http://2010.igem.org/wiki/index.php?title=Special:Contributions/Hugo_87&feed=atom&limit=50&target=Hugo_87&year=&month=2010.igem.org - User contributions [en]2024-03-29T13:26:07ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:TU_Delft/Team/membersTeam:TU Delft/Team/members2010-11-23T04:02:21Z<p>Hugo 87: </p>
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<h2>Team Members</h2><br />
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<p>Why so serious? When it comes to representing Synthetic Biology and iGEM we take things serious. Ofcourse we also had a lot of <a href="https://2010.igem.org/Team:TU_Delft#page=Team/gallery">fun</a>. If you would like to get to know us a bit better, come talk to us at the Jamboree or <a href="https://2010.igem.org/Team:TU_Delft#page=Contact">send us an email</a>!</p><br />
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<h2>Students</h2><br />
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:lbergwerff"><img src="https://static.igem.org/mediawiki/2010/6/69/TU_Delft_teamphoto-luke.jpg" width="120" height="159" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:lbergwerff">View Profile</a></p><br />
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:lbergwerff"><h3>Luke Bergwerff (23)</h3></a><br />
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<td class="name">Education:</td><td class="value">Biochemical Engineering</td><br />
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<td class="name">Main Task:</td><td class="value">Modelling</td><br />
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<td class="name">Sub Project:</td><td class="value">Hydrocarbon Sensing</td><br />
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<td class="name">Half Time:</td><td class="value">66 days</td><br />
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<td class="name">iJAM:</td><td class="value">Keys</td><br />
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<td class="story" colspan=2>"Sometimes turtles turn into ninjas."</td><br />
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:Ptmvanboheemen"><img src="https://static.igem.org/mediawiki/2010/d/d9/TU_Delft_teamphoto-pieter.jpg" width="120" height="159" /></a><br />
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<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:Ptmvanboheemen">View Profile</a></p><br />
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:Ptmvanboheemen"><h3>Pieter van Boheemen (24)</h3></a><br />
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<td class="name">Education:</td><td class="value">Functional Genomics</td><br />
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<td class="name">Main Task:</td><td class="value">Money Manager & Wiki</td><br />
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<td class="name">Sub Project:</td><td class="value">Emulsifier</td><br />
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<td class="name">GC-content:</td><td class="value">156%</td><br />
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<td class="name">iJAM:</td><td class="value">Drums</td><br />
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<td class="story" colspan=2>"I'm blue abedee abedi"</td><br />
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:nadinebongaerts"><img src="https://static.igem.org/mediawiki/2010/2/26/TU_Delft_teamphoto-nadine.jpg" width="120" height="159" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:nadinebongaerts">View Profile</a></p><br />
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:nadinebongaerts"><h3>Nadine Bongaerts (21)</h3></a><br />
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<td class="name">Education:</td><td class="value">Life Science & Technology</td><br />
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<td class="name">Main Task:</td><td class="value">Human Practices</td><br />
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<td class="name">Sub Project:</td><td class="value">Hydrocarbon Sensing</td><br />
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<td class="name">Resistance:</td><td class="value">KAN & AMP</td><br />
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<td class="name">iJAM:</td><td class="value">Singer</td><br />
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<td class="story" colspan=2>"Minimization and simplification is the key to succes"</td><br />
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:Ebrinkman"><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_teamphoto-eva.jpg" width="120" height="159" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:Ebrinkman">View Profile</a></p><br />
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:Ebrinkman"><h3>Eva Brinkman (23)</h3></a><br />
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<td class="name">Education:</td><td class="value">Functional Genomics</td><br />
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<td class="name">Main Task:</td><td class="value">Lab Manager</td><br />
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<td class="name">Sub Project:</td><td class="value">Emulsifier</td><br />
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<td class="name">ORFs:</td><td class="value">3062</td><br />
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<td class="name">iJAM:</td><td class="value">Keys</td><br />
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<td class="story" colspan=2>"Call me Miss Sequencing"</td><br />
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:jcnossen"><img src="https://static.igem.org/mediawiki/2010/4/48/TU_Delft_teamphoto-jelmer.jpg" width="120" height="159" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:jcnossen">View Profile</a></p><br />
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:jcnossen"><h3>Jelmer Cnossen (25)</h3></a><br />
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<td class="name">Education:</td><td class="value">Bioinformatics</td><br />
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<td class="name">Main Task:</td><td class="value">Wiki & Modeling</td><br />
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<td class="name">Sub Project:</td><td class="value">Hydrocarbon Sensing</td><br />
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<td class="name">LD50:</td><td class="value">2 minutes</td><br />
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<td class="name">iJAM:</td><td class="value">Jazz Saxophone</td><br />
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<td class="story" colspan=2>"AlkB back!"</td><br />
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:kschipper"><img src="https://static.igem.org/mediawiki/2010/5/57/TU_Delft_teamphoto-kira.jpg" width="120" height="159" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:kschipper">View Profile</a></p><br />
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<div class="member-description"><br />
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:kschipper"><h3>Kira Schipper (23)</h3></a><br />
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<td class="name">Education:</td><td class="value">Biochemical Engineering</td><br />
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<td class="name">Main Task:</td><td class="value">Organizational Manager</td><br />
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<td class="name">Sub Project:</td><td class="value">Alkane Degradation</td><br />
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<td class="name">Melting Temp:</td><td class="value">87 C</td><br />
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<td class="name">iJAM:</td><td class="value">Producer</td><br />
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<td class="story" colspan=2>"You should buy a Mac"</td><br />
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<div class="member-picture"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:Hugo_87"><img src="https://static.igem.org/mediawiki/2010/8/80/TU_Delft_teamphoto-hugo.jpg" width="120" height="159" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:Hugo_87">View Profile</a></p><br />
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<div class="member-description"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:Hugo_87"><h3>Hugo Cueto Rojas (23)</h3></a><br />
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<td class="name">Education:</td><td class="value">Biochemical Engineering</td><br />
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<td class="name">Main Task:</td><td class="value">The Characterizor</td><br />
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<td class="name">Sub Project:</td><td class="value">Salt & Solvent Tolerance</td><br />
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<td class="name">Glucose:</td><td class="value">200 g/L</td><br />
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<td class="name">iJAM:</td><td class="value">Bass Guitar</td><br />
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<td class="story" colspan=2>"Fiat voluntas Dei", also my well known quote: "IT COULD BE WORSE" </td><br />
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<td class="name">contact:</td><td class="value">h.f.cuetorojas@gmail.com</td><br />
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:ravandervalk"><img src="https://static.igem.org/mediawiki/2010/9/99/TU_Delft_teamphoto-ramon.jpg" width="120" height="159" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:ravandervalk">View Profile</a></p><br />
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<div class="member-description"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:ravandervalk"><h3>Ramon van der Valk (23)</h3></a><br />
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<td class="name">Education:</td><td class="value">Molecular Genetics</td><br />
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<td class="name">Main Task:</td><td class="value">Not-so-safety Manager</td><br />
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<td class="name">Sub Project:</td><td class="value">Salt & Solvent Tolerance</td><br />
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<td class="name">Accession #:</td><td class="value">PWN1337</td><br />
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<td class="name">iJAM:</td><td class="value">Cheer leader</td><br />
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<td class="story" colspan=2>"They made me do it! :'("</td><br />
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:mvoges"><img src="https://static.igem.org/mediawiki/2010/8/82/TU_Delft_teamphoto-thias.jpg" width="120" height="159" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:mvoges">View Profile</a></p><br />
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:mvoges"><h3>Mathias Voges (22)</h3></a><br />
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<td class="name">Education:</td><td class="value">Cell Factory</td><br />
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<td class="name">Main Task:</td><td class="value">Science Manager</td><br />
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<td class="name">Sub Project:</td><td class="value">Alkane Degradation & RBSs</td><br />
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<td class="name">Excitation:</td><td class="value">200 nm</td><br />
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<td class="name">iJAM:</td><td class="value">Lead Guitar</td><br />
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<td class="story" colspan=2>"Do not disturb my circles!"</td><br />
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<h2>Instructors</h2><br />
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:Aljoscha"><img src="https://static.igem.org/mediawiki/2010/4/4a/TU_Delft_Wahl.jpg" width="120" height="120" /></a><br />
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<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:Aljoscha">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:Aljoscha"><h3>Aljoscha Wahl</h3></a><br />
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<td class="name">Department:</td><td class="value">Bioprocessing Technology</td><br />
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<td class="name">Gibbs Free E:</td><td class="value">-300 kJ</td><br />
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<td class="story" colspan=2>"Nein man! ich will noch nicht gehn ich will noch ein bisschen Tanzen"</td><br />
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:Aleabate"><img src="https://static.igem.org/mediawiki/2010/4/41/TU_Delft_Abate.jpg" width="120" height="120" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:Aleabate">View Profile</a></p><br />
</div><br />
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<div class="member-description"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:Aleabate"><h3>Alessandro Abate</h3></a><br />
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<td class="name">Department:</td><td class="value">Delft Center for Systems and Control</td><br />
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<td class="name">10010011:</td><td class="value">FALSE</td><br />
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<td class="story" colspan=2>" there is grandeur in this view of life (c. darwin) "</td><br />
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:Esengul"><img src="https://static.igem.org/mediawiki/2010/7/78/TU_Delft_Yildirim.jpg" width="120" height="120" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:Esengul">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:Esengul"><h3>Esengul Yildirim</h3></a><br />
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<td class="name">Department:</td><td class="value">Enzymology</td><br />
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<td class="name">Conductivity:</td><td class="value">45.2 million S/m</td><br />
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<td class="story" colspan=2>"DNA is life, <br />the rest is just translation..."</td><br />
