http://2009.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=250&target=Verhoeven1981&year=&month=2009.igem.org - User contributions [en]2024-03-29T07:33:17ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Photo_GalleryPhoto Gallery2010-03-10T14:13:59Z<p>Verhoeven1981: </p>
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<div><div style='float:left;width:400px;margin-right:40px;text-align:center;'><br />
[http://www.flickr.com/photos/igemhq/ <font size=4><font color=dodgerblue>'''flick</font><font color=crimson>r&trade;</font> <font color=black>photos'''</font></font>]<br><br />
<small>(Photos are licensed under [http://creativecommons.org/about/licenses Creative Commons - Attribution] license. Please attribute iGEM 2009 Jamboree photos to '''David Appleyard''' and '''iGEM'''.)</small><br />
[http://www.flickr.com/photos/igemhq/ https://static.igem.org/mediawiki/2008/6/6e/Igem_08_blackboard_xsmall.jpg]<br />
<br />
If you upload pictures that you took yourself, use the <font size=3>'''igem'''</font> and <font size=3>'''2009'''</font> tags so that everyone else can find them too.<br />
<div><br />
Post the links to your pictures in the list to the right.<br />
</div><br />
</div><br />
<br />
<br />
<font size=4>'''Pictures taken by the iGEM Community:'''</font><br />
* iGEM Paris [http://picasaweb.google.com/flf.mib/IGEMParisTeam?feat=directlink iGEM Paris]<br />
* iGEM Newcastle [http://www.flickr.com/photos/eridanus/sets/72157622760324890/ iGEM Newcastle]<br />
* iGEM Groningen [http://www.flickr.com/photos/igemgroningen/sets/ iGEM Groningen]</div>Verhoeven1981http://2009.igem.org/Photo_GalleryPhoto Gallery2010-03-10T12:06:36Z<p>Verhoeven1981: </p>
<hr />
<div><div style='float:left;width:400px;margin-right:40px;text-align:center;'><br />
[http://www.flickr.com/photos/igemhq/ <font size=4><font color=dodgerblue>'''flick</font><font color=crimson>r&trade;</font> <font color=black>photos'''</font></font>]<br><br />
<small>(Photos are licensed under [http://creativecommons.org/about/licenses Creative Commons - Attribution] license. Please attribute iGEM 2009 Jamboree photos to '''David Appleyard''' and '''iGEM'''.)</small><br />
[http://www.flickr.com/photos/igemhq/ https://static.igem.org/mediawiki/2008/6/6e/Igem_08_blackboard_xsmall.jpg]<br />
<br />
If you upload pictures that you took yourself, use the <font size=3>'''igem'''</font> and <font size=3>'''2009'''</font> tags so that everyone else can find them too.<br />
<div><br />
Post the links to your pictures in the list to the right.<br />
</div><br />
</div><br />
<br />
<br />
<font size=4>'''Pictures taken by the iGEM Community:'''</font><br />
* iGEM Paris [http://picasaweb.google.com/flf.mib/IGEMParisTeam?feat=directlink iGEM Paris]<br />
* iGEM Newcastle [http://www.flickr.com/photos/eridanus/sets/72157622760324890/ iGEM Newcastle]<br />
* iGEM Groningen [http://www.flickr.com/photos/48231579@N07/sets/ iGEM Groningen]</div>Verhoeven1981http://2009.igem.org/Team:Groningen/TeamTeam:Groningen/Team2009-11-26T13:28:41Z<p>Verhoeven1981: /* 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: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 />
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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>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Submitted_PartsTeam:Groningen/Parts/Submitted Parts2009-10-21T21:34:52Z<p>Verhoeven1981: /* Devices */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Parts}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Parts/Used_Parts}}</div><br />
<br />
<br />
<br />
==Submitted Parts==<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
===Importers===<br />
<br />
'''<partinfo>BBa_K190028</partinfo> GlpF''' <br> [[Team:Groningen/Project/Transport#GlpF|GlpF]] is an aquaglyceroporin channel that facilitates the transport of As(III). his part has been used to import As(III) into the ''E.coli'' cells. It has been used in combination with metal accumulation proteins to make a accumulation device. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190028:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190061</partinfo> GlpF (with strong RBS)''' <br> <partinfo>BBa_K190028</partinfo> [[Team:Groningen/Project/Transport#GlpF|(GlpF)]] with the ribosomal binding site <partinfo>B0034</partinfo>. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190061:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190062</partinfo> GlpF regulated by medium constitutive promotor''' <br> <partinfo>BBa_K190061</partinfo> ([[Team:Groningen/Project/Transport#GlpF|GlpF]] with strong RBS) regulated by the constitutive promotor <partinfo>BBa_J23106</partinfo>.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190062:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190063</partinfo> GlpF regulated by pLacI''' <br> <partinfo>BBa_K190061</partinfo> ([[Team:Groningen/Project/Transport#GlpF|GlpF]]with strong RBS) regulated by the inducible promotor <partinfo>BBa_R0010</partinfo><br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190063:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190018</partinfo> HmtA (contains PstI sites)''' <br> [[Team:Groningen/Project/Transport#HmtA|HmtA]] (heavy metal transporter A) from ''Pseudomonas aeruginosa'' Q9I147 is a P-type ATPase importer. This membrane protein mediates the uptake of copper (Cu) and zinc (Zn). we believe this ATP-driven pump is capable of generating an elevated intracellular concentration of these compounds enabling the harvesting of copper and zinc from the environment.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190018:Design More Information].'''<br />
<br />
===Acummulators===<br />
<br />
'''<partinfo>BBa_K190019</partinfo> fMT''' <br> [[Team:Groningen/Project/Accumulation#fMT|fMT]] is a metallothionein, binding Arsenite(III) and Arsenate(V), it has higher affinity for As(III). As a metallothionein it is also suitable to bind other metals like zinc, copper or cadmium. This biobrick already contains the ribosomal binding site <partinfo>B0034</partinfo>.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190019:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190031</partinfo> fMT regulated by low constitutive promotor''' <br> <partinfo>BBa_K190019</partinfo> ([[Team:Groningen/Project/Accumulation#fMT|fMT]]) regulated by the constitutive promotor <partinfo>BBa_J23109</partinfo>.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190031:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190032</partinfo> fMT regulated by pLacI''' <br><partinfo>BBa_K190019</partinfo> ([[Team:Groningen/Project/Accumulation#fMT|fMT]]) regulated by the inducible promotor <partinfo>BBa_R0010</partinfo>.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190032:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190027</partinfo> MBP-ArsR (fusion protein)''' <br> The negative transcriptional regulator [[Team:Groningen/Project/Accumulation#ArsR|ArsR]] fused with Maltose Binding Protein (MBP) for higher stability. It also carries a His<sub>10</sub> tag. It was designed as an accumulator of As(III).<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190027:Design More Information].'''<br />
<br />
===Promotors===<br />
<br />
'''<partinfo>BBa_K190015</partinfo> Arsenic Promoter''' <br> Promoter sequence with recognition site for ArsR transcriptional regulator protein. ArsR binds to the promoter sequence in the absence of As and releases on binding of As, thereby activating transcription. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190015:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190023</partinfo> Arsenic Promoter (with wild type RBS)''' <br> Promoter sequence containing the recognition site for ArsR transcriptional regulator protein. Downstream from the promoter is the RBS that is attached to this promoter region in the ''E. coli'' genome. ArsR binds to the promoter sequence in the absence of As and releases on binding of As, thereby activating transcription.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190023:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190016</partinfo> Zinc Promoter''' <br> Promoter sequence with recognition site for ZntR transcriptional regulator protein. ZntR activates transcription when Zn(II) is bound. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190016:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190022</partinfo> Zinc Promoter (with wild type RBS)''' <br> The pZntR from ''E. coli'' K.12 has a specific RBS site behind it in the genome. Here the RBS site is attachted to the promoter region. The RBS site might influence the activity of the promoter and will be tested in the same way as <partinfo>BBa_K190016</partinfo>. ZntR activates transcription when Zn(II) is bound.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190022:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190017</partinfo> Copper Promoter''' <br> Promoter sequence with recognition site for the CueR transcription regulating protein. When copper levels in the cell rise CueR activates transcription. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190017:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190024</partinfo> Copper Promoter (with wild type RBS)''' <br> Promoter sequence with recognition site for the CueR transcription regulating protein and its downstream RBS (as it is found in the ''E. coli'' genome). When copper levels in the cell rise CueR activates transcription.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190024:Design More Information].'''<br />
<br />
===GVP Constructs===<br />
<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> GVP consists of 6064 base pairs. It's Backbone is <partinfo>BBa_J61035</partinfo> With a resistance against Ampicillin/Gentamycin <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_I750016:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190025</partinfo> GVP regulated by low constitutive promotor''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the constitutive promoter <partinfo>BBa_J23109</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190025:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190026</partinfo> GVP regulated by medium constitutive promotor''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the constitutive promoter <partinfo>BBa_J23106</partinfo> were combined, and the product ligated into vector pSB1AC3. The construct was used to test and caracterize the gas vesicle formation. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190026:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190033</partinfo> GVP regulated by pArsR''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the metal sensitive promoter <partinfo>BBa_K190015</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190033:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190034</partinfo> GVP regulated by pZntR''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the metal sensitive promoter <partinfo>BBa_K190016</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190034:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190035</partinfo> GVP regulated by pCueO''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the metal sensitive promoter <partinfo>BBa_K190017</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190035:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190036</partinfo> GVP regulated by pLacI''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the LacI and CAP regulated promoter <partinfo>BBa_R0010</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190036:Design More Information].'''<br />
<br />
===Devices===<br />
<br />
'''<partinfo>BBa_K190038</partinfo> Arsenic Accumulation Device (IPTG induced)''' <br> The device consists of a pLacI promoter with fMT (metal binding protein) and GlpF (metal transporter). Upon addition of IPTG the transcription is activated and the fMT gene is expressed in a higher amount compared to the GlpF transporter. Expression of both results in increased metal uptake and accumulation in the cell. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190038:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190039</partinfo> Arsenic Accumulation Device (constitutive)''' <br> The device consists of a constitutive promoter (low expression) with fMT (metal binding protein) and GlpF (metal transporter). Expression of both results in increased metal uptake and accumulation in the cell.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190039:Design More Information].'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-21T21:05:18Z<p>Verhoeven1981: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Parts/Submitted_Parts}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project_Plan}}</div><br />
<br />
==Used Parts==<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===Miscellaneous===<br />
'''<partinfo>BBa_B0034</partinfo> RBS ((Elowitz 1999) -- defines RBS efficiency)''' <br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_K190061 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ligation of part <partinfo>BBa_K190028</partinfo> behind the RBS was successful, confirmed by gel (correct vector size after digestion with EcoRI and PstI) and sequencing with VF2 primer. We used this part in combination with several biobricks for building our constructs e.g. <partinfo>BBa_K190061</partinfo>.<br />
|}<br />
<br />
'''<partinfo>BBa_B0014</partinfo> Double terminator (B0012-B0011)''' <br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_B0014 AddReview 3</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The plasmid containing part <partinfo>BBa_B0014</partinfo> was successfully transformed into ''E. coli'' TOP10 cells (confirmed by single and double digestion). The original plan was to use the terminator behind one of our own designed parts <partinfo>BBa_K190027</partinfo>, and in front of pArsR-RFP. The MBP-ArsR fusion protein was thought to have a regulating effect on the arsenic promotor pArsR in the same way as ArsR regulates the promotor. The terminator separated the two parts on the plasmid. The construct was not used due to time constraints, and not sequenced.<br />
