http://2011.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=20&target=Evanclark&year=&month=2011.igem.org - User contributions [en]2024-03-29T13:19:04ZFrom 2011.igem.orgMediaWiki 1.16.0http://2011.igem.org/Team:Brown-StanfordTeam:Brown-Stanford2012-01-11T19:40:02Z<p>Evanclark: </p>
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= '''APPLY TO JOIN US IN 2012''' =<br />
The Brown and Stanford iGEM teams are planning to continue our transcontinental collaboration in the upcoming year. If you are a student at either of our universities and are interested in participating, please follow the recruitment instructions for your location! <br />
<br />
Still confused about exactly what Synthetic Biology is? Not to worry, here's an awesome video explaining it! http://www.youtube.com/watch?v=rD5uNAMbDaQ&feature=youtu.be<br />
<br />
<br />
== '''For Stanford students''' ==<br />
Please fill out our interest form at https://docs.google.com/spreadsheet/viewform?formkey=dEQyaWdReTN5S2pCcHRVY29hQXh4cHc6MQ.<br />
<br />
Applications will be due Sunday, February 5th at 11:59 PM. Please fill out the application form [http://dl.dropbox.com/u/10558719/2012_iGEM_Application.doc here], and also attach your resume and (unofficial) transcript and email them to stanfordigem@gmail.com. <br />
<br />
We'll be holding some lab technique boot camps and brainstorming/planning sessions in the spring if you make the team, so expect that. If you're chosen, you should also take Dr. Drew Endy's BioE 44 Synthetic Biology Lab in the spring, which is, by the way, an awesome class. <br />
<br />
We will be sending further information to the list above!<br />
<br />
Thanks!<br />
<br />
== '''For Brown students''' ==<br />
(12/5/11)<br />
Brown iGEM is looking for undergrad team members for this upcoming year!<br />
<br />
Have you ever wanted to engineer living organisms to create novel functions and help transform the world? <br />
<br />
Spend a summer working at the Synthetic Biology Initiative at NASA Ames Research Center, and represent Brown at the premiere synthetic biology competition in the world! Opportunities exist for all kinds of microbiology and molecular biology work, computational biology, mathematical modeling, engineering, and much more. Develop incredible research and lab skills as you manage your own project!<br />
<br />
Students of all academic backgrounds and experience levels welcome (this includes Class of '12) <br />
<br />
We are hosting two '''INFO SESSIONS''' this week:<br />
<br />
'''TIME''': 6-7pm on Thursday 12/8, Friday 12/9<br />
<br />
'''LOCATION''': Barus and Holly 190<br />
<br />
-come hear more about iGEM, synthetic biology, and what the award-winning Brown iGEM team does; dinner will be provided!<br />
<br />
<br />
If you can't make it but are still interested, please let us know! Other questions or concerns are welcome as well. Contact us at igem@brown.edu<br />
<br />
<html><br />
<span style="font-size: 20px;"></html>The electronic application at Brown is [http://www.brownigem.com/2012_iGEM_Application.doc available now]. Deadline is 11:59pm on 1/31!<br />
<br />
<br />
Keep up with the 2012 application process on our [https://www.facebook.com/events/146221565480758/ facebook page]!<br />
<html></span></html><br />
<br />
= '''Mars BioTools: Synthetic Biology for Space Exploration''' =<br />
One of the major challenges of space exploration is the limited payload mass that can be launched on a rocket and the difficulty of resupply mid-mission. Any long term settlement will require more resources than astronauts can initially bring with them. Synthetic Biology has the potential to revolutionize space exploration and settlement. Biological tools have a major advantage over classical tools: the ability to self-replicate and regenerate.<br />
<br />
The emerging field of Synthetic Biology will allow us to engineer microbial factories that will largely circumvent the limiting payload factors. These cellular factories will generate fuel, food, medicines and building materials for settlers, but will consist of engineered cells that could be stored in a single test tube, and regrown to production scale on-site, as needed.<br />
<br />
The Brown-Stanford iGEM team is excited to work on three different projects, under the common theme of developing Synthetic Biology applications for space. In addition, as a response to the ethical and philosophical considerations, we interviewed prominent researchers and leaders in the field. '''Click on the astronaut in the corner to view our series.''' <br />
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<h3><img src="https://static.igem.org/mediawiki/2011/3/39/Brown-Stanford_REGObricks-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/REGObricks/Introduction">REGObricks</a></h3><br />
</html><br />
<br />
Any extra-terrestrial settlement will require a habitat to keep its occupants alive, but transport of such a habitat will require a huge amount of payload space. RegoBricks uses bacteria to cement Martian and Lunar regolith (soil) simulant into durable building blocks, similar to concrete bricks, allowing settlements to use in situ resources to build structures.<br />
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<h3><img src="https://static.igem.org/mediawiki/2011/4/4c/Brown-Stanford_DNADamage-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/FRETSensor/Introduction">FRETcetera</a></h3><br />
</html><br />
<br />
Biological interfaces cannot pass information to their users as fast as electronic ones can. FRETcetera develops a novel method of fast-acting biological reporting with changes in cell fluorescence. Bacteria could be used to detect toxic chemicals in the environment, for example, or inform astronauts that their microbial tools are unhealthy.<br />
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<h3><img src="https://static.igem.org/mediawiki/2011/9/9a/Brown-Stanford_PowerCell-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/PowerCell/Introduction">PowerCell</a></h3><br />
</html><br />
<br />
All biological tools need energy to run. PowerCell develops a universal energy source. Engineered photosynthetic bacteria generate carbon and nitrogenous nutrients from sunlight and air and secrete them to power biological tools. These tools will transform the raw materials into fuel, building materials, food, drugs, and other products useful to settlers.<br />
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<div class="pageContent"><br />
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= '''Summary''' =<br />
<br />
=== '''News!''' ===<br />
<br />
===== '''iGEM Awards''' =====<br />
<br />
We did very well at iGEM this year! <br />
<br />
-At the Americas Regional Jamboree, we finished Top 4 and won Best Presentation.<br />
<br />
-At the World Jamboree, we finished in the Sweet 16, and won Best New Application. <br />
<br />
We are extremely grateful to all those who helped us along the way, and our mentors. Thank you so much!<br />
<br />
===== '''Project Summary''' =====<br />
<br />
Here's a minute long teaser of our project!<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/watch?v=b2AkJRTLOcs"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/b2AkJRTLOcs"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Latest Advancements on Projects''' ===<br />
<br />
===== '''Anabaena Transformation and Successful Cell-Type Specific Expression Control''' =====<br />
We successfully transformed our cyanobacterial chassis, Anabaena PCC 7120, with our designed PowerCell genetic construct. Unlike more common cyanobacteria species such as Synechocystis PCC 6803 or Synechococcus elongatus PCC 7942, Anabaena is not naturally competent and does not easily uptake foreign DNA. We performed a triparental conjugation in order to bypass the natural barriers to transformation.<br />
<br />
To demonstrate that expression of our construct is properly co-localized to vegetative cells, we grew cultures of Anabaena in nitrate-free BG11 media to promote heterocyst formation. We demonstrated the successful control of our pSac promoter over our sugar secretion construct through GFP expression. The sugar secretion construct is only expressed in actively photosynthesizing cells. In the first picture, we observe areas of diminished GFP fluorescence at regular intervals (approximately 1 in 10 cells) along our transformed Anabaena filament (the background fluorescence is likely due to chlorophyll). Since our GFP reporter should only express under the pSac promoter and only in non-heterocyst cells, we would expect the spacing of dark spots to match the spacing of heterocysts identified through alcian blue staining. Alcian blue is a dye that selectively binds to polysaccharide chains on the surface of heterocyst cells. As you can see by comparing the two images, our results appear to match up!<br />
<br />
Work to characterize the performance of our cscB sucrose secretion construct is underway. We will assay sucrose levels produced by PowerCell. If they are high enough, we will attempt to grow E. coli W. containing arbitrary biobricks from this nutrient source, in an attempt to show PowerCell can power biological tools.<br />
<br />
<center><br />
<html><object width="400" height="300"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to="></param> <param name="movie" value="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to=" width="400" height="300"></embed></object></html><br />
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<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
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<html><object width="400" height="300"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to="></param> <param name="movie" value="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to=" width="400" height="300"></embed></object></html><br />
<br />
<div style="width: 35%;"><br />
Transformed Anabaena with heterocysts stained by alcian blue. Heterocyst spacing corresponds to the "off" cells from the GFP image, implying that the sucrose secretion construct in not active in heterocysts, as desired.<br />
</div><br />
</center><br />
===== '''Biobricked the urease operon''' =====<br />
<br />
We successfully PCR cloned a 10.7 kb functional urease cassette from the plasmid pBU11, whose genetic sequence has hitherto never been known before. We ligated it onto a backbone, and successfully [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biobrick made a standard BioBrick], characterizing [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Characterization its function] and demonstrating active urease activity. Urease is the catalyst behind the phenomenon known as biocement, which is able to fuse sand into bricks, using nothing more than calcium ions and urea. [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biocementation More about urease.]<br />
<br />
Biobrick part [http://partsregistry.org/Part:BBa_K656013 BBa_K656013] has been submitted to the Registry of Standardized Parts. The sequence of this cassette is unavailable because it currently does not exist in any known registry. At the time of this writing, it is currently being sequenced in the lab of Dr. Chris Mason at Weill Cornell Medical College. As soon as the sequence is available, we will supplement the directory listing.<br />
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<center><br />
<a href="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG"><img src="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%; font-weight:bold;"><br />
Successful transformants on urease test plates.<br />
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<br />
===== '''Stratospheric Balloon Flight''' =====<br />
<br />
We launched samples of our bacteria to the far edge of space (80,000 ft-110,000 ft) to test for their ability to survive simulated Martian environmental conditions. The pressures, temperatures, and radiation conditions at these altitudes are similar to the conditions on the Martian surface.<br />
[[Team:Brown-Stanford/REGObricks/Balloon|Read about our escapades here]]<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/qCQXg8DmZZ0"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/qCQXg8DmZZ0"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Advancements in Human Practices''' ===<br />
===== '''Feature on Synthetic Biology and Space''' =====<br />
We talked with key figures in science and science policy about the implications of human expansion into space, extraterrestrial life and synthetic biology. You can look at our interviews and findings [https://2011.igem.org/Team:Brown-Stanford/SynEthics/Intro here] or by clicking on the astronaut on the upper left!<br />
<br />
===== '''Education and Publicity''' =====<br />
<br />
<html><br />
<table style="width: 100%;"><br />
<tr><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Editor's Choice! x2<br />
</div><br />
</center><br />
</td><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Lunar Science Poster. Click to see full version.<br />
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</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/9YKsa7s8WM4?version=3"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/9YKsa7s8WM4?version=3"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
<br />
<br />
We educated and publicized the power of synthetic biology in a whole suite of venues and mediums, including <br />
<br />
* A crowd of 600+ over two days at [https://2011.igem.org/Team:Brown-Stanford/MakerFaire Maker Faire NYC], resulting in two Editor’s Choice blue ribbons! <br />
* A filming opportunity with the [https://2011.igem.org/Team:Brown-Stanford/BBC BBC] Horizon, to be featured in a documentary on NASA’s Initiative in Synthetic Biology<br />
*Verbal [https://2011.igem.org/Team:Brown-Stanford/NASA presentations] to NASA Administrator Charlie Bolden, NASA Ames Research Center Director Pete Worden, and writer and visionary Stewart Brand <br />
* A poster presentation at NASA’s Lunar Science Forum<br />
* A heart-pounding [https://2011.igem.org/Team:Brown-Stanford/SB5 poem] at the first-ever SB5.0 Synbio Slam<br />
<br />
===== '''Outreach and Collaboration''' =====<br />
<br />
<html><br />
<center><br />
<a href="http://openwetware.org/wiki/IGEM_Outreach"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6f/Brown-Stanford_Cbricks.jpg" style="width: 30%;"><br />
</a><br />
</center><br />
</html><br />
<br />
We spearheaded the '''largest collaborative effort''' in iGEM history to change the way we do outreach, creating [http://openwetware.org/wiki/IGEM_Outreach '''CommunityBricks'''], a database of valuable teaching plans, presentations, community resources and projects for young and old alike.<br />
<br />
===== '''Alumni and Community-Building''' =====<br />
<br />
<br />
[[File:Brown-Stanford alumnigem.png|300px|center|AlumniGEM|link=http://alumni.brownigem.com/]]<br />
<br />
We built the [http://alumni.brownigem.com '''first iGEM Alumni Network,'''] to provide iGEM'ers a growing collection of professional and social resources and keep the spirit of iGEM alive post-Jamborees.<br />
<br />
<html><br />
<div><a href="javascript:toggle();" id="toggleNews"><h3>Show Media and News</h3></a></div><br />
<br />
</div><br />
<div class="pageContent" id="news" style="display: none;"><br />
</html><br />
<br />
== '''Martian Colony''' ==<br />
[[File:Brown-Stanford_MartianColony.png|center|750px]]<br />
<br />
== '''News''' ==<br />
<br />
===In the Lab===<br />
<center><html><object width="600" height="450"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to="></param> <param name="movie" value="http://www.flickr.com/apps/slideshow/show.swf?v=107931"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://www.flickr.com/apps/slideshow/show.swf?v=107931" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to=" width="600" height="450"></embed></object></html></center><br />
<br />
=== '''August 4<sup>th</sup>, 2011''' ===<br />
<center><html><object width="580" height="355"><param name="movie" value="http://www.youtube.com/v/x-MYE4QkjOU"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/x-MYE4QkjOU" type="application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''June 7<sup>th</sup>, 2011''' ===<br />
[[File:Brown-Stanford TeamPicture6-7-2011.jpg|center|500px]]<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-StanfordTeam:Brown-Stanford2011-12-07T10:19:10Z<p>Evanclark: /* Latest Advancements on Projects */</p>
<hr />
<div>{{:Team:Brown-Stanford/Templates/Main}}<br />
{{:Team:Brown-Stanford/Templates/Content}}<br />
{{:Team:Brown-Stanford/Templates/Twitter}}<br />
<br />
= '''APPLYING FOR STANFORD iGEM 2012''' =<br />
<br />
Please fill out our interest form at https://docs.google.com/spreadsheet/viewform?formkey=dEQyaWdReTN5S2pCcHRVY29hQXh4cHc6MQ.<br />
<br />
We will be sending further information to this list!<br />
<br />
Thanks!<br />
<br />
= '''APPLYING FOR BROWN iGEM 2012''' =<br />
(12/5/11)<br />
Brown iGEM is looking for undergrad team members for this upcoming year!<br />
<br />
Have you ever wanted to engineer living organisms to create novel functions and help transform the world? <br />
<br />
Spend a summer working at the Synthetic Biology Initiative at NASA Ames Research Center, and represent Brown at the premiere synthetic biology competition in the world! Opportunities exist for all kinds of microbiology and molecular biology work, computational biology, mathematical modeling, engineering, and much more. Develop incredible research and lab skills as you manage your own project!<br />
<br />
Students of all academic backgrounds and experience levels welcome (this includes Class of '12) <br />
<br />
We are hosting two '''INFO SESSIONS''' this week:<br />
<br />
'''TIME''': 6-7pm on Thursday 12/8, Friday 12/9<br />
<br />
'''LOCATION''': Barus and Holly 190<br />
<br />
-come hear more about iGEM, synthetic biology, and what the award-winning Brown iGEM team does; dinner will be provided!<br />
<br />
<br />
If you can't make it but are still interested, please let us know! Other questions or concerns are welcome as well. Contact us at igem@brown.edu<br />
<br />
<html><br />
<span style="font-size: 20px;"></html>Our electronic application is [http://www.brownigem.com/2012_iGEM_Application.doc available now]. Keep up with the 2012 application process on our [https://www.facebook.com/events/146221565480758/ facebook page]!<br />
<html></span></html><br />
<br />
= '''Mars BioTools: Synthetic Biology for Space Exploration''' =<br />
One of the major challenges of space exploration is the limited payload mass that can be launched on a rocket and the difficulty of resupply mid-mission. Any long term settlement will require more resources than astronauts can initially bring with them. Synthetic Biology has the potential to revolutionize space exploration and settlement. Biological tools have a major advantage over classical tools: the ability to self-replicate and regenerate.<br />
<br />
The emerging field of Synthetic Biology will allow us to engineer microbial factories that will largely circumvent the limiting payload factors. These cellular factories will generate fuel, food, medicines and building materials for settlers, but will consist of engineered cells that could be stored in a single test tube, and regrown to production scale on-site, as needed.<br />
<br />
The Brown-Stanford iGEM team is excited to work on three different projects, under the common theme of developing Synthetic Biology applications for space. In addition, as a response to the ethical and philosophical considerations, we interviewed prominent researchers and leaders in the field. '''Click on the astronaut in the corner to view our series.''' <br />
<br />
<html><br />
</div><br />
<div class="miniContent"><br />
<br />
<div class="miniContentLeft"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/3/39/Brown-Stanford_REGObricks-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/REGObricks/Introduction">REGObricks</a></h3><br />
</html><br />
<br />
Any extra-terrestrial settlement will require a habitat to keep its occupants alive, but transport of such a habitat will require a huge amount of payload space. RegoBricks uses bacteria to cement Martian and Lunar regolith (soil) simulant into durable building blocks, similar to concrete bricks, allowing settlements to use in situ resources to build structures.<br />
<html><br />
</div><br />
<br />
<div class="miniContentRight"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/4/4c/Brown-Stanford_DNADamage-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/FRETSensor/Introduction">FRETcetera</a></h3><br />
</html><br />
<br />
Biological interfaces cannot pass information to their users as fast as electronic ones can. FRETcetera develops a novel method of fast-acting biological reporting with changes in cell fluorescence. Bacteria could be used to detect toxic chemicals in the environment, for example, or inform astronauts that their microbial tools are unhealthy.<br />
<html><br />
<br />
</div><br />
<div class="miniContentCenter"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/9/9a/Brown-Stanford_PowerCell-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/PowerCell/Introduction">PowerCell</a></h3><br />
</html><br />
<br />
All biological tools need energy to run. PowerCell develops a universal energy source. Engineered photosynthetic bacteria generate carbon and nitrogenous nutrients from sunlight and air and secrete them to power biological tools. These tools will transform the raw materials into fuel, building materials, food, drugs, and other products useful to settlers.<br />
<html><br />
</div><br />
<br />
</div><br />
<div class="pageContent"><br />
</html><br />
<br />
= '''Summary''' =<br />
<br />
=== '''News!''' ===<br />
<br />
===== '''iGEM Awards''' =====<br />
<br />
We did very well at iGEM this year! <br />
<br />
-At the Americas Regional Jamboree, we finished Top 4 and won Best Presentation.<br />
<br />
-At the World Jamboree, we finished in the Sweet 16, and won Best New Application. <br />
<br />
We are extremely grateful to all those who helped us along the way, and our mentors. Thank you so much!<br />
<br />
===== '''Project Summary''' =====<br />
<br />
Here's a minute long teaser of our project!<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/watch?v=b2AkJRTLOcs"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/b2AkJRTLOcs"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Latest Advancements on Projects''' ===<br />
<br />
===== '''Anabaena Transformation and Successful Cell-Type Specific Expression Control''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 100%;"><br />
<tr><br />
<br />
<td style="padding-left: 20px; padding-right: 20px; padding-top: 0px; padding-bottom: 0px; width: 100%;"><br />
<br />
<p>We successfully transformed our cyanobacterial chassis, Anabaena PCC 7120, with our designed PowerCell genetic construct. Unlike more common cyanobacteria species such as Synechocystis PCC 6803 or Synechococcus elongatus PCC 7942, Anabaena is not naturally competent and does not easily uptake foreign DNA. We performed a triparental conjugation in order to bypass the natural barriers to transformation.</p><br />
<br />
To demonstrate that expression of our construct is properly co-localized to vegetative cells, we grew cultures of Anabaena in nitrate-free BG11 media to promote heterocyst formation. We demonstrated the successful control of our pSac promoter over our sugar secretion construct through GFP expression. The sugar secretion construct is only expressed in actively photosynthesizing cells. In the first picture, we observe areas of diminished GFP fluorescence at regular intervals (approximately 1 in 10 cells) along our transformed Anabaena filament (the background fluorescence is likely due to chlorophyll). Since our GFP reporter should only express under the pSac promoter and only in non-heterocyst cells, we would expect the spacing of dark spots to match the spacing of heterocysts identified through alcian blue staining. Alcian blue is a dye that selectively binds to polysaccharide chains on the surface of heterocyst cells. As you can see by comparing the two images, our results appear to match up!<br />
<br />
Work to characterize the performance of our cscB sucrose secretion construct is underway. We will assay sucrose levels produced by PowerCell. If they are high enough, we will attempt to grow E. coli W. containing arbitrary biobricks from this nutrient source, in an attempt to show PowerCell can power biological tools. </p><br />
<br />
<center><br />
<html><object width="400" height="300"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to="></param> <param name="movie" value="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to=" width="400" height="300"></embed></object></html><br />
<br />
<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
</div><br />
<br />
<html><object width="400" height="300"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to="></param> <param name="movie" value="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to=" width="400" height="300"></embed></object></html><br />
<br />
<div style="width: 35%;"><br />
Transformed Anabaena with heterocysts stained by alcian blue. Heterocyst spacing corresponds to the "off" cells from the GFP image, implying that the sucrose secretion construct in not active in heterocysts, as desired.<br />
</div><br />
<br />
===== '''Biobricked the urease operon''' =====<br />
<br />
We successfully PCR cloned a 10.7 kb functional urease cassette from the plasmid pBU11, whose genetic sequence has hitherto never been known before. We ligated it onto a backbone, and successfully [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biobrick made a standard BioBrick], characterizing [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Characterization its function] and demonstrating active urease activity. Urease is the catalyst behind the phenomenon known as biocement, which is able to fuse sand into bricks, using nothing more than calcium ions and urea. [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biocementation More about urease.]<br />
<br />
Biobrick part [http://partsregistry.org/Part:BBa_K656013 BBa_K656013] has been submitted to the Registry of Standardized Parts. The sequence of this cassette is unavailable because it currently does not exist in any known registry. At the time of this writing, it is currently being sequenced in the lab of Dr. Chris Mason at Weill Cornell Medical College. As soon as the sequence is available, we will supplement the directory listing.<br />
<html><br />
<br />
<br><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG"><img src="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%; font-weight:bold;"><br />
Successful transformants on urease test plates.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</html><br />
<br />
===== '''Stratospheric Balloon Flight''' =====<br />
<br />
We launched samples of our bacteria to the far edge of space (80,000 ft-110,000 ft) to test for their ability to survive simulated Martian environmental conditions. The pressures, temperatures, and radiation conditions at these altitudes are similar to the conditions on the Martian surface.<br />
[[Team:Brown-Stanford/REGObricks/Balloon|Read about our escapades here]]<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/qCQXg8DmZZ0"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/qCQXg8DmZZ0"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Advancements in Human Practices''' ===<br />
===== '''Feature on Synthetic Biology and Space''' =====<br />
We talked with key figures in science and science policy about the implications of human expansion into space, extraterrestrial life and synthetic biology. You can look at our interviews and findings [https://2011.igem.org/Team:Brown-Stanford/SynEthics/Intro here] or by clicking on the astronaut on the upper left!<br />
<br />
===== '''Education and Publicity''' =====<br />
<br />
<html><br />
<table style="width: 100%;"><br />
<tr><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Editor's Choice! x2<br />
</div><br />
</center><br />
</td><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Lunar Science Poster. Click to see full version.<br />
</div><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/9YKsa7s8WM4?version=3"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/9YKsa7s8WM4?version=3"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
<br />
<br />
We educated and publicized the power of synthetic biology in a whole suite of venues and mediums, including <br />
<br />
* A crowd of 600+ over two days at [https://2011.igem.org/Team:Brown-Stanford/MakerFaire Maker Faire NYC], resulting in two Editor’s Choice blue ribbons! <br />
* A filming opportunity with the [https://2011.igem.org/Team:Brown-Stanford/BBC BBC] Horizon, to be featured in a documentary on NASA’s Initiative in Synthetic Biology<br />
*Verbal [https://2011.igem.org/Team:Brown-Stanford/NASA presentations] to NASA Administrator Charlie Bolden, NASA Ames Research Center Director Pete Worden, and writer and visionary Stewart Brand <br />
* A poster presentation at NASA’s Lunar Science Forum<br />
* A heart-pounding [https://2011.igem.org/Team:Brown-Stanford/SB5 poem] at the first-ever SB5.0 Synbio Slam<br />
<br />
===== '''Outreach and Collaboration''' =====<br />
<br />
<html><br />
<center><br />
<a href="http://openwetware.org/wiki/IGEM_Outreach"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6f/Brown-Stanford_Cbricks.jpg" style="width: 30%;"><br />
</a><br />
</center><br />
</html><br />
<br />
We spearheaded the '''largest collaborative effort''' in iGEM history to change the way we do outreach, creating [http://openwetware.org/wiki/IGEM_Outreach '''CommunityBricks'''], a database of valuable teaching plans, presentations, community resources and projects for young and old alike.<br />
<br />
===== '''Alumni and Community-Building''' =====<br />
<br />
<br />
[[File:Brown-Stanford alumnigem.png|300px|center|AlumniGEM|link=http://alumni.brownigem.com/]]<br />
<br />
We built the [http://alumni.brownigem.com '''first iGEM Alumni Network,'''] to provide iGEM'ers a growing collection of professional and social resources and keep the spirit of iGEM alive post-Jamborees.<br />
<br />
<html><br />
<div><a href="javascript:toggle();" id="toggleNews"><h3>Show Media and News</h3></a></div><br />
<br />
</div><br />
<div class="pageContent" id="news" style="display: none;"><br />
</html><br />
<br />
== '''Martian Colony''' ==<br />
[[File:Brown-Stanford_MartianColony.png|center|750px]]<br />
<br />
== '''News''' ==<br />
<br />
===In the Lab===<br />
<center><html><object width="600" height="450"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to="></param> <param name="movie" value="http://www.flickr.com/apps/slideshow/show.swf?v=107931"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://www.flickr.com/apps/slideshow/show.swf?v=107931" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to=" width="600" height="450"></embed></object></html></center><br />
<br />
=== '''August 4<sup>th</sup>, 2011''' ===<br />
<center><html><object width="580" height="355"><param name="movie" value="http://www.youtube.com/v/x-MYE4QkjOU"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/x-MYE4QkjOU" type="application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''June 7<sup>th</sup>, 2011''' ===<br />
[[File:Brown-Stanford TeamPicture6-7-2011.jpg|center|500px]]<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-StanfordTeam:Brown-Stanford2011-12-07T10:18:24Z<p>Evanclark: /* Anabaena Transformation and Successful Cell-Type Specific Expression Control */</p>
<hr />
<div>{{:Team:Brown-Stanford/Templates/Main}}<br />
{{:Team:Brown-Stanford/Templates/Content}}<br />
{{:Team:Brown-Stanford/Templates/Twitter}}<br />
<br />
= '''APPLYING FOR STANFORD iGEM 2012''' =<br />
<br />
Please fill out our interest form at https://docs.google.com/spreadsheet/viewform?formkey=dEQyaWdReTN5S2pCcHRVY29hQXh4cHc6MQ.<br />
<br />
We will be sending further information to this list!<br />
<br />
Thanks!<br />
<br />
= '''APPLYING FOR BROWN iGEM 2012''' =<br />
(12/5/11)<br />
Brown iGEM is looking for undergrad team members for this upcoming year!<br />
<br />
Have you ever wanted to engineer living organisms to create novel functions and help transform the world? <br />
<br />
Spend a summer working at the Synthetic Biology Initiative at NASA Ames Research Center, and represent Brown at the premiere synthetic biology competition in the world! Opportunities exist for all kinds of microbiology and molecular biology work, computational biology, mathematical modeling, engineering, and much more. Develop incredible research and lab skills as you manage your own project!<br />
<br />
Students of all academic backgrounds and experience levels welcome (this includes Class of '12) <br />
<br />
We are hosting two '''INFO SESSIONS''' this week:<br />
<br />
'''TIME''': 6-7pm on Thursday 12/8, Friday 12/9<br />
<br />
'''LOCATION''': Barus and Holly 190<br />
<br />
-come hear more about iGEM, synthetic biology, and what the award-winning Brown iGEM team does; dinner will be provided!<br />
<br />
<br />
If you can't make it but are still interested, please let us know! Other questions or concerns are welcome as well. Contact us at igem@brown.edu<br />
<br />
<html><br />
<span style="font-size: 20px;"></html>Our electronic application is [http://www.brownigem.com/2012_iGEM_Application.doc available now]. Keep up with the 2012 application process on our [https://www.facebook.com/events/146221565480758/ facebook page]!<br />
<html></span></html><br />
<br />
= '''Mars BioTools: Synthetic Biology for Space Exploration''' =<br />
One of the major challenges of space exploration is the limited payload mass that can be launched on a rocket and the difficulty of resupply mid-mission. Any long term settlement will require more resources than astronauts can initially bring with them. Synthetic Biology has the potential to revolutionize space exploration and settlement. Biological tools have a major advantage over classical tools: the ability to self-replicate and regenerate.<br />
<br />
The emerging field of Synthetic Biology will allow us to engineer microbial factories that will largely circumvent the limiting payload factors. These cellular factories will generate fuel, food, medicines and building materials for settlers, but will consist of engineered cells that could be stored in a single test tube, and regrown to production scale on-site, as needed.<br />
<br />
The Brown-Stanford iGEM team is excited to work on three different projects, under the common theme of developing Synthetic Biology applications for space. In addition, as a response to the ethical and philosophical considerations, we interviewed prominent researchers and leaders in the field. '''Click on the astronaut in the corner to view our series.''' <br />
<br />
<html><br />
</div><br />
<div class="miniContent"><br />
<br />
<div class="miniContentLeft"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/3/39/Brown-Stanford_REGObricks-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/REGObricks/Introduction">REGObricks</a></h3><br />
</html><br />
<br />
Any extra-terrestrial settlement will require a habitat to keep its occupants alive, but transport of such a habitat will require a huge amount of payload space. RegoBricks uses bacteria to cement Martian and Lunar regolith (soil) simulant into durable building blocks, similar to concrete bricks, allowing settlements to use in situ resources to build structures.<br />
<html><br />
</div><br />
<br />
<div class="miniContentRight"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/4/4c/Brown-Stanford_DNADamage-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/FRETSensor/Introduction">FRETcetera</a></h3><br />
</html><br />
<br />
Biological interfaces cannot pass information to their users as fast as electronic ones can. FRETcetera develops a novel method of fast-acting biological reporting with changes in cell fluorescence. Bacteria could be used to detect toxic chemicals in the environment, for example, or inform astronauts that their microbial tools are unhealthy.<br />
<html><br />
<br />
</div><br />
<div class="miniContentCenter"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/9/9a/Brown-Stanford_PowerCell-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/PowerCell/Introduction">PowerCell</a></h3><br />
</html><br />
<br />
All biological tools need energy to run. PowerCell develops a universal energy source. Engineered photosynthetic bacteria generate carbon and nitrogenous nutrients from sunlight and air and secrete them to power biological tools. These tools will transform the raw materials into fuel, building materials, food, drugs, and other products useful to settlers.<br />
<html><br />
</div><br />
<br />
</div><br />
<div class="pageContent"><br />
</html><br />
<br />
= '''Summary''' =<br />
<br />
=== '''News!''' ===<br />
<br />
===== '''iGEM Awards''' =====<br />
<br />
We did very well at iGEM this year! <br />
<br />
-At the Americas Regional Jamboree, we finished Top 4 and won Best Presentation.<br />
<br />
-At the World Jamboree, we finished in the Sweet 16, and won Best New Application. <br />
<br />
We are extremely grateful to all those who helped us along the way, and our mentors. Thank you so much!<br />
<br />
===== '''Project Summary''' =====<br />
<br />
Here's a minute long teaser of our project!<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/watch?v=b2AkJRTLOcs"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/b2AkJRTLOcs"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Latest Advancements on Projects''' ===<br />
<br />
===== '''Anabaena Transformation and Successful Cell-Type Specific Expression Control''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 50%;"><br />
<tr><br />
<br />
<td style="padding-left: 20px; padding-right: 20px; padding-top: 0px; padding-bottom: 0px; width: 50%;"><br />
<br />
<p>We successfully transformed our cyanobacterial chassis, Anabaena PCC 7120, with our designed PowerCell genetic construct. Unlike more common cyanobacteria species such as Synechocystis PCC 6803 or Synechococcus elongatus PCC 7942, Anabaena is not naturally competent and does not easily uptake foreign DNA. We performed a triparental conjugation in order to bypass the natural barriers to transformation.</p><br />
<br />
To demonstrate that expression of our construct is properly co-localized to vegetative cells, we grew cultures of Anabaena in nitrate-free BG11 media to promote heterocyst formation. We demonstrated the successful control of our pSac promoter over our sugar secretion construct through GFP expression. The sugar secretion construct is only expressed in actively photosynthesizing cells. In the first picture, we observe areas of diminished GFP fluorescence at regular intervals (approximately 1 in 10 cells) along our transformed Anabaena filament (the background fluorescence is likely due to chlorophyll). Since our GFP reporter should only express under the pSac promoter and only in non-heterocyst cells, we would expect the spacing of dark spots to match the spacing of heterocysts identified through alcian blue staining. Alcian blue is a dye that selectively binds to polysaccharide chains on the surface of heterocyst cells. As you can see by comparing the two images, our results appear to match up!<br />
<br />
Work to characterize the performance of our cscB sucrose secretion construct is underway. We will assay sucrose levels produced by PowerCell. If they are high enough, we will attempt to grow E. coli W. containing arbitrary biobricks from this nutrient source, in an attempt to show PowerCell can power biological tools. </p><br />
<br />
<center><br />
<html><object width="400" height="300"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to="></param> <param name="movie" value="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to=" width="400" height="300"></embed></object></html><br />
<br />
<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
</div><br />
<br />
<html><object width="400" height="300"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to="></param> <param name="movie" value="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to=" width="400" height="300"></embed></object></html><br />
<br />
<div style="width: 35%;"><br />
Transformed Anabaena with heterocysts stained by alcian blue. Heterocyst spacing corresponds to the "off" cells from the GFP image, implying that the sucrose secretion construct in not active in heterocysts, as desired.<br />
</div><br />
<br />
===== '''Biobricked the urease operon''' =====<br />
<br />
We successfully PCR cloned a 10.7 kb functional urease cassette from the plasmid pBU11, whose genetic sequence has hitherto never been known before. We ligated it onto a backbone, and successfully [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biobrick made a standard BioBrick], characterizing [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Characterization its function] and demonstrating active urease activity. Urease is the catalyst behind the phenomenon known as biocement, which is able to fuse sand into bricks, using nothing more than calcium ions and urea. [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biocementation More about urease.]<br />
<br />
Biobrick part [http://partsregistry.org/Part:BBa_K656013 BBa_K656013] has been submitted to the Registry of Standardized Parts. The sequence of this cassette is unavailable because it currently does not exist in any known registry. At the time of this writing, it is currently being sequenced in the lab of Dr. Chris Mason at Weill Cornell Medical College. As soon as the sequence is available, we will supplement the directory listing.<br />
<html><br />
<br />
<br><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG"><img src="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%; font-weight:bold;"><br />
Successful transformants on urease test plates.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</html><br />
<br />
===== '''Stratospheric Balloon Flight''' =====<br />
<br />
We launched samples of our bacteria to the far edge of space (80,000 ft-110,000 ft) to test for their ability to survive simulated Martian environmental conditions. The pressures, temperatures, and radiation conditions at these altitudes are similar to the conditions on the Martian surface.<br />
[[Team:Brown-Stanford/REGObricks/Balloon|Read about our escapades here]]<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/qCQXg8DmZZ0"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/qCQXg8DmZZ0"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Advancements in Human Practices''' ===<br />
===== '''Feature on Synthetic Biology and Space''' =====<br />
We talked with key figures in science and science policy about the implications of human expansion into space, extraterrestrial life and synthetic biology. You can look at our interviews and findings [https://2011.igem.org/Team:Brown-Stanford/SynEthics/Intro here] or by clicking on the astronaut on the upper left!<br />
<br />
===== '''Education and Publicity''' =====<br />
<br />
<html><br />
<table style="width: 100%;"><br />
<tr><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Editor's Choice! x2<br />
</div><br />
</center><br />
</td><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Lunar Science Poster. Click to see full version.<br />