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<h2>Advisors</h2><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:stefandekok"><img src="https://static.igem.org/mediawiki/2010/b/b2/TU_Delft_Kok.jpg" width="120" height="120" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:stefandekok">View Profile</a></p><br />
</div><br />
<br />
<div class="member-description"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:stefandekok"><h3>Stefan de Kok</h3></a><br />
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<td class="name">Department:</td><td class="value">Fermentation Technology</td><br />
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<td class="name">Yield:</td><td class="value">15 cmol/cmol</td><br />
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<td class="story" colspan=2>"I'm the yeasty boy, yeah!"</td><br />
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</div><br />
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<h3>Domenico Bellomo</h3><br />
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<td class="name">iGEM Medals:</td><td class="value">2 x gold</td><br />
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</html></div>Hugo 87http://2010.igem.org/User:Hugo_87User:Hugo 872010-11-23T04:00:55Z<p>Hugo 87: /* Hugo Cueto Rojas */</p>
<hr />
<div>==Hugo Cueto Rojas==<br />
Hi to all of you!!!<br />
<br />
My name is Hugo, I'll keep it short 'cause my complete name gives a lot of problems. I come from Mexico, currently I'm studying the MSc. programme of LST at TU Delft. This is my second year and probably the last one in the Netherlands, but I will know for sure in another moment.<br />
<br />
I'm a Biotechnology engineer from the National Polytechnique Institute (Mexico city), personally I hate molecular stuff... I'm an engineer more than a Biologist n_n , but I joined to the iGEM team because Synthetic Biology is a great approach for engineering life, which it had been always my ambition (muahahaha). Synthetic Biology promises a lot for us as engineers and iGEM it's been a great experience for me, so far.<br />
<br />
As the only international student in the team, my work is to try to understand some Dutch... =s (it's really hard trust me). Anyway, I'll be working on strain characterizations, conceptual design of parts and perversion of protocols. I hope to see you all on Boston (yay!).<br />
<br />
PLEASE CONTACT ME AT THE FOLLOWING ADDRESS:<br />
<br />
h.f.cuetorojas@gmail.com<br />
<br />
Greetings<br />
<br />
[[Team:TU_Delft/Team/members|Return to the Team Members Page]]</div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/partsTeam:TU Delft/Project/sensing/parts2010-10-27T22:46:07Z<p>Hugo 87: /* BBa_K398331: P(Caif)-RBS-GFP-TT */</p>
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<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
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==Sensing Parts==<br />
<br />
===Introduction===<br />
<br />
'''Catabolite repression control'''<br />
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[[Image:TUDelft_alkS.png|thumb|800px|right|'''Figure 1''' - AlkS Hydrocarbon Regulatory systems from ''Pseudomonas putida'' (F. Rojo, et al. 2009)]]<br />
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In ''Pseudomonas putida'', the AlkS transcriptional regulator activates the expression of its own gene, and that of alkT, from a promoter named PalkS2 in the presence of alkanes. This allows achieving AlkS levels that are high enough to activate the expression of the alkBFGHJKL operon from the PalkB promoter. AlkS recognizes C5–C10 n-alkanes as effectors, but does not respond to shorter or larger alkanes (Rojo et al. 2009) (figure 1)<br />
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In stead of using all the alkBFGHJKL genes, a GFP will be introduced into our system to check the functionality of the PalkB promoter in ''E.coli''. (figure 2)<br />
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Therefore, the first construct is suitable for the analysis of transcriptional activity over the PalkS promoter. A plasmid containing the AlkS-PalkS-PalkB regulatory mechanism will be coupled to GFP and RFP generators in order to determine transcriptional activities of PalkS and PalkB at varying hydrocarbon concentrations by measuring fluorescence.<br />
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[[Image:TU Delft PalkS.PNG|410px|thumb|left|'''Figure 2''' – Schematic description of the alkane sensing pathway with the corresponding genes.]]<br />
[[Image:TU_Delft_PCaif.PNG|410px|thumb|right|'''Figure 3''' – Schematic description of the alkane sensing pathway with the corresponding genes.]]<br />
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'''Control by Crp'''<br />
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The expression of alkane degrading genes in our second ''E.coli'' regulatory system will be regulated by making the alkane-degrading genes sensitive to Crp. The global regulator protein Crp binds to regions of promoters known to activate genes involved in the degradation of non-glucose carbon sources, in this way activating the genes downstream of it. <br />
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For the induction of alkane-degradation genes in this construct the BioBrick consisted out of the P(CaiF) promoter, AlkS gene and PalkB gene. (figure 3)<br />
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<br />
===BBa_K398331: P(Caif)-RBS-GFP-TT===<br />
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pCaiF is a natural promoter found in E. coli K12, pCaiF is part of the translational unit of the protein CaiF which is a transcriptional regulator of carnitine metabolism under anaerobiosis and glucose limitation. From the original sequence, we took the cAMP-Crp and RNApol sigma 70 binding sites. Under low glucose levels, the cellular cAMP (cyclic Adenosine Mononucleotide Phosphate) levels are high and thus Crp (Catabolite gene activator protein) will bind easier to this molecule forming the complex cAMP-Crp. This complex acts as a transcriptional regulator of over 180 proteins involved in metabolism of secondary carbon sources.<br />
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[[Image:TU_Delft_PCaif_ecocyc.jpg|410px|thumb|right|'''Figure 4''' – Schematic drawing of the CaiF regulon in ''E. coli'' K12, the genomic context of our new promoter (taken from [http://BioCyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU141 EcoCyc]).]]<br />
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The original purpose of pCaiF for our project was to serve as a promotor for our proteins when glucose or other carbon sources easier to degrade are not in the medium, in that way cells will grow faster and once a certain cell density is reached they can start to degrade other carbon sources like alkanes.<br />
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Because of the limited amount of time, we just succeeded on the construction of a measurement device in order to characterize pCaiF. An natural occurring E. coli promotor. We hope that other teams will use these sequence in the future for several reasons, among them the necessity of well characterized promoters in the catalog. <br />
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View this part in the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398331 '''part registry''']<br />
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[[Image:TUDelft_pCaif.png|150px]]<br />
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===References===<br />
#'''Canosa, I., J. M. Sanchez-Romero, et al.''' A positive feedback mechanism controls expression of AlkS, the transcriptional regulator of the Pseudomonas oleovorans alkane degradation pathway. ''Molecular Microbiology'' 35(4): 791-799 ('''2000''')<br />
#'''Moreno, R., A. Ruiz-Manzano, et al.''' The Pseudomonas putida Crc global regulator is an RNA binding protein that inhibits translation of the AlkS transcriptional regulator. ''Molecular Microbiology'' 64(3): 665-675 ('''2007''')<br />
#'''van Beilen, J. B., S. Panke, et al.''' Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes. ''Microbiology-Sgm'' 147: 1621-1630 ('''2001''')<br />
#'''Rojo, F.''' , Degradation of alkanes by bacteria. ''Environmental Microbiology'' 11: 2477-2490 ('''2009''')<br />
#'''Kotte, O, Zaugg, J., Heinemann, M. ''', ‘Bacterial adaptation through distributed sensing of metabolic fluxes’, ''Molecular Systems Biology'', 6:355, doi:10.1038/msb.2010.10 ('''2010''')<br />
#'''Kremling, A., Bettenbrock, K., Gilles, E.D.''', ‘Analysis of global control of Escherichia coli carbohydrate uptake’, ''BMC Systems Biology'', 1:42, doi:10.1186/1752-0509-1-42 ('''2007''')<br />
#'''Lin, H. Y., Mathiszik, B., Xu, B., Enfors, S.-O., Neubauer, P.''', ‘Determination of the Maximum Specific Uptake Capacities for Glucose and Oxygen in Glucose-Limited Fed-Batch Cultivations of ''Escherichia coli''’, ''Biotechnology and Bioengineering'', 73, 347-357 ('''2001''')<br />
#'''Alon, U. (ed.)''', An Introduction to Systems Biology: Design Principles of Biological Circuits, ''CRC Press'' ('''2007''')<br />
#EcoCyc: [http://BioCyc.org/ECOLI/NEW-IMAGE?type=ENZYME&object=CPLX0-226 click here]<br />
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<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/tolerance/resultsTeam:TU Delft/Project/tolerance/results2010-10-27T21:58:48Z<p>Hugo 87: /* Survival Results & Conclusions */</p>
<hr />
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<br />
__NOTOC__<br />
==Survival Results & Conclusions==<br />
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===Solvent tolerance===<br />
The solvent tolerance cluster ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K398406 BBa_K398406]) was expressed in ''E. coli'' K12. The growth rate of cells was challenged by different amounts of n-hexane. The results (Fig. 1) suggest that this part indeed improves growth under high n-hexane conditions. The parental strain ''E. coli'' K12 was growing very slowly at 10% (v/v) of n-hexane/M9 mixture.<br />
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[[Image:TU_Delft_406_K12.jpg|500px|thumb|center|'''Fig. 1:''' Growth of ''E. coli'' K12 in M9 medium at different n-hexane concentrations. ]]<br />
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Please note the growth of ''E. coli'' 406 - shows no difficulties under these harsh conditions.<br />
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[[Image:TU_Delft_406_406.jpg|500px|thumb|center|'''Fig. 2:''' Growth of ''E. coli'' 406A (expressing [http://partsregistry.org/Part:BBa_K398406 BBa_K398406] in pSB1A2) in M9 medium at different n-hexane concentrations. ]]<br />
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The growth rates were determined from the exponential phase (using a trendline, Fig. 3).<br />
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[[Image:TU_Delft_Solvent_tolerance.jpg|500px|thumb|center|'''Fig. 3:''' Comparison of the growth rates between ''E. coli'' K12 and ''E. coli'' 406A at different n-hexane concentrations in M9 medium. ]]<br />
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===Salt tolerance===<br />
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We tested the growth of our bbc1 construct ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K398108 BBa_K398108]) under different [https://2010.igem.org/Team:TU_Delft#page=Project/tolerance/characterization salt concentrations].<br />
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The growth rates were determined for the exponential phase (using a trendline, Fig. 4).<br />
<br />
[[Image:TU_Delft_Salt_tolerance.jpg|500px|thumb|center|'''Fig. 4:''' Growth rate in dependence of salt (NaCl).]]<br />
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Up to 0.2 M NaCl effects of salt stress are equally observed for both the negative control and for cells with our biobrick ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K398108 BBa_K398108]). At higher concentrations a significant improvement of growth rate in comparison to the control background ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K398027 BBa_K398027]) is seen. <br />
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===Conclusions===<br />
While at low salt concentrations no phenotype is observed, the resistance to high salt concentrations is significantly improved (up to 35%). The observed behavior can be explained by the vast amount of effects resulting from salt stress. It is possible that our BioBrick assists to reduce one of the inhibiting effects ,and thus leading to a benefit at higher salt stress. <br />
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It was shown, that our BioBrick indeed increases the salt tolerance for concentrations higher than 0.3 M sodium chloride. The general increase in tolerance varies between 10 and 35% depending on the sodium chloride concentration (showing a peak at 0.5 M NaCl which coincides with the concentration of NaCl in sea water).<br />
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===Future prospects===<br />
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We hope that the salt tolerance can be increased further by adding systems (such as ion pumps) to ensure the intracellular threshold and minimize the effects of salt stress even further. As the host organism would then be able to maintain growth at lethal concentrations of both salt and alkanes. This would allow future generations of igemmers to create cultures in a variety of different media, opening a new field of possibilities.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/tolerance/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/tolerance/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/tolerance/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T21:55:03Z<p>Hugo 87: /* Alkane Degradation Results & Conclusions */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1.''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system (AlkB2-system)]===<br />
<br />
<br />
<br />
From the ratios hexadecane/undecane obtained from GC chromatograms, we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. <br />