|}<br />
<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_I750016 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
Part <partinfo>BBa_I750016</partinfo> was one of our main focus points during our project. The part was originally submitted by Melbourne in 2007 with a very limited amount of information (short description and sequence mutations). On their site it was mentioned the cluster was difficult to use and ligation into a plasmid was hard, in contrast we found the cluster to be easily cut and ligated into different plasmids and behind several promotors. We used this part in combination with several biobricks for building our constructs e.g. <partinfo>BBa_K190033</partinfo> and <partinfo>BBa_K190036</partinfo>. Pictures of our cells with induced gas vesicle formation confirmed the production of vesicles in the expected shape and size. In addition, we characterized the part to improve the use in the future and added a lot of information on the registry.<br />
|}<br />
<br />
'''<partinfo>BBa_P1010</partinfo> ccdB cell death gene'''<br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_P1010 AddReview 4</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
P1010 is used when putting BioBrick parts into BioBrick plasmids. The part to be inserted and the plasmid are cut with BioBrick enzymes and mixed. The mixture will include both the original uncut or self ligated plasmid and the desired structure. However, because of CcdB, all of the cells containing the original plasmid die and the surviving colonies are the desired result. The ccdB cell death gene worked as expected killing our ''E. coli'' TOP10 cells, and keeping our ''E. coli'' DB3 cells alive. After noticing the inconsistent sequencing result for the pSB2K3 plasmid with ccdB cell death gene, we decided to choose a different pSB2K3 plasmid with random part to continue with our assemblies. This to minimize the chance of unwanted surprises in the end.<br />
|}<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> Constitutive promoter family member (high expression)''' <br />
{|<br />
|width='10%'|<br />
<partinfo>J23100 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
We used a number of the constitutive promoter family members for testing our biobricks. The constitutive promoters show the expected level of fluorescence when transformed into ''E. coli'' TOP10 cells. Placing parts behind the promoters turned out to be relatively straight forward. We used this part in combination with several biobricks for building our constructs e.g. <partinfo>BBa_I750016</partinfo> was placed behind the promoters.<br />
|} <br />
<br />
'''<partinfo>J23101</partinfo> Constitutive promoter family member (high expression, reference)''' <br />
{|<br />
|width='10%'|<br />
<partinfo>J23101 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
We used a number of the constitutive promoter family members for testing our biobricks. The constitutive promoters show the expected level of fluorescence when transformed into ''E. coli'' TOP10 cells. Placing parts behind the promoters turned out to be relatively straight forward. We used this part in combination with several biobricks for building our constructs e.g. <partinfo>BBa_I750016</partinfo> was placed behind the promoters.<br />
|}<br />
<br />
'''<partinfo>J23106</partinfo> Constitutive promoter family member (medium expression)''' <br />
{|<br />
|width='10%'|<br />
<partinfo>J23106 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
We used a number of the constitutive promoter family members for testing our biobricks. The constitutive promoters show the expected level of fluorescence when transformed into ''E. coli'' TOP10 cells. Placing parts behind the promoters turned out to be relatively straight forward. We used this part in combination with several biobricks for building our constructs e.g. <partinfo>BBa_I750016</partinfo> was placed behind the promoters.<br />
|} <br />
<br />
'''<partinfo>J23109</partinfo> Constitutive promoter family member (low expression)''' <br />
{|<br />
|width='10%'|<br />
<partinfo>J23109 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
We used a number of the constitutive promoter family members for testing our biobricks. The constitutive promoters show the expected level of fluorescence when transformed into ''E. coli'' TOP10 cells. Placing parts behind the promoters turned out to be relatively straight forward. We used this part in combination with several biobricks for building our constructs e.g. <partinfo>BBa_I750016</partinfo> and <partinfo>BBa_K190028</partinfo> were placed behind the promoters.<br />
|} <br />
<br />
'''<partinfo>R0010</partinfo> Promoter (lacI regulated)''' <br> This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first binds the CAP protein, which is generally present in E.coli and is asocciated with cell health and availability of glucose. The second binds LacI protein. <br> <br />
<br />
'''<partinfo>I0500</partinfo> pBad/araC''' <br />
{|<br />
|width='10%'|<br />
<partinfo>I0500 AddReview 0</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The sequence was listed as inconsistent, and the ligations of parts behind the promoter failed. Restriction of isolated plasmids showed fragments of unexpected sizes.<br />
|}<br />
<br />
===Vectors===<br />
'''<partinfo>pSB1AC3</partinfo> High copy BioBrick assembly plasmid'''<br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
pSB1AC3 is a high copy number plasmid carrying ampicillin and chloramphenicol resistance. Together with pSB1A2 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. The transformations with pSB1AC3 (containing different biobricks of own design) into ''E. coli'' TOP10 cells, growth on both antibiotics, and gel analysis (undigested and digested with the EcoRI and PstI) worked as expected. The high (copy) number of plasmids per cell make it an easy to work with plasmid, ideal for cloning and assembly work.<br />
|}<br />
<br />
'''<partinfo>pSB1A2</partinfo> pSB1A2 (Replaced by pSB1A3 in registry) ''' <br> pSB1A2 is a high copy number plasmid carrying ampicillin resistance. Together with pSB1AC3 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br><br />
<br />
'''<partinfo>pSB2K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br><br />
<br />
'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br><br />
<br />
'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a resistance against Ampicillin <br><br />
<br />
'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br></div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-21T20:54:25Z<p>Verhoeven1981: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Parts/Submitted_Parts}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project_Plan}}</div><br />
<br />
==Used Parts==<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===Miscellaneous===<br />
'''<partinfo>BBa_B0034</partinfo> RBS ((Elowitz 1999) -- defines RBS efficiency)''' <br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_K190061 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ligation of part <partinfo>BBa_K190028</partinfo> behind the RBS was successful, confirmed by gel (correct vector size after digestion with EcoRI and PstI) and sequencing with VF2 primer. We used this part in combination with several biobricks for building our constructs e.g. <partinfo>BBa_K190061</partinfo>.<br />
|}<br />
<br />
'''<partinfo>BBa_B0014</partinfo> Double terminator (B0012-B0011)''' <br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_B0014 AddReview 3</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The plasmid containing part <partinfo>BBa_B0014</partinfo> was successfully transformed into ''E. coli'' TOP10 cells (confirmed by single and double digestion). The original plan was to use the terminator behind one of our own designed parts <partinfo>BBa_K190027</partinfo>, and in front of pArsR-RFP. The MBP-ArsR fusion protein was thought to have a regulating effect on the arsenic promotor pArsR in the same way as ArsR regulates the promotor. The terminator separated the two parts on the plasmid. The construct was not used due to time constraints, and not sequenced.<br />
|}<br />
<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_I750016 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
Part <partinfo>BBa_I750016</partinfo> was one of our main focus points during our project. The part was originally submitted by Melbourne in 2007 with a very limited amount of information (short description and sequence mutations). On their site it was mentioned the cluster was difficult to use and ligation into a plasmid was hard, in contrast we found the cluster to be easily cut and ligated into different plasmids and behind several promotors. We used this part in combination with several biobricks for building our constructs e.g. <partinfo>BBa_K190033</partinfo> and <partinfo>BBa_K190036</partinfo>. Pictures of our cells with induced gas vesicle formation confirmed the production of vesicles in the expected shape and size. In addition, we characterized the part to improve the use in the future and added a lot of information on the registry.<br />
|}<br />
<br />
'''<partinfo>BBa_P1010</partinfo> ccdB cell death gene'''<br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_P1010 AddReview 4</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
P1010 is used when putting BioBrick parts into BioBrick plasmids. The part to be inserted and the plasmid are cut with BioBrick enzymes and mixed. The mixture will include both the original uncut or self ligated plasmid and the desired structure. However, because of CcdB, all of the cells containing the original plasmid die and the surviving colonies are the desired result. The ccdB cell death gene worked as expected killing our ''E. coli'' TOP10 cells, and keeping our ''E. coli'' DB3 cells alive. After noticing the inconsistent sequencing result for the pSB2K3 plasmid with ccdB cell death gene, we decided to choose a different pSB2K3 plasmid with random part to continue with our assemblies. This to minimize the chance of unwanted surprises in the end.<br />
|}<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> Constitutive promoter family member (high expression)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> <br />
<br />
'''<partinfo>J23101</partinfo> Constitutive promoter family member (high expression, reference)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> <br />
<br />
'''<partinfo>J23106</partinfo> Constitutive promoter family member (medium expression)''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> <br />
<br />
'''<partinfo>J23109</partinfo> Constitutive promoter family member (low expression)''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> <br />
<br />
'''<partinfo>R0010</partinfo> Promoter (lacI regulated)''' <br> This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first binds the CAP protein, which is generally present in E.coli and is asocciated with cell health and availability of glucose. The second binds LacI protein. <br> <br />
<br />
'''<partinfo>I0500</partinfo> pBad/araC''' <br> This part is <br> <br />
<br />
===Vectors===<br />
'''<partinfo>pSB1AC3</partinfo> High copy BioBrick assembly plasmid'''<br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
pSB1AC3 is a high copy number plasmid carrying ampicillin and chloramphenicol resistance. Together with pSB1A2 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. The transformations with pSB1AC3 (containing different biobricks of own design) into ''E. coli'' TOP10 cells, growth on both antibiotics, and gel analysis (undigested and digested with the EcoRI and PstI) worked as expected. The high (copy) number of plasmids per cell make it an easy to work with plasmid, ideal for cloning and assembly work.<br />
|}<br />
<br />
'''<partinfo>pSB1A2</partinfo> pSB1A2 (Replaced by pSB1A3 in registry) ''' <br> pSB1A2 is a high copy number plasmid carrying ampicillin resistance. Together with pSB1AC3 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br><br />
<br />
'''<partinfo>pSB2K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br><br />
<br />
'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br><br />
<br />
'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a resistance against Ampicillin <br><br />
<br />
'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br></div>Verhoeven1981http://2009.igem.org/Team:Groningen/Safety/LegislationTeam:Groningen/Safety/Legislation2009-10-21T20:51:24Z<p>Verhoeven1981: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
[[Category:Team:Groningen]]<br />
[[Category:Safety]]<br />
__NOTOC__<br />
<br />
==Dutch Legislation==<br />
The legislation around biotechnology in the Netherlands is largely build around European guidelines and decrees. The main subjects in this legislation are to guarantee:<br />
* Safety of humans, animal and environment<br />
* Protecting the consumer and fair trade<br />
Working with genetically modified organisms (GMO's) in the Netherlands is prohibited without a permit. [http://international.vrom.nl/ The Ministry of housing, Spatial Planning and the Environment] grants these permits, on grounds of the [http://wetten.overheid.nl/BWBR0004703/ <i>decree Genetically modified organisms</i>], as it is the primary authority on this subject. The [http://wetten.overheid.nl/BWBR0004703/ <i>decree Genetically modified organisms</i>] is supplemented by the [http://wetten.overheid.nl/BWBR0009653/ <i> sanction Genetically modified organisms</i>] containing specifications for rules, general safety precautions and facility and workrequirement. However the lab facility should also follow the [http://wetten.overheid.nl/BWBR0005829/ <i>decree for arrangement- and permits environmental management</i>].<br />