</div><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/9YKsa7s8WM4?version=3"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/9YKsa7s8WM4?version=3"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
<br />
<br />
We educated and publicized the power of synthetic biology in a whole suite of venues and mediums, including <br />
<br />
* A crowd of 600+ over two days at [https://2011.igem.org/Team:Brown-Stanford/MakerFaire Maker Faire NYC], resulting in two Editor’s Choice blue ribbons! <br />
* A filming opportunity with the [https://2011.igem.org/Team:Brown-Stanford/BBC BBC] Horizon, to be featured in a documentary on NASA’s Initiative in Synthetic Biology<br />
*Verbal [https://2011.igem.org/Team:Brown-Stanford/NASA presentations] to NASA Administrator Charlie Bolden, NASA Ames Research Center Director Pete Worden, and writer and visionary Stewart Brand <br />
* A poster presentation at NASA’s Lunar Science Forum<br />
* A heart-pounding [https://2011.igem.org/Team:Brown-Stanford/SB5 poem] at the first-ever SB5.0 Synbio Slam<br />
<br />
===== '''Outreach and Collaboration''' =====<br />
<br />
<html><br />
<center><br />
<a href="http://openwetware.org/wiki/IGEM_Outreach"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6f/Brown-Stanford_Cbricks.jpg" style="width: 30%;"><br />
</a><br />
</center><br />
</html><br />
<br />
We spearheaded the '''largest collaborative effort''' in iGEM history to change the way we do outreach, creating [http://openwetware.org/wiki/IGEM_Outreach '''CommunityBricks'''], a database of valuable teaching plans, presentations, community resources and projects for young and old alike.<br />
<br />
===== '''Alumni and Community-Building''' =====<br />
<br />
<br />
[[File:Brown-Stanford alumnigem.png|300px|center|AlumniGEM|link=http://alumni.brownigem.com/]]<br />
<br />
We built the [http://alumni.brownigem.com '''first iGEM Alumni Network,'''] to provide iGEM'ers a growing collection of professional and social resources and keep the spirit of iGEM alive post-Jamborees.<br />
<br />
<html><br />
<div><a href="javascript:toggle();" id="toggleNews"><h3>Show Media and News</h3></a></div><br />
<br />
</div><br />
<div class="pageContent" id="news" style="display: none;"><br />
</html><br />
<br />
== '''Martian Colony''' ==<br />
[[File:Brown-Stanford_MartianColony.png|center|750px]]<br />
<br />
== '''News''' ==<br />
<br />
===In the Lab===<br />
<center><html><object width="600" height="450"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to="></param> <param name="movie" value="http://www.flickr.com/apps/slideshow/show.swf?v=107931"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://www.flickr.com/apps/slideshow/show.swf?v=107931" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to=" width="600" height="450"></embed></object></html></center><br />
<br />
=== '''August 4<sup>th</sup>, 2011''' ===<br />
<center><html><object width="580" height="355"><param name="movie" value="http://www.youtube.com/v/x-MYE4QkjOU"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/x-MYE4QkjOU" type="application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''June 7<sup>th</sup>, 2011''' ===<br />
[[File:Brown-Stanford TeamPicture6-7-2011.jpg|center|500px]]<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-StanfordTeam:Brown-Stanford2011-12-07T10:17:18Z<p>Evanclark: /* Latest Advancements on Projects */</p>
<hr />
<div>{{:Team:Brown-Stanford/Templates/Main}}<br />
{{:Team:Brown-Stanford/Templates/Content}}<br />
{{:Team:Brown-Stanford/Templates/Twitter}}<br />
<br />
= '''APPLYING FOR STANFORD iGEM 2012''' =<br />
<br />
Please fill out our interest form at https://docs.google.com/spreadsheet/viewform?formkey=dEQyaWdReTN5S2pCcHRVY29hQXh4cHc6MQ.<br />
<br />
We will be sending further information to this list!<br />
<br />
Thanks!<br />
<br />
= '''APPLYING FOR BROWN iGEM 2012''' =<br />
(12/5/11)<br />
Brown iGEM is looking for undergrad team members for this upcoming year!<br />
<br />
Have you ever wanted to engineer living organisms to create novel functions and help transform the world? <br />
<br />
Spend a summer working at the Synthetic Biology Initiative at NASA Ames Research Center, and represent Brown at the premiere synthetic biology competition in the world! Opportunities exist for all kinds of microbiology and molecular biology work, computational biology, mathematical modeling, engineering, and much more. Develop incredible research and lab skills as you manage your own project!<br />
<br />
Students of all academic backgrounds and experience levels welcome (this includes Class of '12) <br />
<br />
We are hosting two '''INFO SESSIONS''' this week:<br />
<br />
'''TIME''': 6-7pm on Thursday 12/8, Friday 12/9<br />
<br />
'''LOCATION''': Barus and Holly 190<br />
<br />
-come hear more about iGEM, synthetic biology, and what the award-winning Brown iGEM team does; dinner will be provided!<br />
<br />
<br />
If you can't make it but are still interested, please let us know! Other questions or concerns are welcome as well. Contact us at igem@brown.edu<br />
<br />
<html><br />
<span style="font-size: 20px;"></html>Our electronic application is [http://www.brownigem.com/2012_iGEM_Application.doc available now]. Keep up with the 2012 application process on our [https://www.facebook.com/events/146221565480758/ facebook page]!<br />
<html></span></html><br />
<br />
= '''Mars BioTools: Synthetic Biology for Space Exploration''' =<br />
One of the major challenges of space exploration is the limited payload mass that can be launched on a rocket and the difficulty of resupply mid-mission. Any long term settlement will require more resources than astronauts can initially bring with them. Synthetic Biology has the potential to revolutionize space exploration and settlement. Biological tools have a major advantage over classical tools: the ability to self-replicate and regenerate.<br />
<br />
The emerging field of Synthetic Biology will allow us to engineer microbial factories that will largely circumvent the limiting payload factors. These cellular factories will generate fuel, food, medicines and building materials for settlers, but will consist of engineered cells that could be stored in a single test tube, and regrown to production scale on-site, as needed.<br />
<br />
The Brown-Stanford iGEM team is excited to work on three different projects, under the common theme of developing Synthetic Biology applications for space. In addition, as a response to the ethical and philosophical considerations, we interviewed prominent researchers and leaders in the field. '''Click on the astronaut in the corner to view our series.''' <br />
<br />
<html><br />
</div><br />
<div class="miniContent"><br />
<br />
<div class="miniContentLeft"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/3/39/Brown-Stanford_REGObricks-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/REGObricks/Introduction">REGObricks</a></h3><br />
</html><br />
<br />
Any extra-terrestrial settlement will require a habitat to keep its occupants alive, but transport of such a habitat will require a huge amount of payload space. RegoBricks uses bacteria to cement Martian and Lunar regolith (soil) simulant into durable building blocks, similar to concrete bricks, allowing settlements to use in situ resources to build structures.<br />
<html><br />
</div><br />
<br />
<div class="miniContentRight"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/4/4c/Brown-Stanford_DNADamage-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/FRETSensor/Introduction">FRETcetera</a></h3><br />
</html><br />
<br />
Biological interfaces cannot pass information to their users as fast as electronic ones can. FRETcetera develops a novel method of fast-acting biological reporting with changes in cell fluorescence. Bacteria could be used to detect toxic chemicals in the environment, for example, or inform astronauts that their microbial tools are unhealthy.<br />
<html><br />
<br />
</div><br />
<div class="miniContentCenter"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/9/9a/Brown-Stanford_PowerCell-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/PowerCell/Introduction">PowerCell</a></h3><br />
</html><br />
<br />
All biological tools need energy to run. PowerCell develops a universal energy source. Engineered photosynthetic bacteria generate carbon and nitrogenous nutrients from sunlight and air and secrete them to power biological tools. These tools will transform the raw materials into fuel, building materials, food, drugs, and other products useful to settlers.<br />
<html><br />
</div><br />
<br />
</div><br />
<div class="pageContent"><br />
</html><br />
<br />
= '''Summary''' =<br />
<br />
=== '''News!''' ===<br />
<br />
===== '''iGEM Awards''' =====<br />
<br />
We did very well at iGEM this year! <br />
<br />
-At the Americas Regional Jamboree, we finished Top 4 and won Best Presentation.<br />
<br />
-At the World Jamboree, we finished in the Sweet 16, and won Best New Application. <br />
<br />
We are extremely grateful to all those who helped us along the way, and our mentors. Thank you so much!<br />
<br />
===== '''Project Summary''' =====<br />
<br />
Here's a minute long teaser of our project!<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/watch?v=b2AkJRTLOcs"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/b2AkJRTLOcs"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Latest Advancements on Projects''' ===<br />
<br />
===== '''Anabaena Transformation and Successful Cell-Type Specific Expression Control''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 100%;"><br />
<tr><br />
<br />
<td style="padding-left: 20px; padding-right: 20px; padding-top: 0px; padding-bottom: 0px; width: 100%;"><br />
<br />
<p>We successfully transformed our cyanobacterial chassis, Anabaena PCC 7120, with our designed PowerCell genetic construct. Unlike more common cyanobacteria species such as Synechocystis PCC 6803 or Synechococcus elongatus PCC 7942, Anabaena is not naturally competent and does not easily uptake foreign DNA. We performed a triparental conjugation in order to bypass the natural barriers to transformation.</p><br />
<br />
To demonstrate that expression of our construct is properly co-localized to vegetative cells, we grew cultures of Anabaena in nitrate-free BG11 media to promote heterocyst formation. We demonstrated the successful control of our pSac promoter over our sugar secretion construct through GFP expression. The sugar secretion construct is only expressed in actively photosynthesizing cells. In the first picture, we observe areas of diminished GFP fluorescence at regular intervals (approximately 1 in 10 cells) along our transformed Anabaena filament (the background fluorescence is likely due to chlorophyll). Since our GFP reporter should only express under the pSac promoter and only in non-heterocyst cells, we would expect the spacing of dark spots to match the spacing of heterocysts identified through alcian blue staining. Alcian blue is a dye that selectively binds to polysaccharide chains on the surface of heterocyst cells. As you can see by comparing the two images, our results appear to match up!<br />
<br />
Work to characterize the performance of our cscB sucrose secretion construct is underway. We will assay sucrose levels produced by PowerCell. If they are high enough, we will attempt to grow E. coli W. containing arbitrary biobricks from this nutrient source, in an attempt to show PowerCell can power biological tools. </p><br />
<br />
<center><br />
<html><object width="400" height="300"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to="></param> <param name="movie" value="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to=" width="400" height="300"></embed></object></html><br />
<br />
<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
</div><br />
<br />
<html><object width="400" height="300"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to="></param> <param name="movie" value="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to=" width="400" height="300"></embed></object></html><br />
<br />
<div style="width: 35%;"><br />
Transformed Anabaena with heterocysts stained by alcian blue. Heterocyst spacing corresponds to the "off" cells from the GFP image, implying that the sucrose secretion construct in not active in heterocysts, as desired.<br />
</div><br />
<br />
===== '''Biobricked the urease operon''' =====<br />
<br />
We successfully PCR cloned a 10.7 kb functional urease cassette from the plasmid pBU11, whose genetic sequence has hitherto never been known before. We ligated it onto a backbone, and successfully [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biobrick made a standard BioBrick], characterizing [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Characterization its function] and demonstrating active urease activity. Urease is the catalyst behind the phenomenon known as biocement, which is able to fuse sand into bricks, using nothing more than calcium ions and urea. [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biocementation More about urease.]<br />
<br />
Biobrick part [http://partsregistry.org/Part:BBa_K656013 BBa_K656013] has been submitted to the Registry of Standardized Parts. The sequence of this cassette is unavailable because it currently does not exist in any known registry. At the time of this writing, it is currently being sequenced in the lab of Dr. Chris Mason at Weill Cornell Medical College. As soon as the sequence is available, we will supplement the directory listing.<br />
<html><br />
<br />
<br><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG"><img src="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%; font-weight:bold;"><br />
Successful transformants on urease test plates.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</html><br />
<br />
===== '''Stratospheric Balloon Flight''' =====<br />
<br />
We launched samples of our bacteria to the far edge of space (80,000 ft-110,000 ft) to test for their ability to survive simulated Martian environmental conditions. The pressures, temperatures, and radiation conditions at these altitudes are similar to the conditions on the Martian surface.<br />
[[Team:Brown-Stanford/REGObricks/Balloon|Read about our escapades here]]<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/qCQXg8DmZZ0"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/qCQXg8DmZZ0"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Advancements in Human Practices''' ===<br />
===== '''Feature on Synthetic Biology and Space''' =====<br />
We talked with key figures in science and science policy about the implications of human expansion into space, extraterrestrial life and synthetic biology. You can look at our interviews and findings [https://2011.igem.org/Team:Brown-Stanford/SynEthics/Intro here] or by clicking on the astronaut on the upper left!<br />
<br />
===== '''Education and Publicity''' =====<br />
<br />
<html><br />
<table style="width: 100%;"><br />
<tr><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Editor's Choice! x2<br />
</div><br />
</center><br />
</td><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Lunar Science Poster. Click to see full version.<br />
</div><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/9YKsa7s8WM4?version=3"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/9YKsa7s8WM4?version=3"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
<br />
<br />
We educated and publicized the power of synthetic biology in a whole suite of venues and mediums, including <br />
<br />
* A crowd of 600+ over two days at [https://2011.igem.org/Team:Brown-Stanford/MakerFaire Maker Faire NYC], resulting in two Editor’s Choice blue ribbons! <br />
* A filming opportunity with the [https://2011.igem.org/Team:Brown-Stanford/BBC BBC] Horizon, to be featured in a documentary on NASA’s Initiative in Synthetic Biology<br />
*Verbal [https://2011.igem.org/Team:Brown-Stanford/NASA presentations] to NASA Administrator Charlie Bolden, NASA Ames Research Center Director Pete Worden, and writer and visionary Stewart Brand <br />
* A poster presentation at NASA’s Lunar Science Forum<br />
* A heart-pounding [https://2011.igem.org/Team:Brown-Stanford/SB5 poem] at the first-ever SB5.0 Synbio Slam<br />
<br />
===== '''Outreach and Collaboration''' =====<br />
<br />
<html><br />
<center><br />
<a href="http://openwetware.org/wiki/IGEM_Outreach"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6f/Brown-Stanford_Cbricks.jpg" style="width: 30%;"><br />
</a><br />
</center><br />
</html><br />
<br />
We spearheaded the '''largest collaborative effort''' in iGEM history to change the way we do outreach, creating [http://openwetware.org/wiki/IGEM_Outreach '''CommunityBricks'''], a database of valuable teaching plans, presentations, community resources and projects for young and old alike.<br />
<br />
===== '''Alumni and Community-Building''' =====<br />
<br />
<br />
[[File:Brown-Stanford alumnigem.png|300px|center|AlumniGEM|link=http://alumni.brownigem.com/]]<br />
<br />
We built the [http://alumni.brownigem.com '''first iGEM Alumni Network,'''] to provide iGEM'ers a growing collection of professional and social resources and keep the spirit of iGEM alive post-Jamborees.<br />
<br />
<html><br />
<div><a href="javascript:toggle();" id="toggleNews"><h3>Show Media and News</h3></a></div><br />
<br />
</div><br />
<div class="pageContent" id="news" style="display: none;"><br />
</html><br />
<br />
== '''Martian Colony''' ==<br />
[[File:Brown-Stanford_MartianColony.png|center|750px]]<br />
<br />
== '''News''' ==<br />
<br />
===In the Lab===<br />
<center><html><object width="600" height="450"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to="></param> <param name="movie" value="http://www.flickr.com/apps/slideshow/show.swf?v=107931"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://www.flickr.com/apps/slideshow/show.swf?v=107931" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to=" width="600" height="450"></embed></object></html></center><br />
<br />
=== '''August 4<sup>th</sup>, 2011''' ===<br />
<center><html><object width="580" height="355"><param name="movie" value="http://www.youtube.com/v/x-MYE4QkjOU"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/x-MYE4QkjOU" type="application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''June 7<sup>th</sup>, 2011''' ===<br />
[[File:Brown-Stanford TeamPicture6-7-2011.jpg|center|500px]]<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-StanfordTeam:Brown-Stanford2011-12-07T10:14:21Z<p>Evanclark: /* Latest Advancements on Projects */</p>
<hr />
<div>{{:Team:Brown-Stanford/Templates/Main}}<br />
{{:Team:Brown-Stanford/Templates/Content}}<br />
{{:Team:Brown-Stanford/Templates/Twitter}}<br />
<br />
= '''APPLYING FOR STANFORD iGEM 2012''' =<br />
<br />
Please fill out our interest form at https://docs.google.com/spreadsheet/viewform?formkey=dEQyaWdReTN5S2pCcHRVY29hQXh4cHc6MQ.<br />
<br />
We will be sending further information to this list!<br />
<br />
Thanks!<br />
<br />
= '''APPLYING FOR BROWN iGEM 2012''' =<br />
(12/5/11)<br />
Brown iGEM is looking for undergrad team members for this upcoming year!<br />
<br />
Have you ever wanted to engineer living organisms to create novel functions and help transform the world? <br />
<br />
Spend a summer working at the Synthetic Biology Initiative at NASA Ames Research Center, and represent Brown at the premiere synthetic biology competition in the world! Opportunities exist for all kinds of microbiology and molecular biology work, computational biology, mathematical modeling, engineering, and much more. Develop incredible research and lab skills as you manage your own project!<br />
<br />
Students of all academic backgrounds and experience levels welcome (this includes Class of '12) <br />
<br />
We are hosting two '''INFO SESSIONS''' this week:<br />
<br />
'''TIME''': 6-7pm on Thursday 12/8, Friday 12/9<br />
<br />
'''LOCATION''': Barus and Holly 190<br />
<br />
-come hear more about iGEM, synthetic biology, and what the award-winning Brown iGEM team does; dinner will be provided!<br />
<br />
<br />
If you can't make it but are still interested, please let us know! Other questions or concerns are welcome as well. Contact us at igem@brown.edu<br />
<br />
<html><br />
<span style="font-size: 20px;"></html>Our electronic application is [http://www.brownigem.com/2012_iGEM_Application.doc available now]. Keep up with the 2012 application process on our [https://www.facebook.com/events/146221565480758/ facebook page]!<br />
<html></span></html><br />
<br />
= '''Mars BioTools: Synthetic Biology for Space Exploration''' =<br />
One of the major challenges of space exploration is the limited payload mass that can be launched on a rocket and the difficulty of resupply mid-mission. Any long term settlement will require more resources than astronauts can initially bring with them. Synthetic Biology has the potential to revolutionize space exploration and settlement. Biological tools have a major advantage over classical tools: the ability to self-replicate and regenerate.<br />
<br />
The emerging field of Synthetic Biology will allow us to engineer microbial factories that will largely circumvent the limiting payload factors. These cellular factories will generate fuel, food, medicines and building materials for settlers, but will consist of engineered cells that could be stored in a single test tube, and regrown to production scale on-site, as needed.<br />
<br />
The Brown-Stanford iGEM team is excited to work on three different projects, under the common theme of developing Synthetic Biology applications for space. In addition, as a response to the ethical and philosophical considerations, we interviewed prominent researchers and leaders in the field. '''Click on the astronaut in the corner to view our series.''' <br />
<br />
<html><br />
</div><br />
<div class="miniContent"><br />
<br />
<div class="miniContentLeft"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/3/39/Brown-Stanford_REGObricks-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/REGObricks/Introduction">REGObricks</a></h3><br />
</html><br />
<br />
Any extra-terrestrial settlement will require a habitat to keep its occupants alive, but transport of such a habitat will require a huge amount of payload space. RegoBricks uses bacteria to cement Martian and Lunar regolith (soil) simulant into durable building blocks, similar to concrete bricks, allowing settlements to use in situ resources to build structures.<br />
<html><br />
</div><br />
<br />
<div class="miniContentRight"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/4/4c/Brown-Stanford_DNADamage-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/FRETSensor/Introduction">FRETcetera</a></h3><br />
</html><br />
<br />
Biological interfaces cannot pass information to their users as fast as electronic ones can. FRETcetera develops a novel method of fast-acting biological reporting with changes in cell fluorescence. Bacteria could be used to detect toxic chemicals in the environment, for example, or inform astronauts that their microbial tools are unhealthy.<br />
<html><br />
<br />
</div><br />
<div class="miniContentCenter"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/9/9a/Brown-Stanford_PowerCell-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/PowerCell/Introduction">PowerCell</a></h3><br />
</html><br />
<br />
All biological tools need energy to run. PowerCell develops a universal energy source. Engineered photosynthetic bacteria generate carbon and nitrogenous nutrients from sunlight and air and secrete them to power biological tools. These tools will transform the raw materials into fuel, building materials, food, drugs, and other products useful to settlers.<br />
<html><br />
</div><br />
<br />
</div><br />
<div class="pageContent"><br />
</html><br />
<br />
= '''Summary''' =<br />
<br />
=== '''News!''' ===<br />
<br />
===== '''iGEM Awards''' =====<br />
<br />
We did very well at iGEM this year! <br />
<br />
-At the Americas Regional Jamboree, we finished Top 4 and won Best Presentation.<br />
<br />
-At the World Jamboree, we finished in the Sweet 16, and won Best New Application. <br />
<br />
We are extremely grateful to all those who helped us along the way, and our mentors. Thank you so much!<br />
<br />
===== '''Project Summary''' =====<br />
<br />
Here's a minute long teaser of our project!<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/watch?v=b2AkJRTLOcs"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/b2AkJRTLOcs"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Latest Advancements on Projects''' ===<br />
<br />
===== '''Anabaena Transformation and Successful Cell-Type Specific Expression Control''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 100%;"><br />
<tr><br />
<br />
<td style="padding-left: 20px; padding-right: 0px; padding-top: 0px; padding-bottom: 0px; width: 90%;"><br />
<br />
<p>We successfully transformed our cyanobacterial chassis, Anabaena PCC 7120, with our designed PowerCell genetic construct. Unlike more common cyanobacteria species such as Synechocystis PCC 6803 or Synechococcus elongatus PCC 7942, Anabaena is not naturally competent and does not easily uptake foreign DNA. We performed a triparental conjugation in order to bypass the natural barriers to transformation.</p><br />
<br />
To demonstrate that expression of our construct is properly co-localized to vegetative cells, we grew cultures of Anabaena in nitrate-free BG11 media to promote heterocyst formation. We demonstrated the successful control of our pSac promoter over our sugar secretion construct through GFP expression. The sugar secretion construct is only expressed in actively photosynthesizing cells. In the first picture, we observe areas of diminished GFP fluorescence at regular intervals (approximately 1 in 10 cells) along our transformed Anabaena filament (the background fluorescence is likely due to chlorophyll). Since our GFP reporter should only express under the pSac promoter and only in non-heterocyst cells, we would expect the spacing of dark spots to match the spacing of heterocysts identified through alcian blue staining. Alcian blue is a dye that selectively binds to polysaccharide chains on the surface of heterocyst cells. As you can see by comparing the two images, our results appear to match up!<br />
<br />
Work to characterize the performance of our cscB sucrose secretion construct is underway. We will assay sucrose levels produced by PowerCell. If they are high enough, we will attempt to grow E. coli W. containing arbitrary biobricks from this nutrient source, in an attempt to show PowerCell can power biological tools. </p><br />
<br />
<center><br />
<html><object width="400" height="300"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to="></param> <param name="movie" value="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to=" width="400" height="300"></embed></object></html><br />
<br />
<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
</div><br />
<br />
<html><object width="400" height="300"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to="></param> <param name="movie" value="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to=" width="400" height="300"></embed></object></html><br />
<br />
<div style="width: 35%;"><br />
Transformed Anabaena with heterocysts stained by alcian blue. Heterocyst spacing corresponds to the "off" cells from the GFP image, implying that the sucrose secretion construct in not active in heterocysts, as desired.<br />
</div><br />
<br />
===== '''Biobricked the urease operon''' =====<br />
<br />
We successfully PCR cloned a 10.7 kb functional urease cassette from the plasmid pBU11, whose genetic sequence has hitherto never been known before. We ligated it onto a backbone, and successfully [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biobrick made a standard BioBrick], characterizing [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Characterization its function] and demonstrating active urease activity. Urease is the catalyst behind the phenomenon known as biocement, which is able to fuse sand into bricks, using nothing more than calcium ions and urea. [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biocementation More about urease.]<br />
<br />
Biobrick part [http://partsregistry.org/Part:BBa_K656013 BBa_K656013] has been submitted to the Registry of Standardized Parts. The sequence of this cassette is unavailable because it currently does not exist in any known registry. At the time of this writing, it is currently being sequenced in the lab of Dr. Chris Mason at Weill Cornell Medical College. As soon as the sequence is available, we will supplement the directory listing.<br />
<html><br />
<br />
<br><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG"><img src="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%; font-weight:bold;"><br />
Successful transformants on urease test plates.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</html><br />
<br />
===== '''Stratospheric Balloon Flight''' =====<br />
<br />
We launched samples of our bacteria to the far edge of space (80,000 ft-110,000 ft) to test for their ability to survive simulated Martian environmental conditions. The pressures, temperatures, and radiation conditions at these altitudes are similar to the conditions on the Martian surface.<br />
[[Team:Brown-Stanford/REGObricks/Balloon|Read about our escapades here]]<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/qCQXg8DmZZ0"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/qCQXg8DmZZ0"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Advancements in Human Practices''' ===<br />
===== '''Feature on Synthetic Biology and Space''' =====<br />
We talked with key figures in science and science policy about the implications of human expansion into space, extraterrestrial life and synthetic biology. You can look at our interviews and findings [https://2011.igem.org/Team:Brown-Stanford/SynEthics/Intro here] or by clicking on the astronaut on the upper left!<br />
<br />
===== '''Education and Publicity''' =====<br />
<br />
<html><br />
<table style="width: 100%;"><br />
<tr><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Editor's Choice! x2<br />
</div><br />
</center><br />
</td><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Lunar Science Poster. Click to see full version.<br />
</div><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/9YKsa7s8WM4?version=3"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/9YKsa7s8WM4?version=3"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
<br />
<br />
We educated and publicized the power of synthetic biology in a whole suite of venues and mediums, including <br />
<br />
* A crowd of 600+ over two days at [https://2011.igem.org/Team:Brown-Stanford/MakerFaire Maker Faire NYC], resulting in two Editor’s Choice blue ribbons! <br />
* A filming opportunity with the [https://2011.igem.org/Team:Brown-Stanford/BBC BBC] Horizon, to be featured in a documentary on NASA’s Initiative in Synthetic Biology<br />
*Verbal [https://2011.igem.org/Team:Brown-Stanford/NASA presentations] to NASA Administrator Charlie Bolden, NASA Ames Research Center Director Pete Worden, and writer and visionary Stewart Brand <br />
* A poster presentation at NASA’s Lunar Science Forum<br />
* A heart-pounding [https://2011.igem.org/Team:Brown-Stanford/SB5 poem] at the first-ever SB5.0 Synbio Slam<br />
<br />
===== '''Outreach and Collaboration''' =====<br />
<br />
<html><br />
<center><br />
<a href="http://openwetware.org/wiki/IGEM_Outreach"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6f/Brown-Stanford_Cbricks.jpg" style="width: 30%;"><br />
</a><br />
</center><br />
</html><br />
<br />
We spearheaded the '''largest collaborative effort''' in iGEM history to change the way we do outreach, creating [http://openwetware.org/wiki/IGEM_Outreach '''CommunityBricks'''], a database of valuable teaching plans, presentations, community resources and projects for young and old alike.<br />
<br />
===== '''Alumni and Community-Building''' =====<br />
<br />
<br />
[[File:Brown-Stanford alumnigem.png|300px|center|AlumniGEM|link=http://alumni.brownigem.com/]]<br />
<br />
We built the [http://alumni.brownigem.com '''first iGEM Alumni Network,'''] to provide iGEM'ers a growing collection of professional and social resources and keep the spirit of iGEM alive post-Jamborees.<br />
<br />
<html><br />
<div><a href="javascript:toggle();" id="toggleNews"><h3>Show Media and News</h3></a></div><br />
<br />
</div><br />
<div class="pageContent" id="news" style="display: none;"><br />
</html><br />
<br />
== '''Martian Colony''' ==<br />
[[File:Brown-Stanford_MartianColony.png|center|750px]]<br />
<br />
== '''News''' ==<br />
<br />
===In the Lab===<br />
<center><html><object width="600" height="450"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to="></param> <param name="movie" value="http://www.flickr.com/apps/slideshow/show.swf?v=107931"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://www.flickr.com/apps/slideshow/show.swf?v=107931" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to=" width="600" height="450"></embed></object></html></center><br />
<br />
=== '''August 4<sup>th</sup>, 2011''' ===<br />
<center><html><object width="580" height="355"><param name="movie" value="http://www.youtube.com/v/x-MYE4QkjOU"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/x-MYE4QkjOU" type="application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''June 7<sup>th</sup>, 2011''' ===<br />
[[File:Brown-Stanford TeamPicture6-7-2011.jpg|center|500px]]<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-StanfordTeam:Brown-Stanford2011-12-07T10:13:47Z<p>Evanclark: /* Latest Advancements on Projects */</p>
<hr />
<div>{{:Team:Brown-Stanford/Templates/Main}}<br />
{{:Team:Brown-Stanford/Templates/Content}}<br />
{{:Team:Brown-Stanford/Templates/Twitter}}<br />
<br />
= '''APPLYING FOR STANFORD iGEM 2012''' =<br />
<br />
Please fill out our interest form at https://docs.google.com/spreadsheet/viewform?formkey=dEQyaWdReTN5S2pCcHRVY29hQXh4cHc6MQ.<br />
<br />
We will be sending further information to this list!<br />
<br />
Thanks!<br />
<br />
= '''APPLYING FOR BROWN iGEM 2012''' =<br />
(12/5/11)<br />
Brown iGEM is looking for undergrad team members for this upcoming year!<br />
<br />
Have you ever wanted to engineer living organisms to create novel functions and help transform the world? <br />
<br />
Spend a summer working at the Synthetic Biology Initiative at NASA Ames Research Center, and represent Brown at the premiere synthetic biology competition in the world! Opportunities exist for all kinds of microbiology and molecular biology work, computational biology, mathematical modeling, engineering, and much more. Develop incredible research and lab skills as you manage your own project!<br />
<br />
Students of all academic backgrounds and experience levels welcome (this includes Class of '12) <br />
<br />
We are hosting two '''INFO SESSIONS''' this week:<br />
<br />
'''TIME''': 6-7pm on Thursday 12/8, Friday 12/9<br />
<br />
'''LOCATION''': Barus and Holly 190<br />
<br />
-come hear more about iGEM, synthetic biology, and what the award-winning Brown iGEM team does; dinner will be provided!<br />
<br />
<br />
If you can't make it but are still interested, please let us know! Other questions or concerns are welcome as well. Contact us at igem@brown.edu<br />
<br />
<html><br />
<span style="font-size: 20px;"></html>Our electronic application is [http://www.brownigem.com/2012_iGEM_Application.doc available now]. Keep up with the 2012 application process on our [https://www.facebook.com/events/146221565480758/ facebook page]!<br />
<html></span></html><br />
<br />
= '''Mars BioTools: Synthetic Biology for Space Exploration''' =<br />
One of the major challenges of space exploration is the limited payload mass that can be launched on a rocket and the difficulty of resupply mid-mission. Any long term settlement will require more resources than astronauts can initially bring with them. Synthetic Biology has the potential to revolutionize space exploration and settlement. Biological tools have a major advantage over classical tools: the ability to self-replicate and regenerate.<br />
<br />
The emerging field of Synthetic Biology will allow us to engineer microbial factories that will largely circumvent the limiting payload factors. These cellular factories will generate fuel, food, medicines and building materials for settlers, but will consist of engineered cells that could be stored in a single test tube, and regrown to production scale on-site, as needed.<br />
<br />
The Brown-Stanford iGEM team is excited to work on three different projects, under the common theme of developing Synthetic Biology applications for space. In addition, as a response to the ethical and philosophical considerations, we interviewed prominent researchers and leaders in the field. '''Click on the astronaut in the corner to view our series.''' <br />
<br />
<html><br />
</div><br />
<div class="miniContent"><br />
<br />
<div class="miniContentLeft"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/3/39/Brown-Stanford_REGObricks-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/REGObricks/Introduction">REGObricks</a></h3><br />
</html><br />
<br />
Any extra-terrestrial settlement will require a habitat to keep its occupants alive, but transport of such a habitat will require a huge amount of payload space. RegoBricks uses bacteria to cement Martian and Lunar regolith (soil) simulant into durable building blocks, similar to concrete bricks, allowing settlements to use in situ resources to build structures.<br />
<html><br />
</div><br />
<br />
<div class="miniContentRight"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/4/4c/Brown-Stanford_DNADamage-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/FRETSensor/Introduction">FRETcetera</a></h3><br />
</html><br />
<br />
Biological interfaces cannot pass information to their users as fast as electronic ones can. FRETcetera develops a novel method of fast-acting biological reporting with changes in cell fluorescence. Bacteria could be used to detect toxic chemicals in the environment, for example, or inform astronauts that their microbial tools are unhealthy.<br />
<html><br />
<br />
</div><br />
<div class="miniContentCenter"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/9/9a/Brown-Stanford_PowerCell-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/PowerCell/Introduction">PowerCell</a></h3><br />
</html><br />
<br />
All biological tools need energy to run. PowerCell develops a universal energy source. Engineered photosynthetic bacteria generate carbon and nitrogenous nutrients from sunlight and air and secrete them to power biological tools. These tools will transform the raw materials into fuel, building materials, food, drugs, and other products useful to settlers.<br />
<html><br />
</div><br />
<br />
</div><br />
<div class="pageContent"><br />
</html><br />
<br />
= '''Summary''' =<br />
<br />
=== '''News!''' ===<br />
<br />
===== '''iGEM Awards''' =====<br />
<br />
We did very well at iGEM this year! <br />
<br />
-At the Americas Regional Jamboree, we finished Top 4 and won Best Presentation.<br />
<br />
-At the World Jamboree, we finished in the Sweet 16, and won Best New Application. <br />
<br />
We are extremely grateful to all those who helped us along the way, and our mentors. Thank you so much!<br />
<br />
===== '''Project Summary''' =====<br />
<br />
Here's a minute long teaser of our project!<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/watch?v=b2AkJRTLOcs"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/b2AkJRTLOcs"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Latest Advancements on Projects''' ===<br />
<br />
===== '''Anabaena Transformation and Successful Cell-Type Specific Expression Control''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 80%;"><br />
<tr><br />
<br />
<td style="padding-left: 20px; padding-right: 0px; padding-top: 0px; padding-bottom: 0px; width: 90%;"><br />
<br />
<p>We successfully transformed our cyanobacterial chassis, Anabaena PCC 7120, with our designed PowerCell genetic construct. Unlike more common cyanobacteria species such as Synechocystis PCC 6803 or Synechococcus elongatus PCC 7942, Anabaena is not naturally competent and does not easily uptake foreign DNA. We performed a triparental conjugation in order to bypass the natural barriers to transformation.</p><br />
<br />
To demonstrate that expression of our construct is properly co-localized to vegetative cells, we grew cultures of Anabaena in nitrate-free BG11 media to promote heterocyst formation. We demonstrated the successful control of our pSac promoter over our sugar secretion construct through GFP expression. The sugar secretion construct is only expressed in actively photosynthesizing cells. In the first picture, we observe areas of diminished GFP fluorescence at regular intervals (approximately 1 in 10 cells) along our transformed Anabaena filament (the background fluorescence is likely due to chlorophyll). Since our GFP reporter should only express under the pSac promoter and only in non-heterocyst cells, we would expect the spacing of dark spots to match the spacing of heterocysts identified through alcian blue staining. Alcian blue is a dye that selectively binds to polysaccharide chains on the surface of heterocyst cells. As you can see by comparing the two images, our results appear to match up!<br />
<br />
Work to characterize the performance of our cscB sucrose secretion construct is underway. We will assay sucrose levels produced by PowerCell. If they are high enough, we will attempt to grow E. coli W. containing arbitrary biobricks from this nutrient source, in an attempt to show PowerCell can power biological tools. </p><br />
<br />
<center><br />
<html><object width="400" height="300"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to="></param> <param name="movie" value="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to=" width="400" height="300"></embed></object></html><br />
<br />
<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
</div><br />
<br />
<html><object width="400" height="300"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to="></param> <param name="movie" value="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to=" width="400" height="300"></embed></object></html><br />
<br />
<div style="width: 35%;"><br />
Transformed Anabaena with heterocysts stained by alcian blue. Heterocyst spacing corresponds to the "off" cells from the GFP image, implying that the sucrose secretion construct in not active in heterocysts, as desired.<br />
</div><br />
<br />
===== '''Biobricked the urease operon''' =====<br />
<br />
We successfully PCR cloned a 10.7 kb functional urease cassette from the plasmid pBU11, whose genetic sequence has hitherto never been known before. We ligated it onto a backbone, and successfully [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biobrick made a standard BioBrick], characterizing [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Characterization its function] and demonstrating active urease activity. Urease is the catalyst behind the phenomenon known as biocement, which is able to fuse sand into bricks, using nothing more than calcium ions and urea. [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biocementation More about urease.]<br />
<br />