<br />
For more information about our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase|detailed alkane hydroxylase results]] page.<br />
<br />
[[Image:TUDelft_AlkB2_Total.png|600px|thumb|center|'''Figure 2.''' Enzyme activity [U/mg] of alkane hydroxylase system as compared to the negative control ''E.coli'' K12 strain]]<br />
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===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase (LadA)]===<br />
<br />
The analysis of the obtained GC graphs allowed us to estimate the enzymatic activity. We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, read the [[Team:TU_Delft/Project/alkane-degradation/results/LadA|detailed LadA results]] page.<br />
<br />
[[Image:TUDelft_LadA Total.png|600px|thumb|center|'''Figure 3.''' Enzyme activity [U/mg] of the alkane monooxygenase LadA as compared to the negative control ''E.coli'' K12 strain]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of Alcohol Dehydrogenase (ADH)]===<br />
According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain expressing the Biobrick [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control ''Pseudomonas putida''.<br />
<br />
If you are interested in knowing more about our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/ADH|detailed ADH results]] page.<br />
<br />
[[Image:TUDelftADH_final.jpg|600px|thumb|center|'''Figure 4.''' Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of Aldehyde Dehydrogenase (ALDH)]===<br />
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Our results suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks. The expression of ALDH under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold; whereas the expression of the same protein using the part [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] as promoter-rbs combo increases the same activity 3-fold. <br />
<br />
Moreover, the enzymatic activities measured for the constructs [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] and [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] were equivalent to 33.98% and 42.01% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity, respectively. <br />
<br />
If you want to know more about of our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/ALDH|detailed ALDH results]] page. <br />
<br />
It is worthy to mention that our part expresses the protein in a lower amount, meaning that cells express a highly active protein. Thus the cellular resources are spent in a more efficient way than in the strain that overproduces ALDH.<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px|thumb|center|'''Figure 5.''' Comparison of ALDH activities in the different strains tested in this study]]<br />
<br />
<br />
====Useful Literature and References====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T21:54:29Z<p>Hugo 87: /* Characterization of the alkane hydroxylase system (AlkB2-system) */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1.''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system (AlkB2-system)]===<br />
<br />
<br />
<br />
From the ratios hexadecane/undecane obtained from GC chromatograms, we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. <br />
<br />
For more information about our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase|detailed alkane hydroxylase results]] page.<br />
<br />
[[Image:TUDelft_AlkB2_Total.png|600px|thumb|center|'''Figure 2.''' Enzyme activity [U/mg] of alkane hydroxylase system as compared to the negative control ''E.coli'' K12 strain]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase (LadA)]===<br />
<br />
The analysis of the obtained GC graphs allowed us to estimate the enzymatic activity. We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, read the [[Team:TU_Delft/Project/alkane-degradation/results/LadA|detailed LadA results]] page.<br />
<br />
[[Image:TUDelft_LadA Total.png|600px|thumb|center|''Figure 3.''' Enzyme activity [U/mg] of the alkane monooxygenase LadA as compared to the negative control ''E.coli'' K12 strain]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of Alcohol Dehydrogenase (ADH)]===<br />
According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain expressing the Biobrick [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control ''Pseudomonas putida''.<br />
<br />
If you are interested in knowing more about our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/ADH|detailed ADH results]] page.<br />
<br />
[[Image:TUDelftADH_final.jpg|600px|thumb|center|''Figure 4.''' Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of Aldehyde Dehydrogenase (ALDH)]===<br />
<br />
Our results suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks. The expression of ALDH under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold; whereas the expression of the same protein using the part [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] as promoter-rbs combo increases the same activity 3-fold. <br />
<br />
Moreover, the enzymatic activities measured for the constructs [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] and [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] were equivalent to 33.98% and 42.01% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity, respectively. <br />
<br />
If you want to know more about of our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/ALDH|detailed ALDH results]] page. <br />
<br />
It is worthy to mention that our part expresses the protein in a lower amount, meaning that cells express a highly active protein. Thus the cellular resources are spent in a more efficient way than in the strain that overproduces ALDH.<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px|thumb|center|''Figure 5.''' Comparison of ALDH activities in the different strains tested in this study]]<br />
<br />
<br />
====Useful Literature and References====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T21:54:00Z<p>Hugo 87: /* Alkane Degradation Results & Conclusions */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1.''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system (AlkB2-system)]===<br />
<br />
From the ratios hexadecane/undecane we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. <br />
<br />
For more information about our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase|detailed alkane hydroxylase results]] page.<br />
<br />
[[Image:TUDelft_AlkB2_Total.png|600px|thumb|center|''Figure 2.''' Enzyme activity [U/mg] of alkane hydroxylase system as compared to the negative control ''E.coli'' K12 strain]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase (LadA)]===<br />
<br />
The analysis of the obtained GC graphs allowed us to estimate the enzymatic activity. We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, read the [[Team:TU_Delft/Project/alkane-degradation/results/LadA|detailed LadA results]] page.<br />
<br />
[[Image:TUDelft_LadA Total.png|600px|thumb|center|''Figure 3.''' Enzyme activity [U/mg] of the alkane monooxygenase LadA as compared to the negative control ''E.coli'' K12 strain]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of Alcohol Dehydrogenase (ADH)]===<br />
According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain expressing the Biobrick [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control ''Pseudomonas putida''.<br />
<br />
If you are interested in knowing more about our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/ADH|detailed ADH results]] page.<br />
<br />
[[Image:TUDelftADH_final.jpg|600px|thumb|center|''Figure 4.''' Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of Aldehyde Dehydrogenase (ALDH)]===<br />
<br />
Our results suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks. The expression of ALDH under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold; whereas the expression of the same protein using the part [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] as promoter-rbs combo increases the same activity 3-fold. <br />
<br />
Moreover, the enzymatic activities measured for the constructs [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] and [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] were equivalent to 33.98% and 42.01% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity, respectively. <br />
<br />
If you want to know more about of our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/ALDH|detailed ALDH results]] page. <br />
<br />
It is worthy to mention that our part expresses the protein in a lower amount, meaning that cells express a highly active protein. Thus the cellular resources are spent in a more efficient way than in the strain that overproduces ALDH.<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px|thumb|center|''Figure 5.''' Comparison of ALDH activities in the different strains tested in this study]]<br />
<br />
<br />
====Useful Literature and References====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T21:49:40Z<p>Hugo 87: /* Sensing Results and Conclusions */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results and Conclusions ==<br />
[[Image:TU_Delft_pCaiF_RNApol.jpg]]<br />
<br />
===[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] strength===<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center|'''Figure 1.''' GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center|'''Figure 2.''' GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center|'''Figure 3.''' GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center|'''Figure 4.''' GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|'''Figure 5.'''Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
'''NOTE''': Check at the part registry the definition of [http://partsregistry.org/RiPS RiPS]<br />
<br />
=====Conclusions=====<br />
The simple pCaiF promoter with crp-cAMP binding site has shown activity under limiting nutrient conditions. Therefore, '''this promoter can be used to enable catabolic shifts''' from glucose to '''new degradation pathways'''.<br />
<br />
From our point of view, this part will be very useful for future teams. Please also check the results obtained for the model developed for this part: [https://2010.igem.org/Team:TU_Delft#page=Modeling/pcaif-model pCaiF regulation model]<br />
<br />
=====Work for next year's teams=====<br />
We would have liked to express our results according to the protocols [http://partsregistry.org/PoPS in the part registry]. Therefore the output of [http://partsregistry.org/Part:BBa_B0032 B0032] and a standard promoter growing on M9 medium has to be measured.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Notebook/brainstormTeam:TU Delft/Notebook/brainstorm2010-10-27T21:42:21Z<p>Hugo 87: /* Hugo's Risk Scale */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
==Brainstorm==<br />
[[Image:TUDelft_Brainstorm.png|right|400px]]<br />
The key to a succesfull iGEM project is chosing the right subject. It took us from March until early May to decide which idea suited us best. We considered everything from bacterial eyes to plastic soup, and riboswitches to exhaled breath analysis. Our final idea was based on the current events at that time: the BP oil spill. <br />
<br />
===Hugo's Risk Scale===<br />
We used the Hugo Risk Scale (HRS) to determine whether an idea was feasible or not.<br />
<br />
Risk is represented with a value from 0 (no risk) to 10 (very risky)<br />
# Lac operon (or other piece of cake) + our biobrick<br />
# Less than 5 genes or biobricks (copy-paste) + our biobrick<br />
# Less than 10 genes or biobricks (copy-paste) + our biobrick<br />
# Biobrick engineering <5, improvement of things already done<br />
# Multi biobrick engineering >5, improvement of things already done<br />
# Site directed mutagenesis of several genes, results unknown a priori<br />
# Evolutionary engineering involved = sequencing<br />
# Protein engineering involved, results unknown a priori<br />
# A lot of genes and/or biobricks (>20), known genes in other species and characterized. Stress, social friction<br />
# A lot of genes and/or biobricks (>20), Known genes in other species, not fully characterized. Start a project one-eyed = stress, social friction, high chances of collapse<br />
# A lot of genes (>20), Unknown genes= characterization + sequencing + cloning. Start a project from nothing or partially blind, team-member losses + obituraries. Good side: paper in Nature and/or Nobel prize<br />
<br />
===Idea List===<br />
[[Image:TUDelft_Goals.png|right|400px]]<br />
<br />
On this page you see a few of the other ideas we had, that might be an inspiration to future iGEM Teams:<br />
<br />
* Exhaled breath analysis<br />
* Soil Sampling<br />
* Mosquito’s/Parasites<br />
* Cell shrinkage<br />
* Oil remediation (Hydrocarbon biodegradation)<br />
* Bacterial buffer<br />
* Rainbow pH sensor<br />
* Ice melting<br />
* Biofilms (Linked to cell density measurement?)<br />
* Bacterial eye<br />
* GMOs in the Gut<br />
* Algae Bloom solution<br />
* Appetite inhibitor (Caerulein)<br />
* Teeth-plaque<br />
* Cell differentiation<br />
* Vaccines<br />
* Quorum sensing<br />
* Parasites/mosquitoes<br />
* Heavy metals/calcium<br />
* Gut delivery mechanism<br />
* Sea water desalination<br />
* Hydrogen sulfide removal<br />
* Removal of Indool (toilet odor)<br />
* Biological ship coating<br />
* Chewing gum/Grafitti removal<br />
* Motility for yeast cells<br />
* Bacterial solar clock<br />
* Yeast mating factors<br />
* Bacterial battery<br />
* Biological random generator<br />
* Lamp of bacteria<br />
* Polymer production<br />
* Sensors of light, electricity, proteins, pathogens, CO<sub>2</sub>, O<sub>2</sub>, space, glucose, sound, metal, gravity, space, air, nutrients, HTP, magnetic fields, light (wavelength / intensity), temperature, receiver, medium, receptor, radiation, stress, heat, filter, conduction, speed, shear stress, gradient, flux, mutagens, pH, energy, smell, pollutants, products, gas, liquid, osmosis, salinity, prokaryote/eukaryote, DO, hormones, diode, pain, clinical parameters (such as urea => diagnostics), rotation, scan, computer, aging, telomeres, detergents, solvents, viscosity, membranes (and integrity), cell state.</div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/rbs-characterization/resultsTeam:TU Delft/Project/rbs-characterization/results2010-10-27T21:28:57Z<p>Hugo 87: /* The Results */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