<BR><br />
[http://international.vrom.nl/ The Ministry of housing, Spatial Planning and the Environment] shares its responsibilities with the Dutch ministries of:<br />
<br />
*[http://www.minvws.nl/en/ Health, Welfare and Sport], <br />
*[http://www.minlnv.nl/portal/page?_pageid=116,1640354&_dad=portal&_schema=PORTAL Agriculture, Nature and Food Quality], <br />
*[http://www.ez.nl/english/Organisation/ Economic affairs], <br />
*[http://www.minocw.nl/english/index.html Education, culture and science], <br />
*[http://www.minbuza.nl/en Foreign affars] <br />
*[http://english.justitie.nl/ Justice]<br />
<br />
All represented in the <i>Interdepartmental consultation Biotechnology</i>, where actual affairs and progress of biotechnological projects are discussed. <br />
<br />
Technical-scientific advice on the issues concerning the risks for humans and environment with applications of GMO's is given by [http://www.cogem.net/page.ocl?pageid=46&loc=5&version=&mode= The Dutch Committee on Genetic Modification (Cogem)] which also gives advice on the safety precautions to take to protect them against those risks. [http://www.cogem.net/page.ocl?pageid=46&loc=5&version=&mode= Cogem] also gives information on the ethical- and society issues on genetic modification. Cogem already reviewed the Synthetic biology in the Netherlands: [http://www.cogem.net/ContentFiles/CGM080925-01-Biological%20machines1.pdf Biological machines? <i>Anticipating developments in synthetic biology</i>], in which they divided the question about the hazards of synthetic biology in three parts:<br />
# Is there a legislative framework for action?<br />
# Can technical safety measurements be taken to manage risks?<br />
# Can the risks be assesed?<br />
The final conclusion of this report stated that for synthetic the current legislation for GMO's (summarized above) is fully applicable to synthetic biology. A need for new safety legislation especially for synthetic is non-exsting, however this report assumes that the work of synthetic organisms remains in laboratory environments.<br />
<br />
==University of Groningen==<br />
Within the [http://www.rug.nl University of Groningen] the organization of biological safety, including all activities that are carried out with genetically modified organisms (GMOs), is the legal responsibility of the Board of the University of Groningen. The University Board thus acts as the notifier in all GMO-related research where notification is required. The Faculty Boards have been mandated by the Board of the University to implement the rules as set out in the GMO Regulations within their own faculty.<br />
<br />
Within the University of Groningen, biological safety is ensured on the central level by a biological safety organization comprising the biological safety officers (BSO) as well as a staff member of the Health, Safety and Environment Service (HSE). The HSE coordinates the notifications, maintains the register and, in cooperation with the BSO, advises the Faculty Boards and the University Board in the field of biological safety. The central organization is complemented on the local level by responsible officers. <br />
<br />
BSO officers are appointed by the University Board on the recommendation of the Faculty Boards. In order to be considered for this position, BSO officers must have been approved by the [http://international.vrom.nl/ The Ministry of housing, Spatial Planning and the Environment] on the basis of the qualifications as set out in the GMO regulations. BSO officers are members of the staff office and can report directly both to the University board and their own faculty. <br />
<br />
For each project a responsible officer will be appointed by the University Board on the recommendation of the Faculty Board. This officer is responsible for various aspects, including day-to-day matters concerning the activities involving genetically modified organisms. <br />
<br />
<!--==Floating metal uptaking bacteria==<br />
{{todo}} When applying synthetic organisms in the wider environment, beyond a laboratory environment, such as the case would be with water purifying bacteria. The question about novel safety requirements and legislation would need to be reopened.<br />
<BR><br />
Heavy metals<BR><br />
GMO's<BR><br />
Heavy metals & GMO's<BR><br />
--><br />
<br />
==Further reading==<br />
*[http://openwetware.org/wiki/Synthetic_Biology:SB2.0/Biosecurity_resolutions Synthetic Biology:SB2.0/Biosecurity resolutions]<br />
===Global legislation===<br />
*[http://www.biodiv.org/convention/articles.asp?lg=0 Convention on biological diversity]<br />
**[http://www.biodiv.org/doc/legal/cartagena-protocol-en.pdf Cartega (Biosafety) Protocol]<br />
*[http://unece.org/env/pp/gmo.htm Aarhus Convention]<br />
*[http://www.infomil.nl/aarhus Aarhus Convention]<br />
===European legislation===<br />
*[http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexapi!prod!CELEXnumdoc&lg=NL&numdoc=32003R1830&model=guichett Regulation (EG) nr. 1830/2003]: Traceability and labeling GMO's and traceability GMO produced food and feed. <br />
*[http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexapi!prod!CELEXnumdoc&lg=NL&numdoc=32003R1829&model=guichett Regulation (EG) nr. 1829/2003]: Genetically modified food & feed<br />
*[http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexapi!prod!CELEXnumdoc&lg=NL&numdoc=32003R1946&model=guichett Regulation (EG) nr. 1946/2003]: Transport of GMO's accross borders. <br />
*[http://www.vrom.nl/Docs/milieu/Richtlijn_2001_18.pdf Directive 2001/18]: Introduction of GMO's in the environment.<br />
===Dutch legislation===<br />
*[http://www.overheid.nl/biotechnologie/regels Dutch legislations regarding biotechnology]<br />
**[http://www.kennislink.nl/publicaties/bacterien-en-antibiotica-resistentie Bacteria & antibiotic-resistence]<br />
**[http://www.vrom.nl/pagina.html?id=2706&sp=2&dn=w401 Dutch Biotechnology Policy (2000)]<br />
**[http://www.vrom.nl/pagina.html?id=9262 Waste disposal]<br />
<br />
==Involved organisations==<br />
===International organisation===<br />
[http://bch.cbd.int/ The Biosafety Clearing-House]<BR><br />
<br />
===National organisation===<br />
*[http://www.icgeb.org/~bsafesrv/biobiblio.html Biosafety bibliographic database]<BR><br />
*[http://www.en.bvfplatform.nl/home.html The Association BVF Platform]<BR><br />
*[http://www.rivm.nl/milieuportaal/onderwerpen/bodem/ggo-en-bodem/ GMO’s in the environment]<br />
**[http://213.154.234.72/Paginas/bggo.htm Bureau of GMO’s]<BR><br />
**[http://www.cogem.net/page.ocl?pageid=46&loc=5&version=&mode= Committee on Genetic modification]<BR><br />
*[http://international.vrom.nl/ The Ministry of housing, Spatial Planning and the Environment]<br />
**[http://www.vrom.nl/pagina.html?id=8324 biotechnology]<br />
*[http://www.nvmm.nl/nvmm/nvmmcms.nsf/viewdoc/hom-01?opendocument Dutch society for microbiology]<BR><br />
<br />
===Institutional organisation===<br />
*[http://www.rug.nl/Bureau/expertisecentra/amd/milieu/milBiologischeVeiligheid?lang=en Biosafety at the University of Groningen]<BR><br />
<br />
<br />
<html><a href="http://www.cogem.net/page.ocl?pageid=46&loc=5&version=&mode="><img src="http://www.cogem.net/images/logo1.gif"></a></html><br />
<center>http://www.rug.nl/bureau/expertisecentra/amd/milieu/milDataPicture/logoMilieu.gif<br />
http://www.myteacherpages.com/webpages/GSchwab/photos/628423/3logo1.gif http://www.rug.nl/Bureau/expertisecentra/amd/milieu/milDataPicture/logoBioHazard.gif</center>{{Team:Groningen/Footer}}</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-21T20:46:34Z<p>Verhoeven1981: /* Used Parts */</p>
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<div>{{Team:Groningen/Header}}<br />
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<div style="float:left" >{{linkedImage|GroningenPrevious.png|Team:Groningen/Parts/Submitted_Parts}}</div><br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project_Plan}}</div><br />
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==Used Parts==<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===Miscellaneous===<br />
'''<partinfo>BBa_B0034</partinfo> RBS ((Elowitz 1999) -- defines RBS efficiency)''' <br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_K190061 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ligation of part <partinfo>BBa_K190028</partinfo> behind the RBS was successful, confirmed by gel (correct vector size after digestion with EcoRI and PstI) and sequencing with VF2 primer. We used this part in combination with several biobricks for building our constructs e.g. <partinfo>BBa_K190061</partinfo>.<br />
|}<br />
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'''<partinfo>BBa_B0014</partinfo> Double terminator (B0012-B0011)''' <br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_B0014 AddReview 3</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The plasmid containing part <partinfo>BBa_B0014</partinfo> was successfully transformed into ''E. coli'' TOP10 cells (confirmed by single and double digestion). The original plan was to use the terminator behind one of our own designed parts <partinfo>BBa_K190027</partinfo>, and in front of pArsR-RFP. The MBP-ArsR fusion protein was thought to have a regulating effect on the arsenic promotor pArsR in the same way as ArsR regulates the promotor. The terminator separated the two parts on the plasmid. The construct was not used due to time constraints, and not sequenced.<br />
|}<br />
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'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_I750016 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
Part <partinfo>BBa_I750016</partinfo> was one of our main focus points during our project. The part was originally submitted by Melbourne in 2007 with a very limited amount of information (short description and sequence mutations). On their site it was mentioned the cluster was difficult to use and ligation into a plasmid was hard, in contrast we found the cluster to be easily cut and ligated into different plasmids and behind several promotors. We used this part in combination with several biobricks for building our constructs e.g. <partinfo>BBa_K190033</partinfo> and <partinfo>BBa_K190036</partinfo>. Pictures of our cells with induced gas vesicle formation confirmed the production of vesicles in the expected shape and size. In addition, we characterized the part to improve the use in the future and added a lot of information on the registry.<br />
|}<br />
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'''<partinfo>BBa_P1010</partinfo> ccdB cell death gene'''<br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_P1010 AddReview 4</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
P1010 is used when putting BioBrick parts into BioBrick plasmids. The part to be inserted and the plasmid are cut with BioBrick enzymes and mixed. The mixture will include both the original uncut or self ligated plasmid and the desired structure. However, because of CcdB, all of the cells containing the original plasmid die and the surviving colonies are the desired result. The ccdB cell death gene worked as expected killing our ''E. coli'' TOP10 cells, and keeping our ''E. coli'' DB3 cells alive. After noticing the inconsistent sequencing result for the pSB2K3 plasmid with ccdB cell death gene, we decided to choose a different pSB2K3 plasmid with random part to continue with our assemblies. This to minimize the chance of unwanted surprises in the end.<br />
|}<br />
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===Promotors===<br />
'''<partinfo>J23100</partinfo> Constitutive promoter family member (high expression)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> <br />
<br />
'''<partinfo>J23101</partinfo> Constitutive promoter family member (high expression, reference)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> <br />
<br />
'''<partinfo>J23106</partinfo> Constitutive promoter family member (medium expression)''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> <br />
<br />
'''<partinfo>J23109</partinfo> Constitutive promoter family member (low expression)''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> <br />
<br />
'''<partinfo>R0010</partinfo> Promoter (lacI regulated)''' <br> This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first binds the CAP protein, which is generally present in E.coli and is asocciated with cell health and availability of glucose. The second binds LacI protein. <br> <br />
<br />
'''<partinfo>I0500</partinfo> pBad/araC''' <br> This part is <br> <br />
<br />
===Vectors===<br />
'''<partinfo>pSB1AC3</partinfo> High copy BioBrick assembly plasmid'''<br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
pSB1AC3 is a high copy number plasmid carrying ampicillin and chloramphenicol resistance. Together with pSB1A2 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. The transformations with pSB1AC3 (containing different biobricks of own design) into ''E. coli'' TOP10 cells, growth on both antibiotics, and gel analysis (undigested and digested with the EcoRI and PstI) worked as expected. The high (copy) number of plasmids per cell make it an easy to work with plasmid, ideal for cloning and assembly work.<br />
|}<br />
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'''<partinfo>pSB1A2</partinfo> pSB1A2 (Replaced by pSB1A3 in registry) ''' <br> pSB1A2 is a high copy number plasmid carrying ampicillin resistance. Together with pSB1AC3 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br><br />