Biobrick part [http://partsregistry.org/Part:BBa_K656013 BBa_K656013] has been submitted to the Registry of Standardized Parts. The sequence of this cassette is unavailable because it currently does not exist in any known registry. At the time of this writing, it is currently being sequenced in the lab of Dr. Chris Mason at Weill Cornell Medical College. As soon as the sequence is available, we will supplement the directory listing.<br />
<html><br />
<br />
<br><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG"><img src="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%; font-weight:bold;"><br />
Successful transformants on urease test plates.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</html><br />
<br />
===== '''Stratospheric Balloon Flight''' =====<br />
<br />
We launched samples of our bacteria to the far edge of space (80,000 ft-110,000 ft) to test for their ability to survive simulated Martian environmental conditions. The pressures, temperatures, and radiation conditions at these altitudes are similar to the conditions on the Martian surface.<br />
[[Team:Brown-Stanford/REGObricks/Balloon|Read about our escapades here]]<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/qCQXg8DmZZ0"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/qCQXg8DmZZ0"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Advancements in Human Practices''' ===<br />
===== '''Feature on Synthetic Biology and Space''' =====<br />
We talked with key figures in science and science policy about the implications of human expansion into space, extraterrestrial life and synthetic biology. You can look at our interviews and findings [https://2011.igem.org/Team:Brown-Stanford/SynEthics/Intro here] or by clicking on the astronaut on the upper left!<br />
<br />
===== '''Education and Publicity''' =====<br />
<br />
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<table style="width: 100%;"><br />
<tr><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Editor's Choice! x2<br />
</div><br />
</center><br />
</td><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Lunar Science Poster. Click to see full version.<br />
</div><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/9YKsa7s8WM4?version=3"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/9YKsa7s8WM4?version=3"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
<br />
<br />
We educated and publicized the power of synthetic biology in a whole suite of venues and mediums, including <br />
<br />
* A crowd of 600+ over two days at [https://2011.igem.org/Team:Brown-Stanford/MakerFaire Maker Faire NYC], resulting in two Editor’s Choice blue ribbons! <br />
* A filming opportunity with the [https://2011.igem.org/Team:Brown-Stanford/BBC BBC] Horizon, to be featured in a documentary on NASA’s Initiative in Synthetic Biology<br />
*Verbal [https://2011.igem.org/Team:Brown-Stanford/NASA presentations] to NASA Administrator Charlie Bolden, NASA Ames Research Center Director Pete Worden, and writer and visionary Stewart Brand <br />
* A poster presentation at NASA’s Lunar Science Forum<br />
* A heart-pounding [https://2011.igem.org/Team:Brown-Stanford/SB5 poem] at the first-ever SB5.0 Synbio Slam<br />
<br />
===== '''Outreach and Collaboration''' =====<br />
<br />
<html><br />
<center><br />
<a href="http://openwetware.org/wiki/IGEM_Outreach"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6f/Brown-Stanford_Cbricks.jpg" style="width: 30%;"><br />
</a><br />
</center><br />
</html><br />
<br />
We spearheaded the '''largest collaborative effort''' in iGEM history to change the way we do outreach, creating [http://openwetware.org/wiki/IGEM_Outreach '''CommunityBricks'''], a database of valuable teaching plans, presentations, community resources and projects for young and old alike.<br />
<br />
===== '''Alumni and Community-Building''' =====<br />
<br />
<br />
[[File:Brown-Stanford alumnigem.png|300px|center|AlumniGEM|link=http://alumni.brownigem.com/]]<br />
<br />
We built the [http://alumni.brownigem.com '''first iGEM Alumni Network,'''] to provide iGEM'ers a growing collection of professional and social resources and keep the spirit of iGEM alive post-Jamborees.<br />
<br />
<html><br />
<div><a href="javascript:toggle();" id="toggleNews"><h3>Show Media and News</h3></a></div><br />
<br />
</div><br />
<div class="pageContent" id="news" style="display: none;"><br />
</html><br />
<br />
== '''Martian Colony''' ==<br />
[[File:Brown-Stanford_MartianColony.png|center|750px]]<br />
<br />
== '''News''' ==<br />
<br />
===In the Lab===<br />
<center><html><object width="600" height="450"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to="></param> <param name="movie" value="http://www.flickr.com/apps/slideshow/show.swf?v=107931"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://www.flickr.com/apps/slideshow/show.swf?v=107931" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to=" width="600" height="450"></embed></object></html></center><br />
<br />
=== '''August 4<sup>th</sup>, 2011''' ===<br />
<center><html><object width="580" height="355"><param name="movie" value="http://www.youtube.com/v/x-MYE4QkjOU"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/x-MYE4QkjOU" type="application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''June 7<sup>th</sup>, 2011''' ===<br />
[[File:Brown-Stanford TeamPicture6-7-2011.jpg|center|500px]]<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-StanfordTeam:Brown-Stanford2011-12-07T10:11:44Z<p>Evanclark: /* Anabaena Transformation and Successful Cell-Type Specific Expression Control */</p>
<hr />
<div>{{:Team:Brown-Stanford/Templates/Main}}<br />
{{:Team:Brown-Stanford/Templates/Content}}<br />
{{:Team:Brown-Stanford/Templates/Twitter}}<br />
<br />
= '''APPLYING FOR STANFORD iGEM 2012''' =<br />
<br />
Please fill out our interest form at https://docs.google.com/spreadsheet/viewform?formkey=dEQyaWdReTN5S2pCcHRVY29hQXh4cHc6MQ.<br />
<br />
We will be sending further information to this list!<br />
<br />
Thanks!<br />
<br />
= '''APPLYING FOR BROWN iGEM 2012''' =<br />
(12/5/11)<br />
Brown iGEM is looking for undergrad team members for this upcoming year!<br />
<br />
Have you ever wanted to engineer living organisms to create novel functions and help transform the world? <br />
<br />
Spend a summer working at the Synthetic Biology Initiative at NASA Ames Research Center, and represent Brown at the premiere synthetic biology competition in the world! Opportunities exist for all kinds of microbiology and molecular biology work, computational biology, mathematical modeling, engineering, and much more. Develop incredible research and lab skills as you manage your own project!<br />
<br />
Students of all academic backgrounds and experience levels welcome (this includes Class of '12) <br />
<br />
We are hosting two '''INFO SESSIONS''' this week:<br />
<br />
'''TIME''': 6-7pm on Thursday 12/8, Friday 12/9<br />
<br />
'''LOCATION''': Barus and Holly 190<br />
<br />
-come hear more about iGEM, synthetic biology, and what the award-winning Brown iGEM team does; dinner will be provided!<br />
<br />
<br />
If you can't make it but are still interested, please let us know! Other questions or concerns are welcome as well. Contact us at igem@brown.edu<br />
<br />
<html><br />
<span style="font-size: 20px;"></html>Our electronic application is [http://www.brownigem.com/2012_iGEM_Application.doc available now]. Keep up with the 2012 application process on our [https://www.facebook.com/events/146221565480758/ facebook page]!<br />
<html></span></html><br />
<br />
= '''Mars BioTools: Synthetic Biology for Space Exploration''' =<br />
One of the major challenges of space exploration is the limited payload mass that can be launched on a rocket and the difficulty of resupply mid-mission. Any long term settlement will require more resources than astronauts can initially bring with them. Synthetic Biology has the potential to revolutionize space exploration and settlement. Biological tools have a major advantage over classical tools: the ability to self-replicate and regenerate.<br />
<br />
The emerging field of Synthetic Biology will allow us to engineer microbial factories that will largely circumvent the limiting payload factors. These cellular factories will generate fuel, food, medicines and building materials for settlers, but will consist of engineered cells that could be stored in a single test tube, and regrown to production scale on-site, as needed.<br />
<br />
The Brown-Stanford iGEM team is excited to work on three different projects, under the common theme of developing Synthetic Biology applications for space. In addition, as a response to the ethical and philosophical considerations, we interviewed prominent researchers and leaders in the field. '''Click on the astronaut in the corner to view our series.''' <br />
<br />
<html><br />
</div><br />
<div class="miniContent"><br />
<br />
<div class="miniContentLeft"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/3/39/Brown-Stanford_REGObricks-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/REGObricks/Introduction">REGObricks</a></h3><br />
</html><br />
<br />
Any extra-terrestrial settlement will require a habitat to keep its occupants alive, but transport of such a habitat will require a huge amount of payload space. RegoBricks uses bacteria to cement Martian and Lunar regolith (soil) simulant into durable building blocks, similar to concrete bricks, allowing settlements to use in situ resources to build structures.<br />
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</div><br />
<br />
<div class="miniContentRight"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/4/4c/Brown-Stanford_DNADamage-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/FRETSensor/Introduction">FRETcetera</a></h3><br />
</html><br />
<br />
Biological interfaces cannot pass information to their users as fast as electronic ones can. FRETcetera develops a novel method of fast-acting biological reporting with changes in cell fluorescence. Bacteria could be used to detect toxic chemicals in the environment, for example, or inform astronauts that their microbial tools are unhealthy.<br />
<html><br />
<br />
</div><br />
<div class="miniContentCenter"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/9/9a/Brown-Stanford_PowerCell-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/PowerCell/Introduction">PowerCell</a></h3><br />
</html><br />
<br />
All biological tools need energy to run. PowerCell develops a universal energy source. Engineered photosynthetic bacteria generate carbon and nitrogenous nutrients from sunlight and air and secrete them to power biological tools. These tools will transform the raw materials into fuel, building materials, food, drugs, and other products useful to settlers.<br />
<html><br />
</div><br />
<br />
</div><br />
<div class="pageContent"><br />
</html><br />
<br />
= '''Summary''' =<br />
<br />
=== '''News!''' ===<br />
<br />
===== '''iGEM Awards''' =====<br />
<br />
We did very well at iGEM this year! <br />
<br />
-At the Americas Regional Jamboree, we finished Top 4 and won Best Presentation.<br />
<br />
-At the World Jamboree, we finished in the Sweet 16, and won Best New Application. <br />
<br />
We are extremely grateful to all those who helped us along the way, and our mentors. Thank you so much!<br />
<br />
===== '''Project Summary''' =====<br />
<br />
Here's a minute long teaser of our project!<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/watch?v=b2AkJRTLOcs"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/b2AkJRTLOcs"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Latest Advancements on Projects''' ===<br />
<br />
===== '''Anabaena Transformation and Successful Cell-Type Specific Expression Control''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 100%;"><br />
<tr><br />
<br />
<td style="padding-left: 20px; padding-right: 0px; padding-top: 0px; padding-bottom: 0px; width: 90%;"><br />
<br />
<p>We successfully transformed our cyanobacterial chassis, Anabaena PCC 7120, with our designed PowerCell genetic construct. Unlike more common cyanobacteria species such as Synechocystis PCC 6803 or Synechococcus elongatus PCC 7942, Anabaena is not naturally competent and does not easily uptake foreign DNA. We performed a triparental conjugation in order to bypass the natural barriers to transformation.</p><br />
<br />
To demonstrate that expression of our construct is properly co-localized to vegetative cells, we grew cultures of Anabaena in nitrate-free BG11 media to promote heterocyst formation. We demonstrated the successful control of our pSac promoter over our sugar secretion construct through GFP expression. The sugar secretion construct is only expressed in actively photosynthesizing cells. In the first picture, we observe areas of diminished GFP fluorescence at regular intervals (approximately 1 in 10 cells) along our transformed Anabaena filament (the background fluorescence is likely due to chlorophyll). Since our GFP reporter should only express under the pSac promoter and only in non-heterocyst cells, we would expect the spacing of dark spots to match the spacing of heterocysts identified through alcian blue staining. Alcian blue is a dye that selectively binds to polysaccharide chains on the surface of heterocyst cells. As you can see by comparing the two images, our results appear to match up!<br />
<br />
Work to characterize the performance of our cscB sucrose secretion construct is underway. We will assay sucrose levels produced by PowerCell. If they are high enough, we will attempt to grow E. coli W. containing arbitrary biobricks from this nutrient source, in an attempt to show PowerCell can power biological tools. </p><br />
<br />
<center><br />
<html><object width="400" height="300"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to="></param> <param name="movie" value="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to=" width="400" height="300"></embed></object></html><br />
<br />
<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
</div><br />
<br />
<html><object width="400" height="300"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to="></param> <param name="movie" value="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2Fshow%2F&page_show_back_url=%2Fphotos%2Fbrownstanfordigem%2Fsets%2F72157628308638459%2F&set_id=72157628308638459&jump_to=" width="400" height="300"></embed></object></html><br />
<br />
<div style="width: 35%;"><br />
Transformed Anabaena with heterocysts stained by alcian blue. Heterocyst spacing corresponds to the "off" cells from the GFP image, implying that the sucrose secretion construct in not active in heterocysts, as desired.<br />
</div><br />
<br />
===== '''Biobricked the urease operon''' =====<br />
<br />
We successfully PCR cloned a 10.7 kb functional urease cassette from the plasmid pBU11, whose genetic sequence has hitherto never been known before. We ligated it onto a backbone, and successfully [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biobrick made a standard BioBrick], characterizing [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Characterization its function] and demonstrating active urease activity. Urease is the catalyst behind the phenomenon known as biocement, which is able to fuse sand into bricks, using nothing more than calcium ions and urea. [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biocementation More about urease.]<br />
<br />
Biobrick part [http://partsregistry.org/Part:BBa_K656013 BBa_K656013] has been submitted to the Registry of Standardized Parts. The sequence of this cassette is unavailable because it currently does not exist in any known registry. At the time of this writing, it is currently being sequenced in the lab of Dr. Chris Mason at Weill Cornell Medical College. As soon as the sequence is available, we will supplement the directory listing.<br />
<html><br />
<br />
<br><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG"><img src="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%; font-weight:bold;"><br />
Successful transformants on urease test plates.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</html><br />
<br />
===== '''Stratospheric Balloon Flight''' =====<br />
<br />
We launched samples of our bacteria to the far edge of space (80,000 ft-110,000 ft) to test for their ability to survive simulated Martian environmental conditions. The pressures, temperatures, and radiation conditions at these altitudes are similar to the conditions on the Martian surface.<br />
[[Team:Brown-Stanford/REGObricks/Balloon|Read about our escapades here]]<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/qCQXg8DmZZ0"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/qCQXg8DmZZ0"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Advancements in Human Practices''' ===<br />
===== '''Feature on Synthetic Biology and Space''' =====<br />
We talked with key figures in science and science policy about the implications of human expansion into space, extraterrestrial life and synthetic biology. You can look at our interviews and findings [https://2011.igem.org/Team:Brown-Stanford/SynEthics/Intro here] or by clicking on the astronaut on the upper left!<br />
<br />
===== '''Education and Publicity''' =====<br />
<br />
<html><br />
<table style="width: 100%;"><br />
<tr><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Editor's Choice! x2<br />
</div><br />
</center><br />
</td><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Lunar Science Poster. Click to see full version.<br />
</div><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/9YKsa7s8WM4?version=3"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/9YKsa7s8WM4?version=3"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
<br />
<br />
We educated and publicized the power of synthetic biology in a whole suite of venues and mediums, including <br />
<br />
* A crowd of 600+ over two days at [https://2011.igem.org/Team:Brown-Stanford/MakerFaire Maker Faire NYC], resulting in two Editor’s Choice blue ribbons! <br />
* A filming opportunity with the [https://2011.igem.org/Team:Brown-Stanford/BBC BBC] Horizon, to be featured in a documentary on NASA’s Initiative in Synthetic Biology<br />
*Verbal [https://2011.igem.org/Team:Brown-Stanford/NASA presentations] to NASA Administrator Charlie Bolden, NASA Ames Research Center Director Pete Worden, and writer and visionary Stewart Brand <br />
* A poster presentation at NASA’s Lunar Science Forum<br />
* A heart-pounding [https://2011.igem.org/Team:Brown-Stanford/SB5 poem] at the first-ever SB5.0 Synbio Slam<br />
<br />
===== '''Outreach and Collaboration''' =====<br />
<br />
<html><br />
<center><br />
<a href="http://openwetware.org/wiki/IGEM_Outreach"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6f/Brown-Stanford_Cbricks.jpg" style="width: 30%;"><br />
</a><br />
</center><br />
</html><br />
<br />
We spearheaded the '''largest collaborative effort''' in iGEM history to change the way we do outreach, creating [http://openwetware.org/wiki/IGEM_Outreach '''CommunityBricks'''], a database of valuable teaching plans, presentations, community resources and projects for young and old alike.<br />
<br />
===== '''Alumni and Community-Building''' =====<br />
<br />
<br />
[[File:Brown-Stanford alumnigem.png|300px|center|AlumniGEM|link=http://alumni.brownigem.com/]]<br />
<br />
We built the [http://alumni.brownigem.com '''first iGEM Alumni Network,'''] to provide iGEM'ers a growing collection of professional and social resources and keep the spirit of iGEM alive post-Jamborees.<br />
<br />
<html><br />
<div><a href="javascript:toggle();" id="toggleNews"><h3>Show Media and News</h3></a></div><br />
<br />
</div><br />
<div class="pageContent" id="news" style="display: none;"><br />
</html><br />
<br />
== '''Martian Colony''' ==<br />
[[File:Brown-Stanford_MartianColony.png|center|750px]]<br />
<br />
== '''News''' ==<br />
<br />
===In the Lab===<br />
<center><html><object width="600" height="450"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to="></param> <param name="movie" value="http://www.flickr.com/apps/slideshow/show.swf?v=107931"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://www.flickr.com/apps/slideshow/show.swf?v=107931" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to=" width="600" height="450"></embed></object></html></center><br />
<br />
=== '''August 4<sup>th</sup>, 2011''' ===<br />
<center><html><object width="580" height="355"><param name="movie" value="http://www.youtube.com/v/x-MYE4QkjOU"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/x-MYE4QkjOU" type="application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''June 7<sup>th</sup>, 2011''' ===<br />
[[File:Brown-Stanford TeamPicture6-7-2011.jpg|center|500px]]<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-StanfordTeam:Brown-Stanford2011-12-07T10:09:42Z<p>Evanclark: /* Anabaena Transformation and Successful Cell-Type Specific Expression Control */</p>
<hr />
<div>{{:Team:Brown-Stanford/Templates/Main}}<br />
{{:Team:Brown-Stanford/Templates/Content}}<br />
{{:Team:Brown-Stanford/Templates/Twitter}}<br />
<br />
= '''APPLYING FOR STANFORD iGEM 2012''' =<br />
<br />
Please fill out our interest form at https://docs.google.com/spreadsheet/viewform?formkey=dEQyaWdReTN5S2pCcHRVY29hQXh4cHc6MQ.<br />
<br />
We will be sending further information to this list!<br />
<br />
Thanks!<br />
<br />
= '''APPLYING FOR BROWN iGEM 2012''' =<br />
(12/5/11)<br />
Brown iGEM is looking for undergrad team members for this upcoming year!<br />
<br />
Have you ever wanted to engineer living organisms to create novel functions and help transform the world? <br />
<br />
Spend a summer working at the Synthetic Biology Initiative at NASA Ames Research Center, and represent Brown at the premiere synthetic biology competition in the world! Opportunities exist for all kinds of microbiology and molecular biology work, computational biology, mathematical modeling, engineering, and much more. Develop incredible research and lab skills as you manage your own project!<br />
<br />
Students of all academic backgrounds and experience levels welcome (this includes Class of '12) <br />
<br />
We are hosting two '''INFO SESSIONS''' this week:<br />
<br />
'''TIME''': 6-7pm on Thursday 12/8, Friday 12/9<br />
<br />
'''LOCATION''': Barus and Holly 190<br />
<br />
-come hear more about iGEM, synthetic biology, and what the award-winning Brown iGEM team does; dinner will be provided!<br />
<br />
<br />
If you can't make it but are still interested, please let us know! Other questions or concerns are welcome as well. Contact us at igem@brown.edu<br />
<br />
<html><br />
<span style="font-size: 20px;"></html>Our electronic application is [http://www.brownigem.com/2012_iGEM_Application.doc available now]. Keep up with the 2012 application process on our [https://www.facebook.com/events/146221565480758/ facebook page]!<br />
<html></span></html><br />
<br />
= '''Mars BioTools: Synthetic Biology for Space Exploration''' =<br />
One of the major challenges of space exploration is the limited payload mass that can be launched on a rocket and the difficulty of resupply mid-mission. Any long term settlement will require more resources than astronauts can initially bring with them. Synthetic Biology has the potential to revolutionize space exploration and settlement. Biological tools have a major advantage over classical tools: the ability to self-replicate and regenerate.<br />
<br />
The emerging field of Synthetic Biology will allow us to engineer microbial factories that will largely circumvent the limiting payload factors. These cellular factories will generate fuel, food, medicines and building materials for settlers, but will consist of engineered cells that could be stored in a single test tube, and regrown to production scale on-site, as needed.<br />
<br />
The Brown-Stanford iGEM team is excited to work on three different projects, under the common theme of developing Synthetic Biology applications for space. In addition, as a response to the ethical and philosophical considerations, we interviewed prominent researchers and leaders in the field. '''Click on the astronaut in the corner to view our series.''' <br />
<br />
<html><br />
</div><br />
<div class="miniContent"><br />
<br />
<div class="miniContentLeft"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/3/39/Brown-Stanford_REGObricks-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/REGObricks/Introduction">REGObricks</a></h3><br />
</html><br />
<br />
Any extra-terrestrial settlement will require a habitat to keep its occupants alive, but transport of such a habitat will require a huge amount of payload space. RegoBricks uses bacteria to cement Martian and Lunar regolith (soil) simulant into durable building blocks, similar to concrete bricks, allowing settlements to use in situ resources to build structures.<br />
<html><br />
</div><br />
<br />
<div class="miniContentRight"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/4/4c/Brown-Stanford_DNADamage-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/FRETSensor/Introduction">FRETcetera</a></h3><br />
</html><br />
<br />
Biological interfaces cannot pass information to their users as fast as electronic ones can. FRETcetera develops a novel method of fast-acting biological reporting with changes in cell fluorescence. Bacteria could be used to detect toxic chemicals in the environment, for example, or inform astronauts that their microbial tools are unhealthy.<br />
<html><br />
<br />
</div><br />
<div class="miniContentCenter"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/9/9a/Brown-Stanford_PowerCell-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/PowerCell/Introduction">PowerCell</a></h3><br />
</html><br />
<br />
All biological tools need energy to run. PowerCell develops a universal energy source. Engineered photosynthetic bacteria generate carbon and nitrogenous nutrients from sunlight and air and secrete them to power biological tools. These tools will transform the raw materials into fuel, building materials, food, drugs, and other products useful to settlers.<br />
<html><br />
</div><br />
<br />
</div><br />
<div class="pageContent"><br />
</html><br />
<br />
= '''Summary''' =<br />
<br />
=== '''News!''' ===<br />
<br />
===== '''iGEM Awards''' =====<br />
<br />
We did very well at iGEM this year! <br />
<br />
-At the Americas Regional Jamboree, we finished Top 4 and won Best Presentation.<br />
<br />
-At the World Jamboree, we finished in the Sweet 16, and won Best New Application. <br />
<br />
We are extremely grateful to all those who helped us along the way, and our mentors. Thank you so much!<br />
<br />
===== '''Project Summary''' =====<br />
<br />
Here's a minute long teaser of our project!<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/watch?v=b2AkJRTLOcs"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/b2AkJRTLOcs"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Latest Advancements on Projects''' ===<br />
<br />
===== '''Anabaena Transformation and Successful Cell-Type Specific Expression Control''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 100%;"><br />
<tr><br />
<br />
<td style="padding-left: 20px; padding-right: 0px; padding-top: 0px; padding-bottom: 0px; width: 90%;"><br />
<br />
<p>We successfully transformed our cyanobacterial chassis, Anabaena PCC 7120, with our designed PowerCell genetic construct. Unlike more common cyanobacteria species such as Synechocystis PCC 6803 or Synechococcus elongatus PCC 7942, Anabaena is not naturally competent and does not easily uptake foreign DNA. We performed a triparental conjugation in order to bypass the natural barriers to transformation.</p><br />
<br />
To demonstrate that expression of our construct is properly co-localized to vegetative cells, we grew cultures of Anabaena in nitrate-free BG11 media to promote heterocyst formation. We demonstrated the successful control of our pSac promoter over our sugar secretion construct through GFP expression. The sugar secretion construct is only expressed in actively photosynthesizing cells. In the first picture, we observe areas of diminished GFP fluorescence at regular intervals (approximately 1 in 10 cells) along our transformed Anabaena filament (the background fluorescence is likely due to chlorophyll). Since our GFP reporter should only express under the pSac promoter and only in non-heterocyst cells, we would expect the spacing of dark spots to match the spacing of heterocysts identified through alcian blue staining. Alcian blue is a dye that selectively binds to polysaccharide chains on the surface of heterocyst cells. As you can see by comparing the two images, our results appear to match up!<br />
<br />
Work to characterize the performance of our cscB sucrose secretion construct is underway. We will assay sucrose levels produced by PowerCell. If they are high enough, we will attempt to grow E. coli W. containing arbitrary biobricks from this nutrient source, in an attempt to show PowerCell can power biological tools. </p><br />
<br />
<br><br />
<center><br />
<a href="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg"><img src="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
===== '''Biobricked the urease operon''' =====<br />
<br />
We successfully PCR cloned a 10.7 kb functional urease cassette from the plasmid pBU11, whose genetic sequence has hitherto never been known before. We ligated it onto a backbone, and successfully [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biobrick made a standard BioBrick], characterizing [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Characterization its function] and demonstrating active urease activity. Urease is the catalyst behind the phenomenon known as biocement, which is able to fuse sand into bricks, using nothing more than calcium ions and urea. [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biocementation More about urease.]<br />
<br />
Biobrick part [http://partsregistry.org/Part:BBa_K656013 BBa_K656013] has been submitted to the Registry of Standardized Parts. The sequence of this cassette is unavailable because it currently does not exist in any known registry. At the time of this writing, it is currently being sequenced in the lab of Dr. Chris Mason at Weill Cornell Medical College. As soon as the sequence is available, we will supplement the directory listing.<br />
<html><br />
<br />
<br><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG"><img src="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%; font-weight:bold;"><br />
Successful transformants on urease test plates.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</html><br />
<br />
===== '''Stratospheric Balloon Flight''' =====<br />
<br />
We launched samples of our bacteria to the far edge of space (80,000 ft-110,000 ft) to test for their ability to survive simulated Martian environmental conditions. The pressures, temperatures, and radiation conditions at these altitudes are similar to the conditions on the Martian surface.<br />
[[Team:Brown-Stanford/REGObricks/Balloon|Read about our escapades here]]<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/qCQXg8DmZZ0"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/qCQXg8DmZZ0"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Advancements in Human Practices''' ===<br />
===== '''Feature on Synthetic Biology and Space''' =====<br />
We talked with key figures in science and science policy about the implications of human expansion into space, extraterrestrial life and synthetic biology. You can look at our interviews and findings [https://2011.igem.org/Team:Brown-Stanford/SynEthics/Intro here] or by clicking on the astronaut on the upper left!<br />
<br />
===== '''Education and Publicity''' =====<br />
<br />
<html><br />
<table style="width: 100%;"><br />
<tr><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Editor's Choice! x2<br />
</div><br />
</center><br />
</td><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Lunar Science Poster. Click to see full version.<br />
</div><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/9YKsa7s8WM4?version=3"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/9YKsa7s8WM4?version=3"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
<br />
<br />
We educated and publicized the power of synthetic biology in a whole suite of venues and mediums, including <br />
<br />
* A crowd of 600+ over two days at [https://2011.igem.org/Team:Brown-Stanford/MakerFaire Maker Faire NYC], resulting in two Editor’s Choice blue ribbons! <br />
* A filming opportunity with the [https://2011.igem.org/Team:Brown-Stanford/BBC BBC] Horizon, to be featured in a documentary on NASA’s Initiative in Synthetic Biology<br />
*Verbal [https://2011.igem.org/Team:Brown-Stanford/NASA presentations] to NASA Administrator Charlie Bolden, NASA Ames Research Center Director Pete Worden, and writer and visionary Stewart Brand <br />
* A poster presentation at NASA’s Lunar Science Forum<br />
* A heart-pounding [https://2011.igem.org/Team:Brown-Stanford/SB5 poem] at the first-ever SB5.0 Synbio Slam<br />
<br />
===== '''Outreach and Collaboration''' =====<br />
<br />
<html><br />
<center><br />
<a href="http://openwetware.org/wiki/IGEM_Outreach"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6f/Brown-Stanford_Cbricks.jpg" style="width: 30%;"><br />
</a><br />
</center><br />
</html><br />
<br />
We spearheaded the '''largest collaborative effort''' in iGEM history to change the way we do outreach, creating [http://openwetware.org/wiki/IGEM_Outreach '''CommunityBricks'''], a database of valuable teaching plans, presentations, community resources and projects for young and old alike.<br />
<br />
===== '''Alumni and Community-Building''' =====<br />
<br />
<br />
[[File:Brown-Stanford alumnigem.png|300px|center|AlumniGEM|link=http://alumni.brownigem.com/]]<br />
<br />
We built the [http://alumni.brownigem.com '''first iGEM Alumni Network,'''] to provide iGEM'ers a growing collection of professional and social resources and keep the spirit of iGEM alive post-Jamborees.<br />
<br />
<html><br />
<div><a href="javascript:toggle();" id="toggleNews"><h3>Show Media and News</h3></a></div><br />
<br />
</div><br />
<div class="pageContent" id="news" style="display: none;"><br />
</html><br />
<br />
== '''Martian Colony''' ==<br />
[[File:Brown-Stanford_MartianColony.png|center|750px]]<br />
<br />
== '''News''' ==<br />
<br />
===In the Lab===<br />
<center><html><object width="600" height="450"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to="></param> <param name="movie" value="http://www.flickr.com/apps/slideshow/show.swf?v=107931"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://www.flickr.com/apps/slideshow/show.swf?v=107931" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to=" width="600" height="450"></embed></object></html></center><br />
<br />
=== '''August 4<sup>th</sup>, 2011''' ===<br />
<center><html><object width="580" height="355"><param name="movie" value="http://www.youtube.com/v/x-MYE4QkjOU"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/x-MYE4QkjOU" type="application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''June 7<sup>th</sup>, 2011''' ===<br />
[[File:Brown-Stanford TeamPicture6-7-2011.jpg|center|500px]]<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-StanfordTeam:Brown-Stanford2011-12-07T10:09:09Z<p>Evanclark: /* Anabaena Transformation and Successful Cell-Type Specific Expression Control */</p>
<hr />
<div>{{:Team:Brown-Stanford/Templates/Main}}<br />
{{:Team:Brown-Stanford/Templates/Content}}<br />
{{:Team:Brown-Stanford/Templates/Twitter}}<br />
<br />
= '''APPLYING FOR STANFORD iGEM 2012''' =<br />
<br />
Please fill out our interest form at https://docs.google.com/spreadsheet/viewform?formkey=dEQyaWdReTN5S2pCcHRVY29hQXh4cHc6MQ.<br />
<br />
We will be sending further information to this list!<br />
<br />
Thanks!<br />
<br />
= '''APPLYING FOR BROWN iGEM 2012''' =<br />
(12/5/11)<br />
Brown iGEM is looking for undergrad team members for this upcoming year!<br />
<br />
Have you ever wanted to engineer living organisms to create novel functions and help transform the world? <br />
<br />
Spend a summer working at the Synthetic Biology Initiative at NASA Ames Research Center, and represent Brown at the premiere synthetic biology competition in the world! Opportunities exist for all kinds of microbiology and molecular biology work, computational biology, mathematical modeling, engineering, and much more. Develop incredible research and lab skills as you manage your own project!<br />
<br />
Students of all academic backgrounds and experience levels welcome (this includes Class of '12) <br />
<br />
We are hosting two '''INFO SESSIONS''' this week:<br />
<br />
'''TIME''': 6-7pm on Thursday 12/8, Friday 12/9<br />
<br />
'''LOCATION''': Barus and Holly 190<br />
<br />
-come hear more about iGEM, synthetic biology, and what the award-winning Brown iGEM team does; dinner will be provided!<br />
<br />
<br />
If you can't make it but are still interested, please let us know! Other questions or concerns are welcome as well. Contact us at igem@brown.edu<br />
<br />
<html><br />
<span style="font-size: 20px;"></html>Our electronic application is [http://www.brownigem.com/2012_iGEM_Application.doc available now]. Keep up with the 2012 application process on our [https://www.facebook.com/events/146221565480758/ facebook page]!<br />
<html></span></html><br />
<br />
= '''Mars BioTools: Synthetic Biology for Space Exploration''' =<br />
One of the major challenges of space exploration is the limited payload mass that can be launched on a rocket and the difficulty of resupply mid-mission. Any long term settlement will require more resources than astronauts can initially bring with them. Synthetic Biology has the potential to revolutionize space exploration and settlement. Biological tools have a major advantage over classical tools: the ability to self-replicate and regenerate.<br />
<br />
The emerging field of Synthetic Biology will allow us to engineer microbial factories that will largely circumvent the limiting payload factors. These cellular factories will generate fuel, food, medicines and building materials for settlers, but will consist of engineered cells that could be stored in a single test tube, and regrown to production scale on-site, as needed.<br />
<br />
The Brown-Stanford iGEM team is excited to work on three different projects, under the common theme of developing Synthetic Biology applications for space. In addition, as a response to the ethical and philosophical considerations, we interviewed prominent researchers and leaders in the field. '''Click on the astronaut in the corner to view our series.''' <br />
<br />
<html><br />
</div><br />
<div class="miniContent"><br />
<br />
<div class="miniContentLeft"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/3/39/Brown-Stanford_REGObricks-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/REGObricks/Introduction">REGObricks</a></h3><br />
</html><br />
<br />
Any extra-terrestrial settlement will require a habitat to keep its occupants alive, but transport of such a habitat will require a huge amount of payload space. RegoBricks uses bacteria to cement Martian and Lunar regolith (soil) simulant into durable building blocks, similar to concrete bricks, allowing settlements to use in situ resources to build structures.<br />
<html><br />
</div><br />
<br />
<div class="miniContentRight"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/4/4c/Brown-Stanford_DNADamage-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/FRETSensor/Introduction">FRETcetera</a></h3><br />
</html><br />
<br />
Biological interfaces cannot pass information to their users as fast as electronic ones can. FRETcetera develops a novel method of fast-acting biological reporting with changes in cell fluorescence. Bacteria could be used to detect toxic chemicals in the environment, for example, or inform astronauts that their microbial tools are unhealthy.<br />
<html><br />
<br />
</div><br />
<div class="miniContentCenter"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/9/9a/Brown-Stanford_PowerCell-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/PowerCell/Introduction">PowerCell</a></h3><br />
</html><br />
<br />
All biological tools need energy to run. PowerCell develops a universal energy source. Engineered photosynthetic bacteria generate carbon and nitrogenous nutrients from sunlight and air and secrete them to power biological tools. These tools will transform the raw materials into fuel, building materials, food, drugs, and other products useful to settlers.<br />
<html><br />
</div><br />
<br />
</div><br />
<div class="pageContent"><br />
</html><br />
<br />
= '''Summary''' =<br />
<br />
=== '''News!''' ===<br />
<br />
===== '''iGEM Awards''' =====<br />
<br />
We did very well at iGEM this year! <br />
<br />
-At the Americas Regional Jamboree, we finished Top 4 and won Best Presentation.<br />
<br />
-At the World Jamboree, we finished in the Sweet 16, and won Best New Application. <br />
<br />
We are extremely grateful to all those who helped us along the way, and our mentors. Thank you so much!<br />
<br />
===== '''Project Summary''' =====<br />
<br />
Here's a minute long teaser of our project!<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/watch?v=b2AkJRTLOcs"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/b2AkJRTLOcs"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Latest Advancements on Projects''' ===<br />
<br />
===== '''Anabaena Transformation and Successful Cell-Type Specific Expression Control''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 100%;"><br />
<tr><br />
<br />
<td style="padding-left: 20px; padding-right: 0px; padding-top: 0px; padding-bottom: 0px; width: 90%;"><br />
<br />
<p>We successfully transformed our cyanobacterial chassis, Anabaena PCC 7120, with our designed PowerCell genetic construct. Unlike more common cyanobacteria species such as Synechocystis PCC 6803 or Synechococcus elongatus PCC 7942, Anabaena is not naturally competent and does not easily uptake foreign DNA. We performed a triparental conjugation in order to bypass the natural barriers to transformation.</p><br />
<br />
To demonstrate that expression of our construct is properly co-localized to vegetative cells, we grew cultures of Anabaena in nitrate-free BG11 media to promote heterocyst formation. We demonstrated the successful control of our pSac promoter over our sugar secretion construct through GFP expression. The sugar secretion construct is only expressed in actively photosynthesizing cells. In the first picture, we observe areas of diminished GFP fluorescence at regular intervals (approximately 1 in 10 cells) along our transformed Anabaena filament (the background fluorescence is likely due to chlorophyll). Since our GFP reporter should only express under the pSac promoter and only in non-heterocyst cells, we would expect the spacing of dark spots to match the spacing of heterocysts identified through alcian blue staining. Alcian blue is a dye that selectively binds to polysaccharide chains on the surface of heterocyst cells. As you can see by comparing the two images, our results appear to match up!<br />
<br />
Work to characterize the performance of our cscB sucrose secretion construct is underway. We will assay sucrose levels produced by PowerCell. If they are high enough, we will attempt to grow E. coli W. containing arbitrary biobricks from this nutrient source, in an attempt to show PowerCell can power biological tools. </p><br />
<br />
<br><br />