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<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/parts" target="" /></map></html><br />
==The Results==<br />
In the plot below the RBS strengths for the five tested members of the Anderson family are given along with the deviations from the mean. The strengths are given relative to the community RBS [http://partsregistry.org/Part:BBa_B0034 BBa_B0034], which has a strength of unity (1). This relationship could be built by our characterization of the community RBS [http://partsregistry.org/Part:BBa_B0032 BBa_B0032] simultaneously, which is known to have a strength (or efficiency) of 0.3 relative to [http://partsregistry.org/Part:BBa_B0032 BBa_B0034]. Therefore in the plot below, the mean value of BBa_B0032 is set to 0.3. The values have been obtained by the experimental setup described [[Team:TU_Delft/Project/rbs-characterization/parts|here]] and using the [[Team:TU_Delft/Modeling/protein-production-model|model]] to correct for growth dilution.<br />
<br />
[[Image:TUDelft_2010_RBS_strength_graph.PNG|600px|thumb|center|'''Figure 1.''' RBS strength]]<br />
<br />
<html><div id="rbs-results"></html><br />
<br />
==Conclusions==<br />
We've succeeded in characterizing five members of the Anderson ribosome binding site family by 96 well plate cultivation and simultaneous monitoring of GFP fluorescence and biomass absorbance. Fitting the obtained data to our improved [https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization protein production model] yielded RBS strengths values with negligible standard deviations. To recap the following RBS strengths (efficiencies) were found:<br />
<br />
<br />
<html><!-- Original data<br />
J61100 0.019970|0.005119<br />
J61101 0.119333|0.021401 <br />
J61107 0.077024|0.014804 <br />
J61117 0.012552|0.004484 <br />
J61127 0.065226|0.006604 <br />
B0032 0.300000|0.026904<br />
<br />
Rounded, in percentage<br />
J61100 0.020|0.00512<br />
J61101 0.119|0.02140 <br />
J61107 0.077|0.01480 <br />
J61117 0.013|0.00448 <br />
J61127 0.065|0.00660 <br />
B0032 0.300|0.02690<br />
<br />
--></html><br />
<br />
{|<br />
|<b>Ribosome binding site</b><br />
|<b>Mean relative strength</b><br />
|<b>Standard deviation</b><br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61100 J61100]<br />
|0.020 (2.0%)<br />
|0.00512<br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61101 J61101]<br />
|0.119 (11.9%)<br />
|0.02140 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61107 J61107]<br />
|0.077 (7.7%)<br />
|0.01480 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61117 J61117]<br />
|0.013 (1.3%)<br />
|0.00448 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61127 J61127]<br />
|0.065 (6.5%)<br />
|0.00660 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_B0032 B0032]<br />
|0.300 (Reference, 30%)<br />
|0.02690<br />
|}<br />
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<div>__NOTOC__<br />
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==The Results==<br />
In the plot below the RBS strengths for the five tested members of the Anderson family are given along with the deviations from the mean. The strengths are given relative to the community RBS [http://partsregistry.org/Part:BBa_B0034 BBa_B0034], which has a strength of unity (1). This relationship could be built by our characterization of the community RBS [http://partsregistry.org/Part:BBa_B0032 BBa_B0032] simultaneously, which is known to have a strength (or efficiency) of 0.3 relative to [http://partsregistry.org/Part:BBa_B0032 BBa_B0034]. Therefore in the plot below, the mean value of BBa_B0032 is set to 0.3. The values have been obtained by the experimental setup described [[Team:TU_Delft/Project/rbs-characterization/parts|here]] and using the [[Team:TU_Delft/Modeling/protein-production-model|model]] to correct for growth dilution.<br />
<br />
[[Image:TUDelft_2010_RBS_strength_graph.PNG]]<br />
<br />
<html><div id="rbs-results"></html><br />
<br />
==Conclusions==<br />
We've succeeded in characterizing five members of the Anderson ribosome binding site family by 96 well plate cultivation and simultaneous monitoring of GFP fluorescence and biomass absorbance. Fitting the obtained data to our improved [https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization protein production model] yielded RBS strengths values with negligible standard deviations. To recap the following RBS strengths (efficiencies) were found:<br />
<br />
<br />
<html><!-- Original data<br />
J61100 0.019970|0.005119<br />
J61101 0.119333|0.021401 <br />
J61107 0.077024|0.014804 <br />
J61117 0.012552|0.004484 <br />
J61127 0.065226|0.006604 <br />
B0032 0.300000|0.026904<br />
<br />
Rounded, in percentage<br />
J61100 0.020|0.00512<br />
J61101 0.119|0.02140 <br />
J61107 0.077|0.01480 <br />
J61117 0.013|0.00448 <br />
J61127 0.065|0.00660 <br />
B0032 0.300|0.02690<br />
<br />
--></html><br />
<br />
{|<br />
|<b>Ribosome binding site</b><br />
|<b>Mean relative strength</b><br />
|<b>Standard deviation</b><br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61100 J61100]<br />
|0.020 (2.0%)<br />
|0.00512<br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61101 J61101]<br />
|0.119 (11.9%)<br />
|0.02140 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61107 J61107]<br />
|0.077 (7.7%)<br />
|0.01480 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61117 J61117]<br />
|0.013 (1.3%)<br />
|0.00448 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61127 J61127]<br />
|0.065 (6.5%)<br />
|0.00660 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_B0032 B0032]<br />
|0.300 (Reference, 30%)<br />
|0.02690<br />
|}<br />
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<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Team/membersTeam:TU Delft/Team/members2010-10-27T21:17:42Z<p>Hugo 87: </p>
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<a href="https://2010.igem.org/Team:TU_Delft#page=User:Aleabate"><h3>Alessandro Abate</h3></a><br />
<table><br />
<tr><br />
<td class="name">Department:</td><td class="value">Delft Center for Systems and Control</td><br />
</tr><br />
<tr><br />
<br />
<td class="name">10010011:</td><td class="value">FALSE</td><br />
</tr><br />
<tr><br />
<td class="story" colspan=2>" there is grandeur in this view of life (c. darwin) "</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:Esengul"><img src="https://static.igem.org/mediawiki/2010/7/78/TU_Delft_Yildirim.jpg" width="120" height="120" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:Esengul">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:Esengul"><h3>Esengul Yildirim</h3></a><br />
<table><br />
<br />
<tr><br />
<td class="name">Department:</td><td class="value">Enzymology</td><br />
</tr><br />
<tr><br />
<td class="name">Conductivity:</td><td class="value">45.2 million S/m</td><br />
</tr><br />
<tr><br />
<br />
<td class="story" colspan=2>"DNA is life, <br />the rest is just translation..."</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<div style="width: 100%; clear: both;"></div><br />
<h2>Advisors</h2><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:stefandekok"><img src="https://static.igem.org/mediawiki/2010/b/b2/TU_Delft_Kok.jpg" width="120" height="120" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:stefandekok">View Profile</a></p><br />
</div><br />
<br />
<div class="member-description"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:stefandekok"><h3>Stefan de Kok</h3></a><br />
<table><br />
<tr><br />
<td class="name">Department:</td><td class="value">Fermentation Technology</td><br />
</tr><br />
<tr><br />
<br />
<td class="name">Yield:</td><td class="value">15 cmol/cmol</td><br />
</tr><br />
<tr><br />
<td class="story" colspan=2>"I'm the yeasty boy, yeah!"</td><br />
</tr><br />
</table><br />
</div><br />
<br />
</div><br />
<!-- <div class="member-container"><br />
<div class="member-picture"><br />
<a href=""><img src="" width="120" height="120" /></a><br />
<p><a href="">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Domenico Bellomo</h3><br />
<br />
<table><br />
<tr><br />
<td class="name">iGEM Medals:</td><td class="value">2 x gold</td><br />
</tr><br />
<tr><br />
<td class="story" colspan=2>"bla bla bla"</td><br />
</tr><br />
<br />
</table><br />
</div><br />
</div> /--><br />
</div><br />
<!-- Team Overview END /--><br />
</html></div>Hugo 87http://2010.igem.org/File:TU_Delft_pCaiF_RNApol.jpgFile:TU Delft pCaiF RNApol.jpg2010-10-27T20:59:44Z<p>Hugo 87: </p>
<hr />
<div></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:59:17Z<p>Hugo 87: /* Sensing Results and Conclusions */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results and Conclusions ==<br />
[[Image:TU_Delft_pCaiF_RNApol.jpg]]<br />
<br />
===[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] strength===<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
'''NOTE''': Check at the part registry the definition of [http://partsregistry.org/RiPS RiPS]<br />
<br />
=====Conclusions=====<br />
The simple pCaiF promoter with crp-cAMP binding site has shown activity under limiting nutrient conditions. Therefore, '''this promoter can be used to enable catabolic shifts''' from glucose to '''new degradation pathways'''.<br />
<br />
From our point of view, this part will be very useful for future teams. Please also check the results obtained for the model developed for this part: [https://2010.igem.org/Team:TU_Delft#page=Modeling/pcaif-model pCaiF regulation model]<br />
<br />
=====Work for next year's teams=====<br />
We would have liked to express our results according to the protocols [http://partsregistry.org/PoPS in the part registry]. Therefore the output of [http://partsregistry.org/Part:BBa_B0032 B0032] and a standard promoter growing on M9 medium has to be measured.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:53:37Z<p>Hugo 87: /* Sensing Results and Conclusions */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results and Conclusions ==<br />
===[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] strength===<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
'''NOTE''': Check at the part registry the definition of [http://partsregistry.org/RiPS RiPS]<br />
<br />
=====Conclusions=====<br />
The simple pCaiF promoter with crp-cAMP binding site has shown activity under limiting nutrient conditions. Therefore, '''this promoter can be used to enable catabolic shifts''' from glucose to '''new degradation pathways'''.<br />
<br />
From our point of view, this part will be very useful for future teams. Please also check the results obtained for the model developed for this part: [https://2010.igem.org/Team:TU_Delft#page=Modeling/pcaif-model pCaiF regulation model]<br />
<br />
=====Work for next year's teams=====<br />
We would have liked to express our results according to the protocols [http://partsregistry.org/PoPS in the part registry]. Therefore the output of [http://partsregistry.org/Part:BBa_B0032 B0032] and a standard promoter growing on M9 medium has to be measured.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:52:27Z<p>Hugo 87: /* CONCLUSIONS */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results and Conclusions ==<br />
===[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] strength===<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
'''NOTE''': Check at the part registry the definition of [http://partsregistry.org/RiPS RiPS]<br />
<br />
=====Conclusions=====<br />
<br />
The simple pCaiF promoter with crp-cAMP binding site has shown activity under limiting nutrient conditions. Therefore, '''this promoter can be used to enable catabolic shifts''' from glucose to '''new degradation pathways'''.<br />
<br />
From our point of view, this part will be very useful for future teams. Please also check the results obtained for the model developed for this part: [https://2010.igem.org/Team:TU_Delft#page=Modeling/pcaif-model pCaiF regulation model]<br />
<br />
=====Work for next year's teams=====<br />
We would have liked to express our results according to the protocols [http://partsregistry.org/PoPS in the part registry]. Therefore the output of [http://partsregistry.org/Part:BBa_B0032 B0032] and a standard promoter growing on M9 medium has to be measured.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:51:41Z<p>Hugo 87: /* Sensing Results */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results and Conclusions ==<br />
===[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] strength===<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
'''NOTE''': Check at the part registry the definition of [http://partsregistry.org/RiPS RiPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
The simple pCaiF promoter with crp-cAMP binding site has shown activity under limiting nutrient conditions. Therefore, '''this promoter can be used to enable catabolic shifts''' from glucose to '''new degradation pathways'''.<br />
<br />
From our point of view, this part will be very useful for future teams. Please also check the results obtained for the model developed for this part: [https://2010.igem.org/Team:TU_Delft#page=Modeling/pcaif-model pCaiF regulation model]<br />
<br />
<br />
<br />
=====Work for next year's teams=====<br />
We would have liked to express our results according to the protocols [http://partsregistry.org/PoPS in the part registry]. Therefore the output of [http://partsregistry.org/Part:BBa_B0032 B0032] and a standard promoter growing on M9 medium has to be measured.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:51:12Z<p>Hugo 87: /* BBa_K398326 pCaiF strength */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
===[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] strength===<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
