Difference between revisions of "Team:UNITN-Trento/Test"

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<h2>UNITN iGEM 2015</h2>
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<div style="clear:both; width:95%; margin:auto;">
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<div style="background-image:url('https://static.igem.org/mediawiki/2015/d/df/Unitn_pics_main_logo_200.png'); width:170px; height:170px; float:left; background-size:cover;"></div>
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<p>Electron-production with <br /> engineered <i>E. coli</i> and <br /> <strong>proteorhodopsin</strong></p>
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<h2><strong>Work hard, Work safe</strong></h2>
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<h2>Electron production with <strong><i>E. coli</i></strong> and proteorhodopsin </h2>
<p> ? </p>
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<br />
</header>
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<p>The aim of our project is to develop a pseudoautotrophic <span class="italicEnph">E. coli</span>, enabling it to live in the microbial fuel cell anaerobic condition. Our idea is to engineer E. coli with proteorhodopsin, a light-powered proton pump identified in bacteria that live in deep waters. This protein is activated by light and needs retinal molecules to pump protons outward following excitation. Thus we want to engineer our bacteria with blh, that encodes for the enzyme that cleaves beta-carotene and produces retinal.</p>
</section>
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<p>To increase electron production we plan to overexpress pncB that represents the limiting step in the NAD+ synthesis pathway. The more NAD+ is produced, the more electrons are transported by NADH. In short: light activates proteorhodopsin, increases bacterial viability, and drives to a more efficient electron production. Our bacterial device will give rise to a self-sustainable system, powered by sunlight during the day and by a light bulb at night. Daytime-electricity can be either used or stored to be employed as a power source for the lightbulb at night.</p>
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<li style="width: 25%; background-image: url('https://static.igem.org/mediawiki/2015/8/8c/Unitn_pics_slider_1.jpg'); ">
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<p class="pintest">Electricity from E. coli<br/>
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<span>Check out our amazing idea! Have fun!<br/>
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<a class="btn" href="#">Project</a></span></p>
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</li>
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<li style="width: 25%; background-image: url('https://static.igem.org/mediawiki/2015/a/a0/Unitn_pics_slider_2.jpg'); ">
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<p class="pintest">Electricity from E. coli<br/>
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<span>Check out our amazing idea! Have fun!<br/>
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<a class="btn" href="#">Project</a></span></p>
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</li>
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<li style="width: 25%; background-image: url('https://static.igem.org/mediawiki/2015/f/fc/Unitn_pics_slider_3.jpg'); ">
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<p class="pintest">Electricity from E. coli<br/>
 +
<span>Check out our amazing idea! Have fun!<br/>
 +
<a class="btn" href="#">Project</a></span></p>
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</li>
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<li style="width: 25%; background-image: url('https://static.igem.org/mediawiki/2015/a/a7/Unitn_pics_slider_4.jpg'); ">
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<p class="pintest">Meet the team!<br/>
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<span>We are UNITN iGEM 2015 and we are such essence as dNTPs are made on<br/>
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<a class="btn" href="#"> To->Team </a></span></p>
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<li style="width: 25%; background-image: url('https://static.igem.org/mediawiki/2015/0/04/Unitn_pics_slider_5.jpg'); ">
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<p class="pintest">Catch 'em all!<br/>
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<span>Many techniques, different strains and so much fluorescence… Enjoy the Measurement Interlab Study<br/>
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<a class="btn" href="#"> To->Interlab </a></span></p>
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<li style="width: 25%; background-image: url('https://static.igem.org/mediawiki/2015/5/58/Unitn_pics_slider_6.jpg'); ">
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<p class="pintest">Synthetic Biology and mountains<br/>
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<span>Admire our lovely city, surrounded by mountains. Trento is waiting for you<br/>
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<a class="btn" href="#"> To->Interlab </a></span></p>
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<ol class="dots"><li class="dot active">1</li><li class="dot">2</li><li class="dot">3</li><li class="dot">4</li><li class="dot">4</li><li class="dot">4</li></ol></div>
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<h2>Achievements</h2>
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<h4># new BioBricks</h4>
<p>We have reviewed our organisms and parts against the <a class="i_linker" href="https://2015.igem.org/Safety/WhiteList" target="_blank">White List</a>. All the parts and bacterial strains that we used are in the <span class="i_enph">Risk Group 1</span></p>
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<p>We built and submitted a total number of new BioBricks and characterized # of them. Check out our [Parts] page to know more!</p>
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<h4>Improved # Parts</h4>
</header>
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<p>Something existing, reshaped better. # now works!</p>
<p>We have submitted to iGEM headquarters <span class="i_enph">“About Our Lab”</span> and <span class="i_enph">“About Our Project”</span> questionnaires by the June 26<sup>th</sup> deadline</p>
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<h4>Measurements Interlab Study</h4>
</header>
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<p>We are participating also to the Measurements Interlab Study. Check out our progress in the Interlab Section!</p>