<br />
'''<partinfo>pSB2K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br><br />
<br />
'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br><br />
<br />
'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a resistance against Ampicillin <br><br />
<br />
'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br></div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Submitted_PartsTeam:Groningen/Parts/Submitted Parts2009-10-21T19:36:28Z<p>Verhoeven1981: /* Devices */</p>
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<div>{{Team:Groningen/Header}}<br />
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<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Parts/Used_Parts}}</div><br />
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==Submitted Parts==<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
===Importers===<br />
<br />
'''<partinfo>BBa_K190028</partinfo> GlpF''' <br> [[Team:Groningen/Project/Transport#GlpF|GlpF]] is an aquaglyceroporin channel that facilitates the transport of As(III). his part has been used to import As(III) into the ''E.coli'' cells. It has been used in combination with metal accumulation proteins to make a accumulation device. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190028:Design More Information].'''<br />
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'''<partinfo>BBa_K190061</partinfo> GlpF (with strong RBS)''' <br> <partinfo>BBa_K190028</partinfo> [[Team:Groningen/Project/Transport#GlpF|(GlpF)]] with the ribosomal binding site <partinfo>B0034</partinfo>. <br />
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'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190061:Design More Information].'''<br />
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'''<partinfo>BBa_K190062</partinfo> GlpF regulated by medium constitutive promotor''' <br> <partinfo>BBa_K190061</partinfo> ([[Team:Groningen/Project/Transport#GlpF|GlpF]] with strong RBS) regulated by the constitutive promotor <partinfo>BBa_J23106</partinfo>.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190062:Design More Information].'''<br />
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'''<partinfo>BBa_K190063</partinfo> GlpF regulated by pLacI''' <br> <partinfo>BBa_K190061</partinfo> ([[Team:Groningen/Project/Transport#GlpF|GlpF]]with strong RBS) regulated by the inducible promotor <partinfo>BBa_R0010</partinfo><br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190063:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190018</partinfo> HmtA (contains PstI sites)''' <br> [[Team:Groningen/Project/Transport#HmtA|HmtA]] (heavy metal transporter A) from ''Pseudomonas aeruginosa'' Q9I147 is a P-type ATPase importer. This membrane protein mediates the uptake of copper (Cu) and zinc (Zn). we believe this ATP-driven pump is capable of generating an elevated intracellular concentration of these compounds enabling the harvesting of copper and zinc from the environment.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190018:Design More Information].'''<br />
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===Acummulators===<br />
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'''<partinfo>BBa_K190019</partinfo> fMT''' <br> [[Team:Groningen/Project/Accumulation#fMT|fMT]] is a metallothionein, binding Arsenite(III) and Arsenate(V), it has higher affinity for As(III). As a metallothionein it is also suitable to bind other metals like zinc, copper or cadmium. This biobrick already contains the ribosomal binding site <partinfo>B0034</partinfo>.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190019:Design More Information].'''<br />
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'''<partinfo>BBa_K190031</partinfo> fMT regulated by low constitutive promotor''' <br> <partinfo>BBa_K190019</partinfo> ([[Team:Groningen/Project/Accumulation#fMT|fMT]]) regulated by the constitutive promotor <partinfo>BBa_J23109</partinfo>.<br />
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'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190031:Design More Information].'''<br />
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'''<partinfo>BBa_K190032</partinfo> fMT regulated by pLacI''' <br><partinfo>BBa_K190019</partinfo> ([[Team:Groningen/Project/Accumulation#fMT|fMT]]) regulated by the inducible promotor <partinfo>BBa_R0010</partinfo>.<br />
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'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190032:Design More Information].'''<br />
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'''<partinfo>BBa_K190027</partinfo> MBP-ArsR (fusion protein)''' <br> The negative transcriptional regulator [[Team:Groningen/Project/Accumulation#ArsR|ArsR]] fused with Maltose Binding Protein (MBP) for higher stability. It also carries a His<sub>10</sub> tag. It was designed as an accumulator of As(III).<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190027:Design More Information].'''<br />
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===Promotors===<br />
<br />
'''<partinfo>BBa_K190015</partinfo> Arsenic Promoter''' <br> Promoter sequence with recognition site for ArsR transcriptional regulator protein. ArsR binds to the promoter sequence in the absence of As and releases on binding of As, thereby activating transcription. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190015:Design More Information].'''<br />
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'''<partinfo>BBa_K190023</partinfo> Arsenic Promoter (with wild type RBS)''' <br> Promoter sequence containing the recognition site for ArsR transcriptional regulator protein. Downstream from the promoter is the RBS that is attached to this promoter region in the ''E. coli'' genome. ArsR binds to the promoter sequence in the absence of As and releases on binding of As, thereby activating transcription.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190023:Design More Information].'''<br />
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'''<partinfo>BBa_K190016</partinfo> Zinc Promoter''' <br> Promoter sequence with recognition site for ZntR transcriptional regulator protein. ZntR activates transcription when Zn(II) is bound. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190016:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190022</partinfo> Zinc Promoter (with wild type RBS)''' <br> The pZntR from ''E. coli'' K.12 has a specific RBS site behind it in the genome. Here the RBS site is attachted to the promoter region. The RBS site might influence the activity of the promoter and will be tested in the same way as <partinfo>BBa_K190016</partinfo>. ZntR activates transcription when Zn(II) is bound.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190022:Design More Information].'''<br />
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'''<partinfo>BBa_K190017</partinfo> Copper Promoter''' <br> Promoter sequence with recognition site for the CueR transcription regulating protein. When copper levels in the cell rise CueR activates transcription. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190017:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190024</partinfo> Copper Promoter (with wild type RBS)''' <br> Promoter sequence with recognition site for the CueR transcription regulating protein and its downstream RBS (as it is found in the ''E. coli'' genome). When copper levels in the cell rise CueR activates transcription.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190024:Design More Information].'''<br />
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===GVP Constructs===<br />
<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> GVP consists of 6064 base pairs. It's Backbone is <partinfo>BBa_J61035</partinfo> With a resistance against Ampicillin/Gentamycin <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_I750016:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190025</partinfo> GVP regulated by low constitutive promotor''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the constitutive promoter <partinfo>BBa_J23109</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190025:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190026</partinfo> GVP regulated by medium constitutive promotor''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the constitutive promoter <partinfo>BBa_J23106</partinfo> were combined, and the product ligated into vector pSB1AC3. The construct was used to test and caracterize the gas vesicle formation. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190026:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190033</partinfo> GVP regulated by pArsR''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the metal sensitive promoter <partinfo>BBa_K190015</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190033:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190034</partinfo> GVP regulated by pZntR''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the metal sensitive promoter <partinfo>BBa_K190016</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190034:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190035</partinfo> GVP regulated by pCueO''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the metal sensitive promoter <partinfo>BBa_K190017</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190035:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190036</partinfo> GVP regulated by pLacI''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the LacI and CAP regulated promoter <partinfo>BBa_R0010</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190036:Design More Information].'''<br />
<br />
===Devices===<br />
<br />
'''<partinfo>BBa_K190038</partinfo> Arsenic Accumulation Device (IPTG induced)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190038:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190039</partinfo> Arsenic Accumulation Device (constitutive)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190039:Design More Information].'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Submitted_PartsTeam:Groningen/Parts/Submitted Parts2009-10-21T19:31:50Z<p>Verhoeven1981: /* GVP Constructs */</p>
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<div>{{Team:Groningen/Header}}<br />
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<br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Parts/Used_Parts}}</div><br />
<br />
<br />
<br />
==Submitted Parts==<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
===Importers===<br />
<br />
'''<partinfo>BBa_K190028</partinfo> GlpF''' <br> [[Team:Groningen/Project/Transport#GlpF|GlpF]] is an aquaglyceroporin channel that facilitates the transport of As(III). his part has been used to import As(III) into the ''E.coli'' cells. It has been used in combination with metal accumulation proteins to make a accumulation device. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190028:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190061</partinfo> GlpF (with strong RBS)''' <br> <partinfo>BBa_K190028</partinfo> [[Team:Groningen/Project/Transport#GlpF|(GlpF)]] with the ribosomal binding site <partinfo>B0034</partinfo>. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190061:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190062</partinfo> GlpF regulated by medium constitutive promotor''' <br> <partinfo>BBa_K190061</partinfo> ([[Team:Groningen/Project/Transport#GlpF|GlpF]] with strong RBS) regulated by the constitutive promotor <partinfo>BBa_J23106</partinfo>.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190062:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190063</partinfo> GlpF regulated by pLacI''' <br> <partinfo>BBa_K190061</partinfo> ([[Team:Groningen/Project/Transport#GlpF|GlpF]]with strong RBS) regulated by the inducible promotor <partinfo>BBa_R0010</partinfo><br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190063:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190018</partinfo> HmtA (contains PstI sites)''' <br> [[Team:Groningen/Project/Transport#HmtA|HmtA]] (heavy metal transporter A) from ''Pseudomonas aeruginosa'' Q9I147 is a P-type ATPase importer. This membrane protein mediates the uptake of copper (Cu) and zinc (Zn). we believe this ATP-driven pump is capable of generating an elevated intracellular concentration of these compounds enabling the harvesting of copper and zinc from the environment.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190018:Design More Information].'''<br />
<br />
===Acummulators===<br />
<br />
'''<partinfo>BBa_K190019</partinfo> fMT''' <br> [[Team:Groningen/Project/Accumulation#fMT|fMT]] is a metallothionein, binding Arsenite(III) and Arsenate(V), it has higher affinity for As(III). As a metallothionein it is also suitable to bind other metals like zinc, copper or cadmium. This biobrick already contains the ribosomal binding site <partinfo>B0034</partinfo>.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190019:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190031</partinfo> fMT regulated by low constitutive promotor''' <br> <partinfo>BBa_K190019</partinfo> ([[Team:Groningen/Project/Accumulation#fMT|fMT]]) regulated by the constitutive promotor <partinfo>BBa_J23109</partinfo>.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190031:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190032</partinfo> fMT regulated by pLacI''' <br><partinfo>BBa_K190019</partinfo> ([[Team:Groningen/Project/Accumulation#fMT|fMT]]) regulated by the inducible promotor <partinfo>BBa_R0010</partinfo>.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190032:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190027</partinfo> MBP-ArsR (fusion protein)''' <br> The negative transcriptional regulator [[Team:Groningen/Project/Accumulation#ArsR|ArsR]] fused with Maltose Binding Protein (MBP) for higher stability. It also carries a His<sub>10</sub> tag. It was designed as an accumulator of As(III).<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190027:Design More Information].'''<br />