'''NOTE''': Check at the part registry the definition of [http://partsregistry.org/RiPS RiPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
The simple pCaiF promoter with crp-cAMP binding site has shown activity under limiting nutrient conditions. Therefore, '''this promoter can be used to enable catabolic shifts''' from glucose to '''new degradation pathways'''.<br />
<br />
From our point of view, this part will be very useful for future teams. Please also check the results obtained for the model developed for this part: [https://2010.igem.org/Team:TU_Delft#page=Modeling/pcaif-model pCaiF regulation model]<br />
<br />
<br />
<br />
=====Work for next year's teams=====<br />
We would have liked to express our results according to the protocols [http://partsregistry.org/PoPS in the part registry]. Therefore the output of [http://partsregistry.org/Part:BBa_B0032 B0032] and a standard promoter growing on M9 medium has to be measured.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:50:51Z<p>Hugo 87: /* pCaiF strength */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
===[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] strength===<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
'''NOTE''': Check at the part registry the definition of [http://partsregistry.org/RiPS RiPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
The simple pCaiF promoter with crp-cAMP binding site has shown activity under limiting nutrient conditions. Therefore, '''this promoter can be used to enable catabolic shifts''' from glucose to '''new degradation pathways'''.<br />
<br />
From our point of view, this part will be very useful for future teams. Please also check the results obtained for the model developed for this part: [https://2010.igem.org/Team:TU_Delft#page=Modeling/pcaif-model pCaiF regulation model]<br />
<br />
<br />
<br />
=====Work for next year's teams=====<br />
We would have liked to express our results according to the protocols [http://partsregistry.org/PoPS in the part registry]. Therefore the output of [http://partsregistry.org/Part:BBa_B0032 B0032] and a standard promoter growing on M9 medium has to be measured.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:50:04Z<p>Hugo 87: /* Sensing Results */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
'''NOTE''': Check at the part registry the definition of [http://partsregistry.org/RiPS RiPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
The simple pCaiF promoter with crp-cAMP binding site has shown activity under limiting nutrient conditions. Therefore, '''this promoter can be used to enable catabolic shifts''' from glucose to '''new degradation pathways'''.<br />
<br />
From our point of view, this part will be very useful for future teams. Please also check the results obtained for the model developed for this part: [https://2010.igem.org/Team:TU_Delft#page=Modeling/pcaif-model pCaiF regulation model]<br />
<br />
<br />
<br />
=====Work for next year's teams=====<br />
We would have liked to express our results according to the protocols [http://partsregistry.org/PoPS in the part registry]. Therefore the output of [http://partsregistry.org/Part:BBa_B0032 B0032] and a standard promoter growing on M9 medium has to be measured.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:49:45Z<p>Hugo 87: /* WORK FOR NEXT TEAM */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
'''NOTE''': Check at the part registry the definition of [http://partsregistry.org/RiPS RiPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
The simple pCaiF promoter with crp-cAMP binding site has shown activity under limiting nutrient conditions. Therefore, '''this promoter can be used to enable catabolic shifts''' from glucose to '''new degradation pathways'''.<br />
<br />
From our point of view, this part will be very useful for future teams. Please also check the results obtained for the model developed for this part: [https://2010.igem.org/Team:TU_Delft#page=Modeling/pcaif-model pCaiF regulation model]<br />
<br />
<br />
<br />
=====Work for next year's teams=====<br />
We would have liked to express our results according to the protocols [http://partsregistry.org/PoPS in the part registry]. Therefore the output of [http://partsregistry.org/Part:BBa_B0032 B0032] and a standard promoter growing on M9 medium has to be measured.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:48:27Z<p>Hugo 87: /* WORK FOR NEXT TEAM */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
'''NOTE''': Check at the part registry the definition of [http://partsregistry.org/RiPS RiPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
The simple pCaiF promoter with crp-cAMP binding site has shown activity under limiting nutrient conditions. Therefore, '''this promoter can be used to enable catabolic shifts''' from glucose to '''new degradation pathways'''.<br />
<br />
From our point of view, this part will be very useful for future teams. Please also check the results obtained for the model developed for this part: [https://2010.igem.org/Team:TU_Delft#page=Modeling/pcaif-model pCaiF regulation model]<br />
<br />
<br />
<br />
=====WORK FOR NEXT TEAM=====<br />
We would have liked to express our results according to the protocols [http://partsregistry.org/PoPS in the part registry]. Therefore the output of [http://partsregistry.org/Part:BBa_B0032 B0032] and a standard promoter growing on M9 medium has to be measured.<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:44:40Z<p>Hugo 87: /* WORK FOR NEXT TEAM */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
'''NOTE''': Check at the part registry the definition of [http://partsregistry.org/RiPS RiPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
The simple pCaiF promoter with crp-cAMP binding site has shown activity under limiting nutrient conditions. Therefore, '''this promoter can be used to enable catabolic shifts''' from glucose to '''new degradation pathways'''.<br />
<br />
From our point of view, this part will be very useful for future teams. Please also check the results obtained for the model developed for this part: [https://2010.igem.org/Team:TU_Delft#page=Modeling/pcaif-model pCaiF regulation model]<br />
<br />
<br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:44:15Z<p>Hugo 87: /* CONCLUSIONS */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
'''NOTE''': Check at the part registry the definition of [http://partsregistry.org/RiPS RiPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
The simple pCaiF promoter with crp-cAMP binding site has shown activity under limiting nutrient conditions. Therefore, '''this promoter can be used to enable catabolic shifts''' from glucose to '''new degradation pathways'''.<br />
<br />
From our point of view, this part will be very useful for future teams. Please also check the results obtained for the model developed for this part: [https://2010.igem.org/Team:TU_Delft#page=Modeling/pcaif-model pCaiF regulation model]<br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML><br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:43:47Z<p>Hugo 87: /* CONCLUSIONS */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
'''NOTE''': Check at the part registry the definition of [http://partsregistry.org/RiPS RiPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
The simple pCaiF promoter with crp-cAMP binding site has shown activity under limiting nutrient conditions. Therefore, '''this promoter can be used to enable catabolic shifts''' from glucose to '''new degradation pathways'''.<br />
<br />
From our point of view, this part will be very useful for future teams. Please also check the results obtained for the model developed for this part:<br />
<br />
[https://2010.igem.org/Team:TU_Delft#page=Modeling/pcaif-model pCaiF regulation model]<br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML><br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:42:25Z<p>Hugo 87: /* CONCLUSIONS */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
'''NOTE''': Check at the part registry the definition of [http://partsregistry.org/RiPS RiPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
The simple pCaiF promoter with crp-cAMP binding site has shown activity under limiting nutrient conditions. Therefore, '''this promoter can be used to enable catabolic shifts''' from glucose to '''new degradation pathways'''.<br />
<br />
From our point of view, this part will be very useful for future teams. Please also check the results obtained for the model developed for this part:<br />
<br />
[HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=MODELING/PCAIF-MODEL IN SILICO WORK]<br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML><br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:38:59Z<p>Hugo 87: /* BBa_K398326 pCaiF in numbers */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
'''NOTE''': Check at the part registry the definition of [http://partsregistry.org/RiPS RiPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
THE SIMPLE PCAIF PROMOTER WITH CRP-CAMP BINDING SITE HAS SHOWN ACTIVITY UNDER LIMITING NUTRIENT CONDITIONS. THEREFORE THIS PROMOTOR CAN BE USED TO ENABLE A CATABOLIC SHIFT FROM GLUCOSE TO NEW DEGRADATION PATHWAYS. <br />
<br />
FOR US THIS IS A VERY USEFUL PART FOR FUTURE TEAMS. <br />
PLEASE ALSO SEE THE RESULTS OBTAINED BY MODELING: <br />
[HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=MODELING/PCAIF-MODEL IN SILICO WORK]<br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML><br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:38:29Z<p>Hugo 87: /* BBa_K398326 pCaiF in numbers */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
NOTE: Check at the part registry the definition of [http://partsregistry.org/RiPS RiPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
THE SIMPLE PCAIF PROMOTER WITH CRP-CAMP BINDING SITE HAS SHOWN ACTIVITY UNDER LIMITING NUTRIENT CONDITIONS. THEREFORE THIS PROMOTOR CAN BE USED TO ENABLE A CATABOLIC SHIFT FROM GLUCOSE TO NEW DEGRADATION PATHWAYS. <br />
<br />
FOR US THIS IS A VERY USEFUL PART FOR FUTURE TEAMS. <br />
PLEASE ALSO SEE THE RESULTS OBTAINED BY MODELING: <br />
[HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=MODELING/PCAIF-MODEL IN SILICO WORK]<br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML><br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:36:50Z<p>Hugo 87: /* BBa_K398326 pCaiF in numbers */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
NOTE: Check at the part registry the definition of [http://partregistry.org/RIPS RIPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
THE SIMPLE PCAIF PROMOTER WITH CRP-CAMP BINDING SITE HAS SHOWN ACTIVITY UNDER LIMITING NUTRIENT CONDITIONS. THEREFORE THIS PROMOTOR CAN BE USED TO ENABLE A CATABOLIC SHIFT FROM GLUCOSE TO NEW DEGRADATION PATHWAYS. <br />
<br />
FOR US THIS IS A VERY USEFUL PART FOR FUTURE TEAMS. <br />
PLEASE ALSO SEE THE RESULTS OBTAINED BY MODELING: <br />
[HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=MODELING/PCAIF-MODEL IN SILICO WORK]<br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML><br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:35:20Z<p>Hugo 87: /* BBa_K398326 pCaiF IN NUMBERS */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
NOTE: CHECK AT THE PART REGISTRY THE DEFINITION OF [HTTP://PARTSREGISTRY.ORG/RIPS RIPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
THE SIMPLE PCAIF PROMOTER WITH CRP-CAMP BINDING SITE HAS SHOWN ACTIVITY UNDER LIMITING NUTRIENT CONDITIONS. THEREFORE THIS PROMOTOR CAN BE USED TO ENABLE A CATABOLIC SHIFT FROM GLUCOSE TO NEW DEGRADATION PATHWAYS. <br />
<br />
FOR US THIS IS A VERY USEFUL PART FOR FUTURE TEAMS. <br />
PLEASE ALSO SEE THE RESULTS OBTAINED BY MODELING: <br />
[HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=MODELING/PCAIF-MODEL IN SILICO WORK]<br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML><br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:34:06Z<p>Hugo 87: /* Does pCaiF really works under starvation conditions??? */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] IN NUMBERS=====<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
NOTE: CHECK AT THE PART REGISTRY THE DEFINITION OF [HTTP://PARTSREGISTRY.ORG/RIPS RIPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
THE SIMPLE PCAIF PROMOTER WITH CRP-CAMP BINDING SITE HAS SHOWN ACTIVITY UNDER LIMITING NUTRIENT CONDITIONS. THEREFORE THIS PROMOTOR CAN BE USED TO ENABLE A CATABOLIC SHIFT FROM GLUCOSE TO NEW DEGRADATION PATHWAYS. <br />
<br />
FOR US THIS IS A VERY USEFUL PART FOR FUTURE TEAMS. <br />
PLEASE ALSO SEE THE RESULTS OBTAINED BY MODELING: <br />
[HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=MODELING/PCAIF-MODEL IN SILICO WORK]<br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML><br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:32:38Z<p>Hugo 87: /* Diauxic shift */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM (secondary carbon source). This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high GFP expression during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] IN NUMBERS=====<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
NOTE: CHECK AT THE PART REGISTRY THE DEFINITION OF [HTTP://PARTSREGISTRY.ORG/RIPS RIPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
THE SIMPLE PCAIF PROMOTER WITH CRP-CAMP BINDING SITE HAS SHOWN ACTIVITY UNDER LIMITING NUTRIENT CONDITIONS. THEREFORE THIS PROMOTOR CAN BE USED TO ENABLE A CATABOLIC SHIFT FROM GLUCOSE TO NEW DEGRADATION PATHWAYS. <br />
<br />
FOR US THIS IS A VERY USEFUL PART FOR FUTURE TEAMS. <br />
PLEASE ALSO SEE THE RESULTS OBTAINED BY MODELING: <br />
[HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=MODELING/PCAIF-MODEL IN SILICO WORK]<br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML><br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:30:50Z<p>Hugo 87: /* M9 minimal medium */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a reponse by pCaiF.<br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under carbon limitation conditions; which in our case it is detected at the beginning of stationary phase at low initial glucose concentrations, see figure. <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and added Potassium Laurate at a final concentration of 5 mM. This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high expression level during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] IN NUMBERS=====<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
