UCDavis.jpg&quot;</p>
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Lemur3 93
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2013-09-28T01:47:50Z
<p>Lemur3 93: uploaded a new version of &quot;File:Humanpracbanner UCDavis.jpg&quot;</p>
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Lemur3 93
http://2013.igem.org/File:Humanpracbanner_UCDavis.jpg
File:Humanpracbanner UCDavis.jpg
2013-09-28T01:44:48Z
<p>Lemur3 93: uploaded a new version of &quot;File:Humanpracbanner UCDavis.jpg&quot;</p>
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Lemur3 93
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2013-09-28T01:38:52Z
<p>Lemur3 93: uploaded a new version of &quot;File:Attribanner UCDavis.jpg&quot;</p>
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Lemur3 93
http://2013.igem.org/Team:UC_Davis/Gallery
Team:UC Davis/Gallery
2013-09-28T01:34:08Z
<p>Lemur3 93: </p>
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<h1>Gallery</h1><br />
<p>From the wet lab to the dry lab, our team was hard at work this summer. Take a look!</p><br />
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<a href="https://2013.igem.org/Team:UC_Davis/Project"><h3>Project Background</h3></a><br />
<p>Learn about how we combine riboswitches and TALs into robust orthogonal mechanisms for inducible repression. <br />
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<br />
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<p>Check out the cool results of our experiments with RiboTALs. <br />
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<p>Take a look at how we designed a new database for better raw data characterization of Biobricks. <br />
</p><br />
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<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
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Lemur3 93
http://2013.igem.org/Team:UC_Davis/Gallery
Team:UC Davis/Gallery
2013-09-28T01:32:34Z
<p>Lemur3 93: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
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<br />
<img src="https://static.igem.org/mediawiki/2013/d/d3/UCD_2013_Galler_Banner.jpg" class="banner" width="967px" height="226/"><br />
<br />
<div id="twitter"><br />
<a class="twitter-timeline" width=220px height=300px href="https://twitter.com/UCDavisiGEM" data-widget-id="363400889542778880" data-theme="dark"><br />
Tweets by @UCDavisiGEM</a><br />
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<div id="myleftbox"><br />
<div id="myleftbox" class="smallbox"><br />
<h1>Gallery</h1><br />
<p>From the wet lab to the dry lab, our team was hard at work this summer. Take a look!</p><br />
</div><br />
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<img src="https://static.igem.org/mediawiki/2013/f/f8/UCD_Slides_2013_6.JPG" style="width:605px; height:400px; margin-left:50px;" /> <br />
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<li><a href="#n_3">4</a> </li><br />
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<li><a href="#n_2">6</a> </li><br />
<li><a href="#n_3">7</a> </li><br />
<li><a href="#n_1">8</a> </li><br />
<li><a href="#n_2">9</a> </li><br />
<li><a href="#n_3">10</a> </li><br />
<li><a href="#n_1">11</a> </li><br />
<li><a href="#n_2">12</a> </li><br />
<li><a href="#n_3">13</a> </li><br />
<li><a href="#n_1">14</a> </li><br />
<li><a href="#n_2">15</a> </li><br />
<li><a href="#n_3">16</a> </li><br />
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<ul class="forward"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1d/UCD_Big_arrow_right.png"/> <br />
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<td><br />
<br />
<a href="https://2013.igem.org/Team:UC_Davis/Project"><div><br />
<img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"><br />
</div></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project"><h3>Project Background</h3></a><br />
<p>Learn about how we combine riboswitches and TALs into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
<br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br />
</p><br />
</td><br />
<br />
<br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><img src="https://static.igem.org/mediawiki/igem.org/0/00/UCD_Outreach_2013.png" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we designed a new database for better raw data characterization of Biobricks. <br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"</a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
</p><br />
</td><br />
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Lemur3 93
http://2013.igem.org/File:UCD_2013_Galler_Banner.jpg
File:UCD 2013 Galler Banner.jpg
2013-09-28T01:31:30Z
<p>Lemur3 93: </p>
<hr />
<div></div>
Lemur3 93
http://2013.igem.org/File:Protocolsbanner_UCDavis.jpg
File:Protocolsbanner UCDavis.jpg
2013-09-28T00:24:47Z
<p>Lemur3 93: uploaded a new version of &quot;File:Protocolsbanner UCDavis.jpg&quot;</p>
<hr />
<div></div>
Lemur3 93
http://2013.igem.org/File:UCD_2013_Notebook_Overview.jpg
File:UCD 2013 Notebook Overview.jpg
2013-09-28T00:17:21Z
<p>Lemur3 93: uploaded a new version of &quot;File:UCD 2013 Notebook Overview.jpg&quot;</p>
<hr />
<div></div>
Lemur3 93
http://2013.igem.org/Team:UC_Davis/Notebook_Overview
Team:UC Davis/Notebook Overview
2013-09-28T00:06:49Z
<p>Lemur3 93: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
{{Team:UC_Davis/Slideshow}}<br />
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<a href="https://2013.igem.org/Team:UC_Davis/Notebook"><img src="https://static.igem.org/mediawiki/2013/4/47/Notebkbutton_UCDavis.jpg"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Notebook"><h3>Notebook</h3></a><br />
<p>Get to know how we worked on a day by day basis. <br />
</p></a><br />
</td><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Protocols"><img src="https://static.igem.org/mediawiki/2013/f/f0/Protocbutton_UCDavis.jpg"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Protocols"><h3>Protocols</h3></a><br />