<br />
===Promotors===<br />
<br />
'''<partinfo>BBa_K190015</partinfo> Arsenic Promoter''' <br> Promoter sequence with recognition site for ArsR transcriptional regulator protein. ArsR binds to the promoter sequence in the absence of As and releases on binding of As, thereby activating transcription. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190015:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190023</partinfo> Arsenic Promoter (with wild type RBS)''' <br> Promoter sequence containing the recognition site for ArsR transcriptional regulator protein. Downstream from the promoter is the RBS that is attached to this promoter region in the ''E. coli'' genome. ArsR binds to the promoter sequence in the absence of As and releases on binding of As, thereby activating transcription.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190023:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190016</partinfo> Zinc Promoter''' <br> Promoter sequence with recognition site for ZntR transcriptional regulator protein. ZntR activates transcription when Zn(II) is bound. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190016:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190022</partinfo> Zinc Promoter (with wild type RBS)''' <br> The pZntR from ''E. coli'' K.12 has a specific RBS site behind it in the genome. Here the RBS site is attachted to the promoter region. The RBS site might influence the activity of the promoter and will be tested in the same way as <partinfo>BBa_K190016</partinfo>. ZntR activates transcription when Zn(II) is bound.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190022:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190017</partinfo> Copper Promoter''' <br> Promoter sequence with recognition site for the CueR transcription regulating protein. When copper levels in the cell rise CueR activates transcription. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190017:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190024</partinfo> Copper Promoter (with wild type RBS)''' <br> Promoter sequence with recognition site for the CueR transcription regulating protein and its downstream RBS (as it is found in the ''E. coli'' genome). When copper levels in the cell rise CueR activates transcription.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190024:Design More Information].'''<br />
<br />
===GVP Constructs===<br />
<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> GVP consists of 6064 base pairs. It's Backbone is <partinfo>BBa_J61035</partinfo> With a resistance against Ampicillin/Gentamycin <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_I750016:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190025</partinfo> GVP regulated by low constitutive promotor''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the constitutive promoter <partinfo>BBa_J23109</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190025:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190026</partinfo> GVP regulated by medium constitutive promotor''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the constitutive promoter <partinfo>BBa_J23106</partinfo> were combined, and the product ligated into vector pSB1AC3. The construct was used to test and caracterize the gas vesicle formation. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190026:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190033</partinfo> GVP regulated by pArsR''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the metal sensitive promoter <partinfo>BBa_K190015</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190033:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190034</partinfo> GVP regulated by pZntR''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the metal sensitive promoter <partinfo>BBa_K190016</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190034:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190035</partinfo> GVP regulated by pCueO''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the metal sensitive promoter <partinfo>BBa_K190017</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190035:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190036</partinfo> GVP regulated by pLacI''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the LacI and CAP regulated promoter <partinfo>BBa_R0010</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190036:Design More Information].'''<br />
<br />
===Devices===<br />
<br />
'''<partinfo>BBa_K190038</partinfo> Arsenic Accumulation Device (IPTG induced)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190038:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190039</partinfo> Arsenic Accumulation Device (constitutive)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190039:Design More Information].'''<br />
<br />
'''((<partinfo>BBa_K190073</partinfo> Arsenic Accumulation Device (IPTG induced, able to regulate pArsR)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190073:Design More Information].'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Submitted_PartsTeam:Groningen/Parts/Submitted Parts2009-10-21T19:21:59Z<p>Verhoeven1981: /* Acummulators */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<div title="Arsie Says UP TO ACCUMULATION" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Parts/Used_Parts}}</div><br />
<br />
<br />
<br />
==Submitted Parts==<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
===Importers===<br />
<br />
'''<partinfo>BBa_K190028</partinfo> GlpF''' <br> [[Team:Groningen/Project/Transport#GlpF|GlpF]] is an aquaglyceroporin channel that facilitates the transport of As(III). his part has been used to import As(III) into the ''E.coli'' cells. It has been used in combination with metal accumulation proteins to make a accumulation device. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190028:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190061</partinfo> GlpF (with strong RBS)''' <br> <partinfo>BBa_K190028</partinfo> [[Team:Groningen/Project/Transport#GlpF|(GlpF)]] with the ribosomal binding site <partinfo>B0034</partinfo>. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190061:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190062</partinfo> GlpF regulated by medium constitutive promotor''' <br> <partinfo>BBa_K190061</partinfo> ([[Team:Groningen/Project/Transport#GlpF|GlpF]] with strong RBS) regulated by the constitutive promotor <partinfo>BBa_J23106</partinfo>.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190062:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190063</partinfo> GlpF regulated by pLacI''' <br> <partinfo>BBa_K190061</partinfo> ([[Team:Groningen/Project/Transport#GlpF|GlpF]]with strong RBS) regulated by the inducible promotor <partinfo>BBa_R0010</partinfo><br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190063:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190018</partinfo> HmtA (contains PstI sites)''' <br> [[Team:Groningen/Project/Transport#HmtA|HmtA]] (heavy metal transporter A) from ''Pseudomonas aeruginosa'' Q9I147 is a P-type ATPase importer. This membrane protein mediates the uptake of copper (Cu) and zinc (Zn). we believe this ATP-driven pump is capable of generating an elevated intracellular concentration of these compounds enabling the harvesting of copper and zinc from the environment.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190018:Design More Information].'''<br />
<br />
===Acummulators===<br />
<br />
'''<partinfo>BBa_K190019</partinfo> fMT''' <br> [[Team:Groningen/Project/Accumulation#fMT|fMT]] is a metallothionein, binding Arsenite(III) and Arsenate(V), it has higher affinity for As(III). As a metallothionein it is also suitable to bind other metals like zinc, copper or cadmium. This biobrick already contains the ribosomal binding site <partinfo>B0034</partinfo>.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190019:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190031</partinfo> fMT regulated by low constitutive promotor''' <br> <partinfo>BBa_K190019</partinfo> ([[Team:Groningen/Project/Accumulation#fMT|fMT]]) regulated by the constitutive promotor <partinfo>BBa_J23109</partinfo>.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190031:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190032</partinfo> fMT regulated by pLacI''' <br><partinfo>BBa_K190019</partinfo> ([[Team:Groningen/Project/Accumulation#fMT|fMT]]) regulated by the inducible promotor <partinfo>BBa_R0010</partinfo>.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190032:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190027</partinfo> MBP-ArsR (fusion protein)''' <br> The negative transcriptional regulator [[Team:Groningen/Project/Accumulation#ArsR|ArsR]] fused with Maltose Binding Protein (MBP) for higher stability. It also carries a His<sub>10</sub> tag. It was designed as an accumulator of As(III).<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190027:Design More Information].'''<br />
<br />
===Promotors===<br />
<br />
'''<partinfo>BBa_K190015</partinfo> Arsenic Promoter''' <br> Promoter sequence with recognition site for ArsR transcriptional regulator protein. ArsR binds to the promoter sequence in the absence of As and releases on binding of As, thereby activating transcription. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190015:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190023</partinfo> Arsenic Promoter (with wild type RBS)''' <br> Promoter sequence containing the recognition site for ArsR transcriptional regulator protein. Downstream from the promoter is the RBS that is attached to this promoter region in the ''E. coli'' genome. ArsR binds to the promoter sequence in the absence of As and releases on binding of As, thereby activating transcription.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190023:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190016</partinfo> Zinc Promoter''' <br> Promoter sequence with recognition site for ZntR transcriptional regulator protein. ZntR activates transcription when Zn(II) is bound. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190016:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190022</partinfo> Zinc Promoter (with wild type RBS)''' <br> The pZntR from ''E. coli'' K.12 has a specific RBS site behind it in the genome. Here the RBS site is attachted to the promoter region. The RBS site might influence the activity of the promoter and will be tested in the same way as <partinfo>BBa_K190016</partinfo>. ZntR activates transcription when Zn(II) is bound.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190022:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190017</partinfo> Copper Promoter''' <br> Promoter sequence with recognition site for the CueR transcription regulating protein. When copper levels in the cell rise CueR activates transcription. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190017:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190024</partinfo> Copper Promoter (with wild type RBS)''' <br> Promoter sequence with recognition site for the CueR transcription regulating protein and its downstream RBS (as it is found in the ''E. coli'' genome). When copper levels in the cell rise CueR activates transcription.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190024:Design More Information].'''<br />
<br />
===GVP Constructs===<br />
<br />
'''(<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> GVP consists of 6064 base pairs. It's Backbone is <partinfo>BBa_J61035</partinfo> With a resistance against Ampicillin/Gentamycin <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_I750016:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190025</partinfo> GVP regulated by low constitutive promotor''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the constitutive promoter <partinfo>BBa_J23109</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190025:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190026</partinfo> GVP regulated by medium constitutive promotor''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the constitutive promoter <partinfo>BBa_J23106</partinfo> were combined, and the product ligated into vector pSB1AC3. The construct was used to test and caracterize the gas vesicle formation. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190026:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190033</partinfo> GVP regulated by pArsR''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the metal sensitive promoter <partinfo>BBa_K190015</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190033:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190034</partinfo> GVP regulated by pZntR''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the metal sensitive promoter <partinfo>BBa_K190016</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190034:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190035</partinfo> GVP regulated by pCueO''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the metal sensitive promoter <partinfo>BBa_K190017</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation. <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190035:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190036</partinfo> GVP regulated by pLacI''' <br> The Gas Vesicle gene cluster (<partinfo>BBa_I750016</partinfo>) submitted by Melbourne 2007 team and the LacI and CAP regulated promoter <partinfo>BBa_R0010</partinfo> were combined, and the product ligated into vector <partinfo>pSB1AC3</partinfo>. The construct was used to test and caracterize the gas vesicle formation.<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190036:Design More Information].'''<br />
