http://2009.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=100&target=Wilfred&year=&month=2009.igem.org - User contributions [en]2024-03-28T19:03:07ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:Groningen/TeamTeam:Groningen/Team2010-02-01T11:01:01Z<p>Wilfred: /* Where to meet us */</p>
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<div>{{Team:Groningen/Header}}<br />
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<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Future}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Pictures}}</div><br />
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[[Category:Team:Groningen/Disciplines/Project_Management|Team]]<br />
[[Category:Team:Groningen/Roles/Project_Manager|Team]]<br />
<br />
==Our Team At A Glance==<br />
<br />
[[Image:IGEMGroningen_Molen.jpg|400px|thumb|right|[Team:Groningen/Team|Our team!]]<br />
<br />
Welcome to the website of the iGEM Groningen team! We are an interdisciplinary team of 11 enthusiastic students from the [http://www.rug.nl/ University of Groningen] situated in the not-too-big city of [http://portal.groningen.nl/en/startpagina Groningen] in [http://maps.google.com/maps?f=q&source=s_q&hl=en&geocode=&q=Groningen&sll=53.281349,6.689459&sspn=0.007261,0.018926&ie=UTF8&z=12&iwloc=A the north of the Netherlands]. You can contact us by '''[mailto:igemgroningen2009@googlegroups.com mail]'''. <br />
<br />
Our team consists of the following student-members:<br />
<br />
* [[User:JolandaWitteveen|Jolanda Witteveen]] (Biomedical Technology): [[:Category:Team:Groningen/Roles/Chair|Chair]], [[:Category:Team:Groningen/Roles/Project_Manager|Project Manager]]<br />
* [[User:svenjurgens|Sven Jurgens]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [[User:Jaspervdg|Jasper van de Gronde]] (Computational Science and Visualization): [[:Category:Team:Groningen/Roles/Configuration_Manager|Configuration Manager]], [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Verhoeven1981|Michael Verhoeven]] (Chemistry): [[:Category:Team:Groningen/Roles/Public_Relations_Officer|PR Officer]]<br />
* [https://2009.igem.org/User:Nienke Nienke Kuipers] (Molecular Biology): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]] and Lab manager<br />
* [[User:Jelle|Steven Jelle Meijer]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Facility_Manager|Facility Manager Haren]]<br />
* [[User:Wilfred|Wilfred Poppinga]] (Medical Pharmaceutical Sciences): [[:Category:Team:Groningen/Roles/Chair|Vice Chair]], [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [https://2009.igem.org/User:Paulschavemaker Paul Schavemaker] (Molecular Life Sciences): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]]<br />
* [https://2009.igem.org/User:Frans Frans Bianchi] (Molecular Biology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Klaas Bernd Over|Klaas Bernd Over]] (Applied Physics): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Annelies|Annelies van Keulen]] (Molecular Biology/Psychology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
<br />
==Our advisors==<br />
*prof. dr. Oscar Kuipers: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics] (Head)<br />
*prof. dr. Jan Kok: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics]<br />
*prof. dr. Bert Poolman: Biochemistry; [http://www.centreforsyntheticbiology.eu/ Centre for Synthetic Biology] (Director)<br />
*prof. dr. Roel Bovenberg: Synthetic biology and Cell engineering; Corporate Scientist Biotechnology, [http://www.dsm.com/ DSM]<br />
*dr. Dirk Slotboom: Enzymology <br />
*[https://2008.igem.org/Team:Groningen/team.html iGEM Groningen 2008]. Especially Auke van Heel & Martijn Herber<br />
<br />
<br><br><br />
<br />
==Where to hear from us==<br />
===In the media===<br />
Follow us in '''[[Team:Groningen/Publicity| The News]]'''<br />
<br />
Also follow us on '''[http://twitter.com/igemgroningen Twitter]!'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Videos|Videos]]'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Pictures|Pictures]]'''<br />
<br />
==Where to meet us==<br />
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*December 15<sup>th</sup> 2009: Short presentation @ [http://studium.hosting.rug.nl/Studiumgenerale/start.htm Studium Generale] - Groningen<br />
*December 11<sup>th</sup> 2009: Meeting @ [http://www.dsm.com/en_US/html/home/dsm_home.cgi DSM] - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=delft+DSM&fb=1&gl=nl&hq=DSM&hnear=delft&cid=0,0,8723601113946313921&ei=B_jSSrqHIcTz-QbZsNT7Ag&sa=X&oi=local_result&ct=image&resnum=1&ved=0CAoQnwIwAA Delft]<br />
*November 23<sup>rd</sup> 2009: Meeting @ student societies for [http://www.chemische-binding.nl/ Chemistry] and [http://www.fmf.nl/?file=main.html&lang=.en Math, Physics, Computer Science and Astronomy]<br />
*<b>October 30<sup>th</sup> to November 2<sup>nd</sup> 2009: Presentation @ The [https://2009.igem.org/ iGEM] 2009 [https://2009.igem.org/Jamboree Jamboree] - [http://whereis-beta.mit.edu/?mapterms=stata%20center&zoom=15&lat=42.36161990569666&lng=-71.09055519104004&open=object-32 MIT Stata] in Cambridge, MA</b><br />
*October 26<sup>th</sup> 2009: Lecture @ [http://www.hanzeuniversity.eu/home/international Hanze University], Biology & Medical Laboratory Research and Bioinformatics students - room A257 [http://maps.google.nl/maps?q=Zernikeplein+7+Groningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Zernikeplein+7,+9747+Groningen&gl=nl&ei=wwHPSor9A4OF-QaTkL2FAw&sa=X&oi=geocode_result&ct=title&resnum=1 Zernikeplein 11, Groningen] <br />
*October 28<sup>th</sup> 2009: Update Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Haren]<br />
*October 19<sup>th</sup> 2009: [http://www.cs.rug.nl/~biehl/Coll/index.html Colloquium] @ [http://www.rug.nl/informatica/index Institute for Mathematics and Computing Science] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=jGw&resnum=0&q=bernoulliborg%20Groningen%20Nijenborgh%209&um=1&ie=UTF-8&sa=N&tab=wl room 5161.0267 (Bernoulliborg), Groningen]<br />
*October 12<sup>th</sup> 2009: Meeting @ Marine Biology cluster - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 7<sup>th</sup> 2009: Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 2<sup>nd</sup> 2009: Lunch meeting @ [http://www2.dhv.com/default.aspx DHV] - [http://maps.google.com/maps?f=q&source=s_q&hl=nl&geocode=&q=Laan+1914+no+35,+Amersfoort&sll=37.0625,-95.677068&sspn=54.357317,79.013672&ie=UTF8&hq=&hnear=Laan+1914+35,+3818+Amersfoort,+Utrecht,+Nederland&ll=52.134107,5.36828&spn=0.010405,0.01929&t=h&z=16&iwloc=r3 Groene zaal DHV, Amersfoort]<br />
* October 1<sup>st</sup> 2009: Lunch meeting @ Life Science student society [http://www.glv-idun.nl/ GLV Idun] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Groene Zaal, Haren]<br />
*September 29<sup>th</sup> 2009: Meeting @ Applied physics student society [http://www.professorfrancken.nl/ TFV Professor Francken] - [http://maps.google.nl/maps?q=Nijenborgh%204%20NCC%20Complex&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&hl=nl&ie=UTF-8&sa=N&tab=vl NCC complex VIP Room building 16, Groningen]<br />
*[https://2009.igem.org/Team:Groningen/Notebook/24_September_2009 September 24<sup>th</sup> 2009]: Presentation @ 2nd Programme Day of the [http://www.kluyvercentre.nl/ Kluyver Centre] - [http://maps.google.nl/maps?q=Generaal+Foulkesweg+96+6703+DS+Wageningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Generaal+Foulkesweg+96,+6703+Wageningen&gl=nl&ei=giXDSvfgDcrI-Qa53ojvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Wageningse Berg, Wageningen]<br />
*September 11<sup>th</sup> 2009: Presentation @ [http://www.rug.nl/gbb/studyatgbb/generalcourses/gbbsymposium2009 17th Annual] [http://www.rug.nl/gbb/index GBB] Symposium 2009 - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=Hampshire+hotel+Groningen+Radesingel+50,+9711+EK+Groningen&fb=1&gl=nl&hq=Hampshire+hotel&hnear=Groningen+Radesingel+50,+9711+EK+Groningen&cid=0,0,5400363645623663183&ei=eybDSq-jNojj-Qbz1PXuCw&sa=X&oi=local_result&ct=image&resnum=1 Hampshire hotel, Groningen]</div><br />
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{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/TeamTeam:Groningen/Team2010-01-26T12:34:22Z<p>Wilfred: /* Where to meet us */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Future}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Pictures}}</div><br />
<br />
[[Category:Team:Groningen/Disciplines/Project_Management|Team]]<br />
[[Category:Team:Groningen/Roles/Project_Manager|Team]]<br />
<br />
==Our Team At A Glance==<br />
<br />
[[Image:IGEMGroningen_Molen.jpg|400px|thumb|right|[Team:Groningen/Team|Our team!]]<br />
<br />
Welcome to the website of the iGEM Groningen team! We are an interdisciplinary team of 11 enthusiastic students from the [http://www.rug.nl/ University of Groningen] situated in the not-too-big city of [http://portal.groningen.nl/en/startpagina Groningen] in [http://maps.google.com/maps?f=q&source=s_q&hl=en&geocode=&q=Groningen&sll=53.281349,6.689459&sspn=0.007261,0.018926&ie=UTF8&z=12&iwloc=A the north of the Netherlands]. You can contact us by '''[mailto:igemgroningen2009@googlegroups.com mail]'''. <br />
<br />
Our team consists of the following student-members:<br />
<br />
* [[User:JolandaWitteveen|Jolanda Witteveen]] (Biomedical Technology): [[:Category:Team:Groningen/Roles/Chair|Chair]], [[:Category:Team:Groningen/Roles/Project_Manager|Project Manager]]<br />
* [[User:svenjurgens|Sven Jurgens]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [[User:Jaspervdg|Jasper van de Gronde]] (Computational Science and Visualization): [[:Category:Team:Groningen/Roles/Configuration_Manager|Configuration Manager]], [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Verhoeven1981|Michael Verhoeven]] (Chemistry): [[:Category:Team:Groningen/Roles/Public_Relations_Officer|PR Officer]]<br />
* [https://2009.igem.org/User:Nienke Nienke Kuipers] (Molecular Biology): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]] and Lab manager<br />
* [[User:Jelle|Steven Jelle Meijer]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Facility_Manager|Facility Manager Haren]]<br />
* [[User:Wilfred|Wilfred Poppinga]] (Medical Pharmaceutical Sciences): [[:Category:Team:Groningen/Roles/Chair|Vice Chair]], [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [https://2009.igem.org/User:Paulschavemaker Paul Schavemaker] (Molecular Life Sciences): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]]<br />
* [https://2009.igem.org/User:Frans Frans Bianchi] (Molecular Biology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Klaas Bernd Over|Klaas Bernd Over]] (Applied Physics): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Annelies|Annelies van Keulen]] (Molecular Biology/Psychology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
<br />
==Our advisors==<br />
*prof. dr. Oscar Kuipers: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics] (Head)<br />
*prof. dr. Jan Kok: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics]<br />
*prof. dr. Bert Poolman: Biochemistry; [http://www.centreforsyntheticbiology.eu/ Centre for Synthetic Biology] (Director)<br />
*prof. dr. Roel Bovenberg: Synthetic biology and Cell engineering; Corporate Scientist Biotechnology, [http://www.dsm.com/ DSM]<br />
*dr. Dirk Slotboom: Enzymology <br />
*[https://2008.igem.org/Team:Groningen/team.html iGEM Groningen 2008]. Especially Auke van Heel & Martijn Herber<br />
<br />
<br><br><br />
<br />
==Where to hear from us==<br />
===In the media===<br />
Follow us in '''[[Team:Groningen/Publicity| The News]]'''<br />
<br />
Also follow us on '''[http://twitter.com/igemgroningen Twitter]!'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Videos|Videos]]'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Pictures|Pictures]]'''<br />
<br />
==Where to meet us==<br />
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*{{todo}} March 11 & 12, 2010: [http://www.nbc-congress.nl/ 13<sup>th</sup> Netherlands Biotechnology Congress (NBC) ‘Biotechnology for a sustainable society’] @ [http://maps.google.nl/maps?f=q&source=s_q&hl=nl&geocode=&q=De+Reehorst,+Bennekomseweg+24,+6717+LM+Ede,+The+Netherlands&sll=51.997353,5.717354&sspn=0.113935,0.308647&ie=UTF8&hq=De+Reehorst,&hnear=Bennekomseweg+24,+6717+Ede&ll=52.024944,5.671906&spn=0.007117,0.01929&z=16&iwloc=A De Reehorst, Ede]<br />
*December 15<sup>th</sup> 2009: Short presentation @ [http://studium.hosting.rug.nl/Studiumgenerale/start.htm Studium Generale] - Groningen<br />
*December 11<sup>th</sup> 2009: Meeting @ [http://www.dsm.com/en_US/html/home/dsm_home.cgi DSM] - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=delft+DSM&fb=1&gl=nl&hq=DSM&hnear=delft&cid=0,0,8723601113946313921&ei=B_jSSrqHIcTz-QbZsNT7Ag&sa=X&oi=local_result&ct=image&resnum=1&ved=0CAoQnwIwAA Delft]<br />
*November 23<sup>rd</sup> 2009: Meeting @ student societies for [http://www.chemische-binding.nl/ Chemistry] and [http://www.fmf.nl/?file=main.html&lang=.en Math, Physics, Computer Science and Astronomy]<br />
*<b>October 30<sup>th</sup> to November 2<sup>nd</sup> 2009: Presentation @ The [https://2009.igem.org/ iGEM] 2009 [https://2009.igem.org/Jamboree Jamboree] - [http://whereis-beta.mit.edu/?mapterms=stata%20center&zoom=15&lat=42.36161990569666&lng=-71.09055519104004&open=object-32 MIT Stata] in Cambridge, MA</b><br />
*October 26<sup>th</sup> 2009: Lecture @ [http://www.hanzeuniversity.eu/home/international Hanze University], Biology & Medical Laboratory Research and Bioinformatics students - room A257 [http://maps.google.nl/maps?q=Zernikeplein+7+Groningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Zernikeplein+7,+9747+Groningen&gl=nl&ei=wwHPSor9A4OF-QaTkL2FAw&sa=X&oi=geocode_result&ct=title&resnum=1 Zernikeplein 11, Groningen] <br />
*October 28<sup>th</sup> 2009: Update Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Haren]<br />
*October 19<sup>th</sup> 2009: [http://www.cs.rug.nl/~biehl/Coll/index.html Colloquium] @ [http://www.rug.nl/informatica/index Institute for Mathematics and Computing Science] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=jGw&resnum=0&q=bernoulliborg%20Groningen%20Nijenborgh%209&um=1&ie=UTF-8&sa=N&tab=wl room 5161.0267 (Bernoulliborg), Groningen]<br />
*October 12<sup>th</sup> 2009: Meeting @ Marine Biology cluster - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 7<sup>th</sup> 2009: Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 2<sup>nd</sup> 2009: Lunch meeting @ [http://www2.dhv.com/default.aspx DHV] - [http://maps.google.com/maps?f=q&source=s_q&hl=nl&geocode=&q=Laan+1914+no+35,+Amersfoort&sll=37.0625,-95.677068&sspn=54.357317,79.013672&ie=UTF8&hq=&hnear=Laan+1914+35,+3818+Amersfoort,+Utrecht,+Nederland&ll=52.134107,5.36828&spn=0.010405,0.01929&t=h&z=16&iwloc=r3 Groene zaal DHV, Amersfoort]<br />
* October 1<sup>st</sup> 2009: Lunch meeting @ Life Science student society [http://www.glv-idun.nl/ GLV Idun] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Groene Zaal, Haren]<br />
*September 29<sup>th</sup> 2009: Meeting @ Applied physics student society [http://www.professorfrancken.nl/ TFV Professor Francken] - [http://maps.google.nl/maps?q=Nijenborgh%204%20NCC%20Complex&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&hl=nl&ie=UTF-8&sa=N&tab=vl NCC complex VIP Room building 16, Groningen]<br />
*[https://2009.igem.org/Team:Groningen/Notebook/24_September_2009 September 24<sup>th</sup> 2009]: Presentation @ 2nd Programme Day of the [http://www.kluyvercentre.nl/ Kluyver Centre] - [http://maps.google.nl/maps?q=Generaal+Foulkesweg+96+6703+DS+Wageningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Generaal+Foulkesweg+96,+6703+Wageningen&gl=nl&ei=giXDSvfgDcrI-Qa53ojvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Wageningse Berg, Wageningen]<br />
*September 11<sup>th</sup> 2009: Presentation @ [http://www.rug.nl/gbb/studyatgbb/generalcourses/gbbsymposium2009 17th Annual] [http://www.rug.nl/gbb/index GBB] Symposium 2009 - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=Hampshire+hotel+Groningen+Radesingel+50,+9711+EK+Groningen&fb=1&gl=nl&hq=Hampshire+hotel&hnear=Groningen+Radesingel+50,+9711+EK+Groningen&cid=0,0,5400363645623663183&ei=eybDSq-jNojj-Qbz1PXuCw&sa=X&oi=local_result&ct=image&resnum=1 Hampshire hotel, Groningen]</div><br />
|<br />
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{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/PartsTeam:Groningen/Parts2010-01-22T09:41:53Z<p>Wilfred: Added our personal stock list</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
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<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Ethics/Survey}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right">{{linkedImage|Next.JPG|Team:Groningen/Parts/Submitted_Parts}}</div><br />
<br />
<br />
<br />
<br />
*For a comprehensive list of all the parts we used, have a look at our [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Groningen '''parts registry'''].<br />
*Here you can find an informative overview of our [[Team:Groningen/Modelling/Submitted Parts|'''submitted parts''']].<br />
*Experience we had with parts from the registry can be found under [[Team:Groningen/Modelling/Used Parts|'''used parts''']].<br />
*Need anything specific? Take a look in our [[Team:Groningen/Parts/Freezer|freezer]]<br />
<br />
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{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/AcknowledgementsTeam:Groningen/Acknowledgements2009-12-18T16:18:57Z<p>Wilfred: /* Thanks to whom it belongs */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Videos}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Glossary}}</div><br />
<br />
==Thanks to whom it belongs==<br />
<br />
For all the advice, help with getting finances, finding the right place for measurements and assistance,<br />
we would like to thank '''our supervisors''':<br />
{|<br />
|<br />
*Prof. Dr. Oscar Kuipers (head of [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics])<br />
*Prof. Dr. Jan Kok ([http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics])<br />
*Prof. Dr. Bert Poolman (head of [http://www.rug.nl/gbb/research/researchgroups/enzymology/index Membrane Enzymology] and director of [http://www.centreforsyntheticbiology.eu/ Centre for Synthetic Biology]) <br />
*Prof. Dr. Roel Bovenberg ([http://www.dsm.com/ DSM])<br />
*Dr. Dirk-Jan Slotboom ([http://www.rug.nl/gbb/research/researchgroups/enzymology/index Membrane Enzymology])<br />
*Prof. Dr. Marc van der Maarel ([http://www.micfys.fmns.rug.nl/ Microbial Physiology])<br />
*[https://2008.igem.org/Team:Groningen/team.html iGEM Groningen 2008]. Especially Auke van Heel & Martijn Herber<br />
|align="right"|<html><a href="http://molgen.biol.rug.nl"><img style="width: 200px; height: 53px" src="https://static.igem.org/mediawiki/2009/2/2f/MolGen3D.gif"/></a></html><br />
|}<br />
<br />
Other people who gave advice during the project:<br />
{|<br />
|<br />
*Prof. Nigel Brown (University of Edinburgh)<br />
*<br />
|}<br />
<br />
We also would like to thank all the people who were very important for our project, by sending us plasmid samples:<br />
{|<br />
|<br />
*Prof. Carl Nathan and Ben Gold ([http://www.cornell.edu/ Cornell University]) for sending MymT<br />
*Prof. Nigel Robinson and Kevin Waldron ([http://www.ncl.ac.uk/ Newcastle University]) for sending SmtA and SmtA-GST<br />
*Prof. Wilfred Chen ([http://www.ucr.edu/ University of California Riverside]) for sending fMT-GlpF<br />
|}<br />
We also would like to thank all the people who were very important for our project, by doing measurements:<br />
{|<br />
|Dr. Marc C.A. Stuart to make a lot of EM pictures for us ([http://www.rug.nl/gbb/research/researchgroups/electronmicroscopy/index Electron Microscopy], University of Groningen).<br />
|<html><a href="http://www.rug.nl/gbb/research/researchgroups/electronmicroscopy/index"><img style="width: 149px; height: 79px" src="https://static.igem.org/mediawiki/2009/f/fb/Gbblogo.jpg"/></a></html><br />
|-<br />
|Martijn van de Lee and Elly Wijma ([http://www.rikilt.wur.nl/NL/ RIKILT], Wageningen University) for performing ICP-MS arsenic measurements and calculations.<br />
|<html><a href="http://www.rikilt.wur.nl/NL/"><img style="width: 201px; height: 46px" src="https://static.igem.org/mediawiki/2009/a/a9/Rikilt_logo.gif"/></a></html><br />
|-<br />
|Rudi Anakotta ([http://www.vwa.nl/portal/page?_pageid=119,1639634&_dad=portal&_schema=PORTAL Food and Consumer Product Safety Authority], Groningen) for performing ICP-MS measurements.<br />
|<html><a href="http://www.vwa.nl/portal/page?_pageid=119,1639634&_dad=portal&_schema=PORTAL"><img style="width: 201px; height: 46px" src="https://static.igem.org/mediawiki/2009/4/4a/VWA-logo.gif"/></a></html><br />
|}<br />
<br />
<br />
And of course '''our sponsors''', who made this project possible:<br />
{| align="center"<br />
|''Besides being a student city, Groningen is also ahead in the areas of research, innovation and entrepreneurship: a real city of talent. In order to make this known to the rest of the Netherlands, University of Groningen, Hanze University Groningen, the UMCG University Medical Center Groningen and the Province and municipality of Groningen have joined forces. Under the common denominator of ‘Groningen, City of Talent’, in conjunction with Marketing Groningen, they have started a national campaign under the motto of: ‘Here is space for talent. Space for learning, for working and for growing. For further personal development and making the best of yourself. And there is room for living, sports and entertainment. There’s no place like Groningen.’''<br><br><br />
|<html><a id="csb" href="http://www.cityoftalent.nl/"><img style="width: 340px; height: 118px" src="https://static.igem.org/mediawiki/2009/d/db/The_city_of_Talent.jpg"/></a></html><br />
|-<br />
|The Centre for Synthetic Biology (CSB) is a multi-institution effort of the University of Groningen to forge research expertise in the emerging field of synthetic biology. The aim of the initiative is to integrate advanced knowledge within the disciplines of biomolecular sciences, synthetic chemistry, physics and mathematics to (re)design and build integrated biological and (semi-)artificial systems or materials.<br><br><br />
|<html><a id="csb" href="http://www.centreforsyntheticbiology.eu/"><img style="width: 330px; height: 49px" src="https://static.igem.org/mediawiki/igem.org/b/b0/Groningen2008_RUG_CSB.png" /></a></html><br />
|-<br />
|''The DHV Group is a global provider of consultancy and engineering services in the following markets: Transportation(including Aviation), Water, Building and Industry, Spatial Planning and Environment.''<br><br><br />
|<html><a id="DHV" href="http://www2.dhv.com/Default.aspx"><img style="width: 150px; height: 39px" src="http://www2.dhv.com/SiteImages/Over%20DHV/Logos/Logos_DHV/dhv_mk_bl.jpg"></a></html><br />
|-<br />
|Royal DSM N.V. creates innovative products and services in Life Sciences and Materials Sciences that contribute to the quality of life. DSM’s products and services are used globally in a wide range of markets and applications, supporting a healthier, more sustainable and more enjoyable way of life. End markets include human and animal nutrition and health, personal care, pharmaceuticals, automotive, coatings and paint, electrical and electronics, life protection and housing. DSM has annual net sales of EUR 9.3 billion and employs some 23,500 people worldwide. The company is headquartered in the Netherlands, with locations on five continents. DSM is listed on Euronext Amsterdam.<br><br><br />
|<html><a id="DSM" href="http://www.dsm.com"><img style="width: 150px; height: 39px" src="https://static.igem.org/mediawiki/2009/7/7c/DSM-logo.png"></a></html><br />
|-<br />
|''Bij WLN in Glimmen draait alles om water. Het gaat daarbij om water in vele verschijningsvormen, kwaliteiten en toepassingen: van drink- tot afvalwater. Wij onderzoeken, adviseren, ontwikkelen, meten, bewaken en beheren. Door middel van praktijkgerichte cursussen delen we onze kennis en ervaring bovendien met opdrachtgevers.''<br><br><br />
|<html><a id="WLN" href="http://www.wln.nl/Pages/default.aspx"><img style="width: 150px; height: 76px" src="http://www.wln.nl/Style%20Library/images/wln/logo-wln.gif"></a> </html><br />
|-<br />
|Wetsus, centre of excellence for sustainable water technology is a facilitating intermediary for trend-setting knowhow development. Wetsus creates a unique environment and strategic cooperation for development of profitable and sustainable state of the art water treatment technology. The inspiring and multidisciplinary collaboration between some 70 companies and 8 research institutes in Wetsus results in innovations that contribute significantly to the solution of the global water problems. <br><br><br />
|<html><a id="Wetsus" href="http://www.wetsus.nl/"><img src="http://www.competence-research-centres.eu/uploads/tx_extpages/Bild154.gif"></a> </html><br />
|-<br />
|''De SBGG is opgezet om nieuwe business te creëren. Wetenschappelijke uitvindingen met commerciële potentie willen we stimuleren om uit te groeien tot een nieuwe Start-Up. We richten ons vooral op life sciences. We selecteren kansrijke projecten en we stimuleren wetenschappers hun werk niet alleen te publiceren maar ook in de markt te zetten.''<br><br><br />
|-<br />
|''The Kluyver Centre for Genomics of Industrial Fermentation is one of the Centres of Excellence of the Netherlands Genomics Initiative, which pursue the highest standards in genomics research. The Kluyver Centre is a consortium of Delft University of Technology, the universities of Groningen, Leiden and Utrecht, VU University Amsterdam, Wageningen University and Research Centre, NIZO food research and TI Food and Nutrition.''<br><br><br />
|<html><a id="Kluyver" href="http://www.kluyvercentre.nl/"><img style="width: 400px; height: 118px" src="http://www.kluyvercentre.nl/header_4.gif"></a></html><br />
|-<br />
|''Het doel van de Stichting Biotechnologie Nederland (SBN) is het bevorderen van de biotechnologie in Nederland, met name voor en door jonge onderzoekers.''<br><br><br />
|<html><a id="SBN" href="http://nbv.kncv.nl/stichting-biotechnologie-nederland-(sbn).7252.lynkx"><img style="width: 150px; height: 76px" src="http://www.greenchem2007.tudelft.nl/images/SBN.jpg"></a></html><br />
|}<br />
{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/TeamTeam:Groningen/Team2009-12-16T08:37:56Z<p>Wilfred: /* Where to meet us */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Future}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Pictures}}</div><br />
<br />
[[Category:Team:Groningen/Disciplines/Project_Management|Team]]<br />
[[Category:Team:Groningen/Roles/Project_Manager|Team]]<br />
<br />
==Our Team At A Glance==<br />
<br />
[[Image:IGEMGroningen_Molen.jpg|400px|thumb|right|[Team:Groningen/Team|Our team!]]<br />
<br />
Welcome to the website of the iGEM Groningen team! We are an interdisciplinary team of 11 enthusiastic students from the [http://www.rug.nl/ University of Groningen] situated in the not-too-big city of [http://portal.groningen.nl/en/startpagina Groningen] in [http://maps.google.com/maps?f=q&source=s_q&hl=en&geocode=&q=Groningen&sll=53.281349,6.689459&sspn=0.007261,0.018926&ie=UTF8&z=12&iwloc=A the north of the Netherlands]. You can contact us by '''[mailto:igemgroningen2009@googlegroups.com mail]'''. <br />
<br />
Our team consists of the following student-members:<br />
<br />
* [[User:JolandaWitteveen|Jolanda Witteveen]] (Biomedical Technology): [[:Category:Team:Groningen/Roles/Chair|Chair]], [[:Category:Team:Groningen/Roles/Project_Manager|Project Manager]]<br />
* [[User:svenjurgens|Sven Jurgens]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [[User:Jaspervdg|Jasper van de Gronde]] (Computational Science and Visualization): [[:Category:Team:Groningen/Roles/Configuration_Manager|Configuration Manager]], [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Verhoeven1981|Michael Verhoeven]] (Chemistry): [[:Category:Team:Groningen/Roles/Public_Relations_Officer|PR Officer]]<br />
* [https://2009.igem.org/User:Nienke Nienke Kuipers] (Molecular Biology): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]] and Lab manager<br />
* [[User:Jelle|Steven Jelle Meijer]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Facility_Manager|Facility Manager Haren]]<br />
* [[User:Wilfred|Wilfred Poppinga]] (Medical Pharmaceutical Sciences): [[:Category:Team:Groningen/Roles/Chair|Vice Chair]], [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [https://2009.igem.org/User:Paulschavemaker Paul Schavemaker] (Molecular Life Sciences): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]]<br />
* [https://2009.igem.org/User:Frans Frans Bianchi] (Molecular Biology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Klaas Bernd Over|Klaas Bernd Over]] (Applied Physics): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Annelies|Annelies van Keulen]] (Molecular Biology/Psychology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
<br />
==Our advisors==<br />
*prof. dr. Oscar Kuipers: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics] (Head)<br />
*prof. dr. Jan Kok: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics]<br />
*prof. dr. Bert Poolman: Biochemistry; [http://www.centreforsyntheticbiology.eu/ Centre for Synthetic Biology] (Director)<br />
*prof. dr. Roel Bovenberg: Synthetic biology and Cell engineering; Corporate Scientist Biotechnology, [http://www.dsm.com/ DSM]<br />
*dr. Dirk Slotboom: Enzymology <br />
*[https://2008.igem.org/Team:Groningen/team.html iGEM Groningen 2008]. Especially Auke van Heel & Martijn Herber<br />
<br />
<br><br><br />
<br />
==Where to hear from us==<br />
===In the media===<br />
Follow us in '''[[Team:Groningen/Publicity| The News]]'''<br />
<br />
Also follow us on '''[http://twitter.com/igemgroningen Twitter]!'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Videos|Videos]]'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Pictures|Pictures]]'''<br />
<br />
==Where to meet us==<br />
{|<br />
|style="vertical-align:top;"|<br />
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<div class="meet"><br />
*{{todo}} March 11 & 12, 2010: [http://www.nbc-congress.nl/ 13<sup>th</sup> Netherlands Biotechnology Congress (NBC) ‘Biotechnology for a sustainable society’] @ [http://maps.google.nl/maps?f=q&source=s_q&hl=nl&geocode=&q=De+Reehorst,+Bennekomseweg+24,+6717+LM+Ede,+The+Netherlands&sll=51.997353,5.717354&sspn=0.113935,0.308647&ie=UTF8&hq=De+Reehorst,&hnear=Bennekomseweg+24,+6717+Ede&ll=52.024944,5.671906&spn=0.007117,0.01929&z=16&iwloc=A De Reehorst, Ede]<br />
*{{todo}} March 4-6, 2010: [http://www.ibe.org/meetings-and-events.html IBE 2010 Annual Conference] @ [http://maps.google.nl/maps?q=Hyatt+Regency+Cambridge,+MA&ie=UTF8&hl=nl&hq=Hyatt+Regency&hnear=Cambridge,+MA,+USA&ll=42.358671,-71.105232&spn=0.071542,0.154324&z=13&iwloc=A Hyatt Regency Cambridge, MA] <{{todo|TO BE DETERMINED}}><br />
*December 15<sup>th</sup> 2009: Short presentation @ [http://studium.hosting.rug.nl/Studiumgenerale/start.htm Studium Generale] - Groningen<br />
*December 11<sup>th</sup> 2009: Meeting @ [http://www.dsm.com/en_US/html/home/dsm_home.cgi DSM] - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=delft+DSM&fb=1&gl=nl&hq=DSM&hnear=delft&cid=0,0,8723601113946313921&ei=B_jSSrqHIcTz-QbZsNT7Ag&sa=X&oi=local_result&ct=image&resnum=1&ved=0CAoQnwIwAA Delft]<br />
*November 23<sup>rd</sup> 2009: Meeting @ student societies for [http://www.chemische-binding.nl/ Chemistry] and [http://www.fmf.nl/?file=main.html&lang=.en Math, Physics, Computer Science and Astronomy]<br />
*<b>October 30<sup>th</sup> to November 2<sup>nd</sup> 2009: Presentation @ The [https://2009.igem.org/ iGEM] 2009 [https://2009.igem.org/Jamboree Jamboree] - [http://whereis-beta.mit.edu/?mapterms=stata%20center&zoom=15&lat=42.36161990569666&lng=-71.09055519104004&open=object-32 MIT Stata] in Cambridge, MA</b><br />
*October 26<sup>th</sup> 2009: Lecture @ [http://www.hanzeuniversity.eu/home/international Hanze University], Biology & Medical Laboratory Research and Bioinformatics students - room A257 [http://maps.google.nl/maps?q=Zernikeplein+7+Groningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Zernikeplein+7,+9747+Groningen&gl=nl&ei=wwHPSor9A4OF-QaTkL2FAw&sa=X&oi=geocode_result&ct=title&resnum=1 Zernikeplein 11, Groningen] <br />
*October 28<sup>th</sup> 2009: Update Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Haren]<br />
*October 19<sup>th</sup> 2009: [http://www.cs.rug.nl/~biehl/Coll/index.html Colloquium] @ [http://www.rug.nl/informatica/index Institute for Mathematics and Computing Science] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=jGw&resnum=0&q=bernoulliborg%20Groningen%20Nijenborgh%209&um=1&ie=UTF-8&sa=N&tab=wl room 5161.0267 (Bernoulliborg), Groningen]<br />
*October 12<sup>th</sup> 2009: Meeting @ Marine Biology cluster - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 7<sup>th</sup> 2009: Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 2<sup>nd</sup> 2009: Lunch meeting @ [http://www2.dhv.com/default.aspx DHV] - [http://maps.google.com/maps?f=q&source=s_q&hl=nl&geocode=&q=Laan+1914+no+35,+Amersfoort&sll=37.0625,-95.677068&sspn=54.357317,79.013672&ie=UTF8&hq=&hnear=Laan+1914+35,+3818+Amersfoort,+Utrecht,+Nederland&ll=52.134107,5.36828&spn=0.010405,0.01929&t=h&z=16&iwloc=r3 Groene zaal DHV, Amersfoort]<br />
* October 1<sup>st</sup> 2009: Lunch meeting @ Life Science student society [http://www.glv-idun.nl/ GLV Idun] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Groene Zaal, Haren]<br />
*September 29<sup>th</sup> 2009: Meeting @ Applied physics student society [http://www.professorfrancken.nl/ TFV Professor Francken] - [http://maps.google.nl/maps?q=Nijenborgh%204%20NCC%20Complex&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&hl=nl&ie=UTF-8&sa=N&tab=vl NCC complex VIP Room building 16, Groningen]<br />
*[https://2009.igem.org/Team:Groningen/Notebook/24_September_2009 September 24<sup>th</sup> 2009]: Presentation @ 2nd Programme Day of the [http://www.kluyvercentre.nl/ Kluyver Centre] - [http://maps.google.nl/maps?q=Generaal+Foulkesweg+96+6703+DS+Wageningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Generaal+Foulkesweg+96,+6703+Wageningen&gl=nl&ei=giXDSvfgDcrI-Qa53ojvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Wageningse Berg, Wageningen]<br />
*September 11<sup>th</sup> 2009: Presentation @ [http://www.rug.nl/gbb/studyatgbb/generalcourses/gbbsymposium2009 17th Annual] [http://www.rug.nl/gbb/index GBB] Symposium 2009 - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=Hampshire+hotel+Groningen+Radesingel+50,+9711+EK+Groningen&fb=1&gl=nl&hq=Hampshire+hotel&hnear=Groningen+Radesingel+50,+9711+EK+Groningen&cid=0,0,5400363645623663183&ei=eybDSq-jNojj-Qbz1PXuCw&sa=X&oi=local_result&ct=image&resnum=1 Hampshire hotel, Groningen]</div><br />
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{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/TeamTeam:Groningen/Team2009-12-16T08:29:40Z<p>Wilfred: /* Where to meet us */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Future}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Pictures}}</div><br />
<br />
[[Category:Team:Groningen/Disciplines/Project_Management|Team]]<br />
[[Category:Team:Groningen/Roles/Project_Manager|Team]]<br />
<br />
==Our Team At A Glance==<br />
<br />
[[Image:IGEMGroningen_Molen.jpg|400px|thumb|right|[Team:Groningen/Team|Our team!]]<br />
<br />
Welcome to the website of the iGEM Groningen team! We are an interdisciplinary team of 11 enthusiastic students from the [http://www.rug.nl/ University of Groningen] situated in the not-too-big city of [http://portal.groningen.nl/en/startpagina Groningen] in [http://maps.google.com/maps?f=q&source=s_q&hl=en&geocode=&q=Groningen&sll=53.281349,6.689459&sspn=0.007261,0.018926&ie=UTF8&z=12&iwloc=A the north of the Netherlands]. You can contact us by '''[mailto:igemgroningen2009@googlegroups.com mail]'''. <br />
<br />
Our team consists of the following student-members:<br />
<br />
* [[User:JolandaWitteveen|Jolanda Witteveen]] (Biomedical Technology): [[:Category:Team:Groningen/Roles/Chair|Chair]], [[:Category:Team:Groningen/Roles/Project_Manager|Project Manager]]<br />
* [[User:svenjurgens|Sven Jurgens]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [[User:Jaspervdg|Jasper van de Gronde]] (Computational Science and Visualization): [[:Category:Team:Groningen/Roles/Configuration_Manager|Configuration Manager]], [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Verhoeven1981|Michael Verhoeven]] (Chemistry): [[:Category:Team:Groningen/Roles/Public_Relations_Officer|PR Officer]]<br />
* [https://2009.igem.org/User:Nienke Nienke Kuipers] (Molecular Biology): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]] and Lab manager<br />
* [[User:Jelle|Steven Jelle Meijer]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Facility_Manager|Facility Manager Haren]]<br />
* [[User:Wilfred|Wilfred Poppinga]] (Medical Pharmaceutical Sciences): [[:Category:Team:Groningen/Roles/Chair|Vice Chair]], [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [https://2009.igem.org/User:Paulschavemaker Paul Schavemaker] (Molecular Life Sciences): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]]<br />
* [https://2009.igem.org/User:Frans Frans Bianchi] (Molecular Biology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Klaas Bernd Over|Klaas Bernd Over]] (Applied Physics): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Annelies|Annelies van Keulen]] (Molecular Biology/Psychology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
<br />
==Our advisors==<br />
*prof. dr. Oscar Kuipers: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics] (Head)<br />
*prof. dr. Jan Kok: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics]<br />
*prof. dr. Bert Poolman: Biochemistry; [http://www.centreforsyntheticbiology.eu/ Centre for Synthetic Biology] (Director)<br />
*prof. dr. Roel Bovenberg: Synthetic biology and Cell engineering; Corporate Scientist Biotechnology, [http://www.dsm.com/ DSM]<br />
*dr. Dirk Slotboom: Enzymology <br />
*[https://2008.igem.org/Team:Groningen/team.html iGEM Groningen 2008]. Especially Auke van Heel & Martijn Herber<br />
<br />
<br><br><br />
<br />
==Where to hear from us==<br />
===In the media===<br />
Follow us in '''[[Team:Groningen/Publicity| The News]]'''<br />
<br />
Also follow us on '''[http://twitter.com/igemgroningen Twitter]!'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Videos|Videos]]'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Pictures|Pictures]]'''<br />
<br />
==Where to meet us==<br />
{|<br />
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*{{todo}} March 11 & 12, 2010: [http://www.nbc-congress.nl/ 13<sup>th</sup> Netherlands Biotechnology Congress (NBC)‘Biotechnology for a sustainable society’] @ [http://maps.google.nl/maps?f=q&source=s_q&hl=nl&geocode=&q=De+Reehorst,+Bennekomseweg+24,+6717+LM+Ede,+The+Netherlands&sll=51.997353,5.717354&sspn=0.113935,0.308647&ie=UTF8&hq=De+Reehorst,&hnear=Bennekomseweg+24,+6717+Ede&ll=52.024944,5.671906&spn=0.007117,0.01929&z=16&iwloc=A De Reehorst, Ede]<br />
*{{todo}} March 4-6, 2010: [http://www.ibe.org/meetings-and-events.html IBE 2010 Annual Conference] @ [http://maps.google.nl/maps?q=Hyatt+Regency+Cambridge,+MA&ie=UTF8&hl=nl&hq=Hyatt+Regency&hnear=Cambridge,+MA,+USA&ll=42.358671,-71.105232&spn=0.071542,0.154324&z=13&iwloc=A Hyatt Regency Cambridge, MA] <{{todo|TO BE DETERMINED}}><br />
*December 15<sup>th</sup> 2009: Short presentation @ [http://studium.hosting.rug.nl/Studiumgenerale/start.htm Studium Generale] - Groningen<br />
*December 11<sup>th</sup> 2009: Meeting @ [http://www.dsm.com/en_US/html/home/dsm_home.cgi DSM] - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=delft+DSM&fb=1&gl=nl&hq=DSM&hnear=delft&cid=0,0,8723601113946313921&ei=B_jSSrqHIcTz-QbZsNT7Ag&sa=X&oi=local_result&ct=image&resnum=1&ved=0CAoQnwIwAA Delft]<br />
*November 23<sup>rd</sup> 2009: Meeting @ student societies for [http://www.chemische-binding.nl/ Chemistry] and [http://www.fmf.nl/?file=main.html&lang=.en Math, Physics, Computer Science and Astronomy]<br />
*<b>October 30<sup>th</sup> to November 2<sup>nd</sup> 2009: Presentation @ The [https://2009.igem.org/ iGEM] 2009 [https://2009.igem.org/Jamboree Jamboree] - [http://whereis-beta.mit.edu/?mapterms=stata%20center&zoom=15&lat=42.36161990569666&lng=-71.09055519104004&open=object-32 MIT Stata] in Cambridge, MA</b><br />
*October 26<sup>th</sup> 2009: Lecture @ [http://www.hanzeuniversity.eu/home/international Hanze University], Biology & Medical Laboratory Research and Bioinformatics students - room A257 [http://maps.google.nl/maps?q=Zernikeplein+7+Groningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Zernikeplein+7,+9747+Groningen&gl=nl&ei=wwHPSor9A4OF-QaTkL2FAw&sa=X&oi=geocode_result&ct=title&resnum=1 Zernikeplein 11, Groningen] <br />
*October 28<sup>th</sup> 2009: Update Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Haren]<br />
*October 19<sup>th</sup> 2009: [http://www.cs.rug.nl/~biehl/Coll/index.html Colloquium] @ [http://www.rug.nl/informatica/index Institute for Mathematics and Computing Science] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=jGw&resnum=0&q=bernoulliborg%20Groningen%20Nijenborgh%209&um=1&ie=UTF-8&sa=N&tab=wl room 5161.0267 (Bernoulliborg), Groningen]<br />
*October 12<sup>th</sup> 2009: Meeting @ Marine Biology cluster - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 7<sup>th</sup> 2009: Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 2<sup>nd</sup> 2009: Lunch meeting @ [http://www2.dhv.com/default.aspx DHV] - [http://maps.google.com/maps?f=q&source=s_q&hl=nl&geocode=&q=Laan+1914+no+35,+Amersfoort&sll=37.0625,-95.677068&sspn=54.357317,79.013672&ie=UTF8&hq=&hnear=Laan+1914+35,+3818+Amersfoort,+Utrecht,+Nederland&ll=52.134107,5.36828&spn=0.010405,0.01929&t=h&z=16&iwloc=r3 Groene zaal DHV, Amersfoort]<br />
* October 1<sup>st</sup> 2009: Lunch meeting @ Life Science student society [http://www.glv-idun.nl/ GLV Idun] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Groene Zaal, Haren]<br />
*September 29<sup>th</sup> 2009: Meeting @ Applied physics student society [http://www.professorfrancken.nl/ TFV Professor Francken] - [http://maps.google.nl/maps?q=Nijenborgh%204%20NCC%20Complex&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&hl=nl&ie=UTF-8&sa=N&tab=vl NCC complex VIP Room building 16, Groningen]<br />
*[https://2009.igem.org/Team:Groningen/Notebook/24_September_2009 September 24<sup>th</sup> 2009]: Presentation @ 2nd Programme Day of the [http://www.kluyvercentre.nl/ Kluyver Centre] - [http://maps.google.nl/maps?q=Generaal+Foulkesweg+96+6703+DS+Wageningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Generaal+Foulkesweg+96,+6703+Wageningen&gl=nl&ei=giXDSvfgDcrI-Qa53ojvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Wageningse Berg, Wageningen]<br />
*September 11<sup>th</sup> 2009: Presentation @ [http://www.rug.nl/gbb/studyatgbb/generalcourses/gbbsymposium2009 17th Annual] [http://www.rug.nl/gbb/index GBB] Symposium 2009 - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=Hampshire+hotel+Groningen+Radesingel+50,+9711+EK+Groningen&fb=1&gl=nl&hq=Hampshire+hotel&hnear=Groningen+Radesingel+50,+9711+EK+Groningen&cid=0,0,5400363645623663183&ei=eybDSq-jNojj-Qbz1PXuCw&sa=X&oi=local_result&ct=image&resnum=1 Hampshire hotel, Groningen]</div><br />
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{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/TeamTeam:Groningen/Team2009-12-16T08:29:14Z<p>Wilfred: /* Where to meet us */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Future}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Pictures}}</div><br />
<br />
[[Category:Team:Groningen/Disciplines/Project_Management|Team]]<br />
[[Category:Team:Groningen/Roles/Project_Manager|Team]]<br />
<br />
==Our Team At A Glance==<br />
<br />
[[Image:IGEMGroningen_Molen.jpg|400px|thumb|right|[Team:Groningen/Team|Our team!]]<br />
<br />
Welcome to the website of the iGEM Groningen team! We are an interdisciplinary team of 11 enthusiastic students from the [http://www.rug.nl/ University of Groningen] situated in the not-too-big city of [http://portal.groningen.nl/en/startpagina Groningen] in [http://maps.google.com/maps?f=q&source=s_q&hl=en&geocode=&q=Groningen&sll=53.281349,6.689459&sspn=0.007261,0.018926&ie=UTF8&z=12&iwloc=A the north of the Netherlands]. You can contact us by '''[mailto:igemgroningen2009@googlegroups.com mail]'''. <br />
<br />
Our team consists of the following student-members:<br />
<br />
* [[User:JolandaWitteveen|Jolanda Witteveen]] (Biomedical Technology): [[:Category:Team:Groningen/Roles/Chair|Chair]], [[:Category:Team:Groningen/Roles/Project_Manager|Project Manager]]<br />
* [[User:svenjurgens|Sven Jurgens]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [[User:Jaspervdg|Jasper van de Gronde]] (Computational Science and Visualization): [[:Category:Team:Groningen/Roles/Configuration_Manager|Configuration Manager]], [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Verhoeven1981|Michael Verhoeven]] (Chemistry): [[:Category:Team:Groningen/Roles/Public_Relations_Officer|PR Officer]]<br />
* [https://2009.igem.org/User:Nienke Nienke Kuipers] (Molecular Biology): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]] and Lab manager<br />
* [[User:Jelle|Steven Jelle Meijer]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Facility_Manager|Facility Manager Haren]]<br />
* [[User:Wilfred|Wilfred Poppinga]] (Medical Pharmaceutical Sciences): [[:Category:Team:Groningen/Roles/Chair|Vice Chair]], [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [https://2009.igem.org/User:Paulschavemaker Paul Schavemaker] (Molecular Life Sciences): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]]<br />
* [https://2009.igem.org/User:Frans Frans Bianchi] (Molecular Biology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Klaas Bernd Over|Klaas Bernd Over]] (Applied Physics): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Annelies|Annelies van Keulen]] (Molecular Biology/Psychology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
<br />
==Our advisors==<br />
*prof. dr. Oscar Kuipers: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics] (Head)<br />
*prof. dr. Jan Kok: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics]<br />
*prof. dr. Bert Poolman: Biochemistry; [http://www.centreforsyntheticbiology.eu/ Centre for Synthetic Biology] (Director)<br />
*prof. dr. Roel Bovenberg: Synthetic biology and Cell engineering; Corporate Scientist Biotechnology, [http://www.dsm.com/ DSM]<br />
*dr. Dirk Slotboom: Enzymology <br />
*[https://2008.igem.org/Team:Groningen/team.html iGEM Groningen 2008]. Especially Auke van Heel & Martijn Herber<br />
<br />
<br><br><br />
<br />
==Where to hear from us==<br />
===In the media===<br />
Follow us in '''[[Team:Groningen/Publicity| The News]]'''<br />
<br />
Also follow us on '''[http://twitter.com/igemgroningen Twitter]!'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Videos|Videos]]'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Pictures|Pictures]]'''<br />
<br />
==Where to meet us==<br />
{|<br />
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.meet h1 { font-weight:bold; font-size:medium; border:none; margin:0px !important; padding:0px;}<br />
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<div class="meet"><br />
*{{todo}} March 11 & 12, 2010: [http://www.nbc-congress.nl/ 13<sup>th</sup> Netherlands Biotechnology Congress (NBC)‘Biotechnology for a sustainable society’] @ [http://maps.google.nl/maps?f=q&source=s_q&hl=nl&geocode=&q=De+Reehorst,+Bennekomseweg+24,+6717+LM+Ede,+The+Netherlands&sll=51.997353,5.717354&sspn=0.113935,0.308647&ie=UTF8&hq=De+Reehorst,&hnear=Bennekomseweg+24,+6717+Ede&ll=52.024944,5.671906&spn=0.007117,0.01929&z=16&iwloc=A De Reehorst, Ede]<br />
*{{todo}}March 4-6, 2010: [http://www.ibe.org/meetings-and-events.html IBE 2010 Annual Conference] @ [http://maps.google.nl/maps?q=Hyatt+Regency+Cambridge,+MA&ie=UTF8&hl=nl&hq=Hyatt+Regency&hnear=Cambridge,+MA,+USA&ll=42.358671,-71.105232&spn=0.071542,0.154324&z=13&iwloc=A Hyatt Regency Cambridge, MA] <{{todo|TO BE DETERMINED}}><br />
*December 15<sup>th</sup> 2009: Short presentation @ [http://studium.hosting.rug.nl/Studiumgenerale/start.htm Studium Generale] - Groningen<br />
*December 11<sup>th</sup> 2009: Meeting @ [http://www.dsm.com/en_US/html/home/dsm_home.cgi DSM] - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=delft+DSM&fb=1&gl=nl&hq=DSM&hnear=delft&cid=0,0,8723601113946313921&ei=B_jSSrqHIcTz-QbZsNT7Ag&sa=X&oi=local_result&ct=image&resnum=1&ved=0CAoQnwIwAA Delft]<br />
*November 23<sup>rd</sup> 2009: Meeting @ student societies for [http://www.chemische-binding.nl/ Chemistry] and [http://www.fmf.nl/?file=main.html&lang=.en Math, Physics, Computer Science and Astronomy]<br />
*<b>October 30<sup>th</sup> to November 2<sup>nd</sup> 2009: Presentation @ The [https://2009.igem.org/ iGEM] 2009 [https://2009.igem.org/Jamboree Jamboree] - [http://whereis-beta.mit.edu/?mapterms=stata%20center&zoom=15&lat=42.36161990569666&lng=-71.09055519104004&open=object-32 MIT Stata] in Cambridge, MA</b><br />