<p>Find out more about the protocols we followed for each experiment during our project. <br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Attributions"><img src="https://static.igem.org/mediawiki/2013/f/f2/Attributton_UCDavis.jpg"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Attributions"><h3>Attributions</h3></a><br />
<p>Here you can see what we did and everyone that helped us out with our project. <br />
</p><br />
</td><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Gallery"><img src="https://static.igem.org/mediawiki/2013/2/2a/Gallerybutton_UCDavis.jpg"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Gallery"><h3>Gallery</h3></a><br />
<p>Take a look at photos we captured during our project. <br />
</p><br />
</td><br />
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Lemur3 93
http://2013.igem.org/File:UCD_2013_Notebook_Overview.jpg
File:UCD 2013 Notebook Overview.jpg
2013-09-28T00:05:46Z
<p>Lemur3 93: </p>
<hr />
<div></div>
Lemur3 93
http://2013.igem.org/File:UCDavis_databanner.jpg
File:UCDavis databanner.jpg
2013-09-27T22:18:08Z
<p>Lemur3 93: uploaded a new version of &quot;File:UCDavis databanner.jpg&quot;</p>
<hr />
<div></div>
Lemur3 93
http://2013.igem.org/File:UCDavis_databanner.jpg
File:UCDavis databanner.jpg
2013-09-27T22:16:20Z
<p>Lemur3 93: uploaded a new version of &quot;File:UCDavis databanner.jpg&quot;</p>
<hr />
<div></div>
Lemur3 93
http://2013.igem.org/Team:UC_Davis/Safety
Team:UC Davis/Safety
2013-09-27T22:04:56Z
<p>Lemur3 93: </p>
<hr />
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<h1>iGEM Safety Questions <br />
<a href="https://2013.igem.org/Safety">(Safety Page)</a></h1><br />
<br />
<p><br />
<ol><br />
<li>Would any of your project ideas raise safety issues in terms of:</li><br />
<ul><br />
<li>Researcher Safety? </li><br />
<li>Public Safety? </li><br />
<li>Environmental Safety?</li><br />
<br>All the bacterial strains we worked with over the course of this project (E.coli K-12 derivatives DH5alpha, DH10b, MG1655Z1, and BW22826) are non-pathogenic to humans, and all proteins produced are non-toxic. Furthermore, proper safety protocols for the handling of these biological materials were always observed, thus minimizing risks to safety and health of the iGEM team members. The general public may include immunocompromosied individuals that may be more susceptible to E. coli K-12, but the risk of pathogenicity remains minimal.</br><br />
<br> While TALEs are derived from a plant pathogen, Xanthomonas, the TALEs and our chassis do not express pathogenic capabilities. According to the study <a href="http://epa.gov/oppt/biotech/pubs/fra/fra004.htm">'Escherichia coli K-12 Derivatives Final Risk Assessment'</a></hi> published by the United States Environmental Protection Agency in 1997, "the strain E. coli K-12 is a debilitated strain which does not normally colonize the human intestine. It has also been shown to survive poorly in the environment, has a history of safe commercial use, and is not known to have adverse effects on microorganisms or plants." </br><br />
<br></br><br />
</ul><br />
<li>Do any of the new BioBrick parts (or devices) that you made this year raise safety issues? If yes,</li><br />
<ul><br />
<li>Did you document these issues in the Registry?</li><br />
<li>How did you manage to handle the safety issue?</li><br />
<li>How could other teams learn from your experience?</li><br />
<br>The new Biobrick parts that we constructed have minimal potential to generate safety risks. We assembled Biobrick parts that would provide a foundational advance for future iGEM teams and do not pose an intrinsic threat to the environment or to the health of individuals or the public at large. The only concern with the bacteria we used was the small risk that the transformants would genomically acquire the same form antibiotic resistance provided by the plasmids. To mitigate this, we made sure to use sterile conditions when growing these bacteria in cultures and also made sure to safely dispose of any unused cultures. We believe that these precautionary measures are standards by which most labs operate and we hope to remain a model of safety.</br><br />
<br>It is plausible that our construct could generate biosecurity risks through misuse, but only if extensively reengineered for malicious purposes. Our construct itself does not increase the pathogenicity of E. coli K-12 and does not produce any toxins. Likewise, the implementation of our construct in a large-scale industrial setting should not give way to environmental or biosecurity risks. E. coli K-12 has a history of safe use in a large number of industrial applications. Any risks that may arise will be due to the gene(s) controlled by our system, not our system itself. For example, it may be possible to tailor a pathogen to respond to a metabolite through our system, but this is not a risk intrinsic to our system. Furthermore, the risk for horizontal gene transfer in the environment is remote due to the debilitated nature of our chassis. In any case, our construct comes with multiple points of control, so that there is minimal expression of the gene of interest without the addition of an appropriate inducer.</br><br />
<br></br><br />
<br />
</ul><br />
<li>Is there a local biosafety group, committee, or review board at your institution?</li><br />
<ul><br />
<li>If yes, what does your local biosafety group think about your project?</li><br />
<li>If no, which specific biosafety rules or guidelines do you have to consider in your country?</li><br />
</ul><br />
<br><br />
UC Davis has a Safety Services Department on campus that oversees all UCD affiliated research in order to maintain a high standard of safety. We contacted Biosafety Officer M. Malendia Maccree to discuss safety concerns related to our project. She raised no concerns, approves of our project, and has documented it as exempt from <a href="http://oba.od.nih.gove/oba/rac/Guidelines/NIH_Guidelines.htm">NIH Guidelines</a></hi>, which may require initial approval of a project according to Appendices C-II and C-II-A. This exemption is due to the nature of our chassis and the fact that we did not use DNA from Risk Groups 3 or 4.<br />