<br />
===Devices===<br />
<br />
'''<partinfo>BBa_K190038</partinfo> Arsenic Accumulation Device (IPTG induced)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190038:Design More Information].'''<br />
<br />
'''<partinfo>BBa_K190039</partinfo> Arsenic Accumulation Device (constitutive)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190039:Design More Information].'''<br />
<br />
'''((<partinfo>BBa_K190073</partinfo> Arsenic Accumulation Device (IPTG induced, able to regulate pArsR)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190073:Design More Information].'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T17:51:04Z<p>Verhoeven1981: /* Background */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<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 />
<h2>Promotors</h2><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. We take into consideration the following promoters:'''<br />
<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 />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<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. (needs a ref.)<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>Escherichia 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>Escherichia 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 straith 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 it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. 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_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (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 />
The fluorescence (and OD600) was measured as described in [[Team:Groningen/Protocols#Fluorescence_measurement| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased with a relative promoter unit of 2.3 (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). This induction of promoter activity was also found for other metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy, therefore a trendline was used to calculate the relative promoter units(RPU). <br />
<br />
[[Image:RFP over As conc2.PNG]]<br />
:Figure 3: The increase of RFP over an increased intracellular As(III) concentration. The internal arsenic concentration upon induction of cells with 100uM As(III), was calculated by extrapolating the the As(III) uptake curve (incubated 10uM As(III) over 1hr) of ''E. coli'' with pArsR-RFP (in J61002). The polynominal trendline was used to calculate the internal As concentration at the time point used for the fluorescence measurement. <br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<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 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.<br />
<br />
[[Image:Promoters-ArsR.png]]<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 conc of 5000,500,50,5 and 0 &micro;M sodium arsenite.<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>Escherichia 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 />
===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 />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T17:46:08Z<p>Verhoeven1981: /* Zinc Induced Promoters */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<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 />
<h2>Promotors</h2><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. We take into consideration the following promoters:'''<br />
<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 />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
<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. (needs a ref.)<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>Escherichia 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>Escherichia 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 straith 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 it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. 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_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (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 />
The fluorescence (and OD600) was measured as described in [[Team:Groningen/Protocols#Fluorescence_measurement| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased with a relative promoter unit of 2.3 (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). This induction of promoter activity was also found for other metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy, therefore a trendline was used to calculate the relative promoter units(RPU). <br />
<br />
[[Image:RFP over As conc2.PNG]]<br />
:Figure 3: The increase of RFP over an increased intracellular As(III) concentration. The internal arsenic concentration upon induction of cells with 100uM As(III), was calculated by extrapolating the the As(III) uptake curve (incubated 10uM As(III) over 1hr) of ''E. coli'' with pArsR-RFP (in J61002). The polynominal trendline was used to calculate the internal As concentration at the time point used for the fluorescence measurement. <br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<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 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.<br />
<br />
[[Image:Promoters-ArsR.png]]<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 conc of 5000,500,50,5 and 0 &micro;M sodium arsenite.<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>Escherichia 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 />
===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 />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T16:48:10Z<p>Verhoeven1981: /* Zinc Induced Promoters */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<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 />
<h2>Promotors</h2><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. We take into consideration the following promoters:'''<br />
<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 />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
<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. (needs a ref.)<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>Escherichia 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>Escherichia 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 straith 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 it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. 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_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (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 />
The fluorescence (and OD600) was measured as described in [[Team:Groningen/Protocols#Fluorescence_measurement| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased with a relative promoter unit of 2.3 (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). This induction of promoter activity was also found for other metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy, therefore a trendline was used to calculate the relative promoter units(RPU). <br />
<br />
[[Image:RFP over As conc2.PNG]]<br />
:Figure 3: The increase of RFP over an increased intracellular As(III) concentration. The internal arsenic concentration upon induction of cells with 100uM As(III), was calculated by extrapolating the the As(III) uptake curve (incubated 10uM As(III) over 1hr) of ''E. coli'' with pArsR-RFP (in J61002). The polynominal trendline was used to calculate the internal As concentration at the time point used for the fluorescence measurement. <br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<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 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.<br />
<br />
[[Image:Promoters-ArsR.png]]<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 conc of 5000,500,50,5 and 0 &micro;M sodium arsenite.<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>Escherichia 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 />
===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 an essential element that must be maintained at certain levels within all cells. However, like many transition<br />
elements, zinc is also harmful at elevated concentrations. Zinc starvation and zinc toxicity both lead to transcription of a number of recently characterized Escherichia coli genes that encode Zn(II) uptake or export proteins.<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. shown that it is a zinc receptor that is necessary and sufficient to stimulate Zn-responsive transcription at the zntA promoter. Biochemical, DNA footprinting, and in vitro transcription assays indicate that apo-ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to apo-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. We propose that this zinc-sensing homologue of MerR restructures the target promoter in a manner similar to that of other stress-responsive transcription factors. 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 but as yet unknown concentration.<br />
<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 />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-21T16:41:36Z<p>Verhoeven1981: /* cloning strategy */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
<br />
<br />
{| style="clear:both"<br />
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</style></html><br />
<div class="intro"><br />
<h2>Promotors</h2><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. We take into consideration the following promoters:'''<br />
<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 />
|align="center"|[[#Mercury Induced Promoters|<big>Hg</big><br>Mercury Induced Promoters]]<br />
|}<br />
</div><br />
|}<br />
<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
<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. (needs a ref.)<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>Escherichia 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>Escherichia 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 straith 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 it's sequence from the genome database of E.Coli str K12. <br />
It's binding region was established by Lee and co workers. 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_K190015}}<br />
<br />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (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 />
The fluorescence (and OD600) was measured as described in [[Team:Groningen/Protocols#Fluorescence_measurement| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased with a relative promoter unit of 2.3 (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). This induction of promoter activity was also found for other metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy, therefore a trendline was used to calculate the relative promoter units(RPU). <br />
<br />
[[Image:RFP over As conc2.PNG]]<br />
:Figure 3: The increase of RFP over an increased intracellular As(III) concentration. The internal arsenic concentration upon induction of cells with 100uM As(III), was calculated by extrapolating the the As(III) uptake curve (incubated 10uM As(III) over 1hr) of ''E. coli'' with pArsR-RFP (in J61002). The polynominal trendline was used to calculate the internal As concentration at the time point used for the fluorescence measurement. <br />
<br />
The raw data can be found at [https://2009.igem.org/Team:Groningen/Modelling/Downloads| downloads].<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 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.<br />
<br />
[[Image:Promoters-ArsR.png]]<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 conc of 5000,500,50,5 and 0 &micro;M sodium arsenite.<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>Escherichia 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 />
===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 />
====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 />
==Mercury Induced Promoters==<br />
<br />
===MerR===<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-20T04:53:32Z<p>Verhoeven1981: /* Other organisms */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
<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.'''<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
We take into consideration the following promoters:<br />
*Copper Induced Promoters<br />
*Zink Induced Promoters<br />
*Mercury Induced Promoters<br />
*Arsenic Induced Promoters<br />
<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. (needs a ref.)<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>Escherichia 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>Escherichia 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 straith 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 />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png]]<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 />
The fluorescence (and OD600) was measured as described in [[https://2009.igem.org/Team:Groningen/Protocols| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased with 2.26RPU (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2:<br />
<br />
[[Image:RFP over As conc.PNG]]<br />
:Figure 3:<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 />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<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>Escherichia 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 />
===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 />
==Zink Induced Promoters==<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn2+, Co2+, Ni2+ and Cu2+, 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 Zn2+-dependent binding of SczA to a conserved DNA motif. In the absence of Zn2+, 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 Zn2+ stress by activation of the Zn2+-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 Zn2+ and Co2+ (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-20T04:34:17Z<p>Verhoeven1981: /* E. coli */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