*October 26<sup>th</sup> 2009: Lecture @ [http://www.hanzeuniversity.eu/home/international Hanze University], Biology & Medical Laboratory Research and Bioinformatics students - room A257 [http://maps.google.nl/maps?q=Zernikeplein+7+Groningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Zernikeplein+7,+9747+Groningen&gl=nl&ei=wwHPSor9A4OF-QaTkL2FAw&sa=X&oi=geocode_result&ct=title&resnum=1 Zernikeplein 11, Groningen] <br />
*October 28<sup>th</sup> 2009: Update Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Haren]<br />
*October 19<sup>th</sup> 2009: [http://www.cs.rug.nl/~biehl/Coll/index.html Colloquium] @ [http://www.rug.nl/informatica/index Institute for Mathematics and Computing Science] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=jGw&resnum=0&q=bernoulliborg%20Groningen%20Nijenborgh%209&um=1&ie=UTF-8&sa=N&tab=wl room 5161.0267 (Bernoulliborg), Groningen]<br />
*October 12<sup>th</sup> 2009: Meeting @ Marine Biology cluster - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 7<sup>th</sup> 2009: Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 2<sup>nd</sup> 2009: Lunch meeting @ [http://www2.dhv.com/default.aspx DHV] - [http://maps.google.com/maps?f=q&source=s_q&hl=nl&geocode=&q=Laan+1914+no+35,+Amersfoort&sll=37.0625,-95.677068&sspn=54.357317,79.013672&ie=UTF8&hq=&hnear=Laan+1914+35,+3818+Amersfoort,+Utrecht,+Nederland&ll=52.134107,5.36828&spn=0.010405,0.01929&t=h&z=16&iwloc=r3 Groene zaal DHV, Amersfoort]<br />
* October 1<sup>st</sup> 2009: Lunch meeting @ Life Science student society [http://www.glv-idun.nl/ GLV Idun] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Groene Zaal, Haren]<br />
*September 29<sup>th</sup> 2009: Meeting @ Applied physics student society [http://www.professorfrancken.nl/ TFV Professor Francken] - [http://maps.google.nl/maps?q=Nijenborgh%204%20NCC%20Complex&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&hl=nl&ie=UTF-8&sa=N&tab=vl NCC complex VIP Room building 16, Groningen]<br />
*[https://2009.igem.org/Team:Groningen/Notebook/24_September_2009 September 24<sup>th</sup> 2009]: Presentation @ 2nd Programme Day of the [http://www.kluyvercentre.nl/ Kluyver Centre] - [http://maps.google.nl/maps?q=Generaal+Foulkesweg+96+6703+DS+Wageningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Generaal+Foulkesweg+96,+6703+Wageningen&gl=nl&ei=giXDSvfgDcrI-Qa53ojvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Wageningse Berg, Wageningen]<br />
*September 11<sup>th</sup> 2009: Presentation @ [http://www.rug.nl/gbb/studyatgbb/generalcourses/gbbsymposium2009 17th Annual] [http://www.rug.nl/gbb/index GBB] Symposium 2009 - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=Hampshire+hotel+Groningen+Radesingel+50,+9711+EK+Groningen&fb=1&gl=nl&hq=Hampshire+hotel&hnear=Groningen+Radesingel+50,+9711+EK+Groningen&cid=0,0,5400363645623663183&ei=eybDSq-jNojj-Qbz1PXuCw&sa=X&oi=local_result&ct=image&resnum=1 Hampshire hotel, Groningen]</div><br />
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{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/TeamTeam:Groningen/Team2009-12-01T17:30:57Z<p>Wilfred: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Future}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Pictures}}</div><br />
<br />
[[Category:Team:Groningen/Disciplines/Project_Management|Team]]<br />
[[Category:Team:Groningen/Roles/Project_Manager|Team]]<br />
<br />
==Our Team At A Glance==<br />
<br />
[[Image:IGEMGroningen_Molen.jpg|400px|thumb|right|[Team:Groningen/Team|Our team!]]<br />
<br />
Welcome to the website of the iGEM Groningen team! We are an interdisciplinary team of 11 enthusiastic students from the [http://www.rug.nl/ University of Groningen] situated in the not-too-big city of [http://portal.groningen.nl/en/startpagina Groningen] in [http://maps.google.com/maps?f=q&source=s_q&hl=en&geocode=&q=Groningen&sll=53.281349,6.689459&sspn=0.007261,0.018926&ie=UTF8&z=12&iwloc=A the north of the Netherlands]. You can contact us by '''[mailto:igemgroningen2009@googlegroups.com mail]'''. <br />
<br />
Our team consists of the following student-members:<br />
<br />
* [[User:JolandaWitteveen|Jolanda Witteveen]] (Biomedical Technology): [[:Category:Team:Groningen/Roles/Chair|Chair]], [[:Category:Team:Groningen/Roles/Project_Manager|Project Manager]]<br />
* [[User:svenjurgens|Sven Jurgens]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [[User:Jaspervdg|Jasper van de Gronde]] (Computational Science and Visualization): [[:Category:Team:Groningen/Roles/Configuration_Manager|Configuration Manager]], [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Verhoeven1981|Michael Verhoeven]] (Chemistry): [[:Category:Team:Groningen/Roles/Public_Relations_Officer|PR Officer]]<br />
* [https://2009.igem.org/User:Nienke Nienke Kuipers] (Molecular Biology): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]] and Lab manager<br />
* [[User:Jelle|Steven Jelle Meijer]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Facility_Manager|Facility Manager Haren]]<br />
* [[User:Wilfred|Wilfred Poppinga]] (Medical Pharmaceutical Sciences): [[:Category:Team:Groningen/Roles/Chair|Vice Chair]], [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [https://2009.igem.org/User:Paulschavemaker Paul Schavemaker] (Molecular Life Sciences): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]]<br />
* [https://2009.igem.org/User:Frans Frans Bianchi] (Molecular Biology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Klaas Bernd Over|Klaas Bernd Over]] (Applied Physics): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Annelies|Annelies van Keulen]] (Molecular Biology/Psychology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
<br />
==Our advisors==<br />
*prof. dr. Oscar Kuipers: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics] (Head)<br />
*prof. dr. Jan Kok: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics]<br />
*prof. dr. Bert Poolman: Biochemistry; [http://www.centreforsyntheticbiology.eu/ Centre for Synthetic Biology] (Director)<br />
*prof. dr. Roel Bovenberg: Synthetic biology and Cell engineering; Corporate Scientist Biotechnology, [http://www.dsm.com/ DSM]<br />
*dr. Dirk Slotboom: Enzymology <br />
*[https://2008.igem.org/Team:Groningen/team.html iGEM Groningen 2008]. Especially Auke van Heel & Martijn Herber<br />
<br />
<br><br><br />
<br />
==Where to hear from us==<br />
===In the media===<br />
Follow us in '''[[Team:Groningen/Publicity| The News]]'''<br />
<br />
Also follow us on '''[http://twitter.com/igemgroningen Twitter]!'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Videos|Videos]]'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Pictures|Pictures]]'''<br />
<br />
==Where to meet us==<br />
{|<br />
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<div class="meet"><br />
*{{todo}} December 15<sup>th</sup> 2009: Short presentation @ [http://studium.hosting.rug.nl/Studiumgenerale/start.htm Studium Generale] - Groningen<br />
*{{todo}} December 11<sup>th</sup> 2009: Meeting @ [http://www.dsm.com/en_US/html/home/dsm_home.cgi DSM] - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=delft+DSM&fb=1&gl=nl&hq=DSM&hnear=delft&cid=0,0,8723601113946313921&ei=B_jSSrqHIcTz-QbZsNT7Ag&sa=X&oi=local_result&ct=image&resnum=1&ved=0CAoQnwIwAA Delft]<br />
*November 23<sup>rd</sup> 2009: Meeting @ student societies for [http://www.chemische-binding.nl/ Chemistry] and [http://www.fmf.nl/?file=main.html&lang=.en Math, Physics, Computer Science and Astronomy]<br />
*<b>October 30<sup>th</sup> to November 2<sup>nd</sup> 2009: Presentation @ The [https://2009.igem.org/ iGEM] 2009 [https://2009.igem.org/Jamboree Jamboree] - [http://whereis-beta.mit.edu/?mapterms=stata%20center&zoom=15&lat=42.36161990569666&lng=-71.09055519104004&open=object-32 MIT Stata] in Cambridge, MA</b><br />
*October 26<sup>th</sup> 2009: Lecture @ [http://www.hanzeuniversity.eu/home/international Hanze University], Biology & Medical Laboratory Research and Bioinformatics students - room A257 [http://maps.google.nl/maps?q=Zernikeplein+7+Groningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Zernikeplein+7,+9747+Groningen&gl=nl&ei=wwHPSor9A4OF-QaTkL2FAw&sa=X&oi=geocode_result&ct=title&resnum=1 Zernikeplein 11, Groningen] <br />
*October 28<sup>th</sup> 2009: Update Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Haren]<br />
*October 19<sup>th</sup> 2009: [http://www.cs.rug.nl/~biehl/Coll/index.html Colloquium] @ [http://www.rug.nl/informatica/index Institute for Mathematics and Computing Science] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=jGw&resnum=0&q=bernoulliborg%20Groningen%20Nijenborgh%209&um=1&ie=UTF-8&sa=N&tab=wl room 5161.0267 (Bernoulliborg), Groningen]<br />
*October 12<sup>th</sup> 2009: Meeting @ Marine Biology cluster - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 7<sup>th</sup> 2009: Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 2<sup>nd</sup> 2009: Lunch meeting @ [http://www2.dhv.com/default.aspx DHV] - [http://maps.google.com/maps?f=q&source=s_q&hl=nl&geocode=&q=Laan+1914+no+35,+Amersfoort&sll=37.0625,-95.677068&sspn=54.357317,79.013672&ie=UTF8&hq=&hnear=Laan+1914+35,+3818+Amersfoort,+Utrecht,+Nederland&ll=52.134107,5.36828&spn=0.010405,0.01929&t=h&z=16&iwloc=r3 Groene zaal DHV, Amersfoort]<br />
* October 1<sup>st</sup> 2009: Lunch meeting @ Life Science student society [http://www.glv-idun.nl/ GLV Idun] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Groene Zaal, Haren]<br />
*September 29<sup>th</sup> 2009: Meeting @ Applied physics student society [http://www.professorfrancken.nl/ TFV Professor Francken] - [http://maps.google.nl/maps?q=Nijenborgh%204%20NCC%20Complex&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&hl=nl&ie=UTF-8&sa=N&tab=vl NCC complex VIP Room building 16, Groningen]<br />
*[https://2009.igem.org/Team:Groningen/Notebook/24_September_2009 September 24<sup>th</sup> 2009]: Presentation @ 2nd Programme Day of the [http://www.kluyvercentre.nl/ Kluyver Centre] - [http://maps.google.nl/maps?q=Generaal+Foulkesweg+96+6703+DS+Wageningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Generaal+Foulkesweg+96,+6703+Wageningen&gl=nl&ei=giXDSvfgDcrI-Qa53ojvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Wageningse Berg, Wageningen]<br />
*September 11<sup>th</sup> 2009: Presentation @ [http://www.rug.nl/gbb/studyatgbb/generalcourses/gbbsymposium2009 17th Annual] [http://www.rug.nl/gbb/index GBB] Symposium 2009 - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=Hampshire+hotel+Groningen+Radesingel+50,+9711+EK+Groningen&fb=1&gl=nl&hq=Hampshire+hotel&hnear=Groningen+Radesingel+50,+9711+EK+Groningen&cid=0,0,5400363645623663183&ei=eybDSq-jNojj-Qbz1PXuCw&sa=X&oi=local_result&ct=image&resnum=1 Hampshire hotel, Groningen]</div><br />
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{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/TeamTeam:Groningen/Team2009-12-01T17:30:19Z<p>Wilfred: /* Our Team At A Glance */ changing e-mail adress</p>
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<div>{{Team:Groningen/Header}}<br />
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[[Category:Team:Groningen/Disciplines/Project_Management|Team]]<br />
[[Category:Team:Groningen/Roles/Project_Manager|Team]]<br />
<br />
==Our Team At A Glance==<br />
<br />
[[Image:IGEMGroningen_Molen.jpg|400px|thumb|right|[Team:Groningen/Team|Our team!]]<br />
<br />
Welcome to the website of the iGEM Groningen team! We are an interdisciplinary team of 11 enthusiastic students from the [http://www.rug.nl/ University of Groningen] situated in the not-too-big city of [http://portal.groningen.nl/en/startpagina Groningen] in [http://maps.google.com/maps?f=q&source=s_q&hl=en&geocode=&q=Groningen&sll=53.281349,6.689459&sspn=0.007261,0.018926&ie=UTF8&z=12&iwloc=A the north of the Netherlands]. You can contact us by '''[mailto:igemgroningen2009@googlegroups.com]'''. <br />
<br />
Our team consists of the following student-members:<br />
<br />
* [[User:JolandaWitteveen|Jolanda Witteveen]] (Biomedical Technology): [[:Category:Team:Groningen/Roles/Chair|Chair]], [[:Category:Team:Groningen/Roles/Project_Manager|Project Manager]]<br />
* [[User:svenjurgens|Sven Jurgens]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [[User:Jaspervdg|Jasper van de Gronde]] (Computational Science and Visualization): [[:Category:Team:Groningen/Roles/Configuration_Manager|Configuration Manager]], [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Verhoeven1981|Michael Verhoeven]] (Chemistry): [[:Category:Team:Groningen/Roles/Public_Relations_Officer|PR Officer]]<br />
* [https://2009.igem.org/User:Nienke Nienke Kuipers] (Molecular Biology): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]] and Lab manager<br />
* [[User:Jelle|Steven Jelle Meijer]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Facility_Manager|Facility Manager Haren]]<br />
* [[User:Wilfred|Wilfred Poppinga]] (Medical Pharmaceutical Sciences): [[:Category:Team:Groningen/Roles/Chair|Vice Chair]], [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [https://2009.igem.org/User:Paulschavemaker Paul Schavemaker] (Molecular Life Sciences): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]]<br />
* [https://2009.igem.org/User:Frans Frans Bianchi] (Molecular Biology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Klaas Bernd Over|Klaas Bernd Over]] (Applied Physics): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Annelies|Annelies van Keulen]] (Molecular Biology/Psychology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
<br />
==Our advisors==<br />
*prof. dr. Oscar Kuipers: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics] (Head)<br />
*prof. dr. Jan Kok: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics]<br />
*prof. dr. Bert Poolman: Biochemistry; [http://www.centreforsyntheticbiology.eu/ Centre for Synthetic Biology] (Director)<br />
*prof. dr. Roel Bovenberg: Synthetic biology and Cell engineering; Corporate Scientist Biotechnology, [http://www.dsm.com/ DSM]<br />
*dr. Dirk Slotboom: Enzymology <br />
*[https://2008.igem.org/Team:Groningen/team.html iGEM Groningen 2008]. Especially Auke van Heel & Martijn Herber<br />
<br />
<br><br><br />
<br />
==Where to hear from us==<br />
===In the media===<br />
Follow us in '''[[Team:Groningen/Publicity| The News]]'''<br />
<br />
Also follow us on '''[http://twitter.com/igemgroningen Twitter]!'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Videos|Videos]]'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Pictures|Pictures]]'''<br />
<br />
==Where to meet us==<br />
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*{{todo}} December 15<sup>th</sup> 2009: Short presentation @ [http://studium.hosting.rug.nl/Studiumgenerale/start.htm Studium Generale] - Groningen<br />
*{{todo}} December 11<sup>th</sup> 2009: Meeting @ [http://www.dsm.com/en_US/html/home/dsm_home.cgi DSM] - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=delft+DSM&fb=1&gl=nl&hq=DSM&hnear=delft&cid=0,0,8723601113946313921&ei=B_jSSrqHIcTz-QbZsNT7Ag&sa=X&oi=local_result&ct=image&resnum=1&ved=0CAoQnwIwAA Delft]<br />
*November 23<sup>rd</sup> 2009: Meeting @ student societies for [http://www.chemische-binding.nl/ Chemistry] and [http://www.fmf.nl/?file=main.html&lang=.en Math, Physics, Computer Science and Astronomy]<br />
*<b>October 30<sup>th</sup> to November 2<sup>nd</sup> 2009: Presentation @ The [https://2009.igem.org/ iGEM] 2009 [https://2009.igem.org/Jamboree Jamboree] - [http://whereis-beta.mit.edu/?mapterms=stata%20center&zoom=15&lat=42.36161990569666&lng=-71.09055519104004&open=object-32 MIT Stata] in Cambridge, MA</b><br />
*October 26<sup>th</sup> 2009: Lecture @ [http://www.hanzeuniversity.eu/home/international Hanze University], Biology & Medical Laboratory Research and Bioinformatics students - room A257 [http://maps.google.nl/maps?q=Zernikeplein+7+Groningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Zernikeplein+7,+9747+Groningen&gl=nl&ei=wwHPSor9A4OF-QaTkL2FAw&sa=X&oi=geocode_result&ct=title&resnum=1 Zernikeplein 11, Groningen] <br />
*October 28<sup>th</sup> 2009: Update Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Haren]<br />
*October 19<sup>th</sup> 2009: [http://www.cs.rug.nl/~biehl/Coll/index.html Colloquium] @ [http://www.rug.nl/informatica/index Institute for Mathematics and Computing Science] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=jGw&resnum=0&q=bernoulliborg%20Groningen%20Nijenborgh%209&um=1&ie=UTF-8&sa=N&tab=wl room 5161.0267 (Bernoulliborg), Groningen]<br />
*October 12<sup>th</sup> 2009: Meeting @ Marine Biology cluster - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 7<sup>th</sup> 2009: Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 2<sup>nd</sup> 2009: Lunch meeting @ [http://www2.dhv.com/default.aspx DHV] - [http://maps.google.com/maps?f=q&source=s_q&hl=nl&geocode=&q=Laan+1914+no+35,+Amersfoort&sll=37.0625,-95.677068&sspn=54.357317,79.013672&ie=UTF8&hq=&hnear=Laan+1914+35,+3818+Amersfoort,+Utrecht,+Nederland&ll=52.134107,5.36828&spn=0.010405,0.01929&t=h&z=16&iwloc=r3 Groene zaal DHV, Amersfoort]<br />
* October 1<sup>st</sup> 2009: Lunch meeting @ Life Science student society [http://www.glv-idun.nl/ GLV Idun] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Groene Zaal, Haren]<br />
*September 29<sup>th</sup> 2009: Meeting @ Applied physics student society [http://www.professorfrancken.nl/ TFV Professor Francken] - [http://maps.google.nl/maps?q=Nijenborgh%204%20NCC%20Complex&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&hl=nl&ie=UTF-8&sa=N&tab=vl NCC complex VIP Room building 16, Groningen]<br />
*[https://2009.igem.org/Team:Groningen/Notebook/24_September_2009 September 24<sup>th</sup> 2009]: Presentation @ 2nd Programme Day of the [http://www.kluyvercentre.nl/ Kluyver Centre] - [http://maps.google.nl/maps?q=Generaal+Foulkesweg+96+6703+DS+Wageningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Generaal+Foulkesweg+96,+6703+Wageningen&gl=nl&ei=giXDSvfgDcrI-Qa53ojvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Wageningse Berg, Wageningen]<br />
*September 11<sup>th</sup> 2009: Presentation @ [http://www.rug.nl/gbb/studyatgbb/generalcourses/gbbsymposium2009 17th Annual] [http://www.rug.nl/gbb/index GBB] Symposium 2009 - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=Hampshire+hotel+Groningen+Radesingel+50,+9711+EK+Groningen&fb=1&gl=nl&hq=Hampshire+hotel&hnear=Groningen+Radesingel+50,+9711+EK+Groningen&cid=0,0,5400363645623663183&ei=eybDSq-jNojj-Qbz1PXuCw&sa=X&oi=local_result&ct=image&resnum=1 Hampshire hotel, Groningen]</div><br />
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{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/EthicsTeam:Groningen/Ethics2009-12-01T15:41:35Z<p>Wilfred: /* Safety */</p>
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[[Category:Team:Groningen]]<br />
<br />
= Ethical issues in synthetic biology =<br />
<br />
==Introduction==<br />
Synthetic biology is a relatively new field in biology in which engineering and biology are combined. It can be defined as the engineering of biology: the synthesis of complex, biologically based (or inspired) systems, which display functions that do exist in nature [[Team:Groningen/Literature#Serrano, L2007|(Serrano, L2007)]]. There are a number of approaches to synthetic biology; bioengineering, redesigning life and creating alternative life each with their own ethical concerns [[Team:Groningen/Literature#Deplazes, A2009|(Deplazes, A2009)]], [[Team:Groningen/Literature#Chopra, PK, A2006|(Chopra, PK, A2006)]]. <br />
<br />
==Safety==<br />
An important ethical issue for all disciplines is the safety issue, what are the risks posed by synthetic biology? [[Team:Groningen/Literature#Bhutkar, A2005|Bhutkar 2005]] distinguishes three safety risks. First, the risk of negative environmental impact this includes possible side effects, of genetically modified organism (GMO) that have to perform a task outside the lab, which can affect the environment negatively. Another risk is the risk to contaminate the natural genome pool, which includes genetic exchange between a GMO and a natural organism. The third risk mentioned by Bhutkar is the run-off risk, which includes possible unforeseen effects if a GMO has no controlled lifespan outside the lab. So the safety issue concerns the implication that GMOs could have on human health and the environment [[Team:Groningen/Literature#Kelle, A2009|(Kelle, A2009)]], [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]]. Also local [[Team:Groningen/Safety|legislation]] and project specific [[Team:Groningen/Safety|safety]] risks should be considered.<br />
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==Security==<br />
Also security issues are important ethical issues for bioengineering and when defining the difference between bio-safety and bio-security the easiest discrimination is that between mistakes and that of bad intentions. Synthetic biology is a [[Team:Groningen/Glossary#Dual-use|dual-use]] technology; it can be used for good goals or misused and cause harm. Misusage of the techniques and GMO's could result, among other things, in bioweapons and with the threshold for practicing biology getting lower and lower it is creating an unprecedented security problem [[Team:Groningen/Literature#Schmidt2008|(Schmidt, 2008)]]. More and more people will have the possibility to engineer biology and without proper regulatory oversight these socalled [[Team:Groningen/Glossary#Biohacker|biohackers]] can create potential hazards. A list of selected reading about security and possible interventions can be found [http://www.synbioproject.org/topics/synbio101/bibliography/governance/ here], [http://syntheticbiology.org/SB2.0/Biosecurity_resolutions.html here] and [http://www.jcvi.org/cms/research/projects/syngen-options/publications/ here]. Screening of ordered DNA and oligonucleotides for pathogenic agents and to license certain equipment and reagents could make it more controllable. This could be achieved by governmental regulation, however self regulation by the field of synthetic biology will be even better [[Team:Groningen/Literature#Kelle, A2009|(Kelle, A2009)]], [[Team:Groningen/Literature#Samuel, GN, et al.2009|(Samuel, GN, et al.2009)]], [[Team:Groningen/Literature#Nouri, A, et al.2009|(Nouri, A, et al.2009)]], [[Team:Groningen/Literature#Nicholls, H2008|(Nicholls, H2008)]] . <br />
<br />
==Intellectual property==<br />
Another ethical issue is the intellectual property of DNA. Synthetic biology forces rethinking about what should be patentable [[Team:Groningen/Literature#Samuel, GN, et al.2009|(Samuel, GN, et al.2009)]] ? Living organisms have been patented in the past, the first being a purified form of yeast that was patented by Louis Pasteur. The US patent and trademark office (PTO) has, beside the traditional patent requirements, two addition parts, which biotechnological systems should meet in order to be patentable. First is to show that the organism has little change of developing naturally. Second is that it should be shown that natural selection works against the GMO [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]] . The iGEM organisation circumvented this question by using an opensource system in which all Biobricks are documented and freely accessible.<br />
<br />
==Playing God issue==<br />
Another ethical issue, that is most prone in the creating alternative life part of synthetic biology, is the ‘playing God’ issue. This also raises the question what is life? This is an old philosophical question in which different views exists. One is that life is a self-sustained chemical system capable of undergoing Darwinian evolution [[Team:Groningen/Literature#Cleland, CE, et al.2002|(Cleland, CE, et al.2002)]] . Another similar definition states that life is related to possessing metabolic properties, being responsive to the environment, and having the ability to replicate [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]] . Another issue which relates to this question is where a line can be drawn between an engineered machine and a living organism. A possibility to make a distinction is when a ‘value’ is assigned to GMOs. Two values can be assigned, instrumental and intrinsic. Instrumental value is being defined by the use it has for humankind, whereas the intrinsic value, independent of the instrumental value, is assigned to any organism being valuable ‘in and of itself’. The question here is: do bacteria posses an intrinsic value? Also this issue becomes more prominent when the field of synthetic biology develops towards using higher organism and even humans.<br />
<br />
==Responsibilities==<br />
What are the responsibilities of the researchers in this field concerning the above-mentioned ethical issues? First of all safety for themselves and co-workers is a responsibility for all researchers. Also these researchers should think about possible implication of the GMO outside the lab. Especially when the organism has a task to perform outside the lab. Once the implications are clear some actions should be taken to try to minimize the risk of negative effects of the GMO for the environment and human health. Another responsibility for the field of synthetic biology lies in the security issues. Self-regulation and also helping the governmental organisation by making effective governmental regulation could be considered as a responsibility of researchers in the field of synthetic biology. Participation in the public debates about synthetic biology could be important for the public opinion on synthetic biology. It should be avoided that the people outside the synthetic biology field are scared away and, without enough insights, feel that synthetic biology is unethical. Other more delicate ethical issues like the difference between life and nonlife and assigning value to the organism is a more complicated issue, how far does the responsibility of the researchers reach in this matter and who else are responsible? In scope of this question it is advisable that both researchers and students are being taught about the risks and ethical aspects of synthetic biology in order to create awareness.<br />
In line with this the question raises what the ethical responsibilities of iGEM participant are? Obviously safety in working with GMOs is important. However also the safety of the GMO should be considered, what are the implications for the environment and human health and how can the risk of possible negative effects be minimized? Security issues are not directly the responsibility of the iGEM participant however it should not be overlooked. It is the responsibility of the iGEM participant to think about the risk of dual-use for their particular project. The iGEM participant could also give more transparency to the field of synthetic biology towards the public by publication of their project plan and results on their wiki-page. It should be encourage that part of the wiki is written for non-participant and even non-scientist in order to allow the public to gain understanding in the possible applications of synthetic biology. Overall responsibility of iGEM participant lies in gaining awareness of the ethical issues concerning synthetic biology and in particular their own project. The teams should try to gain a clear understanding of the ethical issues of their own project and its application.<br />
<br />
=Ethical issues in Heavy metal scavengers with a vertical gasdrive=<br />
<br />
==Introduction==<br />
It is important to consider ethical issues when introducing a new application, especially when genetic modified organisms are involved. In our survey we included an essay question in which the respondents were asked to give their opinion on ethical isues surrouding our project. We wanted to approach the Delphi method for brainstorming in which participant can speak freely and this will open up the discussion.We used the four ethical issues as described by [[Team:Groningen/Literature#Bhutkar, A2005|(Bhutkar, A2005)]] as a guideline. From these answers, and also from the rest of the survey, it appeared that people are most concerend about the safety for the environment and human health.<br />
<br />
==Safety==<br />
Important issues that should be considerend and solved before the bouyant heavy metal scavengers can be used in application are the possible danger for the natural bacterial population, antibiotic resistance that could spread, the concentrated amounts of a heavy metal, unwanted side effects.<br />
Most of these concerns are only applicable when the bacteria end up in the natural environment. This is obviously something that should be prevented in order to keep the system controllable. Keeping the bacteria in a closed water cleaning facility is a controlable environment, however, there is the risk in traditional water cleaning facilities of overflowing. So since there is still a small risk of bacteria ending up in the natural environment this should be considered.<br />
If bacteria end up in the natural environment many risks are present. One of the most mentioned concerns is the transfer of genes from the GMO to another organism. There is no evidence that gene transfer can happen between two different organisms, however, it is possible between two bacteria of the same strain. It does not happen often, it only happens in a stressful environment [[Team:Groningen/Literature#Mindlin, SZ, et al.2002|(Mindlin, SZ, et al.2002, )]], [[Team:Groningen/Literature#Stephenson, JR, et al.1996|(Stephenson, JR, et al.1996)]] . If a GMO end up in a natural environment it could be very stressful so it is possible that genes are transferred. To prevent this it would be best if the bacterium cannot transfer genes and dies. One way to achieve this is implementing a death-gene. This gene start apoptosis and can be transcribed with a delay. So the bacterium can do its job first and after a certain time or at a certain set-of the death-gene starts apoptosis. In this way the GMO is controllable and the possible unwanted side-effects of the GMO in a natural environment or effect because of a mutation are also decreased.<br />
Another safety issue mentioned in the survey is the escape of a antibiotic resistant strain that can spread this resistant among other bacteria. To overcome this risk other selection mechanisms besides antibiotics should be used like a gene that can make glucose out of lactose.<br />
Another concern has to do with the accumulation of toxic metals. People are afraid that this high concentration of metals can poison other organisms that eat the bacterium or a bacterium with the heavy metal stays behind. In order to make sure that a bacterium that accumulates metals also floats to the surface a feedback mechanism between the accumulation device and the gas vesicle devise could be made.<br />
Yet another concern is the removal of the bacteria and the heavy metals. Some people presume that it is very costly to remove the bacteria from the water cleaning facilitation and that it would be very difficult to remove the heavy metals. The removal of the bacteria does not have to be very expensive. It can be done by using a kind of a sieve. The metals can be removed by destruction of the cells. In case of arsenic antimone can be added and the arsenide importer will export all arsenide from the cell. Another concern is how do you know if the GMOs remove enough or even all of the particular heavy metal from the water or sludge? It can be estimated how much of this particular heavy metal a bacterium can take up and a overload of bacteria should be added so you know for sure that all of that particular heavy metal is removed.<br />
Another problem is what if the amount of the heavy metal is lower than expected and therefor not enough metal is accumulated to float? In this case the death gene should make sure that after a certain amount of time or at a certain signal the bacterium starts apoptosis. <br />
There is a possibility that the bacteria do not only take up the selected heavy metal. Bacteria could take up other metals as well, like copper and zinc. These metals do no harm in the drinking water, they are even useful for humans. However the heavy metals would compete with the copper and zinc for the accumulation proteins so most copper and zinc would be removed. Another way to prevent this from happening is over saturating the bacteria with these other metals before letting them clean the water.<br />
<br />
==Security risk==<br />
Security risks are always present. If the concentrated amounts of heavy metal fall into the wrong hands it could be misused. This risk is minimal since water cleaning facilities are not open for the public and the changes are bigger, and the result more catastrophic, if someone puts toxics in the drinking water.<br />
<br />
==Playing God issue==<br />
Most people do not find modifying bacteria as itself morally incorrect. From our survey it appeared that most people do not find it a problem if these modified bacteria are used in a water or sludge cleaning application. <br />
<br />
==Intellectual property==<br />
No other concerns are of interest in this project apart from the basic question can DNA be patented. Most people find this morally incorrect, however, patenting a system or idea which uses modified bacteria should be possible in order to keep up development.<br />
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{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/IGEM_PublicityIGEM Publicity2009-11-30T19:46:32Z<p>Wilfred: /* team specific */</p>
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<div>__NOTOC__<br />
[https://2008.igem.org/IGEM_Publicity iGEM 2008 Publicity] | [http://parts.mit.edu/igem07/index.php/Media iGEM 2007 Publicity] | [http://parts.mit.edu/wiki/index.php/IGEM_News iGEM 2006 Publicity]<br />
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<span style="color:#1e90ff; font-size:175%">'''blogs </span><span style="color:#3cb371; font-size:175%">covering iGEM 2009'''</span><br />
* The [http://igem.sdu.dk/ <span style="color:#453221; font-size: 130%">'''SDU-Denmark'''</span>] team blog about their iGEM experience.<br />
* A blog about the iGEM project of the [http://aboutgmos.org/iGEM <span style="color:#453221; font-size: 130%">'''Uppsala-Sweden'''</span>] team.<br />
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====<font size=5><font color=dodgerblue>'''video/radio</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
*'''Slovenia, Heidelberg, SDU-Denmark''': [http://www.dradio.de/dlf/sendungen/forschak/1062608/ Die Biobastler von Boston], Nov 2, 2009, Deutschlandfunk radio (in German).<br />
*'''Cambridge, Imperial''': [http://www.sciencefriday.com/program/archives/200911063 Synthetic Biology Competition], Nov 6, 2009, NPR Science Friday (in English).<br />
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====<font size=5><font color=dodgerblue>'''news articles</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
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<br />
If you would like to share an article that was written about iGEM or your iGEM team, please link to it on this page. If you have multiple articles featuring your team, link to them all individually!<br />
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Post the name of your team, the title of your article, where it was featured, and provide a link to it. <br />
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''Example'':<br> <br />
'''Team Example''': ''Title of article'', Nature, [link]<br />
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====<font size=4><font color=dodgerblue>'''general'''</font></font>====<br />
*'''The Economist''': ''Biohacking: Hacking goes squishy'' Sep 3, 2009 [http://www.economist.com/search/displaystory.cfm?story_id=14299634 The Economist]<br />
*'''The Scientist''': ''Brick by Brick'' Feb 1, 2009. [http://www.the-scientist.com/article/display/55378/ The Scientist]<br />
*'''Technology Review''': ''A Genetically Engineered Rainbow of Bacteria'' Nov 03, 2009. [http://www.technologyreview.com/blog/editors/24351/ Technology Review]<br />
*'''Discovery News''': '' Bright Bacteria Wins Synthetic Biology Competition'' Nov 6, 2009. [http://blogs.discoverychannel.co.uk/discovery-news/2009/11/bright-bacteria-wins-synthetic-biology-competition.html Discovery News]<br />
*'''Wired''': '' Building new life forms at the iGEM Jamboree'' Nov 9, 2009. [http://www.wired.co.uk/news/archive/2009-11/09/building-new-life-forms-at-the-igem-jamboree.aspx Wired]<br />
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====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
*'''[[Team:IBB_Pune]]''': "<i>Bio-champs in the making</i>" (English), [http://www.punemirror.in/index.aspx?Page=article&sectname=News%20-%20City&sectid=2&contentid=200909142009091423331646a131eacc#ftr2 (Monday, September 14, 2009 at 11:33:25 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Ganiti Prakriyanmadhe Jeevanuncha Vaapar! (Use of Bacteria in mathematical devices)</i>" (Marathi), [http://beta.esakal.com/2009/09/09220618/pune-use-of-bacterias-in-maths.html(Wednesday, September 09th, 2009 AT 10:09 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Synthetic Biologiteel Bharari ("Advances in Synthetic Biology")</i>" (Marathi), [https://2009.igem.org/Team:IBB_Pune/press (Wednesday 7 October 2009)]<br />
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*'''[[Team:Groningen]]''': "<i>Students from Groningen in competition for best bacteria</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*'''[[Team:Groningen]]''': "<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Team iGEM Groningen has been awarded a Gold Medal</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen iGEM 2009 participants</i>" ([http://www.rug.nl/corporate/nieuws/archief/archief2009/persberichten/174_09?lang=en English]/[http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Dutch]), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09?lang=en Press release University Groningen (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>With a pacmanbacterium in de finals</i>" (Dutch), [http://www.uk.rug.nl/archief/jaargang39/11/12e.php UK (Paper of the Groningen University) (November 12, 2009)]<br />
*'''[[Team:Groningen]]''' & '''[[Team:TUDelft]]''': "<i>iGEM Gold for Dutch</i>" (Dutch), [http://www.bionieuws.nl/artikel.php?id=4918&zoek=iGEM Bionieuws (November 14, 2009)]<br />
*'''[[Team:Paris]]''' : "<i>Un gène éthique qui vaut de l'or</i>" Le Monde, (French), [http://www.lemonde.fr/planete/article/2009/11/27/un-gene-ethique-qui-vaut-de-l-or_1272940_3244.html (November 27, 2009)]<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Un concours organisé par le M.I.T : en route pour Boston ! </i>" (French), [http://www.supbiotech.fr/2009/09/boston-supbiotech-igem.html (September 08, 2009)]<br />
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*'''[[Team:SupBiotech-Paris]]''': "<i>Les étudiants de la biotech interpellent la biologie synthétique</i>" (French), [http://www.vivagora.org/spip.php?breve210 (October 09, 2009)]<br />
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*'''[[Team:UAB-Barcelona]]''': "<i>Un equipo de la UAB, en el concurso de biología sintética del MIT</i>" (Spanish), [http://www.uab.es/servlet/Satellite?cid=1096481466568&pagename=UABDivulga%2FPage%2FTemplatePageDetallArticleInvestigar&param1=1253860327385 (September 23, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>UAB to participate in the synthetic biology competition at MIT</i>" (English), [http://www.uab.es/servlet/Satellite/latest-news/news-detail/uab-to-participate-in-the-synthetic-biology-competition-at-mit-1096476786473.html?noticiaid=1253657853358 (September 23, 2009)]<br />
<br />
*'''[[Team:Valencia]]''': "<i>TheValencia team is awarded the Synthetic Standard Prize</i>" (Spanish), [http://www.elpais.com/articulo/sociedad/Levaduras/funcionan/pixeles/elpepuespval/20091103elpepusoc_9/Tes]<br />
<br />
*'''[[Team:Cambridge]]''': "<i>University of Cambridge team wins iGEM synthetic biology competition </i>" (English), [http://www.biotechniques.com/news/University-of-Cambridge-team-wins-iGEM-synthetic-biology-competition/biotechniques-180278.html (November 5 2009)]<br />
<br />
*'''[[Team:Virginia]]''': ''[http://www.washingtonpost.com/wp-dyn/content/article/2009/10/22/AR2009102204628.html New works of science nonfiction]'', The Washington Post (October 23, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Estafette voor bacterien'',(Dutch) Algemeen Dagblad (November 6, 2009)<br />
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*'''[[Team:TUDelft]]''': ''Bacterie-estafette'', (Dutch) [http://noorderlicht.vpro.nl/themasites/mediaplayer/index.jsp?media=42722491&refernr=42560747&portalnr=3626936&hostname=noorderlicht&mediatype=audio&portalid=noorderlicht# Podcast van Radio 1, VPRO Noorderlicht, Annemieke Smit] (November 9, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette wint goud en award'', (Dutch) [http://www.tudelft.nl/live/pagina.jsp?id=ee44be90-289e-4efe-8a12-4636f1c86b28&lang=nl TNW Today Editie 2] (November 5, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette wint goud en award'', (Dutch) [http://www.tudelft.nl/live/pagina.jsp?id=ee44be90-289e-4efe-8a12-4636f1c86b28&lang=nl TUDelft Nieuws] (November 4, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterial relay race wins gold and award'', [http://www.tudelft.nl/live/pagina.jsp?id=26f61b2e-5a3f-40be-b737-a460b08bced7&lang=en Delft University of Technology Press release] (November 4, 2009)<br />
<br />
*'''[[Team:TorontoMaRSDiscovery]]''': ''A gem of a genetics competition'', [http://www.news.utoronto.ca/science-and-technology/a-gem-of-a-genetics-competition.html University of Toronto eBulletin] (November 23, 2009)<br />
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*'''[[Team:Edinburgh]]''':"<i>Synthetic biology against land mines</i>" (Dutch), [http://www.c2w.nl/synthetische-biologie-tegen-landmijnen.72122.lynkx C<sub>2</sub>W Life Sciences (November 17 2009)]</div>Wilfredhttp://2009.igem.org/Team:Groningen/Brainstorm/Growth_ControlTeam:Groningen/Brainstorm/Growth Control2009-11-28T13:39:42Z<p>Wilfred: </p>
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<div>{{Team:Groningen/Header}}<br />
::::::::::::::::{{todo|<i><b>Under construction</b></i>}}<br />
==Introduction==<br />
Bacteria have a choice between using nutrients for growth or for the production of (commercially) valuable proteins. Being able to control the [http://en.wikipedia.org/wiki/Bacterial_growth bacterial growth cycle] and inducing a premature stationary phase will create the possibility to spend more time producing, using less nutrients for biomass and more for desired production. Stationary phase is nothing more than a stop in an increase of cell numbers by cell death being equal to cell growth. Normally this is induced by the limitation of available nutrients or by means of [http://en.wikipedia.org/wiki/Quorum_sensing quorum sensing] in response to high cell density. With this knowledge, a culture can be created that can respond by limited cell death in response to an added molecule that mimics the response to high cell density.<br />
<br />
See also [http://en.wikipedia.org/wiki/Chemostat Chemostat]<br><br />
One of the most important features of chemostats is that micro-organisms can be grown in a physiological steady state. In steady state, all culture parameters remain constant (culture volume, dissolved oxygen concentration, nutrient and product concentrations, pH, cell density, etc.). Because obtaining a steady state requires at least 5 volume changes, chemostats require large nutrient and waste reservoirs. Creating biological "<i>chemostat</i>" would circumvent these drawbacks.<br />
==Approach==<br />
What we want to reach is an early stationary phase that is inducible and can be relieved to continu onto its natural stationary phase upon depleting its nutrients. To be able to leave to early stationary phase the inductor needs to be degradable or blockable. However, to speak of an actual phase the degradation or inhibition should proceed slowly so not to end the phase too soon. <br />
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In undertaking such an endeavour there are several approaches possible, one is to create a switch that could simply stop cells from reproducing and focus them on to production. The other is to create a culture that upon induction begins to oscilate between cell death and cell growth. Because creating either possibilities should yield some difficulties, the project could be divided into three parts.<br />
#Inducing an early stationary phase (that focusses its metabolism on protein production, visualized by GFP)<br />
#Leaving the early stationary phase (leaving the early stationary phase should be optional in practice)<br />
#Creating an oscilating culture that remains between predefined limits.<br />
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Possibilities to think of might be a time switch so to continu growing after a set period of time, or continu growing as soon as certain demands are fullfilled (certain amount of production). However, both ways of leaving the early stationary phase are then induced intrinsically and not determined by us. The same problem for the idea of adding a substance that induces the production of an autoinducer simultaneous with an slow promotor of an autoinhibitor, as the concentration of the autoinhibitor rises to a certain point the effect of the autoinducer will be block sufficiently to continu growing.<br />
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It would be nice to work with NICE<br />
==Previous contests==<br />
<b>Quorum Sensing</b><br />
:*[https://2007.igem.org/McGill McGill 2007]<br />
:*[https://2007.igem.org/Chase_Simulator Turkey 2007]<br />
:*[https://2007.igem.org/Harvard#Quorum_Sensing Harvard 2007]<br />
:*[https://2007.igem.org/Michigan Michigan 2007]<br />
:*[http://www.openwetware.org/wiki/IGEM:Peking/2007 Peking 2007]<br />
:*[https://2008.igem.org/Team:Calgary_Wetware/Project#SlideFrame_1 Calgary 2008]<br />
:*[https://2008.igem.org/Team:Cambridge/Modelling Cambridge 2008]<br />
:*[https://2008.igem.org/Team:Chiba/Project Chiba 2008]<br />
:*[https://2008.igem.org/Team:Groningen Groningen 2008]<br />
:*[https://2008.igem.org/Team:Montreal Montreal 2008]<br />
<b>Cell cycle</b><br />
:*[https://2006.igem.org/Synchronization_of_Cell_Cycles Bangalore 2006]<br />
:*[https://2008.igem.org/Team:ESBS-Strasbourg Strasbourg 2008]<br />
:*[https://2008.igem.org/Team:ETH_Zurich/Wetlab/Switch_Circuit#Genetic_Experiments_using_Ribosome_Modulation_Factor_.28RMF.29| Zurich 2008]<br />
:*[https://2008.igem.org/Team:University_of_Ottawa/Project Ottowa 2008]<br />
:*[https://2008.igem.org/Team:Paris/Analysis/Design3 Paris 2008]<br />
<b>Cell death</b><br />
:*[https://2008.igem.org/Team:KULeuven/Project/CellDeath Leuven 2008]<br />
:*[https://2008.igem.org/Team:Minnesota/HomeTimeBomb Minnesota 2008]<br />
:*[https://2008.igem.org/Team:NTU-Singapore Singapore 2008]<br />
<br />
==Parts in the [http://partsregistry.org/Main_Page Registry of Standard Parts]:==<br />
<b>Quorum sensing</b><br />
:*[http://partsregistry.org/Part:BBa_K104001 BBa_K104001]: <i>Sensor for small peptide Subtilin</i><br />
:*[http://partsregistry.org/Part:BBa_I13211 BBa_I13211]: <i>Biobricked version of the natural Lux quorum sensing system</i><br />
:*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]: <i>AHL to GFP Converter </i><br />
:*[http://partsregistry.org/Part:BBa_K09100 BBa_K09100] <i>Receiver for AHL and Outputs GFP when AHL is present</i><br />
<br />
<b>Cell cycle</b><br />
:*[http://partsregistry.org/Part:BBa_M31201 BBa_M31201]<br />
:*[http://partsregistry.org/Part:BBa_K105013 BBa_K105013]<br />
:*[http://partsregistry.org/Part:BBa_K105015 BBa_K105015]<br />
:*[http://partsregistry.org/Part:BBa_K101017 BBa_K101017]: <i>a cell-cycle dependent promoter that is repressed before initiation of replication and depressed shortly after</i><br />
:*<del>[http://partsregistry.org/Part:BBa_J22051 BBa_J22051]: <i>an adenylate cyclase promoter, expression is repressed during cell division</i></del><br />
:*[http://partsregistry.org/Part:BBa_J22052 BBa_J22052]: <i>an adenylate cyclase promoter, expression is repressed during cell division</i><br />
:*[http://partsregistry.org/Part:BBa_J22095 BBa_J22095]<br />
:*[http://partsregistry.org/Part:BBa_J22092 BBa_J22092]<br />
:*[http://partsregistry.org/Part:BBa_K142040 BBa_K142040]: <i>Ribosome modulation factor (RMF)</i><br />
:*[http://partsregistry.org/Part:BBa_K142041 BBa_K142041]: <i>Arabinose controlled RMF generator</i><br />
<b>Cell death</b><br />
:*[http://partsregistry.org/Part:BBa_I745006 BBa_I745006]<br />
:*[http://partsregistry.org/Part:BBa_I745007 BBa_I745007]<br />
:*[http://partsregistry.org/Part:BBa_K145008 BBa_K145008]: <i>LuxR Generator</i><br />
:*[http://partsregistry.org/Part:BBa_K145009 BBa_K145009]: <i>ccdB cell death gene under control of an activating Lux PR</i><br />
:*[http://partsregistry.org/Part:BBa_K145109 BBa_K145109]: <i>ccdB cell death gene under the control of a hybrid LuxPR P22 C2 promotor</i><br />