</br><br />
<br>For more information on our country's biosafety guidelines, please refer to <a href="http://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf">Biosafety in Microbiological and Biomedical Laboratories, 5th edition</a></hi>. <br />
<br></br><br />
<li>Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</li><br />
<br>Engineering systems that are orthogonal to the chassis's natural biochemistry could reduce the risk of unintentionally increasing the pathogenicity of the chassis. Systems that are dependent on externally provided metabolites or ligands could also reduce the risk of horizontal gene transfer in the environment. A Biosafety Committee should always be consulted prior to commencing a project that may carry any biosafety risks.</br><br />
<br />
</ol><br />
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Lemur3 93
http://2013.igem.org/File:Safetybanner_UCDavis_2013.jpg
File:Safetybanner UCDavis 2013.jpg
2013-09-27T22:04:22Z
<p>Lemur3 93: </p>
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Lemur3 93
http://2013.igem.org/File:Partsbanner_UCDavis.jpg
File:Partsbanner UCDavis.jpg
2013-09-27T21:55:02Z
<p>Lemur3 93: uploaded a new version of &quot;File:Partsbanner UCDavis.jpg&quot;</p>
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Lemur3 93
http://2013.igem.org/File:Assemblybanner_UCDavis.jpg
File:Assemblybanner UCDavis.jpg
2013-09-27T21:44:32Z
<p>Lemur3 93: uploaded a new version of &quot;File:Assemblybanner UCDavis.jpg&quot;</p>
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Lemur3 93
http://2013.igem.org/File:Modelingbanner_UCDavis.jpg
File:Modelingbanner UCDavis.jpg
2013-09-27T21:40:21Z
<p>Lemur3 93: uploaded a new version of &quot;File:Modelingbanner UCDavis.jpg&quot;</p>
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Lemur3 93
http://2013.igem.org/Team:UC_Davis/Project_Overview
Team:UC Davis/Project Overview
2013-09-27T21:31:13Z
<p>Lemur3 93: </p>
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<div>{{Team:UC_Davis/Head}}<br />
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<div class="floatbox2"> <br />
<h2>Project Links<h2><br />
<ul><br />
<li><a href="#motivation">Project Motivation</a></li><br />
<li><a href="#projbackground">Project Background</a></li><br />
</ul><br />
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<div class="floatbox"><br />
<a id="motivation"><br />
<h1 class="title">PROJECT MOTIVATION</h1><br />
</a><br />
<p><br />
In synthetic biology, every circuit or device contains, at its core, at least one promoter and one protein coding region. While there are countless usefully proteins we could want to create, circuit design is limited by the small number of well characterized inducible promoters at our disposal, and their respective transcription factors. TetR, LacI, AraC, LuxR, and cI...do these sound familiar? <br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/ac/ProjMotive_UCDavis.png" class="centerimg"/><br></br><br />
<center><h3>We need more flexibility in transcriptional and translational control.</h3></center><br />
<p>What if ... <br />
<ul><br />
<li>we had transcriptional regulators that could be used in any strain or any chassis?</li> <br />
<li>we could directly engineer repressors for target sequences, instead of having to assemble parts to place them the under control of an inducible promoter? </li><br />
<li>we could control this repression system with a molecule of choice?</li><br />
<li>we could increase the degrees of freedom that we as researchers have in the control of gene expression pathways?</li><br />
</ul><br />
We would have the ability to host multiple, orthogonal systems within the same chassis. The need to 'bioprospect' metabolites would diminish. A large part of synthetic biology is, ultimately, designing constructs that generate a response to an input stimulus. A construct that is entirely flexible both at its inputs and outputs is the ideal tool to facilitate the engineering of synthetic biology devices. If we decoupled transcription and translation of a repressor device, maintaining fine-tuned control of both processes, and characterized the behavior of all the parts involved, the dynamic range achievable would be stupendous.<br />
</p><br />
</div><br />
<div class="floatbox"><br />
<h1>The Solution</h1><br />
<img src="https://static.igem.org/mediawiki/2013/9/94/Ucdavisoverview2.gif" width=600 height=245 class="centerimg" /><br />
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<a id="projbackground"><br />
<h1 class="title">PROJECT BACKGROUND</h1><br />
</a><br />
<p>Transcription activator-like effectors (TALEs) are proteins secreted by the bacterial pathogen <i>Xanthomonas</i> that contain sequence specific DNA binding domains and can act as transcriptional repressors or activators <a href="#ref">[1]</a>. This binding occurs through hydrogen bonds and van der Waals interactions and is stabilized by the protein's secondary structure. The DNA binding domains are sequence specific due to consecutive protein repeats, the composition of each which corresponds to a certain base preference <a href="#ref">[2]</a>. TAL repressors can therefore be engineered to bind to any DNA sequence of interest, following now well-understood rules for TAL-DNA binding <a href="#ref">[3,4]</a>. TALEs are thus a powerful and modular tool for the control of gene expression in genetic circuits. Current efforts to quantify and predict TALE binding affinities and functionalities are being made in order to create libraries of TALE systems that will serve to streamline research and the development of genetic devices <a href="#ref">[2]</a>.</p><br />
<br />