<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.'''<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
We take into consideration the following promoters:<br />
*Copper Induced Promoters<br />
*Zink Induced Promoters<br />
*Mercury Induced Promoters<br />
*Arsenic Induced Promoters<br />
<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. (needs a ref.)<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>Escherichia 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>Escherichia 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 straith 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 />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png]]<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 />
The fluorescence (and OD600) was measured as described in [[https://2009.igem.org/Team:Groningen/Protocols| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased with 2.26RPU (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2:<br />
<br />
[[Image:RFP over As conc.PNG]]<br />
:Figure 3:<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 />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<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), an 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>Escherichia 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 />
===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 />
==Zink Induced Promoters==<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn2+, Co2+, Ni2+ and Cu2+, 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 Zn2+-dependent binding of SczA to a conserved DNA motif. In the absence of Zn2+, 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 Zn2+ stress by activation of the Zn2+-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 Zn2+ and Co2+ (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-20T04:13:22Z<p>Verhoeven1981: </p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
<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.'''<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
We take into consideration the following promoters:<br />
*Copper Induced Promoters<br />
*Zink Induced Promoters<br />
*Mercury Induced Promoters<br />
*Arsenic Induced Promoters<br />
<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. (needs a ref.)<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>Escherichia 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>Escherichia 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 straith 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 />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png]]<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 />
The fluorescence (and OD600) was measured as described in [[https://2009.igem.org/Team:Groningen/Protocols| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased with 2.26RPU (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2:<br />
<br />
[[Image:RFP over As conc.PNG]]<br />
:Figure 3:<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 />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<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), an 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 />
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>Escherichia 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). All three have the same problem of sensing external copper instead of internal copper. The choice for CusR over CueR is 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.<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 />
==Zink Induced Promoters==<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn2+, Co2+, Ni2+ and Cu2+, 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 Zn2+-dependent binding of SczA to a conserved DNA motif. In the absence of Zn2+, 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 Zn2+ stress by activation of the Zn2+-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 Zn2+ and Co2+ (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-20T03:29:57Z<p>Verhoeven1981: /* Mercury Induced Promoters */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
<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.'''<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
We take into consideration the following promoters:<br />
*Copper Induced Promoters<br />
*Zink Induced Promoters<br />
*Mercury Induced Promoters<br />
*Arsenic Induced Promoters<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 />
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>Escherichia 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). All three have the same problem of sensing external copper instead of internal copper. The choice for CusR over CueR is 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.<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 />
==Zink Induced Promoters==<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn2+, Co2+, Ni2+ and Cu2+, 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 Zn2+-dependent binding of SczA to a conserved DNA motif. In the absence of Zn2+, 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 Zn2+ stress by activation of the Zn2+-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 Zn2+ and Co2+ (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
<br />
<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. (needs a ref.)<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>Escherichia 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>Escherichia 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 straith 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 />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png]]<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 />
The fluorescence (and OD600) was measured as described in [[https://2009.igem.org/Team:Groningen/Protocols| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased with 2.26RPU (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2:<br />
<br />
[[Image:RFP over As conc.PNG]]<br />
:Figure 3:<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), an 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 />
===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 />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-20T03:22:57Z<p>Verhoeven1981: </p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
<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.'''<br />
<br />
==Background==<br />
<br />
Metal sensitive promoters are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promoters activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
We take into consideration the following promoters:<br />
*Copper Induced Promoters<br />
*Zink Induced Promoters<br />
*Mercury Induced Promoters<br />
*Arsenic Induced Promoters<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 />
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>Escherichia 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). All three have the same problem of sensing external copper instead of internal copper. The choice for CusR over CueR is 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.<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 />
==Zink Induced Promoters==<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn2+, Co2+, Ni2+ and Cu2+, 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 Zn2+-dependent binding of SczA to a conserved DNA motif. In the absence of Zn2+, 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 Zn2+ stress by activation of the Zn2+-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 Zn2+ and Co2+ (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<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. (needs a ref.)<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>Escherichia 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>Escherichia 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 straith 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 />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png]]<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 />
The fluorescence (and OD600) was measured as described in [[https://2009.igem.org/Team:Groningen/Protocols| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased with 2.26RPU (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2:<br />
<br />
[[Image:RFP over As conc.PNG]]<br />
:Figure 3:<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), an 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 />
===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 />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-20T03:19:37Z<p>Verhoeven1981: /* Background */</p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
<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. Promotors are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promotors are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promotors varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promotors 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 promotors already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promotors, and the promotor sequence has only to be placed in front of the genes of interest.'''<br />
<br />
==Background==<br />
<br />
Metal sensitive promotors are widely used by bacteria in defence stategies against high concentrations of metals, which would have a destructive result on the cell. The promotors activate transcription of metal binding proteins to encapsule the ions, or transporters to pump the metals outside of the cell. In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
We take into consideration the following promoters:<br />
*Copper Induced Promoters<br />
*Zink Induced Promoters<br />
*Mercury Induced Promoters<br />
*Arsenic Induced Promoters<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 />
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>Escherichia 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). All three have the same problem of sensing external copper instead of internal copper. The choice for CusR over CueR is 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.<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 />
==Zink Induced Promoters==<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn2+, Co2+, Ni2+ and Cu2+, 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 Zn2+-dependent binding of SczA to a conserved DNA motif. In the absence of Zn2+, 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 Zn2+ stress by activation of the Zn2+-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 Zn2+ and Co2+ (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<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. (needs a ref.)<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>Escherichia 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>Escherichia 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 straith 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 />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png]]<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 />
The fluorescence (and OD600) was measured as described in [[https://2009.igem.org/Team:Groningen/Protocols| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased with 2.26RPU (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2:<br />
<br />
[[Image:RFP over As conc.PNG]]<br />
:Figure 3:<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), an 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 />
===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 />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Project/PromotersTeam:Groningen/Project/Promoters2009-10-20T03:13:05Z<p>Verhoeven1981: </p>
<hr />
<div>{{Team:Groningen/Project/Header|}}<br />
<br />
<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. Promotors are used to express genes of interest in cells in either a constitutive or induced manner. The constitutive promotors are used when a constant expression of enzymes is desired, and the amount of activity can be regulated by choosing from a range of promotors varying from low to high expression. If, however, expression is desired at certain points in time, or growth stage, inducible promotors 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 promotors already present in ''E. coli'' cells, the cells contain the necessary components for controlling the promotors, and the promotor sequence has only to be placed in front of the genes of interest.'''<br />
<br />
==Background==<br />
<br />
In order to find different promoters to induce genes in the presence of different heavy metals we used the following list of databases and sites:<br />
{|<br />
|<br />
# [http://www.genome.jp/kegg/kegg2.html KEGG]<br />
# [http://www.ncbi.nlm.nih.gov NCBI]<br />
# [http://regtransbase.lbl.gov Regtransbase]<br />
|}<br />
<br />
We take into consideration the following promoters:<br />
*Copper Induced Promoters<br />
*Zink Induced Promoters<br />
*Mercury Induced Promoters<br />
*Arsenic Induced Promoters<br />
<br />
<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 />
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>Escherichia 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). All three have the same problem of sensing external copper instead of internal copper. The choice for CusR over CueR is 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.<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 />
==Zink Induced Promoters==<br />
<br />
====Other organisms====<br />
''Bacillus subtilis''<br />
<br />