:*[http://partsregistry.org/Part:BBa_K145110 BBa_K145110]: <i>Complete cell death mechanism. Combination of [http://partsregistry.org/Part:BBa_K145108 BBa_K145108] and [http://partsregistry.org/Part:BBa_K145109 Part:BBa_K145109]</i><br />
:*[http://partsregistry.org/Part:BBa_K145151 BBa_K145151]: <i>Coding region for the ccdB (control of cell death) gene</i><br />
:*[http://partsregistry.org/Part:BBa_K145230 BBa_K145230]: <i>A hybrid promoter controls the production of LuxR and ccdB</i><br />
:*[http://partsregistry.org/Part:BBa_K145256 BBa_K145256]: <i>Cell death Part 1</i><br />
:*[http://partsregistry.org/Part:BBa_K145257 BBa_K145257]: <i>Cell death Part 2</i><br />
:*[http://partsregistry.org/Part:BBa_K124003 BBa_K124003]: <i>Induces lysis in E. Coli bacteria</i><br />
:*[http://partsregistry.org/Part:BBa_K124014 BBa_K124014]: <i>Induces lysis faster in E. Coli bacteria</i><br />
:*[http://partsregistry.org/Part:BBa_K124017 BBa_K124017]: <i>Complete casette containing [http://partsregistry.org/Part:BBa_K124014 BBa_K124014]</i><br />
<br />
==Related Literature==<br />
<b>Quorum sensing</b><br />
:*[http://www.ncbi.nlm.nih.gov/pubmed/15374644 Quorum sensing control of lantibiotic production; nisin and subtilin autoregulate their own biosynthesis], Kleerebezem<br />
::<i>In this paper, the molecular mechanism underlying regulation of nisin and subtilin production is reviewed.</i><br />
:*[http://www.ncbi.nlm.nih.gov/pubmed/9675856 Induction of entry into the stationary growth phase in <i>Pseudomonas aeruginosa</i> by N-acylhomoserine lactone], You <i>et al.</i><br />
::<i>Addition of N-acylhomoserine lactone in the exponential growth phase, regardless of cell density, induces a repression of cell growth of P. aeruginosa</i><br />
:*[http://www.ncbi.nlm.nih.gov/pubmed/12032343 Induction of natural competence in <i>Streptococcus pneumoniae</i> triggers lysis and DNA release from a subfraction of the cell population], Steinmoen <i>et al.</i><br />
::<i>In this study Competence stimulating peptide is shown to initiates release of DNA from a subfraction of the bacterial population, probably by cell lysis.</i><br />
:*<b>[http://www.nature.com/msb/journal/v4/n1/full/msb200824.html A synthetic <i>Escherichia coli</i> predator–prey ecosystem], Balagaddé <i>et al.</i><br />
::<i>In this study they created a synthetic ecosystem with bi-directional communication through quorum sensing which regulate each other's gene expression and survival via engineered gene circuits.</i></b><br />
:*<b>[http://www.nature.com/nature/journal/v428/n6985/abs/nature02491.html Programmed population control by cell–cell communication and regulated killing] You <i>et al.</i><br />
::<i>In this study they have built and characterized a 'population control' circuit that autonomously regulates the density of an Escherichia coli population, that is lower than the limits imposed by the environment. The cell density is broadcasted and detected by elements from a bacterial quorum-sensing system, which in turn regulate the death rate</i></b><br />
:*[http://www.ncbi.nlm.nih.gov/pubmed/17959781 Engineered bidirectional communication mediates a consensus in a microbial biofilm consortium], Brenner <i>et al.</i> <br />
::<i>In this study they created two colocalized populations of Escherichia coli that communicate with each other and exhibit a “consensus” gene expression response. Because neither population can respond without the other's signal, this consensus function can be considered a logical AND gate in which the inputs are cell populations.</i><br />
<b>Cell cycle</b><br />
:*[http://www.ncbi.nlm.nih.gov/pubmed/15107854 Just-in-time transcription program in metabolic pathways] Zaslaver <i>et al.</i><br />
::<i>A study that showed certain promotors involved in amino acid biosynthesis being downregulated when compounds of the involved pathways were added.</i><br />
<b>Cell death</b><br />
:*[https://2008.igem.org/Team:Edinburgh/Plan#Bacterial_cell_lysis Edinburgh 2008]<br />
<b>Modelling</b><br />
:*[http://bioinformatics.oxfordjournals.org/cgi/content/full/23/18/2415 Robustness analysis and tuning of synthetic gene networks], Batt <i>et al.</i><br />
::<i>In this work, they demonstrate the biological relevance of a method specifically developed to support the design of synthetic gene networks.</i><br />
:*[http://bib.oxfordjournals.org/cgi/content/abstract/bbp005 Computational systems biology of the cell cycle], Csikász-Nagy<br />
::<i>A review of past and present of computational modeling of cell-cycle regulation</i><br />
{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/PublicityTeam:Groningen/Publicity2009-11-23T08:06:27Z<p>Wilfred: /* News */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
=Websites=<br />
*"<i>Students from Groningen in competition for best bacteria</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*"<i>Students from Groningen are making bacteria to remove heavy metals from water</i>" (Dutch), [http://www.groningenreporter.nl/story.php?title=groninger-studenten-maken-bacteri%3Fn-die-water-zuiveren-van-zware-metalen Groningen Reporter (August 22, 2009)]<br />
*"<i>Team iGEM Groningen has been awarded a Gold Medal</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*"<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
<br />
=News=<br />
*"<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*"<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen iGEM 2009 participants</i>" ([http://www.rug.nl/corporate/nieuws/archief/archief2009/persberichten/174_09?lang=en English]/[http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Dutch]), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09?lang=en Press release University Groningen (November 11, 2009)]<br />
*"<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*"<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*"<i>With a pacmanbacterium in de finals</i>" (Dutch), [http://www.uk.rug.nl/archief/jaargang39/11/12e.php UK (Paper of the Groningen University) (November 12, 2009)]<br />
*"<i>iGEM Gold for Dutch</i>" (Dutch), [http://www.bionieuws.nl/artikel.php?id=4918&zoek=iGEM Bionieuws (November 14, 2009)]<br />
<br />
=Radio=<br />
*Interview [http://www.oogtv.nl/ Oog Radio] - <i>Stad vandaag (News- and background story program)</i> (Dutch) (November 10, 2009)<br />
<br />
=Our Brochure=<br />
<center>[[Image:Inside_Brochure_iGEM_GRONINGEN.jpg|800px]]<br />
[[Image:Outside_Brochure_iGEM_GRONINGEN.jpg|800px]]</center><br />
{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/PublicityTeam:Groningen/Publicity2009-11-23T08:05:07Z<p>Wilfred: /* Websites */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
=Websites=<br />
*"<i>Students from Groningen in competition for best bacteria</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*"<i>Students from Groningen are making bacteria to remove heavy metals from water</i>" (Dutch), [http://www.groningenreporter.nl/story.php?title=groninger-studenten-maken-bacteri%3Fn-die-water-zuiveren-van-zware-metalen Groningen Reporter (August 22, 2009)]<br />
*"<i>Team iGEM Groningen has been awarded a Gold Medal</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*"<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
<br />
=News=<br />
*"<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*"<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Press release University Groningen (November 11, 2009)]<br />
*"<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*"<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*"<i>With a pacmanbacterium in de finals</i>" (Dutch), [http://www.uk.rug.nl/archief/jaargang39/11/12e.php UK (Paper of the Groningen University) (November 12, 2009)]<br />
*"<i>iGEM Gold for Dutch</i>" (Dutch), [http://www.bionieuws.nl/artikel.php?id=4918&zoek=iGEM Bionieuws (November 14, 2009)]<br />
<br />
=Radio=<br />
*Interview [http://www.oogtv.nl/ Oog Radio] - <i>Stad vandaag (News- and background story program)</i> (Dutch) (November 10, 2009)<br />
<br />
=Our Brochure=<br />
<center>[[Image:Inside_Brochure_iGEM_GRONINGEN.jpg|800px]]<br />
[[Image:Outside_Brochure_iGEM_GRONINGEN.jpg|800px]]</center><br />
{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/TeamTeam:Groningen/Team2009-11-23T08:02:39Z<p>Wilfred: /* Presenting */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Future}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Pictures}}</div><br />
<br />
[[Category:Team:Groningen/Disciplines/Project_Management|Team]]<br />
[[Category:Team:Groningen/Roles/Project_Manager|Team]]<br />
<br />
==Our Team At A Glance==<br />
<br />
[[Image:IGEMGroningen_Molen.jpg|400px|thumb|right|[Team:Groningen/Team|Our team!]]<br />
<br />
Welcome to the website of the iGEM Groningen team! We are an interdisciplinary team of 11 enthusiastic students from the [http://www.rug.nl/ University of Groningen] situated in the not-too-big city of [http://portal.groningen.nl/en/startpagina Groningen] in [http://maps.google.com/maps?f=q&source=s_q&hl=en&geocode=&q=Groningen&sll=53.281349,6.689459&sspn=0.007261,0.018926&ie=UTF8&z=12&iwloc=A the north of the Netherlands]. You can contact us by '''[mailto:igemgroningen@googlegroups.com mail]'''. <br />
<br />
Our team consists of the following student-members:<br />
<br />
* [[User:JolandaWitteveen|Jolanda Witteveen]] (Biomedical Technology): [[:Category:Team:Groningen/Roles/Chair|Chair]], [[:Category:Team:Groningen/Roles/Project_Manager|Project Manager]]<br />
* [[User:svenjurgens|Sven Jurgens]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [[User:Jaspervdg|Jasper van de Gronde]] (Computational Science and Visualization): [[:Category:Team:Groningen/Roles/Configuration_Manager|Configuration Manager]], [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Verhoeven1981|Michael Verhoeven]] (Chemistry): [[:Category:Team:Groningen/Roles/Public_Relations_Officer|PR Officer]]<br />
* [https://2009.igem.org/User:Nienke Nienke Kuipers] (Molecular Biology): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]] and Lab manager<br />
* [[User:Jelle|Steven Jelle Meijer]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Facility_Manager|Facility Manager Haren]]<br />
* [[User:Wilfred|Wilfred Poppinga]] (Medical Pharmaceutical Sciences): [[:Category:Team:Groningen/Roles/Chair|Vice Chair]], [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [https://2009.igem.org/User:Paulschavemaker Paul Schavemaker] (Molecular Life Sciences): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]]<br />
* [https://2009.igem.org/User:Frans Frans Bianchi] (Molecular Biology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Klaas Bernd Over|Klaas Bernd Over]] (Applied Physics): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Annelies|Annelies van Keulen]] (Molecular Biology/Psychology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
<br />
==Our advisors==<br />
*prof. dr. Oscar Kuipers: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics] (Head)<br />
*prof. dr. Jan Kok: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics]<br />
*prof. dr. Bert Poolman: Biochemistry; [http://www.centreforsyntheticbiology.eu/ Centre for Synthetic Biology] (Director)<br />
*prof. dr. Roel Bovenberg: Synthetic biology and Cell engineering; Corporate Scientist Biotechnology, [http://www.dsm.com/ DSM]<br />
*dr. Dirk Slotboom: Enzymology <br />
*[https://2008.igem.org/Team:Groningen/team.html iGEM Groningen 2008]. Especially Auke van Heel & Martijn Herber<br />
<br />
<br><br><br />
<br />
==Where to hear from us==<br />
===In the media===<br />
Follow us in '''[[Team:Groningen/Publicity| The News]]'''<br />
<br />
Also follow us on '''[http://twitter.com/igemgroningen Twitter]!'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Videos|Videos]]'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Pictures|Pictures]]'''<br />
<br />
==Where to meet us==<br />
{|<br />
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<div class="meet"><br />
*{{todo}} December 17<sup>th</sup> 2009: Short presentation @ [http://studium.hosting.rug.nl/Studiumgenerale/start.htm Studium Generale] - Groningen<br />
*{{todo}} December 11<sup>th</sup> 2009: Meeting @ [http://www.dsm.com/en_US/html/home/dsm_home.cgi DSM] - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=delft+DSM&fb=1&gl=nl&hq=DSM&hnear=delft&cid=0,0,8723601113946313921&ei=B_jSSrqHIcTz-QbZsNT7Ag&sa=X&oi=local_result&ct=image&resnum=1&ved=0CAoQnwIwAA Delft]<br />
*{{todo}} November 23<sup>rd</sup> 2009: Meeting @ student societies for [http://www.chemische-binding.nl/ Chemistry] and [http://www.fmf.nl/?file=main.html&lang=.en Math, Physics, Computer Science and Astronomy]<br />
*<b>October 30<sup>th</sup> to November 2<sup>nd</sup> 2009: Presentation @ The [https://2009.igem.org/ iGEM] 2009 [https://2009.igem.org/Jamboree Jamboree] - [http://whereis-beta.mit.edu/?mapterms=stata%20center&zoom=15&lat=42.36161990569666&lng=-71.09055519104004&open=object-32 MIT Stata] in Cambridge, MA</b><br />
*October 26<sup>th</sup> 2009: Lecture @ [http://www.hanzeuniversity.eu/home/international Hanze University], Biology & Medical Laboratory Research and Bioinformatics students - room A257 [http://maps.google.nl/maps?q=Zernikeplein+7+Groningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Zernikeplein+7,+9747+Groningen&gl=nl&ei=wwHPSor9A4OF-QaTkL2FAw&sa=X&oi=geocode_result&ct=title&resnum=1 Zernikeplein 11, Groningen] <br />
*October 28<sup>th</sup> 2009: Update Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Haren]<br />
*October 19<sup>th</sup> 2009: [http://www.cs.rug.nl/~biehl/Coll/index.html Colloquium] @ [http://www.rug.nl/informatica/index Institute for Mathematics and Computing Science] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=jGw&resnum=0&q=bernoulliborg%20Groningen%20Nijenborgh%209&um=1&ie=UTF-8&sa=N&tab=wl room 5161.0267 (Bernoulliborg), Groningen]<br />
*October 12<sup>th</sup> 2009: Meeting @ Marine Biology cluster - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 7<sup>th</sup> 2009: Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 2<sup>nd</sup> 2009: Lunch meeting @ [http://www2.dhv.com/default.aspx DHV] - [http://maps.google.com/maps?f=q&source=s_q&hl=nl&geocode=&q=Laan+1914+no+35,+Amersfoort&sll=37.0625,-95.677068&sspn=54.357317,79.013672&ie=UTF8&hq=&hnear=Laan+1914+35,+3818+Amersfoort,+Utrecht,+Nederland&ll=52.134107,5.36828&spn=0.010405,0.01929&t=h&z=16&iwloc=r3 Groene zaal DHV, Amersfoort]<br />
* October 1<sup>st</sup> 2009: Lunch meeting @ Life Science student society [http://www.glv-idun.nl/ GLV Idun] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Groene Zaal, Haren]<br />
*September 29<sup>th</sup> 2009: Meeting @ Applied physics student society [http://www.professorfrancken.nl/ TFV Professor Francken] - [http://maps.google.nl/maps?q=Nijenborgh%204%20NCC%20Complex&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&hl=nl&ie=UTF-8&sa=N&tab=vl NCC complex VIP Room building 16, Groningen]<br />
*[https://2009.igem.org/Team:Groningen/Notebook/24_September_2009 September 24<sup>th</sup> 2009]: Presentation @ 2nd Programme Day of the [http://www.kluyvercentre.nl/ Kluyver Centre] - [http://maps.google.nl/maps?q=Generaal+Foulkesweg+96+6703+DS+Wageningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Generaal+Foulkesweg+96,+6703+Wageningen&gl=nl&ei=giXDSvfgDcrI-Qa53ojvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Wageningse Berg, Wageningen]<br />
*September 11<sup>th</sup> 2009: Presentation @ [http://www.rug.nl/gbb/studyatgbb/generalcourses/gbbsymposium2009 17th Annual] [http://www.rug.nl/gbb/index GBB] Symposium 2009 - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=Hampshire+hotel+Groningen+Radesingel+50,+9711+EK+Groningen&fb=1&gl=nl&hq=Hampshire+hotel&hnear=Groningen+Radesingel+50,+9711+EK+Groningen&cid=0,0,5400363645623663183&ei=eybDSq-jNojj-Qbz1PXuCw&sa=X&oi=local_result&ct=image&resnum=1 Hampshire hotel, Groningen]</div><br />
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{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/TeamTeam:Groningen/Team2009-11-23T08:01:35Z<p>Wilfred: /* Presenting */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Future}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Pictures}}</div><br />
<br />
[[Category:Team:Groningen/Disciplines/Project_Management|Team]]<br />
[[Category:Team:Groningen/Roles/Project_Manager|Team]]<br />
<br />
==Our Team At A Glance==<br />
<br />
[[Image:IGEMGroningen_Molen.jpg|400px|thumb|right|[Team:Groningen/Team|Our team!]]<br />
<br />
Welcome to the website of the iGEM Groningen team! We are an interdisciplinary team of 11 enthusiastic students from the [http://www.rug.nl/ University of Groningen] situated in the not-too-big city of [http://portal.groningen.nl/en/startpagina Groningen] in [http://maps.google.com/maps?f=q&source=s_q&hl=en&geocode=&q=Groningen&sll=53.281349,6.689459&sspn=0.007261,0.018926&ie=UTF8&z=12&iwloc=A the north of the Netherlands]. You can contact us by '''[mailto:igemgroningen@googlegroups.com mail]'''. <br />
<br />
Our team consists of the following student-members:<br />
<br />
* [[User:JolandaWitteveen|Jolanda Witteveen]] (Biomedical Technology): [[:Category:Team:Groningen/Roles/Chair|Chair]], [[:Category:Team:Groningen/Roles/Project_Manager|Project Manager]]<br />
* [[User:svenjurgens|Sven Jurgens]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [[User:Jaspervdg|Jasper van de Gronde]] (Computational Science and Visualization): [[:Category:Team:Groningen/Roles/Configuration_Manager|Configuration Manager]], [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Verhoeven1981|Michael Verhoeven]] (Chemistry): [[:Category:Team:Groningen/Roles/Public_Relations_Officer|PR Officer]]<br />
* [https://2009.igem.org/User:Nienke Nienke Kuipers] (Molecular Biology): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]] and Lab manager<br />
* [[User:Jelle|Steven Jelle Meijer]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Facility_Manager|Facility Manager Haren]]<br />
* [[User:Wilfred|Wilfred Poppinga]] (Medical Pharmaceutical Sciences): [[:Category:Team:Groningen/Roles/Chair|Vice Chair]], [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [https://2009.igem.org/User:Paulschavemaker Paul Schavemaker] (Molecular Life Sciences): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]]<br />
* [https://2009.igem.org/User:Frans Frans Bianchi] (Molecular Biology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Klaas Bernd Over|Klaas Bernd Over]] (Applied Physics): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Annelies|Annelies van Keulen]] (Molecular Biology/Psychology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
<br />
==Our advisors==<br />
*prof. dr. Oscar Kuipers: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics] (Head)<br />
*prof. dr. Jan Kok: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics]<br />
*prof. dr. Bert Poolman: Biochemistry; [http://www.centreforsyntheticbiology.eu/ Centre for Synthetic Biology] (Director)<br />
*prof. dr. Roel Bovenberg: Synthetic biology and Cell engineering; Corporate Scientist Biotechnology, [http://www.dsm.com/ DSM]<br />
*dr. Dirk Slotboom: Enzymology <br />
*[https://2008.igem.org/Team:Groningen/team.html iGEM Groningen 2008]. Especially Auke van Heel & Martijn Herber<br />
<br />
<br><br><br />
<br />
==Where to hear from us==<br />
===In the media===<br />
Follow us in '''[[Team:Groningen/Publicity| The News]]'''<br />
<br />
Also follow us on '''[http://twitter.com/igemgroningen Twitter]!'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Videos|Videos]]'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Pictures|Pictures]]'''<br />
<br />
===Presenting===<br />
{|<br />
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*{{todo}} December 17<sup>th</sup> 2009: Short presentation @ [http://studium.hosting.rug.nl/Studiumgenerale/start.htm Studium Generale] - Groningen<br />
*{{todo}} December 11<sup>th</sup> 2009: Meeting @ [http://www.dsm.com/en_US/html/home/dsm_home.cgi DSM] - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=delft+DSM&fb=1&gl=nl&hq=DSM&hnear=delft&cid=0,0,8723601113946313921&ei=B_jSSrqHIcTz-QbZsNT7Ag&sa=X&oi=local_result&ct=image&resnum=1&ved=0CAoQnwIwAA Delft]<br />
*{{todo}} November 23<sup>rd</sup> 2009: Meeting @ student societies for [http://www.chemische-binding.nl/ Chemistry] and [http://www.fmf.nl/?file=main.html&lang=.en Math, Physics, Computer Science and Astronomy]<br />
*<b>October 30<sup>th</sup> to November 2<sup>nd</sup> 2009: Presentation @ The [https://2009.igem.org/ iGEM] 2009 [https://2009.igem.org/Jamboree Jamboree] - [http://whereis-beta.mit.edu/?mapterms=stata%20center&zoom=15&lat=42.36161990569666&lng=-71.09055519104004&open=object-32 MIT Stata] in Cambridge, MA</b><br />
*October 26<sup>th</sup> 2009: Lecture @ [http://www.hanzeuniversity.eu/home/international Hanze University], Biology & Medical Laboratory Research and Bioinformatics students - room A257 [http://maps.google.nl/maps?q=Zernikeplein+7+Groningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Zernikeplein+7,+9747+Groningen&gl=nl&ei=wwHPSor9A4OF-QaTkL2FAw&sa=X&oi=geocode_result&ct=title&resnum=1 Zernikeplein 11, Groningen] <br />
*October 28<sup>th</sup> 2009: Update Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Haren]<br />
*October 19<sup>th</sup> 2009: [http://www.cs.rug.nl/~biehl/Coll/index.html Colloquium] @ [http://www.rug.nl/informatica/index Institute for Mathematics and Computing Science] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=jGw&resnum=0&q=bernoulliborg%20Groningen%20Nijenborgh%209&um=1&ie=UTF-8&sa=N&tab=wl room 5161.0267 (Bernoulliborg), Groningen]<br />
*October 12<sup>th</sup> 2009: Meeting @ Marine Biology cluster - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 7<sup>th</sup> 2009: Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 2<sup>nd</sup> 2009: Lunch meeting @ [http://www2.dhv.com/default.aspx DHV] - [http://maps.google.com/maps?f=q&source=s_q&hl=nl&geocode=&q=Laan+1914+no+35,+Amersfoort&sll=37.0625,-95.677068&sspn=54.357317,79.013672&ie=UTF8&hq=&hnear=Laan+1914+35,+3818+Amersfoort,+Utrecht,+Nederland&ll=52.134107,5.36828&spn=0.010405,0.01929&t=h&z=16&iwloc=r3 Groene zaal DHV, Amersfoort]<br />
* October 1<sup>st</sup> 2009: Lunch meeting @ Life Science student society [http://www.glv-idun.nl/ GLV Idun] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Groene Zaal, Haren]<br />
*September 29<sup>th</sup> 2009: Meeting @ Applied physics student society [http://www.professorfrancken.nl/ TFV Professor Francken] - [http://maps.google.nl/maps?q=Nijenborgh%204%20NCC%20Complex&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&hl=nl&ie=UTF-8&sa=N&tab=vl NCC complex VIP Room building 16, Groningen]<br />
*[https://2009.igem.org/Team:Groningen/Notebook/24_September_2009 September 24<sup>th</sup> 2009]: Presentation @ 2nd Programme Day of the [http://www.kluyvercentre.nl/ Kluyver Centre] - [http://maps.google.nl/maps?q=Generaal+Foulkesweg+96+6703+DS+Wageningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Generaal+Foulkesweg+96,+6703+Wageningen&gl=nl&ei=giXDSvfgDcrI-Qa53ojvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Wageningse Berg, Wageningen]<br />
*September 11<sup>th</sup> 2009: Presentation @ [http://www.rug.nl/gbb/studyatgbb/generalcourses/gbbsymposium2009 17th Annual] [http://www.rug.nl/gbb/index GBB] Symposium 2009 - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=Hampshire+hotel+Groningen+Radesingel+50,+9711+EK+Groningen&fb=1&gl=nl&hq=Hampshire+hotel&hnear=Groningen+Radesingel+50,+9711+EK+Groningen&cid=0,0,5400363645623663183&ei=eybDSq-jNojj-Qbz1PXuCw&sa=X&oi=local_result&ct=image&resnum=1 Hampshire hotel, Groningen]</div><br />
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{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/IGEM_PublicityIGEM Publicity2009-11-23T07:55:34Z<p>Wilfred: /* team specific */</p>
<hr />
<div>__NOTOC__<br />
[https://2008.igem.org/IGEM_Publicity iGEM 2008 Publicity] | [http://parts.mit.edu/igem07/index.php/Media iGEM 2007 Publicity] | [http://parts.mit.edu/wiki/index.php/IGEM_News iGEM 2006 Publicity]<br />
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<div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [[Press_Kit | iGEM Press Kit]])</div><br />
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<span style="color:#1e90ff; font-size:175%">'''blogs </span><span style="color:#3cb371; font-size:175%">covering iGEM 2009'''</span><br />
* The [http://igem.sdu.dk/ <span style="color:#453221; font-size: 130%">'''SDU-Denmark'''</span>] team blog about their iGEM experience.<br />
* A blog about the iGEM project of the [http://aboutgmos.org/iGEM <span style="color:#453221; font-size: 130%">'''Uppsala-Sweden'''</span>] team.<br />
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====<font size=5><font color=dodgerblue>'''video/radio</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
*'''Slovenia, Heidelberg, SDU-Denmark''': [http://www.dradio.de/dlf/sendungen/forschak/1062608/ Die Biobastler von Boston], Nov 2, 2009, Deutschlandfunk radio (in German).<br />
*'''Cambridge, Imperial''': [http://www.sciencefriday.com/program/archives/200911063 Synthetic Biology Competition], Nov 6, 2009, NPR Science Friday (in English).<br />
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====<font size=5><font color=dodgerblue>'''news articles</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
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If you would like to share an article that was written about iGEM or your iGEM team, please link to it on this page. If you have multiple articles featuring your team, link to them all individually!<br />
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Post the name of your team, the title of your article, where it was featured, and provide a link to it. <br />
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''Example'':<br> <br />
'''Team Example''': ''Title of article'', Nature, [link]<br />
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====<font size=4><font color=dodgerblue>'''general'''</font></font>====<br />
*'''The Economist''': ''Biohacking: Hacking goes squishy'' Sep 3, 2009 [http://www.economist.com/search/displaystory.cfm?story_id=14299634 The Economist]<br />
*'''The Scientist''': ''Brick by Brick'' Feb 1, 2009. [http://www.the-scientist.com/article/display/55378/ The Scientist]<br />
*'''Technology Review''': ''A Genetically Engineered Rainbow of Bacteria'' Nov 03, 2009. [http://www.technologyreview.com/blog/editors/24351/ Technology Review]<br />
*'''Discovery News''': '' Bright Bacteria Wins Synthetic Biology Competition'' Nov 6, 2009. [http://blogs.discoverychannel.co.uk/discovery-news/2009/11/bright-bacteria-wins-synthetic-biology-competition.html Discovery News]<br />
*'''Wired''': '' Building new life forms at the iGEM Jamboree'' Nov 9, 2009. [http://www.wired.co.uk/news/archive/2009-11/09/building-new-life-forms-at-the-igem-jamboree.aspx Wired]<br />
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====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
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*'''[[Team:IBB_Pune]]''': "<i>Bio-champs in the making</i>" (English), [http://www.punemirror.in/index.aspx?Page=article&sectname=News%20-%20City&sectid=2&contentid=200909142009091423331646a131eacc#ftr2 (Monday, September 14, 2009 at 11:33:25 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Ganiti Prakriyanmadhe Jeevanuncha Vaapar! (Use of Bacteria in mathematical devices)</i>" (Marathi), [http://beta.esakal.com/2009/09/09220618/pune-use-of-bacterias-in-maths.html(Wednesday, September 09th, 2009 AT 10:09 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Synthetic Biologiteel Bharari ("Advances in Synthetic Biology")</i>" (Marathi), [https://2009.igem.org/Team:IBB_Pune/press (Wednesday 7 October 2009)]<br />
<br />
*'''[[Team:Groningen]]''': "<i>Students from Groningen in competition for best bacteria</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*'''[[Team:Groningen]]''': "<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Team iGEM Groningen has been awarded a Gold Medal</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen iGEM 2009 participants</i>" ([http://www.rug.nl/corporate/nieuws/archief/archief2009/persberichten/174_09?lang=en English]/[http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Dutch]), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09?lang=en Press release University Groningen (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>With a pacmanbacterium in de finals</i>" (Dutch), [http://www.uk.rug.nl/archief/jaargang39/11/12e.php UK (Paper of the Groningen University) (November 12, 2009)]<br />
*'''[[Team:Groningen]]''' & '''[[Team:TUDelft]]''': "<i>iGEM Gold for Dutch</i>" (Dutch), [http://www.bionieuws.nl/artikel.php?id=4918&zoek=iGEM Bionieuws (November 14, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Un concours organisé par le M.I.T : en route pour Boston ! </i>" (French), [http://www.supbiotech.fr/2009/09/boston-supbiotech-igem.html (September 08, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Les étudiants de la biotech interpellent la biologie synthétique</i>" (French), [http://www.vivagora.org/spip.php?breve210 (October 09, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>Un equipo de la UAB, en el concurso de biología sintética del MIT</i>" (Spanish), [http://www.uab.es/servlet/Satellite?cid=1096481466568&pagename=UABDivulga%2FPage%2FTemplatePageDetallArticleInvestigar&param1=1253860327385 (September 23, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>UAB to participate in the synthetic biology competition at MIT</i>" (English), [http://www.uab.es/servlet/Satellite/latest-news/news-detail/uab-to-participate-in-the-synthetic-biology-competition-at-mit-1096476786473.html?noticiaid=1253657853358 (September 23, 2009)]<br />
<br />
*'''[[Team:Valencia]]''': "<i>TheValencia team is awarded the Synthetic Standard Prize</i>" (Spanish), [http://www.elpais.com/articulo/sociedad/Levaduras/funcionan/pixeles/elpepuespval/20091103elpepusoc_9/Tes]<br />
<br />
*'''[[Team:Cambridge]]''': "<i>University of Cambridge team wins iGEM synthetic biology competition </i>" (English), [http://www.biotechniques.com/news/University-of-Cambridge-team-wins-iGEM-synthetic-biology-competition/biotechniques-180278.html (November 5 2009)]<br />
<br />
*'''[[Team:Virginia]]''': ''[http://www.washingtonpost.com/wp-dyn/content/article/2009/10/22/AR2009102204628.html New works of science nonfiction]'', The Washington Post (October 23, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Estafette voor bacterien'',(Dutch) Algemeen Dagblad (November 6, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette'', (Dutch) [http://noorderlicht.vpro.nl/themasites/mediaplayer/index.jsp?media=42722491&refernr=42560747&portalnr=3626936&hostname=noorderlicht&mediatype=audio&portalid=noorderlicht# Podcast van Radio 1, VPRO Noorderlicht, Annemieke Smit] (November 9, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette wint goud en award'', (Dutch) [http://www.tudelft.nl/live/pagina.jsp?id=ee44be90-289e-4efe-8a12-4636f1c86b28&lang=nl TNW Today Editie 2] (November 5, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette wint goud en award'', (Dutch) [http://www.tudelft.nl/live/pagina.jsp?id=ee44be90-289e-4efe-8a12-4636f1c86b28&lang=nl TUDelft Nieuws] (November 4, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterial relay race wins gold and award'', [http://www.tudelft.nl/live/pagina.jsp?id=26f61b2e-5a3f-40be-b737-a460b08bced7&lang=en Delft University of Technology Press release] (November 4, 2009)</div>Wilfredhttp://2009.igem.org/Team:Groningen/Modelling/ArsenicTeam:Groningen/Modelling/Arsenic2009-11-18T12:26:52Z<p>Wilfred: /* The raw model */</p>
<hr />
<div>{{Team:Groningen/Modelling/Header}}<br />
<div style="clear:both;"></div><br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Modelling}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Modelling/Characterization}}</div><br />
[[Category:Team:Groningen/Disciplines/Analysis_and_Design|Modelling]]<br />
[[Category:Team:Groningen/Roles/Modeller|Modelling]]<br />
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<div class="intro introduction"><br />
==Detailed Model==<br />
Based on the [[#QuasiSteadyState|quasi-steady-state derivation]] below we have made the simplified version of our model shown below. The simplification is based on two key assumptions (which are also illustrated below, next to the table "Breakdown of core substances"):<br />
<br />
*Binding and unbinding of arsenic to/from the transporters occurs on a much smaller time scale than changes in the concentration of arsenic inside and outside the cell. And similarly, we assume that (un)binding of ArsR to/from the ars promoter is much faster than the production of ArsR (for example).<br />
*The concentration of transporters is insignificant compared to the concentration of arsenic inside and outside the cell.<br />
<br />
This leads to the [[Team:Groningen/Glossary#MichaelisMenten|Michaelis-Menten]] equation for import, but also some more general equations for export using ArsB and accumulation with ArsR (for example, the Hill equation can be recognized in the activity of the ars promoter). We explicitly state ''relative abundances'' instead of substituting them into the differential equations. This leads to ''clearer and more insightful'' equations and gives ''more freedom'' to define complicated, interdependent ratios between substances.<br />
</div><br />
<br />
The inexperienced viewer may find the following tables and formulas baffling. We would recommend that one would look at the raw model first to gain an understanding of the basic reactions involved then have a look at the steady-state and the quasi steady-state model. It is not mandatory, but it is probably the the best route to get a better understanding of the model as a whole. Also, perhaps first have a look at [[Team:Groningen/Glossary#MichaelisMenten|Michaelis-Menten]] kinetics before proceeding.<br />
<br />
In contrast to how the quasi-steady-state assumption is normally used we mostly leave the specific states (bound/unbound) of substances intact in the differential equations and explicitly state the relative abundances. This keeps the differential equations shorter and gives more insight into what is actually happening, clearly mapping the "fast" reactions to ratios between substances. This also makes it possible to use quite complicated equations (the Asin and ArsR interdependency is virtually impossible to define using normal methods for example) that would otherwise be unwieldy to handle.<br />
<br />
[[Image:Arsenic_filtering.png|frame|A schematic representation of the processes involved in arsenic filtering (keep in mind that ArsR ''represses'' the expression of the genes behind ars). Note that MBPArsR and fMT are not shown for clarity.<!-- Also, ArsD is not shown here, as it is [[Team:Groningen/BLAST|not present in our E. coli]] and has a role analogous to ArsR.-->]]<br />
<br />
{|class="ourtable"<br />
|+ Reactions<br />
!colspan="2"|Reaction<br />
!Description<br />
!Rate<br />
|-<br />
|colspan="4"|''Transport''<br />
|-<br />
| ||As(III)<sub>ex</sub>T &rarr; As(III)<sub>in</sub>T||Import of arsenic.||(Vc/Vs) v5<sup>&dagger;</sup> As(III)<sub>ex</sub>T / (K5+As(III)<sub>ex</sub>T)<br />
|-<br />
| ||As(III)<sub>in</sub>T &rarr; As(III)<sub>ex</sub>T||Export of arsenic.|| k8 ArsB<sub>As</sub><br />
|-<br />
| ||style="white-space:nowrap;"|ars1T → ars1T + ArsBT||Production of ArsB.|| βB ars1<br />
|-<br />
| ||ArsBT &rarr; null||Degradation of ArsB|| (ln(2)/τB) ArsB<br />
|-<br />
|colspan="4"|''Accumulation''<br />
|-<br />
| ||ars1T → ars1T + ArsRT||From chromosomal operon.|| βRN ars1<br />
|-<br />
| ||proR → proR + ArsRT||Production of ArsR.|| βR pro<br />
|-<br />
| ||style="white-space:nowrap;"|proM → proM + MBPArsRT||Production of MBPArsR.|| βM pro<br />
|-<br />
| ||proF → proF + fMTT||Production of fMT.|| βF pro<br />
|-<br />
| ||ArsRT → null||Degradation of ArsR.|| (ln(2)/τR) ArsR<br />
|-<br />
| ||MBPArsRT → null||Degradation of MBPArsR.|| (ln(2)/τM) MBPArsR<br />
|-<br />
| ||fMTT → null||Degradation of fMT.|| (ln(2)/τF) fMT<br />
|-<br />
|colspan="4"|''Gas vesicles''<br />
|-<br />
| ||ars2T → ars2T + GV||Transcription + translation.|| βG ars2<br />
|-<br />
| ||GV → null||Degradation of gas vesicles.|| (ln(2)/τG) GV<br />
|}<br />
<br />
{|class="ourtable" style="clear:right;"<br />
|+ Core Substances<br />
!colspan="2"|Name<br />
!Description<br />
!Derivative to time<br />
|-<br />
|colspan="4"|''Extracellular''<br />
|-<br />
| ||As(III)<sub>ex</sub>T || As(III) in the solution. || (Vc/Vs) k8 ArsB<sub>As</sub> - (Vc/Vs) v5<sup>&dagger;</sup> As(III)<sub>ex</sub>T / (K5+As(III)<sub>ex</sub>T)<br />
|-<br />
|colspan="4"|''Membrane (all naturally occurring, but we plan to bring GlpF to overexpression)''<br />
|-<br />
| ||GlpFT || Importer of As(III) (concentration w.r.t. the exterior of the cell). || (not used directly in model, assumed to be constant)<br />
|-<br />
| ||ArsBT || Exporter of As(III) (concentration w.r.t. the interior of the cell). || βB ars1 - (ln(2)/τB) ArsB<br />
|-<br />
|colspan="4"|''Intracellular (ars2, pro and GV are introduced)''<br />
|-<br />
| ||As(III)<sub>in</sub>T || As(III) (bound and unbound) in the cell. || v5 As(III)<sub>ex</sub>T / (K5+As(III)<sub>ex</sub>T) - k8 ArsB<sub>As</sub><br />
|-class="estimate"<br />
| ||ars1T || ArsR repressed promoters (bound and unbound) naturally occurring in E. coli. || (concentration is constant = 1.6605nM, one per cell)<br />
|-class="estimate"<br />
| ||ars2T || ArsR repressed promoters in front of gas vesicle genes. || (concentration is constant = 0-166.05nM)<br />
|-class="estimate"<br />
| ||proR || Constitutive promoters in front of arsR. || (concentration is constant = 0-166.05nM)<br />
|-class="estimate"<br />
| ||proM || Constitutive promoters in front of mbp-arsR. || (concentration is constant = 0-166.05nM)<br />
|-class="estimate"<br />
| ||proF || Constitutive promoters in front of fMT. || (concentration is constant = 0-166.05nM)<br />
|-<br />
| ||ArsRT || ArsR in the cell. || βRN ars1 + βR proR - (ln(2)/τR) ArsR<br />
|-<br />
| ||MBPArsRT || MBPArsR in the cell. || βM proM - (ln(2)/τM) MBPArsR<br />
|-<br />
| ||fMTT || fMT in the cell. || βF proF - (ln(2)/τF) fMT<br />
|-<br />
| ||GV || Concentration of gas vesicles. || βG ars2 - (ln(2)/τG) GV<br />
|-style="border:none;"<br />
|colspan="4"|<br />
{|class="ourtable" style="width:100%"<br />
!colspan="5"|<br />
|- style="text-align:center;"<br />
|class="fromPaper" style="padding:0;"|Directly from paper.<br />
|class="selfDerived" style="padding:0;"|Based on data from paper.<br />
|class="experimental" style="padding:0;"|Based on experiment.<br />
|class="estimate" style="padding:0;"|Rough estimate.<br />
|class="unknown" style="padding:0;"|Totally unknown.<br />
|}<br />
|}<br />
<div style="text-align:right;font-size:smaller;"><sup>&dagger;</sup> Note that the "constant" v5 depends on the concentration of GlpF transporters in the cell, and this can depend on whether we bring GlpF to overexpression or not. For simplicity the production/degradation of GlpF is not included explicitly in the model, instead we can vary the constant v5 relative to the value found for wild-type E. coli.</div><br />
<br />
{|<br />
|style="vertical-align:top;"|<br />
{|class="ourtable"<br />
|+ Breakdown of core substances<br />
!Core substance<br />
!Component<br />
!Relative abundance<br />
|-<br />
|rowspan="2"|ArsBT<br />
|style="padding-left:0;"|ArsB<br />
|K7<br />
|-<br />
|ArsB<sub>As</sub><br />
|As(III)in<br />
|-<br />
|rowspan="4"|As(III)inT<br />
|style="padding-left:0;"|As(III)in<br />
|1<br />
|-<br />
|ArsR<sub>As</sub><br />
|ArsR / KR<sub>d</sub><br />
|-<br />
|MBPArsR<sub>As</sub><br />
|MBPArsRT / (KM<sub>d</sub> + As(III)in)<br />
|-<br />
|fMT<sub>As</sub><br />
|n<sub>f</sub> fMTT As(III)<sub>in</sub><sup>n<sub>f</sub>-1</sup> / (KF<sub>d</sub><sup>n<sub>f</sub></sup> + As(III)<sub>in</sub><sup>n<sub>f</sub></sup>)<br />
|-<br />
|rowspan="2"|arsT<br />
|style="padding-left:0;"|ars<br />
|KA<sub>d</sub>²<br />
|-<br />
|ArsR<sub>ars</sub><br />
|ArsR²<br />
|-<br />
|rowspan="2"|ars<br />
|style="padding-left:0;"|ars1<br />
|ars1T<br />
|-<br />
|ars2<br />
|ars2T<br />
|-<br />
|rowspan="3"|ArsRT<br />
|style="padding-left:0;"|ArsR<br />
|1<br />
|-<br />
|ArsR<sub>As</sub><br />
|As(III)<sub>in</sub> / KR<sub>d</sub><br />
|-<br />
|ArsR<sub>ars</sub><br />
|2 ArsR ars / KA<sub>d</sub>²<br />
|-<br />
|rowspan="2"|MBPArsRT<br />
|style="padding-left:0;"|MBPArsR<br />
|KM<sub>d</sub><br />
|-<br />
|MBPArsR<sub>As</sub><br />
|As(III)<sub>in</sub><br />
|-<br />
|rowspan="2"|fMTT<br />
|style="padding-left:0;"|fMT<br />
|KF<sub>d</sub><sup>n<sub>f</sub></sup><br />
|-<br />
|fMT<sub>As</sub><br />
|As(III)<sub>in</sub><sup>n<sub>f</sub></sup><br />
|}<br />
|[[Image:Arsenic Model - Substances.png|frame|Circles correspond to core substances. We consider the reactions between the overlapping substances so fast that we model them by determining the ratios between the substances when the reactions between them are in equilibrium. Also, the complexes formed with <strike>ars,</strike> GlpF and ArsB (the small circles) are considered to have such a low concentration that they are of no importance to the concentrations of As(III)in/-ex and ArsR (the large circles).]]<br />
|}<br />
<br />
{|class="ourtable"<br />
|+ Constants<br />
!Name<br />
!Units<br />
!Value<br />
!Description<br />
|-class="unknown"<br />
|k8<br />
|1/s<br />
|<br />
|Reaction rate constant representing how fast ArsB can export arsenic.<br />
|-class="estimate"<br />
|KR<sub>d</sub><br />
|M<br />
|6&micro;M<br />
|Dissociation constant for ArsR and As(III). Assumed to be about an order of magnitude smaller than KD<sub>d</sub> = 60&micro;M, the corresponding constant for the similar protein ArsD from [[Team:Groningen/Literature#Chen1997|Chen1997]].<br />
|-class="estimate"<br />
|KM<sub>d</sub><br />
|M<br />
|6&micro;M<br />
|Dissociation constant for MBPArsR and As(III). We assume this to be roughly equal to KR<sub>d</sub>.<br />
|-class="unknown"<br />
|KF<sub>d</sub><br />
|M<br />
|<br />
|Dissociation constant for fMT and As(III).<br />
|-class="unknown"<br />
|n<sub>f</sub><br />
|<br />
|<br />
|Hill coefficient for the formation of the complex fMTAs. This is related to the number of arsenic ions that bind to fMT.<br />
|-class="fromPaper"<br />
|KA<sub>d</sub><br />
|M<br />
|0.33&micro;M<br />
|Dissociation constants for ArsR and ars.<br />
* KA<sub>d</sub>² = kA<sub>off</sub>/kA<sub>on</sub> = (0.33&micro;M)²? ([[Team:Groningen/Literature#Chen1997|Chen1997]], suspect as the relevant reference doesn't actually seem to give any value for this)<br />
|-class="selfDerived"<br />
|v5<br />
|mol/(s&middot;L)<br />
|3.1863&micro;mol/(s·L)<br />
|Maximum import rate per liter of cells (see [[Team:Groningen/Glossary#MichaelisMenten|Michaelis-Menten equation]]). Note that we have purposefully chosen to write the units as mol/(s&middot;L) instead of M/s, to emphasize the fact that the rate is per liter of ''cells''.<br />
* v5 = k6 GlpFT (Vs/Vc)<br />
|-class="selfDerived"<br />
|K5<br />
|M<br />
|27.718&micro;M<br />
|Concentration at which import reaches half its maximum import rate (see [[Team:Groningen/Glossary#MichaelisMenten|Michaelis-Menten equation]]).<br />
* K5 = (k5off+k6) / k5on<br />
|-class="unknown"<br />
|K7<br />
|M<br />
|<br />
|Concentration at which export reaches half its maximum export rate (see [[Team:Groningen/Glossary#MichaelisMenten|Michaelis-Menten equation]]).<br />
* K7 = (k7off+k8) / k7on<br />
|-class="unknown"<br />
|&tau;B, &tau;R, &tau;G, etc.<br />
|s<br />
|<br />
|Half-lifes (of ArsB, ArsR and GV, respectively). Degradation rate = ln(2)/&tau; {{infoBox|1=If you take just the degradation into account you will have the equation dC/dt = -k*C, which leads to C(t) = C(0) e<sup>-k t</sup>. So if k = ln(2)/&tau; we get C(t) = C(0) e<sup>-ln(2)/&tau; t</sup> = C(0) 2<sup>-t/&tau;</sup>. In other words &tau; is the time it takes for the concentration to half.}}<br />
|-class="unknown"<br />
|&beta;B, &beta;R, etc.<br />
|1/s<br />
|<br />
|Production rates.<br />
* &beta;RN = the production rate for ArsR behind the ars1 promoter<br />
* &beta;B = the production rate for ArsB behind the ars1 promoter<br />
* &beta;G = the production rate for GV behind the ars2 promoter<br />
* &beta;R = the production rate for ArsR behind a constitutive promoter<br />
* &beta;M = the production rate for MBPArsR behind a constitutive promoter<br />
* &beta;F = the production rate for fMT behind a constitutive promoter<br />
|-<br />
|Vs<br />
|L<br />
|<br />
|Volume of solution (excluding cells).<br />
|-<br />
|Vc<br />
|L<br />
|<br />
|Total volume of cells (in solution) (so Vs+Vc is the total volume).<br />
|-style="border:none;"<br />
|colspan="4"|<br />
{|class="ourtable" style="width:100%"<br />
!colspan="5"|<br />
|- style="text-align:center;"<br />
|class="fromPaper" style="padding:0;"|Directly from paper.<br />
|class="selfDerived" style="padding:0;"|Based on data from paper.<br />
|class="experimental" style="padding:0;"|Based on experiment.<br />
|class="estimate" style="padding:0;"|Rough estimate.<br />
|class="unknown" style="padding:0;"|Totally unknown.<br />
|}<br />
|}<br />
<br />
<br />
==The raw model==<br />
<html><style type="text/css"></html><br />
.import { background: LightGreen; }<br />
.export { background: LightBlue; }<br />
.accumulation { background: LightPink; }<br />
.production { background: LightGoldenRodYellow; }<br />
<html></style></html><br />
<br />
The following table gives all the reactions that take place inside the cell. You can look at the schematic representation of the processes involved to get a good grasp as how every reaction works to the other. Note that proR, ProM and MBPArsR, ProF and Fmt are not displayed in the figure. This has been done for clarity. These reactions are simple constituative promotor reactions. Once you have an insight in the reactions involved you can have a look at the next table.<br />
<br />
[[Image:Arsenic_filtering.png|frame|A schematic representation of the processes involved in arsenic filtering (keep in mind that ArsR ''represses'' the expression of the genes behind ars). Note that MBPArsR and fMT are not shown for clarity.<!-- Also, ArsD is not shown here, as it is [[Team:Groningen/BLAST|not present in our E. coli]] and has a role analogous to ArsR.-->]]<br />
<br />
{|class="ourtable"<br />
|+ Reactions<br />
!colspan="2"|Reaction<br />
!Description<br />
|-<br />
|colspan="3"|''Transport''{{infoBox|In the reactions below you can see the import of arsenic by GlpF and the export of arsenic by ArsB. Only the degradation of ArsB is taken into acount because the ars operon also produces ArsB, as can be seen in the accumulation section. We assume a constant number of GlpF importers. }}(based on [[Team:Groningen/Literature#Rosen1996|Rosen1996]], [[Team:Groningen/Literature#Meng2004|Meng2004]] and [[Team:Groningen/Literature#Rosen2009|Rosen2009]])<br />
|-<br />
| ||<span class="import">As(III)<sub>ex</sub> + GlpF &harr; GlpF<sub>As</sub></span>||The binding and detachment of arsenic to GlpF on the outside of the cell.||<br />
|-<br />
| ||<span class="import">GlpF<sub>As</sub> &rarr; GlpF + As(III)</span>||The release of arsenic on the inside of the cell by GlpF|| <br />
|-<br />
| ||<span class="export">As(III)<sub>in</sub> + ArsB &harr; ArsB<sub>As</sub></span>||The binding and detachment of arsenic to the Exporter ArsB|| <br />
|-<br />
| ||<span class="export">ArsB<sub>As</sub> &rarr; ArsB + As(III)<sub>ex</sub></span>||The release of the bound arsenic by ArsB on the outside of the cell.|| <br />
|-<br />
| ||<span class="export">ArsB &rarr; null</span> ||The degradation of Ars B||<br />
|-<br />
|colspan="3"|''Accumulation''{{infoBox|In the reactions below you can see the production and degradation of all our accumulation proteins. Two things should be noticed: ArsR represses it's own production and that of the GVP clusters and the ars1 operon does not only produce ArsR but also the exporter ArsB}}(mostly based on [[Team:Groningen/Literature#Chen1997|Chen1997]])<br />
|-<br />
| ||As(III)<sub>in</sub> + ArsR &harr; ArsR<sub>As</sub>||The binding and detachment of arsenic to ArsR||<br />
|-<br />
| ||As(III)<sub>in</sub> + MBPArsR &harr; MBPArsR<sub>As</sub>||The binding and detachment of arsenic to MBPArsR || <br />
|-<br />
| ||n<sub>f</sub> As(III)<sub>in</sub> + fMT &harr; fMT<sub>As</sub>||The binding and detachment of arsenic to fMT || <br />
|-<br />
| ||ars1 + 2 ArsR &harr; ArsR<sub>ars1</sub>||the repression of the promotor of the ars1 operon by 2 arsR molecules||<br />
|-<br />
| ||<span class="production">ars2 + 2 ArsR &harr; ArsR<sub>ars2</sub></span>||the repression of the promotor of the ars1 operon by 2 arsR molecules|| <br />
|-<br />
| ||ars1 &rarr; ars1 + ArsR<span class="export"> + ArsB</span> ||The transcription and translation of the ars1 operon to produce ArsR and ArsB||<br />
|-<br />
| ||proR &rarr; proR + ArsR ||The transcription and translation of the proR operon to produce ArsR||<br />
|-<br />
| ||proM &rarr; proM + MBPArsR ||The transcription and translation of the proM operon to produce MBPArsR||<br />