<p><br />Riboswitches, on the other hand, are regulatory structures in the 5’-UTR of mRNA that undergo a conformational change in the presence of a specific ligand that binds to the aptamer domain of the structure <a href="#ref">[5]</a>. This conformational change can regulate the initiation of translation by sequestering the ribosome binding site of the mRNA sequence, making it unavailable for binding <a href="#ref">[5]</a>. Riboswitches have been shown to work in diverse bacterial species and many natural examples have been found for riboswitches that turn off translation in the presence of the target ligand as well <a href="#ref">[6,7]</a>. Riboswitches have also been well characterized, are dose-dependent and have been engineered to respond to non-natural ligands thus providing an orthogonal control system <a href="#ref">[8]</a>. These RNA-based devices, like the TALE proteins, are also modular and powerful tools for the control of gene expression.</p><br />
<br />
<p><br />The fusion of these two devices--placing the TAL repressors under the control of riboswitches--offers a means by which to control the expression of any gene of interest, using wider variety of inducers than has been common to date. As our understanding of riboswitches and our ability to engineer aptamer binding domains develop it will be possible to develop a fully orthogonal, highly versatile systems for control of gene expression. There is a potential for multiplexing, as riboswitches designed to respond to different molecules and fused to different TAL repressors can be used in parallel within a single chassis, or can be induced in a temporally sequential manner for applications such as developmental research. We have demonstrated that RiboTALes function as synthetic transcription factors that are orthogonal to the natural biochemistry of the cell and increase the degrees of freedom available to us in the control of genetic circuits.</p><br />
</div><br />
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<div class="floatbox2"><br />
<h2>TALE binding to DNA <a href="#ref">[1]</a></h2><br />
<img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" width=195 height=195 /><br />
<h2>Riboswitch with inducer bound <a href="#ref">[9]</a></h2><br />
<img src="https://static.igem.org/mediawiki/2013/7/7e/Riboswitchpic_UCDavis.jpg" width=195 height=195 /><br />
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<table class="showbox"><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project"><img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project"><h3>Project Background</h3></a><br />
<p>Learn about how we combine riboswitches and TAL's into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br />
</p><br />
</td><br />
<br />
<br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><img src="https://static.igem.org/mediawiki/igem.org/0/00/UCD_Outreach_2013.png" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we designed a new database for better raw data characterization of Biobricks. <br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
</p><br />
</td><br />
<br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="ref"><h3>References</h3></a><br />
<p><br />
[1] D. Dong, Y. Chuangye, P. Xiaojing, M. Mahfouz, W. Jiawei, Z. Jian-Kang, et al., "Structural Basis for Sequence-Specific Recognition of DNA by TAL Effectors," Science, vol. 335, pp. 720-723, 10 2012.<br />
<br /><br />
[2] J. F. Meckler, M. S. Bhakta, M. S. Kim, R. Ovadia, C. H. Habrian, A. Zykovich, et al., "Quantitative analysis of TALE-DNA interactions suggests polarity effects," Nucleic Acids Res, vol. 41, pp. 4118-28, Apr 2013.<br />
<br /><br />
[3] J. Boch, H. Scholze, S. Schornack, A. Landgraf, S. Hahn, S. Kay, et al., "Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors," Science, vol. 326, pp. 1509-1512, Dec 2009.<br />
<br /><br />
[4] M. J. Moscou and A. J. Bogdanove, "A Simple Cipher Governs DNA Recognition by TAL Effectors," Science, vol. 326, pp. 1501-1501, Dec 11 2009.<br />
<br /><br />
[5] M. P. Caron, L. Bastet, A. Lussier, M. Simoneau-Roy, E. Masse, and D. A. Lafontaine, "Dual-acting riboswitch control of translation initiation and mRNA decay," Proceedings of the National Academy of Sciences of the United States of America, vol. 109, pp. E3444-E3453, Dec 2012.<br />
<br /><br />
[6] S. Topp, C. M. K. Reynoso, J. C. Seeliger, I. S. Goldlust, S. K. Desai, D. Murat, et al., "Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species (vol 76, pg 7881, 2010)," Applied and Environmental Microbiology, vol. 77, pp. 2199-2199, Mar 2011.<br />
<br /><br />
[7] N. Muranaka, K. Abe, and Y. Yokobayashi, "Mechanism-Guided Library Design and Dual Genetic Selection of Synthetic OFF Riboswitches," Chembiochem, vol. 10, pp. 2375-2381, Sep 2009.<br />
<br /><br />
[8] N. Dixon, J. N. Duncan, T. Geerlings, M. S. Dunstan, J. E. G. McCarthy, D. Leys, et al., "Reengineering orthogonally selective riboswitches," Proceedings of the National Academy of Sciences of the United States of America, vol. 107, pp. 2830-2835, Feb 16 2010.<br />
<br /><br />
[9] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.<br />
</p><br />
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Lemur3 93
http://2013.igem.org/File:UC_Davis_contactbanner.jpg
File:UC Davis contactbanner.jpg
2013-09-27T21:15:31Z
<p>Lemur3 93: uploaded a new version of &quot;File:UC Davis contactbanner.jpg&quot;</p>
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Lemur3 93
http://2013.igem.org/File:UC_Davis_contactbanner.jpg
File:UC Davis contactbanner.jpg
2013-09-27T21:15:00Z
<p>Lemur3 93: uploaded a new version of &quot;File:UC Davis contactbanner.jpg&quot;: Reverted to version as of 21:12, 27 September 2013</p>
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Lemur3 93
http://2013.igem.org/File:UC_Davis_contactbanner.jpg
File:UC Davis contactbanner.jpg
2013-09-27T21:13:34Z
<p>Lemur3 93: uploaded a new version of &quot;File:UC Davis contactbanner.jpg&quot;</p>
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Lemur3 93
http://2013.igem.org/File:UC_Davis_contactbanner.jpg
File:UC Davis contactbanner.jpg
2013-09-27T21:12:05Z
<p>Lemur3 93: uploaded a new version of &quot;File:UC Davis contactbanner.jpg&quot;</p>
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Lemur3 93
http://2013.igem.org/File:Teambiobanner_UCDavis.jpg