NOTE: CHECK AT THE PART REGISTRY THE DEFINITION OF [HTTP://PARTSREGISTRY.ORG/RIPS RIPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
THE SIMPLE PCAIF PROMOTER WITH CRP-CAMP BINDING SITE HAS SHOWN ACTIVITY UNDER LIMITING NUTRIENT CONDITIONS. THEREFORE THIS PROMOTOR CAN BE USED TO ENABLE A CATABOLIC SHIFT FROM GLUCOSE TO NEW DEGRADATION PATHWAYS. <br />
<br />
FOR US THIS IS A VERY USEFUL PART FOR FUTURE TEAMS. <br />
PLEASE ALSO SEE THE RESULTS OBTAINED BY MODELING: <br />
[HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=MODELING/PCAIF-MODEL IN SILICO WORK]<br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML><br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:28:27Z<p>Hugo 87: /* LB medium */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence signal produced was weak, moreover we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time.<br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a pCaiF response. <br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under limitation (entry of stationary phase at low glucose concentrations, see figure). <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and added Potassium Laurate at a final concentration of 5 mM. This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high expression level during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] IN NUMBERS=====<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
NOTE: CHECK AT THE PART REGISTRY THE DEFINITION OF [HTTP://PARTSREGISTRY.ORG/RIPS RIPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
THE SIMPLE PCAIF PROMOTER WITH CRP-CAMP BINDING SITE HAS SHOWN ACTIVITY UNDER LIMITING NUTRIENT CONDITIONS. THEREFORE THIS PROMOTOR CAN BE USED TO ENABLE A CATABOLIC SHIFT FROM GLUCOSE TO NEW DEGRADATION PATHWAYS. <br />
<br />
FOR US THIS IS A VERY USEFUL PART FOR FUTURE TEAMS. <br />
PLEASE ALSO SEE THE RESULTS OBTAINED BY MODELING: <br />
[HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=MODELING/PCAIF-MODEL IN SILICO WORK]<br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML><br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:25:28Z<p>Hugo 87: /* Sensing Results */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
An organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studied the regulatory Biobrick [http://partsregistry.org/Part:BBa_K398326 BBa_K398326] using [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] in order to measure the output given by our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence produced is a really low signal and we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time. <br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a pCaiF response. <br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under limitation (entry of stationary phase at low glucose concentrations, see figure). <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and added Potassium Laurate at a final concentration of 5 mM. This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high expression level during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] IN NUMBERS=====<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
NOTE: CHECK AT THE PART REGISTRY THE DEFINITION OF [HTTP://PARTSREGISTRY.ORG/RIPS RIPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
THE SIMPLE PCAIF PROMOTER WITH CRP-CAMP BINDING SITE HAS SHOWN ACTIVITY UNDER LIMITING NUTRIENT CONDITIONS. THEREFORE THIS PROMOTOR CAN BE USED TO ENABLE A CATABOLIC SHIFT FROM GLUCOSE TO NEW DEGRADATION PATHWAYS. <br />
<br />
FOR US THIS IS A VERY USEFUL PART FOR FUTURE TEAMS. <br />
PLEASE ALSO SEE THE RESULTS OBTAINED BY MODELING: <br />
[HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=MODELING/PCAIF-MODEL IN SILICO WORK]<br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML><br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:13:12Z<p>Hugo 87: /* CONCLUSIONS */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
<br />
GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
NOTE: CHECK AT THE PART REGISTRY THE DEFINITION OF [HTTP://PARTSREGISTRY.ORG/RIPS RIPS]<br />
<br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
<br />
<br />
As an organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studies the regulatory part using a proxy system with GFP. We attached a GFP generator in order to measure the output of our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence produced is a really low signal and we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time. <br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a pCaiF response. <br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under limitation (entry of stationary phase at low glucose concentrations, see figure). <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and added Potassium Laurate at a final concentration of 5 mM. This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high expression level during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] IN NUMBERS=====<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
NOTE: CHECK AT THE PART REGISTRY THE DEFINITION OF [HTTP://PARTSREGISTRY.ORG/RIPS RIPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
THE SIMPLE PCAIF PROMOTER WITH CRP-CAMP BINDING SITE HAS SHOWN ACTIVITY UNDER LIMITING NUTRIENT CONDITIONS. THEREFORE THIS PROMOTOR CAN BE USED TO ENABLE A CATABOLIC SHIFT FROM GLUCOSE TO NEW DEGRADATION PATHWAYS. <br />
<br />
FOR US THIS IS A VERY USEFUL PART FOR FUTURE TEAMS. <br />
PLEASE ALSO SEE THE RESULTS OBTAINED BY MODELING: <br />
[HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=MODELING/PCAIF-MODEL IN SILICO WORK]<br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML><br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:12:05Z<p>Hugo 87: /* Sensing Results */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
<br />
GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
NOTE: CHECK AT THE PART REGISTRY THE DEFINITION OF [HTTP://PARTSREGISTRY.ORG/RIPS RIPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
THE SIMPLE PCAIF PROMOTER WITH CRP-CAMP BINDING SITE HAS SHOWN ACTIVITY UNDER LIMITING NUTRIENT CONDITIONS. THEREFORE THIS PROMOTOR CAN BE USED TO ENABLE A CATABOLIC SHIFT FROM GLUCOSE TO NEW DEGRADATION PATHWAYS. <br />
<br />
FOR US THIS IS A VERY USEFUL PART FOR FUTURE TEAMS. <br />
PLEASE ALSO SEE THE RESULTS OBTAINED BY MODELING: <br />
[HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=MODELING/PCAIF-MODEL IN SILICO WORK]<br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:10:50Z<p>Hugo 87: /* Sensing Results */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
<br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project; as we explained before we looked for a promoter that could enable the expression of proteins under low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: once glucose becomes a limiting factor, the expression of alkane degradation genes under pCaiF control will (theoreticallly) enable the cells to shift from glucose metabolism to alkane degradation. This all could be achieved just by a adding a piece of DNA of just 51 base pairs. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp binding domain, cAMP-crp is known as transcriptional regulator. When glucose concentrations are high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells; however during starvation periods cAMP levels increase and this also increases the concentration of the complex cAMP-crp. This activates at least 180 genes related to starvation response, among which a protein used during Carnitine anaerobic metabolism. <br />
<br />
=====LB medium=====<br />
<br />
Due to time constraints we couldn't make a more elegant circuit or express our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)], however we wanted to show that the part does what we expected: Protein production at low glucose concentrations. We attached a GFP generator in order to measure the output of our part. First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is a slight improvement on GFP production when the LB medium is diluted 50%. However the fluorescence produced is a really low signal and we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time. <br />
<br />
=====M9 minimal medium=====<br />
<br />
We also tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see how pCaiF responds when the starvation phase starts earlier than normal during ''E. coli'' cultures. <br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under starvation periods (stationary phase at low glucose concentrations). We overlapped the plots in order to show the differences between the different conditions tested.<br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a secondary carbon source in the medium, we decided to lower the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM. With this experiment we expected to see how the GFP profile behaves when cAMP levels drop in presence of a second carbon source.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, because as we expected the GFP production decreases when the catabolism of the secondary carbon source starts. You can check that in the plot because of the change in the slope for the GFP profile.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really work under starvation conditions???=====<br />
We had some doubts about the activity of pCaiF under starvation periods because we saw a leaky production of GFP that followed the biomass profile. We decided to compare the GFP production during the exponential phase and the stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. This is especially clear when the initial glucose concentration is 2 g/L. For the Laurate growth phase in the diauxic experiment we saw a remaining activity from the glucose starvation period and during Laurate consumption we saw a reduction in the GFP production as we expected.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
In order to convert arbitrary fluorescence units to something meaningful, we used the parameters that were suggested in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040], we divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back to the number of moles per well, knowing that our reaction volume was 100 µL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules present in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (glucose phase)</td><td>7.4517E+06</td><td>2.1543E+07</td></tr> <tr><td>Diauxic growth (Laurate phase)</td><td>1.1435E+07</td><td>6.6428E+06</td></tr></table><br />
<br />
NOTE: Check the part registry for the definition of [http://partsregistry.org/RiPS RiPS]<br />
<br />
Properly, we should express everything in terms of [http://partsregistry.org/PoPS Polymerase Per Second] (PoPS); however we lack the data about the output of B0032 and a standard promoter growing the cells in M9 medium. Thus, the mRNA production by this promoter remains unknown; nevertheless with these results we are reporting the protein production using a Promoter-RBS combination that could become standard for other teams who decide to express their proteins under glucose starvation.<br />
<br />
We developed a simple model, in order to explain in silico what happens with the cAMP levels. If you want to know more about this check our [https://2010.igem.org/Team:TU_Delft#page=Modeling/pcaif-model in silico work]. ;-)<br />
<br />
We reported here the biological function of a new promoter for the part registry that works under starvation periods regulated by cAMP levels, a very useful part for future teams. However, some data is still missing like the mRNA production by this part. Other teams interested on this part should measure this parameter in order to make a complete characterization of our part. <br />
<br />
<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:10:27Z<p>Hugo 87: /* pCaiF strength */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
==Sensing Results==<br />
<br />
__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project; as we explained before we looked for a promoter that could enable the expression of proteins under low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: once glucose becomes a limiting factor, the expression of alkane degradation genes under pCaiF control will (theoreticallly) enable the cells to shift from glucose metabolism to alkane degradation. This all could be achieved just by a adding a piece of DNA of just 51 base pairs. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp binding domain, cAMP-crp is known as transcriptional regulator. When glucose concentrations are high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells; however during starvation periods cAMP levels increase and this also increases the concentration of the complex cAMP-crp. This activates at least 180 genes related to starvation response, among which a protein used during Carnitine anaerobic metabolism. <br />
<br />
=====LB medium=====<br />
<br />
Due to time constraints we couldn't make a more elegant circuit or express our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)], however we wanted to show that the part does what we expected: Protein production at low glucose concentrations. We attached a GFP generator in order to measure the output of our part. First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is a slight improvement on GFP production when the LB medium is diluted 50%. However the fluorescence produced is a really low signal and we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time. <br />
<br />
=====M9 minimal medium=====<br />
<br />
We also tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see how pCaiF responds when the starvation phase starts earlier than normal during ''E. coli'' cultures. <br />