|-<br />
| ||proF &rarr; proF + fMT ||The transcription and translation of the proF operon to produce fMT||<br />
|-<br />
| ||ArsR &rarr; null ||The degradation of ArsR||<br />
|-<br />
| ||MBPArsR &rarr; null ||The degradation of MBPArsR||<br />
|-<br />
| ||fMT &rarr; null ||The degradation of fMT||<br />
|-<br />
|colspan="3"|''Gas vesicles''{{infoBox|These two reactions give the production and degradation rate of the GVP clusters. Keep in mind that ars2 is repressed by the accumulation protein ArsR. This reaction can be found under accumulation part.}}<br />
|-<br />
| ||ars2 &rarr; ars2<span class="production"> + GV</span> ||The transcription and translation of the ars2 operon to produce GVP clusters wich will make the cell float||<br />
|-<br />
| ||<span class="production">GV &rarr; null</span> ||The degradation of GVP||<br />
|-<br />
|colspan="3"|<br />
{|class="ourtable" style="width:100%"<br />
!colspan="5"|<br />
|- style="text-align:center;"<br />
|class="fromPaper" style="padding:0;"|Import related.<br />
|class="fromPaper" style="padding:0;"|Import related.<br />
|class="experimental" style="padding:0;"|Export related.<br />
|class="experimental" style="padding:0;"|Export related.<br />
|class="estimate" style="padding:0;"|GVP Production related.<br />
|}<br />
|}<br />
<br />
Here you can find the time derivatives for each substance we derived. The constants are explained in the next teble. After one has a full understanding of all the constants and derivatives and and reactions. One can begin the process of simplifying the model and thus one can have a look at the quasi steady-state model and the steady-state model. <br />
<br />
{|class="ourtable"<br />
|+ Core substances<br />
!colspan="2"|substance<br />
!Description<br />
!Derivative to time<br />
|-<br />
|colspan="4"|''Extracellular''<br />
|-<br />
| ||As(III)<sub>ex</sub>||As(III) in the solution||(d/dt) As(III)<sub>ex</sub> = <span class="import">- (d/dt) GlpF<sub>As</sub> - k6 GlpF<sub>As</sub></span><span class="export"> + (Vc/Vs) k8 ArsB<sub>As</sub></span><br />
|-<br />
|colspan="4"|''Membrane'' (all naturally occurring, but we plan to bring GlpF to overexpression)<br />
|-<br />
| ||GlpF||concentration w.r.t. the exterior of the cell||(d/dt) GlpF = <span class="import">- (d/dt) GlpF<sub>As</sub></span><br />
|-<br />
| ||GlpF<sub>As</sub>||concentration w.r.t. the exterior of the cell||(d/dt) GlpF<sub>As</sub> = <span class="import">k5<sub>on</sub> As(III)<sub>ex</sub> GlpF - (k5<sub>off</sub>+k6) GlpF<sub>As</sub></span><br />
|-<br />
| ||ArsB||concentration w.r.t. the interior of the cell||(d/dt) ArsB = <span class="export">- (d/dt) ArsB<sub>As</sub> + &beta;4 ars1 - ln(2)/&tau;B ArsB</span><br />
|-<br />
| ||ArsB<sub>As</sub> ||concentration w.r.t. the interior of the cell||(d/dt) ArsB<sub>As</sub> = <span class="export">k7<sub>on</sub> As(III)<sub>in</sub> ArsB - (k7<sub>off</sub>+k8) ArsB<sub>As</sub></span><br />
|-<br />
|colspan="4"|''Intracellular'' (ars2, pro and GV are introduced)<br />
|-<br />
| ||As(III)<sub>in</sub>||concentration of As(III) inside the cell||(d/dt) As(III)<sub>in</sub> = - (d/dt) ArsR<sub>As</sub> - (d/dt) MBPArsR<sub>As</sub> - n<sub>f</sub> (d/dt) fMT<sub>As</sub><span class="export"> - (d/dt) ArsB<sub>As</sub> - k8 ArsB<sub>As</sub></span><span class="import"> + (Vs/Vc) k6 GlpF<sub>As</sub></span><br />
|-<br />
| ||ars1 {{infoBox|ars1 stands for the promotor in front of the operon which contains the information for the production of the accumulation protein ArsR and the exporter ArsB. It is selfregulatory in the sence that it produces it's own repressor in the form of ArsR}} ||concentration of unbound promoters naturally occurring in <i>E. coli</i>||(d/dt) ars1 = - (d/dt) ArsR<sub>ars1</sub><br />
|-<br />
| ||ars2 {{infoBox|ars2 stands for the promotor in front of the operon which contains the information for the production of Gas Vesicles. Unlike ars 1 it is not selfregulatory, but the if everything goes correctly the production of gas vesicles will only start if there arsenic inside the cell}}||concentration of unbound promoters in front of gas vesicle genes||(d/dt) ars2 = <span class="production">- (d/dt) ArsR<sub>ars2</sub></span><br />
|-<br />
| ||proR ||concentration of constitutive promoters in front of arsR|| (d/dt)proR = 0 in our model<br />
|-<br />
| ||proM ||concentration of constitutive promoters in front of mbp-arsR|| (d/dt)proM = 0 in our model<br />
|-<br />
| ||proF ||concentration of constitutive promoters in front of fMT|| (d/dt)proF = 0 in our model<br />
|-<br />
| ||ArsR {{infoBox|ArsR binds to ars to repress production of the genes they regulate, and binds to As(III) to make it less of a problem for the cell.}}||concentration of the accumulation protein ArsR||(d/dt) ArsR = &beta;RN ars1 + &beta;R proR - (ln(2)/&tau;R) ArsR - (d/dt) ArsR<sub>As</sub> - 2 (d/dt) ArsR<sub>ars1</sub><span class="production"> - 2 (d/dt) ArsR<sub>ars2</sub></span> <br />
|-<br />
| ||ArsR<sub>As</sub> || the concentration of ArsR bound to As(III)||(d/dt) ArsR<sub>As</sub> = kR<sub>on</sub> ArsR As(III)<sub>in</sub> - kR<sub>off</sub> ArsR<sub>As</sub><br />
|-<br />
| ||ArsR<sub>ars1</sub> ||the concentration of ArsR bound to ars1||(d/dt) ArsR<sub>ars1</sub> = kA<sub>on</sub> ArsR&sup2; ars1 - kA<sub>off</sub> ArsR<sub>ars1</sub><br />
|-<br />
| ||ArsR<sub>ars2</sub> ||the concentration of ArsR bound to ars2||(d/dt) ArsR<sub>ars2</sub> = <span class="production">kA<sub>on</sub> ArsR&sup2; ars2 - kA<sub>off</sub> ArsR<sub>ars2</sub></span><br />
|-<br />
| ||MBPArsR {{infoBox|A fusion of maltose binding protein and ArsR. It is more stable than the normal ArsR variant, but it is no longer able to act as a repressor for the ars promotor.}}|| a fusion of maltose binding protein and ArsR||(d/dt) MBPArsR = &beta;M proM - (ln(2)/&tau;M) MBPArsR - (d/dt) MBPArsR<sub>As</sub><br />
|-<br />
| ||MBPArsR<sub>As</sub> ||bound to As(III)||(d/dt) MBPArsR<sub>As</sub> = kM<sub>on</sub> MBPArsR As(III)<sub>in</sub> - kM<sub>off</sub> MBPArsR<sub>As</sub><br />
|-<br />
| ||fMT {{infoBox|It is another binding protein. Unlike it's counterpart it capeble of containing up to five As(III) particles or one As(V) particle }} || Arsenic binding metallotein ||(d/dt) fMT = &beta;F proF - (ln(2)/&tau;F) fMT - (d/dt) fMT<sub>As</sub><br />
|-<br />
| ||fMT<sub>As</sub> ||bound to multiple As(III)||fMT<sub>As</sub> = kF<sub>on</sub> fMT As(III)<sub>in</sub><sup>n<sub>f</sub></sup> - kF<sub>off</sub> fMT<sub>As</sub><br />
|-<br />
| ||ArsR<sub>As</sub> ||bound to As(III)<br />
|-<br />
| ||GV ||concentration of gas vesicles||(d/dt) GV = <span class="production">&beta;G ars2 - ln(2)/&tau;G GV</span><br />
|-<br />
|colspan="4"|<br />
{|class="ourtable" style="width:100%"<br />
!colspan="5"|<br />
|- style="text-align:center;"<br />
|class="fromPaper" style="padding:0;"|Import related.<br />
|class="fromPaper" style="padding:0;"|Import related.<br />
|class="experimental" style="padding:0;"|Export related.<br />
|class="experimental" style="padding:0;"|Export related.<br />
|class="estimate" style="padding:0;"|GVP Production related.<br />
|}<br />
|}<br />
<br />
The variables above can be related to each other through the following "reactions" (color coding is continued below to show which parts of the differential equations refer to which groups of reactions):<br />
<br />
<br />
Using the following constants/definitions:<br />
{|class="ourtable"<br />
|-<br />
!Name<br />
!Units<br />
!Description<br />
|-<br />
|kRon, kMon, k5on, etc.<br />
|1/(M&middot;s)<br />
|Reaction rate constants for reactions to a complex.<br />
|-<br />
|kAon<br />
|1/(M²&middot;s)<br />
|Reaction rate constants for reactions to a complex.<br />
|-<br />
|kFon<br />
|1/(M<sup>n<sub>f</sub></sup>&middot;s)<br />
|Reaction rate constants for reactions to a complex.<br />
|-<br />
|kRoff, kMoff, kFoff, kAoff, k5off, etc.<br />
|1/s<br />
|Reaction rate constants for reactions from a complex.<br />
|-<br />
|k6, k8<br />
|1/s<br />
|Reaction rate constants representing how fast transporters transport their cargo to "the other side".<br />
|-<br />
|&tau;B, &tau;R, &tau;M, &tau;F, &tau;G<br />
|s<br />
|Half-lifes (of ArsB, ArsR, MBPArsR, fMT and GV, respectively). Degradation rate = ln(2)/&tau; {{infoBox|1=If you take just the degradation into account you will have the equation dC/dt = -k*C, which leads to C(t) = C(0) e<sup>-k t</sup>. So if k = ln(2)/&tau; we get C(t) = C(0) e<sup>-ln(2)/&tau; t</sup> = C(0) 2<sup>-t/&tau;</sup>. In other words &tau; is the time it takes for the concentration to half.}}<br />
|-<br />
|&beta;RN, &beta;R, etc.<br />
|1/s<br />
|Production rates.<br />
* &beta;RN = the production rate for ArsR behind the ars1 promoter<br />
* &beta;B = the production rate for ArsB behind the ars1 promoter<br />
* &beta;G = the production rate for GV behind the ars2 promoter<br />
* &beta;R = the production rate for ArsR behind a constitutive promoter<br />
* &beta;M = the production rate for MBPArsR behind a constitutive promoter<br />
* &beta;F = the production rate for fMT behind a constitutive promoter<br />
|-<br />
|Vs<br />
|L<br />
|Volume of solution (excluding cells).<br />
|-<br />
|Vc<br />
|L<br />
|Total volume of cells (in solution) (so Vs+Vc is the total volume).<br />
|}<br />
See [[Team:Groningen/Literature#Chen1997|Chen1997]] for the interplay between ArsR and ArsD (the latter has a role similar to ArsR, but we do not treat it, as it is [[Team:Groningen/BLAST|not present in our system]]).<br />
<br />
==Quasi steady state{{anchor|QuasiSteadyState}}==<br />
First of all, we assume the concentration of transporters is quite low compared to the concentration of the transported substances. After all, if this were not the case the transporters would act more like "storage" proteins than transporters (note that this can be even more rigorously justified if, for example, GlpFT<<K5). This leads to:<br />
<br />
<pre><br />
As(III)exT &asymp; As(III)ex<br />
As(III)inT &asymp; As(III)in + ArsRAs + MBPArsRAs + nf fMTAs<br />
</pre><br />
<br />
Also, we assume the binding and unbinding of molecules to the transporters occurs on a much finer time-scale than any actual changes to the concentrations inside and outside the cell. Similarly, within the cell we assume diffusion processes are very fast and binding/unbinding of substances is quite fast compared to the production of proteins. This leads us to assume that the following ratios between substances are constantly in equilibrium:<br />
<br />
{{frame|1=<br />
<div style="text-align:left;"><br />
We use the following when grouping the ars promoters:<br />
<pre><br />
arsT = ars + ArsRars<br />
ars1 / ars1T = ars2 / ars2T<br />
<br />
ars = ars1 + ars2<br />
ars = ars1 (1 + ars2T / ars1T)<br />
ars1 = ars / (1 + ars2T / ars1T)<br />
ars1 = ars ars1T / arsT<br />
<br />
ars2 = ars ars2T / arsT<br />
</pre><br />
</div><br />
}}<br />
<br />
<pre><br />
As(III)ex : GlpFAs &asymp; As(III)ex : 0<br />
GlpF : GlpFAs<br />
ArsB : ArsBAs<br />
As(III)in : ArsRAs : MBPArsRAs : nf fMTAs : ArsBAs &asymp; As(III)in : ArsRAs : MBPArsRAs : nf fMTAs : 0<br />
ArsR : ArsRAs : 2 ArsRars<br />
ars : ArsRars<br />
</pre><br />
<br />
To determine what the unknown ratios are we can set the following derivatives to zero (these are the derivatives of the complexes corresponding to the four overlapping regions in the diagram):<br />
<br />
<pre><br />
0 = (d/dt) GlpFAs = k5on As(III)ex GlpF - (k5off+k6) GlpFAs<br />
0 = (d/dt) ArsBAs = k7on As(III)in ArsB - (k7off+k8) ArsBAs<br />
0 = (d/dt) ArsRars = kAon ArsR² ars - kAoff ArsRars<br />
0 = (d/dt) ArsRAs = kRon ArsR As(III)in - kRoff ArsRAs<br />
0 = (d/dt) MBPArsRAs = kMon MBPArsR As(III)in - kMoff MBPArsRAs<br />
0 = (d/dt) fMTAs = kFon fMT As(III)in^nf - kFoff fMTAs<br />
</pre><br />
<br />
The first two derivates let us determine the ratios between bound and unbound transporters:<br />
<br />
<pre><br />
0 = (d/dt) GlpFAs = k5on As(III)ex GlpF - (k5off+k6) GlpFAs<br />
<br />
k5on As(III)ex GlpF = (k5off+k6) GlpFAs<br />
GlpF = (k5off+k6)/k5on GlpFAs / As(III)ex<br />
GlpF = K5 GlpFAs / As(III)ex<br />
<br />
GlpF : GlpFAs<br />
K5 GlpFAs / As(III)ex : GlpFAs<br />
K5 : As(III)ex<br />
<br />
0 = (d/dt) ArsBAs = k7on As(III)in ArsB - (k7off+k8) ArsBAs<br />
<br />
k7on As(III)in ArsB = (k7off+k8) ArsBAs<br />
ArsB = (k7off+k8)/k7on ArsBAs / As(III)in<br />
ArsB = K7 ArsBAs / As(III)in<br />
<br />
ArsB : ArsBAs<br />
K7 ArsBAs / As(III)in : ArsBAs<br />
K7 : As(III)in<br />
</pre><br />
<br />
The next two differential equations can be used to determine the relative abundances of ArsR and ArsRAs, and ars and ArsRars:<br />
<br />
<pre><br />
0 = (d/dt) ArsRAs = kRon ArsR As(III)in - kRoff ArsRAs<br />
<br />
kRon ArsR As(III)in = kRoff ArsRAs<br />
ArsRAs = kRon/kRoff ArsR As(III)in<br />
ArsRAs = ArsR As(III)in / KRd<br />
<br />
ArsR : ArsRAs<br />
ArsR : ArsR As(III)in / KRd<br />
KRd : As(III)in<br />
<br />
0 = (d/dt) ArsRars = kAon ArsR² ars - kAoff ArsRars<br />
<br />
kAon ArsR² ars = kAoff ArsRars<br />
ArsRars = kAon/kAoff ArsR² ars<br />
ArsRars = ArsR² ars / KAd²<br />
<br />
ArsR : 2 ArsRars<br />
ArsR : 2 ArsR² ars / KAd²<br />
KAd² : 2 ArsR ars<br />
<br />
ars : ArsRars<br />
ars : ArsR² ars / KAd²<br />
KAd² : ArsR²<br />
</pre><br />
<br />
For MBPArsR and fMT we find:<br />
<br />
<pre><br />
0 = (d/dt) MBPArsRAs = kMon MBPArsR As(III)in - kMoff MBPArsRAs<br />
<br />
MBPArsR : MBPArsRAs = KMd : As(III)in<br />
<br />
0 = (d/dt) fMTAs = kFon fMT As(III)in^nf - kFoff fMTAs<br />
<br />
fMT : fMTAs = KFd^nf : As(III)in^nf<br />
</pre><br />
<br />
And finally the relative abundances of arsenic:<br />
<br />
<pre><br />
ArsRAs = ArsR As(III)in / KRd<br />
<br />
As(III)in : ArsRAs : MBPArsRAs : n fMTAs<br />
As(III)in : ArsR As(III)in / KRd : MBPArsRT As(III)in / (KMd+As(III)in) : n fMTT As(III)in^nf / (KFd^nf+As(III)in^nf)<br />
1 : ArsR / KRd : MBPArsRT / (KMd+As(III)in) : n fMTT As(III)in^(nf-1) / (KFd^nf+As(III)in^nf)<br />
</pre><br />
<br />
Summarizing:<br />
<br />
<pre><br />
GlpF : GlpFAs = K5 : As(III)ex<br />
ArsB : ArsBAs = K7 : As(III)in<br />
As(III)in : ArsRAs : MBPArsRAs : n fMTAs &asymp; 1 : ArsR / KRd : MBPArsRT / (KMd+As(III)in) : n fMTT As(III)in^(nf-1) / (KFd^nf+As(III)in^nf)<br />
ars : ArsRars = KAd² : ArsR²<br />
ArsR : ArsRAs : 2 ArsRars &asymp; 1 : As(III)in / KRd : 2 ArsR ars / KAd²<br />
MBPArsR : MBPArsRAs = KMd : As(III)in<br />
fMT : fMTAs = KFd^nf : As(III)in^nf<br />
</pre><br />
<br />
Now we can look at the differential equations for the totals of ArsB (so ArsBT=ArsB+ArsBAs), ArsR, As(III)in and As(III)ex (GlpFT and arsT are assumed to be constant):<br />
<br />
<pre><br />
(d/dt) As(III)exT = (d/dt) As(III)ex + (d/dt) GlpFAs<br />
= - (d/dt) GlpFAs - k6 GlpFAs + (Vc/Vs) k8 ArsBAs + (d/dt) GlpFAs<br />
= (Vc/Vs) k8 ArsBAs - k6 GlpFAs<br />
= (Vc/Vs) k8 ArsBAs - (Vc/Vs) v5 GlpFAs / GlpFT<br />
= (Vc/Vs) k8 ArsBAs - (Vc/Vs) v5 As(III)ex / (K5+As(III)ex)<br />
= (Vc/Vs) k8 ArsBAs - (Vc/Vs) v5 As(III)exT / (K5+As(III)exT)<br />
(d/dt) ArsBT = (d/dt) ArsB + (d/dt) ArsBAs<br />
= - (d/dt) ArsBAs + βB ars1 - ln(2)/τB ArsB + (d/dt) ArsBAs<br />
= βB ars1 - ln(2)/τB ArsB<br />
(d/dt) As(III)inT = -(Vs/Vc) (d/dt) As(III)exT<br />
= v5 As(III)exT / (K5+As(III)exT) - k8 ArsBT As(III)in / (K7+As(III)in)<br />
(d/dt) ArsRT = (d/dt) ArsR + (d/dt) ArsRAs + 2 (d/dt) ArsRars<br />
= βRN ars1 + βR proR - (ln(2)/τR) ArsR - (d/dt) ArsRAs - 2 (d/dt) ArsRars + (d/dt) ArsRAs + 2 (d/dt) ArsRars<br />
= βRN ars1 + βR proR - (ln(2)/τR) ArsR<br />
(d/dt) MBPArsRT = (d/dt) MBPArsR + (d/dt) MBPArsRAs<br />
= βM proM - (ln(2)/τM) MBPArsR<br />
(d/dt) fMTT = (d/dt) fMT + (d/dt) fMTAs<br />
= βF proF - (ln(2)/τF) fMT<br />
</pre><br />
<br />
==Steady state==<br />
By looking at the steady state of the system we can say something about its long-term behaviour. This also makes it easier to analyze relations between variables. To derive the steady state solution we take the quasi steady state solution and simplify it further by setting additional derivatives to zero:<br />
<br />
<pre><br />
0 = (d/dt) ArsBT = βB ars1 - ln(2)/τB ArsB<br />
0 = (d/dt) As(III)inT = v5 As(III)exT / (K5+As(III)exT) - k8 ArsBAs<br />
0 = (d/dt) ArsRT = βRN ars1 + βR pro - (ln(2)/τR) ArsR<br />
0 = (d/dt) MBPArsRT = βM proM - (ln(2)/τM) MBPArsR<br />
0 = (d/dt) fMTT = βF proF - (ln(2)/τF) fMT<br />
0 = (d/dt) GV = βG ars2 - ln(2)/τG GV<br />
</pre><br />
<br />
This directly leads to:<br />
<br />
<pre><br />
0 = βB ars1 - ln(2)/τB ArsB<br />
ArsB = βB (τB/ln(2)) ars1<br />
ArsB = βB (τB/ln(2)) ars1T KAd²/(KAd²+ArsR²)<br />
<br />
0 = βM proM - (ln(2)/τM) MBPArsR<br />
MBPArsR = βM (τM/ln(2)) proM<br />
<br />
0 = βF proF - (ln(2)/τF) fMT<br />
fMT = βF (τF/ln(2)) proF<br />
<br />
0 = βG ars2 - ln(2)/τG GV<br />
GV = βG (τB/ln(2)) ars2<br />
GV = βG (τB/ln(2)) ars2T KAd²/(KAd²+ArsR²)<br />
</pre><br />
<br />
For the intra- and extracellular concentrations we can find the following equation, giving a maximum for As(III)in of <code>K7 v5/(k8 ArsB)</code> (as As(III)exT cannot be negative){{infoBox|Conveniently the function <code>x/(c-x)</code> is non-negative and non-decreasing for x&isin;[0,c&rang;.}}:<br />
<br />
<pre><br />
0 = v5 As(III)exT / (K5+As(III)exT) - k8 ArsBAs<br />
0 = v5 As(III)exT / (K5+As(III)exT) - k8 ArsB As(III)in / K7<br />
0 = v5 As(III)exT - k8 ArsB As(III)in / K7 (K5+As(III)exT)<br />
0 = v5 As(III)exT - k8 ArsB As(III)in As(III)exT / K7 - k8 ArsB As(III)in K5 / K7<br />
0 = As(III)exT (v5 - k8 ArsB As(III)in / K7) - k8 ArsB As(III)in K5 / K7<br />
As(III)exT = k8 ArsB As(III)in K5 / (v5 K7 - k8 ArsB As(III)in)<br />
As(III)exT = K5 As(III)in / (K7 v5/(k8 ArsB) - As(III)in)<br />
</pre><br />
<br />
As we can safely assume arsenic neither disappears into nothingness nor appears from nothingness, we can use this to derive (As(III)T is the total amount of arsenic):<br />
<br />
<pre><br />
As(III)inT = As(III)in (1 + ArsR/KRd + MBPArsR/KMd + fMT As(III)in^(nf-1)/KFd^nf)<br />
<br />
As(III)T = Vs As(III)exT + Vc As(III)inT<br />
0 = Vs As(III)exT + Vc As(III)inT - As(III)T<br />
0 = Vs K5 As(III)in / (K7 v5/(k8 ArsB) - As(III)in) + Vc As(III)in (1 + ArsR/KRd + MBPArsR/KMd + fMT As(III)in^(nf-1)/KFd^nf) - As(III)T<br />
</pre><br />
<br />
As the function on the right-hand side is non-decreasing for <code>As(III)in&isin;[0,K7 v5/(k8 ArsB)&rang;</code> it at most has one zero on this interval (and it has one, as it starts at a negative value and gets arbitrarily large as As(III)in approaches the end of its range). So this zero can safely be found using any number of numerical methods.<br />
<br />
Finally, for ArsR we can find the following third-order equation:<br />
<br />
<pre><br />
0 = βRN ars1 + βR pro - (ln(2)/τR) ArsR<br />
0 = βRN ars1T KAd²/(KAd²+ArsR²) + βR pro - (ln(2)/τR) ArsR<br />
0 = βRN ars1T KAd² + βR pro (KAd²+ArsR²) - (ln(2)/τR) ArsR (KAd²+ArsR²)<br />
0 = βRN ars1T KAd² + βR pro KAd² + βR pro ArsR² - (ln(2)/τR) ArsR KAd² - (ln(2)/τR) ArsR³<br />
0 = (βRN ars1T + βR pro) KAd² - (ln(2)/τR) KAd² ArsR + βR pro ArsR² - (ln(2)/τR) ArsR³<br />
0 = (βRN ars1T + βR pro) (τR/ln(2)) KAd² - KAd² ArsR + βR (τR/ln(2)) pro ArsR² - ArsR³<br />
</pre><br />
<br />
According to Mathematica's solution of <code>Reduce[eq && KAd > 0 && arsT >= 0 && pro >= 0 && &beta;1 > 0 && &beta;3 > 0 && &tau;R > 0, ArsR, Reals]</code> (where eq is the equation shown above) there is only one real solution (examining the discriminant of eq confirms this), so we can solve the equation safely using Newton's (or Halley's) method.<br />
<br />
{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/PublicityTeam:Groningen/Publicity2009-11-17T18:27:08Z<p>Wilfred: /* News */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
=Websites=<br />
*"<i>Synthetic biology students from Groningen in the race for best bacterium</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*"<i>Students from Groningen are making bacteria to remove heavy metals from water</i>" (Dutch), [http://www.groningenreporter.nl/story.php?title=groninger-studenten-maken-bacteri%3Fn-die-water-zuiveren-van-zware-metalen Groningen Reporter (August 22, 2009)]<br />
*"<i>Team iGEM Groningen wins golden medal</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*"<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
<br />
=News=<br />
*"<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*"<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Press release University Groningen (November 11, 2009)]<br />
*"<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*"<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*"<i>With a pacmanbacterium in de finals</i>" (Dutch), [http://www.uk.rug.nl/archief/jaargang39/11/12e.php UK (Paper of the Groningen University) (November 12, 2009)]<br />
*"<i>iGEM Gold for Dutch</i>" (Dutch), [http://www.bionieuws.nl/artikel.php?id=4918&zoek=iGEM Bionieuws (November 14, 2009)]<br />
<br />
=Radio=<br />
*Interview [http://www.oogtv.nl/ Oog Radio] - <i>Stad vandaag (News- and background story program)</i> (Dutch) (November 10, 2009)<br />
<br />
=Our Brochure=<br />
<center>[[Image:Inside_Brochure_iGEM_GRONINGEN.jpg|800px]]<br />
[[Image:Outside_Brochure_iGEM_GRONINGEN.jpg|800px]]</center><br />
{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/IGEM_PublicityIGEM Publicity2009-11-17T18:25:57Z<p>Wilfred: Groningen & Delft in Bionieuws</p>
<hr />
<div>__NOTOC__<br />
[https://2008.igem.org/IGEM_Publicity iGEM 2008 Publicity] | [http://parts.mit.edu/igem07/index.php/Media iGEM 2007 Publicity] | [http://parts.mit.edu/wiki/index.php/IGEM_News iGEM 2006 Publicity]<br />
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<span style="color:#1e90ff; font-size:175%">'''blogs </span><span style="color:#3cb371; font-size:175%">covering iGEM 2009'''</span><br />
* The [http://igem.sdu.dk/ <span style="color:#453221; font-size: 130%">'''SDU-Denmark'''</span>] team blog about their iGEM experience.<br />
* A blog about the iGEM project of the [http://aboutgmos.org/iGEM <span style="color:#453221; font-size: 130%">'''Uppsala-Sweden'''</span>] team.<br />
<br />
<br />
<br />
<br />
====<font size=5><font color=dodgerblue>'''video/radio</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
*'''Slovenia, Heidelberg, SDU-Denmark''': [http://www.dradio.de/dlf/sendungen/forschak/1062608/ Die Biobastler von Boston], Nov 2, 2009, Deutschlandfunk radio (in German).<br />
*'''Cambridge, Imperial''': [http://www.sciencefriday.com/program/archives/200911063 Synthetic Biology Competition], Nov 6, 2009, NPR Science Friday (in English).<br />
<br />
<br />
<br />
<br />
====<font size=5><font color=dodgerblue>'''news articles</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
<br />
<br />
If you would like to share an article that was written about iGEM or your iGEM team, please link to it on this page. If you have multiple articles featuring your team, link to them all individually!<br />
<br />
Post the name of your team, the title of your article, where it was featured, and provide a link to it. <br />
<br />
''Example'':<br> <br />
'''Team Example''': ''Title of article'', Nature, [link]<br />
<br />
====<font size=4><font color=dodgerblue>'''general'''</font></font>====<br />
*'''The Economist''': ''Biohacking: Hacking goes squishy'' Sep 3, 2009 [http://www.economist.com/search/displaystory.cfm?story_id=14299634 The Economist]<br />
*'''The Scientist''': ''Brick by Brick'' Feb 1, 2009. [http://www.the-scientist.com/article/display/55378/ The Scientist]<br />
*'''Technology Review''': ''A Genetically Engineered Rainbow of Bacteria'' Nov 03, 2009. [http://www.technologyreview.com/blog/editors/24351/ Technology Review]<br />
*'''Discovery News''': '' Bright Bacteria Wins Synthetic Biology Competition'' Nov 6, 2009. [http://blogs.discoverychannel.co.uk/discovery-news/2009/11/bright-bacteria-wins-synthetic-biology-competition.html Discovery News]<br />
*'''Wired''': '' Building new life forms at the iGEM Jamboree'' Nov 9, 2009. [http://www.wired.co.uk/news/archive/2009-11/09/building-new-life-forms-at-the-igem-jamboree.aspx Wired]<br />
<br />
====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
<br />
*'''[[Team:IBB_Pune]]''': "<i>Bio-champs in the making</i>" (English), [http://www.punemirror.in/index.aspx?Page=article&sectname=News%20-%20City&sectid=2&contentid=200909142009091423331646a131eacc#ftr2 (Monday, September 14, 2009 at 11:33:25 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Ganiti Prakriyanmadhe Jeevanuncha Vaapar! (Use of Bacteria in mathematical devices)</i>" (Marathi), [http://beta.esakal.com/2009/09/09220618/pune-use-of-bacterias-in-maths.html(Wednesday, September 09th, 2009 AT 10:09 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Synthetic Biologiteel Bharari ("Advances in Synthetic Biology")</i>" (Marathi), [https://2009.igem.org/Team:IBB_Pune/press (Wednesday 7 October 2009)]<br />
<br />
*'''[[Team:Groningen]]''': "<i>Synthetic biology students from Groningen in the race for best bacterium</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*'''[[Team:Groningen]]''': "<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Team iGEM Groningen wins golden medal</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Press release University Groningen (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>With a pacmanbacterium in de finals</i>" (Dutch), [http://www.uk.rug.nl/archief/jaargang39/11/12e.php UK (Paper of the Groningen University) (November 12, 2009)]<br />
*'''[[Team:Groningen]]''' & '''[[Team:TUDelft]]''': "<i>iGEM Gold for Dutch</i>" (Dutch), [http://www.bionieuws.nl/artikel.php?id=4918&zoek=iGEM Bionieuws (November 14, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Un concours organisé par le M.I.T : en route pour Boston ! </i>" (French), [http://www.supbiotech.fr/2009/09/boston-supbiotech-igem.html (September 08, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Les étudiants de la biotech interpellent la biologie synthétique</i>" (French), [http://www.vivagora.org/spip.php?breve210 (October 09, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>Un equipo de la UAB, en el concurso de biología sintética del MIT</i>" (Spanish), [http://www.uab.es/servlet/Satellite?cid=1096481466568&pagename=UABDivulga%2FPage%2FTemplatePageDetallArticleInvestigar&param1=1253860327385 (September 23, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>UAB to participate in the synthetic biology competition at MIT</i>" (English), [http://www.uab.es/servlet/Satellite/latest-news/news-detail/uab-to-participate-in-the-synthetic-biology-competition-at-mit-1096476786473.html?noticiaid=1253657853358 (September 23, 2009)]<br />
<br />
*'''[[Team:Valencia]]''': "<i>TheValencia team is awarded the Synthetic Standard Prize</i>" (Spanish), [http://www.elpais.com/articulo/sociedad/Levaduras/funcionan/pixeles/elpepuespval/20091103elpepusoc_9/Tes]<br />
<br />
*'''[[Team:Cambridge]]''': "<i>University of Cambridge team wins iGEM synthetic biology competition </i>" (English), [http://www.biotechniques.com/news/University-of-Cambridge-team-wins-iGEM-synthetic-biology-competition/biotechniques-180278.html (November 5 2009)]<br />
<br />
*'''[[Team:Virginia]]''': ''[http://www.washingtonpost.com/wp-dyn/content/article/2009/10/22/AR2009102204628.html New works of science nonfiction]'', The Washington Post (October 23, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Estafette voor bacterien'',(Dutch) Algemeen Dagblad (November 6, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette'', (Dutch) [http://noorderlicht.vpro.nl/themasites/mediaplayer/index.jsp?media=42722491&refernr=42560747&portalnr=3626936&hostname=noorderlicht&mediatype=audio&portalid=noorderlicht# Podcast van Radio 1, VPRO Noorderlicht, Annemieke Smit] (November 9, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette wint goud en award'', (Dutch) [http://www.tudelft.nl/live/pagina.jsp?id=ee44be90-289e-4efe-8a12-4636f1c86b28&lang=nl TNW Today Editie 2] (November 5, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette wint goud en award'', (Dutch) [http://www.tudelft.nl/live/pagina.jsp?id=ee44be90-289e-4efe-8a12-4636f1c86b28&lang=nl TUDelft Nieuws] (November 4, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterial relay race wins gold and award'', [http://www.tudelft.nl/live/pagina.jsp?id=26f61b2e-5a3f-40be-b737-a460b08bced7&lang=en Delft University of Technology Press release] (November 4, 2009)</div>Wilfredhttp://2009.igem.org/Team:Groningen/PublicityTeam:Groningen/Publicity2009-11-14T13:52:58Z<p>Wilfred: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
=Websites=<br />
*"<i>Synthetic biology students from Groningen in the race for best bacterium</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*"<i>Students from Groningen are making bacteria to remove heavy metals from water</i>" (Dutch), [http://www.groningenreporter.nl/story.php?title=groninger-studenten-maken-bacteri%3Fn-die-water-zuiveren-van-zware-metalen Groningen Reporter (August 22, 2009)]<br />
*"<i>Team iGEM Groningen wins golden medal</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*"<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
<br />
=News=<br />
*"<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*"<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Press release University Groningen (November 11, 2009)]<br />
*"<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*"<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*"<i>With a pacmanbacterium in de finals</i>" (Dutch), [http://www.uk.rug.nl/archief/jaargang39/11/12e.php UK (Paper of the Groningen University) (November 12, 2009)]<br />
<br />
=Radio=<br />
*Interview [http://www.oogtv.nl/ Oog Radio] - <i>Stad vandaag (News- and background story program)</i> (Dutch) (November 10, 2009)<br />
<br />
=Our Brochure=<br />
<center>[[Image:Inside_Brochure_iGEM_GRONINGEN.jpg|800px]]<br />
[[Image:Outside_Brochure_iGEM_GRONINGEN.jpg|800px]]</center><br />
{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/IGEM_PublicityIGEM Publicity2009-11-12T16:26:52Z<p>Wilfred: /* team specific */</p>
<hr />
<div>__NOTOC__<br />
[https://2008.igem.org/IGEM_Publicity iGEM 2008 Publicity] | [http://parts.mit.edu/igem07/index.php/Media iGEM 2007 Publicity] | [http://parts.mit.edu/wiki/index.php/IGEM_News iGEM 2006 Publicity]<br />
<br />
<div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [[Press_Kit | iGEM Press Kit]])</div><br />
<br />
<html><br />
<iframe frameborder="0" width="728" height="90" marginwidth="0" marginheight="0" style="margin:0px 0px 25px 100px;"<br />
src="http://www.google.com/uds/modules/elements/newsshow/iframe.html?q=iGEM&rsz=large&format=728x90"><br />
</iframe><br />
</html><br />
<br />
<br />
<span style="color:#1e90ff; font-size:175%">'''blogs </span><span style="color:#3cb371; font-size:175%">covering iGEM 2009'''</span><br />
* The [http://igem.sdu.dk/ <span style="color:#453221; font-size: 130%">'''SDU-Denmark'''</span>] team blog about their iGEM experience.<br />
* A blog about the iGEM project of the [http://aboutgmos.org/iGEM <span style="color:#453221; font-size: 130%">'''Uppsala-Sweden'''</span>] team.<br />
<br />
<br />
<br />
<br />
====<font size=5><font color=dodgerblue>'''video/radio</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
*'''Slovenia, Heidelberg, SDU-Denmark''': [http://www.dradio.de/dlf/sendungen/forschak/1062608/ Die Biobastler von Boston], Nov 2, 2009, Deutschlandfunk radio (in German).<br />
*'''Cambridge, Imperial''': [http://www.sciencefriday.com/program/archives/200911063 Synthetic Biology Competition], Nov 6, 2009, NPR Science Friday (in English).<br />
<br />
====<font size=5><font color=dodgerblue>'''news articles</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
<br />
<br />
If you would like to share an article that was written about iGEM or your iGEM team, please link to it on this page. If you have multiple articles featuring your team, link to them all individually!<br />
<br />
Post the name of your team, the title of your article, where it was featured, and provide a link to it. <br />
<br />
''Example'':<br> <br />
'''Team Example''': ''Title of article'', Nature, [link]<br />
<br />
====<font size=4><font color=dodgerblue>'''general'''</font></font>====<br />
*'''The Economist''': ''Biohacking: Hacking goes squishy'' Sep 3, 2009 [http://www.economist.com/search/displaystory.cfm?story_id=14299634 The Economist]<br />
*'''The Scientist''': ''Brick by Brick'' Feb 1, 2009. [http://www.the-scientist.com/article/display/55378/ The Scientist]<br />
*'''Technology Review''': ''A Genetically Engineered Rainbow of Bacteria'' Nov 03, 2009. [http://www.technologyreview.com/blog/editors/24351/ Technology Review]<br />
*'''Discovery News''': '' Bright Bacteria Wins Synthetic Biology Competition'' Nov 6, 2009. [http://blogs.discoverychannel.co.uk/discovery-news/2009/11/bright-bacteria-wins-synthetic-biology-competition.html Discovery News]<br />
<br />
====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
<br />
*'''[[Team:IBB_Pune]]''': "<i>Bio-champs in the making</i>" (English), [http://www.punemirror.in/index.aspx?Page=article&sectname=News%20-%20City&sectid=2&contentid=200909142009091423331646a131eacc#ftr2 (Monday, September 14, 2009 at 11:33:25 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Ganiti Prakriyanmadhe Jeevanuncha Vaapar! (Use of Bacteria in mathematical devices)</i>" (Marathi), [http://beta.esakal.com/2009/09/09220618/pune-use-of-bacterias-in-maths.html(Wednesday, September 09th, 2009 AT 10:09 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Synthetic Biologiteel Bharari ("Advances in Synthetic Biology")</i>" (Marathi), [https://2009.igem.org/Team:IBB_Pune/press (Wednesday 7 October 2009)]<br />
<br />
*'''[[Team:Groningen]]''': "<i>Synthetic biology students from Groningen in the race for best bacterium</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*'''[[Team:Groningen]]''': "<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Team iGEM Groningen wins golden medal</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Press release University Groningen (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>With a pacmanbacterium in de finals</i>" (Dutch), [http://www.uk.rug.nl/archief/jaargang39/11/12e.php UK (Paper of the Groningen University) (November 12, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Un concours organisé par le M.I.T : en route pour Boston ! </i>" (French), [http://www.supbiotech.fr/2009/09/boston-supbiotech-igem.html (September 08, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Les étudiants de la biotech interpellent la biologie synthétique</i>" (French), [http://www.vivagora.org/spip.php?breve210 (October 09, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>Un equipo de la UAB, en el concurso de biología sintética del MIT</i>" (Spanish), [http://www.uab.es/servlet/Satellite?cid=1096481466568&pagename=UABDivulga%2FPage%2FTemplatePageDetallArticleInvestigar&param1=1253860327385 (September 23, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>UAB to participate in the synthetic biology competition at MIT</i>" (English), [http://www.uab.es/servlet/Satellite/latest-news/news-detail/uab-to-participate-in-the-synthetic-biology-competition-at-mit-1096476786473.html?noticiaid=1253657853358 (September 23, 2009)]<br />
<br />
*'''[[Team:Valencia]]''': "<i>TheValencia team is awarded the Synthetic Standard Prize</i>" (Spanish), [http://www.elpais.com/articulo/sociedad/Levaduras/funcionan/pixeles/elpepuespval/20091103elpepusoc_9/Tes]<br />
<br />
*'''[[Team:Cambridge]]''': "<i>University of Cambridge team wins iGEM synthetic biology competition </i>" (English), [http://www.biotechniques.com/news/University-of-Cambridge-team-wins-iGEM-synthetic-biology-competition/biotechniques-180278.html (November 5 2009)]<br />
<br />
*'''[[Team:Virginia]]''': ''[http://www.washingtonpost.com/wp-dyn/content/article/2009/10/22/AR2009102204628.html New works of science nonfiction]'', The Washington Post (October 23, 2009)</div>Wilfredhttp://2009.igem.org/Team:Groningen/PublicityTeam:Groningen/Publicity2009-11-12T16:26:29Z<p>Wilfred: /* News */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
=Websites=<br />
*"<i>Synthetic biology students from Groningen in the race for best bacterium</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*"<i>Students from Groningen are making bacteria to remove heavy metals from water</i>" (Dutch), [http://www.groningenreporter.nl/story.php?title=groninger-studenten-maken-bacteri%3Fn-die-water-zuiveren-van-zware-metalen Groningen Reporter (August 22, 2009)]<br />
*"<i>Team iGEM Groningen wins golden medal</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*"<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
<br />
=News=<br />
*"<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*"<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Press release University Groningen (November 11, 2009)]<br />
*"<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*"<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*"<i>With a pacmanbacterium in de finals</i>" (Dutch), [http://www.uk.rug.nl/archief/jaargang39/11/12e.php UK (Paper of the Groningen University) (November 12, 2009)]<br />
<br />
=Our Brochure=<br />
<center>[[Image:Inside_Brochure_iGEM_GRONINGEN.jpg|800px]]<br />
[[Image:Outside_Brochure_iGEM_GRONINGEN.jpg|800px]]</center><br />
{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/PublicityTeam:Groningen/Publicity2009-11-12T16:24:13Z<p>Wilfred: /* News */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
=Websites=<br />
*"<i>Synthetic biology students from Groningen in the race for best bacterium</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*"<i>Students from Groningen are making bacteria to remove heavy metals from water</i>" (Dutch), [http://www.groningenreporter.nl/story.php?title=groninger-studenten-maken-bacteri%3Fn-die-water-zuiveren-van-zware-metalen Groningen Reporter (August 22, 2009)]<br />
*"<i>Team iGEM Groningen wins golden medal</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*"<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
<br />
=News=<br />
*"<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*"<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Press release University Groningen (November 11, 2009)]<br />
*"<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*"<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*"<i>With a pacmanbacteria in de finals</i>" (Dutch), [http://www.uk.rug.nl/archief/jaargang39/11/12e.php UK (Paper of the Groningen University) (November 12, 2009)]<br />
<br />
=Our Brochure=<br />
<center>[[Image:Inside_Brochure_iGEM_GRONINGEN.jpg|800px]]<br />
[[Image:Outside_Brochure_iGEM_GRONINGEN.jpg|800px]]</center><br />
{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/IGEM_PublicityIGEM Publicity2009-11-12T16:23:12Z<p>Wilfred: /* team specific */</p>
<hr />
<div>__NOTOC__<br />
[https://2008.igem.org/IGEM_Publicity iGEM 2008 Publicity] | [http://parts.mit.edu/igem07/index.php/Media iGEM 2007 Publicity] | [http://parts.mit.edu/wiki/index.php/IGEM_News iGEM 2006 Publicity]<br />
<br />
<div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [[Press_Kit | iGEM Press Kit]])</div><br />
<br />
<html><br />
<iframe frameborder="0" width="728" height="90" marginwidth="0" marginheight="0" style="margin:0px 0px 25px 100px;"<br />
src="http://www.google.com/uds/modules/elements/newsshow/iframe.html?q=iGEM&rsz=large&format=728x90"><br />
</iframe><br />
</html><br />
<br />
<br />
<span style="color:#1e90ff; font-size:175%">'''blogs </span><span style="color:#3cb371; font-size:175%">covering iGEM 2009'''</span><br />
* The [http://igem.sdu.dk/ <span style="color:#453221; font-size: 130%">'''SDU-Denmark'''</span>] team blog about their iGEM experience.<br />
* A blog about the iGEM project of the [http://aboutgmos.org/iGEM <span style="color:#453221; font-size: 130%">'''Uppsala-Sweden'''</span>] team.<br />
<br />
<br />
<br />
<br />
====<font size=5><font color=dodgerblue>'''video/radio</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
*'''Slovenia, Heidelberg, SDU-Denmark''': [http://www.dradio.de/dlf/sendungen/forschak/1062608/ Die Biobastler von Boston], Nov 2, 2009, Deutschlandfunk radio (in German).<br />
*'''Cambridge, Imperial''': [http://www.sciencefriday.com/program/archives/200911063 Synthetic Biology Competition], Nov 6, 2009, NPR Science Friday (in English).<br />
<br />
====<font size=5><font color=dodgerblue>'''news articles</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
<br />
<br />
If you would like to share an article that was written about iGEM or your iGEM team, please link to it on this page. If you have multiple articles featuring your team, link to them all individually!<br />
<br />
Post the name of your team, the title of your article, where it was featured, and provide a link to it. <br />
<br />
''Example'':<br> <br />
'''Team Example''': ''Title of article'', Nature, [link]<br />
<br />
====<font size=4><font color=dodgerblue>'''general'''</font></font>====<br />
*'''The Economist''': ''Biohacking: Hacking goes squishy'' Sep 3, 2009 [http://www.economist.com/search/displaystory.cfm?story_id=14299634 The Economist]<br />
*'''The Scientist''': ''Brick by Brick'' Feb 1, 2009. [http://www.the-scientist.com/article/display/55378/ The Scientist]<br />
*'''Technology Review''': ''A Genetically Engineered Rainbow of Bacteria'' Nov 03, 2009. [http://www.technologyreview.com/blog/editors/24351/ Technology Review]<br />
*'''Discovery News''': '' Bright Bacteria Wins Synthetic Biology Competition'' Nov 6, 2009. [http://blogs.discoverychannel.co.uk/discovery-news/2009/11/bright-bacteria-wins-synthetic-biology-competition.html Discovery News]<br />
<br />
====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
<br />
*'''[[Team:IBB_Pune]]''': "<i>Bio-champs in the making</i>" (English), [http://www.punemirror.in/index.aspx?Page=article&sectname=News%20-%20City&sectid=2&contentid=200909142009091423331646a131eacc#ftr2 (Monday, September 14, 2009 at 11:33:25 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Ganiti Prakriyanmadhe Jeevanuncha Vaapar! (Use of Bacteria in mathematical devices)</i>" (Marathi), [http://beta.esakal.com/2009/09/09220618/pune-use-of-bacterias-in-maths.html(Wednesday, September 09th, 2009 AT 10:09 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Synthetic Biologiteel Bharari ("Advances in Synthetic Biology")</i>" (Marathi), [https://2009.igem.org/Team:IBB_Pune/press (Wednesday 7 October 2009)]<br />
<br />
*'''[[Team:Groningen]]''': "<i>Synthetic biology students from Groningen in the race for best bacterium</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*'''[[Team:Groningen]]''': "<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Team iGEM Groningen wins golden medal</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Press release University Groningen (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>With a pacmanbacteria in de finals</i>" (Dutch), [http://www.uk.rug.nl/archief/jaargang39/11/12e.php UK (Paper of the Groningen University) (November 12, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Un concours organisé par le M.I.T : en route pour Boston ! </i>" (French), [http://www.supbiotech.fr/2009/09/boston-supbiotech-igem.html (September 08, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Les étudiants de la biotech interpellent la biologie synthétique</i>" (French), [http://www.vivagora.org/spip.php?breve210 (October 09, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>Un equipo de la UAB, en el concurso de biología sintética del MIT</i>" (Spanish), [http://www.uab.es/servlet/Satellite?cid=1096481466568&pagename=UABDivulga%2FPage%2FTemplatePageDetallArticleInvestigar&param1=1253860327385 (September 23, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>UAB to participate in the synthetic biology competition at MIT</i>" (English), [http://www.uab.es/servlet/Satellite/latest-news/news-detail/uab-to-participate-in-the-synthetic-biology-competition-at-mit-1096476786473.html?noticiaid=1253657853358 (September 23, 2009)]<br />
<br />
*'''[[Team:Valencia]]''': "<i>TheValencia team is awarded the Synthetic Standard Prize</i>" (Spanish), [http://www.elpais.com/articulo/sociedad/Levaduras/funcionan/pixeles/elpepuespval/20091103elpepusoc_9/Tes]<br />
<br />
*'''[[Team:Cambridge]]''': "<i>University of Cambridge team wins iGEM synthetic biology competition </i>" (English), [http://www.biotechniques.com/news/University-of-Cambridge-team-wins-iGEM-synthetic-biology-competition/biotechniques-180278.html (November 5 2009)]<br />
<br />
*'''[[Team:Virginia]]''': ''[http://www.washingtonpost.com/wp-dyn/content/article/2009/10/22/AR2009102204628.html New works of science nonfiction]'', The Washington Post (October 23, 2009)</div>Wilfredhttp://2009.igem.org/Team:Groningen/PublicityTeam:Groningen/Publicity2009-11-12T10:04:55Z<p>Wilfred: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
=Websites=<br />
*"<i>Synthetic biology students from Groningen in the race for best bacterium</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*"<i>Students from Groningen are making bacteria to remove heavy metals from water</i>" (Dutch), [http://www.groningenreporter.nl/story.php?title=groninger-studenten-maken-bacteri%3Fn-die-water-zuiveren-van-zware-metalen Groningen Reporter (August 22, 2009)]<br />
*"<i>Team iGEM Groningen wins golden medal</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*"<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
<br />
=News=<br />
*"<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*"<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Press release University Groningen (November 11, 2009)]<br />
*"<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*"<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
=Our Brochure=<br />
<center>[[Image:Inside_Brochure_iGEM_GRONINGEN.jpg|800px]]<br />
[[Image:Outside_Brochure_iGEM_GRONINGEN.jpg|800px]]</center><br />
{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/PublicityTeam:Groningen/Publicity2009-11-12T10:04:08Z<p>Wilfred: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
=Websites=<br />
*"<i>Synthetic biology students from Groningen in the race for best bacterium</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*"<i>Students from Groningen are making bacteria to remove heavy metals from water</i>" (Dutch), [http://www.groningenreporter.nl/story.php?title=groninger-studenten-maken-bacteri%3Fn-die-water-zuiveren-van-zware-metalen Groningen Reporter (August 22, 2009)]<br />
*"<i>Team iGEM Groningen wins golden medal</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*"<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
<br />
=News=<br />
*"<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*"<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Press release University Groningen (November 11, 2009)]<br />
*"<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*"<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
=Our Brochure=<br />
<center>[[Image:Inside_Brochure_iGEM_GRONINGEN.jpg|800px]]<br />
[[Image:Outside_Brochure_iGEM_GRONINGEN.jpg|800px]]</center></div>Wilfredhttp://2009.igem.org/Team:Groningen/PublicityTeam:Groningen/Publicity2009-11-12T10:01:02Z<p>Wilfred: </p>
<hr />
<div>=Websites=<br />
*"<i>Synthetic biology students from Groningen in the race for best bacterium</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*"<i>Students from Groningen are making bacteria to remove heavy metals from water</i>" (Dutch), [http://www.groningenreporter.nl/story.php?title=groninger-studenten-maken-bacteri%3Fn-die-water-zuiveren-van-zware-metalen Groningen Reporter (August 22, 2009)]<br />
*"<i>Team iGEM Groningen wins golden medal</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*"<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
<br />
=News=<br />
*"<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*"<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Press release University Groningen (November 11, 2009)]<br />
*"<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*"<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
=Our Brochure=<br />
<center>[[Image:Inside_Brochure_iGEM_GRONINGEN.jpg|800px]]<br />
[[Image:Outside_Brochure_iGEM_GRONINGEN.jpg|800px]]</center></div>Wilfredhttp://2009.igem.org/Team:Groningen/PublicityTeam:Groningen/Publicity2009-11-12T09:58:33Z<p>Wilfred: /* Websites */</p>
<hr />
<div>=Websites=<br />
*"<i>Synthetic biology students from Groningen in the race for best bacterium</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*"<i>Students from Groningen are making bacteria to remove heavy metals from water</i>" (Dutch), [http://www.groningenreporter.nl/story.php?title=groninger-studenten-maken-bacteri%3Fn-die-water-zuiveren-van-zware-metalen Groningen Reporter (August 22, 2009)]<br />
*"<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*"<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*"<i>Team iGEM Groningen wins golden medal</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Press release University Groningen (November 11, 2009)]<br />