<center><br />
<a href="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg"><img src="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<br><br />
<center><br />
<a href="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg"><img src="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
===== '''Biobricked the urease operon''' =====<br />
<br />
We successfully PCR cloned a 10.7 kb functional urease cassette from the plasmid pBU11, whose genetic sequence has hitherto never been known before. We ligated it onto a backbone, and successfully [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biobrick made a standard BioBrick], characterizing [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Characterization its function] and demonstrating active urease activity. Urease is the catalyst behind the phenomenon known as biocement, which is able to fuse sand into bricks, using nothing more than calcium ions and urea. [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biocementation More about urease.]<br />
<br />
Biobrick part [http://partsregistry.org/Part:BBa_K656013 BBa_K656013] has been submitted to the Registry of Standardized Parts. The sequence of this cassette is unavailable because it currently does not exist in any known registry. At the time of this writing, it is currently being sequenced in the lab of Dr. Chris Mason at Weill Cornell Medical College. As soon as the sequence is available, we will supplement the directory listing.<br />
<html><br />
<br />
<br><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG"><img src="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%; font-weight:bold;"><br />
Successful transformants on urease test plates.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</html><br />
<br />
===== '''Stratospheric Balloon Flight''' =====<br />
<br />
We launched samples of our bacteria to the far edge of space (80,000 ft-110,000 ft) to test for their ability to survive simulated Martian environmental conditions. The pressures, temperatures, and radiation conditions at these altitudes are similar to the conditions on the Martian surface.<br />
[[Team:Brown-Stanford/REGObricks/Balloon|Read about our escapades here]]<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/qCQXg8DmZZ0"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/qCQXg8DmZZ0"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Advancements in Human Practices''' ===<br />
===== '''Feature on Synthetic Biology and Space''' =====<br />
We talked with key figures in science and science policy about the implications of human expansion into space, extraterrestrial life and synthetic biology. You can look at our interviews and findings [https://2011.igem.org/Team:Brown-Stanford/SynEthics/Intro here] or by clicking on the astronaut on the upper left!<br />
<br />
===== '''Education and Publicity''' =====<br />
<br />
<html><br />
<table style="width: 100%;"><br />
<tr><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Editor's Choice! x2<br />
</div><br />
</center><br />
</td><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Lunar Science Poster. Click to see full version.<br />
</div><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/9YKsa7s8WM4?version=3"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/9YKsa7s8WM4?version=3"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
<br />
<br />
We educated and publicized the power of synthetic biology in a whole suite of venues and mediums, including <br />
<br />
* A crowd of 600+ over two days at [https://2011.igem.org/Team:Brown-Stanford/MakerFaire Maker Faire NYC], resulting in two Editor’s Choice blue ribbons! <br />
* A filming opportunity with the [https://2011.igem.org/Team:Brown-Stanford/BBC BBC] Horizon, to be featured in a documentary on NASA’s Initiative in Synthetic Biology<br />
*Verbal [https://2011.igem.org/Team:Brown-Stanford/NASA presentations] to NASA Administrator Charlie Bolden, NASA Ames Research Center Director Pete Worden, and writer and visionary Stewart Brand <br />
* A poster presentation at NASA’s Lunar Science Forum<br />
* A heart-pounding [https://2011.igem.org/Team:Brown-Stanford/SB5 poem] at the first-ever SB5.0 Synbio Slam<br />
<br />
===== '''Outreach and Collaboration''' =====<br />
<br />
<html><br />
<center><br />
<a href="http://openwetware.org/wiki/IGEM_Outreach"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6f/Brown-Stanford_Cbricks.jpg" style="width: 30%;"><br />
</a><br />
</center><br />
</html><br />
<br />
We spearheaded the '''largest collaborative effort''' in iGEM history to change the way we do outreach, creating [http://openwetware.org/wiki/IGEM_Outreach '''CommunityBricks'''], a database of valuable teaching plans, presentations, community resources and projects for young and old alike.<br />
<br />
===== '''Alumni and Community-Building''' =====<br />
<br />
<br />
[[File:Brown-Stanford alumnigem.png|300px|center|AlumniGEM|link=http://alumni.brownigem.com/]]<br />
<br />
We built the [http://alumni.brownigem.com '''first iGEM Alumni Network,'''] to provide iGEM'ers a growing collection of professional and social resources and keep the spirit of iGEM alive post-Jamborees.<br />
<br />
<html><br />
<div><a href="javascript:toggle();" id="toggleNews"><h3>Show Media and News</h3></a></div><br />
<br />
</div><br />
<div class="pageContent" id="news" style="display: none;"><br />
</html><br />
<br />
== '''Martian Colony''' ==<br />
[[File:Brown-Stanford_MartianColony.png|center|750px]]<br />
<br />
== '''News''' ==<br />
<br />
===In the Lab===<br />
<center><html><object width="600" height="450"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to="></param> <param name="movie" value="http://www.flickr.com/apps/slideshow/show.swf?v=107931"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://www.flickr.com/apps/slideshow/show.swf?v=107931" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to=" width="600" height="450"></embed></object></html></center><br />
<br />
=== '''August 4<sup>th</sup>, 2011''' ===<br />
<center><html><object width="580" height="355"><param name="movie" value="http://www.youtube.com/v/x-MYE4QkjOU"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/x-MYE4QkjOU" type="application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''June 7<sup>th</sup>, 2011''' ===<br />
[[File:Brown-Stanford TeamPicture6-7-2011.jpg|center|500px]]<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-StanfordTeam:Brown-Stanford2011-12-07T10:08:41Z<p>Evanclark: /* Latest Advancements on Projects */</p>
<hr />
<div>{{:Team:Brown-Stanford/Templates/Main}}<br />
{{:Team:Brown-Stanford/Templates/Content}}<br />
{{:Team:Brown-Stanford/Templates/Twitter}}<br />
<br />
= '''APPLYING FOR STANFORD iGEM 2012''' =<br />
<br />
Please fill out our interest form at https://docs.google.com/spreadsheet/viewform?formkey=dEQyaWdReTN5S2pCcHRVY29hQXh4cHc6MQ.<br />
<br />
We will be sending further information to this list!<br />
<br />
Thanks!<br />
<br />
= '''APPLYING FOR BROWN iGEM 2012''' =<br />
(12/5/11)<br />
Brown iGEM is looking for undergrad team members for this upcoming year!<br />
<br />
Have you ever wanted to engineer living organisms to create novel functions and help transform the world? <br />
<br />
Spend a summer working at the Synthetic Biology Initiative at NASA Ames Research Center, and represent Brown at the premiere synthetic biology competition in the world! Opportunities exist for all kinds of microbiology and molecular biology work, computational biology, mathematical modeling, engineering, and much more. Develop incredible research and lab skills as you manage your own project!<br />
<br />
Students of all academic backgrounds and experience levels welcome (this includes Class of '12) <br />
<br />
We are hosting two '''INFO SESSIONS''' this week:<br />
<br />
'''TIME''': 6-7pm on Thursday 12/8, Friday 12/9<br />
<br />
'''LOCATION''': Barus and Holly 190<br />
<br />
-come hear more about iGEM, synthetic biology, and what the award-winning Brown iGEM team does; dinner will be provided!<br />
<br />
<br />
If you can't make it but are still interested, please let us know! Other questions or concerns are welcome as well. Contact us at igem@brown.edu<br />
<br />
<html><br />
<span style="font-size: 20px;"></html>Our electronic application is [http://www.brownigem.com/2012_iGEM_Application.doc available now]. Keep up with the 2012 application process on our [https://www.facebook.com/events/146221565480758/ facebook page]!<br />
<html></span></html><br />
<br />
= '''Mars BioTools: Synthetic Biology for Space Exploration''' =<br />
One of the major challenges of space exploration is the limited payload mass that can be launched on a rocket and the difficulty of resupply mid-mission. Any long term settlement will require more resources than astronauts can initially bring with them. Synthetic Biology has the potential to revolutionize space exploration and settlement. Biological tools have a major advantage over classical tools: the ability to self-replicate and regenerate.<br />
<br />
The emerging field of Synthetic Biology will allow us to engineer microbial factories that will largely circumvent the limiting payload factors. These cellular factories will generate fuel, food, medicines and building materials for settlers, but will consist of engineered cells that could be stored in a single test tube, and regrown to production scale on-site, as needed.<br />
<br />
The Brown-Stanford iGEM team is excited to work on three different projects, under the common theme of developing Synthetic Biology applications for space. In addition, as a response to the ethical and philosophical considerations, we interviewed prominent researchers and leaders in the field. '''Click on the astronaut in the corner to view our series.''' <br />
<br />
<html><br />
</div><br />
<div class="miniContent"><br />
<br />
<div class="miniContentLeft"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/3/39/Brown-Stanford_REGObricks-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/REGObricks/Introduction">REGObricks</a></h3><br />
</html><br />
<br />
Any extra-terrestrial settlement will require a habitat to keep its occupants alive, but transport of such a habitat will require a huge amount of payload space. RegoBricks uses bacteria to cement Martian and Lunar regolith (soil) simulant into durable building blocks, similar to concrete bricks, allowing settlements to use in situ resources to build structures.<br />
<html><br />
</div><br />
<br />
<div class="miniContentRight"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/4/4c/Brown-Stanford_DNADamage-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/FRETSensor/Introduction">FRETcetera</a></h3><br />
</html><br />
<br />
Biological interfaces cannot pass information to their users as fast as electronic ones can. FRETcetera develops a novel method of fast-acting biological reporting with changes in cell fluorescence. Bacteria could be used to detect toxic chemicals in the environment, for example, or inform astronauts that their microbial tools are unhealthy.<br />
<html><br />
<br />
</div><br />
<div class="miniContentCenter"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/9/9a/Brown-Stanford_PowerCell-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/PowerCell/Introduction">PowerCell</a></h3><br />
</html><br />
<br />
All biological tools need energy to run. PowerCell develops a universal energy source. Engineered photosynthetic bacteria generate carbon and nitrogenous nutrients from sunlight and air and secrete them to power biological tools. These tools will transform the raw materials into fuel, building materials, food, drugs, and other products useful to settlers.<br />
<html><br />
</div><br />
<br />
</div><br />
<div class="pageContent"><br />
</html><br />
<br />
= '''Summary''' =<br />
<br />
=== '''News!''' ===<br />
<br />
===== '''iGEM Awards''' =====<br />
<br />
We did very well at iGEM this year! <br />
<br />
-At the Americas Regional Jamboree, we finished Top 4 and won Best Presentation.<br />
<br />
-At the World Jamboree, we finished in the Sweet 16, and won Best New Application. <br />
<br />
We are extremely grateful to all those who helped us along the way, and our mentors. Thank you so much!<br />
<br />
===== '''Project Summary''' =====<br />
<br />
Here's a minute long teaser of our project!<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/watch?v=b2AkJRTLOcs"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/b2AkJRTLOcs"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Latest Advancements on Projects''' ===<br />
<br />
===== '''Anabaena Transformation and Successful Cell-Type Specific Expression Control''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 100%;"><br />
<tr><br />
<br />
<td style="padding-left: 20px; padding-right: 0px; padding-top: 0px; padding-bottom: 0px; width: 90%;"><br />
<br />
<p>We successfully transformed our cyanobacterial chassis, Anabaena PCC 7120, with our designed PowerCell genetic construct. Unlike more common cyanobacteria species such as Synechocystis PCC 6803 or Synechococcus elongatus PCC 7942, Anabaena is not naturally competent and does not easily uptake foreign DNA. We performed a triparental conjugation in order to bypass the natural barriers to transformation.</p><br />
<br />
To demonstrate that expression of our construct is properly co-localized to vegetative cells, we grew cultures of Anabaena in nitrate-free BG11 media to promote heterocyst formation. We demonstrated the successful control of our pSac promoter over our sugar secretion construct through GFP expression. The sugar secretion construct is only expressed in actively photosynthesizing cells. In the first picture, we observe areas of diminished GFP fluorescence at regular intervals (approximately 1 in 10 cells) along our transformed Anabaena filament (the background fluorescence is likely due to chlorophyll). Since our GFP reporter should only express under the pSac promoter and only in non-heterocyst cells, we would expect the spacing of dark spots to match the spacing of heterocysts identified through alcian blue staining. Alcian blue is a dye that selectively binds to polysaccharide chains on the surface of heterocyst cells. As you can see by comparing the two images, our results appear to match up!<br />
<br />
Work to characterize the performance of our cscB sucrose secretion construct is underway. We will assay sucrose levels produced by PowerCell. If they are high enough, we will attempt to grow E. coli W. containing arbitrary biobricks from this nutrient source, in an attempt to show PowerCell can power biological tools. </p><br />
<br />
<br><br />
<center><br />
<a href="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg"><img src="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<br />
===== '''Biobricked the urease operon''' =====<br />
<br />
We successfully PCR cloned a 10.7 kb functional urease cassette from the plasmid pBU11, whose genetic sequence has hitherto never been known before. We ligated it onto a backbone, and successfully [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biobrick made a standard BioBrick], characterizing [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Characterization its function] and demonstrating active urease activity. Urease is the catalyst behind the phenomenon known as biocement, which is able to fuse sand into bricks, using nothing more than calcium ions and urea. [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biocementation More about urease.]<br />
<br />
Biobrick part [http://partsregistry.org/Part:BBa_K656013 BBa_K656013] has been submitted to the Registry of Standardized Parts. The sequence of this cassette is unavailable because it currently does not exist in any known registry. At the time of this writing, it is currently being sequenced in the lab of Dr. Chris Mason at Weill Cornell Medical College. As soon as the sequence is available, we will supplement the directory listing.<br />
<html><br />
<br />
<br><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG"><img src="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG" style="width:40%"><br />
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Successful transformants on urease test plates.<br />
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<br />
===== '''Stratospheric Balloon Flight''' =====<br />
<br />
We launched samples of our bacteria to the far edge of space (80,000 ft-110,000 ft) to test for their ability to survive simulated Martian environmental conditions. The pressures, temperatures, and radiation conditions at these altitudes are similar to the conditions on the Martian surface.<br />
[[Team:Brown-Stanford/REGObricks/Balloon|Read about our escapades here]]<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/qCQXg8DmZZ0"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/qCQXg8DmZZ0"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Advancements in Human Practices''' ===<br />
===== '''Feature on Synthetic Biology and Space''' =====<br />
We talked with key figures in science and science policy about the implications of human expansion into space, extraterrestrial life and synthetic biology. You can look at our interviews and findings [https://2011.igem.org/Team:Brown-Stanford/SynEthics/Intro here] or by clicking on the astronaut on the upper left!<br />
<br />
===== '''Education and Publicity''' =====<br />
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<a href="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg"><br />
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<div style="width: 70%; font-weight: bold;"><br />
Editor's Choice! x2<br />
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<a href="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg" style="width:80%;"><br />
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<div style="width: 70%; font-weight: bold;"><br />
Lunar Science Poster. Click to see full version.<br />
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<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/9YKsa7s8WM4?version=3"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/9YKsa7s8WM4?version=3"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
<br />
<br />
We educated and publicized the power of synthetic biology in a whole suite of venues and mediums, including <br />
<br />
* A crowd of 600+ over two days at [https://2011.igem.org/Team:Brown-Stanford/MakerFaire Maker Faire NYC], resulting in two Editor’s Choice blue ribbons! <br />
* A filming opportunity with the [https://2011.igem.org/Team:Brown-Stanford/BBC BBC] Horizon, to be featured in a documentary on NASA’s Initiative in Synthetic Biology<br />
*Verbal [https://2011.igem.org/Team:Brown-Stanford/NASA presentations] to NASA Administrator Charlie Bolden, NASA Ames Research Center Director Pete Worden, and writer and visionary Stewart Brand <br />
* A poster presentation at NASA’s Lunar Science Forum<br />
* A heart-pounding [https://2011.igem.org/Team:Brown-Stanford/SB5 poem] at the first-ever SB5.0 Synbio Slam<br />
<br />
===== '''Outreach and Collaboration''' =====<br />
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<center><br />
<a href="http://openwetware.org/wiki/IGEM_Outreach"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6f/Brown-Stanford_Cbricks.jpg" style="width: 30%;"><br />
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<br />
We spearheaded the '''largest collaborative effort''' in iGEM history to change the way we do outreach, creating [http://openwetware.org/wiki/IGEM_Outreach '''CommunityBricks'''], a database of valuable teaching plans, presentations, community resources and projects for young and old alike.<br />
<br />
===== '''Alumni and Community-Building''' =====<br />
<br />
<br />
[[File:Brown-Stanford alumnigem.png|300px|center|AlumniGEM|link=http://alumni.brownigem.com/]]<br />
<br />
We built the [http://alumni.brownigem.com '''first iGEM Alumni Network,'''] to provide iGEM'ers a growing collection of professional and social resources and keep the spirit of iGEM alive post-Jamborees.<br />
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<div><a href="javascript:toggle();" id="toggleNews"><h3>Show Media and News</h3></a></div><br />
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<br />
== '''Martian Colony''' ==<br />
[[File:Brown-Stanford_MartianColony.png|center|750px]]<br />
<br />
== '''News''' ==<br />
<br />
===In the Lab===<br />
<center><html><object width="600" height="450"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to="></param> <param name="movie" value="http://www.flickr.com/apps/slideshow/show.swf?v=107931"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://www.flickr.com/apps/slideshow/show.swf?v=107931" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to=" width="600" height="450"></embed></object></html></center><br />
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=== '''August 4<sup>th</sup>, 2011''' ===<br />
<center><html><object width="580" height="355"><param name="movie" value="http://www.youtube.com/v/x-MYE4QkjOU"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/x-MYE4QkjOU" type="application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''June 7<sup>th</sup>, 2011''' ===<br />
[[File:Brown-Stanford TeamPicture6-7-2011.jpg|center|500px]]<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-Stanford/PowerCell/NutrientSecretionTeam:Brown-Stanford/PowerCell/NutrientSecretion2011-12-07T09:55:32Z<p>Evanclark: /* PowerCell Transformation */</p>
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<li><a href="/Team:Brown-Stanford/PowerCell/Introduction">Introduction</a></li><br />
<li><a href="/Team:Brown-Stanford/PowerCell/Cyanobacteria">Cyanobacteria</a></li><br />
<li><a href="/Team:Brown-Stanford/PowerCell/Background">Photosynthesis on Mars</a></li><br />
<li id="active"><a href="#" id="current">Nutrient Secretion and Utilization</a></li><br />
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{{:Team:Brown-Stanford/Templates/Content}}<br />
<br />
== '''Nutrient Secretion and Utilization''' ==<br />
<br />
<br />
<br />
PowerCell is the generator that will power all of the BioTools in a Martian settlement. PowerCell will secrete nutrients for use by other biological tools a source of energy and raw materials to construct useful products.<br />
<br />
[[File:Brown-Stanford_Battery.png|200px|right]]<br />
<br />
Like any other tools, biological tools need energy to run and raw materials to work with. The two major nutrient sources needed for biological tools are carbon sources (sugars) and nitrogenous nutrients (ammonia). In a Martian settlement, PowerCell will harness the energy of the sun to harvest these nutrients directly from the atmosphere and provide them to the settlement's biological tools.<br />
<br />
This summer, we tackled only the half involving sugar secretion. This was partially to due to the time constraints of a summer project, and partially because there is evidence that ammonia secretion occurs in cyanobacteria by means of passive diffusion without the need for genetic tinkering ([http://www.sciencedirect.com/science/article/pii/0378109791906924 ''Ammonia translocation in cyanobacteria'']). More research needs to be done to determine if this level of diffusion is sufficient to sustain other biological tools. In any case, we designed our system on a [https://2011.igem.org/Team:Brown-Stanford/PowerCell/Cyanobacteria platform] (''Anabaena 7120'') with the capability to fix atmospheric nitrogen, so that this avenue is open for exploration in the future.<br />
<br />
=== Sucrose ===<br />
<br />
The sugar we chose to secrete is sucrose. The major inspiration for our project was the paper [http://aem.asm.org/cgi/content/abstract/76/11/3462 ''Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products'']. This work was done in Dr. Pamela Silver's lab, and describes a glucose/fructose secretion device, achieved in the single-celled bacterium ''S. elongatus''.<br />
<br />
[[File:Brown-Stanford invA.png|300px|right|thumb|Sugar secretion ''Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products''. ''Niederholtmeyer et al.'']]<br />
<br />
In a nutshell, ''S. elongatus'' were forced under salt stress to produce sucrose, which was broken down into glucose and fructose by the glf enzyme, then transported out of the cell by the invA transporter.<br />
<br />
At the Fifth International Conference on Synthetic Biology, [https://2011.igem.org/Team:Brown-Stanford/SB5 SB5.0], we spoke with a member of Dr. Silver's lab, Danny Ducat. He advised us that one of the problems with the glucose/fructose secretion system is its low yield. He suspected that because glucose and fructose are directly metabolizable by the cell, much of these sugars are consumed by the cell before they ever have a chance to be secreted. For this reason, it may be better to directly secrete sucrose, which is not metabolizable by the cell, and worry about breaking it down later. This is what we did, although the choice does have some ramifications.<br />
<br />
Because ''E. coli'' is the a primary host species for genetic modification, it is likely that many biological tools for space exploration will be designed in ''E. coli''. The most common laboratory strains of ''E. coli'' , TOP10 and K12, cannot directly metabolize sucrose. For this reason, they cannot make use of PowerCell's output directly. In order to utilize this output, sucrose will need to be broken down into glucose and fructose , or biological tools that intend to utilize PowerCell should be hosted by a strain that can directly metabolize sucrose. ''E. coli W'' is a safe and easily transformable laboratory strain with this ability, and, as outlined in [http://www.biomedcentral.com/1471-2164/12/9#IDAJ1FOQ ''The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli''], may confer some other advantages to biological tools to be used in space, including including fast growth, stress tolerance, growth to high cell densities. Furthermore, there is some indication that sucrose as a feedstock can confer other benefits, such as oxidative, heat, and acid stress ([http://www.springerlink.com/content/b80870618103w67l/ ''Development of sucrose-utilizing Escherichia coli K-12 strain by cloning β-fructofuranosidases and its application for l-threonine production'']).<br />
<br />
=== PowerCell Contruct Design ===<br />
<br />
We wanted to isolate sucrose secretion to just vegetative cells, as heterocysts do not participate in production of sucrose through photosynthesis. A similar cell-type-specific gene expression was was achieved in [http://www.sciencedirect.com/science/article/pii/S0167701204001745 ''Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression in Nostoc punctiforme'']. Arguetta et al. confined expression of a fluorescent marker to only vegetative cells in a similar filamentous diazotrophic cyanobacterium, ''Nostoc Punctiforme''. They placed the GFP gene behind a promoter present in the Photosystem I of ''Nostoc punctiforme'', psaC. This way, GFP expression was only turned on in photosynthesizing cells, and not in heterocysts. They did not know the exact sequence of the psaC promoter, so they just took the 400 base pairs upstream of the psaC gene as a unit containing the promoter. We have done similarly to isolate the psaC promoter from ''Anabaena 7120''. Below is an image showing cell-type-specific GFP expression achieved in the paper. <br />
<br />
<br />
[[File:Brown-Stanford Cell-Type-Specific.png|672px|center|thumb|Cell-type-specific GFP expression in ''Nostoc punctiforme''. Arguetta et al.]]<br />
<br />
<br />
We placed a sucrose symporter gene, cscB, behind our ''Anabaena 7120'' psaC promoter in order to confine sucrose secretion to only vegetative cells. Registry standard RBSes and terminators were used for ease of assembly and compatibility. Below is a diagram of our sucrose secretion device. A discussion of the inner workings of the cscB gene can be found in [http://www.sciencedirect.com/science/article/pii/S0006291X85714490 ''Active Transport by the CscB Permease in Escherichia coli K-12''].<br />
<br />
<br />
[[Image:Brown-Stanford cscB.jpg|700px|center]]<br />
<br />
<br />
We have also created several other constructs containing GFP for debugging purposes. We used a modified version of GFP, GFPmut3B because normal florescence markers are easily lost among background chlorophyll pigments in cyanobacteria ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860132/ ''Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology'']). <br />
<br />
<br />
[[File:Brown-Stanford GFP.jpg|700px|center]]<br />
<br />
<br />
[[File:Brown-Stanford cscB GFP.jpg|700px|center|thumb|Final PowerCell Construct: http://partsregistry.org/Part:BBa_K656012]]<br />
<br />
=='''Tranformation into Anabaena 7120'''==<br />
<br />
Cyanobacterial transformation can be difficult.<br />
<br />
There are cyanobacteria which will accept DNA without complaint. Many model cyanobacterium species, such as ''Synechococcus elongatus'' or ''Synechocystis'' PCC6803, for example, will simply take up naked DNA in solution and express it. <br />
<br />
''Anabaena 7120'', on the other hand, must take its DNA through a rather circuitous path; the DNA construct must first be placed in a cargo plasmid and transformed into ''E. coli'' by traditional means. Transfer to ''Anabaena'' takes place by conjugation, facilitated by a second ''E. coli'' strain carrying a plasmid encoding the machinery for bacterial conjugation. <br />
<br />
Another problem is the propensity of ''Anabaena'' to slice and dice foreign DNA with isoschizomers of the restriction enzymes AvaI, AvaII and AvaIII. This has been addressed with methyltransferases targeting the same sequences; that means a third ''E. coli'' strain carrying these methyltransferases (a helper plasmid) participates in the conjugation. At the end of all this, a certain number of cyanobacterial cells take up the DNA, and are selected for with neomycin on minimal media. As soon as the unsuccessful exconjugates and the bacterial parental strains die off, transformant colonies can be picked. One significant hindrance associated with this process is the slow cyanobacterial doubling time; the transformants can take upwards of a week to grow.<br />
<br />
Our first trials were performed with the helper plasmid pDS4101, the conjugative plasmid pRL443 and the cargo plasmid pRL25. pDS4101 does not possess the methyltransferases which can epigenetically protect the incoming DNA from restriction, and so can only be used to transfer plasmids lacking restriction sites, such as pRL25, as a proof of concept. <br />
<br />
Towards the end of the summer we were able to obtain the helper plasmid pRL623 containing the three methyltransferases which together offer complete protection from restriction inside ''Anabaena''. There was an added obstacle to this new route, however: pRL623 can only be carried against certain ''E. coli'' genetic backgrounds lacking methyl-restricting enzymes, in order to prevent restriction of the host genome. This meant that transfer of the helper plasmid into the cargo plasmid strain before conjugation into ''Anabaena'' results in the suicide of that cargo strain, and no transformation. The solution to this was transformation by more traditional means of the cargo plasmid into the strain already carrying the helper plasmid; the cargo was then be delivered into ''Anabaena'' without transfer of the dangerous helper plasmid, and the helper plasmid had its opportunity to protectively methylate the cargo prior to transformation.<br />
<br />
We are sincerely grateful to Dr. Jeff Elhai (Virginia Commonwealth University), Dr. Peter Wolk (Michigan State University), James Golden (University of California, San Diego) for their crucial help and guidance with cyanobacterial transformation.<br />
<br />
[[File:Brown-Stanford Triparental mating.JPG|500px||center|thumb|Triparental mating: Our desired construct (from the '''donor strain''') and a helper plasmid are inserted into a '''helper strain'''. The '''helper strain''' and '''conjugative strain''' are spotted with the '''recipient''' Anabaena for the three-parent mating]]<br />
<br />
=='''PowerCell Transformation'''==<br />
<br />
[[File:Brown-Stanford Transformed Anabaena brightfield.JPG|400px||center|thumb|Anabaena culture after triparental mating (bright field light micrograph)]]<br />
<br />
[[File:Brown-Stanford Transformed Anabaena fluorescent.JPG|400px||center|thumb|Same Anabaena culture showing GFP expression in successful transformant]]<br />
<br />
We transformed the final PowerCell construct into Anabeana using the methods described above. Expression of GFP is clearly visible, indicating the transformation was successful. Some non-transformants can be seen in the background.<br />
<br />
We selected for our transformants using neomycin, and let the cultures grow.<br />
<br />
[[File:Anabaena Selection.jpeg|400px||center|thumb|Left: BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Middle: BG11(N-) with neomycin + 50µl of the original conjugation mixture. Right: BG11(N-) with neomycin + 50µl of WT Anabaena.<br />
]]<br />
<br />
The initial selection looked promising. The left-most culture tube contains BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Both transformed and untransformed Anabaena should be able to grow in this mixture. As expected, this culture tube has the highest cell density. The middle culture tube contains BG11(N-) with neomycin + 50µl of the original conjugation mixture. Only Anabaena containing our plasmid should be able to grow in this mixture. As desired, growth is still visible, but is less dense. The right-most tube contains BG11(N-) with neomycin + 50µl of WT Anabaena. Nothing should be able to grow here. As expected, everything in this tube is dead.<br />
<br />
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<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
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<div style="width: 35%;"><br />
Transformed Anabaena with heterocysts stained by alcian blue. Heterocyst spacing corresponds to the "off" cells from the GFP image, implying that the sucrose secretion construct in not active in heterocysts, as desired.<br />
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<br />
<br />
After about 2 weeks of growth, we can confirm the correct regulation of our sugar secretion construct by our pSac promoter by observing the presence of the GFP reporter. GFP expression is on in vegetative cells, and off in heterocysts, which implies that the sugar secretion construct is only active in vegetative cells, as we desire. Work is currently underway to assay the sucrose concentrations that are generated by our construct. We hope that these concentrations will be high enough to support growth of E. coli W; our experiments suggest the minimum concentration is around 5 mM (see section below). If the concentrations are high enough, we intend to grow E. coli W hosting several arbitrary active BioBrick plasmids to show that PowerCell can power general biological tools. Updates will be posted here as they become available! <br />
<br />
----<br />
<br />
== '''Utilizing sucrose from PowerCell''' ==<br />
<br />
==='''E. coli W''' ===<br />
<br />
[[File:Brown-Stanford Sugar Cane.jpg|300px|thumb|Sugarcane is an extremely low-cost source of bulk sucrose used in production]]<br />
<br />
An essential aspect of the PowerCell project lies in microorganisms being able to survive off the nutrients being secreted by our genetically engineered ''Anabaena''. Our carbon source, sucrose, is a disaccharide that is not necessarily able to be metabolized by all species.<br />
<br />
Here our project intersects with current trends of bioproduction on Earth; sucrose is a cheap and easily obtained sugar, so there is interest in engineerable strains of microorganisms able to survive on it as a sole carbon source. <br />
<br />
We came across research suggesting that ''E. coli'' W is one such promising strain. It is one of the few strains of non-pathogenic ''E. coli'' able to metabolize sucrose, has good tolerance for environmental stresses, and grows rapidly. (Archer 2011) There have been papers describing the process of adapting ''E. coli'' W as a bioreactor organism, and methods of growing them to high density and productivity using fed-batch cultures. (Lee 1993, Lee 1997). <br />
<br />
In fact, the strain has already been used to produce a number of useful products. These include D(-)-lactate, a precursor to the formation of certain biodegradable plastic polymers, and L-alanine, a food additive and nutritional supplement. (Shukla 2004, Zhang 2007) With these examples, it is not difficult to imagine the production role of ''E. coli'' W in our Martian colony.<br />
<br />
=== '''E. coli W Sucrose Metabolism Experiment''' ===<br />
<br />
In the application of our project, PowerCell would grow alongside and support a productive microorganism strain such as E. coli W. We were thus interested in seeing whether ''E. coli'' W could grow in the conditions generated by PowerCell. This meant culturing on minimal BG-11 media with added sucrose to simulate secretion by ''Anabaena''.<br />
<br />
[[File:Brown-Stanford E coli W colony count.jpg|300px|left|thumb|Growth of our ''E. coli'' W varies by amount of sucrose (there was no 200ul plate for 80mM sucrose)]] <br />
<br />
Based on literature (Niederholtmeyer, et al,2010) and correspondence with Mr. Ducat, we predicted that our ''Anabaena'' culture would produce no more than 8mM of sucrose. It would be difficult to support substantial E. coli growth on such low levels without a means of concentrating sugar via in the bioreactor design. <br />
<br />
[[File:Brown-Stanford E coli W plate.jpg|300px|right|thumb|E. coli W colonies on BG-11 agar + 60 mM sucrose]]<br />
<br />
Initially we attempted to compare growth on BG-11 + sucrose with liquid cultures. However, multiple attempts yielded no appreciable change in O.D. after several days of incubation. We suspect that this was because ''E. coli'' growth was too small to detect via spectrophotometer. <br />
<br />
Next, we moved to agar plates as a more sensitive means of detecting cell growth. ''E. coli'' W with Amp resistance were grown, washed and resuspended in PBS to eliminate residual LB media, and streaked them on BG-11 Amp+ plates with varying concentrations of sugar (5mM, 10mM, 30mM, 60mM, and 80mM).<br />
<br />
The cells were incubated at 37C and observed regularly before colonies appeared on the fifth day. Visible colonies were observed on plates with sucrose concentrations as low as 5mM. These data suggest that, with the sugar secreted by PowerCell expressing cscB and no additional metabolic engineering, it is possible to support another organism.<br />
<br />
----<br />
<br />
==References==<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|1|Sammy Boussiba, Jane Gibson, Ammonia translocation in cyanobacteria, FEMS Microbiology Letters, Volume 88, Issue 1, July 1991, Pages 1-14, ISSN 0378-1097, 10.1016/0378-1097(91)90692-4<br />
Niederholtmeyer, Henrike, Wolfstadter, Bernd T., Savage, David F., Silver, Pamela A., Way, Jeffrey C. Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products. Appl. Environ. Microbiol. 2010 76: 3462-3466}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|2|Kim, Jihyun F , Lars K Nielsen, Colin T Archer, Sang Yup Lee, Claudia E Vickers, Jin Hwan Park, and Haeyoung Jeong. "The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli ."BioMed Central 12 (2011).}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|3|Lee, Jeong Wook, Sol Choi, Jin Hwan Park, Claudia E. Vickers, Lars K. Nielsen, and Sang Yup Lee. "Development of Sucrose-utilizing Escherichia Coli K-12 Strain by Cloning β-fructofuranosidases and Its Application for L-threonine Production."Applied Microbiology and Biotechnology 88.4 (2010): 905-13. Print.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|4|Claudia Argueta, Kamile Yuksek, Michael Summers, Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression in Nostoc punctiforme, Journal of Microbiological Methods, Volume 59, Issue 2, November 2004, Pages 181-188, ISSN 0167-7012, 10.1016/j.mimet.2004.06.009.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|5|M. Sahintoth, S. Frillingos, J.W. Lengeler, H.R. Kaback, Active Transport by the CscB Permease in Escherichia coli K-12, Biochemical and Biophysical Research Communications, Volume 208, Issue 3, 28 March 1995, Pages 1116-1123, ISSN 0006-291X, 10.1006/bbrc.1995.1449.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|6|Huang, H. H., D. Camsund, P. Lindblad, and T. Heidorn. "Design and Characterization of Molecular Tools for a Synthetic Biology Approach towards Developing Cyanobacterial Biotechnology." Nucleic Acids Research 38.8 (2010): 2577-593. Print.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|7|Lee, SY and Ho Nam Chang. High cell density cultivation of Escherichia coli W using sucrose as a carbon source. Biotechnology Letters 15:9 (1993) 971--974.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|8|Lee JS, Lee SY and Sunwon Park. Fed-batch culture of Escherichia coli W by exponential feeding of sucrose as a carbon source. Biotechnology Techniques 11:1 (1997) 59-62.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|9|Shukla, VB et al. Production of D(-)-lactate from sucrose and molasses. Biotechnology Letters 26 (2004) 689-693.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-Stanford/PowerCell/NutrientSecretionTeam:Brown-Stanford/PowerCell/NutrientSecretion2011-12-07T09:54:33Z<p>Evanclark: /* PowerCell Transformation */</p>
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<div>{{:Team:Brown-Stanford/Templates/Main}}<br />
<html><br />
<div id="subHeader"><br />
<ul id="subHeaderList"><br />
<li><a href="/Team:Brown-Stanford/PowerCell/Introduction">Introduction</a></li><br />
<li><a href="/Team:Brown-Stanford/PowerCell/Cyanobacteria">Cyanobacteria</a></li><br />
<li><a href="/Team:Brown-Stanford/PowerCell/Background">Photosynthesis on Mars</a></li><br />
<li id="active"><a href="#" id="current">Nutrient Secretion and Utilization</a></li><br />
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</div><br />
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{{:Team:Brown-Stanford/Templates/Content}}<br />
<br />
== '''Nutrient Secretion and Utilization''' ==<br />
<br />
<br />
<br />
PowerCell is the generator that will power all of the BioTools in a Martian settlement. PowerCell will secrete nutrients for use by other biological tools a source of energy and raw materials to construct useful products.<br />
<br />
[[File:Brown-Stanford_Battery.png|200px|right]]<br />
<br />
Like any other tools, biological tools need energy to run and raw materials to work with. The two major nutrient sources needed for biological tools are carbon sources (sugars) and nitrogenous nutrients (ammonia). In a Martian settlement, PowerCell will harness the energy of the sun to harvest these nutrients directly from the atmosphere and provide them to the settlement's biological tools.<br />
<br />
This summer, we tackled only the half involving sugar secretion. This was partially to due to the time constraints of a summer project, and partially because there is evidence that ammonia secretion occurs in cyanobacteria by means of passive diffusion without the need for genetic tinkering ([http://www.sciencedirect.com/science/article/pii/0378109791906924 ''Ammonia translocation in cyanobacteria'']). More research needs to be done to determine if this level of diffusion is sufficient to sustain other biological tools. In any case, we designed our system on a [https://2011.igem.org/Team:Brown-Stanford/PowerCell/Cyanobacteria platform] (''Anabaena 7120'') with the capability to fix atmospheric nitrogen, so that this avenue is open for exploration in the future.<br />