File:Teambiobanner UCDavis.jpg
2013-09-27T20:27:30Z
<p>Lemur3 93: uploaded a new version of &quot;File:Teambiobanner UCDavis.jpg&quot;</p>
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Lemur3 93
http://2013.igem.org/File:Teambanner_UCDavis.jpg
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2013-09-27T20:12:42Z
<p>Lemur3 93: uploaded a new version of &quot;File:Teambanner UCDavis.jpg&quot;</p>
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Lemur3 93
http://2013.igem.org/Team:UC_Davis/Database
Team:UC Davis/Database
2013-09-27T19:07:11Z
<p>Lemur3 93: </p>
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<div class="floatboxwide"><br />
<br />
The BioBrick Characterization Data Depot (the Depot) is a way for teams to share their characterization data with the world. It uses a <a href="https://2013.igem.org/Team:UC_Davis/Database/Standards">semi-standardized</a> database designed to be both flexible and structured. Originally intended to share the multivariate characterization data of riboTALs, the Depot was repurposed for sharing characterization data of all part types.<br />
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<a href="http://dilbert.cs.ucdavis.edu/Depot/"><img src="https://static.igem.org/mediawiki/2013/4/4e/Depotbutton_UCDavis.jpg" class="blur"></a><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot/"><h1 id="randcss5">Visit <br> the <br> Depot</h1></a><br />
</td><br />
</tr><br />
</table><br />
<h2>Links<h2><br />
<ul><br />
<li><a href="#motivation">Motivation</a></li><br />
<li><a href="#approaches">Approaches</a></li><br />
<li><a href="#example_upload">Example Upload</a></li><br />
<li><a href="#example_search">Example Search</a></li><br />
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<a id="motivation"><br />
<h1 class="title">Motivation: Empowerment and Openness</h1><br />
</a><br />
<p><br />
The from the iGEM DNA distribution kit and registry, to the eagerness of faculty and universities across the globe, the iGEM competition empowers students to make a raw idea into a tangible, presentable project. This fact is made clear depth by the number and depth of projects in past years.<a href="#external_links"><sup>1,2</sup></a><br />
</p><br />
<p><br />
However, as BioBrick circuits become more complex, their design process can be dramatically slowed by the large number of relationships and dependencies between their individual parts. Empowering iGEM-ers with an standardized, accessible database for the characterization of BioBrick parts is the clear solution to address problems in high complexity circuits.<a href="#ref">[1]</a> <br />
</p><br />
<p><br />
In addition to empowering iGEM-ers, a uploading data to a standardized characterization database promotes openness. In other biological applications, such as genomic sequencing<a href="#external_links"><sup>3</sup></a> and protein structuring<a href="#external_links"><sup>4</sup></a>, open data is the norm for strong reasons; open data allows communities to validate successes and understand failures, while supporting future researchers downstream. For these reasons, iGEM is founded upon an open community. Open data through the Depot is another way for iGEM-ers to contribute to an open community. <br />
</p><br />
<br />
<a id="approaches"><br />
<h1 class="title">Approaches: Past, and the Depot</h1><br />
</a><br />
<p><br />
Past approaches to designing a BioBrick characterization database dealt with difficult questions<a href="#external_links"><sup>5</sup></a> dealing with two main topics: what are reasonable standards that a database can enforce, and how to make characterizations across labs or experiments comparable. To answer these questions, one would have to make many, many decisions. Decisions like choosing between the use of different characterization protocols, lab equipment, machine settings, data format, and on and on for each part type. Additionally, these decisions are posed in a global scale, where accessibility and timetables are major concerns. More so, because synthetic biology is a relatively young field, many facets of part characterizations are likely to change. The complexity of decisions and synthetic biology's youth make shelving the idea of a completely standardized database very appealing. <br />
</p><br />
<p><br />
We agreed, at least in some senses. An enforced, strictly standardized database for all part types is unlike our approach we took for the Depot. In our approach, we instead created data and protocol standards for the promoters, a simple part that also have a history<a href="#external_links"><sup>6</sup></a> of data standardization; for the other part types, we created a flexible data format to accommodate different methods of characterization and data types that teams could possibly use. <br />
<p><br />
totally unpolished: For our new part part RiboTal, we wanted to see how it functions before making data standards for it. Go see how we went about answering some data standards issues here: link.<br />
</p><br />
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<a id="example_upload"><br />
<h1 class="title">Example Upload</h1><br />
</a><br />
<p><br />
The process is straightforward. Users register, then upload their four files that adhere to Depot <a href="https://2013.igem.org/Team:UC_Davis/Database/Standards">standards</a>, as well as other relevant information to their experiment. A prototype page that shows the basic functional elements is shown here. <p><img src="https://static.igem.org/mediawiki/2013/4/46/UCD_depot_upload.png" class="genpic"></p><br />
</p><br />
<br />
<a id="example_search"><br />
<h1 class="title">Example Search</h1><br />
</a><br />
<p><br />
</p><br />
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<a href="http://dilbert.cs.ucdavis.edu/Depot/"><img src="https://static.igem.org/mediawiki/2013/4/4e/Depotbutton_UCDavis.jpg" class="blur"></a><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot/"><h3>Visit the Depot</h3></a><br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Database/Standards"><img src="https://static.igem.org/mediawiki/2013/c/c6/UCD_2013_BCDD_Icon.PNG" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Database/Standards"><h3>Data Standards</h3></a><br />