*"<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*"<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*"<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*"<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
<br />
=News=<br />
=Our Brochure=<br />
<center>[[Image:Inside_Brochure_iGEM_GRONINGEN.jpg|800px]]<br />
[[Image:Outside_Brochure_iGEM_GRONINGEN.jpg|800px]]</center></div>Wilfredhttp://2009.igem.org/IGEM_PublicityIGEM Publicity2009-11-12T09:56:04Z<p>Wilfred: /* general */</p>
<hr />
<div>__NOTOC__<br />
[https://2008.igem.org/IGEM_Publicity iGEM 2008 Publicity] | [http://parts.mit.edu/igem07/index.php/Media iGEM 2007 Publicity] | [http://parts.mit.edu/wiki/index.php/IGEM_News iGEM 2006 Publicity]<br />
<br />
<div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [[Press_Kit | iGEM Press Kit]])</div><br />
<br />
<html><br />
<iframe frameborder="0" width="728" height="90" marginwidth="0" marginheight="0" style="margin:0px 0px 25px 100px;"<br />
src="http://www.google.com/uds/modules/elements/newsshow/iframe.html?q=iGEM&rsz=large&format=728x90"><br />
</iframe><br />
</html><br />
<br />
<br />
<span style="color:#1e90ff; font-size:175%">'''blogs </span><span style="color:#3cb371; font-size:175%">covering iGEM 2009'''</span><br />
* The [http://igem.sdu.dk/ <span style="color:#453221; font-size: 130%">'''SDU-Denmark'''</span>] team blog about their iGEM experience.<br />
* A blog about the iGEM project of the [http://aboutgmos.org/iGEM <span style="color:#453221; font-size: 130%">'''Uppsala-Sweden'''</span>] team.<br />
<br />
<br />
<br />
<br />
====<font size=5><font color=dodgerblue>'''video/radio</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
*'''Slovenia, Heidelberg, SDU-Denmark''': [http://www.dradio.de/dlf/sendungen/forschak/1062608/ Die Biobastler von Boston], Nov 2, 2009, Deutschlandfunk radio (in German).<br />
*'''Cambridge, Imperial''': [http://www.sciencefriday.com/program/archives/200911063 Synthetic Biology Competition], Nov 6, 2009, NPR Science Friday (in English).<br />
<br />
====<font size=5><font color=dodgerblue>'''news articles</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
<br />
<br />
If you would like to share an article that was written about iGEM or your iGEM team, please link to it on this page. If you have multiple articles featuring your team, link to them all individually!<br />
<br />
Post the name of your team, the title of your article, where it was featured, and provide a link to it. <br />
<br />
''Example'':<br> <br />
'''Team Example''': ''Title of article'', Nature, [link]<br />
<br />
====<font size=4><font color=dodgerblue>'''general'''</font></font>====<br />
*'''The Economist''': ''Biohacking: Hacking goes squishy'' Sep 3, 2009 [http://www.economist.com/search/displaystory.cfm?story_id=14299634 The Economist]<br />
*'''The Scientist''': ''Brick by Brick'' Feb 1, 2009. [http://www.the-scientist.com/article/display/55378/ The Scientist]<br />
*'''Technology Review''': ''A Genetically Engineered Rainbow of Bacteria'' Nov 03, 2009. [http://www.technologyreview.com/blog/editors/24351/ Technology Review]<br />
*'''Discovery News''': '' Bright Bacteria Wins Synthetic Biology Competition'' Nov 6, 2009. [http://blogs.discoverychannel.co.uk/discovery-news/2009/11/bright-bacteria-wins-synthetic-biology-competition.html Discovery News]<br />
<br />
====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
<br />
*'''[[Team:IBB_Pune]]''': "<i>Bio-champs in the making</i>" (English), [http://www.punemirror.in/index.aspx?Page=article&sectname=News%20-%20City&sectid=2&contentid=200909142009091423331646a131eacc#ftr2 (Monday, September 14, 2009 at 11:33:25 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Ganiti Prakriyanmadhe Jeevanuncha Vaapar! (Use of Bacteria in mathematical devices)</i>" (Marathi), [http://beta.esakal.com/2009/09/09220618/pune-use-of-bacterias-in-maths.html(Wednesday, September 09th, 2009 AT 10:09 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Synthetic Biologiteel Bharari ("Advances in Synthetic Biology")</i>" (Marathi), [https://2009.igem.org/Team:IBB_Pune/press (Wednesday 7 October 2009)]<br />
<br />
*'''[[Team:Groningen]]''': "<i>Synthetic biology students from Groningen in the race for best bacterium</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*'''[[Team:Groningen]]''': "Talent of the Purest Water" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*'''[[Team:Groningen]]''': "<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Team iGEM Groningen wins golden medal</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Press release University Groningen (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i> Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i> Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Un concours organisé par le M.I.T : en route pour Boston ! </i>" (French), [http://www.supbiotech.fr/2009/09/boston-supbiotech-igem.html (September 08, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Les étudiants de la biotech interpellent la biologie synthétique</i>" (French), [http://www.vivagora.org/spip.php?breve210 (October 09, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>Un equipo de la UAB, en el concurso de biología sintética del MIT</i>" (Spanish), [http://www.uab.es/servlet/Satellite?cid=1096481466568&pagename=UABDivulga%2FPage%2FTemplatePageDetallArticleInvestigar&param1=1253860327385 (September 23, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>UAB to participate in the synthetic biology competition at MIT</i>" (English), [http://www.uab.es/servlet/Satellite/latest-news/news-detail/uab-to-participate-in-the-synthetic-biology-competition-at-mit-1096476786473.html?noticiaid=1253657853358 (September 23, 2009)]<br />
<br />
*'''[[Team:Valencia]]''': "<i>TheValencia team is awarded the Synthetic Standard Prize</i>" (Spanish), [http://www.elpais.com/articulo/sociedad/Levaduras/funcionan/pixeles/elpepuespval/20091103elpepusoc_9/Tes]<br />
<br />
*'''[[Team:Cambridge]]''': "<i>University of Cambridge team wins iGEM synthetic biology competition </i>" (English), [http://www.biotechniques.com/news/University-of-Cambridge-team-wins-iGEM-synthetic-biology-competition/biotechniques-180278.html (November 5 2009)]<br />
<br />
*'''[[Team:Virginia]]''': ''[http://www.washingtonpost.com/wp-dyn/content/article/2009/10/22/AR2009102204628.html New works of science nonfiction]'', The Washington Post (October 23, 2009)</div>Wilfredhttp://2009.igem.org/IGEM_PublicityIGEM Publicity2009-11-12T09:54:48Z<p>Wilfred: /* team specific */ Groningen</p>
<hr />
<div>__NOTOC__<br />
[https://2008.igem.org/IGEM_Publicity iGEM 2008 Publicity] | [http://parts.mit.edu/igem07/index.php/Media iGEM 2007 Publicity] | [http://parts.mit.edu/wiki/index.php/IGEM_News iGEM 2006 Publicity]<br />
<br />
<div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [[Press_Kit | iGEM Press Kit]])</div><br />
<br />
<html><br />
<iframe frameborder="0" width="728" height="90" marginwidth="0" marginheight="0" style="margin:0px 0px 25px 100px;"<br />
src="http://www.google.com/uds/modules/elements/newsshow/iframe.html?q=iGEM&rsz=large&format=728x90"><br />
</iframe><br />
</html><br />
<br />
<br />
<span style="color:#1e90ff; font-size:175%">'''blogs </span><span style="color:#3cb371; font-size:175%">covering iGEM 2009'''</span><br />
* The [http://igem.sdu.dk/ <span style="color:#453221; font-size: 130%">'''SDU-Denmark'''</span>] team blog about their iGEM experience.<br />
* A blog about the iGEM project of the [http://aboutgmos.org/iGEM <span style="color:#453221; font-size: 130%">'''Uppsala-Sweden'''</span>] team.<br />
<br />
<br />
<br />
<br />
====<font size=5><font color=dodgerblue>'''video/radio</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
*'''Slovenia, Heidelberg, SDU-Denmark''': [http://www.dradio.de/dlf/sendungen/forschak/1062608/ Die Biobastler von Boston], Nov 2, 2009, Deutschlandfunk radio (in German).<br />
*'''Cambridge, Imperial''': [http://www.sciencefriday.com/program/archives/200911063 Synthetic Biology Competition], Nov 6, 2009, NPR Science Friday (in English).<br />
<br />
====<font size=5><font color=dodgerblue>'''news articles</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
<br />
<br />
If you would like to share an article that was written about iGEM or your iGEM team, please link to it on this page. If you have multiple articles featuring your team, link to them all individually!<br />
<br />
Post the name of your team, the title of your article, where it was featured, and provide a link to it. <br />
<br />
''Example'':<br> <br />
'''Team Example''': ''Title of article'', Nature, [link]<br />
<br />
====<font size=4><font color=dodgerblue>'''general'''</font></font>====<br />
*'''The Economist''': ''Biohacking: Hacking goes squishy'' Sep 3, 2009 [http://www.economist.com/search/displaystory.cfm?story_id=14299634 The Economist]<br />
*'''The Scientist''': ''Brick by Brick'' Feb 1, 2009. [http://www.the-scientist.com/article/display/55378/ The Scientist]<br />
*'''Technology Review''': ''A Genetically Engineered Rainbow of Bacteria'' November 03, 2009. [http://www.technologyreview.com/blog/editors/24351/ Technology Review]<br />
<br />
====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
<br />
*'''[[Team:IBB_Pune]]''': "<i>Bio-champs in the making</i>" (English), [http://www.punemirror.in/index.aspx?Page=article&sectname=News%20-%20City&sectid=2&contentid=200909142009091423331646a131eacc#ftr2 (Monday, September 14, 2009 at 11:33:25 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Ganiti Prakriyanmadhe Jeevanuncha Vaapar! (Use of Bacteria in mathematical devices)</i>" (Marathi), [http://beta.esakal.com/2009/09/09220618/pune-use-of-bacterias-in-maths.html(Wednesday, September 09th, 2009 AT 10:09 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Synthetic Biologiteel Bharari ("Advances in Synthetic Biology")</i>" (Marathi), [https://2009.igem.org/Team:IBB_Pune/press (Wednesday 7 October 2009)]<br />
<br />
*'''[[Team:Groningen]]''': "<i>Synthetic biology students from Groningen in the race for best bacterium</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*'''[[Team:Groningen]]''': "Talent of the Purest Water" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*'''[[Team:Groningen]]''': "<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Team iGEM Groningen wins golden medal</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Press release University Groningen (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i> Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i> Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Un concours organisé par le M.I.T : en route pour Boston ! </i>" (French), [http://www.supbiotech.fr/2009/09/boston-supbiotech-igem.html (September 08, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Les étudiants de la biotech interpellent la biologie synthétique</i>" (French), [http://www.vivagora.org/spip.php?breve210 (October 09, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>Un equipo de la UAB, en el concurso de biología sintética del MIT</i>" (Spanish), [http://www.uab.es/servlet/Satellite?cid=1096481466568&pagename=UABDivulga%2FPage%2FTemplatePageDetallArticleInvestigar&param1=1253860327385 (September 23, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>UAB to participate in the synthetic biology competition at MIT</i>" (English), [http://www.uab.es/servlet/Satellite/latest-news/news-detail/uab-to-participate-in-the-synthetic-biology-competition-at-mit-1096476786473.html?noticiaid=1253657853358 (September 23, 2009)]<br />
<br />
*'''[[Team:Valencia]]''': "<i>TheValencia team is awarded the Synthetic Standard Prize</i>" (Spanish), [http://www.elpais.com/articulo/sociedad/Levaduras/funcionan/pixeles/elpepuespval/20091103elpepusoc_9/Tes]<br />
<br />
*'''[[Team:Cambridge]]''': "<i>University of Cambridge team wins iGEM synthetic biology competition </i>" (English), [http://www.biotechniques.com/news/University-of-Cambridge-team-wins-iGEM-synthetic-biology-competition/biotechniques-180278.html (November 5 2009)]<br />
<br />
*'''[[Team:Virginia]]''': ''[http://www.washingtonpost.com/wp-dyn/content/article/2009/10/22/AR2009102204628.html New works of science nonfiction]'', The Washington Post (October 23, 2009)</div>Wilfredhttp://2009.igem.org/Team:Groningen/PublicityTeam:Groningen/Publicity2009-11-10T11:21:42Z<p>Wilfred: /* Websites */</p>
<hr />
<div>=Websites=<br />
*"<i>Synthetic biology students from Groningen in the race for best bacterium</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*"<i>Students from Groningen are making bacteria to remove heavy metals from water</i>" (Dutch), [http://www.groningenreporter.nl/story.php?title=groninger-studenten-maken-bacteri%3Fn-die-water-zuiveren-van-zware-metalen Groningen Reporter (August 22, 2009)]<br />
*"<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*"<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
<br />
=News=<br />
=Our Brochure=<br />
<center>[[Image:Inside_Brochure_iGEM_GRONINGEN.jpg|800px]]<br />
[[Image:Outside_Brochure_iGEM_GRONINGEN.jpg|800px]]</center></div>Wilfredhttp://2009.igem.org/IGEM_PublicityIGEM Publicity2009-11-10T11:07:16Z<p>Wilfred: /* team specific */ Groningen</p>
<hr />
<div>__NOTOC__<br />
[https://2008.igem.org/IGEM_Publicity iGEM 2008 Publicity] | [http://parts.mit.edu/igem07/index.php/Media iGEM 2007 Publicity] | [http://parts.mit.edu/wiki/index.php/IGEM_News iGEM 2006 Publicity]<br />
<br />
<div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [[Press_Kit | iGEM Press Kit]])</div><br />
<br />
<html><br />
<iframe frameborder="0" width="728" height="90" marginwidth="0" marginheight="0" style="margin:0px 0px 25px 100px;"<br />
src="http://www.google.com/uds/modules/elements/newsshow/iframe.html?q=iGEM&rsz=large&format=728x90"><br />
</iframe><br />
</html><br />
<br />
<br />
<span style="color:#1e90ff; font-size:175%">'''blogs </span><span style="color:#3cb371; font-size:175%">covering iGEM 2009'''</span><br />
* The [http://igem.sdu.dk/ <span style="color:#453221; font-size: 130%">'''SDU-Denmark'''</span>] team blog about their iGEM experience.<br />
* A blog about the iGEM project of the [http://aboutgmos.org/iGEM <span style="color:#453221; font-size: 130%">'''Uppsala-Sweden'''</span>] team.<br />
<br />
<br />
<br />
<br />
====<font size=5><font color=dodgerblue>'''video/radio</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
*'''Slovenia, Heidelberg, SDU-Denmark''': [http://www.dradio.de/dlf/sendungen/forschak/1062608/ Die Biobastler von Boston], Nov 2, 2009, Deutschlandfunk radio (in German).<br />
*'''Cambridge, Imperial''': [http://www.sciencefriday.com/program/archives/200911063 Synthetic Biology Competition], Nov 6, 2009, NPR Science Friday (in English).<br />
<br />
====<font size=5><font color=dodgerblue>'''news articles</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
<br />
<br />
If you would like to share an article that was written about iGEM or your iGEM team, please link to it on this page. If you have multiple articles featuring your team, link to them all individually!<br />
<br />
Post the name of your team, the title of your article, where it was featured, and provide a link to it. <br />
<br />
''Example'':<br> <br />
'''Team Example''': ''Title of article'', Nature, [link]<br />
<br />
====<font size=4><font color=dodgerblue>'''general'''</font></font>====<br />
*'''The Economist''': ''Biohacking: Hacking goes squishy'' Sep 3, 2009 [http://www.economist.com/search/displaystory.cfm?story_id=14299634 The Economist]<br />
*'''The Scientist''': ''Brick by Brick'' Feb 1, 2009. [http://www.the-scientist.com/article/display/55378/ The Scientist]<br />
*'''Technology Review''': ''A Genetically Engineered Rainbow of Bacteria'' November 03, 2009. [http://www.technologyreview.com/blog/editors/24351/ Technology Review]<br />
<br />
====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
<br />
*'''[[Team:IBB_Pune]]''': "<i>Bio-champs in the making</i>" (English), [http://www.punemirror.in/index.aspx?Page=article&sectname=News%20-%20City&sectid=2&contentid=200909142009091423331646a131eacc#ftr2 (Monday, September 14, 2009 at 11:33:25 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Ganiti Prakriyanmadhe Jeevanuncha Vaapar! (Use of Bacteria in mathematical devices)</i>" (Marathi), [http://beta.esakal.com/2009/09/09220618/pune-use-of-bacterias-in-maths.html(Wednesday, September 09th, 2009 AT 10:09 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Synthetic Biologiteel Bharari ("Advances in Synthetic Biology")</i>" (Marathi), [https://2009.igem.org/Team:IBB_Pune/press (Wednesday 7 October 2009)]<br />
<br />
*'''[[Team:Groningen]]''': "<i>Synthetic biology students from Groningen in the race for best bacterium</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*'''[[Team:Groningen]]''': "Talent of the Purest Water" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*'''[[Team:Groningen]]''': "<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Un concours organisé par le M.I.T : en route pour Boston ! </i>" (French), [http://www.supbiotech.fr/2009/09/boston-supbiotech-igem.html (September 08, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Les étudiants de la biotech interpellent la biologie synthétique</i>" (French), [http://www.vivagora.org/spip.php?breve210 (October 09, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>Un equipo de la UAB, en el concurso de biología sintética del MIT</i>" (Spanish), [http://www.uab.es/servlet/Satellite?cid=1096481466568&pagename=UABDivulga%2FPage%2FTemplatePageDetallArticleInvestigar&param1=1253860327385 (September 23, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>UAB to participate in the synthetic biology competition at MIT</i>" (English), [http://www.uab.es/servlet/Satellite/latest-news/news-detail/uab-to-participate-in-the-synthetic-biology-competition-at-mit-1096476786473.html?noticiaid=1253657853358 (September 23, 2009)]<br />
<br />
*'''[[Team:Valencia]]''': "<i>TheValencia team is awarded the Synthetic Standard Prize</i>" (Spanish), [http://www.elpais.com/articulo/sociedad/Levaduras/funcionan/pixeles/elpepuespval/20091103elpepusoc_9/Tes]<br />
<br />
*'''[[Team:Cambridge]]''': "<i>University of Cambridge team wins iGEM synthetic biology competition </i>" (English), [http://www.biotechniques.com/news/University-of-Cambridge-team-wins-iGEM-synthetic-biology-competition/biotechniques-180278.html (November 5 2009)]<br />
<br />
*'''[[Team:Virginia]]''': ''[http://www.washingtonpost.com/wp-dyn/content/article/2009/10/22/AR2009102204628.html New works of science nonfiction]'', The Washington Post (October 23, 2009)</div>Wilfredhttp://2009.igem.org/IGEM_PublicityIGEM Publicity2009-11-10T10:54:30Z<p>Wilfred: /* team specific */</p>
<hr />
<div>__NOTOC__<br />
[https://2008.igem.org/IGEM_Publicity iGEM 2008 Publicity] | [http://parts.mit.edu/igem07/index.php/Media iGEM 2007 Publicity] | [http://parts.mit.edu/wiki/index.php/IGEM_News iGEM 2006 Publicity]<br />
<br />
<div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [[Press_Kit | iGEM Press Kit]])</div><br />
<br />
<html><br />
<iframe frameborder="0" width="728" height="90" marginwidth="0" marginheight="0" style="margin:0px 0px 25px 100px;"<br />
src="http://www.google.com/uds/modules/elements/newsshow/iframe.html?q=iGEM&rsz=large&format=728x90"><br />
</iframe><br />
</html><br />
<br />
<br />
<span style="color:#1e90ff; font-size:175%">'''blogs </span><span style="color:#3cb371; font-size:175%">covering iGEM 2009'''</span><br />
* The [http://igem.sdu.dk/ <span style="color:#453221; font-size: 130%">'''SDU-Denmark'''</span>] team blog about their iGEM experience.<br />
* A blog about the iGEM project of the [http://aboutgmos.org/iGEM <span style="color:#453221; font-size: 130%">'''Uppsala-Sweden'''</span>] team.<br />
<br />
<br />
<br />
<br />
====<font size=5><font color=dodgerblue>'''video/radio</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
*'''Slovenia, Heidelberg, SDU-Denmark''': [http://www.dradio.de/dlf/sendungen/forschak/1062608/ Die Biobastler von Boston], Nov 2, 2009, Deutschlandfunk radio (in German).<br />
*'''Cambridge, Imperial''': [http://www.sciencefriday.com/program/archives/200911063 Synthetic Biology Competition], Nov 6, 2009, NPR Science Friday (in English).<br />
<br />
====<font size=5><font color=dodgerblue>'''news articles</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
<br />
<br />
If you would like to share an article that was written about iGEM or your iGEM team, please link to it on this page. If you have multiple articles featuring your team, link to them all individually!<br />
<br />
Post the name of your team, the title of your article, where it was featured, and provide a link to it. <br />
<br />
''Example'':<br> <br />
'''Team Example''': ''Title of article'', Nature, [link]<br />
<br />
====<font size=4><font color=dodgerblue>'''general'''</font></font>====<br />
*'''The Economist''': ''Biohacking: Hacking goes squishy'' Sep 3, 2009 [http://www.economist.com/search/displaystory.cfm?story_id=14299634 The Economist]<br />
*'''The Scientist''': ''Brick by Brick'' Feb 1, 2009. [http://www.the-scientist.com/article/display/55378/ The Scientist]<br />
*'''Technology Review''': ''A Genetically Engineered Rainbow of Bacteria'' November 03, 2009. [http://www.technologyreview.com/blog/editors/24351/ Technology Review]<br />
<br />
====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
<br />
*'''[[Team:IBB_Pune]]''': "<i>Bio-champs in the making</i>" (English), [http://www.punemirror.in/index.aspx?Page=article&sectname=News%20-%20City&sectid=2&contentid=200909142009091423331646a131eacc#ftr2 (Monday, September 14, 2009 at 11:33:25 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Ganiti Prakriyanmadhe Jeevanuncha Vaapar! (Use of Bacteria in mathematical devices)</i>" (Marathi), [http://beta.esakal.com/2009/09/09220618/pune-use-of-bacterias-in-maths.html(Wednesday, September 09th, 2009 AT 10:09 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Synthetic Biologiteel Bharari ("Advances in Synthetic Biology")</i>" (Marathi), [https://2009.igem.org/Team:IBB_Pune/press (Wednesday 7 October 2009)]<br />
<br />
*'''[[Team:Groningen]]''': "<i>Synthetic biology students from Groningen in the race for best bacterium</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Un concours organisé par le M.I.T : en route pour Boston ! </i>" (French), [http://www.supbiotech.fr/2009/09/boston-supbiotech-igem.html (September 08, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Les étudiants de la biotech interpellent la biologie synthétique</i>" (French), [http://www.vivagora.org/spip.php?breve210 (October 09, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>Un equipo de la UAB, en el concurso de biología sintética del MIT</i>" (Spanish), [http://www.uab.es/servlet/Satellite?cid=1096481466568&pagename=UABDivulga%2FPage%2FTemplatePageDetallArticleInvestigar&param1=1253860327385 (September 23, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>UAB to participate in the synthetic biology competition at MIT</i>" (English), [http://www.uab.es/servlet/Satellite/latest-news/news-detail/uab-to-participate-in-the-synthetic-biology-competition-at-mit-1096476786473.html?noticiaid=1253657853358 (September 23, 2009)]<br />
<br />
*'''[[Team:Valencia]]''': "<i>TheValencia team is awarded the Synthetic Standard Prize</i>" (Spanish), [http://www.elpais.com/articulo/sociedad/Levaduras/funcionan/pixeles/elpepuespval/20091103elpepusoc_9/Tes]<br />
<br />
*'''[[Team:Cambridge]]''': "<i>University of Cambridge team wins iGEM synthetic biology competition </i>" (English), [http://www.biotechniques.com/news/University-of-Cambridge-team-wins-iGEM-synthetic-biology-competition/biotechniques-180278.html (November 5 2009)]<br />
<br />
*'''[[Team:Virginia]]''': ''[http://www.washingtonpost.com/wp-dyn/content/article/2009/10/22/AR2009102204628.html New works of science nonfiction]'', The Washington Post (October 23, 2009)</div>Wilfredhttp://2009.igem.org/Team:Groningen/PublicityTeam:Groningen/Publicity2009-11-10T10:53:13Z<p>Wilfred: /* Websites */</p>
<hr />
<div>=Websites=<br />
*"<i>Synthetic biology students from Groningen in the race for best bacterium</i>" (Dutch), [http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*"<i>Students from Groningen are making bacteria to remove heavy metals from water</i>" (Dutch), [http://www.groningenreporter.nl/story.php?title=groninger-studenten-maken-bacteri%3Fn-die-water-zuiveren-van-zware-metalen Groningen Reporter (August 22, 2009)]<br />
*"<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
<br />
=News=<br />
=Our Brochure=<br />
<center>[[Image:Inside_Brochure_iGEM_GRONINGEN.jpg|800px]]<br />
[[Image:Outside_Brochure_iGEM_GRONINGEN.jpg|800px]]</center></div>Wilfredhttp://2009.igem.org/Team:Groningen/TeamTeam:Groningen/Team2009-11-10T10:49:16Z<p>Wilfred: /* Presenting */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Future}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Pictures}}</div><br />
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[[Category:Team:Groningen/Disciplines/Project_Management|Team]]<br />
[[Category:Team:Groningen/Roles/Project_Manager|Team]]<br />
<br />
==Our Team At A Glance==<br />
<br />
[[Image:IGEMGroningen_Molen.jpg|400px|thumb|right|[Team:Groningen/Team|Our team!]]<br />
<br />
Welcome to the website of the iGEM Groningen team! We are an interdisciplinary team of 11 enthusiastic students from the [http://www.rug.nl/ University of Groningen] situated in the not-too-big city of [http://portal.groningen.nl/en/startpagina Groningen] in [http://maps.google.com/maps?f=q&source=s_q&hl=en&geocode=&q=Groningen&sll=53.281349,6.689459&sspn=0.007261,0.018926&ie=UTF8&z=12&iwloc=A the north of the Netherlands]. You can contact us by '''[mailto:igemgroningen@googlegroups.com mail]'''. <br />
<br />
Our team consists of the following student-members:<br />
<br />
* [[User:JolandaWitteveen|Jolanda Witteveen]] (Biomedical Technology): [[:Category:Team:Groningen/Roles/Chair|Chair]], [[:Category:Team:Groningen/Roles/Project_Manager|Project Manager]]<br />
* [[User:svenjurgens|Sven Jurgens]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [[User:Jaspervdg|Jasper van de Gronde]] (Computational Science and Visualization): [[:Category:Team:Groningen/Roles/Configuration_Manager|Configuration Manager]], [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Verhoeven1981|Michael Verhoeven]] (Chemistry): [[:Category:Team:Groningen/Roles/Public_Relations_Officer|PR Officer]]<br />
* [https://2009.igem.org/User:Nienke Nienke Kuipers] (Molecular Biology): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]] and Lab manager<br />
* [[User:Jelle|Steven Jelle Meijer]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Facility_Manager|Facility Manager Haren]]<br />
* [[User:Wilfred|Wilfred Poppinga]] (Medical Pharmaceutical Sciences): [[:Category:Team:Groningen/Roles/Chair|Vice Chair]], [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [https://2009.igem.org/User:Paulschavemaker Paul Schavemaker] (Molecular Life Sciences): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]]<br />
* [https://2009.igem.org/User:Frans Frans Bianchi] (Molecular Biology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Klaas Bernd Over|Klaas Bernd Over]] (Applied Physics): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Annelies|Annelies van Keulen]] (Molecular Biology/Psychology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
<br />
==Our advisors==<br />
*prof. dr. Oscar Kuipers: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics] (Head)<br />
*prof. dr. Jan Kok: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics]<br />
*prof. dr. Bert Poolman: Biochemistry; [http://www.centreforsyntheticbiology.eu/ Centre for Synthetic Biology] (Director)<br />
*prof. dr. Roel Bovenberg: Synthetic biology and Cell engineering; Corporate Scientist Biotechnology, [http://www.dsm.com/ DSM]<br />
*dr. Dirk Slotboom: Enzymology <br />
*[https://2008.igem.org/Team:Groningen/team.html iGEM Groningen 2008]. Especially Auke van Heel & Martijn Herber<br />
<br />
<br><br><br />
<br />
==Where to hear from us==<br />
===In the media===<br />
Follow us in '''[[Team:Groningen/Publicity| The News]]'''<br />
<br />
Also follow us on '''[http://twitter.com/igemgroningen Twitter]!'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Videos|Videos]]'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Pictures|Pictures]]'''<br />
<br />
===Presenting===<br />
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*{{todo}} December 11<sup>th</sup> 2009: Meeting @ [http://www.dsm.com/en_US/html/home/dsm_home.cgi DSM] - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=delft+DSM&fb=1&gl=nl&hq=DSM&hnear=delft&cid=0,0,8723601113946313921&ei=B_jSSrqHIcTz-QbZsNT7Ag&sa=X&oi=local_result&ct=image&resnum=1&ved=0CAoQnwIwAA Delft]<br />
*{{todo}} November 23<sup>rd</sup> 2009: Meeting @ student societies for [http://www.chemische-binding.nl/ Chemistry] and [http://www.fmf.nl/?file=main.html&lang=.en Math, Physics, Computer Science and Astronomy]<br />
*<b>October 30<sup>th</sup> to November 2<sup>nd</sup> 2009: Presentation @ The [https://2009.igem.org/ iGEM] 2009 [https://2009.igem.org/Jamboree Jamboree] - [http://whereis-beta.mit.edu/?mapterms=stata%20center&zoom=15&lat=42.36161990569666&lng=-71.09055519104004&open=object-32 MIT Stata] in Cambridge, MA</b><br />
*October 26<sup>th</sup> 2009: Lecture @ [http://www.hanzeuniversity.eu/home/international Hanze University], Biology & Medical Laboratory Research and Bioinformatics students - room A257 [http://maps.google.nl/maps?q=Zernikeplein+7+Groningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Zernikeplein+7,+9747+Groningen&gl=nl&ei=wwHPSor9A4OF-QaTkL2FAw&sa=X&oi=geocode_result&ct=title&resnum=1 Zernikeplein 11, Groningen] <br />
*October 28<sup>th</sup> 2009: Update Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Haren]<br />
*October 19<sup>th</sup> 2009: [http://www.cs.rug.nl/~biehl/Coll/index.html Colloquium] @ [http://www.rug.nl/informatica/index Institute for Mathematics and Computing Science] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=jGw&resnum=0&q=bernoulliborg%20Groningen%20Nijenborgh%209&um=1&ie=UTF-8&sa=N&tab=wl room 5161.0267 (Bernoulliborg), Groningen]<br />
*October 12<sup>th</sup> 2009: Meeting @ Marine Biology cluster - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 7<sup>th</sup> 2009: Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 2<sup>nd</sup> 2009: Lunch meeting @ [http://www2.dhv.com/default.aspx DHV] - [http://maps.google.com/maps?f=q&source=s_q&hl=nl&geocode=&q=Laan+1914+no+35,+Amersfoort&sll=37.0625,-95.677068&sspn=54.357317,79.013672&ie=UTF8&hq=&hnear=Laan+1914+35,+3818+Amersfoort,+Utrecht,+Nederland&ll=52.134107,5.36828&spn=0.010405,0.01929&t=h&z=16&iwloc=r3 Groene zaal DHV, Amersfoort]<br />
* October 1<sup>st</sup> 2009: Lunch meeting @ Life Science student society [http://www.glv-idun.nl/ GLV Idun] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Groene Zaal, Haren]<br />
*September 29<sup>th</sup> 2009: Meeting @ Applied physics student society [http://www.professorfrancken.nl/ TFV Professor Francken] - [http://maps.google.nl/maps?q=Nijenborgh%204%20NCC%20Complex&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&hl=nl&ie=UTF-8&sa=N&tab=vl NCC complex VIP Room building 16, Groningen]<br />
*[https://2009.igem.org/Team:Groningen/Notebook/24_September_2009 September 24<sup>th</sup> 2009]: Presentation @ 2nd Programme Day of the [http://www.kluyvercentre.nl/ Kluyver Centre] - [http://maps.google.nl/maps?q=Generaal+Foulkesweg+96+6703+DS+Wageningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Generaal+Foulkesweg+96,+6703+Wageningen&gl=nl&ei=giXDSvfgDcrI-Qa53ojvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Wageningse Berg, Wageningen]<br />
*September 11<sup>th</sup> 2009: Presentation @ [http://www.rug.nl/gbb/studyatgbb/generalcourses/gbbsymposium2009 17th Annual] [http://www.rug.nl/gbb/index GBB] Symposium 2009 - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=Hampshire+hotel+Groningen+Radesingel+50,+9711+EK+Groningen&fb=1&gl=nl&hq=Hampshire+hotel&hnear=Groningen+Radesingel+50,+9711+EK+Groningen&cid=0,0,5400363645623663183&ei=eybDSq-jNojj-Qbz1PXuCw&sa=X&oi=local_result&ct=image&resnum=1 Hampshire hotel, Groningen]</div><br />
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{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Category:Team:Groningen/Roles/TreasurerCategory:Team:Groningen/Roles/Treasurer2009-10-22T01:03:38Z<p>Wilfred: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
[[Category:Team:Groningen]]<br />
[[Category:Team:Groningen/Roles|Roles/Treasurer]]<br />
===In charge of all matters financial.===<br />
<br />
[[User:svenjurgens|Sven Jurgens]] and [[User:Wilfred|Wilfred Poppinga]] are responsible for keeping track of all expenses made and for making sure that sufficient funding is coming in. <br />
<br />
Interested to help [[Team:Groningen|iGEM Groningen 2009]] obtain the <b><u><i>gold</i></u></b> once more? Or do you know someone who might be interested? We are <u>always</u> looking for <b>sponsors</b>! <br />
----<br />
===[mailto:igemgroningen@googlegroups.com E-mail us!]===<br />
<center>[[Image:Inside_Brochure_iGEM_GRONINGEN.jpg|800px]]<br />
[[Image:Outside_Brochure_iGEM_GRONINGEN.jpg|800px]]</center><br />
<br />
==[mailto:igemgroningen@googlegroups.com YOUR LOGO HERE?]== <br />
[[Image:Sponsor_igem_xsmall.png|center|Groningen]]<br />
:::::::::::::::::<b>-GRONINGEN-</b><br />
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[[Image:The_city_of_Talent.jpg|[http://www.cityoftalent.nl/en The City of Talent]| 250px]]<br />
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<html><a id="Wetsus" href="http://www.wetsus.nl/"><img src="http://www.competence-research-centres.eu/uploads/tx_extpages/Bild154.gif"></a></center></html></div>Wilfredhttp://2009.igem.org/User:WilfredUser:Wilfred2009-10-22T00:53:59Z<p>Wilfred: </p>
<hr />
<div>[[Category:Team:Groningen/Roles/Treasurer]]<br />
[[Category:Team:Groningen/Roles/Chair]]<br />
[[Category:Team:Groningen/Roles/Implementer]]<br />
{{Team:Groningen/Header}}<br />
==Wilfred Poppinga== <br />
[[Image:Wilfred_Poppinga.jpg|thumb|This is me]]<br />
<b>About me</b><br><br />
With my background in [http://en.wikipedia.org/wiki/Biotechnology Biotechnology] and currently as a Master student in [http://www.rug.nl/levenswetenschappen/informatievoor/studiekiezers_msc/iv_mfw/index?lang=en Medical Pharmaceutical Sciences] at the [http://www.rug.nl/ University of Groningen] you will find me [[:Category:Team:Groningen/Roles/Implementer| in the lab]]. Besides the technical skills I keep busy in [[:Category:Team:Groningen/Roles/Treasurer| fund raising]] and act as [[:Category:Team:Groningen/Roles/Chair| second in command]] during our team meetings.<br />
With a long lasting passion for Synthetic Biology joining [[Team:Groningen/Team| iGEM Groningen 2009]] was only natural when the occasion presented itself. I am looking forward to getting deep into the field, and meeting all our fellow iGEM participants!<br />
<br><br><br />
<!--<b><i>Events</i></b><br><br />
[[Jamboree/Social_Event|iCEM]]<BR><br />
[https://2009.igem.org/Workshops/Europe London] was great! [https://2009.igem.org/User:Jaspervdg Jasper] and I really enjoyed meeting the other teams, look for a small report in our [https://2009.igem.org/Team:Groningen/Notebook notebook] on the corresponding dates.<br />
:<b> Presented at [http://www2.dhv.com/default.aspx/ DHV]!</b><br />
:<b> Presented at [http://www.kluyvercentre.nl/ the Kluyver centre]!</b><br />
::Add and edit info on [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Groningen our sandbox]<br />
:<b> Have to write [[Team:Groningen/Safety| the Safety]] part!</b><br />
:<b>Currently working on finding ways to [[Team:Groningen/Project/Accumulation| keep heavy metals in cells]] for the project of [[Team:Groningen/Project| Buoyant bacteria]]</b><br />
::<b> Participated in [[Team:Groningen/Brainstorm/Glucose_Sensing| Glucose sensing]]</b><br />
:::<b>Going finishing the wiki for [[Team:Groningen/Growth Control| Growth control]]</b><br />
Trying to create [[Template:Team:Groningen/HumanPractice/Image]]<br />
Trying to create [[https://2009.igem.org/Team:Groningen/Protocols]]<br />
--><br />
<b><i>Note</i></b>:<br />
While visiting potentials [https://2009.igem.org/Category:Team:Groningen/Roles/Treasurer#YOUR_LOGO_HERE_? sponsors], people always tend to ask me why we publish everything before the competition is over. And somehow it's very difficult to convince that it isn't a competition as per the definition they use. I try to make clear that the real competition is within ourselves, trying to reach new hights. Going where we could never go on our own, together with our [https://2009.igem.org/Team:Groningen/Team team] and as an [https://2009.igem.org/Main_Page iGEM] community. I think they rarely understand it completely, but I guess they see the enthusiasm and realize whatever kind of competition it is, it must be good enough to sponsor. If only I had the time to visit everyone in person..<br />
<br>Keeping busy with fundraising? Please [mailto:W.J.Poppinga@student.rug.nl contact me] to share your experience, <u>how to explain a competition where you try to work together with your rivals?</u><br />
----<br />
<!--==='''''Check back soon!'''''===--><br />
<center><i>Folle nocht en wille kin een protte tille</i></center><br />
[mailto:W.J.Poppinga@student.rug.nl E-mail me]<BR><br />
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{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/TeamTeam:Groningen/Team2009-10-22T00:51:43Z<p>Wilfred: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Future}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Pictures}}</div><br />
<br />
[[Category:Team:Groningen/Disciplines/Project_Management|Team]]<br />
[[Category:Team:Groningen/Roles/Project_Manager|Team]]<br />
<br />
==Our Team At A Glance==<br />
<br />
[[Image:IGEMGroningen_Molen.jpg|400px|thumb|right|[Team:Groningen/Team|Our team!]]<br />
<br />
Welcome to the website of the iGEM Groningen team! We are an interdisciplinary team of 11 enthusiastic students from the [http://www.rug.nl/ University of Groningen] situated in the not-too-big city of [http://portal.groningen.nl/en/startpagina Groningen] in [http://maps.google.com/maps?f=q&source=s_q&hl=en&geocode=&q=Groningen&sll=53.281349,6.689459&sspn=0.007261,0.018926&ie=UTF8&z=12&iwloc=A the north of the Netherlands]. You can contact us by '''[mailto:igemgroningen@googlegroups.com mail]'''. <br />
<br />
Our team consists of the following student-members:<br />
<br />
* [[User:JolandaWitteveen|Jolanda Witteveen]] (Biomedical Technology): [[:Category:Team:Groningen/Roles/Chair|Chair]], [[:Category:Team:Groningen/Roles/Project_Manager|Project Manager]]<br />
* [[User:svenjurgens|Sven Jurgens]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [[User:Jaspervdg|Jasper van de Gronde]] (Computational Science and Visualization): [[:Category:Team:Groningen/Roles/Configuration_Manager|Configuration Manager]], [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Verhoeven1981|Michael Verhoeven]] (Chemistry): [[:Category:Team:Groningen/Roles/Public_Relations_Officer|PR Officer]]<br />
* [https://2009.igem.org/User:Nienke Nienke Kuipers] (Molecular Biology): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]] and Lab manager<br />
* [[User:Jelle|Steven Jelle Meijer]] (Molecular Biology): [[:Category:Team:Groningen/Roles/Facility_Manager|Facility Manager Haren]]<br />
* [[User:Wilfred|Wilfred Poppinga]] (Medical Pharmaceutical Sciences): [[:Category:Team:Groningen/Roles/Chair|Vice Chair]], [[:Category:Team:Groningen/Roles/Treasurer|Treasurer]]<br />
* [https://2009.igem.org/User:Paulschavemaker Paul Schavemaker] (Molecular Life Sciences): [[:Category:Team:Groningen/Roles/Scribe|Minutes secretary]]<br />
* [https://2009.igem.org/User:Frans Frans Bianchi] (Molecular Biology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Klaas Bernd Over|Klaas Bernd Over]] (Applied Physics): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
* [[User:Annelies|Annelies van Keulen]] (Molecular Biology/Psychology): [[:Category:Team:Groningen/Roles/Modeller|Modeller]]<br />
<br />
==Our advisors==<br />
*prof. dr. Oscar Kuipers: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics] (Head)<br />
*prof. dr. Jan Kok: [http://molgen.biol.rug.nl/molgen/index.php Molecular Genetics]<br />
*prof. dr. Bert Poolman: Biochemistry; [http://www.centreforsyntheticbiology.eu/ Centre for Synthetic Biology] (Director)<br />
*prof. dr. Roel Bovenberg: Synthetic biology and Cell engineering; Corporate Scientist Biotechnology, [http://www.dsm.com/ DSM]<br />
*dr. Dirk Slotboom: Enzymology <br />
*[https://2008.igem.org/Team:Groningen/team.html iGEM Groningen 2008]. Especially Auke van Heel & Martijn Herber<br />
<br />
<br><br><br />
<br />
==Where to hear from us==<br />
===In the media===<br />
Follow us in '''[[Team:Groningen/Publicity| The News]]'''<br />
<br />
Also follow us on '''[http://twitter.com/igemgroningen Twitter]!'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Videos|Videos]]'''<br />
<br />
Check out some interesting '''[[Team:Groningen/Pictures|Pictures]]'''<br />
<br />
===Presenting===<br />
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<div class="meet"><br />
*{{todo}} December 11<sup>th</sup> 2009: Meeting @ [http://www.dsm.com/en_US/html/home/dsm_home.cgi DSM] - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=delft+DSM&fb=1&gl=nl&hq=DSM&hnear=delft&cid=0,0,8723601113946313921&ei=B_jSSrqHIcTz-QbZsNT7Ag&sa=X&oi=local_result&ct=image&resnum=1&ved=0CAoQnwIwAA Delft]<br />
*{{todo}} November 23<sup>rd</sup> 2009: Meeting @ student societies for [http://www.chemische-binding.nl/ Chemistry] and [http://www.fmf.nl/?file=main.html&lang=.en Math, Physics, Computer Science and Astronomy]<br />
*{{todo}} <b>October 30<sup>th</sup> to November 2<sup>nd</sup> 2009: Presentation @ The [https://2009.igem.org/ iGEM] 2009 [https://2009.igem.org/Jamboree Jamboree] - [http://whereis-beta.mit.edu/?mapterms=stata%20center&zoom=15&lat=42.36161990569666&lng=-71.09055519104004&open=object-32 MIT Stata] and [http://whereis-beta.mit.edu/?mapterms=lobby%2013&zoom=15&lat=42.35993922977393&lng=-71.092529296875&open=object-13 Lobby 13] in Cambridge, MA</b><br />
*{{todo}} October 26<sup>th</sup> 2009: Lecture @ [http://www.hanzeuniversity.eu/home/international Hanze University], Biology & Medical Laboratory Research and Bioinformatics students - room A257 [http://maps.google.nl/maps?q=Zernikeplein+7+Groningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Zernikeplein+7,+9747+Groningen&gl=nl&ei=wwHPSor9A4OF-QaTkL2FAw&sa=X&oi=geocode_result&ct=title&resnum=1 Zernikeplein 11, Groningen] <br />
*{{todo}} October 23<sup>rd</sup> 2009: Update Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Haren]<br />
*October 19<sup>th</sup> 2009: [http://www.cs.rug.nl/~biehl/Coll/index.html Colloquium] @ [http://www.rug.nl/informatica/index Institute for Mathematics and Computing Science] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=jGw&resnum=0&q=bernoulliborg%20Groningen%20Nijenborgh%209&um=1&ie=UTF-8&sa=N&tab=wl room 5161.0267 (Bernoulliborg), Groningen]<br />
*October 12<sup>th</sup> 2009: Meeting @ Marine Biology cluster - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 7<sup>th</sup> 2009: Lecture @ the Bachelor course [http://www.rug.nl/ocasys/fwn/vak/show?code=WLB07010 Genes & Behaviour] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 D225, Haren]<br />
* October 2<sup>nd</sup> 2009: Lunch meeting @ [http://www2.dhv.com/default.aspx DHV] - [http://maps.google.com/maps?f=q&source=s_q&hl=nl&geocode=&q=Laan+1914+no+35,+Amersfoort&sll=37.0625,-95.677068&sspn=54.357317,79.013672&ie=UTF8&hq=&hnear=Laan+1914+35,+3818+Amersfoort,+Utrecht,+Nederland&ll=52.134107,5.36828&spn=0.010405,0.01929&t=h&z=16&iwloc=r3 Groene zaal DHV, Amersfoort]<br />
* October 1<sup>st</sup> 2009: Lunch meeting @ Life Science student society [http://www.glv-idun.nl/ GLV Idun] - [http://maps.google.nl/maps?hl=nl&client=firefox-a&rls=org.mozilla:nl:official&hs=7wv&q=Haren+groningen&um=1&ie=UTF-8&hq=&hnear=Haren&gl=nl&ei=CSXDSsXoLJTc-Qbd7IXvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Groene Zaal, Haren]<br />
*September 29<sup>th</sup> 2009: Meeting @ Applied physics student society [http://www.professorfrancken.nl/ TFV Professor Francken] - [http://maps.google.nl/maps?q=Nijenborgh%204%20NCC%20Complex&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&hl=nl&ie=UTF-8&sa=N&tab=vl NCC complex VIP Room building 16, Groningen]<br />
*[https://2009.igem.org/Team:Groningen/Notebook/24_September_2009 September 24<sup>th</sup> 2009]: Presentation @ 2nd Programme Day of the [http://www.kluyvercentre.nl/ Kluyver Centre] - [http://maps.google.nl/maps?q=Generaal+Foulkesweg+96+6703+DS+Wageningen&oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&hq=&hnear=Generaal+Foulkesweg+96,+6703+Wageningen&gl=nl&ei=giXDSvfgDcrI-Qa53ojvCw&sa=X&oi=geocode_result&ct=image&resnum=1 Wageningse Berg, Wageningen]<br />
*September 11<sup>th</sup> 2009: Presentation @ [http://www.rug.nl/gbb/studyatgbb/generalcourses/gbbsymposium2009 17th Annual] [http://www.rug.nl/gbb/index GBB] Symposium 2009 - [http://maps.google.nl/maps?oe=utf-8&rls=org.mozilla:nl:official&client=firefox-a&um=1&ie=UTF-8&q=Hampshire+hotel+Groningen+Radesingel+50,+9711+EK+Groningen&fb=1&gl=nl&hq=Hampshire+hotel&hnear=Groningen+Radesingel+50,+9711+EK+Groningen&cid=0,0,5400363645623663183&ei=eybDSq-jNojj-Qbz1PXuCw&sa=X&oi=local_result&ct=image&resnum=1 Hampshire hotel, Groningen]</div><br />
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{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/FutureTeam:Groningen/Future2009-10-22T00:43:20Z<p>Wilfred: /* Labwork */</p>
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<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Brainstorm}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Team}}</div><br />
<br />
==Labwork==<br />
A large amount of progress has been obtained in the research line of arsenic accumulation, however several other interesting metals (e.g. copper, zinc) had to be abandoned which might provide more interesting applications. For this to occur, the constructs that were abandoned along the way (e.g. SmtA, HmtA) should be finished and characterized. But also for the existing arsenic devices more characterizing can be performed, ''e''.''g''. more detailed characterization of the uptake capacity of the transporter (using higher metal concentrations in death assays). In this project fermentation was used to supply the culture with medium saturated with air in higher amount than can be attained in shake cultures. This was to ensure a higher amount of gas to be available to enter the gas vesicles, for a future project it might be of interest to sparge nitrogen into the broth to obtain anaerobic conditions. Anaerobic condition will influence the oxidation state of arsenic, probably having an effect on the uptake efficiency. A nice addition to the gas vesicle cluster would be the removal of the 10 time repeat, which was attempted in our project without success.<br />
<br />
==Modelling==<br />
===JavaScript===<br />
To make it possible to have interactive graphs and calculators on our Wiki, based on our model, we have implemented our entire model in JavaScript. This included developing our own ODE solver (among other things). We feel that having our model accessible on the Wiki has many advantages and it would be very useful to develop a library to handle ODE (and perhaps stochastic) models using JavaScript. Such a library could support:<br />
<br />
*Graphs that work well in pretty much any browser, including a legend (and possibly 3D functionality). Linked graphs would be a plus.<br />
*Reading from various sources, like an HTML table, Google Docs, csv files, etc.<br />
*Integration of ODE models defined by a function that returns the gradient based on the current values.<br />
*Equilibrium computations. Preferably based on the same function used for integrating ODE models.<br />
*Fitting a model to experimental data.<br />
<br />
==="Dumbed-down" user interface===<br />
Normally a model is used only "internally" to, for example, predict results and/or analyse them. We have made our model a more integral part of our project by making (parts of) it available right next to our theoretical texts and results, but this could be taken much further. It would be interesting to make an interface to our model that is suited for someone without an extensive background in molecular biology and/or modelling, with easy-to-use sliders and clear visual representations.<br />
<br />
A "dumbed-down" user interface would likely not be aimed at doing any of the normal modelling tasks, but would be more of an educational tool, allowing an interactive exploration of a project. This could be immensely useful in giving people insight into an iGEM project like our own.<br />
<br />
===Parameters===<br />