<br />
=== Sucrose ===<br />
<br />
The sugar we chose to secrete is sucrose. The major inspiration for our project was the paper [http://aem.asm.org/cgi/content/abstract/76/11/3462 ''Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products'']. This work was done in Dr. Pamela Silver's lab, and describes a glucose/fructose secretion device, achieved in the single-celled bacterium ''S. elongatus''.<br />
<br />
[[File:Brown-Stanford invA.png|300px|right|thumb|Sugar secretion ''Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products''. ''Niederholtmeyer et al.'']]<br />
<br />
In a nutshell, ''S. elongatus'' were forced under salt stress to produce sucrose, which was broken down into glucose and fructose by the glf enzyme, then transported out of the cell by the invA transporter.<br />
<br />
At the Fifth International Conference on Synthetic Biology, [https://2011.igem.org/Team:Brown-Stanford/SB5 SB5.0], we spoke with a member of Dr. Silver's lab, Danny Ducat. He advised us that one of the problems with the glucose/fructose secretion system is its low yield. He suspected that because glucose and fructose are directly metabolizable by the cell, much of these sugars are consumed by the cell before they ever have a chance to be secreted. For this reason, it may be better to directly secrete sucrose, which is not metabolizable by the cell, and worry about breaking it down later. This is what we did, although the choice does have some ramifications.<br />
<br />
Because ''E. coli'' is the a primary host species for genetic modification, it is likely that many biological tools for space exploration will be designed in ''E. coli''. The most common laboratory strains of ''E. coli'' , TOP10 and K12, cannot directly metabolize sucrose. For this reason, they cannot make use of PowerCell's output directly. In order to utilize this output, sucrose will need to be broken down into glucose and fructose , or biological tools that intend to utilize PowerCell should be hosted by a strain that can directly metabolize sucrose. ''E. coli W'' is a safe and easily transformable laboratory strain with this ability, and, as outlined in [http://www.biomedcentral.com/1471-2164/12/9#IDAJ1FOQ ''The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli''], may confer some other advantages to biological tools to be used in space, including including fast growth, stress tolerance, growth to high cell densities. Furthermore, there is some indication that sucrose as a feedstock can confer other benefits, such as oxidative, heat, and acid stress ([http://www.springerlink.com/content/b80870618103w67l/ ''Development of sucrose-utilizing Escherichia coli K-12 strain by cloning β-fructofuranosidases and its application for l-threonine production'']).<br />
<br />
=== PowerCell Contruct Design ===<br />
<br />
We wanted to isolate sucrose secretion to just vegetative cells, as heterocysts do not participate in production of sucrose through photosynthesis. A similar cell-type-specific gene expression was was achieved in [http://www.sciencedirect.com/science/article/pii/S0167701204001745 ''Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression in Nostoc punctiforme'']. Arguetta et al. confined expression of a fluorescent marker to only vegetative cells in a similar filamentous diazotrophic cyanobacterium, ''Nostoc Punctiforme''. They placed the GFP gene behind a promoter present in the Photosystem I of ''Nostoc punctiforme'', psaC. This way, GFP expression was only turned on in photosynthesizing cells, and not in heterocysts. They did not know the exact sequence of the psaC promoter, so they just took the 400 base pairs upstream of the psaC gene as a unit containing the promoter. We have done similarly to isolate the psaC promoter from ''Anabaena 7120''. Below is an image showing cell-type-specific GFP expression achieved in the paper. <br />
<br />
<br />
[[File:Brown-Stanford Cell-Type-Specific.png|672px|center|thumb|Cell-type-specific GFP expression in ''Nostoc punctiforme''. Arguetta et al.]]<br />
<br />
<br />
We placed a sucrose symporter gene, cscB, behind our ''Anabaena 7120'' psaC promoter in order to confine sucrose secretion to only vegetative cells. Registry standard RBSes and terminators were used for ease of assembly and compatibility. Below is a diagram of our sucrose secretion device. A discussion of the inner workings of the cscB gene can be found in [http://www.sciencedirect.com/science/article/pii/S0006291X85714490 ''Active Transport by the CscB Permease in Escherichia coli K-12''].<br />
<br />
<br />
[[Image:Brown-Stanford cscB.jpg|700px|center]]<br />
<br />
<br />
We have also created several other constructs containing GFP for debugging purposes. We used a modified version of GFP, GFPmut3B because normal florescence markers are easily lost among background chlorophyll pigments in cyanobacteria ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860132/ ''Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology'']). <br />
<br />
<br />
[[File:Brown-Stanford GFP.jpg|700px|center]]<br />
<br />
<br />
[[File:Brown-Stanford cscB GFP.jpg|700px|center|thumb|Final PowerCell Construct: http://partsregistry.org/Part:BBa_K656012]]<br />
<br />
=='''Tranformation into Anabaena 7120'''==<br />
<br />
Cyanobacterial transformation can be difficult.<br />
<br />
There are cyanobacteria which will accept DNA without complaint. Many model cyanobacterium species, such as ''Synechococcus elongatus'' or ''Synechocystis'' PCC6803, for example, will simply take up naked DNA in solution and express it. <br />
<br />
''Anabaena 7120'', on the other hand, must take its DNA through a rather circuitous path; the DNA construct must first be placed in a cargo plasmid and transformed into ''E. coli'' by traditional means. Transfer to ''Anabaena'' takes place by conjugation, facilitated by a second ''E. coli'' strain carrying a plasmid encoding the machinery for bacterial conjugation. <br />
<br />
Another problem is the propensity of ''Anabaena'' to slice and dice foreign DNA with isoschizomers of the restriction enzymes AvaI, AvaII and AvaIII. This has been addressed with methyltransferases targeting the same sequences; that means a third ''E. coli'' strain carrying these methyltransferases (a helper plasmid) participates in the conjugation. At the end of all this, a certain number of cyanobacterial cells take up the DNA, and are selected for with neomycin on minimal media. As soon as the unsuccessful exconjugates and the bacterial parental strains die off, transformant colonies can be picked. One significant hindrance associated with this process is the slow cyanobacterial doubling time; the transformants can take upwards of a week to grow.<br />
<br />
Our first trials were performed with the helper plasmid pDS4101, the conjugative plasmid pRL443 and the cargo plasmid pRL25. pDS4101 does not possess the methyltransferases which can epigenetically protect the incoming DNA from restriction, and so can only be used to transfer plasmids lacking restriction sites, such as pRL25, as a proof of concept. <br />
<br />
Towards the end of the summer we were able to obtain the helper plasmid pRL623 containing the three methyltransferases which together offer complete protection from restriction inside ''Anabaena''. There was an added obstacle to this new route, however: pRL623 can only be carried against certain ''E. coli'' genetic backgrounds lacking methyl-restricting enzymes, in order to prevent restriction of the host genome. This meant that transfer of the helper plasmid into the cargo plasmid strain before conjugation into ''Anabaena'' results in the suicide of that cargo strain, and no transformation. The solution to this was transformation by more traditional means of the cargo plasmid into the strain already carrying the helper plasmid; the cargo was then be delivered into ''Anabaena'' without transfer of the dangerous helper plasmid, and the helper plasmid had its opportunity to protectively methylate the cargo prior to transformation.<br />
<br />
We are sincerely grateful to Dr. Jeff Elhai (Virginia Commonwealth University), Dr. Peter Wolk (Michigan State University), James Golden (University of California, San Diego) for their crucial help and guidance with cyanobacterial transformation.<br />
<br />
[[File:Brown-Stanford Triparental mating.JPG|500px||center|thumb|Triparental mating: Our desired construct (from the '''donor strain''') and a helper plasmid are inserted into a '''helper strain'''. The '''helper strain''' and '''conjugative strain''' are spotted with the '''recipient''' Anabaena for the three-parent mating]]<br />
<br />
=='''PowerCell Transformation'''==<br />
<br />
[[File:Brown-Stanford Transformed Anabaena brightfield.JPG|400px||center|thumb|Anabaena culture after triparental mating (bright field light micrograph)]]<br />
<br />
[[File:Brown-Stanford Transformed Anabaena fluorescent.JPG|400px||center|thumb|Same Anabaena culture showing GFP expression in successful transformant]]<br />
<br />
We transformed the final PowerCell construct into Anabeana using the methods described above. Expression of GFP is clearly visible, indicating the transformation was successful. Some non-transformants can be seen in the background.<br />
<br />
We selected for our transformants using neomycin, and let the cultures grow.<br />
<br />
[[File:Anabaena Selection.jpeg|400px||center|thumb|Left: BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Middle: BG11(N-) with neomycin + 50µl of the original conjugation mixture. Right: BG11(N-) with neomycin + 50µl of WT Anabaena.<br />
]]<br />
<br />
The initial selection looked promising. The left-most culture tube contains BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Both transformed and untransformed Anabaena should be able to grow in this mixture. As expected, this culture tube has the highest cell density. The middle culture tube contains BG11(N-) with neomycin + 50µl of the original conjugation mixture. Only Anabaena containing our plasmid should be able to grow in this mixture. As desired, growth is still visible, but is less dense. The right-most tube contains BG11(N-) with neomycin + 50µl of WT Anabaena. Nothing should be able to grow here. As expected, everything in this tube is dead.<br />
<br />
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<br />
<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
</div><br />
<br />
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<br />
<div style="width: 35%;"><br />
Transformed Anabaena with heterocysts stained by alcian blue. Heterocyst spacing corresponds to the "off" cells from the GFP image, implying that the sucrose secretion construct in not active in heterocysts, as desired.<br />
</div><br />
<br />
</center><br />
<br />
<br />
After about 2 weeks of growth, we can confirm the correct regulation of our sugar secretion construct by our pSac promoter by observing the presence of the GFP reporter. GFP expression is on in vegetative cells, and off in heterocysts, which implies that the sugar secretion construct is only active in vegetative cells, as we desire. Work is currently underway to assay the sucrose concentrations that are generated by our construct. We hope that these concentrations will be high enough to support growth of E. coli W; our experiments suggest the minimum concentration is around 5 mM (see section below). If the concentrations are high enough, we intend to grow E. coli W hosting several arbitrary active BioBrick plasmids, to show that PowerCell can power general biological tools. Updates will be posted here as they become available! <br />
<br />
----<br />
<br />
== '''Utilizing sucrose from PowerCell''' ==<br />
<br />
==='''E. coli W''' ===<br />
<br />
[[File:Brown-Stanford Sugar Cane.jpg|300px|thumb|Sugarcane is an extremely low-cost source of bulk sucrose used in production]]<br />
<br />
An essential aspect of the PowerCell project lies in microorganisms being able to survive off the nutrients being secreted by our genetically engineered ''Anabaena''. Our carbon source, sucrose, is a disaccharide that is not necessarily able to be metabolized by all species.<br />
<br />
Here our project intersects with current trends of bioproduction on Earth; sucrose is a cheap and easily obtained sugar, so there is interest in engineerable strains of microorganisms able to survive on it as a sole carbon source. <br />
<br />
We came across research suggesting that ''E. coli'' W is one such promising strain. It is one of the few strains of non-pathogenic ''E. coli'' able to metabolize sucrose, has good tolerance for environmental stresses, and grows rapidly. (Archer 2011) There have been papers describing the process of adapting ''E. coli'' W as a bioreactor organism, and methods of growing them to high density and productivity using fed-batch cultures. (Lee 1993, Lee 1997). <br />
<br />
In fact, the strain has already been used to produce a number of useful products. These include D(-)-lactate, a precursor to the formation of certain biodegradable plastic polymers, and L-alanine, a food additive and nutritional supplement. (Shukla 2004, Zhang 2007) With these examples, it is not difficult to imagine the production role of ''E. coli'' W in our Martian colony.<br />
<br />
=== '''E. coli W Sucrose Metabolism Experiment''' ===<br />
<br />
In the application of our project, PowerCell would grow alongside and support a productive microorganism strain such as E. coli W. We were thus interested in seeing whether ''E. coli'' W could grow in the conditions generated by PowerCell. This meant culturing on minimal BG-11 media with added sucrose to simulate secretion by ''Anabaena''.<br />
<br />
[[File:Brown-Stanford E coli W colony count.jpg|300px|left|thumb|Growth of our ''E. coli'' W varies by amount of sucrose (there was no 200ul plate for 80mM sucrose)]] <br />
<br />
Based on literature (Niederholtmeyer, et al,2010) and correspondence with Mr. Ducat, we predicted that our ''Anabaena'' culture would produce no more than 8mM of sucrose. It would be difficult to support substantial E. coli growth on such low levels without a means of concentrating sugar via in the bioreactor design. <br />
<br />
[[File:Brown-Stanford E coli W plate.jpg|300px|right|thumb|E. coli W colonies on BG-11 agar + 60 mM sucrose]]<br />
<br />
Initially we attempted to compare growth on BG-11 + sucrose with liquid cultures. However, multiple attempts yielded no appreciable change in O.D. after several days of incubation. We suspect that this was because ''E. coli'' growth was too small to detect via spectrophotometer. <br />
<br />
Next, we moved to agar plates as a more sensitive means of detecting cell growth. ''E. coli'' W with Amp resistance were grown, washed and resuspended in PBS to eliminate residual LB media, and streaked them on BG-11 Amp+ plates with varying concentrations of sugar (5mM, 10mM, 30mM, 60mM, and 80mM).<br />
<br />
The cells were incubated at 37C and observed regularly before colonies appeared on the fifth day. Visible colonies were observed on plates with sucrose concentrations as low as 5mM. These data suggest that, with the sugar secreted by PowerCell expressing cscB and no additional metabolic engineering, it is possible to support another organism.<br />
<br />
----<br />
<br />
==References==<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|1|Sammy Boussiba, Jane Gibson, Ammonia translocation in cyanobacteria, FEMS Microbiology Letters, Volume 88, Issue 1, July 1991, Pages 1-14, ISSN 0378-1097, 10.1016/0378-1097(91)90692-4<br />
Niederholtmeyer, Henrike, Wolfstadter, Bernd T., Savage, David F., Silver, Pamela A., Way, Jeffrey C. Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products. Appl. Environ. Microbiol. 2010 76: 3462-3466}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|2|Kim, Jihyun F , Lars K Nielsen, Colin T Archer, Sang Yup Lee, Claudia E Vickers, Jin Hwan Park, and Haeyoung Jeong. "The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli ."BioMed Central 12 (2011).}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|3|Lee, Jeong Wook, Sol Choi, Jin Hwan Park, Claudia E. Vickers, Lars K. Nielsen, and Sang Yup Lee. "Development of Sucrose-utilizing Escherichia Coli K-12 Strain by Cloning β-fructofuranosidases and Its Application for L-threonine Production."Applied Microbiology and Biotechnology 88.4 (2010): 905-13. Print.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|4|Claudia Argueta, Kamile Yuksek, Michael Summers, Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression in Nostoc punctiforme, Journal of Microbiological Methods, Volume 59, Issue 2, November 2004, Pages 181-188, ISSN 0167-7012, 10.1016/j.mimet.2004.06.009.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|5|M. Sahintoth, S. Frillingos, J.W. Lengeler, H.R. Kaback, Active Transport by the CscB Permease in Escherichia coli K-12, Biochemical and Biophysical Research Communications, Volume 208, Issue 3, 28 March 1995, Pages 1116-1123, ISSN 0006-291X, 10.1006/bbrc.1995.1449.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|6|Huang, H. H., D. Camsund, P. Lindblad, and T. Heidorn. "Design and Characterization of Molecular Tools for a Synthetic Biology Approach towards Developing Cyanobacterial Biotechnology." Nucleic Acids Research 38.8 (2010): 2577-593. Print.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|7|Lee, SY and Ho Nam Chang. High cell density cultivation of Escherichia coli W using sucrose as a carbon source. Biotechnology Letters 15:9 (1993) 971--974.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|8|Lee JS, Lee SY and Sunwon Park. Fed-batch culture of Escherichia coli W by exponential feeding of sucrose as a carbon source. Biotechnology Techniques 11:1 (1997) 59-62.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|9|Shukla, VB et al. Production of D(-)-lactate from sucrose and molasses. Biotechnology Letters 26 (2004) 689-693.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-Stanford/PowerCell/NutrientSecretionTeam:Brown-Stanford/PowerCell/NutrientSecretion2011-12-07T09:49:44Z<p>Evanclark: /* PowerCell Transformation */</p>
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<html><br />
<div id="subHeader"><br />
<ul id="subHeaderList"><br />
<li><a href="/Team:Brown-Stanford/PowerCell/Introduction">Introduction</a></li><br />
<li><a href="/Team:Brown-Stanford/PowerCell/Cyanobacteria">Cyanobacteria</a></li><br />
<li><a href="/Team:Brown-Stanford/PowerCell/Background">Photosynthesis on Mars</a></li><br />
<li id="active"><a href="#" id="current">Nutrient Secretion and Utilization</a></li><br />
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{{:Team:Brown-Stanford/Templates/Content}}<br />
<br />
== '''Nutrient Secretion and Utilization''' ==<br />
<br />
<br />
<br />
PowerCell is the generator that will power all of the BioTools in a Martian settlement. PowerCell will secrete nutrients for use by other biological tools a source of energy and raw materials to construct useful products.<br />
<br />
[[File:Brown-Stanford_Battery.png|200px|right]]<br />
<br />
Like any other tools, biological tools need energy to run and raw materials to work with. The two major nutrient sources needed for biological tools are carbon sources (sugars) and nitrogenous nutrients (ammonia). In a Martian settlement, PowerCell will harness the energy of the sun to harvest these nutrients directly from the atmosphere and provide them to the settlement's biological tools.<br />
<br />
This summer, we tackled only the half involving sugar secretion. This was partially to due to the time constraints of a summer project, and partially because there is evidence that ammonia secretion occurs in cyanobacteria by means of passive diffusion without the need for genetic tinkering ([http://www.sciencedirect.com/science/article/pii/0378109791906924 ''Ammonia translocation in cyanobacteria'']). More research needs to be done to determine if this level of diffusion is sufficient to sustain other biological tools. In any case, we designed our system on a [https://2011.igem.org/Team:Brown-Stanford/PowerCell/Cyanobacteria platform] (''Anabaena 7120'') with the capability to fix atmospheric nitrogen, so that this avenue is open for exploration in the future.<br />
<br />
=== Sucrose ===<br />
<br />
The sugar we chose to secrete is sucrose. The major inspiration for our project was the paper [http://aem.asm.org/cgi/content/abstract/76/11/3462 ''Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products'']. This work was done in Dr. Pamela Silver's lab, and describes a glucose/fructose secretion device, achieved in the single-celled bacterium ''S. elongatus''.<br />
<br />
[[File:Brown-Stanford invA.png|300px|right|thumb|Sugar secretion ''Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products''. ''Niederholtmeyer et al.'']]<br />
<br />
In a nutshell, ''S. elongatus'' were forced under salt stress to produce sucrose, which was broken down into glucose and fructose by the glf enzyme, then transported out of the cell by the invA transporter.<br />
<br />
At the Fifth International Conference on Synthetic Biology, [https://2011.igem.org/Team:Brown-Stanford/SB5 SB5.0], we spoke with a member of Dr. Silver's lab, Danny Ducat. He advised us that one of the problems with the glucose/fructose secretion system is its low yield. He suspected that because glucose and fructose are directly metabolizable by the cell, much of these sugars are consumed by the cell before they ever have a chance to be secreted. For this reason, it may be better to directly secrete sucrose, which is not metabolizable by the cell, and worry about breaking it down later. This is what we did, although the choice does have some ramifications.<br />
<br />
Because ''E. coli'' is the a primary host species for genetic modification, it is likely that many biological tools for space exploration will be designed in ''E. coli''. The most common laboratory strains of ''E. coli'' , TOP10 and K12, cannot directly metabolize sucrose. For this reason, they cannot make use of PowerCell's output directly. In order to utilize this output, sucrose will need to be broken down into glucose and fructose , or biological tools that intend to utilize PowerCell should be hosted by a strain that can directly metabolize sucrose. ''E. coli W'' is a safe and easily transformable laboratory strain with this ability, and, as outlined in [http://www.biomedcentral.com/1471-2164/12/9#IDAJ1FOQ ''The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli''], may confer some other advantages to biological tools to be used in space, including including fast growth, stress tolerance, growth to high cell densities. Furthermore, there is some indication that sucrose as a feedstock can confer other benefits, such as oxidative, heat, and acid stress ([http://www.springerlink.com/content/b80870618103w67l/ ''Development of sucrose-utilizing Escherichia coli K-12 strain by cloning β-fructofuranosidases and its application for l-threonine production'']).<br />
<br />
=== PowerCell Contruct Design ===<br />
<br />
We wanted to isolate sucrose secretion to just vegetative cells, as heterocysts do not participate in production of sucrose through photosynthesis. A similar cell-type-specific gene expression was was achieved in [http://www.sciencedirect.com/science/article/pii/S0167701204001745 ''Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression in Nostoc punctiforme'']. Arguetta et al. confined expression of a fluorescent marker to only vegetative cells in a similar filamentous diazotrophic cyanobacterium, ''Nostoc Punctiforme''. They placed the GFP gene behind a promoter present in the Photosystem I of ''Nostoc punctiforme'', psaC. This way, GFP expression was only turned on in photosynthesizing cells, and not in heterocysts. They did not know the exact sequence of the psaC promoter, so they just took the 400 base pairs upstream of the psaC gene as a unit containing the promoter. We have done similarly to isolate the psaC promoter from ''Anabaena 7120''. Below is an image showing cell-type-specific GFP expression achieved in the paper. <br />
<br />
<br />
[[File:Brown-Stanford Cell-Type-Specific.png|672px|center|thumb|Cell-type-specific GFP expression in ''Nostoc punctiforme''. Arguetta et al.]]<br />
<br />
<br />
We placed a sucrose symporter gene, cscB, behind our ''Anabaena 7120'' psaC promoter in order to confine sucrose secretion to only vegetative cells. Registry standard RBSes and terminators were used for ease of assembly and compatibility. Below is a diagram of our sucrose secretion device. A discussion of the inner workings of the cscB gene can be found in [http://www.sciencedirect.com/science/article/pii/S0006291X85714490 ''Active Transport by the CscB Permease in Escherichia coli K-12''].<br />
<br />
<br />
[[Image:Brown-Stanford cscB.jpg|700px|center]]<br />
<br />
<br />
We have also created several other constructs containing GFP for debugging purposes. We used a modified version of GFP, GFPmut3B because normal florescence markers are easily lost among background chlorophyll pigments in cyanobacteria ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860132/ ''Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology'']). <br />
<br />
<br />
[[File:Brown-Stanford GFP.jpg|700px|center]]<br />
<br />
<br />
[[File:Brown-Stanford cscB GFP.jpg|700px|center|thumb|Final PowerCell Construct: http://partsregistry.org/Part:BBa_K656012]]<br />
<br />
=='''Tranformation into Anabaena 7120'''==<br />
<br />
Cyanobacterial transformation can be difficult.<br />
<br />
There are cyanobacteria which will accept DNA without complaint. Many model cyanobacterium species, such as ''Synechococcus elongatus'' or ''Synechocystis'' PCC6803, for example, will simply take up naked DNA in solution and express it. <br />
<br />
''Anabaena 7120'', on the other hand, must take its DNA through a rather circuitous path; the DNA construct must first be placed in a cargo plasmid and transformed into ''E. coli'' by traditional means. Transfer to ''Anabaena'' takes place by conjugation, facilitated by a second ''E. coli'' strain carrying a plasmid encoding the machinery for bacterial conjugation. <br />
<br />
Another problem is the propensity of ''Anabaena'' to slice and dice foreign DNA with isoschizomers of the restriction enzymes AvaI, AvaII and AvaIII. This has been addressed with methyltransferases targeting the same sequences; that means a third ''E. coli'' strain carrying these methyltransferases (a helper plasmid) participates in the conjugation. At the end of all this, a certain number of cyanobacterial cells take up the DNA, and are selected for with neomycin on minimal media. As soon as the unsuccessful exconjugates and the bacterial parental strains die off, transformant colonies can be picked. One significant hindrance associated with this process is the slow cyanobacterial doubling time; the transformants can take upwards of a week to grow.<br />
<br />
Our first trials were performed with the helper plasmid pDS4101, the conjugative plasmid pRL443 and the cargo plasmid pRL25. pDS4101 does not possess the methyltransferases which can epigenetically protect the incoming DNA from restriction, and so can only be used to transfer plasmids lacking restriction sites, such as pRL25, as a proof of concept. <br />
<br />
Towards the end of the summer we were able to obtain the helper plasmid pRL623 containing the three methyltransferases which together offer complete protection from restriction inside ''Anabaena''. There was an added obstacle to this new route, however: pRL623 can only be carried against certain ''E. coli'' genetic backgrounds lacking methyl-restricting enzymes, in order to prevent restriction of the host genome. This meant that transfer of the helper plasmid into the cargo plasmid strain before conjugation into ''Anabaena'' results in the suicide of that cargo strain, and no transformation. The solution to this was transformation by more traditional means of the cargo plasmid into the strain already carrying the helper plasmid; the cargo was then be delivered into ''Anabaena'' without transfer of the dangerous helper plasmid, and the helper plasmid had its opportunity to protectively methylate the cargo prior to transformation.<br />
<br />
We are sincerely grateful to Dr. Jeff Elhai (Virginia Commonwealth University), Dr. Peter Wolk (Michigan State University), James Golden (University of California, San Diego) for their crucial help and guidance with cyanobacterial transformation.<br />
<br />
[[File:Brown-Stanford Triparental mating.JPG|500px||center|thumb|Triparental mating: Our desired construct (from the '''donor strain''') and a helper plasmid are inserted into a '''helper strain'''. The '''helper strain''' and '''conjugative strain''' are spotted with the '''recipient''' Anabaena for the three-parent mating]]<br />
<br />
=='''PowerCell Transformation'''==<br />
<br />
[[File:Brown-Stanford Transformed Anabaena brightfield.JPG|400px||center|thumb|Anabaena culture after triparental mating (bright field light micrograph)]]<br />
<br />
[[File:Brown-Stanford Transformed Anabaena fluorescent.JPG|400px||center|thumb|Same Anabaena culture showing GFP expression in successful transformant]]<br />
<br />
We transformed the final PowerCell construct into Anabeana using the methods described above. Expression of GFP is clearly visible, indicating the transformation was successful. Some non-transformants can be seen in the background.<br />
<br />
We selected for our transformants using neomycin, and let the cultures grow.<br />
<br />
[[File:Anabaena Selection.jpeg|400px||center|thumb|Left: BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Middle: BG11(N-) with neomycin + 50µl of the original conjugation mixture. Right: BG11(N-) with neomycin + 50µl of WT Anabaena.<br />
]]<br />
<br />
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<br />
<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
</div><br />
</center><br />
<br />
<br />
The initial selection looked promising. The left-most culture tube contains BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Both transformed and untransformed Anabaena should be able to grow in this mixture. As expected, this culture tube has the highest cell density. The middle culture tube contains BG11(N-) with neomycin + 50µl of the original conjugation mixture. Only Anabaena containing our plasmid should be able to grow in this mixture. As desired, growth is still visible, but is less dense. The right-most tube contains BG11(N-) with neomycin + 50µl of WT Anabaena. Nothing should be able to grow here. As expected, everything in this tube is dead.<br />
<br />
After about 2 weeks of growth, we can confirm the correct regulation of our sugar secretion construct by our pSac promoter by observing the presence of the GFP reporter. GFP expression is on in vegetative cells, and off in heterocysts, which implies that the sugar secretion construct is only active in vegetative cells, as we desire. Work is currently underway to assay the sucrose concentrations that are generated by our construct. We hope that these concentrations will be high enough to support growth of E. coli W; our experiments suggest the minimum concentration is around 5 mM (see section below). If the concentrations are high enough, we intend to grow E. coli W hosting several arbitrary active BioBrick plasmids, to show that PowerCell can power general biological tools. Updates will be posted here as they become available! <br />
<br />
<br />
<br />
<br />
----<br />
<br />
== '''Utilizing sucrose from PowerCell''' ==<br />
<br />
==='''E. coli W''' ===<br />
<br />
[[File:Brown-Stanford Sugar Cane.jpg|300px|thumb|Sugarcane is an extremely low-cost source of bulk sucrose used in production]]<br />
<br />
An essential aspect of the PowerCell project lies in microorganisms being able to survive off the nutrients being secreted by our genetically engineered ''Anabaena''. Our carbon source, sucrose, is a disaccharide that is not necessarily able to be metabolized by all species.<br />
<br />
Here our project intersects with current trends of bioproduction on Earth; sucrose is a cheap and easily obtained sugar, so there is interest in engineerable strains of microorganisms able to survive on it as a sole carbon source. <br />
<br />
We came across research suggesting that ''E. coli'' W is one such promising strain. It is one of the few strains of non-pathogenic ''E. coli'' able to metabolize sucrose, has good tolerance for environmental stresses, and grows rapidly. (Archer 2011) There have been papers describing the process of adapting ''E. coli'' W as a bioreactor organism, and methods of growing them to high density and productivity using fed-batch cultures. (Lee 1993, Lee 1997). <br />
<br />
In fact, the strain has already been used to produce a number of useful products. These include D(-)-lactate, a precursor to the formation of certain biodegradable plastic polymers, and L-alanine, a food additive and nutritional supplement. (Shukla 2004, Zhang 2007) With these examples, it is not difficult to imagine the production role of ''E. coli'' W in our Martian colony.<br />
<br />
=== '''E. coli W Sucrose Metabolism Experiment''' ===<br />
<br />
In the application of our project, PowerCell would grow alongside and support a productive microorganism strain such as E. coli W. We were thus interested in seeing whether ''E. coli'' W could grow in the conditions generated by PowerCell. This meant culturing on minimal BG-11 media with added sucrose to simulate secretion by ''Anabaena''.<br />
<br />
[[File:Brown-Stanford E coli W colony count.jpg|300px|left|thumb|Growth of our ''E. coli'' W varies by amount of sucrose (there was no 200ul plate for 80mM sucrose)]] <br />
<br />
Based on literature (Niederholtmeyer, et al,2010) and correspondence with Mr. Ducat, we predicted that our ''Anabaena'' culture would produce no more than 8mM of sucrose. It would be difficult to support substantial E. coli growth on such low levels without a means of concentrating sugar via in the bioreactor design. <br />
<br />
[[File:Brown-Stanford E coli W plate.jpg|300px|right|thumb|E. coli W colonies on BG-11 agar + 60 mM sucrose]]<br />
<br />
Initially we attempted to compare growth on BG-11 + sucrose with liquid cultures. However, multiple attempts yielded no appreciable change in O.D. after several days of incubation. We suspect that this was because ''E. coli'' growth was too small to detect via spectrophotometer. <br />
<br />
Next, we moved to agar plates as a more sensitive means of detecting cell growth. ''E. coli'' W with Amp resistance were grown, washed and resuspended in PBS to eliminate residual LB media, and streaked them on BG-11 Amp+ plates with varying concentrations of sugar (5mM, 10mM, 30mM, 60mM, and 80mM).<br />
<br />
The cells were incubated at 37C and observed regularly before colonies appeared on the fifth day. Visible colonies were observed on plates with sucrose concentrations as low as 5mM. These data suggest that, with the sugar secreted by PowerCell expressing cscB and no additional metabolic engineering, it is possible to support another organism.<br />
<br />
----<br />
<br />
==References==<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|1|Sammy Boussiba, Jane Gibson, Ammonia translocation in cyanobacteria, FEMS Microbiology Letters, Volume 88, Issue 1, July 1991, Pages 1-14, ISSN 0378-1097, 10.1016/0378-1097(91)90692-4<br />
Niederholtmeyer, Henrike, Wolfstadter, Bernd T., Savage, David F., Silver, Pamela A., Way, Jeffrey C. Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products. Appl. Environ. Microbiol. 2010 76: 3462-3466}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|2|Kim, Jihyun F , Lars K Nielsen, Colin T Archer, Sang Yup Lee, Claudia E Vickers, Jin Hwan Park, and Haeyoung Jeong. "The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli ."BioMed Central 12 (2011).}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|3|Lee, Jeong Wook, Sol Choi, Jin Hwan Park, Claudia E. Vickers, Lars K. Nielsen, and Sang Yup Lee. "Development of Sucrose-utilizing Escherichia Coli K-12 Strain by Cloning β-fructofuranosidases and Its Application for L-threonine Production."Applied Microbiology and Biotechnology 88.4 (2010): 905-13. Print.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|4|Claudia Argueta, Kamile Yuksek, Michael Summers, Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression in Nostoc punctiforme, Journal of Microbiological Methods, Volume 59, Issue 2, November 2004, Pages 181-188, ISSN 0167-7012, 10.1016/j.mimet.2004.06.009.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|5|M. Sahintoth, S. Frillingos, J.W. Lengeler, H.R. Kaback, Active Transport by the CscB Permease in Escherichia coli K-12, Biochemical and Biophysical Research Communications, Volume 208, Issue 3, 28 March 1995, Pages 1116-1123, ISSN 0006-291X, 10.1006/bbrc.1995.1449.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|6|Huang, H. H., D. Camsund, P. Lindblad, and T. Heidorn. "Design and Characterization of Molecular Tools for a Synthetic Biology Approach towards Developing Cyanobacterial Biotechnology." Nucleic Acids Research 38.8 (2010): 2577-593. Print.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|7|Lee, SY and Ho Nam Chang. High cell density cultivation of Escherichia coli W using sucrose as a carbon source. Biotechnology Letters 15:9 (1993) 971--974.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|8|Lee JS, Lee SY and Sunwon Park. Fed-batch culture of Escherichia coli W by exponential feeding of sucrose as a carbon source. Biotechnology Techniques 11:1 (1997) 59-62.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|9|Shukla, VB et al. Production of D(-)-lactate from sucrose and molasses. Biotechnology Letters 26 (2004) 689-693.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-Stanford/PowerCell/NutrientSecretionTeam:Brown-Stanford/PowerCell/NutrientSecretion2011-12-07T09:48:29Z<p>Evanclark: /* PowerCell Transformation */</p>
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<ul id="subHeaderList"><br />
<li><a href="/Team:Brown-Stanford/PowerCell/Introduction">Introduction</a></li><br />
<li><a href="/Team:Brown-Stanford/PowerCell/Cyanobacteria">Cyanobacteria</a></li><br />
<li><a href="/Team:Brown-Stanford/PowerCell/Background">Photosynthesis on Mars</a></li><br />
<li id="active"><a href="#" id="current">Nutrient Secretion and Utilization</a></li><br />
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{{:Team:Brown-Stanford/Templates/Content}}<br />
<br />
== '''Nutrient Secretion and Utilization''' ==<br />
<br />
<br />
<br />
PowerCell is the generator that will power all of the BioTools in a Martian settlement. PowerCell will secrete nutrients for use by other biological tools a source of energy and raw materials to construct useful products.<br />
<br />
[[File:Brown-Stanford_Battery.png|200px|right]]<br />
<br />
Like any other tools, biological tools need energy to run and raw materials to work with. The two major nutrient sources needed for biological tools are carbon sources (sugars) and nitrogenous nutrients (ammonia). In a Martian settlement, PowerCell will harness the energy of the sun to harvest these nutrients directly from the atmosphere and provide them to the settlement's biological tools.<br />
<br />
This summer, we tackled only the half involving sugar secretion. This was partially to due to the time constraints of a summer project, and partially because there is evidence that ammonia secretion occurs in cyanobacteria by means of passive diffusion without the need for genetic tinkering ([http://www.sciencedirect.com/science/article/pii/0378109791906924 ''Ammonia translocation in cyanobacteria'']). More research needs to be done to determine if this level of diffusion is sufficient to sustain other biological tools. In any case, we designed our system on a [https://2011.igem.org/Team:Brown-Stanford/PowerCell/Cyanobacteria platform] (''Anabaena 7120'') with the capability to fix atmospheric nitrogen, so that this avenue is open for exploration in the future.<br />
<br />
=== Sucrose ===<br />
<br />
The sugar we chose to secrete is sucrose. The major inspiration for our project was the paper [http://aem.asm.org/cgi/content/abstract/76/11/3462 ''Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products'']. This work was done in Dr. Pamela Silver's lab, and describes a glucose/fructose secretion device, achieved in the single-celled bacterium ''S. elongatus''.<br />
<br />
[[File:Brown-Stanford invA.png|300px|right|thumb|Sugar secretion ''Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products''. ''Niederholtmeyer et al.'']]<br />
<br />
In a nutshell, ''S. elongatus'' were forced under salt stress to produce sucrose, which was broken down into glucose and fructose by the glf enzyme, then transported out of the cell by the invA transporter.<br />
<br />
At the Fifth International Conference on Synthetic Biology, [https://2011.igem.org/Team:Brown-Stanford/SB5 SB5.0], we spoke with a member of Dr. Silver's lab, Danny Ducat. He advised us that one of the problems with the glucose/fructose secretion system is its low yield. He suspected that because glucose and fructose are directly metabolizable by the cell, much of these sugars are consumed by the cell before they ever have a chance to be secreted. For this reason, it may be better to directly secrete sucrose, which is not metabolizable by the cell, and worry about breaking it down later. This is what we did, although the choice does have some ramifications.<br />
<br />
Because ''E. coli'' is the a primary host species for genetic modification, it is likely that many biological tools for space exploration will be designed in ''E. coli''. The most common laboratory strains of ''E. coli'' , TOP10 and K12, cannot directly metabolize sucrose. For this reason, they cannot make use of PowerCell's output directly. In order to utilize this output, sucrose will need to be broken down into glucose and fructose , or biological tools that intend to utilize PowerCell should be hosted by a strain that can directly metabolize sucrose. ''E. coli W'' is a safe and easily transformable laboratory strain with this ability, and, as outlined in [http://www.biomedcentral.com/1471-2164/12/9#IDAJ1FOQ ''The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli''], may confer some other advantages to biological tools to be used in space, including including fast growth, stress tolerance, growth to high cell densities. Furthermore, there is some indication that sucrose as a feedstock can confer other benefits, such as oxidative, heat, and acid stress ([http://www.springerlink.com/content/b80870618103w67l/ ''Development of sucrose-utilizing Escherichia coli K-12 strain by cloning β-fructofuranosidases and its application for l-threonine production'']).<br />
<br />
=== PowerCell Contruct Design ===<br />
<br />