<br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under starvation periods (stationary phase at low glucose concentrations). We overlapped the plots in order to show the differences between the different conditions tested.<br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a secondary carbon source in the medium, we decided to lower the initial glucose amount to 1 g/L and we added Potassium Laurate at a final concentration of 5 mM. With this experiment we expected to see how the GFP profile behaves when cAMP levels drop in presence of a second carbon source.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, because as we expected the GFP production decreases when the catabolism of the secondary carbon source starts. You can check that in the plot because of the change in the slope for the GFP profile.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really work under starvation conditions???=====<br />
We had some doubts about the activity of pCaiF under starvation periods because we saw a leaky production of GFP that followed the biomass profile. We decided to compare the GFP production during the exponential phase and the stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. This is especially clear when the initial glucose concentration is 2 g/L. For the Laurate growth phase in the diauxic experiment we saw a remaining activity from the glucose starvation period and during Laurate consumption we saw a reduction in the GFP production as we expected.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] in numbers=====<br />
In order to convert arbitrary fluorescence units to something meaningful, we used the parameters that were suggested in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040], we divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back to the number of moles per well, knowing that our reaction volume was 100 µL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules present in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (glucose phase)</td><td>7.4517E+06</td><td>2.1543E+07</td></tr> <tr><td>Diauxic growth (Laurate phase)</td><td>1.1435E+07</td><td>6.6428E+06</td></tr></table><br />
<br />
NOTE: Check the part registry for the definition of [http://partsregistry.org/RiPS RiPS]<br />
<br />
Properly, we should express everything in terms of [http://partsregistry.org/PoPS Polymerase Per Second] (PoPS); however we lack the data about the output of B0032 and a standard promoter growing the cells in M9 medium. Thus, the mRNA production by this promoter remains unknown; nevertheless with these results we are reporting the protein production using a Promoter-RBS combination that could become standard for other teams who decide to express their proteins under glucose starvation.<br />
<br />
We developed a simple model, in order to explain in silico what happens with the cAMP levels. If you want to know more about this check our [https://2010.igem.org/Team:TU_Delft#page=Modeling/pcaif-model in silico work]. ;-)<br />
<br />
We reported here the biological function of a new promoter for the part registry that works under starvation periods regulated by cAMP levels, a very useful part for future teams. However, some data is still missing like the mRNA production by this part. Other teams interested on this part should measure this parameter in order to make a complete characterization of our part. <br />
<br />
<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:27:38Z<p>Hugo 87: /* Characterization of the ALdehyde DeHydrogenase system */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate. <br />
From the ratios we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. For more information about our findings [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase CLICK HERE].<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Ratio octane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Octane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.832<br />
|6.16<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control<br />
|0.821<br />
|13.3<br />
|1.34<br />
|1.51<br />
|1.23E-3<br />
|-<br />
|''E.coli'' K12 strain #1 (AH-system)<br />
|0.652<br />
|14.4<br />
|21.6<br />
|1.70<br />
|1.77E-2<br />
|-<br />
|''E.coli'' K12 strain #2 (AH-system)<br />
|0.439<br />
|3.96<br />
|47.0<br />
|1.46<br />
|4.49E-2<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
<br />
We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA CLICK ON THIS LINK]<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Average ratio hexadecane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Hexadecane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.900<br />
|3.19<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002])<br />
|0.807<br />
|3.48<br />
|1.76<br />
|4.46<br />
|5.49E-04<br />
|-<br />
|''E.coli'' K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.600<br />
|15.4<br />
|5.68<br />
|4.58<br />
|1.72E-03<br />
|-<br />
|''E.coli'' K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.532<br />
|4.25<br />
|6.95<br />
|3.18<br />
|3.03E-03<br />
|-<br />
|''E.coli'' TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027])<br />
|0.492<br />
|2.98<br />
|7.71<br />
|3.22<br />
|3.33E-03<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain expressing the Biobrick [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control ''Pseudomonas putida''.<br />
<br />
If you are interested in knowing more about our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH CLICK ON THIS LINK].<br />
<br />
[[Image:TUDelftADH_final.jpg|600px|thumb|center|Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
Our results suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks. The expression of ALDH under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold; whereas the expression of the same protein using the part [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] as promoter-rbs combo increases the same activity 3-fold. <br />
<br />
Moreover, the enzymatic activities measured for the constructs [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] and [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] were equivalent to 33.98% and 42.01% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity, respectively. If you want to know more about of our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH CLICK ON THIS LINK]. <br />
<br />
It is worthy to mention that our part expresses the protein in a lower amount, meaning that cells express a highly active protein. Thus the cellular resources are spent in a more efficient way than in the strain that overproduces ALDH.<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px|thumb|center|Comparison of ALDH activities in the different strains tested in this study]]<br />
<br />
<br />
====USEFUL LITERATURE AND REFERENCES====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:26:54Z<p>Hugo 87: /* Characterization of the ALdehyde DeHydrogenase system */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate. <br />
From the ratios we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. For more information about our findings [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase CLICK HERE].<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Ratio octane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Octane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.832<br />
|6.16<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control<br />
|0.821<br />
|13.3<br />
|1.34<br />
|1.51<br />
|1.23E-3<br />
|-<br />
|''E.coli'' K12 strain #1 (AH-system)<br />
|0.652<br />
|14.4<br />
|21.6<br />
|1.70<br />
|1.77E-2<br />
|-<br />
|''E.coli'' K12 strain #2 (AH-system)<br />
|0.439<br />
|3.96<br />
|47.0<br />
|1.46<br />
|4.49E-2<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
<br />
We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA CLICK ON THIS LINK]<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Average ratio hexadecane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Hexadecane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.900<br />
|3.19<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002])<br />
|0.807<br />
|3.48<br />
|1.76<br />
|4.46<br />
|5.49E-04<br />
|-<br />
|''E.coli'' K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.600<br />
|15.4<br />
|5.68<br />
|4.58<br />
|1.72E-03<br />
|-<br />
|''E.coli'' K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.532<br />
|4.25<br />
|6.95<br />
|3.18<br />
|3.03E-03<br />
|-<br />
|''E.coli'' TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027])<br />
|0.492<br />
|2.98<br />
|7.71<br />
|3.22<br />
|3.33E-03<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain expressing the Biobrick [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control ''Pseudomonas putida''.<br />
<br />
If you are interested in knowing more about our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH CLICK ON THIS LINK].<br />
<br />
[[Image:TUDelftADH_final.jpg|600px|thumb|center|Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
Our results suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks. The expression of ALDH under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold; whereas the expression of the same protein using the part [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] as promoter-rbs combo increases the same activity 3-fold. <br />
<br />
Moreover, the enzymatic activities measured for the constructs [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] and [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] were equivalent to 33.98% and 42.01% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity, respectively. If you want to know more about of our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH CLICK ON THIS LINK]. <br />
<br />
It is worthy to mention that our part expresses the protein in a lower amount, meaning that cells express a highly active protein. Thus the cellular resources are spent in a ... way.<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px|thumb|center|Comparison of ALDH activities in the different strains tested in this study]]<br />
<br />
<br />
====USEFUL LITERATURE AND REFERENCES====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:26:10Z<p>Hugo 87: /* Characterization of the ALdehyde DeHydrogenase system */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate. <br />
From the ratios we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. For more information about our findings [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase CLICK HERE].<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Ratio octane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Octane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.832<br />
|6.16<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control<br />
|0.821<br />
|13.3<br />
|1.34<br />
|1.51<br />
|1.23E-3<br />
|-<br />
|''E.coli'' K12 strain #1 (AH-system)<br />
|0.652<br />
|14.4<br />
|21.6<br />
|1.70<br />
|1.77E-2<br />
|-<br />
|''E.coli'' K12 strain #2 (AH-system)<br />
|0.439<br />
|3.96<br />
|47.0<br />
|1.46<br />
|4.49E-2<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
<br />
We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA CLICK ON THIS LINK]<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Average ratio hexadecane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Hexadecane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.900<br />
|3.19<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002])<br />
|0.807<br />
|3.48<br />
|1.76<br />
|4.46<br />
|5.49E-04<br />
|-<br />
|''E.coli'' K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.600<br />
|15.4<br />
|5.68<br />
|4.58<br />
|1.72E-03<br />
|-<br />
|''E.coli'' K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.532<br />
|4.25<br />
|6.95<br />
|3.18<br />
|3.03E-03<br />
|-<br />
|''E.coli'' TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027])<br />
|0.492<br />
|2.98<br />
|7.71<br />
|3.22<br />
|3.33E-03<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain expressing the Biobrick [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control ''Pseudomonas putida''.<br />
<br />
If you are interested in knowing more about our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH CLICK ON THIS LINK].<br />
<br />
[[Image:TUDelftADH_final.jpg|600px|thumb|center|Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
Our results suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks. The expression of ALDH under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold; whereas the expression of the same protein using the part [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] as promoter-rbs combo increases the same activity 3-fold. <br />
<br />
Moreover, the enzymatic activities measured for the constructs [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] and [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] were equivalent to 33.98% and 42.01% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity, respectively. If you want to know more about of our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH CLICK ON THIS LINK]. <br />
<br />
It is worthy to mention that our part expresses the protein in a lower amount, meaning that cells express a highly active protein.<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px|thumb|center|Comparison of ALDH activities in the different strains tested in this study]]<br />
<br />
<br />
====USEFUL LITERATURE AND REFERENCES====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:22:12Z<p>Hugo 87: /* Characterization of the Alcohol DeHydrogenase (ADH) system */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate. <br />
From the ratios we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. For more information about our findings [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase CLICK HERE].<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Ratio octane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Octane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.832<br />
|6.16<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control<br />
|0.821<br />
|13.3<br />
|1.34<br />
|1.51<br />
|1.23E-3<br />
|-<br />
|''E.coli'' K12 strain #1 (AH-system)<br />
|0.652<br />
|14.4<br />
|21.6<br />
|1.70<br />