Our determination of the import rate of arsenic is reasonably accurate, but many more parameters of which we are much less certain remain. On [[Team:Groningen/Modelling/Characterization|our characterization page]] several different ways to find these parameters are discussed.<br />
<br />
{{Team:Groningen/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/Project/VesicleTeam:Groningen/Project/Vesicle2009-10-21T23:48:09Z<p>Wilfred: /* Buoyancy Tests */</p>
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<h1>Gas vesicles</h1><br />
'''Our goal in this project is to make cells bouyant in the presence of certain concentrations of metals like copper, zinc and arsenic. Metal induced gas vesicle production can provide our cells with this bouyancy. Gas vesicles are bacterial organelles consisting entirely of proteins that envelope a gas filled space. We made, and send to the registry, parts in which the metal sensitive promoters for copper, zinc and arsenic were cloned in front of the ''gvp'' (Gas Vesicle Protein) gene cluster. For further characterization of the ''gvp'' gene cluster inducible and constitutive promoters were also cloned in front of this cluster. Buoyancy tests showed that our constructs were able to increase cell buoyancy and electron micrographs showed the presence of gas vesicles. A model was made to predict what volume fraction of a cell would have to be gas vesicle for this cell to have a density equal to that of water.'''<br />
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<br />
==Background==<br />
<br />
Gas vesicles are organelles made entirely out of proteins that envelope a gas filled space. Because only gas can penetrate the gas vesicles the total density of the cell is lowered. This lower cell density leads in turn to a buoyancy phenotype. Outside of the laboratory this buoyancy is used by microorganisms to vertically position themselves in the water column or simply to reduce their sinking rates. Organisms can regulate buoyancy by reducing gas vesicle production or by accumulation of denser compounds like carbohydrates. For certain cyanobacteria this regulation depends on light intensities. <br />
<br />
For a number of organisms it has been shown that all proteins important for the expression of gas vesicle are part of a single gene cluster. The ''gvp'' gene cluster used in this project was cloned from ''Bacillus megaterium'' into ''E. coli'' ([[Team:Groningen/Literature#Li1998|Li & Cannon 1998]]). This gene cluster, now containing 11 genes was turned into a biobrick by Melbourne 2007 (<partinfo>BBa_I750016</partinfo>). Figure 1 shows the gene cluster as it was send in by Melbourne in 2007.<br />
<br />
For more info see: [[Team:Groningen/Literature#Walsby1994|Walsby 1994]].<br />
<br />
[[Image:Gas vesicle cluster bba I750016.PNG|frame|Figure 1: Gas vesicle gene cluster (<partinfo>BBa_I750016</partinfo>)<br />
]]<br />
<br />
==Goal==<br />
<br />
The goal of our project is to engineer an organism that can remove heavy metals from water. To facilitate easy separation the cells that have taken up metals should float so they can be removed from the water. The introduction of ''gvp'' gene cluster provides the cell with the required buoyancy. <br />
<br />
To make the buoyancy level of the cell responsive to the metal concentration inside the cell, metal sensitive promoters would have to be cloned in front of the ''gvp'' gene cluster. In this project these metal sensitive promoters were responsive to zinc, copper and arsenic. We also wanted to make a construct with constitutive and inducible promoters in front of the GVP cluster to show a proof of principle and as a back-up if our metal induced construct would fail. <br />
<br />
Finally we wanted to improve the Melbourne 2007 biobrick (<partinfo>BBa_I750016</partinfo>) by removing a repeat that was accidentally introduced during the removal of forbidden restriction sites.<br />
<br />
==Cloning strategy== <br />
For our msGVP (metal sensitive GVP) constructs we ordered oligo's containing the promoter region and the necessary restriction sites. When annealed these pieces of DNA have ''Eco''RI and ''Spe''I sticky ends. The vector containing GVP (<partinfo>BBa_I750016</partinfo>) was cut with ''Eco''RI and ''Xba''I and was ligated to the promoter. ([[Team:Groningen/Protocols#Annealing synthetic oligo’s|Protocol]])<br />
<br />
The metal sensitive GVP constructs are: <partinfo>BBa_K190033</partinfo>, <partinfo>BBa_K190034</partinfo> and <partinfo>BBa_K190035</partinfo>.<br />
<br />
Then because of compatibility issues when our entire system has to be assembled into one cell the whole metal sensitive promoter and GVP part were transfered to a different vector.<br />
<br />
<center>[[Image:Cloning strategy floating device1.PNG]]</center><br />
:Figure 2: The floating device will be built up of an inducible promoter which can be induced by a certain intracellular concentration of metal-ions, and a gas vesicle cluster.<br />
<br />
==Buoyancy Tests==<br />
<br />
The buoyancy of GVP was tested by using the [[Team:Groningen/Protocols#Buoyancy_test|buoyancy test protocol]]. The cells were grown in medium and induced and were resuspended in a salt solution (0.15 mM NaCl) in a test tube and were left for a while in order to give the cells time to sink or float. <br />
<br />
''Different circumstances''<br />
<br />
Several different circumstances and small changes to the protocol were made in order to find the perfect circumstance for the buoyancy test. It appeared that with a low cell density the difference between floating and sinking could not be seen very well. The results were best visible with and cell density of OD<sub>600</sub>=1.5. Also we tried to do the buoyancy test in a longer tube since it was expected that the difference between floating and sinking would be more obvious. This, however, did not appear to be the case, unfortunately. Also doing the buoyancy test in a higher saline concentration did not have an enhanced floating effect. <br />
Another adaption we tried was the way of induction. In the standard protocol the cells were induced in the overnight culture. It was also tried if induction in the saline or at the exponential phase of growth or even induction on plate would make any difference. Unfortunately this did not make a huge difference.<br />
<br />
''Fermentor test''<br />
<br />
[[Image:Buoyancy pNL29.jpg|800px|thumb|center|Figure 3. Buoyancy test with pNL29, cells were induced in exponential phase and resuspended in NaCl in a OD<sub>600</sub> of 1.5 A) Fermentor buoyancy test, samples taken after 1 hour, 2.5 hours, 6 hours, 8.5 hours and 22.5 hours. The cells were induced after 1 hour, at exponential phase. Photgraph taken 1 day after resuspension. B) normal, non-fermentor buoyancy test, samples taken at t=1 t/m 4. Photograph taken 1 day after resuspension. C) Same as A, photograph was taken 2 days after resuspension. D) same as C, photograph taken 2 days after resuspension.]]<br />
<br />
{|<br />
|[[Image:Buoyancy arsR GVP.jpg|500px|thumb|Figure 4. Buoyancy test with pSB1AC3 containing pArsR-GVP, cells were induced in exponential phase and resuspended in NaCl in a OD<sub>600</sub> of 1.5 A) Fermentor buoyancy test, samples taken after 1 hour, 2 hours, 6 hours, 7.5 hours and 22 hours. The cells were induced after 1 hour, at exponential phase. Photgraph taken 1 day after resuspension. B) normal, non-fermentor buoyancy test, samples taken at t=1 t/m 4. Photograph taken 1 day after resuspension.]]<br />
|[[Image:Groningen_ODFerAndNonFer.PNG|400px|thumb|Figure 5. Graph of OD measurements at 600nm of both the fermentor and non-fermentor tests. Solid lines and points represent actual measurements, dotted lines represent the expected curve between the last two measurements]]<br />
|}<br />
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<br />
So the length of the tube, the saline concentration and the time of induction did work for the buoyancy test. It was also suggested that there was not enough gas in the surrounding of the cells and a better result could be achieved if this could be improve or another gas could be used. To test this we tried to grow the cells in a [[Team:Groningen/Protocols#Fermentation|fermentor]]. It was also suggested that the fermentor test could be done with helium, however, modelling showed that it would not make a difference in floating which gas is used as long as it is lighter than water (check this yourself by changing the density (ρ) of the gas [[Team:Groningen/Project/Vesicle#Modelling|here]]). Therefore the fermentor test was done by using air. This resulted in better buoyancy results. As can be seen in figure 3A the positive control pNL29 showed better buoyancy over time. After 2 hours the cells were in exponential phase and were induced with IPTG. After 8,5 hours the buoyancy is best, after 22.5 hours the buoyancy the cell level is declining. This suggests that there is an optimum after 8,5 hours. The cells at t=22.5 are probably in stationary phase whereas the cells at t=8.5h could still be in exponential phase, this could explain the difference in buoyancy found. It suggests that in stationary phase less gas vesicles are produced. Figure 3C shows the same tubes 24 hours later. This still shows buoyancy for the t=6 and t=8 tubes and no buoyancy for the others. This suggest that the buoyancy last for at least 24 hours. Simultaneously a normal, non-fermentor, buoyancy test was also performed with the same construct. In figure 3B these results at day 1 can be seen, this shows nothing. After a day no buoyancy can be seen for t=1 and t=2 a more dense cell suspension can be seen for t=3 and t=4 (figure 3D), however still no confincing buoyancy can be seen.<br />
Figure 4 shows the results from one of our own constructs, pArsR-GvP (<partinfo>BBa_K190033</partinfo>) grown in a fermentor. This shows an increase in buoyancy in time, however, at t=22.5h no buoyant cells can be seen. A buoyancy test done at the same time without a fermentor shows the same increase in buoyancy but does show buoyancy at t=22.5h (figure 4B). This difference can be explained since the cells in the fermentor are probably already dead or dying. In a fermentor the cell density is large this causes the cells to die.<br />
<br />
==Electron Microscopy==<br />
<br />
To check whether gas vesicles really were present in the cells we did some electron microscopy.<br />
<br />
In Figure 5 a picture of gas vesicles in a protoplast can be seen. This protoplast comes from an ''E. coli'' cell that contained a plasmid with the ''gvp'' gene cluster behind an arsenic sensitive promoter (<partinfo>BBa_K190033</partinfo>).<br />
<br />
<br />
[[Image:GasvesiclesEM.jpg|thumb|500px|left|Figure 5. Gas vesicles in ''E. coli'' protoplasts (<partinfo>BBa_K190033</partinfo>). The cells were treated with Lysozyme and SDS to create the protoplasts, uranyl acetate was used for staining. Magnification: 11500x.]]<br />
<br />
<div style="clear:both"></div><br />
<br />
==Modelling==<br />
<br />
===Buoyancy===<br />
The gas vesicles are shaped roughly like a cylinder with a cone at each end, whose cross-section we model as (based mostly on [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]):<br />
<br />
[[Image:Vesicle_Shape.png]]<br />
<br />
We assume the interior of the wall of the gas vesicle is similarly shaped to the exterior, just slightly smaller (the right-most part of the image above illustrates this situation for the left tip of the gas vesicle). This means the different dimensions are related through the equations below. To determine the total volume, just use them with the given width/diameter (at least for the dimensions given in [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]). To determine the gas volume, use them with w<sub>gas</sub> and d<sub>gas</sub>.<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
w = <input type="text" id="w" value="300"/> nm (</html>[[:Image:Ars-lyzo-007.png|TEM picture]]<html>)<br/><br />
d = <input type="text" id="d" value="75"/> nm (</html>[[:Image:Ars-lyzo-007.png|TEM picture]]<html>)<br/><br />
tw = <input type="text" id="tw" value="1.8"/> nm (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/><br />
a = <input type="text" id="a" value="77"/> &deg; (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/><br />
&rho;<sub>gas</sub> = <input type="text" id="rhogas" value="1.2"/> kg/m<sup>3</sup> (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/> <!-- Walsby1994, for moist air at atmospheric pressure --><br />
&rho;<sub>wall</sub> = <input type="text" id="rhowall" value="1320"/> kg/m<sup>3</sup> (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/> <!-- Walsby1994 --><br />
<br />
<button onClick="computeVolumes()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="volumeError" style="color:red"></div><br />
V<sub>gas</sub> = <span id="Vgas"></span> nm<sup>3</sup><br/><br />
M<sub>gas</sub> = <span id="Mgas"></span> yg<br/><br />
V<sub>wall</sub> = <span id="Vwall"></span> nm<sup>3</sup><br/><br />
M<sub>wall</sub> = <span id="Mwall"></span> yg<br/><br />
V<sub>vesicle</sub> = <span id="Vvesicle"></span> nm<sup>3</sup><br/><br />
M<sub>vesicle</sub> = <span id="Mvesicle"></span> yg<br/><br />
<b>&rho;<sub>vesicle</sub> = <span id="rhovesicle"></span> kg/m<sup>3</sup></b><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeVolumes);<br />
<br />
function computeVolumes() {<br />
// Input<br />
var wNode = document.getElementById("w");<br />
var twNode = document.getElementById("tw");<br />
var dNode = document.getElementById("d");<br />
var aNode = document.getElementById("a");<br />
var rhogasNode = document.getElementById("rhogas");<br />
var rhowallNode = document.getElementById("rhowall");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var volumeErrorNode = document.getElementById("volumeError");<br />
var wwtNode = document.getElementById("wwt");<br />
volumeErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var VgasNode = document.getElementById("Vgas");<br />
var VwallNode = document.getElementById("Vwall");<br />
var MgasNode = document.getElementById("Mgas");<br />
var MwallNode = document.getElementById("Mwall");<br />
var VvesicleNode = document.getElementById("Vvesicle");<br />
var MvesicleNode = document.getElementById("Mvesicle");<br />
var rhovesicleNode = document.getElementById("rhovesicle");<br />
<br />
// Read inputs<br />
var w = Number(wNode.value);<br />
var tw = Number(twNode.value);<br />
var d = Number(dNode.value);<br />
var a = Number(aNode.value) * Math.PI / 180.0;<br />
var rhogas = Number(rhogasNode.value);<br />
var rhowall = Number(rhowallNode.value);<br />
<br />
// Compute Vgas and Vwall<br />
try {<br />
var wwt = tw/Math.sin(a/2);<br />
var Vvesicle = computeVolume(w, d, a);<br />
var Vgas = computeVolume(w-2*wwt,d-2*tw,a);<br />
var Vwall = Vvesicle - Vgas;<br />
var Mgas = rhogas*Vgas;<br />
var Mwall = rhowall*Vwall;<br />
var Mvesicle = Mgas+Mwall;<br />
var rhovesicle = Mvesicle/Vvesicle;<br />
} catch(err) {<br />
volumeErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
if (wwtNode) setOutput(wwtNode, wwt);<br />
<br />
// Set outputs<br />
setOutput(VgasNode, Vgas);<br />
setOutput(VwallNode, Vwall);<br />
setOutput(MgasNode, Mgas);<br />
setOutput(MwallNode, Mwall);<br />
setOutput(VvesicleNode, Vvesicle);<br />
setOutput(MvesicleNode, Mvesicle);<br />
setOutput(rhovesicleNode, rhovesicle);<br />
}<br />
<br />
function computeVolume(w,d,a) {<br />
// This computes the volume of cylinder with a cone at each end as defined in the text.<br />
var wt = (1/2)*d/Math.tan(a/2);<br />
var wc = w-2*wt;<br />
var Vc = (1/4)*Math.PI*Math.pow(d,2)*wc;<br />
var Vt = (1/12)*Math.PI*Math.pow(d,2)*wt;<br />
if (wc<0) throw Error("The given diameter would imply a larger width.<br/>(Do not trust the computed volumes!)");<br />
return Vc+2*Vt;<br />
}<br />
<br />
function formatNumberToHTML(v,p) {<br />
if (p===undefined) p = 5;<br />
return v.toPrecision(p)<br />
.replace(/e\+([0-9]+)$/i,'&middot;10<sup>$1</sup>')<br />
.replace(/e\-([0-9]+)$/i,'&middot;10<sup>-$1</sup>');<br />
}<br />
<br />
function setOutput(node,v,p) {<br />
node.innerHTML = formatNumberToHTML(v);<br />
node.value = v;<br />
}<br />
</script><br />
</html><br />
|style="vertical-align:top;"|<pre><br />
w = total width<br />
tw = thickness of wall (1.8-1.95nm)<br />
d = diameter<br />
a = 77 degrees<br />
&rho;gas = density of gas in vesicle (kg/m^3 = yg/nm^3)<br />
&rho;wall = density of vesicle wall (kg/m^3)<br />
wwt = tw/sin(a/2)<br />
wt = (1/2)*d/tan(a/2)<br />
wc = w - 2*wt<br />
Vc = (1/4)*pi*d^2*wc<br />
Vt = (1/12)*pi*d^2*wt<br />
V = Vc+2*Vt<br />
M = &rho;*V<br />
<br />
wgas = w-2*wwt = width of gas space<br />
dgas = d-2*tw = diameter of gas space<br />
V = Vgas + Vwall<br />
</pre><br />
|}<br />
<br />
Now we can consider the buoyant density of <i>E. coli</i> with gas vesicles. We have chosen to approach this problem using densities and volume ratios. According to [[Team:Groningen/Literature#Baldwin1995|Baldwin 1995]], [[Team:Groningen/Literature#Bylund1991|Bylund 1991]] and [[Team:Groningen/Literature#Poole1977|Poole 1977]], the density of (wild-type) <i>E. coli</i> is 1100 kg/m<sup>3</sup> &plusmn;3% under wildly varying conditions. This makes our method easier than trying to directly compute the density of a single cell, due to the fact that the volume can differ wildly (both during the life cycle and from strain to strain) and a lack of concrete data on the number of gas vesicles produced (in <i>E. coli</i>). Note that the computations below assume that the gas vesicles simply add to the existing structures.<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<nobr>&rho;<sub>medium</sub> = <input type="text" id="rhomedium" value="1000"/> kg/m<sup>3</sup></nobr><br/><br />
&rho;<sub>cell</sub> = <input type="text" id="rhocell" value="1100"/> kg/m<sup>3</sup><br/> <!-- Reasonable estimate, TODO: more precision+reference --><br />
<br />
<button onClick="computeEColiDensity()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="densityError" style="color:red"></div><br />
V<sub>v</sub> / V<sub>cv</sub> > <span id="relVvesicles"></span><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeEColiDensity);<br />
<br />
function computeEColiDensity() {<br />
// Input<br />
var rhomediumNode = document.getElementById("rhomedium");<br />
var rhovesicleNode = document.getElementById("rhovesicle");<br />
var rhocellNode = document.getElementById("rhocell");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var densityErrorNode = document.getElementById("densityError");<br />
densityErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var relVvesiclesNode = document.getElementById("relVvesicles");<br />
<br />
// Read inputs<br />
var rhomedium = Number(rhomediumNode.value);<br />
var rhovesicle = Number(rhovesicleNode.value);<br />
var rhocell = Number(rhocellNode.value);<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var relVvesicles = 1.0 - (rhomedium-rhovesicle)/(rhocell-rhovesicle);<br />
if (rhovesicle>=rhocell) throw Error("Vesicle denser than cell, > should be <.");<br />
} catch(err) {<br />
densityErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
<br />
// Set outputs<br />
setOutput(relVvesiclesNode, relVvesicles);<br />
document.getElementById('densityLimitGraph').refresh();<br />
}<br />
</script><br />
</html><br />
{{graph|Team:Groningen/Graphs/DensityLimit|id=densityLimitGraph}}<br />
|style="vertical-align:top;"|<pre><br />
Vc = volume of a cell without gas vesicles<br />
Vv = volume of gas vesicles in cell<br />
Vcv = volume of a cell with gas vesicles (assumed to be Vc+Vv)<br />
&rho;c = density of a cell without gas vesicles<br />
&rho;v = density of gas vesicles<br />
&rho;m = density of medium<br />
<br />
The following has to be true if the cell floats:<br />
Vc*&rho;c + Vv*&rho;v < Vcv*&rho;m<br />
(Vcv-Vv)*&rho;c + Vv*&rho;v < Vcv*&rho;m<br />
&rho;c + (Vv/Vcv)*(&rho;v-&rho;c) < &rho;m<br />
Assume (&rho;v - &rho;c)<0<br />
Vv/Vcv > (&rho;m - &rho;c)/(&rho;v - &rho;c)<br />
Vv/Vcv > 1 - (&rho;m - &rho;v)/(&rho;c - &rho;v)<br />
</pre><br />
'''Explanation of the graph'''<br />
<br />
Four curves are shown, corresponding to how many gas vesicles a cell needs with "our" gas vesicles (unless you changed the constants in the calculator above), the gas vesicles documented in [[Team:Groningen/Literature#Li1998|Li 1998]]{{infoBox|Using a width and diameter of 75nm and 50nm, respectively. Here we assume that their "width" should be interpreted as our diameter, as doing it the other way around would leave no room for a cylinder and they specifically mention that the vesicles appear to be shaped like cylinders with conical ends.}}, the gas vesicles from Anabaena in [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]{{infoBox|Using a width and diameter of 500nm and 84nm, respectively.}} and our gas vesicles when the medium has the density of seawater.<br />
<br />
'''The X-axis''' depicts the cell density of the part of the cell not occupied by gas vesicles.<br />
<br />
'''The Y-axis''' depicts the minimum volume fraction of the cell that should consist of gas vesicles to make the cell float.<br />
|}<br />
<br />
{{GraphHeader}}<br />
<br />
==Conclusion & Discussion==<br />
<br />
We have experimented with two different constructs containing the ''gvp'' gene cluster i.e. pNL29 containing the 6 kb gene cluster from ''Bacillus megaterium'' ([[Team:Groningen/Literature#Li1998|Li & Cannon 1998]]) and [http://partsregistry.org/wiki/index.php/Part:BBa_I750016 BBa_I750016] from the [https://2007.igem.org/Melbourne Melbourne 2007] iGEM team.<br />
We observed that it is best to have an OD600 of 1.5 when doing bouyancy tests, for withnessing differences with lower values is difficult. Furthermore buoyancy tests carried out in sea water or normal (LB) medium also give rise to difficult to interpret results. <br />
<br />
For cells cultivated in a more aerobic environment, such as the ones carried out in a fermentor, an enhanced bouyancy phenotype is observed. The extra O<sub>2</sub> added probably causes a higher concentration of intracellular oxygen, that can diffuse to the gas vesicles that are produced. The best buoyancy phenotype is withnessed at t=8.5 hours, however at t=22.5 hours no buoyancy can be seen. This suggests that there is an optimum after 8,5 hours.The cells at t=22.5 are probably in stationary phase whereas the cells at t=8.5h could still be in exponential phase, this could explain the difference in buoyancy found. It suggests that in stationary phase less gas vesicles are produced. Buoyancy is observed after 1 day, and also still after 2 days, which suggest that the buoyancy last for at least 24 hours. This is in accordance with experimental data from [[Team:Groningen/Literature#Walsby1994|Walsby, 1994]], who also still observed a buoyant phenotype after 2 days.<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/Project/VesicleTeam:Groningen/Project/Vesicle2009-10-21T23:43:42Z<p>Wilfred: /* Cloning strategy */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Project/Promoters}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Modelling}}</div><br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h1>Gas vesicles</h1><br />
'''Our goal in this project is to make cells bouyant in the presence of certain concentrations of metals like copper, zinc and arsenic. Metal induced gas vesicle production can provide our cells with this bouyancy. Gas vesicles are bacterial organelles consisting entirely of proteins that envelope a gas filled space. We made, and send to the registry, parts in which the metal sensitive promoters for copper, zinc and arsenic were cloned in front of the ''gvp'' (Gas Vesicle Protein) gene cluster. For further characterization of the ''gvp'' gene cluster inducible and constitutive promoters were also cloned in front of this cluster. Buoyancy tests showed that our constructs were able to increase cell buoyancy and electron micrographs showed the presence of gas vesicles. A model was made to predict what volume fraction of a cell would have to be gas vesicle for this cell to have a density equal to that of water.'''<br />
<br><br><br><br />
</div><br />
|}<br />
<br />
<br />
==Background==<br />
<br />
Gas vesicles are organelles made entirely out of proteins that envelope a gas filled space. Because only gas can penetrate the gas vesicles the total density of the cell is lowered. This lower cell density leads in turn to a buoyancy phenotype. Outside of the laboratory this buoyancy is used by microorganisms to vertically position themselves in the water column or simply to reduce their sinking rates. Organisms can regulate buoyancy by reducing gas vesicle production or by accumulation of denser compounds like carbohydrates. For certain cyanobacteria this regulation depends on light intensities. <br />
<br />
For a number of organisms it has been shown that all proteins important for the expression of gas vesicle are part of a single gene cluster. The ''gvp'' gene cluster used in this project was cloned from ''Bacillus megaterium'' into ''E. coli'' ([[Team:Groningen/Literature#Li1998|Li & Cannon 1998]]). This gene cluster, now containing 11 genes was turned into a biobrick by Melbourne 2007 (<partinfo>BBa_I750016</partinfo>). Figure 1 shows the gene cluster as it was send in by Melbourne in 2007.<br />
<br />
For more info see: [[Team:Groningen/Literature#Walsby1994|Walsby 1994]].<br />
<br />
[[Image:Gas vesicle cluster bba I750016.PNG|frame|Figure 1: Gas vesicle gene cluster (<partinfo>BBa_I750016</partinfo>)<br />
]]<br />
<br />
==Goal==<br />
<br />
The goal of our project is to engineer an organism that can remove heavy metals from water. To facilitate easy separation the cells that have taken up metals should float so they can be removed from the water. The introduction of ''gvp'' gene cluster provides the cell with the required buoyancy. <br />
<br />
To make the buoyancy level of the cell responsive to the metal concentration inside the cell, metal sensitive promoters would have to be cloned in front of the ''gvp'' gene cluster. In this project these metal sensitive promoters were responsive to zinc, copper and arsenic. We also wanted to make a construct with constitutive and inducible promoters in front of the GVP cluster to show a proof of principle and as a back-up if our metal induced construct would fail. <br />
<br />
Finally we wanted to improve the Melbourne 2007 biobrick (<partinfo>BBa_I750016</partinfo>) by removing a repeat that was accidentally introduced during the removal of forbidden restriction sites.<br />
<br />
==Cloning strategy== <br />
For our msGVP (metal sensitive GVP) constructs we ordered oligo's containing the promoter region and the necessary restriction sites. When annealed these pieces of DNA have ''Eco''RI and ''Spe''I sticky ends. The vector containing GVP (<partinfo>BBa_I750016</partinfo>) was cut with ''Eco''RI and ''Xba''I and was ligated to the promoter. ([[Team:Groningen/Protocols#Annealing synthetic oligo’s|Protocol]])<br />
<br />
The metal sensitive GVP constructs are: <partinfo>BBa_K190033</partinfo>, <partinfo>BBa_K190034</partinfo> and <partinfo>BBa_K190035</partinfo>.<br />
<br />
Then because of compatibility issues when our entire system has to be assembled into one cell the whole metal sensitive promoter and GVP part were transfered to a different vector.<br />
<br />
<center>[[Image:Cloning strategy floating device1.PNG]]</center><br />
:Figure 2: The floating device will be built up of an inducible promoter which can be induced by a certain intracellular concentration of metal-ions, and a gas vesicle cluster.<br />
<br />
==Buoyancy Tests==<br />
<br />
The buoyancy of GVP was tested by using the [[Team:Groningen/Protocols#Bouyancytest|buoyancy test protocol]]. The cells were grown in medium and induced and were resuspended in a salt solution (0.15 mM NaCl) in a test tube and were left for a while in order to give the cells time to sink or float. <br />
<br />
''Different circumstances''<br />
<br />
Several different circumstances and small changes to the protocol were made in order to find the perfect circumstance for the buoyancy test. It appeared that with a low cell density the difference between floating and sinking could not be seen very well. The results were best visible with and cell density of OD<sub>600</sub>=1.5. Also we tried to do the buoyancy test in a longer tube since it was expected that the difference between floating and sinking would be more obvious. This, however, did not appear to be the case, unfortunately. Also doing the buoyancy test in a higher saline concentration did not have an enhanced floating effect. <br />
Another adaption we tried was the way of induction. In the standard protocol the cells were induced in the overnight culture. It was also tried if induction in the saline or at the exponential phase of growth or even induction on plate would make any difference. Unfortunately this did not make a huge difference.<br />
<br />
''Fermentor test''<br />
<br />
[[Image:Buoyancy pNL29.jpg|800px|thumb|center|Figure 3. Buoyancy test with pNL29, cells were induced in exponential phase and resuspended in NaCl in a OD600 of 1.5 A) Fermentor buoyancy test, samples taken after 1 hour, 2.5 hours, 6 hours, 8.5 hours and 22.5 hours. The cells were induced after 1 hour, at exponential phase. Photgraph taken 1 day after resuspension. B) normal, non-fermentor buoyancy test, samples taken at t=1 t/m 4. Photograph taken 1 day after resuspension. C) Same as A, photograph was taken 2 days after resuspension. D) same as C, photograph taken 2 days after resuspension.]]<br />
<br />
{|<br />
|[[Image:Buoyancy arsR GVP.jpg|500px|thumb|Figure 4. Buoyancy test with pSB1AC3 containing pArsR-GVP, cells were induced in exponential phase and resuspended in NaCl in a OD600 of 1.5 A) Fermentor buoyancy test, samples taken after 1 hour, 2 hours, 6 hours, 7.5 hours and 22 hours. The cells were induced after 1 hour, at exponential phase. Photgraph taken 1 day after resuspension. B) normal, non-fermentor buoyancy test, samples taken at t=1 t/m 4. Photograph taken 1 day after resuspension.]]<br />
|[[Image:Groningen_ODFerAndNonFer.PNG|400px|thumb|Figure 5. Graph of OD measurements at 600nm of both the fermentor and non-fermentor tests. Solid lines and points represent actual measurements, dotted lines represent the expected curve between the last two measurements]]<br />
|}<br />
<div style="clear:both"></div><br />
<br />
So the length of the tube, the saline concentration and the time of induction did work for the buoyancy test. It was also suggested that there was not enough gas in the surrounding of the cells and a better result could be achieved if this could be improve or another gas could be used. To test this we tried to grow the cells in a [[Team:Groningen/Protocols#Fermentation|fermentor]]. It was also suggested that the fermentor test could be done with helium, however, modelling showed that it would not make a difference in floating which gas is used as long as it is lighter than water (check this yourself by changing the density (ρ) of the gas [[Team:Groningen/Project/Vesicle#Modelling|here]]). Therefore the fermentor test was done by using O<sub>2</sub>. This resulted in better buoyancy results. As can be seen in figure 3A the positive control pNL29 showed better buoyancy over time. After 2 hours the cells were in exponential phase and were induced with IPTG. After 8,5 hours the buoyancy is best, after 22.5 hours the buoyancy the cell level is declining. This suggests that there is an optimum after 8,5 hours. The cells at t=22.5 are probably in stationary phase whereas the cells at t=8.5h could still be in exponential phase, this could explain the difference in buoyancy found. It suggests that in stationary phase less gas vesicles are produced. Figure 3C shows the same tubes 24 hours later. This still shows buoyancy for the t=6 and t=8 tubes and no buoyancy for the others. This suggest that the buoyancy last for at least 24 hours. Simultaneously a normal, non-fermentor, buoyancy test was also performed with the same construct. In figure 3B these results at day 1 can be seen, this shows nothing. After a day no buoyancy can be seen for t=1 and t=2 a more dense cell suspension can be seen for t=3 and t=4 (figure 3D), however still no confincing buoyancy can be seen.<br />
Figure 4 shows the results from one of our own constructs, pArsR-GvP (<partinfo>BBa_K190033</partinfo>) grown in a fermentor. This shows an increase in buoyancy in time, however, at t=22.5h no buoyant cells can be seen. A buoyancy test done at the same time without a fermentor shows the same increase in buoyancy but does show buoyancy at t=22.5h (figure 4B). This difference can be explained since the cells in the fermentor are probably already dead or dying. In a fermentor the cell density is large this causes the cells to die.<br />
<br />
==Electron Microscopy==<br />
<br />
To check whether gas vesicles really were present in the cells we did some electron microscopy.<br />
<br />
In Figure 5 a picture of gas vesicles in a protoplast can be seen. This protoplast comes from an ''E. coli'' cell that contained a plasmid with the ''gvp'' gene cluster behind an arsenic sensitive promoter (<partinfo>BBa_K190033</partinfo>).<br />
<br />
<br />
[[Image:GasvesiclesEM.jpg|thumb|500px|left|Figure 5. Gas vesicles in ''E. coli'' protoplasts (<partinfo>BBa_K190033</partinfo>). The cells were treated with Lysozyme and SDS to create the protoplasts, uranyl acetate was used for staining. Magnification: 11500x.]]<br />
<br />
<div style="clear:both"></div><br />
<br />
==Modelling==<br />
<br />
===Buoyancy===<br />
The gas vesicles are shaped roughly like a cylinder with a cone at each end, whose cross-section we model as (based mostly on [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]):<br />
<br />
[[Image:Vesicle_Shape.png]]<br />
<br />
We assume the interior of the wall of the gas vesicle is similarly shaped to the exterior, just slightly smaller (the right-most part of the image above illustrates this situation for the left tip of the gas vesicle). This means the different dimensions are related through the equations below. To determine the total volume, just use them with the given width/diameter (at least for the dimensions given in [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]). To determine the gas volume, use them with w<sub>gas</sub> and d<sub>gas</sub>.<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
w = <input type="text" id="w" value="300"/> nm (</html>[[:Image:Ars-lyzo-007.png|TEM picture]]<html>)<br/><br />
d = <input type="text" id="d" value="75"/> nm (</html>[[:Image:Ars-lyzo-007.png|TEM picture]]<html>)<br/><br />
tw = <input type="text" id="tw" value="1.8"/> nm (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/><br />
a = <input type="text" id="a" value="77"/> &deg; (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/><br />
&rho;<sub>gas</sub> = <input type="text" id="rhogas" value="1.2"/> kg/m<sup>3</sup> (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/> <!-- Walsby1994, for moist air at atmospheric pressure --><br />
&rho;<sub>wall</sub> = <input type="text" id="rhowall" value="1320"/> kg/m<sup>3</sup> (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/> <!-- Walsby1994 --><br />
<br />
<button onClick="computeVolumes()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="volumeError" style="color:red"></div><br />
V<sub>gas</sub> = <span id="Vgas"></span> nm<sup>3</sup><br/><br />
M<sub>gas</sub> = <span id="Mgas"></span> yg<br/><br />
V<sub>wall</sub> = <span id="Vwall"></span> nm<sup>3</sup><br/><br />
M<sub>wall</sub> = <span id="Mwall"></span> yg<br/><br />
V<sub>vesicle</sub> = <span id="Vvesicle"></span> nm<sup>3</sup><br/><br />
M<sub>vesicle</sub> = <span id="Mvesicle"></span> yg<br/><br />
<b>&rho;<sub>vesicle</sub> = <span id="rhovesicle"></span> kg/m<sup>3</sup></b><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeVolumes);<br />
<br />
function computeVolumes() {<br />
// Input<br />
var wNode = document.getElementById("w");<br />
var twNode = document.getElementById("tw");<br />
var dNode = document.getElementById("d");<br />
var aNode = document.getElementById("a");<br />
var rhogasNode = document.getElementById("rhogas");<br />
var rhowallNode = document.getElementById("rhowall");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var volumeErrorNode = document.getElementById("volumeError");<br />
var wwtNode = document.getElementById("wwt");<br />
volumeErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var VgasNode = document.getElementById("Vgas");<br />
var VwallNode = document.getElementById("Vwall");<br />
var MgasNode = document.getElementById("Mgas");<br />
var MwallNode = document.getElementById("Mwall");<br />
var VvesicleNode = document.getElementById("Vvesicle");<br />
var MvesicleNode = document.getElementById("Mvesicle");<br />
var rhovesicleNode = document.getElementById("rhovesicle");<br />
<br />
// Read inputs<br />
var w = Number(wNode.value);<br />
var tw = Number(twNode.value);<br />
var d = Number(dNode.value);<br />
var a = Number(aNode.value) * Math.PI / 180.0;<br />
var rhogas = Number(rhogasNode.value);<br />
var rhowall = Number(rhowallNode.value);<br />
<br />
// Compute Vgas and Vwall<br />
try {<br />
var wwt = tw/Math.sin(a/2);<br />
var Vvesicle = computeVolume(w, d, a);<br />
var Vgas = computeVolume(w-2*wwt,d-2*tw,a);<br />
var Vwall = Vvesicle - Vgas;<br />
var Mgas = rhogas*Vgas;<br />
var Mwall = rhowall*Vwall;<br />
var Mvesicle = Mgas+Mwall;<br />
var rhovesicle = Mvesicle/Vvesicle;<br />
} catch(err) {<br />
volumeErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
if (wwtNode) setOutput(wwtNode, wwt);<br />
<br />
// Set outputs<br />
setOutput(VgasNode, Vgas);<br />
setOutput(VwallNode, Vwall);<br />
setOutput(MgasNode, Mgas);<br />
setOutput(MwallNode, Mwall);<br />
setOutput(VvesicleNode, Vvesicle);<br />
setOutput(MvesicleNode, Mvesicle);<br />
setOutput(rhovesicleNode, rhovesicle);<br />
}<br />
<br />
function computeVolume(w,d,a) {<br />
// This computes the volume of cylinder with a cone at each end as defined in the text.<br />
var wt = (1/2)*d/Math.tan(a/2);<br />
var wc = w-2*wt;<br />
var Vc = (1/4)*Math.PI*Math.pow(d,2)*wc;<br />
var Vt = (1/12)*Math.PI*Math.pow(d,2)*wt;<br />
if (wc<0) throw Error("The given diameter would imply a larger width.<br/>(Do not trust the computed volumes!)");<br />
return Vc+2*Vt;<br />
}<br />
<br />
function formatNumberToHTML(v,p) {<br />
if (p===undefined) p = 5;<br />
return v.toPrecision(p)<br />
.replace(/e\+([0-9]+)$/i,'&middot;10<sup>$1</sup>')<br />
.replace(/e\-([0-9]+)$/i,'&middot;10<sup>-$1</sup>');<br />
}<br />
<br />
function setOutput(node,v,p) {<br />
node.innerHTML = formatNumberToHTML(v);<br />
node.value = v;<br />
}<br />
</script><br />
</html><br />
|style="vertical-align:top;"|<pre><br />
w = total width<br />
tw = thickness of wall (1.8-1.95nm)<br />
d = diameter<br />
a = 77 degrees<br />
&rho;gas = density of gas in vesicle (kg/m^3 = yg/nm^3)<br />
&rho;wall = density of vesicle wall (kg/m^3)<br />
wwt = tw/sin(a/2)<br />
wt = (1/2)*d/tan(a/2)<br />
wc = w - 2*wt<br />
Vc = (1/4)*pi*d^2*wc<br />
Vt = (1/12)*pi*d^2*wt<br />
V = Vc+2*Vt<br />
M = &rho;*V<br />
<br />
wgas = w-2*wwt = width of gas space<br />
dgas = d-2*tw = diameter of gas space<br />
V = Vgas + Vwall<br />
</pre><br />
|}<br />
<br />
Now we can consider the buoyant density of <i>E. coli</i> with gas vesicles. We have chosen to approach this problem using densities and volume ratios. According to [[Team:Groningen/Literature#Baldwin1995|Baldwin 1995]], [[Team:Groningen/Literature#Bylund1991|Bylund 1991]] and [[Team:Groningen/Literature#Poole1977|Poole 1977]], the density of (wild-type) <i>E. coli</i> is 1100 kg/m<sup>3</sup> &plusmn;3% under wildly varying conditions. This makes our method easier than trying to directly compute the density of a single cell, due to the fact that the volume can differ wildly (both during the life cycle and from strain to strain) and a lack of concrete data on the number of gas vesicles produced (in <i>E. coli</i>). Note that the computations below assume that the gas vesicles simply add to the existing structures.<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<nobr>&rho;<sub>medium</sub> = <input type="text" id="rhomedium" value="1000"/> kg/m<sup>3</sup></nobr><br/><br />
&rho;<sub>cell</sub> = <input type="text" id="rhocell" value="1100"/> kg/m<sup>3</sup><br/> <!-- Reasonable estimate, TODO: more precision+reference --><br />
<br />
<button onClick="computeEColiDensity()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="densityError" style="color:red"></div><br />
V<sub>v</sub> / V<sub>cv</sub> > <span id="relVvesicles"></span><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeEColiDensity);<br />
<br />
function computeEColiDensity() {<br />
// Input<br />
var rhomediumNode = document.getElementById("rhomedium");<br />
var rhovesicleNode = document.getElementById("rhovesicle");<br />
var rhocellNode = document.getElementById("rhocell");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var densityErrorNode = document.getElementById("densityError");<br />
densityErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var relVvesiclesNode = document.getElementById("relVvesicles");<br />
<br />
// Read inputs<br />
var rhomedium = Number(rhomediumNode.value);<br />
var rhovesicle = Number(rhovesicleNode.value);<br />
var rhocell = Number(rhocellNode.value);<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var relVvesicles = 1.0 - (rhomedium-rhovesicle)/(rhocell-rhovesicle);<br />
if (rhovesicle>=rhocell) throw Error("Vesicle denser than cell, > should be <.");<br />
} catch(err) {<br />
densityErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
<br />
// Set outputs<br />
setOutput(relVvesiclesNode, relVvesicles);<br />
document.getElementById('densityLimitGraph').refresh();<br />
}<br />
</script><br />
</html><br />
{{graph|Team:Groningen/Graphs/DensityLimit|id=densityLimitGraph}}<br />
|style="vertical-align:top;"|<pre><br />
Vc = volume of a cell without gas vesicles<br />
Vv = volume of gas vesicles in cell<br />
Vcv = volume of a cell with gas vesicles (assumed to be Vc+Vv)<br />
&rho;c = density of a cell without gas vesicles<br />
&rho;v = density of gas vesicles<br />
&rho;m = density of medium<br />
<br />
The following has to be true if the cell floats:<br />
Vc*&rho;c + Vv*&rho;v < Vcv*&rho;m<br />
(Vcv-Vv)*&rho;c + Vv*&rho;v < Vcv*&rho;m<br />
&rho;c + (Vv/Vcv)*(&rho;v-&rho;c) < &rho;m<br />
Assume (&rho;v - &rho;c)<0<br />
Vv/Vcv > (&rho;m - &rho;c)/(&rho;v - &rho;c)<br />
Vv/Vcv > 1 - (&rho;m - &rho;v)/(&rho;c - &rho;v)<br />
</pre><br />
'''Explanation of the graph'''<br />
<br />
Four curves are shown, corresponding to how many gas vesicles a cell needs with "our" gas vesicles (unless you changed the constants in the calculator above), the gas vesicles documented in [[Team:Groningen/Literature#Li1998|Li 1998]]{{infoBox|Using a width and diameter of 75nm and 50nm, respectively. Here we assume that their "width" should be interpreted as our diameter, as doing it the other way around would leave no room for a cylinder and they specifically mention that the vesicles appear to be shaped like cylinders with conical ends.}}, the gas vesicles from Anabaena in [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]{{infoBox|Using a width and diameter of 500nm and 84nm, respectively.}} and our gas vesicles when the medium has the density of seawater.<br />
<br />
'''The X-axis''' depicts the cell density of the part of the cell not occupied by gas vesicles.<br />
<br />
'''The Y-axis''' depicts the minimum volume fraction of the cell that should consist of gas vesicles to make the cell float.<br />
|}<br />
<br />
{{GraphHeader}}<br />
<br />
==Conclusion & Discussion==<br />
<br />
We have experimented with two different constructs containing the ''gvp'' gene cluster i.e. pNL29 containing the 6 kb gene cluster from ''Bacillus megaterium'' ([[Team:Groningen/Literature#Li1998|Li & Cannon 1998]]) and [http://partsregistry.org/wiki/index.php/Part:BBa_I750016 BBa_I750016] from the [http://parts.mit.edu/igem07/index.php/Melbourne Melbourne 2007] iGEM team.<br />
We observed that it is best to have an OD600 of 1.5 when doing bouyancy tests, for withnessing differences with lower values is difficult. Furthermore buoyancy tests carried out in sea water or normal (LB) medium also give rise to difficult to interpret results. <br />
<br />
For cells cultivated in a more aerobic environment, such as the ones carried out in a fermentor, an enhanced bouyancy phenotype is observed. The extra O<sub>2</sub> added probably causes a higher concentration of intracellular oxygen, that can diffuse to the gas vesicles that are produced. The best buoyancy phenotype is withnessed at t=8.5 hours, however at t=22.5 hours no buoyancy can be seen. This suggests that there is an optimum after 8,5 hours.The cells at t=22.5 are probably in stationary phase whereas the cells at t=8.5h could still be in exponential phase, this could explain the difference in buoyancy found. It suggests that in stationary phase less gas vesicles are produced. Buoyancy is observed after 1 day, and also still after 2 days, which suggest that the buoyancy last for at least 24 hours. This is in accordance with experimental data from [[Team:Groningen/Literature#Walsby1994|Walsby, 1994]], who also still observed a buoyant phenotype after 2 days.<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/Project/VesicleTeam:Groningen/Project/Vesicle2009-10-21T23:43:04Z<p>Wilfred: </p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Project/Promoters}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Modelling}}</div><br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h1>Gas vesicles</h1><br />
'''Our goal in this project is to make cells bouyant in the presence of certain concentrations of metals like copper, zinc and arsenic. Metal induced gas vesicle production can provide our cells with this bouyancy. Gas vesicles are bacterial organelles consisting entirely of proteins that envelope a gas filled space. We made, and send to the registry, parts in which the metal sensitive promoters for copper, zinc and arsenic were cloned in front of the ''gvp'' (Gas Vesicle Protein) gene cluster. For further characterization of the ''gvp'' gene cluster inducible and constitutive promoters were also cloned in front of this cluster. Buoyancy tests showed that our constructs were able to increase cell buoyancy and electron micrographs showed the presence of gas vesicles. A model was made to predict what volume fraction of a cell would have to be gas vesicle for this cell to have a density equal to that of water.'''<br />
<br><br><br><br />
</div><br />
|}<br />
<br />
<br />
==Background==<br />
<br />
Gas vesicles are organelles made entirely out of proteins that envelope a gas filled space. Because only gas can penetrate the gas vesicles the total density of the cell is lowered. This lower cell density leads in turn to a buoyancy phenotype. Outside of the laboratory this buoyancy is used by microorganisms to vertically position themselves in the water column or simply to reduce their sinking rates. Organisms can regulate buoyancy by reducing gas vesicle production or by accumulation of denser compounds like carbohydrates. For certain cyanobacteria this regulation depends on light intensities. <br />
<br />
For a number of organisms it has been shown that all proteins important for the expression of gas vesicle are part of a single gene cluster. The ''gvp'' gene cluster used in this project was cloned from ''Bacillus megaterium'' into ''E. coli'' ([[Team:Groningen/Literature#Li1998|Li & Cannon 1998]]). This gene cluster, now containing 11 genes was turned into a biobrick by Melbourne 2007 (<partinfo>BBa_I750016</partinfo>). Figure 1 shows the gene cluster as it was send in by Melbourne in 2007.<br />
<br />
For more info see: [[Team:Groningen/Literature#Walsby1994|Walsby 1994]].<br />
<br />
[[Image:Gas vesicle cluster bba I750016.PNG|frame|Figure 1: Gas vesicle gene cluster (<partinfo>BBa_I750016</partinfo>)<br />
]]<br />
<br />
==Goal==<br />
<br />
The goal of our project is to engineer an organism that can remove heavy metals from water. To facilitate easy separation the cells that have taken up metals should float so they can be removed from the water. The introduction of ''gvp'' gene cluster provides the cell with the required buoyancy. <br />
<br />
To make the buoyancy level of the cell responsive to the metal concentration inside the cell, metal sensitive promoters would have to be cloned in front of the ''gvp'' gene cluster. In this project these metal sensitive promoters were responsive to zinc, copper and arsenic. We also wanted to make a construct with constitutive and inducible promoters in front of the GVP cluster to show a proof of principle and as a back-up if our metal induced construct would fail. <br />
<br />
Finally we wanted to improve the Melbourne 2007 biobrick (<partinfo>BBa_I750016</partinfo>) by removing a repeat that was accidentally introduced during the removal of forbidden restriction sites.<br />
<br />
==Cloning strategy== <br />