<p>See the Depot's standards and data requirements so you can share your data as well.<br />
</p><br />
</td> <br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Database/Initial_Population"><br />
<img src="https://static.igem.org/mediawiki/2013/6/65/Depotpopbutton_UCDavis.jpg" class="blur /><br />
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<a href="https://2013.igem.org/Team:UC_Davis/Database/Initial_Population"><h3>Initial Population</h3></a><br />
<p>See how we initially populated the Depot.<br />
</p><br />
</td><br />
<td><br />
</td><br />
</tr><br />
</tbody></table><br />
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<div class="floatbox"><br />
<a id="ref"><h3>References</h3></a><br />
<p><br />
[1] M Galdzicki, D Chandran, JH Gennari, HM Sauro, "Design and Analysis of Bio-molecular Circuits", pp. 281, 2011<br />
</p><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="external_links"><h3>External Links</h3></a><br />
<p><br />
<sup>1 </sup><a href="https://2011.igem.org/Jamborees">https://2011.igem.org/Jamborees</a><br><br />
<sup>2 </sup><a href="https://2012.igem.org/Jamborees">https://2012.igem.org/Jamborees</a><br><br />
<sup>3 </sup><a href="http://www.ncbi.nlm.nih.gov/genbank">http://www.ncbi.nlm.nih.gov/genbank</a><br><br />
<sup>4 </sup><a href="http://www.pdb.org/pdb/home/home.do">http://www.pdb.org/pdb/home/home.do</a><br><br />
<sup>5 </sup><a href="http://openwetware.org/wiki/Parts_characterization#Discussion_Topics">http://openwetware.org/wiki/Parts_characterization#Discussion_Topics</a><br><br />
<sup>6 </sup><a href="http://www.jbioleng.org/content/3/1/4">http://www.jbioleng.org/content/3/1/4</a><br />
<br />
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Lemur3 93
http://2013.igem.org/Team:UC_Davis/Database
Team:UC Davis/Database
2013-09-27T19:05:38Z
<p>Lemur3 93: </p>
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<div class="floatboxwide"><br />
<br />
The BioBrick Characterization Data Depot (the Depot) is a way for teams to share their characterization data with the world. It uses a <a href="https://2013.igem.org/Team:UC_Davis/Database/Standards">semi-standardized</a> database designed to be both flexible and structured. Originally intended to share the multivariate characterization data of riboTALs, the Depot was repurposed for sharing characterization data of all part types.<br />
</div><br />
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<tbody><br />
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<a href="http://dilbert.cs.ucdavis.edu/Depot/"><img src="https://static.igem.org/mediawiki/2013/4/4e/Depotbutton_UCDavis.jpg" class="blur"></a><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot/"><h1 id="randcss5">Visit <br> the <br> Depot</h1></a><br />
</td><br />
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</table><br />
<h2>Links<h2><br />
<ul><br />
<li><a href="#motivation">Motivation</a></li><br />
<li><a href="#approaches">Approaches</a></li><br />
<li><a href="#example_upload">Example Upload</a></li><br />
<li><a href="#example_search">Example Search</a></li><br />
</ul><br />
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<br />
<div class="floatbox"><br />
<a id="motivation"><br />
<h1 class="title">Motivation: Empowerment and Openness</h1><br />
</a><br />
<p><br />
The from the iGEM DNA distribution kit and registry, to the eagerness of faculty and universities across the globe, the iGEM competition empowers students to make a raw idea into a tangible, presentable project. This fact is made clear depth by the number and depth of projects in past years.<a href="#external_links"><sup>1,2</sup></a><br />
</p><br />
<p><br />
However, as BioBrick circuits become more complex, their design process can be dramatically slowed by the large number of relationships and dependencies between their individual parts. Empowering iGEM-ers with an standardized, accessible database for the characterization of BioBrick parts is the clear solution to address problems in high complexity circuits.<a href="#ref">[1]</a> <br />
</p><br />
<p><br />
In addition to empowering iGEM-ers, a uploading data to a standardized characterization database promotes openness. In other biological applications, such as genomic sequencing<a href="#external_links"><sup>3</sup></a> and protein structuring<a href="#external_links"><sup>4</sup></a>, open data is the norm for strong reasons; open data allows communities to validate successes and understand failures, while supporting future researchers downstream. For these reasons, iGEM is founded upon an open community. Open data through the Depot is another way for iGEM-ers to contribute to an open community. <br />
</p><br />
<br />
<a id="approaches"><br />
<h1 class="title">Approaches: Past, and the Depot</h1><br />
</a><br />
<p><br />
Past approaches to designing a BioBrick characterization database dealt with difficult questions<a href="#external_links"><sup>5</sup></a> dealing with two main topics: what are reasonable standards that a database can enforce, and how to make characterizations across labs or experiments comparable. To answer these questions, one would have to make many, many decisions. Decisions like choosing between the use of different characterization protocols, lab equipment, machine settings, data format, and on and on for each part type. Additionally, these decisions are posed in a global scale, where accessibility and timetables are major concerns. More so, because synthetic biology is a relatively young field, many facets of part characterizations are likely to change. The complexity of decisions and synthetic biology's youth make shelving the idea of a completely standardized database very appealing. <br />
</p><br />
<p><br />