<p>We have submitted to iGEM headquarters the <span class="i_enph">“Final Safety Form”</span> by the August 28<sup>th</sup> deadline</p>
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<h3>About Safety</h3>
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<p>We, the UNITN iGEM Team want to follow high standards of safe and responsible biological engineering. Before entering the lab we attended mandatory on-line courses regarding lab safety with final tests. In particular we focused on the following topics:</p>
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<ul class="checklist">
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<li>General considerations on risks and prevention</li>
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<li>Electrical Hazard</li>
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<li>Chemical Hazard</li>
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<li>Biological Hazard</li>
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<li>Compressed gasses and cryogenic liquids</li>
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</ul>
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<p>We also attended the “Health and safety at the workplace” course, which is mandatory for all students and workers at the University of Trento.</p>
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<h3>Safe Project Design</h3>
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<p class="question">Who will use your product? What opinions do these people have about your project?</p>
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<p class="question">Where will your product be used? On a farm, in a factory, inside human bodies, in the ocean?</p>
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<p class="question">If your product is successful, who will receive benefits and who will be harmed?</p>
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<p class="question">What happens when it's all used up? Will it be sterilized, discarded, or recycled?</p>
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<p class="question">Is it safer, cheaper, or better than other technologies that do the same thing?</p>
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<p>Answer</p>
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<h3>Working Safely... with Parts</h3>
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<p>All the parts we used throughout the project are NOT toxic or dangerous for  humans or the environment. We neither worked with proteins toxic themselves, nor we used enzymes that synthesize toxic molecules. In addition to basic parts from the registry (i.e. RBS, promoters, β-carotene, etc), we also used two genes from the SAR86 uncultured bacteria. These genes are proteorhodopsin, which was extracted from the Registry (BBa_K773002) and blh (15,15’-β-carotene dioxygenase), which was synthesized by Genescript. There is no associated risk level with the uncultured bacteria SAR86, however we are using only DNA sequences that encodes for genes, which carry distinct functions that are no harmful to humans, or the environment. We have clarified this issue with iGEM Safety Office.</p>
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<p>We did <span class="i_enph">NOT</span> use any virulence factors in our organisms.</p>
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<p>The parts we used are to be considered safe as single parts and also as combined. Environmental dispersion of those parts would not represent a biological hazard or danger.</p>
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<header>
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<h3>Working Safely... with Organisms</h3>
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</header>
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<p>All the bacterial strains used are from Risk Group 1. We used the following strains:</p>
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<ul class="checklist">
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<li> One Shot® TOP10 Chemically Competent E. coli</li>
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<li> K12 - NEB 10-beta E. coli</li>
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<li> K12 - JM109 E. coli</li>
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<li> BL21 - NEB Express E. coli</li>
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<h3>Personal Protective Equipment (PPE)</h3>
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<p>Last but non least we adopted the appropriate PPE during the lab work.  We always used lab coats, nitrile gloves and goggles.  We safely operated with biological material under the laminar flow cabinet BioAir SAFEMATE Series Class II 1.8.</p>
+
<p>We used chemicals that are associated with hazard (ethidium bromide, azide,  organic solvents) under the chemical hood Fume Hood MOD. ASEM® 120EN New - Class 0.</p>
+
<p>We carefully revised all the Material Safety Datasheet of each compound that was  used prior to start an experiment.</p> 
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Revision as of 12:14, 10 August 2015

Electron production with E. coli and proteorhodopsin


The aim of our project is to develop a pseudoautotrophic E. coli, enabling it to live in the microbial fuel cell anaerobic condition. Our idea is to engineer E. coli with proteorhodopsin, a light-powered proton pump identified in bacteria that live in deep waters. This protein is activated by light and needs retinal molecules to pump protons outward following excitation. Thus we want to engineer our bacteria with blh, that encodes for the enzyme that cleaves beta-carotene and produces retinal.

To increase electron production we plan to overexpress pncB that represents the limiting step in the NAD+ synthesis pathway. The more NAD+ is produced, the more electrons are transported by NADH. In short: light activates proteorhodopsin, increases bacterial viability, and drives to a more efficient electron production. Our bacterial device will give rise to a self-sustainable system, powered by sunlight during the day and by a light bulb at night. Daytime-electricity can be either used or stored to be employed as a power source for the lightbulb at night.

Achievements

# new BioBricks

We built and submitted a total number of new BioBricks and characterized # of them. Check out our [Parts] page to know more!

Improved # Parts

Something existing, reshaped better. # now works!

Measurements Interlab Study

We are participating also to the Measurements Interlab Study. Check out our progress in the Interlab Section!