For our msGVP (metal sensitive GVP) constructs we ordered oligo's containing the promoter region and the necessary restriction sites. When annealed these pieces of DNA have ''Eco''RI and ''Spe''I sticky ends. The vector containing GVP (<partinfo>BBa_I750016</partinfo>) was cut with ''Eco''RI and ''Xba''I and was ligated to the promoter. ([[Team:Groningen/Protocols#Annealing synthetic oligo’s|Protocol]])<br />
<br />
The metal sensitive GVP constructs are: <partinfo>BBa_K190033</partinfo>, <partinfo>BBa_K190034</partinfo> and <partinfo>BBa_K190035</partinfo>.<br />
<br />
Then because of compatibility issues when our entire system has to be assembled into one cell the whole metal sensitive promoter and GVP part were transfered to a different vector.<br />
<br />
[[Image:Cloning strategy floating device1.PNG]]<br />
:Figure 2: The floating device will be built up of an inducible promoter which can be induced by a certain intracellular concentration of metal-ions, and a gas vesicle cluster.<br />
<br />
==Buoyancy Tests==<br />
<br />
The buoyancy of GVP was tested by using the [[Team:Groningen/Protocols#Bouyancytest|buoyancy test protocol]]. The cells were grown in medium and induced and were resuspended in a salt solution (0.15 mM NaCl) in a test tube and were left for a while in order to give the cells time to sink or float. <br />
<br />
''Different circumstances''<br />
<br />
Several different circumstances and small changes to the protocol were made in order to find the perfect circumstance for the buoyancy test. It appeared that with a low cell density the difference between floating and sinking could not be seen very well. The results were best visible with and cell density of OD<sub>600</sub>=1.5. Also we tried to do the buoyancy test in a longer tube since it was expected that the difference between floating and sinking would be more obvious. This, however, did not appear to be the case, unfortunately. Also doing the buoyancy test in a higher saline concentration did not have an enhanced floating effect. <br />
Another adaption we tried was the way of induction. In the standard protocol the cells were induced in the overnight culture. It was also tried if induction in the saline or at the exponential phase of growth or even induction on plate would make any difference. Unfortunately this did not make a huge difference.<br />
<br />
''Fermentor test''<br />
<br />
[[Image:Buoyancy pNL29.jpg|800px|thumb|center|Figure 3. Buoyancy test with pNL29, cells were induced in exponential phase and resuspended in NaCl in a OD600 of 1.5 A) Fermentor buoyancy test, samples taken after 1 hour, 2.5 hours, 6 hours, 8.5 hours and 22.5 hours. The cells were induced after 1 hour, at exponential phase. Photgraph taken 1 day after resuspension. B) normal, non-fermentor buoyancy test, samples taken at t=1 t/m 4. Photograph taken 1 day after resuspension. C) Same as A, photograph was taken 2 days after resuspension. D) same as C, photograph taken 2 days after resuspension.]]<br />
<br />
{|<br />
|[[Image:Buoyancy arsR GVP.jpg|500px|thumb|Figure 4. Buoyancy test with pSB1AC3 containing pArsR-GVP, cells were induced in exponential phase and resuspended in NaCl in a OD600 of 1.5 A) Fermentor buoyancy test, samples taken after 1 hour, 2 hours, 6 hours, 7.5 hours and 22 hours. The cells were induced after 1 hour, at exponential phase. Photgraph taken 1 day after resuspension. B) normal, non-fermentor buoyancy test, samples taken at t=1 t/m 4. Photograph taken 1 day after resuspension.]]<br />
|[[Image:Groningen_ODFerAndNonFer.PNG|400px|thumb|Figure 5. Graph of OD measurements at 600nm of both the fermentor and non-fermentor tests. Solid lines and points represent actual measurements, dotted lines represent the expected curve between the last two measurements]]<br />
|}<br />
<div style="clear:both"></div><br />
<br />
So the length of the tube, the saline concentration and the time of induction did work for the buoyancy test. It was also suggested that there was not enough gas in the surrounding of the cells and a better result could be achieved if this could be improve or another gas could be used. To test this we tried to grow the cells in a [[Team:Groningen/Protocols#Fermentation|fermentor]]. It was also suggested that the fermentor test could be done with helium, however, modelling showed that it would not make a difference in floating which gas is used as long as it is lighter than water (check this yourself by changing the density (ρ) of the gas [[Team:Groningen/Project/Vesicle#Modelling|here]]). Therefore the fermentor test was done by using O<sub>2</sub>. This resulted in better buoyancy results. As can be seen in figure 3A the positive control pNL29 showed better buoyancy over time. After 2 hours the cells were in exponential phase and were induced with IPTG. After 8,5 hours the buoyancy is best, after 22.5 hours the buoyancy the cell level is declining. This suggests that there is an optimum after 8,5 hours. The cells at t=22.5 are probably in stationary phase whereas the cells at t=8.5h could still be in exponential phase, this could explain the difference in buoyancy found. It suggests that in stationary phase less gas vesicles are produced. Figure 3C shows the same tubes 24 hours later. This still shows buoyancy for the t=6 and t=8 tubes and no buoyancy for the others. This suggest that the buoyancy last for at least 24 hours. Simultaneously a normal, non-fermentor, buoyancy test was also performed with the same construct. In figure 3B these results at day 1 can be seen, this shows nothing. After a day no buoyancy can be seen for t=1 and t=2 a more dense cell suspension can be seen for t=3 and t=4 (figure 3D), however still no confincing buoyancy can be seen.<br />
Figure 4 shows the results from one of our own constructs, pArsR-GvP (<partinfo>BBa_K190033</partinfo>) grown in a fermentor. This shows an increase in buoyancy in time, however, at t=22.5h no buoyant cells can be seen. A buoyancy test done at the same time without a fermentor shows the same increase in buoyancy but does show buoyancy at t=22.5h (figure 4B). This difference can be explained since the cells in the fermentor are probably already dead or dying. In a fermentor the cell density is large this causes the cells to die.<br />
<br />
==Electron Microscopy==<br />
<br />
To check whether gas vesicles really were present in the cells we did some electron microscopy.<br />
<br />
In Figure 5 a picture of gas vesicles in a protoplast can be seen. This protoplast comes from an ''E. coli'' cell that contained a plasmid with the ''gvp'' gene cluster behind an arsenic sensitive promoter (<partinfo>BBa_K190033</partinfo>).<br />
<br />
<br />
[[Image:GasvesiclesEM.jpg|thumb|500px|left|Figure 5. Gas vesicles in ''E. coli'' protoplasts (<partinfo>BBa_K190033</partinfo>). The cells were treated with Lysozyme and SDS to create the protoplasts, uranyl acetate was used for staining. Magnification: 11500x.]]<br />
<br />
<div style="clear:both"></div><br />
<br />
==Modelling==<br />
<br />
===Buoyancy===<br />
The gas vesicles are shaped roughly like a cylinder with a cone at each end, whose cross-section we model as (based mostly on [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]):<br />
<br />
[[Image:Vesicle_Shape.png]]<br />
<br />
We assume the interior of the wall of the gas vesicle is similarly shaped to the exterior, just slightly smaller (the right-most part of the image above illustrates this situation for the left tip of the gas vesicle). This means the different dimensions are related through the equations below. To determine the total volume, just use them with the given width/diameter (at least for the dimensions given in [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]). To determine the gas volume, use them with w<sub>gas</sub> and d<sub>gas</sub>.<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
w = <input type="text" id="w" value="300"/> nm (</html>[[:Image:Ars-lyzo-007.png|TEM picture]]<html>)<br/><br />
d = <input type="text" id="d" value="75"/> nm (</html>[[:Image:Ars-lyzo-007.png|TEM picture]]<html>)<br/><br />
tw = <input type="text" id="tw" value="1.8"/> nm (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/><br />
a = <input type="text" id="a" value="77"/> &deg; (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/><br />
&rho;<sub>gas</sub> = <input type="text" id="rhogas" value="1.2"/> kg/m<sup>3</sup> (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/> <!-- Walsby1994, for moist air at atmospheric pressure --><br />
&rho;<sub>wall</sub> = <input type="text" id="rhowall" value="1320"/> kg/m<sup>3</sup> (</html>[[Team:Groningen/Literature#Walsby1994|Walsby1994]]<html>)<br/> <!-- Walsby1994 --><br />
<br />
<button onClick="computeVolumes()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="volumeError" style="color:red"></div><br />
V<sub>gas</sub> = <span id="Vgas"></span> nm<sup>3</sup><br/><br />
M<sub>gas</sub> = <span id="Mgas"></span> yg<br/><br />
V<sub>wall</sub> = <span id="Vwall"></span> nm<sup>3</sup><br/><br />
M<sub>wall</sub> = <span id="Mwall"></span> yg<br/><br />
V<sub>vesicle</sub> = <span id="Vvesicle"></span> nm<sup>3</sup><br/><br />
M<sub>vesicle</sub> = <span id="Mvesicle"></span> yg<br/><br />
<b>&rho;<sub>vesicle</sub> = <span id="rhovesicle"></span> kg/m<sup>3</sup></b><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeVolumes);<br />
<br />
function computeVolumes() {<br />
// Input<br />
var wNode = document.getElementById("w");<br />
var twNode = document.getElementById("tw");<br />
var dNode = document.getElementById("d");<br />
var aNode = document.getElementById("a");<br />
var rhogasNode = document.getElementById("rhogas");<br />
var rhowallNode = document.getElementById("rhowall");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var volumeErrorNode = document.getElementById("volumeError");<br />
var wwtNode = document.getElementById("wwt");<br />
volumeErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var VgasNode = document.getElementById("Vgas");<br />
var VwallNode = document.getElementById("Vwall");<br />
var MgasNode = document.getElementById("Mgas");<br />
var MwallNode = document.getElementById("Mwall");<br />
var VvesicleNode = document.getElementById("Vvesicle");<br />
var MvesicleNode = document.getElementById("Mvesicle");<br />
var rhovesicleNode = document.getElementById("rhovesicle");<br />
<br />
// Read inputs<br />
var w = Number(wNode.value);<br />
var tw = Number(twNode.value);<br />
var d = Number(dNode.value);<br />
var a = Number(aNode.value) * Math.PI / 180.0;<br />
var rhogas = Number(rhogasNode.value);<br />
var rhowall = Number(rhowallNode.value);<br />
<br />
// Compute Vgas and Vwall<br />
try {<br />
var wwt = tw/Math.sin(a/2);<br />
var Vvesicle = computeVolume(w, d, a);<br />
var Vgas = computeVolume(w-2*wwt,d-2*tw,a);<br />
var Vwall = Vvesicle - Vgas;<br />
var Mgas = rhogas*Vgas;<br />
var Mwall = rhowall*Vwall;<br />
var Mvesicle = Mgas+Mwall;<br />
var rhovesicle = Mvesicle/Vvesicle;<br />
} catch(err) {<br />
volumeErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
if (wwtNode) setOutput(wwtNode, wwt);<br />
<br />
// Set outputs<br />
setOutput(VgasNode, Vgas);<br />
setOutput(VwallNode, Vwall);<br />
setOutput(MgasNode, Mgas);<br />
setOutput(MwallNode, Mwall);<br />
setOutput(VvesicleNode, Vvesicle);<br />
setOutput(MvesicleNode, Mvesicle);<br />
setOutput(rhovesicleNode, rhovesicle);<br />
}<br />
<br />
function computeVolume(w,d,a) {<br />
// This computes the volume of cylinder with a cone at each end as defined in the text.<br />
var wt = (1/2)*d/Math.tan(a/2);<br />
var wc = w-2*wt;<br />
var Vc = (1/4)*Math.PI*Math.pow(d,2)*wc;<br />
var Vt = (1/12)*Math.PI*Math.pow(d,2)*wt;<br />
if (wc<0) throw Error("The given diameter would imply a larger width.<br/>(Do not trust the computed volumes!)");<br />
return Vc+2*Vt;<br />
}<br />
<br />
function formatNumberToHTML(v,p) {<br />
if (p===undefined) p = 5;<br />
return v.toPrecision(p)<br />
.replace(/e\+([0-9]+)$/i,'&middot;10<sup>$1</sup>')<br />
.replace(/e\-([0-9]+)$/i,'&middot;10<sup>-$1</sup>');<br />
}<br />
<br />
function setOutput(node,v,p) {<br />
node.innerHTML = formatNumberToHTML(v);<br />
node.value = v;<br />
}<br />
</script><br />
</html><br />
|style="vertical-align:top;"|<pre><br />
w = total width<br />
tw = thickness of wall (1.8-1.95nm)<br />
d = diameter<br />
a = 77 degrees<br />
&rho;gas = density of gas in vesicle (kg/m^3 = yg/nm^3)<br />
&rho;wall = density of vesicle wall (kg/m^3)<br />
wwt = tw/sin(a/2)<br />
wt = (1/2)*d/tan(a/2)<br />
wc = w - 2*wt<br />
Vc = (1/4)*pi*d^2*wc<br />
Vt = (1/12)*pi*d^2*wt<br />
V = Vc+2*Vt<br />
M = &rho;*V<br />
<br />
wgas = w-2*wwt = width of gas space<br />
dgas = d-2*tw = diameter of gas space<br />
V = Vgas + Vwall<br />
</pre><br />
|}<br />
<br />
Now we can consider the buoyant density of <i>E. coli</i> with gas vesicles. We have chosen to approach this problem using densities and volume ratios. According to [[Team:Groningen/Literature#Baldwin1995|Baldwin 1995]], [[Team:Groningen/Literature#Bylund1991|Bylund 1991]] and [[Team:Groningen/Literature#Poole1977|Poole 1977]], the density of (wild-type) <i>E. coli</i> is 1100 kg/m<sup>3</sup> &plusmn;3% under wildly varying conditions. This makes our method easier than trying to directly compute the density of a single cell, due to the fact that the volume can differ wildly (both during the life cycle and from strain to strain) and a lack of concrete data on the number of gas vesicles produced (in <i>E. coli</i>). Note that the computations below assume that the gas vesicles simply add to the existing structures.<br />
<br />
{|<br />
|style="vertical-align:top;"|<html><br />
<div style="background:#efe;border:1px solid #9c9;padding:1em;"><br />
<table style="border-collapse:collapse;background:none;"><tr><br />
<td style="border-right:1px solid #9c9;padding-right:1em;"><br />
<nobr>&rho;<sub>medium</sub> = <input type="text" id="rhomedium" value="1000"/> kg/m<sup>3</sup></nobr><br/><br />
&rho;<sub>cell</sub> = <input type="text" id="rhocell" value="1100"/> kg/m<sup>3</sup><br/> <!-- Reasonable estimate, TODO: more precision+reference --><br />
<br />
<button onClick="computeEColiDensity()">Compute</button><br/><br />
</td><br />
<br />
<td style="padding-left:1em;"><br />
<div id="densityError" style="color:red"></div><br />
V<sub>v</sub> / V<sub>cv</sub> > <span id="relVvesicles"></span><br/><br />
</td><br />
</tr></table><br />
</div><br />
<script type="text/javascript"><br />
<br />
addOnloadHook(computeEColiDensity);<br />
<br />
function computeEColiDensity() {<br />
// Input<br />
var rhomediumNode = document.getElementById("rhomedium");<br />
var rhovesicleNode = document.getElementById("rhovesicle");<br />
var rhocellNode = document.getElementById("rhocell");<br />
<br />
// Intermediates (mostly useful for debugging)<br />
var densityErrorNode = document.getElementById("densityError");<br />
densityErrorNode.innerHTML = '';<br />
<br />
// Outputs<br />
var relVvesiclesNode = document.getElementById("relVvesicles");<br />
<br />
// Read inputs<br />
var rhomedium = Number(rhomediumNode.value);<br />
var rhovesicle = Number(rhovesicleNode.value);<br />
var rhocell = Number(rhocellNode.value);<br />
<br />
// Compute density(/-ies)<br />
try {<br />
var relVvesicles = 1.0 - (rhomedium-rhovesicle)/(rhocell-rhovesicle);<br />
if (rhovesicle>=rhocell) throw Error("Vesicle denser than cell, > should be <.");<br />
} catch(err) {<br />
densityErrorNode.innerHTML = err.message;<br />
}<br />
<br />
// Set intermediates if they exist<br />
<br />
// Set outputs<br />
setOutput(relVvesiclesNode, relVvesicles);<br />
document.getElementById('densityLimitGraph').refresh();<br />
}<br />
</script><br />
</html><br />
{{graph|Team:Groningen/Graphs/DensityLimit|id=densityLimitGraph}}<br />
|style="vertical-align:top;"|<pre><br />
Vc = volume of a cell without gas vesicles<br />
Vv = volume of gas vesicles in cell<br />
Vcv = volume of a cell with gas vesicles (assumed to be Vc+Vv)<br />
&rho;c = density of a cell without gas vesicles<br />
&rho;v = density of gas vesicles<br />
&rho;m = density of medium<br />
<br />
The following has to be true if the cell floats:<br />
Vc*&rho;c + Vv*&rho;v < Vcv*&rho;m<br />
(Vcv-Vv)*&rho;c + Vv*&rho;v < Vcv*&rho;m<br />
&rho;c + (Vv/Vcv)*(&rho;v-&rho;c) < &rho;m<br />
Assume (&rho;v - &rho;c)<0<br />
Vv/Vcv > (&rho;m - &rho;c)/(&rho;v - &rho;c)<br />
Vv/Vcv > 1 - (&rho;m - &rho;v)/(&rho;c - &rho;v)<br />
</pre><br />
'''Explanation of the graph'''<br />
<br />
Four curves are shown, corresponding to how many gas vesicles a cell needs with "our" gas vesicles (unless you changed the constants in the calculator above), the gas vesicles documented in [[Team:Groningen/Literature#Li1998|Li 1998]]{{infoBox|Using a width and diameter of 75nm and 50nm, respectively. Here we assume that their "width" should be interpreted as our diameter, as doing it the other way around would leave no room for a cylinder and they specifically mention that the vesicles appear to be shaped like cylinders with conical ends.}}, the gas vesicles from Anabaena in [[Team:Groningen/Literature#Walsby1994|Walsby 1994]]{{infoBox|Using a width and diameter of 500nm and 84nm, respectively.}} and our gas vesicles when the medium has the density of seawater.<br />
<br />
'''The X-axis''' depicts the cell density of the part of the cell not occupied by gas vesicles.<br />
<br />
'''The Y-axis''' depicts the minimum volume fraction of the cell that should consist of gas vesicles to make the cell float.<br />
|}<br />
<br />
{{GraphHeader}}<br />
<br />
==Conclusion & Discussion==<br />
<br />
We have experimented with two different constructs containing the ''gvp'' gene cluster i.e. pNL29 containing the 6 kb gene cluster from ''Bacillus megaterium'' ([[Team:Groningen/Literature#Li1998|Li & Cannon 1998]]) and [http://partsregistry.org/wiki/index.php/Part:BBa_I750016 BBa_I750016] from the [http://parts.mit.edu/igem07/index.php/Melbourne Melbourne 2007] iGEM team.<br />
We observed that it is best to have an OD600 of 1.5 when doing bouyancy tests, for withnessing differences with lower values is difficult. Furthermore buoyancy tests carried out in sea water or normal (LB) medium also give rise to difficult to interpret results. <br />
<br />
For cells cultivated in a more aerobic environment, such as the ones carried out in a fermentor, an enhanced bouyancy phenotype is observed. The extra O<sub>2</sub> added probably causes a higher concentration of intracellular oxygen, that can diffuse to the gas vesicles that are produced. The best buoyancy phenotype is withnessed at t=8.5 hours, however at t=22.5 hours no buoyancy can be seen. This suggests that there is an optimum after 8,5 hours.The cells at t=22.5 are probably in stationary phase whereas the cells at t=8.5h could still be in exponential phase, this could explain the difference in buoyancy found. It suggests that in stationary phase less gas vesicles are produced. Buoyancy is observed after 1 day, and also still after 2 days, which suggest that the buoyancy last for at least 24 hours. This is in accordance with experimental data from [[Team:Groningen/Literature#Walsby1994|Walsby, 1994]], who also still observed a buoyant phenotype after 2 days.<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T23:30:21Z<p>Wilfred: </p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Project/Accumulation}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle}}</div><br />
<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h1>Promoters</h1><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest.By cloning the ArsR and CueO promotor in front of RFP we have shown that by induction with respectively Arsenite and Copper repression of the promotor is reduced and expression of RFP enhanced. We took the following promoters into consideration:'''<br />
<center><br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|}<br />
</center><br />
</div><br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters.<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>E. coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>E. coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straight forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting its sequence from the genome database of ''E. Coli'' str K12. <br />
Its binding region was established by Lee and co workers. The promotor region was designed ''in silico'' with its own RBS and the pre and suffix were ''in silico'' cuted with ''Eco''RI and ''Spe''I creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR (<partinfo>Bba_K190015</partinfo>) was tested by using a test construct, composed of pArsR and RFP on <partinfo>Bba_J61002</partinfo> (Figure 1).<br />
<br />
<center>[[Image:Promoter measurement device.png|200px]]</center><br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence of the red fluorescent protein was measured as described in [[Team:Groningen/Protocols#Fluorescence_of_resting_cells_with_J61002-pArsR|protocols]]. Upon induction of the ArsR promoter the expression of RFP increased, as seen in figure 2. From the enhanced fluorescence a value for the relative promoter unit (RPU) was calculated according to [[Team:Groningen/Literature#Kelly2009|Kelly 2009]] (formula 9). Thereby an induction of 2.3 RPU was found, which was in consensus with the promoter activity found for arsenic metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The arsenite uptake in ''E. coli'' with J61002-<partinfo>Bba_K190015</partinfo> over time was measured using the [[Team:Groningen/Protocols#Metal_uptake_assay_for_E._coliKostal_2004|arsenite uptake assay]], this was done upon incubation with 10µM NaAsO<sub>2</sub>. This data was multiplied by the following ratio: As(III) uptake upon induction for 1hr with 100µM As(III) devided by As(III) uptake upon induction for with 10 µM As(III). The increasing intracellular concentration is shown in figure 3. <br />
<br />
<center>[[Image:UptakeRPU.png]]</center><br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100 µM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter unit with. <br />
<br />
<center>[[Image:Uptake100um.png]]</center><br />
:Figure 3: The internal arsenic concentration, calculated from experimental data for ''E. coli'' with J61002-<partinfo>Bba_K190015</partinfo>. The resting cells were incubated with As(III). For further information see text. (The computed fit is <code>(0.152465*x)/(1 + 0.121918*x)</code>.)<br />
<br />
The raw data can be found at [[Team:Groningen/Modelling/Downloads|downloads]].<br />
<br />
=====Fluorescence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22 h. see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000, 500, 50, 5 and 0 &micro;M. The fluorescence normalized to the OD<sub>600</sub> is plotted in figure 4. In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
<center>[[Image:Promoters-ArsR.png]]</center><br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentrations of 5000, 500, 50, 5 and 0 &micro;M sodium arsenite. <partinfo>Bba_J23101</partinfo> is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD<sub>600</sub> to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0 &micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50 &micro;M arsenite which is as high as 85% of the fluorescence from reference promotor <partinfo>Bba_J23101</partinfo>. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4 &deg;C. Induction to a final concentration of 5000 &micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000 &micro;M is the poisoning of the cells with Arsenite. The poisoning of the cells is best seen in the OD plotted against time as shown in figure 5. The cells induced to a concentration of 5000 &micro;M Arsenite shows a big decrease in OD between 5 and 22 hours after induction due to Arsenite poisoning.<br />
<br />
<center>[[Image:Promoters-ArsR-OD.png]]</center><br />
:Figure 5: Shows the OD plotted against time of ''E.coli'' with plasmid J61002 containing the pArsR RFP construct.<br />
<br />
===Conclusion===<br />
Both promoter tests, with resting cells and growing cells, show clearly that the pArsR promoter is functional. The negative transcriptional regulator ArsR releases the promoter region upon induction with arsenite. The promoter strength was calculated in relative promoter units, upon induction of resting cells with 100 &micro;M As(III) an increase of 2.3 was found. A disadvantage of the usage of pArsR, also clearly shown by the two measurements, is that the negative regulation is leaky as there is already some RFP expressed without addition of arsenite. The OD<sub>600</sub> measurements of the growing cell measurements showed that concentrations as high as 5000 &micro;M Arsenite are poisonous for '' E. coli'' TOP 10 cells.<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1 &micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
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arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
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<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids. Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>E. coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the buoyancy genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce ''gvp'' transcription at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
====Cloning strategy====<br />
<br />
The CueR CueO sensitive promotor was designed by substracting its sequence from the genome database of ''E. coli'' str K12.It's binding region was established by Yamamoto and co worker. The promotor region was designed in silico with its own RBS and the pre and suffix were ''in silico'' cuted with ''Eco''RI and ''Spe''I creating sticky ends. See parts registry {{Part|BBa_K190024}}<br />
<br />
====Results====<br />
In order characterize the CueO promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22 h. see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000, 500, 50, 5 and 0 &micro;M. The fluorescence normalized to the OD<sub>600</sub> is plotted in figure 4. In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
<center>[[Image:Promoters-CueO.png]]</center><br />
:Figure 6: Shows the fluorescence of RFP expressed with the CueO promotor. The fluorescence is normalized to 1 and plotted against time. The ArsR promotor is induced to concentration of 5000, 500, 50, 5 and 0 &micro;M CuSO<sub>4</sub>. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 6 is normalized to the OD<sub>600</sub> to correct for differences in cell concentration. As can be seen in figure 6 non induced CueO RFP (0 &micro;M) shows no fluorescence meaning that the promotor is not leaking. <br />
The Fluorescence for CuSO<sub>4</sub> induced cells shows only slight increase in the order of 0 < 5000 < 5 < 50 < 500<br />
&micro;M CuSO<sub>4</sub>. The cells induced to a concentration of 5000 &micro;M CuSO<sub>4</sub> show no increase in fluorescence which could be due to poisoning of the cells by the CuSO<sub>4</sub>. In figure 7 can be seen that the OD<sub>600</sub> of the copper induced cells is increasing in first 5 hours and then stabilizes or even decreases in case of induction to 5000 &micro;M CuSO<sub>4</sub>.<br />
<br />
<center>[[Image:Promoters-CueO-OD.png]]</center><br />
:Figure 7: Shows the OD<sub>600</sub> plotted against time of ''E.coli'' with plasmid J61002 containing the pCueO RFP construct.<br />
<br />
===Conclusion===<br />
The fluorescence measurements of the CueR promotor show that there is no fluorescence without induction of CuSO<sub>4</sub>. Upon induction with CuSO<sub>4</sub> the cells show an increase in RFP fluorescence which keeps increasing over 22 hours after induction.<br />
<!--<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
--><br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with ''Eco''RI and ''Spe''I restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<!--<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
<br />
--><br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T23:15:07Z<p>Wilfred: /* Results */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Project/Accumulation}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle}}</div><br />
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<div class="intro"><br />
<h1>Promoters</h1><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest.By cloning the ArsR and CueO promotor in front of RFP we have shown that by induction with respectively Arsenite and Copper repression of the promotor is reduced and expression of RFP enhanced. We took the following promoters into consideration:'''<br />
<center><br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|}<br />
</center><br />
</div><br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters.<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>E. coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>E. coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straight forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting its sequence from the genome database of ''E. Coli'' str K12. <br />
Its binding region was established by Lee and co workers. The promotor region was designed ''in silico'' with its own RBS and the pre and suffix were ''in silico'' cuted with ''Eco''RI and ''Spe''I creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR (<partinfo>Bba_K190015</partinfo>) was tested by using a test construct, composed of pArsR and RFP on <partinfo>Bba_J61002</partinfo> (Figure 1).<br />
<br />
<center>[[Image:Promoter measurement device.png|200px]]</center><br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence of the red fluorescent protein was measured as described in [[Team:Groningen/Protocols#Fluorescence_of_resting_cells_with_J61002-pArsR|protocols]]. Upon induction of the ArsR promoter the expression of RFP increased, as seen in figure 2. From the enhanced fluorescence a value for the relative promoter unit (RPU) was calculated according to [[Team:Groningen/Literature#Kelly2009|Kelly 2009]] (formula 9). Thereby an induction of 2.3 RPU was found, which was in consensus with the promoter activity found for arsenic metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The arsenite uptake in ''E. coli'' with J61002-<partinfo>Bba_K190015</partinfo> over time was measured using the [[Team:Groningen/Protocols#Metal_uptake_assay_for_E._coliKostal_2004|arsenite uptake assay]], this was done upon incubation with 10µM NaAsO<sub>2</sub>. This data was multiplied by the following ratio: As(III) uptake upon induction for 1hr with 100µM As(III) devided by As(III) uptake upon induction for with 10 µM As(III). The increasing intracellular concentration is shown in figure 3. <br />
<br />
<center>[[Image:UptakeRPU.png]]</center><br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100 µM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter unit with. <br />
<br />
<center>[[Image:Uptake100um.png]]</center><br />
:Figure 3: The internal arsenic concentration, calculated from experimental data for ''E. coli'' with J61002-<partinfo>Bba_K190015</partinfo>. The resting cells were incubated with As(III). For further information see text. (The computed fit is <code>(0.152465*x)/(1 + 0.121918*x)</code>.)<br />
<br />
The raw data can be found at [[Team:Groningen/Modelling/Downloads|downloads]].<br />
<br />
=====Fluorescence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22 h. see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000, 500, 50, 5 and 0 &micro;M. The fluorescence normalized to the OD<sub>600</sub> is plotted in figure 4. In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
<center>[[Image:Promoters-ArsR.png]]</center><br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentrations of 5000, 500, 50, 5 and 0 &micro;M sodium arsenite. <partinfo>Bba_J23101</partinfo> is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD<sub>600</sub> to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0 &micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50 &micro;M arsenite which is as high as 85% of the fluorescence from reference promotor <partinfo>Bba_J23101</partinfo>. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4 &deg;C. Induction to a final concentration of 5000 &micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000 &micro;M is the poisoning of the cells with Arsenite. The poisoning of the cells is best seen in the OD plotted against time as shown in figure 5. The cells induced to a concentration of 5000 &micro;M Arsenite shows a big decrease in OD between 5 and 22 hours after induction due to Arsenite poisoning.<br />
<br />
<center>[[Image:Promoters-ArsR-OD.png]]</center><br />
:Figure 5: Shows the OD plotted against time of ''E.coli'' with plasmid J61002 containing the pArsR RFP construct.<br />
<br />
===Conclusion===<br />
Both promoter tests, with resting cells and growing cells, show clearly that the pArsR promoter is functional. The negative transcriptional regulator ArsR releases the promoter region upon induction with arsenite. The promoter strength was calculated in relative promoter units, upon induction of resting cells with 100 &micro;M As(III) an increase of 2.3 was found. A disadvantage of the usage of pArsR, also clearly shown by the two measurements, is that the negative regulation is leaky as there is already some RFP expressed without addition of arsenite. The OD<sub>600</sub> measurements of the growing cell measurements showed that concentrations as high as 5000 &micro;M Arsenite are poisonous for '' E. coli'' TOP 10 cells.<br />
<br />
===Modelling===<br />
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<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1 &micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids. Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>E. coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the buoyancy genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce ''gvp'' transcription at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
====Cloning strategy====<br />
<br />
The CueR CueO sensitive promotor was designed by substracting its sequence from the genome database of ''E. coli'' str K12.It's binding region was established by Yamamoto and co worker. The promotor region was designed in silico with its own RBS and the pre and suffix were ''in silico'' cuted with ''Eco''RI and ''Spe''I creating sticky ends. See parts registry {{Part|BBa_K190024}}<br />
<br />
====Results====<br />
In order characterize the CueO promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22 h. see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000, 500, 50, 5 and 0 &micro;M. The fluorescence normalized to the OD<sub>600</sub> is plotted in figure 4. In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
<center>[[Image:Promoters-CueO.png]]</center><br />
:Figure 6: Shows the fluorescence of RFP expressed with the CueO promotor. The fluorescence is normalized to 1 and plotted against time. The ArsR promotor is induced to concentration of 5000, 500, 50, 5 and 0 &micro;M CuSO<sub>4</sub>. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 6 is normalized to the OD<sub>600</sub> to correct for differences in cell concentration. As can be seen in figure 6 non induced CueO RFP (0 &micro;M) shows no fluorescence meaning that the promotor is not leaking. <br />
The Fluorescence for CuSO<sub>4</sub> induced cells shows only slight increase in the order of 0 < 5000 < 5 < 50 < 500<br />
&micro;M CuSO<sub>4</sub>. The cells induced to a concentration of 5000 &micro;M CuSO<sub>4</sub> show no increase in fluorescence which could be due to poisoning of the cells by the CuSO<sub>4</sub>. In figure 7 can be seen that the OD<sub>600</sub> of the copper induced cells is increasing in first 5 hours and then stabilizes or even decreases in case of induction to 5000 &micro;M CuSO<sub>4</sub>.<br />
<br />
<center>[[Image:Promoters-CueO-OD.png]]</center><br />
:Figure 7: Shows the OD<sub>600</sub> plotted against time of ''E.coli'' with plasmid J61002 containing the pCueO RFP construct.<br />
<br />
===Conclusion===<br />
The fluorescence measurements of the CueR promotor show that there is no fluorescence without induction of CuSO<sub>4</sub>. Upon induction with CuSO<sub>4</sub> the cells show an increase in RFP fluorescence which keeps increasing over 22 hours after induction.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
<br />
<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T23:11:25Z<p>Wilfred: /* Results */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Project/Accumulation}}</div><br />
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<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
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<div class="intro"><br />
<h1>Promoters</h1><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest.By cloning the ArsR and CueO promotor in front of RFP we have shown that by induction with respectively Arsenite and Copper repression of the promotor is reduced and expression of RFP enhanced. We took the following promoters into consideration:'''<br />
<center><br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|}<br />
</center><br />
</div><br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters.<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>E. coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>E. coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straight forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting its sequence from the genome database of ''E. Coli'' str K12. <br />
Its binding region was established by Lee and co workers. The promotor region was designed ''in silico'' with its own RBS and the pre and suffix were ''in silico'' cuted with ''Eco''RI and ''Spe''I creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR (<partinfo>Bba_K190015</partinfo>) was tested by using a test construct, composed of pArsR and RFP on <partinfo>Bba_J61002</partinfo> (Figure 1).<br />
<br />
<center>[[Image:Promoter measurement device.png|200px]]</center><br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence of the red fluorescent protein was measured as described in [[Team:Groningen/Protocols#Fluorescence_of_resting_cells_with_J61002-pArsR|protocols]]. Upon induction of the ArsR promoter the expression of RFP increased, as seen in figure 2. From the enhanced fluorescence a value for the relative promoter unit (RPU) was calculated according to [[Team:Groningen/Literature#Kelly2009|Kelly 2009]] (formula 9). Thereby an induction of 2.3 RPU was found, which was in consensus with the promoter activity found for arsenic metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The arsenite uptake in ''E. coli'' with J61002-<partinfo>Bba_K190015</partinfo> over time was measured using the [[Team:Groningen/Protocols#Metal_uptake_assay_for_E._coliKostal_2004|arsenite uptake assay]], this was done upon incubation with 10µM NaAsO<sub>2</sub>. This data was multiplied by the following ratio: As(III) uptake upon induction for 1hr with 100µM As(III) devided by As(III) uptake upon induction for with 10 µM As(III). The increasing intracellular concentration is shown in figure 3. <br />
<br />
<center>[[Image:UptakeRPU.png]]</center><br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100 µM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter unit with. <br />
<br />
<center>[[Image:Uptake100um.png]]</center><br />
:Figure 3: The internal arsenic concentration, calculated from experimental data for ''E. coli'' with J61002-<partinfo>Bba_K190015</partinfo>. The resting cells were incubated with As(III). For further information see text. (The computed fit is <code>(0.152465*x)/(1 + 0.121918*x)</code>.)<br />
<br />
The raw data can be found at [[Team:Groningen/Modelling/Downloads|downloads]].<br />
<br />
=====Fluorescence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22 h. see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000, 500, 50, 5 and 0 &micro;M. The fluorescence normalized to the OD<sub>600</sub> is plotted in figure 4. In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
<center>[[Image:Promoters-ArsR.png]]</center><br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentrations of 5000, 500, 50, 5 and 0 &micro;M sodium arsenite. <partinfo>Bba_J23101</partinfo> is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD<sub>600</sub> to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0 &micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50 &micro;M arsenite which is as high as 85% of the fluorescence from reference promotor <partinfo>Bba_J23101</partinfo>. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4 &deg;C. Induction to a final concentration of 5000 &micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000 &micro;M is the poisoning of the cells with Arsenite. The poisoning of the cells is best seen in the OD plotted against time as shown in figure 5. The cells induced to a concentration of 5000 &micro;M Arsenite shows a big decrease in OD between 5 and 22 hours after induction due to Arsenite poisoning.<br />
<br />
<center>[[Image:Promoters-ArsR-OD.png]]</center><br />
:Figure 5: Shows the OD plotted against time of ''E.coli'' with plasmid J61002 containing the pArsR RFP construct.<br />
<br />
===Conclusion===<br />
Both promoter tests, with resting cells and growing cells, show clearly that the pArsR promoter is functional. The negative transcriptional regulator ArsR releases the promoter region upon induction with arsenite. The promoter strength was calculated in relative promoter units, upon induction of resting cells with 100 &micro;M As(III) an increase of 2.3 was found. A disadvantage of the usage of pArsR, also clearly shown by the two measurements, is that the negative regulation is leaky as there is already some RFP expressed without addition of arsenite. The OD<sub>600</sub> measurements of the growing cell measurements showed that concentrations as high as 5000 &micro;M Arsenite are poisonous for '' E. coli'' TOP 10 cells.<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1 &micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids. Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>E. coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the buoyancy genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce ''gvp'' transcription at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
====Cloning strategy====<br />
<br />
The CueR CueO sensitive promotor was designed by substracting its sequence from the genome database of ''E. coli'' str K12.It's binding region was established by Yamamoto and co worker. The promotor region was designed in silico with its own RBS and the pre and suffix were ''in silico'' cuted with ''Eco''RI and ''Spe''I creating sticky ends. See parts registry {{Part|BBa_K190024}}<br />
<br />
====Results====<br />
In order characterize the CueO promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22 h. see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000, 500, 50, 5 and 0 &micro;M. The fluorescence normalized to the OD<sub>600</sub> is plotted in figure 4. In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
<center>[[Image:Promoters-CueO.png]]</center><br />
:Figure 6: Shows the fluorescence of RFP expressed with the CueO promotor. The fluorescence is normalized to 1 and plotted against time. The ArsR promotor is induced to concentration of 5000, 500, 50, 5 and 0 &micro;M CuSO<sub>4</sub>. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 6 is normalized to the OD<sub>600</sub> to correct for differences in cell concentration. As can be seen in figure 6 non induced CueO RFP (0 &micro;M) shows no fluorescence meaning that the promotor is not leaking. <br />
The Fluorescence for CuSO<sub>4</sub> induced cells shows only slight increase in the order of 0 < 5000 < 5 < 50 < 500<br />
&micro;M CuSO<sub>4</sub>. The cells induced to a concentration of 5000 &micro;M CuSO<sub>4</sub> show no increase in fluorescence which could be due to poisoning of the cells by the CuSO<sub>4</sub>. In figure 7 can be seen that the OD<sub>600</sub? of the copper induced cells is increasing in first 5 hours and then stabilizes or even decreases in case of induction to 5000 &micro;M CuSO<sub>4</sub>.<br />
<br />
<center>[[Image:Promoters-CueO-OD.png]]</center><br />
:Figure 7: Shows the OD<sub>600</sub> plotted against time of ''E.coli'' with plasmid J61002 containing the pCueO RFP construct.<br />
<br />
===Conclusion===<br />
The fluorescence measurements of the CueR promotor show that there is no fluorescence without induction of CuSO<sub>4</sub>. Upon induction with CuSO<sub>4</sub> the cells show an increase in RFP fluorescence which keeps increasing over 22 hours after induction.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
<br />
<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T23:10:16Z<p>Wilfred: /* Results */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Project/Accumulation}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle}}</div><br />
<br />
<br />
{| style="clear:both"<br />
|<html><style type="text/css"><br />
.intro { margin-left:0px; margin-top:10px; padding:10px; border-left:solid 5px #FFF6D5; border-right:solid 5px #FFF6D5; text-align:justify;background:#FFFFE5; }<br />
</style></html><br />
<div class="intro"><br />
<h1>Promoters</h1><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest.By cloning the ArsR and CueO promotor in front of RFP we have shown that by induction with respectively Arsenite and Copper repression of the promotor is reduced and expression of RFP enhanced. We took the following promoters into consideration:'''<br />
<center><br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|}<br />
</center><br />
</div><br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters.<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>E. coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>E. coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straight forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting its sequence from the genome database of ''E. Coli'' str K12. <br />
Its binding region was established by Lee and co workers. The promotor region was designed ''in silico'' with its own RBS and the pre and suffix were ''in silico'' cuted with ''Eco''RI and ''Spe''I creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR (<partinfo>Bba_K190015</partinfo>) was tested by using a test construct, composed of pArsR and RFP on <partinfo>Bba_J61002</partinfo> (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence of the red fluorescent protein was measured as described in [[Team:Groningen/Protocols#Fluorescence_of_resting_cells_with_J61002-pArsR|protocols]]. Upon induction of the ArsR promoter the expression of RFP increased, as seen in figure 2. From the enhanced fluorescence a value for the relative promoter unit (RPU) was calculated according to [[Team:Groningen/Literature#Kelly2009|Kelly 2009]] (formula 9). Thereby an induction of 2.3 RPU was found, which was in consensus with the promoter activity found for arsenic metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The arsenite uptake in ''E. coli'' with J61002-<partinfo>Bba_K190015</partinfo> over time was measured using the [[Team:Groningen/Protocols#Metal_uptake_assay_for_E._coliKostal_2004|arsenite uptake assay]], this was done upon incubation with 10µM NaAsO<sub>2</sub>. This data was multiplied by the following ratio: As(III) uptake upon induction for 1hr with 100µM As(III) devided by As(III) uptake upon induction for with 10 µM As(III). The increasing intracellular concentration is shown in figure 3. <br />
<br />
<center>[[Image:UptakeRPU.png]]</center><br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100 µM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter unit with. <br />
<br />
<center>[[Image:Uptake100um.png]]</center><br />
:Figure 3: The internal arsenic concentration, calculated from experimental data for ''E. coli'' with J61002-<partinfo>Bba_K190015</partinfo>. The resting cells were incubated with As(III). For further information see text. (The computed fit is <code>(0.152465*x)/(1 + 0.121918*x)</code>.)<br />
<br />
The raw data can be found at [[Team:Groningen/Modelling/Downloads|downloads]].<br />
<br />
=====Fluorescence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22 h. see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000, 500, 50, 5 and 0 &micro;M. The fluorescence normalized to the OD<sub>600</sub> is plotted in figure 4. In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
<center>[[Image:Promoters-ArsR.png]]</center><br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentrations of 5000, 500, 50, 5 and 0 &micro;M sodium arsenite. <partinfo>Bba_J23101</partinfo> is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD<sub>600</sub> to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0 &micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50 &micro;M arsenite which is as high as 85% of the fluorescence from reference promotor <partinfo>Bba_J23101</partinfo>. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4 &deg;C. Induction to a final concentration of 5000 &micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000 &micro;M is the poisoning of the cells with Arsenite. The poisoning of the cells is best seen in the OD plotted against time as shown in figure 5. The cells induced to a concentration of 5000 &micro;M Arsenite shows a big decrease in OD between 5 and 22 hours after induction due to Arsenite poisoning.<br />