We agreed, at least in some senses. An enforced, strictly standardized database for all part types is unlike our approach we took for the Depot. In our approach, we instead created data and protocol standards for the promoters, a simple part that also have a history<a href="#external_links"><sup>6</sup></a> of data standardization; for the other part types, we created a flexible data format to accommodate different methods of characterization and data types that teams could possibly use. <br />
<p><br />
totally unpolished: For our new part part RiboTal, we wanted to see how it functions before making data standards for it. Go see how we went about answering some data standards issues here: link.<br />
</p><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="example_upload"><br />
<h1 class="title">Example Upload</h1><br />
</a><br />
<p><br />
The process is straightforward. Users register, then upload their four files that adhere to Depot <a href="https://2013.igem.org/Team:UC_Davis/Database/Standards">standards</a>, as well as other relevant information to their experiment. A prototype page that shows the basic functional elements is shown here. <p><img src="https://static.igem.org/mediawiki/2013/4/46/UCD_depot_upload.png"></p><br />
</p><br />
<br />
<a id="example_search"><br />
<h1 class="title">Example Search</h1><br />
</a><br />
<p><br />
</p><br />
</div><br />
<br />
<div class="floatbox"><br />
<table class="showbox"><br />
<tbody><br />
<tr><br />
<td><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot/"><img src="https://static.igem.org/mediawiki/2013/4/4e/Depotbutton_UCDavis.jpg" class="blur"></a><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot/"><h3>Visit the Depot</h3></a><br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Database/Standards"><img src="https://static.igem.org/mediawiki/2013/c/c6/UCD_2013_BCDD_Icon.PNG" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Database/Standards"><h3>Data Standards</h3></a><br />
<p>See the Depot's standards and data requirements so you can share your data as well.<br />
</p><br />
</td> <br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Database/Initial_Population"><br />
<img src="https://static.igem.org/mediawiki/2013/6/65/Depotpopbutton_UCDavis.jpg" class="blur /><br />
<!--img src="https://static.igem.org/mediawiki/2013/6/63/UCD_2013_BCDD_Icon2.PNG" class="blur"--></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Database/Initial_Population"><h3>Initial Population</h3></a><br />
<p>See how we initially populated the Depot.<br />
</p><br />
</td><br />
<td><br />
</td><br />
</tr><br />
</tbody></table><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="ref"><h3>References</h3></a><br />
<p><br />
[1] M Galdzicki, D Chandran, JH Gennari, HM Sauro, "Design and Analysis of Bio-molecular Circuits", pp. 281, 2011<br />
</p><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="external_links"><h3>External Links</h3></a><br />
<p><br />
<sup>1 </sup><a href="https://2011.igem.org/Jamborees">https://2011.igem.org/Jamborees</a><br><br />
<sup>2 </sup><a href="https://2012.igem.org/Jamborees">https://2012.igem.org/Jamborees</a><br><br />
<sup>3 </sup><a href="http://www.ncbi.nlm.nih.gov/genbank">http://www.ncbi.nlm.nih.gov/genbank</a><br><br />
<sup>4 </sup><a href="http://www.pdb.org/pdb/home/home.do">http://www.pdb.org/pdb/home/home.do</a><br><br />
<sup>5 </sup><a href="http://openwetware.org/wiki/Parts_characterization#Discussion_Topics">http://openwetware.org/wiki/Parts_characterization#Discussion_Topics</a><br><br />
<sup>6 </sup><a href="http://www.jbioleng.org/content/3/1/4">http://www.jbioleng.org/content/3/1/4</a><br />
<br />
</p><br />
</div><br />
<div id="sitemapbox" class="floatbox"><br />
</div><br />
<br />
</body><br />
<br />
<script type="text/javascript"><br />
$("#sitemapbox").load("https://2013.igem.org/Template:Team:UC_Davis/site_map #sitemap1");<br />
</script><br />
</html></div>
Lemur3 93
http://2013.igem.org/Team:UC_Davis/Database
Team:UC Davis/Database
2013-09-27T19:05:21Z
<p>Lemur3 93: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
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#randcss5 {<br />
width:62px;<br />
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<body><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot/"><img src="https://static.igem.org/mediawiki/2013/9/95/UCD_2013_BCDDv2_Banner.jpg" class="banner" width=967px height=226/></a><br />
<div class="floatboxwide"><br />
<br />
The BioBrick Characterization Data Depot (the Depot) is a way for teams to share their characterization data with the world. It uses a <a href="https://2013.igem.org/Team:UC_Davis/Database/Standards">semi-standardized</a> database designed to be both flexible and structured. Originally intended to share the multivariate characterization data of riboTALs, the Depot was repurposed for sharing characterization data of all part types.<br />
</div><br />
<br />
<div class="floatbox2"> <br />
<table class="showbox"><br />
<tbody><br />
<tr><br />
<td><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot/"><img src="https://static.igem.org/mediawiki/2013/4/4e/Depotbutton_UCDavis.jpg" class="blur"></a><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot/"><h1 id="randcss5">Visit <br> the <br> Depot</h1></a><br />
</td><br />
</tr><br />
</table><br />
<h2>Links<h2><br />
<ul><br />
<li><a href="#motivation">Motivation</a></li><br />
<li><a href="#approaches">Approaches</a></li><br />
<li><a href="#example_upload">Example Upload</a></li><br />
<li><a href="#example_search">Example Search</a></li><br />
</ul><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="motivation"><br />
<h1 class="title">Motivation: Empowerment and Openness</h1><br />
</a><br />
<p><br />
The from the iGEM DNA distribution kit and registry, to the eagerness of faculty and universities across the globe, the iGEM competition empowers students to make a raw idea into a tangible, presentable project. This fact is made clear depth by the number and depth of projects in past years.<a href="#external_links"><sup>1,2</sup></a><br />
</p><br />