We wanted to isolate sucrose secretion to just vegetative cells, as heterocysts do not participate in production of sucrose through photosynthesis. A similar cell-type-specific gene expression was was achieved in [http://www.sciencedirect.com/science/article/pii/S0167701204001745 ''Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression in Nostoc punctiforme'']. Arguetta et al. confined expression of a fluorescent marker to only vegetative cells in a similar filamentous diazotrophic cyanobacterium, ''Nostoc Punctiforme''. They placed the GFP gene behind a promoter present in the Photosystem I of ''Nostoc punctiforme'', psaC. This way, GFP expression was only turned on in photosynthesizing cells, and not in heterocysts. They did not know the exact sequence of the psaC promoter, so they just took the 400 base pairs upstream of the psaC gene as a unit containing the promoter. We have done similarly to isolate the psaC promoter from ''Anabaena 7120''. Below is an image showing cell-type-specific GFP expression achieved in the paper. <br />
<br />
<br />
[[File:Brown-Stanford Cell-Type-Specific.png|672px|center|thumb|Cell-type-specific GFP expression in ''Nostoc punctiforme''. Arguetta et al.]]<br />
<br />
<br />
We placed a sucrose symporter gene, cscB, behind our ''Anabaena 7120'' psaC promoter in order to confine sucrose secretion to only vegetative cells. Registry standard RBSes and terminators were used for ease of assembly and compatibility. Below is a diagram of our sucrose secretion device. A discussion of the inner workings of the cscB gene can be found in [http://www.sciencedirect.com/science/article/pii/S0006291X85714490 ''Active Transport by the CscB Permease in Escherichia coli K-12''].<br />
<br />
<br />
[[Image:Brown-Stanford cscB.jpg|700px|center]]<br />
<br />
<br />
We have also created several other constructs containing GFP for debugging purposes. We used a modified version of GFP, GFPmut3B because normal florescence markers are easily lost among background chlorophyll pigments in cyanobacteria ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860132/ ''Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology'']). <br />
<br />
<br />
[[File:Brown-Stanford GFP.jpg|700px|center]]<br />
<br />
<br />
[[File:Brown-Stanford cscB GFP.jpg|700px|center|thumb|Final PowerCell Construct: http://partsregistry.org/Part:BBa_K656012]]<br />
<br />
=='''Tranformation into Anabaena 7120'''==<br />
<br />
Cyanobacterial transformation can be difficult.<br />
<br />
There are cyanobacteria which will accept DNA without complaint. Many model cyanobacterium species, such as ''Synechococcus elongatus'' or ''Synechocystis'' PCC6803, for example, will simply take up naked DNA in solution and express it. <br />
<br />
''Anabaena 7120'', on the other hand, must take its DNA through a rather circuitous path; the DNA construct must first be placed in a cargo plasmid and transformed into ''E. coli'' by traditional means. Transfer to ''Anabaena'' takes place by conjugation, facilitated by a second ''E. coli'' strain carrying a plasmid encoding the machinery for bacterial conjugation. <br />
<br />
Another problem is the propensity of ''Anabaena'' to slice and dice foreign DNA with isoschizomers of the restriction enzymes AvaI, AvaII and AvaIII. This has been addressed with methyltransferases targeting the same sequences; that means a third ''E. coli'' strain carrying these methyltransferases (a helper plasmid) participates in the conjugation. At the end of all this, a certain number of cyanobacterial cells take up the DNA, and are selected for with neomycin on minimal media. As soon as the unsuccessful exconjugates and the bacterial parental strains die off, transformant colonies can be picked. One significant hindrance associated with this process is the slow cyanobacterial doubling time; the transformants can take upwards of a week to grow.<br />
<br />
Our first trials were performed with the helper plasmid pDS4101, the conjugative plasmid pRL443 and the cargo plasmid pRL25. pDS4101 does not possess the methyltransferases which can epigenetically protect the incoming DNA from restriction, and so can only be used to transfer plasmids lacking restriction sites, such as pRL25, as a proof of concept. <br />
<br />
Towards the end of the summer we were able to obtain the helper plasmid pRL623 containing the three methyltransferases which together offer complete protection from restriction inside ''Anabaena''. There was an added obstacle to this new route, however: pRL623 can only be carried against certain ''E. coli'' genetic backgrounds lacking methyl-restricting enzymes, in order to prevent restriction of the host genome. This meant that transfer of the helper plasmid into the cargo plasmid strain before conjugation into ''Anabaena'' results in the suicide of that cargo strain, and no transformation. The solution to this was transformation by more traditional means of the cargo plasmid into the strain already carrying the helper plasmid; the cargo was then be delivered into ''Anabaena'' without transfer of the dangerous helper plasmid, and the helper plasmid had its opportunity to protectively methylate the cargo prior to transformation.<br />
<br />
We are sincerely grateful to Dr. Jeff Elhai (Virginia Commonwealth University), Dr. Peter Wolk (Michigan State University), James Golden (University of California, San Diego) for their crucial help and guidance with cyanobacterial transformation.<br />
<br />
[[File:Brown-Stanford Triparental mating.JPG|500px||center|thumb|Triparental mating: Our desired construct (from the '''donor strain''') and a helper plasmid are inserted into a '''helper strain'''. The '''helper strain''' and '''conjugative strain''' are spotted with the '''recipient''' Anabaena for the three-parent mating]]<br />
<br />
=='''PowerCell Transformation'''==<br />
<br />
[[File:Brown-Stanford Transformed Anabaena brightfield.JPG|400px||center|thumb|Anabaena culture after triparental mating (bright field light micrograph)]]<br />
<br />
[[File:Brown-Stanford Transformed Anabaena fluorescent.JPG|400px||center|thumb|Same Anabaena culture showing GFP expression in successful transformant]]<br />
<br />
We transformed the final PowerCell construct into Anabeana using the methods described above. Expression of GFP is clearly visible, indicating the transformation was successful. Some non-transformants can be seen in the background.<br />
<br />
We selected for our transformants using neomycin, and let the cultures grow.<br />
<br />
[[File:Anabaena Selection.jpeg|400px||center|thumb|Left: BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Middle: BG11(N-) with neomycin + 50µl of the original conjugation mixture. Right: BG11(N-) with neomycin + 50µl of WT Anabaena.<br />
]]<br />
<br />
<br />
The initial selection looked promising. The left-most culture tube contains BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Both transformed and untransformed Anabaena should be able to grow in this mixture. As expected, this culture tube has the highest cell density. The middle culture tube contains BG11(N-) with neomycin + 50µl of the original conjugation mixture. Only Anabaena containing our plasmid should be able to grow in this mixture. As desired, growth is still visible, but is less dense. The right-most tube contains BG11(N-) with neomycin + 50µl of WT Anabaena. Nothing should be able to grow here. As expected, everything in this tube is dead.<br />
<br />
After about 2 weeks of growth, we can confirm the correct regulation of our sugar secretion construct by our pSac promoter by observing the presence of the GFP reporter. GFP expression is on in vegetative cells, and off in heterocysts, which implies that the sugar secretion construct is only active in vegetative cells, as we desire. Work is currently underway to assay the sucrose concentrations that are generated by our construct. We hope that these concentrations will be high enough to support growth of E. coli W; our experiments suggest the minimum concentration is around 5 mM (see section below). If the concentrations are high enough, we intend to grow E. coli W hosting several arbitrary active BioBrick plasmids, to show that PowerCell can power general biological tools. Updates will be posted here as they become available! <br />
<br />
<br />
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<br />
<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
</div><br />
<br />
<br />
----<br />
<br />
== '''Utilizing sucrose from PowerCell''' ==<br />
<br />
==='''E. coli W''' ===<br />
<br />
[[File:Brown-Stanford Sugar Cane.jpg|300px|thumb|Sugarcane is an extremely low-cost source of bulk sucrose used in production]]<br />
<br />
An essential aspect of the PowerCell project lies in microorganisms being able to survive off the nutrients being secreted by our genetically engineered ''Anabaena''. Our carbon source, sucrose, is a disaccharide that is not necessarily able to be metabolized by all species.<br />
<br />
Here our project intersects with current trends of bioproduction on Earth; sucrose is a cheap and easily obtained sugar, so there is interest in engineerable strains of microorganisms able to survive on it as a sole carbon source. <br />
<br />
We came across research suggesting that ''E. coli'' W is one such promising strain. It is one of the few strains of non-pathogenic ''E. coli'' able to metabolize sucrose, has good tolerance for environmental stresses, and grows rapidly. (Archer 2011) There have been papers describing the process of adapting ''E. coli'' W as a bioreactor organism, and methods of growing them to high density and productivity using fed-batch cultures. (Lee 1993, Lee 1997). <br />
<br />
In fact, the strain has already been used to produce a number of useful products. These include D(-)-lactate, a precursor to the formation of certain biodegradable plastic polymers, and L-alanine, a food additive and nutritional supplement. (Shukla 2004, Zhang 2007) With these examples, it is not difficult to imagine the production role of ''E. coli'' W in our Martian colony.<br />
<br />
=== '''E. coli W Sucrose Metabolism Experiment''' ===<br />
<br />
In the application of our project, PowerCell would grow alongside and support a productive microorganism strain such as E. coli W. We were thus interested in seeing whether ''E. coli'' W could grow in the conditions generated by PowerCell. This meant culturing on minimal BG-11 media with added sucrose to simulate secretion by ''Anabaena''.<br />
<br />
[[File:Brown-Stanford E coli W colony count.jpg|300px|left|thumb|Growth of our ''E. coli'' W varies by amount of sucrose (there was no 200ul plate for 80mM sucrose)]] <br />
<br />
Based on literature (Niederholtmeyer, et al,2010) and correspondence with Mr. Ducat, we predicted that our ''Anabaena'' culture would produce no more than 8mM of sucrose. It would be difficult to support substantial E. coli growth on such low levels without a means of concentrating sugar via in the bioreactor design. <br />
<br />
[[File:Brown-Stanford E coli W plate.jpg|300px|right|thumb|E. coli W colonies on BG-11 agar + 60 mM sucrose]]<br />
<br />
Initially we attempted to compare growth on BG-11 + sucrose with liquid cultures. However, multiple attempts yielded no appreciable change in O.D. after several days of incubation. We suspect that this was because ''E. coli'' growth was too small to detect via spectrophotometer. <br />
<br />
Next, we moved to agar plates as a more sensitive means of detecting cell growth. ''E. coli'' W with Amp resistance were grown, washed and resuspended in PBS to eliminate residual LB media, and streaked them on BG-11 Amp+ plates with varying concentrations of sugar (5mM, 10mM, 30mM, 60mM, and 80mM).<br />
<br />
The cells were incubated at 37C and observed regularly before colonies appeared on the fifth day. Visible colonies were observed on plates with sucrose concentrations as low as 5mM. These data suggest that, with the sugar secreted by PowerCell expressing cscB and no additional metabolic engineering, it is possible to support another organism.<br />
<br />
----<br />
<br />
==References==<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|1|Sammy Boussiba, Jane Gibson, Ammonia translocation in cyanobacteria, FEMS Microbiology Letters, Volume 88, Issue 1, July 1991, Pages 1-14, ISSN 0378-1097, 10.1016/0378-1097(91)90692-4<br />
Niederholtmeyer, Henrike, Wolfstadter, Bernd T., Savage, David F., Silver, Pamela A., Way, Jeffrey C. Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products. Appl. Environ. Microbiol. 2010 76: 3462-3466}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|2|Kim, Jihyun F , Lars K Nielsen, Colin T Archer, Sang Yup Lee, Claudia E Vickers, Jin Hwan Park, and Haeyoung Jeong. "The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli ."BioMed Central 12 (2011).}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|3|Lee, Jeong Wook, Sol Choi, Jin Hwan Park, Claudia E. Vickers, Lars K. Nielsen, and Sang Yup Lee. "Development of Sucrose-utilizing Escherichia Coli K-12 Strain by Cloning β-fructofuranosidases and Its Application for L-threonine Production."Applied Microbiology and Biotechnology 88.4 (2010): 905-13. Print.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|4|Claudia Argueta, Kamile Yuksek, Michael Summers, Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression in Nostoc punctiforme, Journal of Microbiological Methods, Volume 59, Issue 2, November 2004, Pages 181-188, ISSN 0167-7012, 10.1016/j.mimet.2004.06.009.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|5|M. Sahintoth, S. Frillingos, J.W. Lengeler, H.R. Kaback, Active Transport by the CscB Permease in Escherichia coli K-12, Biochemical and Biophysical Research Communications, Volume 208, Issue 3, 28 March 1995, Pages 1116-1123, ISSN 0006-291X, 10.1006/bbrc.1995.1449.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|6|Huang, H. H., D. Camsund, P. Lindblad, and T. Heidorn. "Design and Characterization of Molecular Tools for a Synthetic Biology Approach towards Developing Cyanobacterial Biotechnology." Nucleic Acids Research 38.8 (2010): 2577-593. Print.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|7|Lee, SY and Ho Nam Chang. High cell density cultivation of Escherichia coli W using sucrose as a carbon source. Biotechnology Letters 15:9 (1993) 971--974.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|8|Lee JS, Lee SY and Sunwon Park. Fed-batch culture of Escherichia coli W by exponential feeding of sucrose as a carbon source. Biotechnology Techniques 11:1 (1997) 59-62.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|9|Shukla, VB et al. Production of D(-)-lactate from sucrose and molasses. Biotechnology Letters 26 (2004) 689-693.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-Stanford/PowerCell/NutrientSecretionTeam:Brown-Stanford/PowerCell/NutrientSecretion2011-12-07T09:47:02Z<p>Evanclark: /* PowerCell Transformation */</p>
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<div>{{:Team:Brown-Stanford/Templates/Main}}<br />
<html><br />
<div id="subHeader"><br />
<ul id="subHeaderList"><br />
<li><a href="/Team:Brown-Stanford/PowerCell/Introduction">Introduction</a></li><br />
<li><a href="/Team:Brown-Stanford/PowerCell/Cyanobacteria">Cyanobacteria</a></li><br />
<li><a href="/Team:Brown-Stanford/PowerCell/Background">Photosynthesis on Mars</a></li><br />
<li id="active"><a href="#" id="current">Nutrient Secretion and Utilization</a></li><br />
</ul><br />
</div><br />
</html><br />
{{:Team:Brown-Stanford/Templates/Content}}<br />
<br />
== '''Nutrient Secretion and Utilization''' ==<br />
<br />
<br />
<br />
PowerCell is the generator that will power all of the BioTools in a Martian settlement. PowerCell will secrete nutrients for use by other biological tools a source of energy and raw materials to construct useful products.<br />
<br />
[[File:Brown-Stanford_Battery.png|200px|right]]<br />
<br />
Like any other tools, biological tools need energy to run and raw materials to work with. The two major nutrient sources needed for biological tools are carbon sources (sugars) and nitrogenous nutrients (ammonia). In a Martian settlement, PowerCell will harness the energy of the sun to harvest these nutrients directly from the atmosphere and provide them to the settlement's biological tools.<br />
<br />
This summer, we tackled only the half involving sugar secretion. This was partially to due to the time constraints of a summer project, and partially because there is evidence that ammonia secretion occurs in cyanobacteria by means of passive diffusion without the need for genetic tinkering ([http://www.sciencedirect.com/science/article/pii/0378109791906924 ''Ammonia translocation in cyanobacteria'']). More research needs to be done to determine if this level of diffusion is sufficient to sustain other biological tools. In any case, we designed our system on a [https://2011.igem.org/Team:Brown-Stanford/PowerCell/Cyanobacteria platform] (''Anabaena 7120'') with the capability to fix atmospheric nitrogen, so that this avenue is open for exploration in the future.<br />
<br />
=== Sucrose ===<br />
<br />
The sugar we chose to secrete is sucrose. The major inspiration for our project was the paper [http://aem.asm.org/cgi/content/abstract/76/11/3462 ''Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products'']. This work was done in Dr. Pamela Silver's lab, and describes a glucose/fructose secretion device, achieved in the single-celled bacterium ''S. elongatus''.<br />
<br />
[[File:Brown-Stanford invA.png|300px|right|thumb|Sugar secretion ''Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products''. ''Niederholtmeyer et al.'']]<br />
<br />
In a nutshell, ''S. elongatus'' were forced under salt stress to produce sucrose, which was broken down into glucose and fructose by the glf enzyme, then transported out of the cell by the invA transporter.<br />
<br />
At the Fifth International Conference on Synthetic Biology, [https://2011.igem.org/Team:Brown-Stanford/SB5 SB5.0], we spoke with a member of Dr. Silver's lab, Danny Ducat. He advised us that one of the problems with the glucose/fructose secretion system is its low yield. He suspected that because glucose and fructose are directly metabolizable by the cell, much of these sugars are consumed by the cell before they ever have a chance to be secreted. For this reason, it may be better to directly secrete sucrose, which is not metabolizable by the cell, and worry about breaking it down later. This is what we did, although the choice does have some ramifications.<br />
<br />
Because ''E. coli'' is the a primary host species for genetic modification, it is likely that many biological tools for space exploration will be designed in ''E. coli''. The most common laboratory strains of ''E. coli'' , TOP10 and K12, cannot directly metabolize sucrose. For this reason, they cannot make use of PowerCell's output directly. In order to utilize this output, sucrose will need to be broken down into glucose and fructose , or biological tools that intend to utilize PowerCell should be hosted by a strain that can directly metabolize sucrose. ''E. coli W'' is a safe and easily transformable laboratory strain with this ability, and, as outlined in [http://www.biomedcentral.com/1471-2164/12/9#IDAJ1FOQ ''The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli''], may confer some other advantages to biological tools to be used in space, including including fast growth, stress tolerance, growth to high cell densities. Furthermore, there is some indication that sucrose as a feedstock can confer other benefits, such as oxidative, heat, and acid stress ([http://www.springerlink.com/content/b80870618103w67l/ ''Development of sucrose-utilizing Escherichia coli K-12 strain by cloning β-fructofuranosidases and its application for l-threonine production'']).<br />
<br />
=== PowerCell Contruct Design ===<br />
<br />
We wanted to isolate sucrose secretion to just vegetative cells, as heterocysts do not participate in production of sucrose through photosynthesis. A similar cell-type-specific gene expression was was achieved in [http://www.sciencedirect.com/science/article/pii/S0167701204001745 ''Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression in Nostoc punctiforme'']. Arguetta et al. confined expression of a fluorescent marker to only vegetative cells in a similar filamentous diazotrophic cyanobacterium, ''Nostoc Punctiforme''. They placed the GFP gene behind a promoter present in the Photosystem I of ''Nostoc punctiforme'', psaC. This way, GFP expression was only turned on in photosynthesizing cells, and not in heterocysts. They did not know the exact sequence of the psaC promoter, so they just took the 400 base pairs upstream of the psaC gene as a unit containing the promoter. We have done similarly to isolate the psaC promoter from ''Anabaena 7120''. Below is an image showing cell-type-specific GFP expression achieved in the paper. <br />
<br />
<br />
[[File:Brown-Stanford Cell-Type-Specific.png|672px|center|thumb|Cell-type-specific GFP expression in ''Nostoc punctiforme''. Arguetta et al.]]<br />
<br />
<br />
We placed a sucrose symporter gene, cscB, behind our ''Anabaena 7120'' psaC promoter in order to confine sucrose secretion to only vegetative cells. Registry standard RBSes and terminators were used for ease of assembly and compatibility. Below is a diagram of our sucrose secretion device. A discussion of the inner workings of the cscB gene can be found in [http://www.sciencedirect.com/science/article/pii/S0006291X85714490 ''Active Transport by the CscB Permease in Escherichia coli K-12''].<br />
<br />
<br />
[[Image:Brown-Stanford cscB.jpg|700px|center]]<br />
<br />
<br />
We have also created several other constructs containing GFP for debugging purposes. We used a modified version of GFP, GFPmut3B because normal florescence markers are easily lost among background chlorophyll pigments in cyanobacteria ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860132/ ''Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology'']). <br />
<br />
<br />
[[File:Brown-Stanford GFP.jpg|700px|center]]<br />
<br />
<br />
[[File:Brown-Stanford cscB GFP.jpg|700px|center|thumb|Final PowerCell Construct: http://partsregistry.org/Part:BBa_K656012]]<br />
<br />
=='''Tranformation into Anabaena 7120'''==<br />
<br />
Cyanobacterial transformation can be difficult.<br />
<br />
There are cyanobacteria which will accept DNA without complaint. Many model cyanobacterium species, such as ''Synechococcus elongatus'' or ''Synechocystis'' PCC6803, for example, will simply take up naked DNA in solution and express it. <br />
<br />
''Anabaena 7120'', on the other hand, must take its DNA through a rather circuitous path; the DNA construct must first be placed in a cargo plasmid and transformed into ''E. coli'' by traditional means. Transfer to ''Anabaena'' takes place by conjugation, facilitated by a second ''E. coli'' strain carrying a plasmid encoding the machinery for bacterial conjugation. <br />
<br />
Another problem is the propensity of ''Anabaena'' to slice and dice foreign DNA with isoschizomers of the restriction enzymes AvaI, AvaII and AvaIII. This has been addressed with methyltransferases targeting the same sequences; that means a third ''E. coli'' strain carrying these methyltransferases (a helper plasmid) participates in the conjugation. At the end of all this, a certain number of cyanobacterial cells take up the DNA, and are selected for with neomycin on minimal media. As soon as the unsuccessful exconjugates and the bacterial parental strains die off, transformant colonies can be picked. One significant hindrance associated with this process is the slow cyanobacterial doubling time; the transformants can take upwards of a week to grow.<br />
<br />
Our first trials were performed with the helper plasmid pDS4101, the conjugative plasmid pRL443 and the cargo plasmid pRL25. pDS4101 does not possess the methyltransferases which can epigenetically protect the incoming DNA from restriction, and so can only be used to transfer plasmids lacking restriction sites, such as pRL25, as a proof of concept. <br />
<br />
Towards the end of the summer we were able to obtain the helper plasmid pRL623 containing the three methyltransferases which together offer complete protection from restriction inside ''Anabaena''. There was an added obstacle to this new route, however: pRL623 can only be carried against certain ''E. coli'' genetic backgrounds lacking methyl-restricting enzymes, in order to prevent restriction of the host genome. This meant that transfer of the helper plasmid into the cargo plasmid strain before conjugation into ''Anabaena'' results in the suicide of that cargo strain, and no transformation. The solution to this was transformation by more traditional means of the cargo plasmid into the strain already carrying the helper plasmid; the cargo was then be delivered into ''Anabaena'' without transfer of the dangerous helper plasmid, and the helper plasmid had its opportunity to protectively methylate the cargo prior to transformation.<br />
<br />
We are sincerely grateful to Dr. Jeff Elhai (Virginia Commonwealth University), Dr. Peter Wolk (Michigan State University), James Golden (University of California, San Diego) for their crucial help and guidance with cyanobacterial transformation.<br />
<br />
[[File:Brown-Stanford Triparental mating.JPG|500px||center|thumb|Triparental mating: Our desired construct (from the '''donor strain''') and a helper plasmid are inserted into a '''helper strain'''. The '''helper strain''' and '''conjugative strain''' are spotted with the '''recipient''' Anabaena for the three-parent mating]]<br />
<br />
=='''PowerCell Transformation'''==<br />
<br />
[[File:Brown-Stanford Transformed Anabaena brightfield.JPG|400px||center|thumb|Anabaena culture after triparental mating (bright field light micrograph)]]<br />
<br />
[[File:Brown-Stanford Transformed Anabaena fluorescent.JPG|400px||center|thumb|Same Anabaena culture showing GFP expression in successful transformant]]<br />
<br />
We transformed the final PowerCell construct into Anabeana using the methods described above. Expression of GFP is clearly visible, indicating the transformation was successful. Some non-transformants can be seen in the background.<br />
<br />
We selected for our transformants using neomycin, and let the cultures grow.<br />
<br />
[[File:Anabaena Selection.jpeg|400px||center|thumb|Left: BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Middle: BG11(N-) with neomycin + 50µl of the original conjugation mixture. Right: BG11(N-) with neomycin + 50µl of WT Anabaena.<br />
]]<br />
<br />
<br />
The initial selection looked promising. The left-most culture tube contains BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Both transformed and untransformed Anabaena should be able to grow in this mixture. As expected, this culture tube has the highest cell density. The middle culture tube contains BG11(N-) with neomycin + 50µl of the original conjugation mixture. Only Anabaena containing our plasmid should be able to grow in this mixture. As desired, growth is still visible, but is less dense. The right-most tube contains BG11(N-) with neomycin + 50µl of WT Anabaena. Nothing should be able to grow here. As expected, everything in this tube is dead.<br />
<br />
After about 2 weeks of growth, we can confirm the correct regulation of our sugar secretion construct by our pSac promoter by observing the presence of the GFP reporter. GFP expression is on in vegetative cells, and off in heterocysts, which implies that the sugar secretion construct is only active in vegetative cells, as we desire. Work is currently underway to assay the sucrose concentrations that are generated by our construct. We hope that these concentrations will be high enough to support growth of E. coli W; our experiments suggest the minimum concentration is around 5 mM (see section below). If the concentrations are high enough, we intend to grow E. coli W hosting several arbitrary active BioBrick plasmids, to show that PowerCell can power general biological tools. Updates will be posted here as they become available! <br />
<br />
<br />
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<br />
<br />
----<br />
<br />
== '''Utilizing sucrose from PowerCell''' ==<br />
<br />
==='''E. coli W''' ===<br />
<br />
[[File:Brown-Stanford Sugar Cane.jpg|300px|thumb|Sugarcane is an extremely low-cost source of bulk sucrose used in production]]<br />
<br />
An essential aspect of the PowerCell project lies in microorganisms being able to survive off the nutrients being secreted by our genetically engineered ''Anabaena''. Our carbon source, sucrose, is a disaccharide that is not necessarily able to be metabolized by all species.<br />
<br />
Here our project intersects with current trends of bioproduction on Earth; sucrose is a cheap and easily obtained sugar, so there is interest in engineerable strains of microorganisms able to survive on it as a sole carbon source. <br />
<br />
We came across research suggesting that ''E. coli'' W is one such promising strain. It is one of the few strains of non-pathogenic ''E. coli'' able to metabolize sucrose, has good tolerance for environmental stresses, and grows rapidly. (Archer 2011) There have been papers describing the process of adapting ''E. coli'' W as a bioreactor organism, and methods of growing them to high density and productivity using fed-batch cultures. (Lee 1993, Lee 1997). <br />
<br />
In fact, the strain has already been used to produce a number of useful products. These include D(-)-lactate, a precursor to the formation of certain biodegradable plastic polymers, and L-alanine, a food additive and nutritional supplement. (Shukla 2004, Zhang 2007) With these examples, it is not difficult to imagine the production role of ''E. coli'' W in our Martian colony.<br />
<br />
=== '''E. coli W Sucrose Metabolism Experiment''' ===<br />
<br />
In the application of our project, PowerCell would grow alongside and support a productive microorganism strain such as E. coli W. We were thus interested in seeing whether ''E. coli'' W could grow in the conditions generated by PowerCell. This meant culturing on minimal BG-11 media with added sucrose to simulate secretion by ''Anabaena''.<br />
<br />
[[File:Brown-Stanford E coli W colony count.jpg|300px|left|thumb|Growth of our ''E. coli'' W varies by amount of sucrose (there was no 200ul plate for 80mM sucrose)]] <br />
<br />
Based on literature (Niederholtmeyer, et al,2010) and correspondence with Mr. Ducat, we predicted that our ''Anabaena'' culture would produce no more than 8mM of sucrose. It would be difficult to support substantial E. coli growth on such low levels without a means of concentrating sugar via in the bioreactor design. <br />
<br />
[[File:Brown-Stanford E coli W plate.jpg|300px|right|thumb|E. coli W colonies on BG-11 agar + 60 mM sucrose]]<br />
<br />
Initially we attempted to compare growth on BG-11 + sucrose with liquid cultures. However, multiple attempts yielded no appreciable change in O.D. after several days of incubation. We suspect that this was because ''E. coli'' growth was too small to detect via spectrophotometer. <br />
<br />
Next, we moved to agar plates as a more sensitive means of detecting cell growth. ''E. coli'' W with Amp resistance were grown, washed and resuspended in PBS to eliminate residual LB media, and streaked them on BG-11 Amp+ plates with varying concentrations of sugar (5mM, 10mM, 30mM, 60mM, and 80mM).<br />
<br />
The cells were incubated at 37C and observed regularly before colonies appeared on the fifth day. Visible colonies were observed on plates with sucrose concentrations as low as 5mM. These data suggest that, with the sugar secreted by PowerCell expressing cscB and no additional metabolic engineering, it is possible to support another organism.<br />
<br />
----<br />
<br />
==References==<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|1|Sammy Boussiba, Jane Gibson, Ammonia translocation in cyanobacteria, FEMS Microbiology Letters, Volume 88, Issue 1, July 1991, Pages 1-14, ISSN 0378-1097, 10.1016/0378-1097(91)90692-4<br />
Niederholtmeyer, Henrike, Wolfstadter, Bernd T., Savage, David F., Silver, Pamela A., Way, Jeffrey C. Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products. Appl. Environ. Microbiol. 2010 76: 3462-3466}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|2|Kim, Jihyun F , Lars K Nielsen, Colin T Archer, Sang Yup Lee, Claudia E Vickers, Jin Hwan Park, and Haeyoung Jeong. "The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli ."BioMed Central 12 (2011).}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|3|Lee, Jeong Wook, Sol Choi, Jin Hwan Park, Claudia E. Vickers, Lars K. Nielsen, and Sang Yup Lee. "Development of Sucrose-utilizing Escherichia Coli K-12 Strain by Cloning β-fructofuranosidases and Its Application for L-threonine Production."Applied Microbiology and Biotechnology 88.4 (2010): 905-13. Print.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|4|Claudia Argueta, Kamile Yuksek, Michael Summers, Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression in Nostoc punctiforme, Journal of Microbiological Methods, Volume 59, Issue 2, November 2004, Pages 181-188, ISSN 0167-7012, 10.1016/j.mimet.2004.06.009.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|5|M. Sahintoth, S. Frillingos, J.W. Lengeler, H.R. Kaback, Active Transport by the CscB Permease in Escherichia coli K-12, Biochemical and Biophysical Research Communications, Volume 208, Issue 3, 28 March 1995, Pages 1116-1123, ISSN 0006-291X, 10.1006/bbrc.1995.1449.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|6|Huang, H. H., D. Camsund, P. Lindblad, and T. Heidorn. "Design and Characterization of Molecular Tools for a Synthetic Biology Approach towards Developing Cyanobacterial Biotechnology." Nucleic Acids Research 38.8 (2010): 2577-593. Print.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|7|Lee, SY and Ho Nam Chang. High cell density cultivation of Escherichia coli W using sucrose as a carbon source. Biotechnology Letters 15:9 (1993) 971--974.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|8|Lee JS, Lee SY and Sunwon Park. Fed-batch culture of Escherichia coli W by exponential feeding of sucrose as a carbon source. Biotechnology Techniques 11:1 (1997) 59-62.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|9|Shukla, VB et al. Production of D(-)-lactate from sucrose and molasses. Biotechnology Letters 26 (2004) 689-693.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-Stanford/PowerCell/NutrientSecretionTeam:Brown-Stanford/PowerCell/NutrientSecretion2011-12-07T09:43:57Z<p>Evanclark: /* PowerCell Transformation */</p>
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<li><a href="/Team:Brown-Stanford/PowerCell/Introduction">Introduction</a></li><br />
<li><a href="/Team:Brown-Stanford/PowerCell/Cyanobacteria">Cyanobacteria</a></li><br />
<li><a href="/Team:Brown-Stanford/PowerCell/Background">Photosynthesis on Mars</a></li><br />
<li id="active"><a href="#" id="current">Nutrient Secretion and Utilization</a></li><br />
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<br />
== '''Nutrient Secretion and Utilization''' ==<br />
<br />
<br />
<br />
PowerCell is the generator that will power all of the BioTools in a Martian settlement. PowerCell will secrete nutrients for use by other biological tools a source of energy and raw materials to construct useful products.<br />
<br />
[[File:Brown-Stanford_Battery.png|200px|right]]<br />
<br />
Like any other tools, biological tools need energy to run and raw materials to work with. The two major nutrient sources needed for biological tools are carbon sources (sugars) and nitrogenous nutrients (ammonia). In a Martian settlement, PowerCell will harness the energy of the sun to harvest these nutrients directly from the atmosphere and provide them to the settlement's biological tools.<br />
<br />
This summer, we tackled only the half involving sugar secretion. This was partially to due to the time constraints of a summer project, and partially because there is evidence that ammonia secretion occurs in cyanobacteria by means of passive diffusion without the need for genetic tinkering ([http://www.sciencedirect.com/science/article/pii/0378109791906924 ''Ammonia translocation in cyanobacteria'']). More research needs to be done to determine if this level of diffusion is sufficient to sustain other biological tools. In any case, we designed our system on a [https://2011.igem.org/Team:Brown-Stanford/PowerCell/Cyanobacteria platform] (''Anabaena 7120'') with the capability to fix atmospheric nitrogen, so that this avenue is open for exploration in the future.<br />
<br />
=== Sucrose ===<br />
<br />
The sugar we chose to secrete is sucrose. The major inspiration for our project was the paper [http://aem.asm.org/cgi/content/abstract/76/11/3462 ''Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products'']. This work was done in Dr. Pamela Silver's lab, and describes a glucose/fructose secretion device, achieved in the single-celled bacterium ''S. elongatus''.<br />
<br />
[[File:Brown-Stanford invA.png|300px|right|thumb|Sugar secretion ''Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products''. ''Niederholtmeyer et al.'']]<br />
<br />
In a nutshell, ''S. elongatus'' were forced under salt stress to produce sucrose, which was broken down into glucose and fructose by the glf enzyme, then transported out of the cell by the invA transporter.<br />
<br />
At the Fifth International Conference on Synthetic Biology, [https://2011.igem.org/Team:Brown-Stanford/SB5 SB5.0], we spoke with a member of Dr. Silver's lab, Danny Ducat. He advised us that one of the problems with the glucose/fructose secretion system is its low yield. He suspected that because glucose and fructose are directly metabolizable by the cell, much of these sugars are consumed by the cell before they ever have a chance to be secreted. For this reason, it may be better to directly secrete sucrose, which is not metabolizable by the cell, and worry about breaking it down later. This is what we did, although the choice does have some ramifications.<br />
<br />
Because ''E. coli'' is the a primary host species for genetic modification, it is likely that many biological tools for space exploration will be designed in ''E. coli''. The most common laboratory strains of ''E. coli'' , TOP10 and K12, cannot directly metabolize sucrose. For this reason, they cannot make use of PowerCell's output directly. In order to utilize this output, sucrose will need to be broken down into glucose and fructose , or biological tools that intend to utilize PowerCell should be hosted by a strain that can directly metabolize sucrose. ''E. coli W'' is a safe and easily transformable laboratory strain with this ability, and, as outlined in [http://www.biomedcentral.com/1471-2164/12/9#IDAJ1FOQ ''The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli''], may confer some other advantages to biological tools to be used in space, including including fast growth, stress tolerance, growth to high cell densities. Furthermore, there is some indication that sucrose as a feedstock can confer other benefits, such as oxidative, heat, and acid stress ([http://www.springerlink.com/content/b80870618103w67l/ ''Development of sucrose-utilizing Escherichia coli K-12 strain by cloning β-fructofuranosidases and its application for l-threonine production'']).<br />
<br />
=== PowerCell Contruct Design ===<br />
<br />
We wanted to isolate sucrose secretion to just vegetative cells, as heterocysts do not participate in production of sucrose through photosynthesis. A similar cell-type-specific gene expression was was achieved in [http://www.sciencedirect.com/science/article/pii/S0167701204001745 ''Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression in Nostoc punctiforme'']. Arguetta et al. confined expression of a fluorescent marker to only vegetative cells in a similar filamentous diazotrophic cyanobacterium, ''Nostoc Punctiforme''. They placed the GFP gene behind a promoter present in the Photosystem I of ''Nostoc punctiforme'', psaC. This way, GFP expression was only turned on in photosynthesizing cells, and not in heterocysts. They did not know the exact sequence of the psaC promoter, so they just took the 400 base pairs upstream of the psaC gene as a unit containing the promoter. We have done similarly to isolate the psaC promoter from ''Anabaena 7120''. Below is an image showing cell-type-specific GFP expression achieved in the paper. <br />
<br />
<br />
[[File:Brown-Stanford Cell-Type-Specific.png|672px|center|thumb|Cell-type-specific GFP expression in ''Nostoc punctiforme''. Arguetta et al.]]<br />
<br />
<br />
We placed a sucrose symporter gene, cscB, behind our ''Anabaena 7120'' psaC promoter in order to confine sucrose secretion to only vegetative cells. Registry standard RBSes and terminators were used for ease of assembly and compatibility. Below is a diagram of our sucrose secretion device. A discussion of the inner workings of the cscB gene can be found in [http://www.sciencedirect.com/science/article/pii/S0006291X85714490 ''Active Transport by the CscB Permease in Escherichia coli K-12''].<br />
<br />
<br />
[[Image:Brown-Stanford cscB.jpg|700px|center]]<br />
<br />
<br />
We have also created several other constructs containing GFP for debugging purposes. We used a modified version of GFP, GFPmut3B because normal florescence markers are easily lost among background chlorophyll pigments in cyanobacteria ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860132/ ''Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology'']). <br />
<br />
<br />
[[File:Brown-Stanford GFP.jpg|700px|center]]<br />
<br />
<br />
[[File:Brown-Stanford cscB GFP.jpg|700px|center|thumb|Final PowerCell Construct: http://partsregistry.org/Part:BBa_K656012]]<br />
<br />
=='''Tranformation into Anabaena 7120'''==<br />
<br />
Cyanobacterial transformation can be difficult.<br />
<br />
There are cyanobacteria which will accept DNA without complaint. Many model cyanobacterium species, such as ''Synechococcus elongatus'' or ''Synechocystis'' PCC6803, for example, will simply take up naked DNA in solution and express it. <br />
<br />
''Anabaena 7120'', on the other hand, must take its DNA through a rather circuitous path; the DNA construct must first be placed in a cargo plasmid and transformed into ''E. coli'' by traditional means. Transfer to ''Anabaena'' takes place by conjugation, facilitated by a second ''E. coli'' strain carrying a plasmid encoding the machinery for bacterial conjugation. <br />
<br />
Another problem is the propensity of ''Anabaena'' to slice and dice foreign DNA with isoschizomers of the restriction enzymes AvaI, AvaII and AvaIII. This has been addressed with methyltransferases targeting the same sequences; that means a third ''E. coli'' strain carrying these methyltransferases (a helper plasmid) participates in the conjugation. At the end of all this, a certain number of cyanobacterial cells take up the DNA, and are selected for with neomycin on minimal media. As soon as the unsuccessful exconjugates and the bacterial parental strains die off, transformant colonies can be picked. One significant hindrance associated with this process is the slow cyanobacterial doubling time; the transformants can take upwards of a week to grow.<br />
<br />
Our first trials were performed with the helper plasmid pDS4101, the conjugative plasmid pRL443 and the cargo plasmid pRL25. pDS4101 does not possess the methyltransferases which can epigenetically protect the incoming DNA from restriction, and so can only be used to transfer plasmids lacking restriction sites, such as pRL25, as a proof of concept. <br />
<br />