<br />
<center>[[Image:Promoters-ArsR-OD.png]]</center><br />
:Figure 5: Shows the OD plotted against time of ''E.coli'' with plasmid J61002 containing the pArsR RFP construct.<br />
<br />
===Conclusion===<br />
Both promoter tests, with resting cells and growing cells, show clearly that the pArsR promoter is functional. The negative transcriptional regulator ArsR releases the promoter region upon induction with arsenite. The promoter strength was calculated in relative promoter units, upon induction of resting cells with 100 &micro;M As(III) an increase of 2.3 was found. A disadvantage of the usage of pArsR, also clearly shown by the two measurements, is that the negative regulation is leaky as there is already some RFP expressed without addition of arsenite. The OD<sub>600</sub> measurements of the growing cell measurements showed that concentrations as high as 5000 &micro;M Arsenite are poisonous for '' E. coli'' TOP 10 cells.<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1 &micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids. Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>E. coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the buoyancy genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce ''gvp'' transcription at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
====Cloning strategy====<br />
<br />
The CueR CueO sensitive promotor was designed by substracting its sequence from the genome database of ''E. coli'' str K12.It's binding region was established by Yamamoto and co worker. The promotor region was designed in silico with its own RBS and the pre and suffix were ''in silico'' cuted with ''Eco''RI and ''Spe''I creating sticky ends. See parts registry {{Part|BBa_K190024}}<br />
<br />
====Results====<br />
In order characterize the CueO promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22 h. see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000, 500, 50, 5 and 0 &micro;M. The fluorescence normalized to the OD<sub>600</sub> is plotted in figure 4. In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
<center>[[Image:Promoters-CueO.png]]</center><br />
:Figure 6: Shows the fluorescence of RFP expressed with the CueO promotor. The fluorescence is normalized to 1 and plotted against time. The ArsR promotor is induced to concentration of 5000, 500, 50, 5 and 0 &micro;M CuSO<sub>4</sub>. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 6 is normalized to the OD<sub>600</sub> to correct for differences in cell concentration. As can be seen in figure 6 non induced CueO RFP (0 &micro;M) shows no fluorescence meaning that the promotor is not leaking. <br />
The Fluorescence for CuSO<sub>4</sub> induced cells shows only slight increase in the order of 0 < 5000 < 5 < 50 < 500<br />
&micro;M CuSO<sub>4</sub>. The cells induced to a concentration of 5000 &micro;M CuSO<sub>4</sub> show no increase in fluorescence which could be due to poisoning of the cells by the CuSO<sub>4</sub>. In figure 7 can be seen that the OD<sub>600</sub? of the copper induced cells is increasing in first 5 hours and then stabilizes or even decreases in case of induction to 5000 &micro;M CuSO<sub>4</sub>.<br />
<br />
<center>[[Image:Promoters-CueO-OD.png]]</center><br />
:Figure 7: Shows the OD<sub>600</sub> plotted against time of ''E.coli'' with plasmid J61002 containing the pCueO RFP construct.<br />
<br />
===Conclusion===<br />
The fluorescence measurements of the CueR promotor show that there is no fluorescence without induction of CuSO<sub>4</sub>. Upon induction with CuSO<sub>4</sub> the cells show an increase in RFP fluorescence which keeps increasing over 22 hours after induction.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
<br />
<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T23:05:36Z<p>Wilfred: /* Copper Induced Promoters */</p>
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<div class="intro"><br />
<h1>Promotors</h1><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest.By cloning the ArsR and CueO promotor in front of RFP we have shown that by induction with respectively Arsenite and Copper repression of the promotor is reduced and expression of RFP enhanced. We took the following promoters into consideration:'''<br />
<center><br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|}<br />
</center><br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters.<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>E. coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>E. coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straight forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting its sequence from the genome database of ''E. Coli'' str K12. <br />
Its binding region was established by Lee and co workers. The promotor region was designed ''in silico'' with its own RBS and the pre and suffix were ''in silico'' cuted with ''Eco''RI and ''Spe''I creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR (<partinfo>Bba_K190015</partinfo>) was tested by using a test construct, composed of pArsR and RFP on <partinfo>Bba_J61002</partinfo> (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence of the red fluorescent protein was measured as described in [[Team:Groningen/Protocols#Fluorescence_of_resting_cells_with_J61002-pArsR|protocols]]. Upon induction of the ArsR promoter the expression of RFP increased, as seen in figure 2. From the enhanced fluorescence a value for the relative promoter unit (RPU) was calculated according to [[Team:Groningen/Literature#Kelly2009|Kelly 2009]] (formula 9). Thereby an induction of 2.3 RPU was found, which was in consensus with the promoter activity found for arsenic metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The arsenite uptake in ''E. coli'' with J61002-<partinfo>Bba_K190015</partinfo> over time was measured using the [[Team:Groningen/Protocols#Metal_uptake_assay_for_E._coliKostal_2004|arsenite uptake assay]], this was done upon incubation with 10µM NaAsO<sub>2</sub>. This data was multiplied by the following ratio: As(III) uptake upon induction for 1hr with 100µM As(III) devided by As(III) uptake upon induction for with 10 µM As(III). The increasing intracellular concentration is shown in figure 3. <br />
<br />
<center>[[Image:UptakeRPU.png]]</center><br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100 µM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter unit with. <br />
<br />
<center>[[Image:Uptake100um.png]]</center><br />
:Figure 3: The internal arsenic concentration, calculated from experimental data for ''E. coli'' with J61002-<partinfo>Bba_K190015</partinfo>. The resting cells were incubated with As(III). For further information see text.<br />
<br />
The raw data can be found at [[Team:Groningen/Modelling/Downloads|downloads]].<br />
<br />
=====Fluorescence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22 h. see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000, 500, 50, 5 and 0 &micro;M. The fluorescence normalized to the OD<sub>600</sub> is plotted in figure 4. In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
<center>[[Image:Promoters-ArsR.png]]</center><br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentrations of 5000, 500, 50, 5 and 0 &micro;M sodium arsenite. <partinfo>Bba_J23101</partinfo> is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD<sub>600</sub> to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0 &micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50 &micro;M arsenite which is as high as 85% of the fluorescence from reference promotor <partinfo>Bba_J23101</partinfo>. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4 &deg;C. Induction to a final concentration of 5000 &micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000 &micro;M is the poisoning of the cells with Arsenite. The poisoning of the cells is best seen in the OD plotted against time as shown in figure 5. The cells induced to a concentration of 5000 &micro;M Arsenite shows a big decrease in OD between 5 and 22 hours after induction due to Arsenite poisoning.<br />
<br />
<center>[[Image:Promoters-ArsR-OD.png]]</center><br />
:Figure 5: Shows the OD plotted against time of ''E.coli'' with plasmid J61002 containing the pArsR RFP construct.<br />
<br />
===Conclusion===<br />
Both promoter tests, with resting cells and growing cells, show clearly that the pArsR promoter is functional. The negative transcriptional regulator ArsR releases the promoter region upon induction with arsenite. The promoter strength was calculated in relative promoter units, upon induction of resting cells with 100 &micro;M As(III) an increase of 2.3 was found. A disadvantage of the usage of pArsR, also clearly shown by the two measurements, is that the negative regulation is leaky as there is already some RFP expressed without addition of arsenite. The OD<sub>600</sub> measurements of the growing cell measurements showed that concentrations as high as 5000 &micro;M Arsenite are poisonous for '' E. coli'' TOP 10 cells.<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1 &micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids. Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>E. coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the buoyancy genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce ''gvp'' transcription at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
====Cloning strategy====<br />
<br />
The CueR CueO sensitive promotor was designed by substracting its sequence from the genome database of ''E. coli'' str K12.It's binding region was established by Yamamoto and co worker. The promotor region was designed in silico with its own RBS and the pre and suffix were ''in silico'' cuted with ''Eco''RI and ''Spe''I creating sticky ends. See parts registry {{Part|BBa_K190024}}<br />
<br />
====Results====<br />
In order characterize the CueO promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22 h. see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000, 500, 50, 5 and 0 &micro;M. The fluorescence normalized to the OD<sub>600</sub> is plotted in figure 4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
<center>[[Image:Promoters-CueO.png]]</center><br />
:Figure 6: Shows the fluorescence of RFP expressed with the CueO promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentration of 5000,500,50,5 and 0 &micro;M CuSO<sub>4</sub>. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 6 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 6 non induced CueO RFP (0&micro;M)shows no fluorescence meaning that the promotor is not leaking. <br />
The Fluorescence for CuSO<sub>4</sub> induced cells shows only slight increase in the order of 0 < 5000 < 5 < 50 < 500<br />
&micro;M CuSO<sub>4</sub>. The cells induced to a concentration of 5000&micro;M CuSO<sub>4</sub> show no increase in fluorescence which could be due to poisoning of the cells by the CuSO<sub>4</sub>. In figure 7 can be seen that the OD of the Copper induced cells is increasing in first 5 hours and then stabilizes or even decreases in case of induction to 5000&micro;M CuSO<sub>4</sub>.<br />
<br />
<center>[[Image:Promoters-CueO-OD.png]]</center><br />
:Figure 7: Shows the OD plotted against time of ''E.coli'' with plasmid J61002 containing the pCueO RFP construct.<br />
<br />
===Conclusion===<br />
The fluorescence measurements of the CueR promotor show that there is no fluorescence without induction of CuSO<sub>4</sub>. Upon induction with CuSO<sub>4</sub> the cells show an increase in RFP fluorescence which keeps increasing over 22 hours after induction.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
<br />
<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T22:47:45Z<p>Wilfred: /* Modelling */</p>
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<div class="intro"><br />
<h1>Promotors</h1><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest.By cloning the ArsR and CueO promotor in front of RFP we have shown that by induction with respectively Arsenite and Copper repression of the promotor is reduced and expression of RFP enhanced. We took the following promoters into consideration:'''<br />
<center><br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|}<br />
</center><br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters.<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>E. coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>E. coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straight forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting its sequence from the genome database of ''E. Coli'' str K12. <br />
Its binding region was established by Lee and co workers. The promotor region was designed ''in silico'' with its own RBS and the pre and suffix were ''in silico'' cuted with ''Eco''RI and ''Spe''I creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR (<partinfo>Bba_K190015</partinfo>) was tested by using a test construct, composed of pArsR and RFP on <partinfo>Bba_J61002</partinfo> (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence of the red fluorescent protein was measured as described in [[Team:Groningen/Protocols#Fluorescence_of_resting_cells_with_J61002-pArsR|protocols]]. Upon induction of the ArsR promoter the expression of RFP increased, as seen in figure 2. From the enhanced fluorescence a value for the relative promoter unit (RPU) was calculated according to [[Team:Groningen/Literature#Kelly2009|Kelly 2009]] (formula 9). Thereby an induction of 2.3 RPU was found, which was in consensus with the promoter activity found for arsenic metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The arsenite uptake in ''E. coli'' with J61002-<partinfo>Bba_K190015</partinfo> over time was measured using the [[Team:Groningen/Protocols#Metal_uptake_assay_for_E._coliKostal_2004|arsenite uptake assay]], this was done upon incubation with 10µM NaAsO<sub>2</sub>. This data was multiplied by the following ratio: As(III) uptake upon induction for 1hr with 100µM As(III) devided by As(III) uptake upon induction for with 10 µM As(III). The increasing intracellular concentration is shown in figure 3. <br />
<br />
<center>[[Image:UptakeRPU.png]]</center><br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100 µM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter unit with. <br />
<br />
<center>[[Image:Uptake100um.png]]</center><br />
:Figure 3: The internal arsenic concentration, calculated from experimental data for ''E. coli'' with J61002-<partinfo>Bba_K190015</partinfo>. The resting cells were incubated with As(III). For further information see text.<br />
<br />
The raw data can be found at [[Team:Groningen/Modelling/Downloads|downloads]].<br />
<br />
=====Fluorescence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22 h. see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000, 500, 50, 5 and 0 &micro;M. The fluorescence normalized to the OD<sub>600</sub> is plotted in figure 4. In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
<center>[[Image:Promoters-ArsR.png]]</center><br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentrations of 5000, 500, 50, 5 and 0 &micro;M sodium arsenite. <partinfo>Bba_J23101</partinfo> is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD<sub>600</sub> to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0 &micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50 &micro;M arsenite which is as high as 85% of the fluorescence from reference promotor <partinfo>Bba_J23101</partinfo>. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4 &deg;C. Induction to a final concentration of 5000 &micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000 &micro;M is the poisoning of the cells with Arsenite. The poisoning of the cells is best seen in the OD plotted against time as shown in figure 5. The cells induced to a concentration of 5000 &micro;M Arsenite shows a big decrease in OD between 5 and 22 hours after induction due to Arsenite poisoning.<br />
<br />
<center>[[Image:Promoters-ArsR-OD.png]]</center><br />
:Figure 5: Shows the OD plotted against time of ''E.coli'' with plasmid J61002 containing the pArsR RFP construct.<br />
<br />
===Conclusion===<br />
Both promoter tests, with resting cells and growing cells, show clearly that the pArsR promoter is functional. The negative transcriptional regulator ArsR releases the promoter region upon induction with arsenite. The promoter strength was calculated in relative promoter units, upon induction of resting cells with 100 &micro;M As(III) an increase of 2.3 was found. A disadvantage of the usage of pArsR, also clearly shown by the two measurements, is that the negative regulation is leaky as there is already some RFP expressed without addition of arsenite. The OD<sub>600</sub> measurements of the growing cell measurements showed that concentrations as high as 5000 &micro;M Arsenite are poisonous for '' E. coli'' TOP 10 cells.<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1 &micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>E. coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
====Cloning strategy====<br />
<br />
The CueR CueO sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12.It's binding region was established by Yamamoto and co worker. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190024}}<br />
<br />
====Results====<br />
<br />
In order characterize the CueO promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000, 500, 50, 5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-CueO.png]]<br />
:Figure 6: Shows the fluorescence of RFP expressed with the CueO promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentration of 5000,500,50,5 and 0 &micro;M CuSO<sub>4</sub>. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 6 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 6 non induced CueO RFP (0&micro;M)shows no fluorescence meaning that the promotor is not leaking. <br />
The Fluorescence for CuSO<sub>4</sub> induced cells shows only slight increase in the order of 0 < 5000 < 5 < 50 < 500<br />
&micro;M CuSO<sub>4</sub>. The cells induced to a concentration of 5000&micro;M CuSO<sub>4</sub> show no increase in fluorescence which could be due to poisoning of the cells by the CuSO<sub>4</sub>. In figure 7 can be seen that the OD of the Copper induced cells is increasing in first 5 hours and then stabilizes or even decreases in case of induction to 5000&micro;M CuSO<sub>4</sub>.<br />
<br />
[[Image:Promoters-CueO-OD.png]]<br />
:Figure 7: Shows the OD plotted against time of ''E.coli'' with plasmid J61002 containing the pCueO RFP construct.<br />
<br />
===Conclusion===<br />
The fluorescence measurements of the CueR promotor show that there is no fluorescence without induction of CuSO<sub>4</sub>. Upon induction with CuSO<sub>4</sub> the cells show an increase in RFP fluorescence which keeps increasing over 22 hours after induction.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
<br />
<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T22:46:31Z<p>Wilfred: /* Conclusion */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Project/Accumulation}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle}}</div><br />
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<div class="intro"><br />
<h1>Promotors</h1><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest.By cloning the ArsR and CueO promotor in front of RFP we have shown that by induction with respectively Arsenite and Copper repression of the promotor is reduced and expression of RFP enhanced. We took the following promoters into consideration:'''<br />
<center><br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|}<br />
</center><br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters.<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>E. coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>E. coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straight forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting its sequence from the genome database of ''E. Coli'' str K12. <br />
Its binding region was established by Lee and co workers. The promotor region was designed ''in silico'' with its own RBS and the pre and suffix were ''in silico'' cuted with ''Eco''RI and ''Spe''I creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR (<partinfo>Bba_K190015</partinfo>) was tested by using a test construct, composed of pArsR and RFP on <partinfo>Bba_J61002</partinfo> (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence of the red fluorescent protein was measured as described in [[Team:Groningen/Protocols#Fluorescence_of_resting_cells_with_J61002-pArsR|protocols]]. Upon induction of the ArsR promoter the expression of RFP increased, as seen in figure 2. From the enhanced fluorescence a value for the relative promoter unit (RPU) was calculated according to [[Team:Groningen/Literature#Kelly2009|Kelly 2009]] (formula 9). Thereby an induction of 2.3 RPU was found, which was in consensus with the promoter activity found for arsenic metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The arsenite uptake in ''E. coli'' with J61002-<partinfo>Bba_K190015</partinfo> over time was measured using the [[Team:Groningen/Protocols#Metal_uptake_assay_for_E._coliKostal_2004|arsenite uptake assay]], this was done upon incubation with 10µM NaAsO<sub>2</sub>. This data was multiplied by the following ratio: As(III) uptake upon induction for 1hr with 100µM As(III) devided by As(III) uptake upon induction for with 10 µM As(III). The increasing intracellular concentration is shown in figure 3. <br />
<br />
<center>[[Image:UptakeRPU.png]]</center><br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100 µM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter unit with. <br />
<br />
<center>[[Image:Uptake100um.png]]</center><br />
:Figure 3: The internal arsenic concentration, calculated from experimental data for ''E. coli'' with J61002-<partinfo>Bba_K190015</partinfo>. The resting cells were incubated with As(III). For further information see text.<br />
<br />
The raw data can be found at [[Team:Groningen/Modelling/Downloads|downloads]].<br />
<br />
=====Fluorescence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22 h. see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000, 500, 50, 5 and 0 &micro;M. The fluorescence normalized to the OD<sub>600</sub> is plotted in figure 4. In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
<center>[[Image:Promoters-ArsR.png]]</center><br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentrations of 5000, 500, 50, 5 and 0 &micro;M sodium arsenite. <partinfo>Bba_J23101</partinfo> is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD<sub>600</sub> to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0 &micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50 &micro;M arsenite which is as high as 85% of the fluorescence from reference promotor <partinfo>Bba_J23101</partinfo>. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4 &deg;C. Induction to a final concentration of 5000 &micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000 &micro;M is the poisoning of the cells with Arsenite. The poisoning of the cells is best seen in the OD plotted against time as shown in figure 5. The cells induced to a concentration of 5000 &micro;M Arsenite shows a big decrease in OD between 5 and 22 hours after induction due to Arsenite poisoning.<br />
<br />
<center>[[Image:Promoters-ArsR-OD.png]]</center><br />
:Figure 5: Shows the OD plotted against time of ''E.coli'' with plasmid J61002 containing the pArsR RFP construct.<br />
<br />
===Conclusion===<br />
Both promoter tests, with resting cells and growing cells, show clearly that the pArsR promoter is functional. The negative transcriptional regulator ArsR releases the promoter region upon induction with arsenite. The promoter strength was calculated in relative promoter units, upon induction of resting cells with 100 &micro;M As(III) an increase of 2.3 was found. A disadvantage of the usage of pArsR, also clearly shown by the two measurements, is that the negative regulation is leaky as there is already some RFP expressed without addition of arsenite. The OD<sub>600</sub> measurements of the growing cell measurements showed that concentrations as high as 5000 &micro;M Arsenite are poisonous for '' E. coli'' TOP 10 cells.<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
var graphNodes = [document.getElementById("promoterActivationGraph"),<br />
document.getElementById("promoterActivationGraphNP"),<br />
document.getElementById("promoterActivationGraphS"),<br />
document.getElementById("promoterActivationGraphG"),<br />
document.getElementById("promoterActivationGraph2")];<br />
for(var i in graphNodes) if (graphNodes[i]) graphNodes[i].refresh();<br />
}<br />
</script><br />
</html><br />
<span id="promoterActivationData"></span><br />
{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>E. coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
====Cloning strategy====<br />
<br />
The CueR CueO sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12.It's binding region was established by Yamamoto and co worker. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190024}}<br />
<br />
====Results====<br />
<br />
In order characterize the CueO promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000, 500, 50, 5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-CueO.png]]<br />
:Figure 6: Shows the fluorescence of RFP expressed with the CueO promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentration of 5000,500,50,5 and 0 &micro;M CuSO<sub>4</sub>. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 6 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 6 non induced CueO RFP (0&micro;M)shows no fluorescence meaning that the promotor is not leaking. <br />
The Fluorescence for CuSO<sub>4</sub> induced cells shows only slight increase in the order of 0 < 5000 < 5 < 50 < 500<br />
&micro;M CuSO<sub>4</sub>. The cells induced to a concentration of 5000&micro;M CuSO<sub>4</sub> show no increase in fluorescence which could be due to poisoning of the cells by the CuSO<sub>4</sub>. In figure 7 can be seen that the OD of the Copper induced cells is increasing in first 5 hours and then stabilizes or even decreases in case of induction to 5000&micro;M CuSO<sub>4</sub>.<br />
<br />
[[Image:Promoters-CueO-OD.png]]<br />
:Figure 7: Shows the OD plotted against time of ''E.coli'' with plasmid J61002 containing the pCueO RFP construct.<br />
<br />
===Conclusion===<br />
The fluorescence measurements of the CueR promotor show that there is no fluorescence without induction of CuSO<sub>4</sub>. Upon induction with CuSO<sub>4</sub> the cells show an increase in RFP fluorescence which keeps increasing over 22 hours after induction.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
<br />
<br />
<br />
<br />
{{Team:Groningen/Project/Footer}}</div>Wilfredhttp://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T22:44:18Z<p>Wilfred: /* Fluorescence of growing cells */</p>
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<div>{{Team:Groningen/Project/Header|}}<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Project/Accumulation}}</div><br />
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<div class="intro"><br />
<h1>Promotors</h1><br />
'''A promoter is a part of DNA involved in the regulation of gene transcription by RNA polymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong. Promoters are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promoters are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promoters varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promoters are the best choice for regulating gene expression. In our system, we want to induce GVP production when the concentration of desired metal in the cells reaches a certain level. By choosing metal sensitive promoters already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promoters, and the promoter sequence has only to be placed in front of the genes of interest.By cloning the ArsR and CueO promotor in front of RFP we have shown that by induction with respectively Arsenite and Copper repression of the promotor is reduced and expression of RFP enhanced. We took the following promoters into consideration:'''<br />
<center><br />
{| cellpadding="30"<br />
|align="center"|[[#Arsenic Induced Promoters|<big>As</big><br>Arsenic Induced Promoters]]<br />
|align="center"|[[#Copper Induced Promoters|<big>Cu</big><br>Copper Induced Promoters]]<br />
|align="center"|[[#Zinc Induced Promoters|<big>Zn</big><br>Zinc Induced Promoters]]<br />
|}<br />
</center><br />
==Arsenic Induced Promoters==<br />
<br />
Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including <i>E. coli</i>, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters.<br />
<br />
====<i>E. coli</i>====<br />
<br />
Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of <i>E. coli</i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].<br />
<br />
*ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).<br />
<br />
*ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in <i>E. coli</i> has been used for removal of arsenite from contaminated water (KEGG).<br />
<br />
(ArsR)<sub>2</sub>-DNA &rarr; ArsR-Ar + ArsR-Ar + DNA &rarr; Activation of transription<br />
<br />
The presence of all genes and promoters on the chromosome of <i>E. coli</i> makes the use of the arsRp for induction of the GVP cluster relatively straight forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.<br />
<br />
====Cloning strategy====<br />
<br />
The ArsR sensitive promotor was designed by substracting its sequence from the genome database of ''E. Coli'' str K12. <br />
Its binding region was established by Lee and co workers. The promotor region was designed ''in silico'' with its own RBS and the pre and suffix were ''in silico'' cuted with ''Eco''RI and ''Spe''I creating sticky ends. See parts registry {{Part|BBa_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR (<partinfo>Bba_K190015</partinfo>) was tested by using a test construct, composed of pArsR and RFP on <partinfo>Bba_J61002</partinfo> (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png|200px]]<br />
:Figure 1: The promoter testing device in J61002, where RFP expression is under control of the promoter which is placed in front of it. <br />
<br />
=====Fluorescence of resting cells=====<br />
<br />
The fluorescence of the red fluorescent protein was measured as described in [[Team:Groningen/Protocols#Fluorescence_of_resting_cells_with_J61002-pArsR|protocols]]. Upon induction of the ArsR promoter the expression of RFP increased, as seen in figure 2. From the enhanced fluorescence a value for the relative promoter unit (RPU) was calculated according to [[Team:Groningen/Literature#Kelly2009|Kelly 2009]] (formula 9). Thereby an induction of 2.3 RPU was found, which was in consensus with the promoter activity found for arsenic metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The arsenite uptake in ''E. coli'' with J61002-<partinfo>Bba_K190015</partinfo> over time was measured using the [[Team:Groningen/Protocols#Metal_uptake_assay_for_E._coliKostal_2004|arsenite uptake assay]], this was done upon incubation with 10µM NaAsO<sub>2</sub>. This data was multiplied by the following ratio: As(III) uptake upon induction for 1hr with 100µM As(III) devided by As(III) uptake upon induction for with 10 µM As(III). The increasing intracellular concentration is shown in figure 3. <br />
<br />
<center>[[Image:UptakeRPU.png]]</center><br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100 µM As(III). The data was a bit noisy therefore a trendline was calculated and used to calculate the relative promoter unit with. <br />
<br />
<center>[[Image:Uptake100um.png]]</center><br />
:Figure 3: The internal arsenic concentration, calculated from experimental data for ''E. coli'' with J61002-<partinfo>Bba_K190015</partinfo>. The resting cells were incubated with As(III). For further information see text.<br />
<br />
The raw data can be found at [[Team:Groningen/Modelling/Downloads|downloads]].<br />
<br />
=====Fluorescence of growing cells=====<br />
<br />
In order to further characterize the ArsR promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22 h. see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000, 500, 50, 5 and 0 &micro;M. The fluorescence normalized to the OD<sub>600</sub> is plotted in figure 4. In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
<center>[[Image:Promoters-ArsR.png]]</center><br />
:Figure 4: Shows the fluorescence of RFP expressed with the ArsR promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentrations of 5000, 500, 50, 5 and 0 &micro;M sodium arsenite. <partinfo>Bba_J23101</partinfo> is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 4 is normalized to the OD<sub>600</sub> to correct for differences in cell concentration. As can be seen in figure 4 non induced ArsR RFP (0 &micro;M)is already fluorescent at the time of induction, meaning that the promotor is leaking. What figure 4 also shows is that upon induction the fluorescence increases meaning that the promotor although leaking is less suppresed in the presence of Arsenite. The highest increase in fluorescence is upon induction to a concentration of 50 &micro;M arsenite which is as high as 85% of the fluorescence from reference promotor <partinfo>Bba_J23101</partinfo>. Almost all plots show a slight decrease of fluorescence in the beginning due to the recovery of resuspending the cells at 4 &deg;C. Induction to a final concentration of 5000 &micro;M of Arsenite gives after 1 hour already an increase but decreases after 2 hours and shows only a slow increase in fluorescence after 5 hours. Reason for the lower fluorescence intensity of induction to 5000 &micro;M is the poisoning of the cells with Arsenite. The poisoning of the cells is best seen in the OD plotted against time as shown in figure 5. The cells induced to a concentration of 5000 &micro;M Arsenite shows a big decrease in OD between 5 and 22 hours after induction due to Arsenite poisoning.<br />
<br />
<center>[[Image:Promoters-ArsR-OD.png]]</center><br />
:Figure 5: Shows the OD plotted against time of ''E.coli'' with plasmid J61002 containing the pArsR RFP construct.<br />
<br />
===Conclusion===<br />
Both promoter test, with resting cells and growing cells, show clearly that the pArsR promoter is functional. The negative transcriptional regulator ArsR releases the promoter region upon induction with arsenite. The promoter strength was calculated in relative promoter units, upon induction of resting cells with 100uM As(III) an increase of 2.3 was found. A disadvantage of the usage of pArsR, also clearly shown by the two measurements, is that the negative regulation is leaky as there is already some RFP expressed without addition of arsenite. The OD measurements of the growing cell measurements showed that concentrations as high as 5000&micro;M Arsenite are poisonous for E.Coli top 10 cells.<br />
<br />
===Modelling===<br />
{{GraphHeader}}<br />
<html><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Model.js?action=raw"></script><br />
<script type="text/javascript" src="/Team:Groningen/Modelling/Arsenic.js?action=raw"></script><br />
</html><br />
<br />
The three graphs below illustrate the promoter response after induction with arsenic (directly in the cell, with the equivalent of 1&micro;M in the solution) with and without constitutive expression of ArsR (the first two graphs) and with slower production and degradation of ArsR (the two left graphs). Also, each graph has a line showing the formation of a product behind the ars promoter that does not degrade (and has production rate 1), subtracting the production that would have occurred without induction to show the effect of adding arsenic. Some conclusions:<br />
<br />
* Constitutive expression of ArsR greatly reduces (and slows) the promoter response.<br />
* On the other hand, if we divide the production and degradation rates of ArsR by ten the promoter response is ten times slower, producing ten times as much product.<br />
* In the bottom-right graph the induction is done gradually (the amount of arsenic increases linearly during the first five minutes), showing the high-pass behaviour of the promoter and that this can negatively impact product formation.<br />
<br />
<html><br />
<script type="text/javascript"><br />
addOnloadHook(computePromoterActivation);<br />
<br />
function computePromoterActivation() {<br />
// Set up constants<br />
var maxt = 600;<br />
var c = arsenicModelConstants();<br />
var cNP = {}, cS = {}, cG = {};<br />
c.v5 = 0;<br />
c.k8 = 0;<br />
c.pro = 0;<br />
c.ars2T = 0;<br />
for(var a in c) {<br />
cNP[a] = c[a];<br />
cS[a] = c[a];<br />
cG[a] = c[a];<br />
}<br />
<br />
var Vcell = 1 * 1e-15; // micrometer^3/cell -> liter/cell<br />
var avogadro = 6.02214179e23; // 1/mol<br />
c.pro = 2/(avogadro*Vcell); // 1/cell -> mol/L<br />
cS.tauR *= 10;<br />
cS.beta1 /= 10;<br />
cS.beta3 /= 10;<br />
cG.ars2T = 100*cG.ars1T;<br />
<br />
// Initialize<br />
var x0 = arsenicModelInitialization(c,0);<br />
var xNP0 = arsenicModelInitialization(cNP,0);<br />
var xS0 = arsenicModelInitialization(cS,0);<br />
var x20 = arsenicModelInitialization(c,0);<br />
var xG0 = arsenicModelInitialization(cG,0);<br />
var AsT = 1e-6*c.Vs;<br />
x0.AsinT = AsT/c.Vc;<br />
xNP0.AsinT = AsT/c.Vc;<br />
xS0.AsinT = AsT/c.Vc;<br />
x20.AsinT = 0;<br />
xG0.AsinT = AsT/c.Vc;<br />
<br />
// Simulate<br />
var x = simulate(x0,maxt,function(t,d){return arsenicModelGradient(c,d);});<br />
var xNP = simulate(xNP0,maxt,function(t,d){return arsenicModelGradient(cNP,d);});<br />
var xS = simulate(xS0,maxt*10,function(t,d){return arsenicModelGradient(cS,d);});<br />
var xG = simulate(xG0,maxt,function(t,d){return arsenicModelGradient(cG,d);});<br />
var x2 = simulate(x0,maxt,function(t,d){<br />
var Dx = arsenicModelGradient(c,d);<br />
if (t<maxt/2) Dx.AsinT += (AsT/c.Vc)*2/maxt;<br />
return Dx;<br />
});<br />
<br />
// Output<br />
function convertToSeries(c,x0,x) {<br />
var bAsin, cAsin, ArsR, ars, arsP, arsE;<br />
var arsInt = 0;<br />
var series = [[],[]];<br />
var preTime = -x.time[x._arsF.length-1]/(60*20);<br />
arsE = x0._arsF;<br />
series[0].push({x:preTime,y:100*arsE});<br />
series[0].push({x:0,y:100*arsE});<br />
series[1].push({x:preTime,y:0});<br />
for(var i=0; i<x._arsF.length; i++) {<br />
ars = x._arsF[i];<br />
if (i>0) arsInt += (x.time[i]-x.time[i-1])*(ars+arsP)/2;<br />
series[0].push({x:x.time[i]/60,y:100*ars});<br />
series[1].push({x:x.time[i]/60,y:(arsInt-x.time[i]*arsE)});<br />
arsP = ars;<br />
}<br />
return series;<br />
}<br />
document.getElementById("promoterActivationData").data = {<br />
ars:convertToSeries(c,x0,x),<br />
arsNP:convertToSeries(cNP,xNP0,xNP),<br />
arsS:convertToSeries(cS,xS0,xS),<br />
arsG:convertToSeries(cG,xG0,xG),<br />
ars2:convertToSeries(c,x20,x2)};<br />
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{|<br />
!Wild-type<br />
!+ ArsR overexpression<br />
!+ extra ars promoters<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
!Slower response<br />
!Gradual induction<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
===Other organisms===<br />
''Bacillus subtilis''<br />
<br />
In <i>B. subtilis</i>, an ArsR family repressor (ArsR<sub>BS</sub>) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), and arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsR<sub>BS</sub> recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).<br />
<br />
A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V).<br />
<br />
==Copper Induced Promoters==<br />
<br />
Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.<br />
<br />
====<i>E. coli </i>====<br />
<br />
"The intracellular level of copper in ''E. coli'' is controlled by the export of excess copper, but the entire systems of copper uptake and intracellular copper delivery are not fully understood. Two regulatory systems, the<br />
CueR and CusR systems, have been identified to be involved in transcription regulation of the genes for copper<br />
homeostasis (Rensing et al., 2000; Rensing and Grass, 2003). CueR, a MerR-family transcription factor, stimulates<br />
copper-induced transcription of both copA encoding Cu(I)-translocating P-type ATPase pump (exporter), that is the central component for maintenance of the copper homeostasis, and cueO encoding a periplasmic multicopper<br />
oxidase for detoxification (Outten et al., 2000; Petersen and Moller, 2000)." (from Yamamoto K., 2005)<br />
<br />
Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of <i>E. coli </i> str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link]. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-1821 link].<br />
<br />
*CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0570 here] along with other information.<br />
<br />
*CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under '''anaerobic growth''' and under '''extreme copper stress''' in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found [http://www.genome.jp/dbget-bin/www_bget?eco+b0571 here] along with other information. <br />
<br />
Cu &rarr; CusS &rarr; +P &rarr; CusR &rarr; Activation of transription<br />
<br />
The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). Both CpxR and YedW have the same problem of sensing external copper instead of internal copper, CueR is thought to respond to intracellular concentrations of copper. The choice for CusR over CueR would be based on the frequency of binding sites of both on the genome of <i>E. coli</i> (1 vs. 197 times), which gives CusR more chance of binding to our promoter. However, the idea behind our project is to induce GVP transtriction at a high intracellular concentration, and results in the CueR related promoter.<br />
<br />
====Cloning strategy====<br />
<br />
The CueR CueO sensitive promotor was designed by substracting it's sequence from the genome database of E.Coli str K12.It's binding region was established by Yamamoto and co worker. The promotor region was designed in silico with it's own RBS and the pre and suffix were in silico cuted with EcoRI and SpeI creating sticky ends. See parts registry {{Part|BBa_K190024}}<br />
<br />
====Results====<br />
<br />
In order characterize the CueO promotor, measurements were done by inducing cells in the exponential phase. After induction the fluorescence was measured for 22hr see [[Team:Groningen/Protocols#fluorescence_measurement| protocols]]. The RFP was excited at 580 nm and emission was measured at 600 nm. In order to have a significant high enough signal cells were resuspended at OD<sub>600</sub>=0.5 in half the volume. The cells were induced to an end concentration of 5000, 500, 50, 5 and 0 &micro;M. The fluorescence normalized to the OD is plotted in figure4.In all measurements {{Part|BBa_J23101|BBa_J23101}} was taken along to serve as a reference.<br />
<br />
[[Image:Promoters-CueO.png]]<br />
:Figure 6: Shows the fluorescence of RFP expressed with the CueO promotor. The fluorescence is normalized to 1 and p plotted against time. The ArsR promotor is induced to concentration of 5000,500,50,5 and 0 &micro;M CuSO<sub>4</sub>. Bba_J23101 is a constitutive promotor which is used as a reference for asigning promotor strength.<br />
<br />
The fluorescence in figure 6 is normalized to the OD to correct for differences in cell concentration. As can be seen in figure 6 non induced CueO RFP (0&micro;M)shows no fluorescence meaning that the promotor is not leaking. <br />
The Fluorescence for CuSO<sub>4</sub> induced cells shows only slight increase in the order of 0 < 5000 < 5 < 50 < 500<br />
&micro;M CuSO<sub>4</sub>. The cells induced to a concentration of 5000&micro;M CuSO<sub>4</sub> show no increase in fluorescence which could be due to poisoning of the cells by the CuSO<sub>4</sub>. In figure 7 can be seen that the OD of the Copper induced cells is increasing in first 5 hours and then stabilizes or even decreases in case of induction to 5000&micro;M CuSO<sub>4</sub>.<br />
<br />
[[Image:Promoters-CueO-OD.png]]<br />
:Figure 7: Shows the OD plotted against time of ''E.coli'' with plasmid J61002 containing the pCueO RFP construct.<br />
<br />
===Conclusion===<br />
The fluorescence measurements of the CueR promotor show that there is no fluorescence without induction of CuSO<sub>4</sub>. Upon induction with CuSO<sub>4</sub> the cells show an increase in RFP fluorescence which keeps increasing over 22 hours after induction.<br />
<br />
===Parts Registry===<br />
<br />
Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>CusR/CusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before).<br />
<br />
'''Abs''': This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in <i>E. coli</i>. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of <i>cusA</i>, Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusB</i>, <i>cusC</i> and Promoter from the copper-sensitive CusR/CusS two component signal system in <i>E. coli</i> (the <i>cusR/cusS</i> genes are not in parts registry, and are for external Cu concentration as mentioned before). <i>CusF</i> genes coding the proteins of copper metabolic system were used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.<br />
*{{part|BBa_I760005}}<br />
*Cu-sensitive promoter <br />
*Part-only sequence (16 bp):<br />
::atgacaaaattgtcat<br />
<br />
====Other organisms====<br />
<br />
''Mycobacterium tuberculosis'' <br><br />
'''Abs.''': Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA [[Team:Groningen/Literature#Liu2006|Liu 2006]].<br />
<br />
''Pseudomonas syringae'' <br><br />
'''Abs.''': The copper resistance (cop) operon promoter (Pcop) of <i>Pseudomonas syringae</i> is copper-inducible, and requires the regulatory genes <i>copR</i> and <i>copS</i>. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of <i>copA</i> [[Team:Groningen/Literature#Mills1994|Mills 1994]].<br />
<br />
''Sulfolobus solfataricus'' <br><br />
'''Abs.''': That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the <i>copMA</i> gene cluster [[Team:Groningen/Literature#Ettema2006|Ettema 2006]].<br />
<br />
''Lactococcus lactis'' <br><br />
'''Abs.''': Two regulatory genes (<i>lcoR</i> and <i>lcoS</i>) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (<i>cat</i>). The transcription start site involved in copper induction was mapped by primer extension [[Team:Groningen/Literature#Khunajakr1999|Khunajakr 1999]].<br />
<br />
==Zinc Induced Promoters==<br />
<br />
Zinc is essential for the functioning of cells, and must be maintained at certain levels within the cell. However, apart from its function, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized ''E. coli'' genes that encode Zn(II) uptake or export proteins. (from Outten C.E. et al, 1999)<br />
<br />
ZntR protein found in ''E. coli'', a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. ZntR functions as a zinc receptor that is necessary to activate Zn-responsive transcription at the zntA promoter. ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal concentration. (from Outten C.E. et al, 1999)<br />
<br />
The sequence of zntRp has been used to design synthetic oligos ending in biobrick pre- and suffix with EcoRI and SpeI restriction overhangs. The promoter sequence contains the -35 and -10 sequence with the atypical 20-base pair spacer region for binding of ZntR ([http://partsregistry.org/wiki/index.php/Part:BBa_K190016 BBa_K190016]). In addition, the promoter was designed with a RBS found before the zntA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K190022 BBa_K190022]). The commonly used RBS part ([http://partsregistry.org/wiki/index.php/Part:BBa_B0034 BBa_B0034]) might be to strong and give unwanted leakage of the promoter.<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup> and Cu<sup>2+</sup>, is regulated by an ArsR-type repressor (CzrABS) as well [[Team:Groningen/Literature#Moore2005|Moore 2005]].<br />
<br />
''Streptococcus pneumoniae''<br />
<br />
'''Abs.''': Activation of the czcD promoter by SczA is shown to proceed by Zn<sup>2+</sup>-dependent binding of SczA to a conserved DNA motif. In the absence of Zn<sup>2+</sup>, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family<br />
Kloosterman T.G., et al. (O.P. Kuipers), The novel transcriptional regulator SczA mediates protection against Zn<sup>2+</sup> stress by activation of the Zn<sup>2+</sup>-resistance gene czcD in ''Streptococcus pneumoniae'', Molecular Microbiology, 2007, 65(4), 1049–1063. Retrieved from "https://2009.igem.org/Team:Groningen/Project/Promoters" <br />
<br />
<br />
''Staphylococcus aureus''<br />
<br />
'''Abs.''': In ''Staphylococcus aureus'' CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn<sup>2+</sup> and Co<sup>2+</sup> (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
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{{Team:Groningen/Project/Footer}}</div>Wilfred