<p><br />
However, as BioBrick circuits become more complex, their design process can be dramatically slowed by the large number of relationships and dependencies between their individual parts. Empowering iGEM-ers with an standardized, accessible database for the characterization of BioBrick parts is the clear solution to address problems in high complexity circuits.<a href="#ref">[1]</a> <br />
</p><br />
<p><br />
In addition to empowering iGEM-ers, a uploading data to a standardized characterization database promotes openness. In other biological applications, such as genomic sequencing<a href="#external_links"><sup>3</sup></a> and protein structuring<a href="#external_links"><sup>4</sup></a>, open data is the norm for strong reasons; open data allows communities to validate successes and understand failures, while supporting future researchers downstream. For these reasons, iGEM is founded upon an open community. Open data through the Depot is another way for iGEM-ers to contribute to an open community. <br />
</p><br />
<br />
<a id="approaches"><br />
<h1 class="title">Approaches: Past, and the Depot</h1><br />
</a><br />
<p><br />
Past approaches to designing a BioBrick characterization database dealt with difficult questions<a href="#external_links"><sup>5</sup></a> dealing with two main topics: what are reasonable standards that a database can enforce, and how to make characterizations across labs or experiments comparable. To answer these questions, one would have to make many, many decisions. Decisions like choosing between the use of different characterization protocols, lab equipment, machine settings, data format, and on and on for each part type. Additionally, these decisions are posed in a global scale, where accessibility and timetables are major concerns. More so, because synthetic biology is a relatively young field, many facets of part characterizations are likely to change. The complexity of decisions and synthetic biology's youth make shelving the idea of a completely standardized database very appealing. <br />
</p><br />
<p><br />
We agreed, at least in some senses. An enforced, strictly standardized database for all part types is unlike our approach we took for the Depot. In our approach, we instead created data and protocol standards for the promoters, a simple part that also have a history<a href="#external_links"><sup>6</sup></a> of data standardization; for the other part types, we created a flexible data format to accommodate different methods of characterization and data types that teams could possibly use. <br />
<p><br />
totally unpolished: For our new part part RiboTal, we wanted to see how it functions before making data standards for it. Go see how we went about answering some data standards issues here: link.<br />
</p><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="example_upload"><br />
<h1 class="title">Example Upload</h1><br />
</a><br />
<p><br />
The process is straightforward. Users register, then upload their four files that adhere to Depot <a href="https://2013.igem.org/Team:UC_Davis/Database/Standards">standards</a>, as well as other relevant information to their experiment. A prototype page that shows the basic functional elements is shown here. <p><img src="https://static.igem.org/mediawiki/2013/4/46/UCD_depot_upload.png"></p><br />
</p><br />
<br />
<a id="example_search"><br />
<h1 class="title">Example Search</h1><br />
</a><br />
<p><br />
</p><br />
</div><br />
<br />
<div class="floatbox"><br />
<table class="showbox"><br />
<tbody><br />
<tr><br />
<td><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot/"><img src="https://static.igem.org/mediawiki/2013/4/4e/Depotbutton_UCDavis.jpg" class="blur"></a><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot/"><h3>Visit the Depot</h3></a><br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Database/Standards"><img src="https://static.igem.org/mediawiki/2013/c/c6/UCD_2013_BCDD_Icon.PNG" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Database/Standards"><h3>Data Standards</h3></a><br />
<p>See the Depot's standards and data requirements so you can share your data as well.<br />
</p><br />
</td> <br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Database/Initial_Population"><br />
<img src="https://static.igem.org/mediawiki/2013/6/65/Depotpopbutton_UCDavis.jpg" class="blur /><br />
<!--img src="https://static.igem.org/mediawiki/2013/6/63/UCD_2013_BCDD_Icon2.PNG" class="blur"--></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Database/Initial_Population"><h3>Initial Population</h3></a><br />
<p>See how we initially populated the Depot.<br />
</p><br />
</td><br />
<td><br />
</td><br />
</tr><br />
</tbody></table><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="ref"><h3>References</h3></a><br />
<p><br />
[1] M Galdzicki, D Chandran, JH Gennari, HM Sauro, "Design and Analysis of Bio-molecular Circuits", pp. 281, 2011<br />
</p><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="external_links"><h3>External Links</h3></a><br />
<p><br />
<sup>1 </sup><a href="https://2011.igem.org/Jamborees">https://2011.igem.org/Jamborees</a><br><br />
<sup>2 </sup><a href="https://2012.igem.org/Jamborees">https://2012.igem.org/Jamborees</a><br><br />
<sup>3 </sup><a href="http://www.ncbi.nlm.nih.gov/genbank">http://www.ncbi.nlm.nih.gov/genbank</a><br><br />
<sup>4 </sup><a href="http://www.pdb.org/pdb/home/home.do">http://www.pdb.org/pdb/home/home.do</a><br><br />
<sup>5 </sup><a href="http://openwetware.org/wiki/Parts_characterization#Discussion_Topics">http://openwetware.org/wiki/Parts_characterization#Discussion_Topics</a><br><br />
<sup>6 </sup><a href="http://www.jbioleng.org/content/3/1/4">http://www.jbioleng.org/content/3/1/4</a><br />
<br />
</p><br />
</div><br />
<div id="sitemapbox" class="floatbox"><br />
</div><br />
<br />
</body><br />
<br />
<script type="text/javascript"><br />
$("#sitemapbox").load("https://2013.igem.org/Template:Team:UC_Davis/site_map #sitemap1");<br />
</script><br />
</html></div>
Lemur3 93