Towards the end of the summer we were able to obtain the helper plasmid pRL623 containing the three methyltransferases which together offer complete protection from restriction inside ''Anabaena''. There was an added obstacle to this new route, however: pRL623 can only be carried against certain ''E. coli'' genetic backgrounds lacking methyl-restricting enzymes, in order to prevent restriction of the host genome. This meant that transfer of the helper plasmid into the cargo plasmid strain before conjugation into ''Anabaena'' results in the suicide of that cargo strain, and no transformation. The solution to this was transformation by more traditional means of the cargo plasmid into the strain already carrying the helper plasmid; the cargo was then be delivered into ''Anabaena'' without transfer of the dangerous helper plasmid, and the helper plasmid had its opportunity to protectively methylate the cargo prior to transformation.<br />
<br />
We are sincerely grateful to Dr. Jeff Elhai (Virginia Commonwealth University), Dr. Peter Wolk (Michigan State University), James Golden (University of California, San Diego) for their crucial help and guidance with cyanobacterial transformation.<br />
<br />
[[File:Brown-Stanford Triparental mating.JPG|500px||center|thumb|Triparental mating: Our desired construct (from the '''donor strain''') and a helper plasmid are inserted into a '''helper strain'''. The '''helper strain''' and '''conjugative strain''' are spotted with the '''recipient''' Anabaena for the three-parent mating]]<br />
<br />
=='''PowerCell Transformation'''==<br />
<br />
[[File:Brown-Stanford Transformed Anabaena brightfield.JPG|400px||center|thumb|Anabaena culture after triparental mating (bright field light micrograph)]]<br />
<br />
[[File:Brown-Stanford Transformed Anabaena fluorescent.JPG|400px||center|thumb|Same Anabaena culture showing GFP expression in successful transformant]]<br />
<br />
We transformed the final PowerCell construct into Anabeana using the methods described above. Expression of GFP is clearly visible, indicating the transformation was successful. Some non-transformants can be seen in the background.<br />
<br />
We selected for our transformants using neomycin, and let the cultures grow.<br />
<br />
[[File:Anabaena Selection.jpeg|400px||center|thumb|Left: BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Middle: BG11(N-) with neomycin + 50µl of the original conjugation mixture. Right: BG11(N-) with neomycin + 50µl of WT Anabaena.<br />
]]<br />
<br />
<br />
The initial selection looked promising. The left-most culture tube contains BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Both transformed and untransformed Anabaena should be able to grow in this mixture. As expected, this culture tube has the highest cell density. The middle culture tube contains BG11(N-) with neomycin + 50µl of the original conjugation mixture. Only Anabaena containing our plasmid should be able to grow in this mixture. As desired, growth is still visible, but is less dense. The right-most tube contains BG11(N-) with neomycin + 50µl of WT Anabaena. Nothing should be able to grow here. As expected, everything in this tube is dead.<br />
<br />
After about 2 weeks of growth, we can confirm the correct regulation of our sugar secretion construct by our pSac promoter by observing the presence of the GFP reporter. GFP expression is on in vegetative cells, and off in heterocysts, which implies that the sugar secretion construct is only active in vegetative cells, as we desire. Work is currently underway to assay the sucrose concentrations that are generated by our construct. We hope that these concentrations will be high enough to support growth of E. coli W; our experiments suggest the minimum concentration is around 5 mM (see section below). If the concentrations are high enough, we intend to grow E. coli W hosting several arbitrary active BioBrick plasmids, to show that PowerCell can power general biological tools. Updates will be posted here as they become available! <br />
<br />
<br><br />
<center><br />
<a href="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg"><img src="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg" style="width:40%"><br />
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Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
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<a href="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg"><img src="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg" style="width:40%"><br />
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Transformed Anabaena with heterocysts stained with alcian blue<br />
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----<br />
<br />
== '''Utilizing sucrose from PowerCell''' ==<br />
<br />
==='''E. coli W''' ===<br />
<br />
[[File:Brown-Stanford Sugar Cane.jpg|300px|thumb|Sugarcane is an extremely low-cost source of bulk sucrose used in production]]<br />
<br />
An essential aspect of the PowerCell project lies in microorganisms being able to survive off the nutrients being secreted by our genetically engineered ''Anabaena''. Our carbon source, sucrose, is a disaccharide that is not necessarily able to be metabolized by all species.<br />
<br />
Here our project intersects with current trends of bioproduction on Earth; sucrose is a cheap and easily obtained sugar, so there is interest in engineerable strains of microorganisms able to survive on it as a sole carbon source. <br />
<br />
We came across research suggesting that ''E. coli'' W is one such promising strain. It is one of the few strains of non-pathogenic ''E. coli'' able to metabolize sucrose, has good tolerance for environmental stresses, and grows rapidly. (Archer 2011) There have been papers describing the process of adapting ''E. coli'' W as a bioreactor organism, and methods of growing them to high density and productivity using fed-batch cultures. (Lee 1993, Lee 1997). <br />
<br />
In fact, the strain has already been used to produce a number of useful products. These include D(-)-lactate, a precursor to the formation of certain biodegradable plastic polymers, and L-alanine, a food additive and nutritional supplement. (Shukla 2004, Zhang 2007) With these examples, it is not difficult to imagine the production role of ''E. coli'' W in our Martian colony.<br />
<br />
=== '''E. coli W Sucrose Metabolism Experiment''' ===<br />
<br />
In the application of our project, PowerCell would grow alongside and support a productive microorganism strain such as E. coli W. We were thus interested in seeing whether ''E. coli'' W could grow in the conditions generated by PowerCell. This meant culturing on minimal BG-11 media with added sucrose to simulate secretion by ''Anabaena''.<br />
<br />
[[File:Brown-Stanford E coli W colony count.jpg|300px|left|thumb|Growth of our ''E. coli'' W varies by amount of sucrose (there was no 200ul plate for 80mM sucrose)]] <br />
<br />
Based on literature (Niederholtmeyer, et al,2010) and correspondence with Mr. Ducat, we predicted that our ''Anabaena'' culture would produce no more than 8mM of sucrose. It would be difficult to support substantial E. coli growth on such low levels without a means of concentrating sugar via in the bioreactor design. <br />
<br />
[[File:Brown-Stanford E coli W plate.jpg|300px|right|thumb|E. coli W colonies on BG-11 agar + 60 mM sucrose]]<br />
<br />
Initially we attempted to compare growth on BG-11 + sucrose with liquid cultures. However, multiple attempts yielded no appreciable change in O.D. after several days of incubation. We suspect that this was because ''E. coli'' growth was too small to detect via spectrophotometer. <br />
<br />
Next, we moved to agar plates as a more sensitive means of detecting cell growth. ''E. coli'' W with Amp resistance were grown, washed and resuspended in PBS to eliminate residual LB media, and streaked them on BG-11 Amp+ plates with varying concentrations of sugar (5mM, 10mM, 30mM, 60mM, and 80mM).<br />
<br />
The cells were incubated at 37C and observed regularly before colonies appeared on the fifth day. Visible colonies were observed on plates with sucrose concentrations as low as 5mM. These data suggest that, with the sugar secreted by PowerCell expressing cscB and no additional metabolic engineering, it is possible to support another organism.<br />
<br />
----<br />
<br />
==References==<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|1|Sammy Boussiba, Jane Gibson, Ammonia translocation in cyanobacteria, FEMS Microbiology Letters, Volume 88, Issue 1, July 1991, Pages 1-14, ISSN 0378-1097, 10.1016/0378-1097(91)90692-4<br />
Niederholtmeyer, Henrike, Wolfstadter, Bernd T., Savage, David F., Silver, Pamela A., Way, Jeffrey C. Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products. Appl. Environ. Microbiol. 2010 76: 3462-3466}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|2|Kim, Jihyun F , Lars K Nielsen, Colin T Archer, Sang Yup Lee, Claudia E Vickers, Jin Hwan Park, and Haeyoung Jeong. "The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli ."BioMed Central 12 (2011).}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|3|Lee, Jeong Wook, Sol Choi, Jin Hwan Park, Claudia E. Vickers, Lars K. Nielsen, and Sang Yup Lee. "Development of Sucrose-utilizing Escherichia Coli K-12 Strain by Cloning β-fructofuranosidases and Its Application for L-threonine Production."Applied Microbiology and Biotechnology 88.4 (2010): 905-13. Print.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|4|Claudia Argueta, Kamile Yuksek, Michael Summers, Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression in Nostoc punctiforme, Journal of Microbiological Methods, Volume 59, Issue 2, November 2004, Pages 181-188, ISSN 0167-7012, 10.1016/j.mimet.2004.06.009.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|5|M. Sahintoth, S. Frillingos, J.W. Lengeler, H.R. Kaback, Active Transport by the CscB Permease in Escherichia coli K-12, Biochemical and Biophysical Research Communications, Volume 208, Issue 3, 28 March 1995, Pages 1116-1123, ISSN 0006-291X, 10.1006/bbrc.1995.1449.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|6|Huang, H. H., D. Camsund, P. Lindblad, and T. Heidorn. "Design and Characterization of Molecular Tools for a Synthetic Biology Approach towards Developing Cyanobacterial Biotechnology." Nucleic Acids Research 38.8 (2010): 2577-593. Print.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|7|Lee, SY and Ho Nam Chang. High cell density cultivation of Escherichia coli W using sucrose as a carbon source. Biotechnology Letters 15:9 (1993) 971--974.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|8|Lee JS, Lee SY and Sunwon Park. Fed-batch culture of Escherichia coli W by exponential feeding of sucrose as a carbon source. Biotechnology Techniques 11:1 (1997) 59-62.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Footnote|9|Shukla, VB et al. Production of D(-)-lactate from sucrose and molasses. Biotechnology Letters 26 (2004) 689-693.}}<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-StanfordTeam:Brown-Stanford2011-12-07T09:21:53Z<p>Evanclark: /* APPLYING FOR STANFORD iGEM 2012 */</p>
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= '''APPLYING FOR STANFORD iGEM 2012''' =<br />
<br />
Please fill out our interest form at https://docs.google.com/spreadsheet/viewform?formkey=dEQyaWdReTN5S2pCcHRVY29hQXh4cHc6MQ.<br />
<br />
We will be sending further information to this list!<br />
<br />
Thanks!<br />
<br />
= '''APPLYING FOR BROWN iGEM 2012''' =<br />
(12/5/11)<br />
Brown iGEM is looking for undergrad team members for this upcoming year!<br />
<br />
Have you ever wanted to engineer living organisms to create novel functions and help transform the world? <br />
<br />
Spend a summer working at the Synthetic Biology Initiative at NASA Ames Research Center, and represent Brown at the premiere synthetic biology competition in the world! Opportunities exist for all kinds of microbiology and molecular biology work, computational biology, mathematical modeling, engineering, and much more. Develop incredible research and lab skills as you manage your own project!<br />
<br />
Students of all academic backgrounds and experience levels welcome (this includes Class of '12) <br />
<br />
We are hosting two '''INFO SESSIONS''' this week:<br />
<br />
'''TIME''': 6-7pm on Thursday 12/8, Friday 12/9<br />
<br />
'''LOCATION''': Barus and Holly 190<br />
<br />
-come hear more about iGEM, synthetic biology, and what the award-winning Brown iGEM team does; dinner will be provided!<br />
<br />
<br />
If you can't make it but are still interested, please let us know! Other questions or concerns are welcome as well. Contact us at igem@brown.edu<br />
<br />
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<span style="font-size: 20px;"></html>Our electronic application is [http://www.brownigem.com/2012_iGEM_Application.doc available now]. Keep up with the 2012 application process on our [https://www.facebook.com/events/146221565480758/ facebook page]!<br />
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= '''Mars BioTools: Synthetic Biology for Space Exploration''' =<br />
One of the major challenges of space exploration is the limited payload mass that can be launched on a rocket and the difficulty of resupply mid-mission. Any long term settlement will require more resources than astronauts can initially bring with them. Synthetic Biology has the potential to revolutionize space exploration and settlement. Biological tools have a major advantage over classical tools: the ability to self-replicate and regenerate.<br />
<br />
The emerging field of Synthetic Biology will allow us to engineer microbial factories that will largely circumvent the limiting payload factors. These cellular factories will generate fuel, food, medicines and building materials for settlers, but will consist of engineered cells that could be stored in a single test tube, and regrown to production scale on-site, as needed.<br />
<br />
The Brown-Stanford iGEM team is excited to work on three different projects, under the common theme of developing Synthetic Biology applications for space. In addition, as a response to the ethical and philosophical considerations, we interviewed prominent researchers and leaders in the field. '''Click on the astronaut in the corner to view our series.''' <br />
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<h3><img src="https://static.igem.org/mediawiki/2011/3/39/Brown-Stanford_REGObricks-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/REGObricks/Introduction">REGObricks</a></h3><br />
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Any extra-terrestrial settlement will require a habitat to keep its occupants alive, but transport of such a habitat will require a huge amount of payload space. RegoBricks uses bacteria to cement Martian and Lunar regolith (soil) simulant into durable building blocks, similar to concrete bricks, allowing settlements to use in situ resources to build structures.<br />
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<h3><img src="https://static.igem.org/mediawiki/2011/4/4c/Brown-Stanford_DNADamage-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/FRETSensor/Introduction">FRETcetera</a></h3><br />
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Biological interfaces cannot pass information to their users as fast as electronic ones can. FRETcetera develops a novel method of fast-acting biological reporting with changes in cell fluorescence. Bacteria could be used to detect toxic chemicals in the environment, for example, or inform astronauts that their microbial tools are unhealthy.<br />
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<h3><img src="https://static.igem.org/mediawiki/2011/9/9a/Brown-Stanford_PowerCell-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/PowerCell/Introduction">PowerCell</a></h3><br />
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<br />
All biological tools need energy to run. PowerCell develops a universal energy source. Engineered photosynthetic bacteria generate carbon and nitrogenous nutrients from sunlight and air and secrete them to power biological tools. These tools will transform the raw materials into fuel, building materials, food, drugs, and other products useful to settlers.<br />
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<br />
= '''Summary''' =<br />
<br />
=== '''News!''' ===<br />
<br />
===== '''iGEM Awards''' =====<br />
<br />
We did very well at iGEM this year! <br />
<br />
-At the Americas Regional Jamboree, we finished Top 4 and won Best Presentation.<br />
<br />
-At the World Jamboree, we finished in the Sweet 16, and won Best New Application. <br />
<br />
We are extremely grateful to all those who helped us along the way, and our mentors. Thank you so much!<br />
<br />
===== '''Project Summary''' =====<br />
<br />
Here's a minute long teaser of our project!<br />
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=== '''Latest Advancements on Projects''' ===<br />
<br />
===== '''Further evidence of pSac promoter specificity''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 100%;"><br />
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<br />
<td style="padding-left: 20px; padding-right: 0px; padding-top: 0px; padding-bottom: 0px; width: 90%;"><br />
<br />
<p> To further demonstrate that expression of our construct is properly co-localized to vegetative cells, we grew cultures of Anabaena in nitrate-free BG11 media to promote heterocyst formation. In the first picture, we observed areas of diminished GFP fluorescence at regular intervals (approximately 1 in 10 cells) along our transformed Anabaena filament. We designed our GFP reporter to express under the pSac promoter and only in non-heterocyst cells, so we would expect the spacing of our dark spots to match the spacing of heterocysts identified through alcian blue staining. Alcian blue is a dye that selectively binds to polysaccharide chains on the surface of heterocyst cells. As you can see by comparing the two images, our results appear to match up!<br />
<br />
<br><br />
<center><br />
<a href="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg"><img src="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%;"><br />
Transformed Anabaena with heterocysts stained with alcian blue<br />
</div><br />
<br><br />
<br><br />
</center><br />
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</table><br />
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<br />
===== '''Cyanobacteria Transformation''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 100%;"><br />
<tr><br />
<br />
<td style="padding-left: 20px; padding-right: 0px; padding-top: 0px; padding-bottom: 0px; width: 90%;"><br />
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<p>We successfully transformed our cyanobacterial chassis, Anabaena PCC 7120, with our designed PowerCell genetic construct. Unlike more common cyanobacteria species such as Synechocystis PCC 6803 or Synechococcus elongatus PCC 7942, Anabaena is not naturally competent and does not easily uptake foreign DNA. </p><br />
<br />
<p>We performed a triparental conjugation in order to bypass the natural barriers to transformation. We demonstrated the successful control of our pSac promoter over our sugar secretion construct through GFP expression. The sugar secretion construct is only activated in actively photosynthesizing cells (not heterocysts). Work to characterize the performance of our cscB sucrose secretion construct is underway. We will assay sucrose levels produced by PowerCell. If they are high enough, we will attempt to grow E. coli W. containing arbitrary biobricks from this nutrient source, in an attempt to show PowerCell can power biological tools. </p><br />
<br />
<br />
<br><br />
<center><br />
<a href="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg"><img src="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
</div><br />
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</center><br />
</td><br />
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</table><br />
</html><br />
<br />
===== '''Biobricked the urease operon''' =====<br />
<br />
We successfully PCR cloned a 10.7 kb functional urease cassette from the plasmid pBU11, whose genetic sequence has hitherto never been known before. We ligated it onto a backbone, and successfully [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biobrick made a standard BioBrick], characterizing [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Characterization its function] and demonstrating active urease activity. Urease is the catalyst behind the phenomenon known as biocement, which is able to fuse sand into bricks, using nothing more than calcium ions and urea. [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biocementation More about urease.]<br />
<br />
Biobrick part [http://partsregistry.org/Part:BBa_K656013 BBa_K656013] has been submitted to the Registry of Standardized Parts. The sequence of this cassette is unavailable because it currently does not exist in any known registry. At the time of this writing, it is currently being sequenced in the lab of Dr. Chris Mason at Weill Cornell Medical College. As soon as the sequence is available, we will supplement the directory listing.<br />
<html><br />
<br />
<br><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG"><img src="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%; font-weight:bold;"><br />
Successful transformants on urease test plates.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</html><br />
<br />
===== '''Stratospheric Balloon Flight''' =====<br />
<br />
We launched samples of our bacteria to the far edge of space (80,000 ft-110,000 ft) to test for their ability to survive simulated Martian environmental conditions. The pressures, temperatures, and radiation conditions at these altitudes are similar to the conditions on the Martian surface.<br />
[[Team:Brown-Stanford/REGObricks/Balloon|Read about our escapades here]]<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/qCQXg8DmZZ0"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/qCQXg8DmZZ0"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Advancements in Human Practices''' ===<br />
===== '''Feature on Synthetic Biology and Space''' =====<br />
We talked with key figures in science and science policy about the implications of human expansion into space, extraterrestrial life and synthetic biology. You can look at our interviews and findings [https://2011.igem.org/Team:Brown-Stanford/SynEthics/Intro here] or by clicking on the astronaut on the upper left!<br />
<br />
===== '''Education and Publicity''' =====<br />
<br />
<html><br />
<table style="width: 100%;"><br />
<tr><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Editor's Choice! x2<br />
</div><br />
</center><br />
</td><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Lunar Science Poster. Click to see full version.<br />
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</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/9YKsa7s8WM4?version=3"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/9YKsa7s8WM4?version=3"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
<br />
<br />
We educated and publicized the power of synthetic biology in a whole suite of venues and mediums, including <br />
<br />
* A crowd of 600+ over two days at [https://2011.igem.org/Team:Brown-Stanford/MakerFaire Maker Faire NYC], resulting in two Editor’s Choice blue ribbons! <br />
* A filming opportunity with the [https://2011.igem.org/Team:Brown-Stanford/BBC BBC] Horizon, to be featured in a documentary on NASA’s Initiative in Synthetic Biology<br />
*Verbal [https://2011.igem.org/Team:Brown-Stanford/NASA presentations] to NASA Administrator Charlie Bolden, NASA Ames Research Center Director Pete Worden, and writer and visionary Stewart Brand <br />
* A poster presentation at NASA’s Lunar Science Forum<br />
* A heart-pounding [https://2011.igem.org/Team:Brown-Stanford/SB5 poem] at the first-ever SB5.0 Synbio Slam<br />
<br />
===== '''Outreach and Collaboration''' =====<br />
<br />
<html><br />
<center><br />
<a href="http://openwetware.org/wiki/IGEM_Outreach"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6f/Brown-Stanford_Cbricks.jpg" style="width: 30%;"><br />
</a><br />
</center><br />
</html><br />
<br />
We spearheaded the '''largest collaborative effort''' in iGEM history to change the way we do outreach, creating [http://openwetware.org/wiki/IGEM_Outreach '''CommunityBricks'''], a database of valuable teaching plans, presentations, community resources and projects for young and old alike.<br />
<br />
===== '''Alumni and Community-Building''' =====<br />
<br />
<br />
[[File:Brown-Stanford alumnigem.png|300px|center|AlumniGEM|link=http://alumni.brownigem.com/]]<br />
<br />
We built the [http://alumni.brownigem.com '''first iGEM Alumni Network,'''] to provide iGEM'ers a growing collection of professional and social resources and keep the spirit of iGEM alive post-Jamborees.<br />
<br />
<html><br />
<div><a href="javascript:toggle();" id="toggleNews"><h3>Show Media and News</h3></a></div><br />
<br />
</div><br />
<div class="pageContent" id="news" style="display: none;"><br />
</html><br />
<br />
== '''Martian Colony''' ==<br />
[[File:Brown-Stanford_MartianColony.png|center|750px]]<br />
<br />
== '''News''' ==<br />
<br />
===In the Lab===<br />
<center><html><object width="600" height="450"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to="></param> <param name="movie" value="http://www.flickr.com/apps/slideshow/show.swf?v=107931"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://www.flickr.com/apps/slideshow/show.swf?v=107931" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to=" width="600" height="450"></embed></object></html></center><br />
<br />
=== '''August 4<sup>th</sup>, 2011''' ===<br />
<center><html><object width="580" height="355"><param name="movie" value="http://www.youtube.com/v/x-MYE4QkjOU"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/x-MYE4QkjOU" type="application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''June 7<sup>th</sup>, 2011''' ===<br />
[[File:Brown-Stanford TeamPicture6-7-2011.jpg|center|500px]]<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-StanfordTeam:Brown-Stanford2011-12-07T09:21:13Z<p>Evanclark: /* APPLYING FOR BROWN iGEM 2012 */</p>
<hr />
<div>{{:Team:Brown-Stanford/Templates/Main}}<br />
{{:Team:Brown-Stanford/Templates/Content}}<br />
{{:Team:Brown-Stanford/Templates/Twitter}}<br />
<br />
= '''APPLYING FOR STANFORD iGEM 2012''' =<br />
<br />
Please fill out our form at https://docs.google.com/spreadsheet/viewform?formkey=dEQyaWdReTN5S2pCcHRVY29hQXh4cHc6MQ.<br />
<br />
We will be sending further information to this list!<br />
<br />
= '''APPLYING FOR BROWN iGEM 2012''' =<br />
(12/5/11)<br />
Brown iGEM is looking for undergrad team members for this upcoming year!<br />
<br />
Have you ever wanted to engineer living organisms to create novel functions and help transform the world? <br />
<br />
Spend a summer working at the Synthetic Biology Initiative at NASA Ames Research Center, and represent Brown at the premiere synthetic biology competition in the world! Opportunities exist for all kinds of microbiology and molecular biology work, computational biology, mathematical modeling, engineering, and much more. Develop incredible research and lab skills as you manage your own project!<br />
<br />
Students of all academic backgrounds and experience levels welcome (this includes Class of '12) <br />
<br />
We are hosting two '''INFO SESSIONS''' this week:<br />
<br />
'''TIME''': 6-7pm on Thursday 12/8, Friday 12/9<br />
<br />
'''LOCATION''': Barus and Holly 190<br />
<br />
-come hear more about iGEM, synthetic biology, and what the award-winning Brown iGEM team does; dinner will be provided!<br />
<br />
<br />
If you can't make it but are still interested, please let us know! Other questions or concerns are welcome as well. Contact us at igem@brown.edu<br />
<br />
<html><br />
<span style="font-size: 20px;"></html>Our electronic application is [http://www.brownigem.com/2012_iGEM_Application.doc available now]. Keep up with the 2012 application process on our [https://www.facebook.com/events/146221565480758/ facebook page]!<br />
<html></span></html><br />
<br />
= '''Mars BioTools: Synthetic Biology for Space Exploration''' =<br />
One of the major challenges of space exploration is the limited payload mass that can be launched on a rocket and the difficulty of resupply mid-mission. Any long term settlement will require more resources than astronauts can initially bring with them. Synthetic Biology has the potential to revolutionize space exploration and settlement. Biological tools have a major advantage over classical tools: the ability to self-replicate and regenerate.<br />
<br />
The emerging field of Synthetic Biology will allow us to engineer microbial factories that will largely circumvent the limiting payload factors. These cellular factories will generate fuel, food, medicines and building materials for settlers, but will consist of engineered cells that could be stored in a single test tube, and regrown to production scale on-site, as needed.<br />
<br />
The Brown-Stanford iGEM team is excited to work on three different projects, under the common theme of developing Synthetic Biology applications for space. In addition, as a response to the ethical and philosophical considerations, we interviewed prominent researchers and leaders in the field. '''Click on the astronaut in the corner to view our series.''' <br />
<br />
<html><br />
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<div class="miniContent"><br />
<br />
<div class="miniContentLeft"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/3/39/Brown-Stanford_REGObricks-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/REGObricks/Introduction">REGObricks</a></h3><br />
</html><br />
<br />
Any extra-terrestrial settlement will require a habitat to keep its occupants alive, but transport of such a habitat will require a huge amount of payload space. RegoBricks uses bacteria to cement Martian and Lunar regolith (soil) simulant into durable building blocks, similar to concrete bricks, allowing settlements to use in situ resources to build structures.<br />
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</div><br />
<br />
<div class="miniContentRight"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/4/4c/Brown-Stanford_DNADamage-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/FRETSensor/Introduction">FRETcetera</a></h3><br />
</html><br />
<br />
Biological interfaces cannot pass information to their users as fast as electronic ones can. FRETcetera develops a novel method of fast-acting biological reporting with changes in cell fluorescence. Bacteria could be used to detect toxic chemicals in the environment, for example, or inform astronauts that their microbial tools are unhealthy.<br />
<html><br />
<br />
</div><br />
<div class="miniContentCenter"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/9/9a/Brown-Stanford_PowerCell-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/PowerCell/Introduction">PowerCell</a></h3><br />
</html><br />
<br />
All biological tools need energy to run. PowerCell develops a universal energy source. Engineered photosynthetic bacteria generate carbon and nitrogenous nutrients from sunlight and air and secrete them to power biological tools. These tools will transform the raw materials into fuel, building materials, food, drugs, and other products useful to settlers.<br />
<html><br />
</div><br />
<br />
</div><br />
<div class="pageContent"><br />
</html><br />
<br />
= '''Summary''' =<br />
<br />
=== '''News!''' ===<br />
<br />
===== '''iGEM Awards''' =====<br />
<br />
We did very well at iGEM this year! <br />
<br />
-At the Americas Regional Jamboree, we finished Top 4 and won Best Presentation.<br />
<br />
-At the World Jamboree, we finished in the Sweet 16, and won Best New Application. <br />
<br />
We are extremely grateful to all those who helped us along the way, and our mentors. Thank you so much!<br />
<br />
===== '''Project Summary''' =====<br />
<br />
Here's a minute long teaser of our project!<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/watch?v=b2AkJRTLOcs"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/b2AkJRTLOcs"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Latest Advancements on Projects''' ===<br />
<br />
===== '''Further evidence of pSac promoter specificity''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 100%;"><br />
<tr><br />
<br />
<td style="padding-left: 20px; padding-right: 0px; padding-top: 0px; padding-bottom: 0px; width: 90%;"><br />
<br />
<p> To further demonstrate that expression of our construct is properly co-localized to vegetative cells, we grew cultures of Anabaena in nitrate-free BG11 media to promote heterocyst formation. In the first picture, we observed areas of diminished GFP fluorescence at regular intervals (approximately 1 in 10 cells) along our transformed Anabaena filament. We designed our GFP reporter to express under the pSac promoter and only in non-heterocyst cells, so we would expect the spacing of our dark spots to match the spacing of heterocysts identified through alcian blue staining. Alcian blue is a dye that selectively binds to polysaccharide chains on the surface of heterocyst cells. As you can see by comparing the two images, our results appear to match up!<br />
<br />
<br><br />
<center><br />
<a href="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg"><img src="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%;"><br />
Transformed Anabaena with heterocysts stained with alcian blue<br />
</div><br />
<br><br />
<br><br />
</center><br />
</td><br />
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</table><br />
</html><br />
<br />
===== '''Cyanobacteria Transformation''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 100%;"><br />
<tr><br />
<br />
<td style="padding-left: 20px; padding-right: 0px; padding-top: 0px; padding-bottom: 0px; width: 90%;"><br />
<br />
<p>We successfully transformed our cyanobacterial chassis, Anabaena PCC 7120, with our designed PowerCell genetic construct. Unlike more common cyanobacteria species such as Synechocystis PCC 6803 or Synechococcus elongatus PCC 7942, Anabaena is not naturally competent and does not easily uptake foreign DNA. </p><br />
<br />
<p>We performed a triparental conjugation in order to bypass the natural barriers to transformation. We demonstrated the successful control of our pSac promoter over our sugar secretion construct through GFP expression. The sugar secretion construct is only activated in actively photosynthesizing cells (not heterocysts). Work to characterize the performance of our cscB sucrose secretion construct is underway. We will assay sucrose levels produced by PowerCell. If they are high enough, we will attempt to grow E. coli W. containing arbitrary biobricks from this nutrient source, in an attempt to show PowerCell can power biological tools. </p><br />
<br />
<br />
<br><br />
<center><br />
<a href="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg"><img src="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
===== '''Biobricked the urease operon''' =====<br />
<br />
We successfully PCR cloned a 10.7 kb functional urease cassette from the plasmid pBU11, whose genetic sequence has hitherto never been known before. We ligated it onto a backbone, and successfully [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biobrick made a standard BioBrick], characterizing [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Characterization its function] and demonstrating active urease activity. Urease is the catalyst behind the phenomenon known as biocement, which is able to fuse sand into bricks, using nothing more than calcium ions and urea. [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biocementation More about urease.]<br />
<br />
Biobrick part [http://partsregistry.org/Part:BBa_K656013 BBa_K656013] has been submitted to the Registry of Standardized Parts. The sequence of this cassette is unavailable because it currently does not exist in any known registry. At the time of this writing, it is currently being sequenced in the lab of Dr. Chris Mason at Weill Cornell Medical College. As soon as the sequence is available, we will supplement the directory listing.<br />
<html><br />
<br />
<br><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG"><img src="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%; font-weight:bold;"><br />
Successful transformants on urease test plates.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</html><br />
<br />
===== '''Stratospheric Balloon Flight''' =====<br />
<br />
We launched samples of our bacteria to the far edge of space (80,000 ft-110,000 ft) to test for their ability to survive simulated Martian environmental conditions. The pressures, temperatures, and radiation conditions at these altitudes are similar to the conditions on the Martian surface.<br />
[[Team:Brown-Stanford/REGObricks/Balloon|Read about our escapades here]]<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/qCQXg8DmZZ0"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/qCQXg8DmZZ0"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Advancements in Human Practices''' ===<br />
===== '''Feature on Synthetic Biology and Space''' =====<br />
We talked with key figures in science and science policy about the implications of human expansion into space, extraterrestrial life and synthetic biology. You can look at our interviews and findings [https://2011.igem.org/Team:Brown-Stanford/SynEthics/Intro here] or by clicking on the astronaut on the upper left!<br />
<br />
===== '''Education and Publicity''' =====<br />
<br />
<html><br />
<table style="width: 100%;"><br />
<tr><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Editor's Choice! x2<br />
</div><br />
</center><br />
</td><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Lunar Science Poster. Click to see full version.<br />
</div><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/9YKsa7s8WM4?version=3"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/9YKsa7s8WM4?version=3"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
<br />
<br />
We educated and publicized the power of synthetic biology in a whole suite of venues and mediums, including <br />
<br />
* A crowd of 600+ over two days at [https://2011.igem.org/Team:Brown-Stanford/MakerFaire Maker Faire NYC], resulting in two Editor’s Choice blue ribbons! <br />
* A filming opportunity with the [https://2011.igem.org/Team:Brown-Stanford/BBC BBC] Horizon, to be featured in a documentary on NASA’s Initiative in Synthetic Biology<br />
*Verbal [https://2011.igem.org/Team:Brown-Stanford/NASA presentations] to NASA Administrator Charlie Bolden, NASA Ames Research Center Director Pete Worden, and writer and visionary Stewart Brand <br />
* A poster presentation at NASA’s Lunar Science Forum<br />
* A heart-pounding [https://2011.igem.org/Team:Brown-Stanford/SB5 poem] at the first-ever SB5.0 Synbio Slam<br />
<br />
===== '''Outreach and Collaboration''' =====<br />
<br />
<html><br />
<center><br />
<a href="http://openwetware.org/wiki/IGEM_Outreach"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6f/Brown-Stanford_Cbricks.jpg" style="width: 30%;"><br />
</a><br />
</center><br />
</html><br />
<br />
We spearheaded the '''largest collaborative effort''' in iGEM history to change the way we do outreach, creating [http://openwetware.org/wiki/IGEM_Outreach '''CommunityBricks'''], a database of valuable teaching plans, presentations, community resources and projects for young and old alike.<br />
<br />
===== '''Alumni and Community-Building''' =====<br />
<br />
<br />
[[File:Brown-Stanford alumnigem.png|300px|center|AlumniGEM|link=http://alumni.brownigem.com/]]<br />
<br />
We built the [http://alumni.brownigem.com '''first iGEM Alumni Network,'''] to provide iGEM'ers a growing collection of professional and social resources and keep the spirit of iGEM alive post-Jamborees.<br />
<br />
<html><br />
<div><a href="javascript:toggle();" id="toggleNews"><h3>Show Media and News</h3></a></div><br />
<br />
</div><br />
<div class="pageContent" id="news" style="display: none;"><br />
</html><br />
<br />
== '''Martian Colony''' ==<br />
[[File:Brown-Stanford_MartianColony.png|center|750px]]<br />
<br />
== '''News''' ==<br />
<br />
===In the Lab===<br />
<center><html><object width="600" height="450"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to="></param> <param name="movie" value="http://www.flickr.com/apps/slideshow/show.swf?v=107931"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://www.flickr.com/apps/slideshow/show.swf?v=107931" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to=" width="600" height="450"></embed></object></html></center><br />
<br />
=== '''August 4<sup>th</sup>, 2011''' ===<br />
<center><html><object width="580" height="355"><param name="movie" value="http://www.youtube.com/v/x-MYE4QkjOU"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/x-MYE4QkjOU" type="application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''June 7<sup>th</sup>, 2011''' ===<br />
[[File:Brown-Stanford TeamPicture6-7-2011.jpg|center|500px]]<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-StanfordTeam:Brown-Stanford2011-12-07T09:17:52Z<p>Evanclark: /* Further evidence of pSac promoter specificity */</p>
<hr />
<div>{{:Team:Brown-Stanford/Templates/Main}}<br />
{{:Team:Brown-Stanford/Templates/Content}}<br />
{{:Team:Brown-Stanford/Templates/Twitter}}<br />
<br />
= '''APPLYING FOR BROWN iGEM 2012''' =<br />
(12/5/11)<br />
Brown iGEM is looking for undergrad team members for this upcoming year!<br />
<br />
Have you ever wanted to engineer living organisms to create novel functions and help transform the world? <br />
<br />
Spend a summer working at the Synthetic Biology Initiative at NASA Ames Research Center, and represent Brown at the premiere synthetic biology competition in the world! Opportunities exist for all kinds of microbiology and molecular biology work, computational biology, mathematical modeling, engineering, and much more. Develop incredible research and lab skills as you manage your own project!<br />
<br />
Students of all academic backgrounds and experience levels welcome (this includes Class of '12) <br />
<br />
We are hosting two '''INFO SESSIONS''' this week:<br />
<br />
'''TIME''': 6-7pm on Thursday 12/8, Friday 12/9<br />
<br />
'''LOCATION''': Barus and Holly 190<br />
<br />
-come hear more about iGEM, synthetic biology, and what the award-winning Brown iGEM team does; dinner will be provided!<br />
<br />
<br />
If you can't make it but are still interested, please let us know! Other questions or concerns are welcome as well. Contact us at igem@brown.edu<br />
<br />
<html><br />
<span style="font-size: 20px;"></html>Our electronic application is [http://www.brownigem.com/2012_iGEM_Application.doc available now]. Keep up with the 2012 application process on our [https://www.facebook.com/events/146221565480758/ facebook page]!<br />
<html></span></html><br />
<br />
= '''Mars BioTools: Synthetic Biology for Space Exploration''' =<br />
One of the major challenges of space exploration is the limited payload mass that can be launched on a rocket and the difficulty of resupply mid-mission. Any long term settlement will require more resources than astronauts can initially bring with them. Synthetic Biology has the potential to revolutionize space exploration and settlement. Biological tools have a major advantage over classical tools: the ability to self-replicate and regenerate.<br />
<br />