|1.77E-2<br />
|-<br />
|''E.coli'' K12 strain #2 (AH-system)<br />
|0.439<br />
|3.96<br />
|47.0<br />
|1.46<br />
|4.49E-2<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
<br />
We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA CLICK ON THIS LINK]<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Average ratio hexadecane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Hexadecane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.900<br />
|3.19<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002])<br />
|0.807<br />
|3.48<br />
|1.76<br />
|4.46<br />
|5.49E-04<br />
|-<br />
|''E.coli'' K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.600<br />
|15.4<br />
|5.68<br />
|4.58<br />
|1.72E-03<br />
|-<br />
|''E.coli'' K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.532<br />
|4.25<br />
|6.95<br />
|3.18<br />
|3.03E-03<br />
|-<br />
|''E.coli'' TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027])<br />
|0.492<br />
|2.98<br />
|7.71<br />
|3.22<br />
|3.33E-03<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain expressing the Biobrick [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control ''Pseudomonas putida''.<br />
<br />
If you are interested in knowing more about our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH CLICK ON THIS LINK].<br />
<br />
[[Image:TUDelftADH_final.jpg|600px|thumb|center|Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
<br />
====USEFUL LITERATURE AND REFERENCES====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:21:46Z<p>Hugo 87: /* Characterization of the long-chain alkane monooxygenase; LadA */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate. <br />
From the ratios we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. For more information about our findings [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase CLICK HERE].<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Ratio octane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Octane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.832<br />
|6.16<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control<br />
|0.821<br />
|13.3<br />
|1.34<br />
|1.51<br />
|1.23E-3<br />
|-<br />
|''E.coli'' K12 strain #1 (AH-system)<br />
|0.652<br />
|14.4<br />
|21.6<br />
|1.70<br />
|1.77E-2<br />
|-<br />
|''E.coli'' K12 strain #2 (AH-system)<br />
|0.439<br />
|3.96<br />
|47.0<br />
|1.46<br />
|4.49E-2<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
<br />
We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA CLICK ON THIS LINK]<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Average ratio hexadecane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Hexadecane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.900<br />
|3.19<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002])<br />
|0.807<br />
|3.48<br />
|1.76<br />
|4.46<br />
|5.49E-04<br />
|-<br />
|''E.coli'' K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.600<br />
|15.4<br />
|5.68<br />
|4.58<br />
|1.72E-03<br />
|-<br />
|''E.coli'' K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.532<br />
|4.25<br />
|6.95<br />
|3.18<br />
|3.03E-03<br />
|-<br />
|''E.coli'' TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027])<br />
|0.492<br />
|2.98<br />
|7.71<br />
|3.22<br />
|3.33E-03<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain expressing the Biobrick [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control ''Pseudomonas putida''.<br />
<br />
[[Image:TUDelftADH_final.jpg|600px|thumb|center|Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
<br />
If you are interested in knowing more about our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH CLICK ON THIS LINK].<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
<br />
====USEFUL LITERATURE AND REFERENCES====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:21:16Z<p>Hugo 87: /* Characterization of the alkane hydroxylase system */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate. <br />
From the ratios we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. For more information about our findings [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase CLICK HERE].<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Ratio octane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Octane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.832<br />
|6.16<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control<br />
|0.821<br />
|13.3<br />
|1.34<br />
|1.51<br />
|1.23E-3<br />
|-<br />
|''E.coli'' K12 strain #1 (AH-system)<br />
|0.652<br />
|14.4<br />
|21.6<br />
|1.70<br />
|1.77E-2<br />
|-<br />
|''E.coli'' K12 strain #2 (AH-system)<br />
|0.439<br />
|3.96<br />
|47.0<br />
|1.46<br />
|4.49E-2<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Average ratio hexadecane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Hexadecane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.900<br />
|3.19<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002])<br />
|0.807<br />
|3.48<br />
|1.76<br />
|4.46<br />
|5.49E-04<br />
|-<br />
|''E.coli'' K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.600<br />
|15.4<br />
|5.68<br />
|4.58<br />
|1.72E-03<br />
|-<br />
|''E.coli'' K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.532<br />
|4.25<br />
|6.95<br />
|3.18<br />
|3.03E-03<br />
|-<br />
|''E.coli'' TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027])<br />
|0.492<br />
|2.98<br />
|7.71<br />
|3.22<br />
|3.33E-03<br />
|}<br />
<br />
We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA CLICK ON THIS LINK]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain expressing the Biobrick [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control ''Pseudomonas putida''.<br />
<br />
[[Image:TUDelftADH_final.jpg|600px|thumb|center|Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
<br />
If you are interested in knowing more about our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH CLICK ON THIS LINK].<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
<br />
====USEFUL LITERATURE AND REFERENCES====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:20:15Z<p>Hugo 87: /* Characterization of the Alcohol DeHydrogenase (ADH) system */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Ratio octane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Octane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.832<br />
|6.16<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control<br />
|0.821<br />
|13.3<br />
|1.34<br />
|1.51<br />
|1.23E-3<br />
|-<br />
|''E.coli'' K12 strain #1 (AH-system)<br />
|0.652<br />
|14.4<br />
|21.6<br />
|1.70<br />
|1.77E-2<br />
|-<br />
|''E.coli'' K12 strain #2 (AH-system)<br />
|0.439<br />
|3.96<br />
|47.0<br />
|1.46<br />
|4.49E-2<br />
|}<br />
<br />
From the ratios we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. For more information about our findings [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase CLICK HERE].<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Average ratio hexadecane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Hexadecane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.900<br />
|3.19<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002])<br />
|0.807<br />
|3.48<br />
|1.76<br />
|4.46<br />
|5.49E-04<br />
|-<br />
|''E.coli'' K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.600<br />
|15.4<br />
|5.68<br />
|4.58<br />
|1.72E-03<br />
|-<br />
|''E.coli'' K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.532<br />
|4.25<br />
|6.95<br />
|3.18<br />
|3.03E-03<br />
|-<br />
|''E.coli'' TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027])<br />
|0.492<br />
|2.98<br />
|7.71<br />
|3.22<br />
|3.33E-03<br />
|}<br />
<br />
We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA CLICK ON THIS LINK]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain expressing the Biobrick [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control ''Pseudomonas putida''.<br />
<br />
[[Image:TUDelftADH_final.jpg|600px|thumb|center|Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
<br />
If you are interested in knowing more about our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH CLICK ON THIS LINK].<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
<br />
====USEFUL LITERATURE AND REFERENCES====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:18:20Z<p>Hugo 87: /* Characterization of the Alcohol DeHydrogenase (ADH) system */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Ratio octane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Octane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.832<br />
|6.16<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control<br />
|0.821<br />
|13.3<br />
|1.34<br />
|1.51<br />
|1.23E-3<br />
|-<br />
|''E.coli'' K12 strain #1 (AH-system)<br />
|0.652<br />
|14.4<br />
|21.6<br />
|1.70<br />
|1.77E-2<br />
|-<br />
|''E.coli'' K12 strain #2 (AH-system)<br />
|0.439<br />
|3.96<br />
|47.0<br />
|1.46<br />
|4.49E-2<br />
|}<br />
<br />
From the ratios we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. For more information about our findings [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase CLICK HERE].<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Average ratio hexadecane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Hexadecane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.900<br />
|3.19<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002])<br />
|0.807<br />
|3.48<br />
|1.76<br />
|4.46<br />
|5.49E-04<br />
|-<br />
|''E.coli'' K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.600<br />
|15.4<br />
|5.68<br />
|4.58<br />
|1.72E-03<br />
|-<br />
|''E.coli'' K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.532<br />
|4.25<br />
|6.95<br />
|3.18<br />
|3.03E-03<br />
|-<br />
|''E.coli'' TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027])<br />
|0.492<br />
|2.98<br />
|7.71<br />
|3.22<br />
|3.33E-03<br />
|}<br />
<br />
We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA CLICK ON THIS LINK]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain 018A has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control ''Pseudomonas putida''.<br />
<br />
[[Image:TUDelftADH_final.jpg|600px|thumb|center|Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
<br />
====USEFUL LITERATURE AND REFERENCES====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:13:49Z<p>Hugo 87: /* Characterization of the long-chain alkane monooxygenase; LadA */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Ratio octane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Octane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.832<br />
|6.16<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control<br />
|0.821<br />
|13.3<br />
|1.34<br />
|1.51<br />
|1.23E-3<br />
|-<br />
|''E.coli'' K12 strain #1 (AH-system)<br />
|0.652<br />
|14.4<br />
|21.6<br />
|1.70<br />
|1.77E-2<br />
|-<br />
|''E.coli'' K12 strain #2 (AH-system)<br />
|0.439<br />
|3.96<br />
|47.0<br />
|1.46<br />
|4.49E-2<br />
|}<br />
<br />
From the ratios we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. For more information about our findings [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase CLICK HERE].<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Average ratio hexadecane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Hexadecane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.900<br />
|3.19<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002])<br />
|0.807<br />
|3.48<br />
|1.76<br />
|4.46<br />
|5.49E-04<br />
|-<br />
|''E.coli'' K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.600<br />
|15.4<br />
|5.68<br />
|4.58<br />
|1.72E-03<br />
|-<br />
|''E.coli'' K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.532<br />
|4.25<br />
|6.95<br />
|3.18<br />
|3.03E-03<br />
|-<br />
|''E.coli'' TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027])<br />
|0.492<br />
|2.98<br />
|7.71<br />
|3.22<br />
|3.33E-03<br />
|}<br />
<br />
We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA CLICK ON THIS LINK]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
<br />
====USEFUL LITERATURE AND REFERENCES====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:08:53Z<p>Hugo 87: /* Characterization of the alkane hydroxylase system */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Ratio octane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Octane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.832<br />
|6.16<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control<br />
|0.821<br />
|13.3<br />
|1.34<br />
|1.51<br />
|1.23E-3<br />
|-<br />
|''E.coli'' K12 strain #1 (AH-system)<br />
|0.652<br />
|14.4<br />
|21.6<br />
|1.70<br />
|1.77E-2<br />
|-<br />
|''E.coli'' K12 strain #2 (AH-system)<br />
|0.439<br />
|3.96<br />
|47.0<br />
|1.46<br />
|4.49E-2<br />
|}<br />
<br />
From the ratios we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. For more information about our findings [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase CLICK HERE].<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
<br />
====USEFUL LITERATURE AND REFERENCES====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:07:00Z<p>Hugo 87: /* Characterization of the alkane hydroxylase system */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
We've succeeded in characterizing the activity of the alkane hydroxylase system using gas chromatography. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. For more information about our findings [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase CLICK HERE].<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
<br />
====USEFUL LITERATURE AND REFERENCES====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Hugo 87http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:03:35Z<p>Hugo 87: /* Characterization of the long-chain alkane monooxygenase; LadA */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
<br />
====USEFUL LITERATURE AND REFERENCES====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Hugo 87