The emerging field of Synthetic Biology will allow us to engineer microbial factories that will largely circumvent the limiting payload factors. These cellular factories will generate fuel, food, medicines and building materials for settlers, but will consist of engineered cells that could be stored in a single test tube, and regrown to production scale on-site, as needed.<br />
<br />
The Brown-Stanford iGEM team is excited to work on three different projects, under the common theme of developing Synthetic Biology applications for space. In addition, as a response to the ethical and philosophical considerations, we interviewed prominent researchers and leaders in the field. '''Click on the astronaut in the corner to view our series.''' <br />
<br />
<html><br />
</div><br />
<div class="miniContent"><br />
<br />
<div class="miniContentLeft"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/3/39/Brown-Stanford_REGObricks-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/REGObricks/Introduction">REGObricks</a></h3><br />
</html><br />
<br />
Any extra-terrestrial settlement will require a habitat to keep its occupants alive, but transport of such a habitat will require a huge amount of payload space. RegoBricks uses bacteria to cement Martian and Lunar regolith (soil) simulant into durable building blocks, similar to concrete bricks, allowing settlements to use in situ resources to build structures.<br />
<html><br />
</div><br />
<br />
<div class="miniContentRight"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/4/4c/Brown-Stanford_DNADamage-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/FRETSensor/Introduction">FRETcetera</a></h3><br />
</html><br />
<br />
Biological interfaces cannot pass information to their users as fast as electronic ones can. FRETcetera develops a novel method of fast-acting biological reporting with changes in cell fluorescence. Bacteria could be used to detect toxic chemicals in the environment, for example, or inform astronauts that their microbial tools are unhealthy.<br />
<html><br />
<br />
</div><br />
<div class="miniContentCenter"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/9/9a/Brown-Stanford_PowerCell-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/PowerCell/Introduction">PowerCell</a></h3><br />
</html><br />
<br />
All biological tools need energy to run. PowerCell develops a universal energy source. Engineered photosynthetic bacteria generate carbon and nitrogenous nutrients from sunlight and air and secrete them to power biological tools. These tools will transform the raw materials into fuel, building materials, food, drugs, and other products useful to settlers.<br />
<html><br />
</div><br />
<br />
</div><br />
<div class="pageContent"><br />
</html><br />
<br />
= '''Summary''' =<br />
<br />
=== '''News!''' ===<br />
<br />
===== '''iGEM Awards''' =====<br />
<br />
We did very well at iGEM this year! <br />
<br />
-At the Americas Regional Jamboree, we finished Top 4 and won Best Presentation.<br />
<br />
-At the World Jamboree, we finished in the Sweet 16, and won Best New Application. <br />
<br />
We are extremely grateful to all those who helped us along the way, and our mentors. Thank you so much!<br />
<br />
===== '''Project Summary''' =====<br />
<br />
Here's a minute long teaser of our project!<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/watch?v=b2AkJRTLOcs"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/b2AkJRTLOcs"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Latest Advancements on Projects''' ===<br />
<br />
===== '''Further evidence of pSac promoter specificity''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 100%;"><br />
<tr><br />
<br />
<td style="padding-left: 20px; padding-right: 0px; padding-top: 0px; padding-bottom: 0px; width: 90%;"><br />
<br />
<p> To further demonstrate that expression of our construct is properly co-localized to vegetative cells, we grew cultures of Anabaena in nitrate-free BG11 media to promote heterocyst formation. In the first picture, we observed areas of diminished GFP fluorescence at regular intervals (approximately 1 in 10 cells) along our transformed Anabaena filament. We designed our GFP reporter to express under the pSac promoter and only in non-heterocyst cells, so we would expect the spacing of our dark spots to match the spacing of heterocysts identified through alcian blue staining. Alcian blue is a dye that selectively binds to polysaccharide chains on the surface of heterocyst cells. As you can see by comparing the two images, our results appear to match up!<br />
<br />
<br><br />
<center><br />
<a href="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg"><img src="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%;"><br />
Transformed Anabaena with heterocysts stained with alcian blue<br />
</div><br />
<br><br />
<br><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
===== '''Cyanobacteria Transformation''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 100%;"><br />
<tr><br />
<br />
<td style="padding-left: 20px; padding-right: 0px; padding-top: 0px; padding-bottom: 0px; width: 90%;"><br />
<br />
<p>We successfully transformed our cyanobacterial chassis, Anabaena PCC 7120, with our designed PowerCell genetic construct. Unlike more common cyanobacteria species such as Synechocystis PCC 6803 or Synechococcus elongatus PCC 7942, Anabaena is not naturally competent and does not easily uptake foreign DNA. </p><br />
<br />
<p>We performed a triparental conjugation in order to bypass the natural barriers to transformation. We demonstrated the successful control of our pSac promoter over our sugar secretion construct through GFP expression. The sugar secretion construct is only activated in actively photosynthesizing cells (not heterocysts). Work to characterize the performance of our cscB sucrose secretion construct is underway. We will assay sucrose levels produced by PowerCell. If they are high enough, we will attempt to grow E. coli W. containing arbitrary biobricks from this nutrient source, in an attempt to show PowerCell can power biological tools. </p><br />
<br />
<br />
<br><br />
<center><br />
<a href="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg"><img src="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
===== '''Biobricked the urease operon''' =====<br />
<br />
We successfully PCR cloned a 10.7 kb functional urease cassette from the plasmid pBU11, whose genetic sequence has hitherto never been known before. We ligated it onto a backbone, and successfully [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biobrick made a standard BioBrick], characterizing [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Characterization its function] and demonstrating active urease activity. Urease is the catalyst behind the phenomenon known as biocement, which is able to fuse sand into bricks, using nothing more than calcium ions and urea. [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biocementation More about urease.]<br />
<br />
Biobrick part [http://partsregistry.org/Part:BBa_K656013 BBa_K656013] has been submitted to the Registry of Standardized Parts. The sequence of this cassette is unavailable because it currently does not exist in any known registry. At the time of this writing, it is currently being sequenced in the lab of Dr. Chris Mason at Weill Cornell Medical College. As soon as the sequence is available, we will supplement the directory listing.<br />
<html><br />
<br />
<br><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG"><img src="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%; font-weight:bold;"><br />
Successful transformants on urease test plates.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</html><br />
<br />
===== '''Stratospheric Balloon Flight''' =====<br />
<br />
We launched samples of our bacteria to the far edge of space (80,000 ft-110,000 ft) to test for their ability to survive simulated Martian environmental conditions. The pressures, temperatures, and radiation conditions at these altitudes are similar to the conditions on the Martian surface.<br />
[[Team:Brown-Stanford/REGObricks/Balloon|Read about our escapades here]]<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/qCQXg8DmZZ0"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/qCQXg8DmZZ0"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Advancements in Human Practices''' ===<br />
===== '''Feature on Synthetic Biology and Space''' =====<br />
We talked with key figures in science and science policy about the implications of human expansion into space, extraterrestrial life and synthetic biology. You can look at our interviews and findings [https://2011.igem.org/Team:Brown-Stanford/SynEthics/Intro here] or by clicking on the astronaut on the upper left!<br />
<br />
===== '''Education and Publicity''' =====<br />
<br />
<html><br />
<table style="width: 100%;"><br />
<tr><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Editor's Choice! x2<br />
</div><br />
</center><br />
</td><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Lunar Science Poster. Click to see full version.<br />
</div><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/9YKsa7s8WM4?version=3"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/9YKsa7s8WM4?version=3"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
<br />
<br />
We educated and publicized the power of synthetic biology in a whole suite of venues and mediums, including <br />
<br />
* A crowd of 600+ over two days at [https://2011.igem.org/Team:Brown-Stanford/MakerFaire Maker Faire NYC], resulting in two Editor’s Choice blue ribbons! <br />
* A filming opportunity with the [https://2011.igem.org/Team:Brown-Stanford/BBC BBC] Horizon, to be featured in a documentary on NASA’s Initiative in Synthetic Biology<br />
*Verbal [https://2011.igem.org/Team:Brown-Stanford/NASA presentations] to NASA Administrator Charlie Bolden, NASA Ames Research Center Director Pete Worden, and writer and visionary Stewart Brand <br />
* A poster presentation at NASA’s Lunar Science Forum<br />
* A heart-pounding [https://2011.igem.org/Team:Brown-Stanford/SB5 poem] at the first-ever SB5.0 Synbio Slam<br />
<br />
===== '''Outreach and Collaboration''' =====<br />
<br />
<html><br />
<center><br />
<a href="http://openwetware.org/wiki/IGEM_Outreach"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6f/Brown-Stanford_Cbricks.jpg" style="width: 30%;"><br />
</a><br />
</center><br />
</html><br />
<br />
We spearheaded the '''largest collaborative effort''' in iGEM history to change the way we do outreach, creating [http://openwetware.org/wiki/IGEM_Outreach '''CommunityBricks'''], a database of valuable teaching plans, presentations, community resources and projects for young and old alike.<br />
<br />
===== '''Alumni and Community-Building''' =====<br />
<br />
<br />
[[File:Brown-Stanford alumnigem.png|300px|center|AlumniGEM|link=http://alumni.brownigem.com/]]<br />
<br />
We built the [http://alumni.brownigem.com '''first iGEM Alumni Network,'''] to provide iGEM'ers a growing collection of professional and social resources and keep the spirit of iGEM alive post-Jamborees.<br />
<br />
<html><br />
<div><a href="javascript:toggle();" id="toggleNews"><h3>Show Media and News</h3></a></div><br />
<br />
</div><br />
<div class="pageContent" id="news" style="display: none;"><br />
</html><br />
<br />
== '''Martian Colony''' ==<br />
[[File:Brown-Stanford_MartianColony.png|center|750px]]<br />
<br />
== '''News''' ==<br />
<br />
===In the Lab===<br />
<center><html><object width="600" height="450"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to="></param> <param name="movie" value="http://www.flickr.com/apps/slideshow/show.swf?v=107931"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://www.flickr.com/apps/slideshow/show.swf?v=107931" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to=" width="600" height="450"></embed></object></html></center><br />
<br />
=== '''August 4<sup>th</sup>, 2011''' ===<br />
<center><html><object width="580" height="355"><param name="movie" value="http://www.youtube.com/v/x-MYE4QkjOU"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/x-MYE4QkjOU" type="application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''June 7<sup>th</sup>, 2011''' ===<br />
[[File:Brown-Stanford TeamPicture6-7-2011.jpg|center|500px]]<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-StanfordTeam:Brown-Stanford2011-12-07T09:16:53Z<p>Evanclark: /* Further evidence of pSac promoter specificity */</p>
<hr />
<div>{{:Team:Brown-Stanford/Templates/Main}}<br />
{{:Team:Brown-Stanford/Templates/Content}}<br />
{{:Team:Brown-Stanford/Templates/Twitter}}<br />
<br />
= '''APPLYING FOR BROWN iGEM 2012''' =<br />
(12/5/11)<br />
Brown iGEM is looking for undergrad team members for this upcoming year!<br />
<br />
Have you ever wanted to engineer living organisms to create novel functions and help transform the world? <br />
<br />
Spend a summer working at the Synthetic Biology Initiative at NASA Ames Research Center, and represent Brown at the premiere synthetic biology competition in the world! Opportunities exist for all kinds of microbiology and molecular biology work, computational biology, mathematical modeling, engineering, and much more. Develop incredible research and lab skills as you manage your own project!<br />
<br />
Students of all academic backgrounds and experience levels welcome (this includes Class of '12) <br />
<br />
We are hosting two '''INFO SESSIONS''' this week:<br />
<br />
'''TIME''': 6-7pm on Thursday 12/8, Friday 12/9<br />
<br />
'''LOCATION''': Barus and Holly 190<br />
<br />
-come hear more about iGEM, synthetic biology, and what the award-winning Brown iGEM team does; dinner will be provided!<br />
<br />
<br />
If you can't make it but are still interested, please let us know! Other questions or concerns are welcome as well. Contact us at igem@brown.edu<br />
<br />
<html><br />
<span style="font-size: 20px;"></html>Our electronic application is [http://www.brownigem.com/2012_iGEM_Application.doc available now]. Keep up with the 2012 application process on our [https://www.facebook.com/events/146221565480758/ facebook page]!<br />
<html></span></html><br />
<br />
= '''Mars BioTools: Synthetic Biology for Space Exploration''' =<br />
One of the major challenges of space exploration is the limited payload mass that can be launched on a rocket and the difficulty of resupply mid-mission. Any long term settlement will require more resources than astronauts can initially bring with them. Synthetic Biology has the potential to revolutionize space exploration and settlement. Biological tools have a major advantage over classical tools: the ability to self-replicate and regenerate.<br />
<br />
The emerging field of Synthetic Biology will allow us to engineer microbial factories that will largely circumvent the limiting payload factors. These cellular factories will generate fuel, food, medicines and building materials for settlers, but will consist of engineered cells that could be stored in a single test tube, and regrown to production scale on-site, as needed.<br />
<br />
The Brown-Stanford iGEM team is excited to work on three different projects, under the common theme of developing Synthetic Biology applications for space. In addition, as a response to the ethical and philosophical considerations, we interviewed prominent researchers and leaders in the field. '''Click on the astronaut in the corner to view our series.''' <br />
<br />
<html><br />
</div><br />
<div class="miniContent"><br />
<br />
<div class="miniContentLeft"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/3/39/Brown-Stanford_REGObricks-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/REGObricks/Introduction">REGObricks</a></h3><br />
</html><br />
<br />
Any extra-terrestrial settlement will require a habitat to keep its occupants alive, but transport of such a habitat will require a huge amount of payload space. RegoBricks uses bacteria to cement Martian and Lunar regolith (soil) simulant into durable building blocks, similar to concrete bricks, allowing settlements to use in situ resources to build structures.<br />
<html><br />
</div><br />
<br />
<div class="miniContentRight"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/4/4c/Brown-Stanford_DNADamage-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/FRETSensor/Introduction">FRETcetera</a></h3><br />
</html><br />
<br />
Biological interfaces cannot pass information to their users as fast as electronic ones can. FRETcetera develops a novel method of fast-acting biological reporting with changes in cell fluorescence. Bacteria could be used to detect toxic chemicals in the environment, for example, or inform astronauts that their microbial tools are unhealthy.<br />
<html><br />
<br />
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<div class="miniContentCenter"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/9/9a/Brown-Stanford_PowerCell-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/PowerCell/Introduction">PowerCell</a></h3><br />
</html><br />
<br />
All biological tools need energy to run. PowerCell develops a universal energy source. Engineered photosynthetic bacteria generate carbon and nitrogenous nutrients from sunlight and air and secrete them to power biological tools. These tools will transform the raw materials into fuel, building materials, food, drugs, and other products useful to settlers.<br />
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<br />
</div><br />
<div class="pageContent"><br />
</html><br />
<br />
= '''Summary''' =<br />
<br />
=== '''News!''' ===<br />
<br />
===== '''iGEM Awards''' =====<br />
<br />
We did very well at iGEM this year! <br />
<br />
-At the Americas Regional Jamboree, we finished Top 4 and won Best Presentation.<br />
<br />
-At the World Jamboree, we finished in the Sweet 16, and won Best New Application. <br />
<br />
We are extremely grateful to all those who helped us along the way, and our mentors. Thank you so much!<br />
<br />
===== '''Project Summary''' =====<br />
<br />
Here's a minute long teaser of our project!<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/watch?v=b2AkJRTLOcs"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/b2AkJRTLOcs"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Latest Advancements on Projects''' ===<br />
<br />
===== '''Further evidence of pSac promoter specificity''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 100%;"><br />
<tr><br />
<br />
<td style="padding-left: 20px; padding-right: 0px; padding-top: 0px; padding-bottom: 0px; width: 90%;"><br />
<br />
<p> To further demonstrate that expression of our construct is properly co-localized to vegetative cells, we grew cultures of Anabaena in nitrate-free BG11 media to promote heterocyst formation. In the first picture, we observed areas of diminished GFP fluorescence at regular intervals (approximately 1 in 10 cells) along our transformed Anabaena filament. We designed our GFP reporter to express under the pSac promoter and only in non-heterocyst cells, so we would expect the spacing of our dark spots to match the spacing of heterocysts identified through alcian blue staining. Alcian blue is a dye that selectively binds to polysaccharide chains on the surface of heterocyst cells. As you can see by comparing the two images, our results appear to match up!<br />
<br />
<br><br />
<center><br />
<a href="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg"><img src="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%; font-weight:bold;"><br />
Transformed Anabaena after staining with alcian blue<br />
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</center><br />
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</table><br />
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<br />
===== '''Cyanobacteria Transformation''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 100%;"><br />
<tr><br />
<br />
<td style="padding-left: 20px; padding-right: 0px; padding-top: 0px; padding-bottom: 0px; width: 90%;"><br />
<br />
<p>We successfully transformed our cyanobacterial chassis, Anabaena PCC 7120, with our designed PowerCell genetic construct. Unlike more common cyanobacteria species such as Synechocystis PCC 6803 or Synechococcus elongatus PCC 7942, Anabaena is not naturally competent and does not easily uptake foreign DNA. </p><br />
<br />
<p>We performed a triparental conjugation in order to bypass the natural barriers to transformation. We demonstrated the successful control of our pSac promoter over our sugar secretion construct through GFP expression. The sugar secretion construct is only activated in actively photosynthesizing cells (not heterocysts). Work to characterize the performance of our cscB sucrose secretion construct is underway. We will assay sucrose levels produced by PowerCell. If they are high enough, we will attempt to grow E. coli W. containing arbitrary biobricks from this nutrient source, in an attempt to show PowerCell can power biological tools. </p><br />
<br />
<br />
<br><br />
<center><br />
<a href="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg"><img src="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
</div><br />
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<br><br />
</center><br />
</td><br />
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</table><br />
</html><br />
<br />
===== '''Biobricked the urease operon''' =====<br />
<br />
We successfully PCR cloned a 10.7 kb functional urease cassette from the plasmid pBU11, whose genetic sequence has hitherto never been known before. We ligated it onto a backbone, and successfully [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biobrick made a standard BioBrick], characterizing [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Characterization its function] and demonstrating active urease activity. Urease is the catalyst behind the phenomenon known as biocement, which is able to fuse sand into bricks, using nothing more than calcium ions and urea. [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biocementation More about urease.]<br />
<br />
Biobrick part [http://partsregistry.org/Part:BBa_K656013 BBa_K656013] has been submitted to the Registry of Standardized Parts. The sequence of this cassette is unavailable because it currently does not exist in any known registry. At the time of this writing, it is currently being sequenced in the lab of Dr. Chris Mason at Weill Cornell Medical College. As soon as the sequence is available, we will supplement the directory listing.<br />
<html><br />
<br />
<br><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG"><img src="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%; font-weight:bold;"><br />
Successful transformants on urease test plates.<br />
</div><br />
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</center><br />
</html><br />
<br />
===== '''Stratospheric Balloon Flight''' =====<br />
<br />
We launched samples of our bacteria to the far edge of space (80,000 ft-110,000 ft) to test for their ability to survive simulated Martian environmental conditions. The pressures, temperatures, and radiation conditions at these altitudes are similar to the conditions on the Martian surface.<br />
[[Team:Brown-Stanford/REGObricks/Balloon|Read about our escapades here]]<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/qCQXg8DmZZ0"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/qCQXg8DmZZ0"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Advancements in Human Practices''' ===<br />
===== '''Feature on Synthetic Biology and Space''' =====<br />
We talked with key figures in science and science policy about the implications of human expansion into space, extraterrestrial life and synthetic biology. You can look at our interviews and findings [https://2011.igem.org/Team:Brown-Stanford/SynEthics/Intro here] or by clicking on the astronaut on the upper left!<br />
<br />
===== '''Education and Publicity''' =====<br />
<br />
<html><br />
<table style="width: 100%;"><br />
<tr><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Editor's Choice! x2<br />
</div><br />
</center><br />
</td><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg" style="width:80%;"><br />
</a><br />
<br><br />
<div style="width: 70%; font-weight: bold;"><br />
Lunar Science Poster. Click to see full version.<br />
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</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
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<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/9YKsa7s8WM4?version=3"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/9YKsa7s8WM4?version=3"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
<br />
<br />
We educated and publicized the power of synthetic biology in a whole suite of venues and mediums, including <br />
<br />
* A crowd of 600+ over two days at [https://2011.igem.org/Team:Brown-Stanford/MakerFaire Maker Faire NYC], resulting in two Editor’s Choice blue ribbons! <br />
* A filming opportunity with the [https://2011.igem.org/Team:Brown-Stanford/BBC BBC] Horizon, to be featured in a documentary on NASA’s Initiative in Synthetic Biology<br />
*Verbal [https://2011.igem.org/Team:Brown-Stanford/NASA presentations] to NASA Administrator Charlie Bolden, NASA Ames Research Center Director Pete Worden, and writer and visionary Stewart Brand <br />
* A poster presentation at NASA’s Lunar Science Forum<br />
* A heart-pounding [https://2011.igem.org/Team:Brown-Stanford/SB5 poem] at the first-ever SB5.0 Synbio Slam<br />
<br />
===== '''Outreach and Collaboration''' =====<br />
<br />
<html><br />
<center><br />
<a href="http://openwetware.org/wiki/IGEM_Outreach"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6f/Brown-Stanford_Cbricks.jpg" style="width: 30%;"><br />
</a><br />
</center><br />
</html><br />
<br />
We spearheaded the '''largest collaborative effort''' in iGEM history to change the way we do outreach, creating [http://openwetware.org/wiki/IGEM_Outreach '''CommunityBricks'''], a database of valuable teaching plans, presentations, community resources and projects for young and old alike.<br />
<br />
===== '''Alumni and Community-Building''' =====<br />
<br />
<br />
[[File:Brown-Stanford alumnigem.png|300px|center|AlumniGEM|link=http://alumni.brownigem.com/]]<br />
<br />
We built the [http://alumni.brownigem.com '''first iGEM Alumni Network,'''] to provide iGEM'ers a growing collection of professional and social resources and keep the spirit of iGEM alive post-Jamborees.<br />
<br />
<html><br />
<div><a href="javascript:toggle();" id="toggleNews"><h3>Show Media and News</h3></a></div><br />
<br />
</div><br />
<div class="pageContent" id="news" style="display: none;"><br />
</html><br />
<br />
== '''Martian Colony''' ==<br />
[[File:Brown-Stanford_MartianColony.png|center|750px]]<br />
<br />
== '''News''' ==<br />
<br />
===In the Lab===<br />
<center><html><object width="600" height="450"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to="></param> <param name="movie" value="http://www.flickr.com/apps/slideshow/show.swf?v=107931"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://www.flickr.com/apps/slideshow/show.swf?v=107931" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2Fshow%2F&page_show_back_url=%2Fphotos%2F67993117%40N08%2Fsets%2F72157627630556315%2F&set_id=72157627630556315&jump_to=" width="600" height="450"></embed></object></html></center><br />
<br />
=== '''August 4<sup>th</sup>, 2011''' ===<br />
<center><html><object width="580" height="355"><param name="movie" value="http://www.youtube.com/v/x-MYE4QkjOU"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/x-MYE4QkjOU" type="application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''June 7<sup>th</sup>, 2011''' ===<br />
[[File:Brown-Stanford TeamPicture6-7-2011.jpg|center|500px]]<br />
<br />
{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclarkhttp://2011.igem.org/Team:Brown-StanfordTeam:Brown-Stanford2011-12-07T09:15:56Z<p>Evanclark: /* Cyanobacteria Transformation */</p>
<hr />
<div>{{:Team:Brown-Stanford/Templates/Main}}<br />
{{:Team:Brown-Stanford/Templates/Content}}<br />
{{:Team:Brown-Stanford/Templates/Twitter}}<br />
<br />
= '''APPLYING FOR BROWN iGEM 2012''' =<br />
(12/5/11)<br />
Brown iGEM is looking for undergrad team members for this upcoming year!<br />
<br />
Have you ever wanted to engineer living organisms to create novel functions and help transform the world? <br />
<br />
Spend a summer working at the Synthetic Biology Initiative at NASA Ames Research Center, and represent Brown at the premiere synthetic biology competition in the world! Opportunities exist for all kinds of microbiology and molecular biology work, computational biology, mathematical modeling, engineering, and much more. Develop incredible research and lab skills as you manage your own project!<br />
<br />
Students of all academic backgrounds and experience levels welcome (this includes Class of '12) <br />
<br />
We are hosting two '''INFO SESSIONS''' this week:<br />
<br />
'''TIME''': 6-7pm on Thursday 12/8, Friday 12/9<br />
<br />
'''LOCATION''': Barus and Holly 190<br />
<br />
-come hear more about iGEM, synthetic biology, and what the award-winning Brown iGEM team does; dinner will be provided!<br />
<br />
<br />
If you can't make it but are still interested, please let us know! Other questions or concerns are welcome as well. Contact us at igem@brown.edu<br />
<br />
<html><br />
<span style="font-size: 20px;"></html>Our electronic application is [http://www.brownigem.com/2012_iGEM_Application.doc available now]. Keep up with the 2012 application process on our [https://www.facebook.com/events/146221565480758/ facebook page]!<br />
<html></span></html><br />
<br />
= '''Mars BioTools: Synthetic Biology for Space Exploration''' =<br />
One of the major challenges of space exploration is the limited payload mass that can be launched on a rocket and the difficulty of resupply mid-mission. Any long term settlement will require more resources than astronauts can initially bring with them. Synthetic Biology has the potential to revolutionize space exploration and settlement. Biological tools have a major advantage over classical tools: the ability to self-replicate and regenerate.<br />
<br />
The emerging field of Synthetic Biology will allow us to engineer microbial factories that will largely circumvent the limiting payload factors. These cellular factories will generate fuel, food, medicines and building materials for settlers, but will consist of engineered cells that could be stored in a single test tube, and regrown to production scale on-site, as needed.<br />
<br />
The Brown-Stanford iGEM team is excited to work on three different projects, under the common theme of developing Synthetic Biology applications for space. In addition, as a response to the ethical and philosophical considerations, we interviewed prominent researchers and leaders in the field. '''Click on the astronaut in the corner to view our series.''' <br />
<br />
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</div><br />
<div class="miniContent"><br />
<br />
<div class="miniContentLeft"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/3/39/Brown-Stanford_REGObricks-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/REGObricks/Introduction">REGObricks</a></h3><br />
</html><br />
<br />
Any extra-terrestrial settlement will require a habitat to keep its occupants alive, but transport of such a habitat will require a huge amount of payload space. RegoBricks uses bacteria to cement Martian and Lunar regolith (soil) simulant into durable building blocks, similar to concrete bricks, allowing settlements to use in situ resources to build structures.<br />
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<br />
<div class="miniContentRight"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/4/4c/Brown-Stanford_DNADamage-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/FRETSensor/Introduction">FRETcetera</a></h3><br />
</html><br />
<br />
Biological interfaces cannot pass information to their users as fast as electronic ones can. FRETcetera develops a novel method of fast-acting biological reporting with changes in cell fluorescence. Bacteria could be used to detect toxic chemicals in the environment, for example, or inform astronauts that their microbial tools are unhealthy.<br />
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<br />
</div><br />
<div class="miniContentCenter"><br />
<h3><img src="https://static.igem.org/mediawiki/2011/9/9a/Brown-Stanford_PowerCell-Thumb.png" width="30px"> <a href="/Team:Brown-Stanford/PowerCell/Introduction">PowerCell</a></h3><br />
</html><br />
<br />
All biological tools need energy to run. PowerCell develops a universal energy source. Engineered photosynthetic bacteria generate carbon and nitrogenous nutrients from sunlight and air and secrete them to power biological tools. These tools will transform the raw materials into fuel, building materials, food, drugs, and other products useful to settlers.<br />
<html><br />
</div><br />
<br />
</div><br />
<div class="pageContent"><br />
</html><br />
<br />
= '''Summary''' =<br />
<br />
=== '''News!''' ===<br />
<br />
===== '''iGEM Awards''' =====<br />
<br />
We did very well at iGEM this year! <br />
<br />
-At the Americas Regional Jamboree, we finished Top 4 and won Best Presentation.<br />
<br />
-At the World Jamboree, we finished in the Sweet 16, and won Best New Application. <br />
<br />
We are extremely grateful to all those who helped us along the way, and our mentors. Thank you so much!<br />
<br />
===== '''Project Summary''' =====<br />
<br />
Here's a minute long teaser of our project!<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/watch?v=b2AkJRTLOcs"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/b2AkJRTLOcs"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Latest Advancements on Projects''' ===<br />
<br />
===== '''Further evidence of pSac promoter specificity''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 100%;"><br />
<tr><br />
<br />
<td style="padding-left: 20px; padding-right: 0px; padding-top: 0px; padding-bottom: 0px; width: 90%;"><br />
<br />
<p> To further demonstrate that expression of our construct is properly co-localized to vegetative cells, we grew cultures of Anabaena in nitrate-free BG11 media to promote heterocyst formation. In the first picture, we observed areas of diminished GFP fluorescence at regular intervals (approximately 1 in 10 cells) along our transformed Anabaena filament. We designed our GFP reporter to express under the pSac promoter and only in non-heterocyst cells, so we would expect the spacing of our dark spots to match the spacing of heterocysts identified through alcian blue staining. Alcian blue is a dye that selectively binds to polysaccharide chains on the surface of heterocyst cells. As you can see by comparing the two images, our results appear to match up!<br />
<br />
<br><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/ae/Brown-Stanford_Transformed_Anabaena_alcian.JPG"><img src="https://static.igem.org/mediawiki/2011/a/ae/Brown-Stanford_Transformed_Anabaena_alcian.JPG" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%; font-weight:bold;"><br />
Transformed Anabaena after staining with alcian blue<br />
</div><br />
<br><br />
<br><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<br />
<br />
===== '''Cyanobacteria Transformation''' =====<br />
<br />
<html><br />
<table style="padding: 0px; background: #DDD; width: 100%;"><br />
<tr><br />
<br />
<td style="padding-left: 20px; padding-right: 0px; padding-top: 0px; padding-bottom: 0px; width: 90%;"><br />
<br />
<p>We successfully transformed our cyanobacterial chassis, Anabaena PCC 7120, with our designed PowerCell genetic construct. Unlike more common cyanobacteria species such as Synechocystis PCC 6803 or Synechococcus elongatus PCC 7942, Anabaena is not naturally competent and does not easily uptake foreign DNA. </p><br />
<br />
<p>We performed a triparental conjugation in order to bypass the natural barriers to transformation. We demonstrated the successful control of our pSac promoter over our sugar secretion construct through GFP expression. The sugar secretion construct is only activated in actively photosynthesizing cells (not heterocysts). Work to characterize the performance of our cscB sucrose secretion construct is underway. We will assay sucrose levels produced by PowerCell. If they are high enough, we will attempt to grow E. coli W. containing arbitrary biobricks from this nutrient source, in an attempt to show PowerCell can power biological tools. </p><br />
<br />
<br />
<br><br />
<center><br />
<a href="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg"><img src="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%;"><br />
Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
===== '''Biobricked the urease operon''' =====<br />
<br />
We successfully PCR cloned a 10.7 kb functional urease cassette from the plasmid pBU11, whose genetic sequence has hitherto never been known before. We ligated it onto a backbone, and successfully [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biobrick made a standard BioBrick], characterizing [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Characterization its function] and demonstrating active urease activity. Urease is the catalyst behind the phenomenon known as biocement, which is able to fuse sand into bricks, using nothing more than calcium ions and urea. [https://2011.igem.org/Team:Brown-Stanford/REGObricks/Biocementation More about urease.]<br />
<br />
Biobrick part [http://partsregistry.org/Part:BBa_K656013 BBa_K656013] has been submitted to the Registry of Standardized Parts. The sequence of this cassette is unavailable because it currently does not exist in any known registry. At the time of this writing, it is currently being sequenced in the lab of Dr. Chris Mason at Weill Cornell Medical College. As soon as the sequence is available, we will supplement the directory listing.<br />
<html><br />
<br />
<br><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG"><img src="https://static.igem.org/mediawiki/2011/8/81/Transformant_urease_plates.JPG" style="width:40%"><br />
</a><br />
<br><br />
<div style="width: 35%; font-weight:bold;"><br />
Successful transformants on urease test plates.<br />
</div><br />
<br><br />
<br><br />
</center><br />
</html><br />
<br />
===== '''Stratospheric Balloon Flight''' =====<br />
<br />
We launched samples of our bacteria to the far edge of space (80,000 ft-110,000 ft) to test for their ability to survive simulated Martian environmental conditions. The pressures, temperatures, and radiation conditions at these altitudes are similar to the conditions on the Martian surface.<br />
[[Team:Brown-Stanford/REGObricks/Balloon|Read about our escapades here]]<br />
<br />
<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/qCQXg8DmZZ0"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/qCQXg8DmZZ0"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
<br />
=== '''Advancements in Human Practices''' ===<br />
===== '''Feature on Synthetic Biology and Space''' =====<br />
We talked with key figures in science and science policy about the implications of human expansion into space, extraterrestrial life and synthetic biology. You can look at our interviews and findings [https://2011.igem.org/Team:Brown-Stanford/SynEthics/Intro here] or by clicking on the astronaut on the upper left!<br />
<br />
===== '''Education and Publicity''' =====<br />
<br />
<html><br />
<table style="width: 100%;"><br />
<tr><br />
<td style="width: 50%;"><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/c/cd/Brown-Stanford_Maker.jpg"><br />
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Editor's Choice! x2<br />
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<a href="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a5/Brown-Stanford_Outreach_Poster_for_Lunar_2.jpg" style="width:80%;"><br />
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<div style="width: 70%; font-weight: bold;"><br />
Lunar Science Poster. Click to see full version.<br />
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<center><html><object width="480" height="280"><param name="movie" value="http://www.youtube.com/v/9YKsa7s8WM4?version=3"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/9YKsa7s8WM4?version=3"application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
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We educated and publicized the power of synthetic biology in a whole suite of venues and mediums, including <br />
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* A crowd of 600+ over two days at [https://2011.igem.org/Team:Brown-Stanford/MakerFaire Maker Faire NYC], resulting in two Editor’s Choice blue ribbons! <br />
* A filming opportunity with the [https://2011.igem.org/Team:Brown-Stanford/BBC BBC] Horizon, to be featured in a documentary on NASA’s Initiative in Synthetic Biology<br />
*Verbal [https://2011.igem.org/Team:Brown-Stanford/NASA presentations] to NASA Administrator Charlie Bolden, NASA Ames Research Center Director Pete Worden, and writer and visionary Stewart Brand <br />
* A poster presentation at NASA’s Lunar Science Forum<br />
* A heart-pounding [https://2011.igem.org/Team:Brown-Stanford/SB5 poem] at the first-ever SB5.0 Synbio Slam<br />
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===== '''Outreach and Collaboration''' =====<br />
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<a href="http://openwetware.org/wiki/IGEM_Outreach"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6f/Brown-Stanford_Cbricks.jpg" style="width: 30%;"><br />
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We spearheaded the '''largest collaborative effort''' in iGEM history to change the way we do outreach, creating [http://openwetware.org/wiki/IGEM_Outreach '''CommunityBricks'''], a database of valuable teaching plans, presentations, community resources and projects for young and old alike.<br />
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===== '''Alumni and Community-Building''' =====<br />
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[[File:Brown-Stanford alumnigem.png|300px|center|AlumniGEM|link=http://alumni.brownigem.com/]]<br />
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We built the [http://alumni.brownigem.com '''first iGEM Alumni Network,'''] to provide iGEM'ers a growing collection of professional and social resources and keep the spirit of iGEM alive post-Jamborees.<br />
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<div><a href="javascript:toggle();" id="toggleNews"><h3>Show Media and News</h3></a></div><br />
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== '''Martian Colony''' ==<br />
[[File:Brown-Stanford_MartianColony.png|center|750px]]<br />
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== '''News''' ==<br />
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===In the Lab===<br />
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=== '''August 4<sup>th</sup>, 2011''' ===<br />
<center><html><object width="580" height="355"><param name="movie" value="http://www.youtube.com/v/x-MYE4QkjOU"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/x-MYE4QkjOU" type="application/x-shockwave-flash" wmode="transparent" width="580" height="355"></embed></object></html></center><br />
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=== '''June 7<sup>th</sup>, 2011''' ===<br />
[[File:Brown-Stanford TeamPicture6-7-2011.jpg|center|500px]]<br />
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{{:Team:Brown-Stanford/Templates/Foot}}</div>Evanclark