'''Abs.''': The ''Bacillus subtilis'' cation efflux pump czcD, which mediates resistance against Zn2+, Co2+, Ni2+ and Cu2+, 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 Zn2+-dependent binding of SczA to a conserved DNA motif. In the absence of Zn2+, 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 Zn2+ stress by activation of the Zn2+-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 Zn2+ and Co2+ (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).<br />
<br />
==Mercury Induced Promoters==<br />
<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. (needs a ref.)<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>Escherichia 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>Escherichia 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 straith 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 />
====Results====<br />
The functionality of pArsR was tested by using a test construct, composed of pArsR and RFP (Figure 1).<br />
<br />
[[Image:Promoter measurement device.png]]<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 />
The fluorescence (and OD600) was measured as described in [[https://2009.igem.org/Team:Groningen/Protocols| protocols]]. Upon induction of the ArsR promoter the expression of RFP increased with 2.26RPU (calculated according to formula 9 as described by [[Team:Groningen/Literature#Kelly2009|Kelly 2009]]). The increase in fluorescence over time is shown in figure 2 and the fluorescence change due to a change in the internal as(III) concentration in figure 3. <br />
<br />
[[Image:Fluorescence over time.PNG]]<br />
:Figure 2:<br />
<br />
[[Image:RFP over As conc.PNG]]<br />
:Figure 3:<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), an 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 />
===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 />
|{{graph|Team:Groningen/Graphs/PromoterActivationNP|promoterActivitationGraphNP}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation|promoterActivitationGraph}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationG|promoterActivitationGraphG}}<br />
|-<br />
|{{graph|Team:Groningen/Graphs/PromoterActivationSlow|promoterActivitationGraphS}}<br />
|{{graph|Team:Groningen/Graphs/PromoterActivation2|promoterActivitationGraph2}}<br />
|}<br />
<br />
<div title="Arsie Says UP TO GAS VESICLES" style="float:right" >{{linkedImage|Next.JPG|Team:Groningen/Project/Vesicle|}}</div><br />
{{Team:Groningen/Project/Footer}}</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-18T06:59:18Z<p>Verhoeven1981: /* Miscellaneous */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Used Parts==<br />
{{todo}}Maybe some general information of parts used from the registry.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===Miscellaneous===<br />
'''<partinfo>BBa_B0034</partinfo> RBS ((Elowitz 1999) -- defines RBS efficiency)''' <br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_K190061 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ligation of part <partinfo>BBa_K190028</partinfo> behind the RBS was successful, confirmed by gel (correct vector size after digestion with EcoRI and PstI) and sequencing with VF2 primer. We used this part in combination with several biobricks for building our constructs e.g. <partinfo>BBa_K190061</partinfo>.<br />
|}<br />
<br />
'''<partinfo>BBa_B0014</partinfo> Double terminator (B0012-B0011)''' <br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_B0014 AddReview 3</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The plasmid containing part <partinfo>BBa_B0014</partinfo> was successfully transformed into ''E. coli'' TOP10 cells (confirmed by single and double digestion). The original plan was to use the terminator behind one of our own designed parts <partinfo>BBa_K190027</partinfo>, and in front of pArsR-RFP. The MBP-ArsR fusion protein was thought to have a regulating effect on the arsenic promotor pArsR in the same way as ArsR regulates the promotor. The terminator separated the two parts on the plasmid. The construct was not used due to time constraints, and not sequenced.<br />
|}<br />
<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_I750016 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
Part <partinfo>BBa_I750016</partinfo> was one of our main focus points during our project. The part was originally submitted by Melbourne in 2007 with a very limited amount of information (short description and sequence mutations). On their site it was mentioned the cluster was difficult to use and ligation into a plasmid was hard, in contrast we found the cluster to be easily cut and ligated into different plasmids and behind several promotors. We used this part in combination with several biobricks for building our constructs e.g. <partinfo>BBa_K190033</partinfo> and <partinfo>BBa_K190036</partinfo>. Pictures of our cells with induced gas vesicle formation confirmed the production of vesicles in the expected shape and size. In addition, we characterized the part to improve the use in the future and added a lot of information on the registry.<br />
|}<br />
<br />
'''<partinfo>BBa_P1010</partinfo> ccdB cell death gene'''<br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_P1010 AddReview 4</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
P1010 is used when putting BioBrick parts into BioBrick plasmids. The part to be inserted and the plasmid are cut with BioBrick enzymes and mixed. The mixture will include both the original uncut or self ligated plasmid and the desired structure. However, because of CcdB, all of the cells containing the original plasmid die and the surviving colonies are the desired result. The ccdB cell death gene worked as expected killing our ''E. coli'' TOP10 cells, and keeping our ''E. coli'' DB3 cells alive. After noticing the inconsistent sequencing result for the pSB2K3 plasmid with ccdB cell death gene, we decided to choose a different pSB2K3 plasmid with random part to continue with our assemblies. This to minimize the chance of unwanted surprises in the end.<br />
|}<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> Constitutive promoter family member (high expression)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> <br />
<br />
'''<partinfo>J23101</partinfo> Constitutive promoter family member (high expression, reference)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> <br />
<br />
'''<partinfo>J23106</partinfo> Constitutive promoter family member (medium expression)''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> <br />
<br />
'''<partinfo>J23109</partinfo> Constitutive promoter family member (low expression)''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> <br />
<br />
'''<partinfo>R0010</partinfo> Promoter (lacI regulated)''' <br> This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first binds the CAP protein, which is generally present in E.coli and is asocciated with cell health and availability of glucose. The second binds LacI protein. <br> <br />
<br />
'''<partinfo>I0500</partinfo> pBad/araC''' <br> This part is <br> <br />
<br />
===Vectors===<br />
'''<partinfo>pSB1AC3</partinfo> High copy BioBrick assembly plasmid'''<br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
pSB1AC3 is a high copy number plasmid carrying ampicillin and chloramphenicol resistance. Together with pSB1A2 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. The transformations with pSB1AC3 (containing different biobricks of own design) into ''E. coli'' TOP10 cells, growth on both antibiotics, and gel analysis (undigested and digested with the EcoRI and PstI) worked as expected. The high (copy) number of plasmids per cell make it an easy to work with plasmid, ideal for cloning and assembly work.<br />
|}<br />
<br />
'''<partinfo>pSB1A2</partinfo> pSB1A2 (Replaced by pSB1A3 in registry) ''' <br> pSB1A2 is a high copy number plasmid carrying ampicillin resistance. Together with pSB1AC3 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br><br />
<br />
'''<partinfo>pSB2K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br><br />
<br />
'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br><br />
<br />
'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a rsistance against Ampicillin <br><br />
<br />
'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br></div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-18T06:31:55Z<p>Verhoeven1981: /* Miscellaneous */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Used Parts==<br />
{{todo}}Maybe some general information of parts used from the registry.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===Miscellaneous===<br />
'''<partinfo>BBa_B0034</partinfo> RBS ((Elowitz 1999) -- defines RBS efficiency)''' <br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_K190061 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ligation of part <partinfo>BBa_K190028</partinfo> behind the RBS was successful, confirmed by gel (correct vector size after digestion with EcoRI and PstI) and sequencing with VF2 primer. We used this part in combination with several genes for building our biobricks e.g. <partinfo>BBa_K190061</partinfo>.<br />
|}<br />
<br />
'''<partinfo>BBa_B0014</partinfo> Double terminator (B0012-B0011)''' <br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_B0014 AddReview 3</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The plasmid containing part <partinfo>BBa_B0014</partinfo> was successfully transformed into ''E. coli'' TOP10 cells (confirmed by single and double digestion). The original plan was to use the terminator behind one of our own designed parts <partinfo>BBa_K190027</partinfo>, and in front of pArsR-RFP. The MBP-ArsR fusion protein was thought to have a regulating effect on the arsenic promotor pArsR in the same way as ArsR regulates the promotor. The terminator separated the two parts on the plasmid. The construct was not used due to time constraints, and not sequenced.<br />
|}<br />
<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_I750016 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
We used this part in combination with several genes for building our biobricks e.g. <partinfo>BBa_K190061</partinfo>.<br />
|}<br />
<br />
'''<partinfo>BBa_P1010</partinfo> ccdB cell death gene'''<br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_P1010 AddReview 4</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
P1010 is used when putting BioBrick parts into BioBrick plasmids. The part to be inserted and the plasmid are cut with BioBrick enzymes and mixed. The mixture will include both the original uncut or self ligated plasmid and the desired structure. However, because of CcdB, all of the cells containing the original plasmid die and the surviving colonies are the desired result. The ccdB cell death gene worked as expected killing our ''E. coli'' TOP10 cells, and keeping our ''E. coli'' DB3 cells alive. After noticing the inconsistent sequencing result for the pSB2K3 plasmid with ccdB cell death gene, we decided to choose a different pSB2K3 plasmid with random part to continue with our assemblies. This to minimize the chance of unwanted surprises in the end.<br />
|}<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> Constitutive promoter family member (high expression)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> <br />
<br />
'''<partinfo>J23101</partinfo> Constitutive promoter family member (high expression, reference)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> <br />
<br />
'''<partinfo>J23106</partinfo> Constitutive promoter family member (medium expression)''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> <br />
<br />
'''<partinfo>J23109</partinfo> Constitutive promoter family member (low expression)''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> <br />
<br />
'''<partinfo>R0010</partinfo> Promoter (lacI regulated)''' <br> This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first binds the CAP protein, which is generally present in E.coli and is asocciated with cell health and availability of glucose. The second binds LacI protein. <br> <br />
<br />
'''<partinfo>I0500</partinfo> pBad/araC''' <br> This part is <br> <br />
<br />
===Vectors===<br />
'''<partinfo>pSB1AC3</partinfo> High copy BioBrick assembly plasmid'''<br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
pSB1AC3 is a high copy number plasmid carrying ampicillin and chloramphenicol resistance. Together with pSB1A2 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. The transformations with pSB1AC3 (containing different biobricks of own design) into ''E. coli'' TOP10 cells, growth on both antibiotics, and gel analysis (undigested and digested with the EcoRI and PstI) worked as expected. The high (copy) number of plasmids per cell make it an easy to work with plasmid, ideal for cloning and assembly work.<br />
|}<br />
<br />
'''<partinfo>pSB1A2</partinfo> pSB1A2 (Replaced by pSB1A3 in registry) ''' <br> pSB1A2 is a high copy number plasmid carrying ampicillin resistance. Together with pSB1AC3 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br><br />
<br />
'''<partinfo>pSB2K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br><br />
<br />
'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br><br />
<br />
'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a rsistance against Ampicillin <br><br />
<br />
'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br></div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-17T17:18:19Z<p>Verhoeven1981: /* Used Parts */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Used Parts==<br />
{{todo}}Maybe some general information of parts used from the registry.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===Miscellaneous===<br />
'''<partinfo>BBa_B0034</partinfo> RBS ((Elowitz 1999) -- defines RBS efficiency)''' <br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ligation of a part behind the RBS succeded, and was confirmed by gel (correct vector size) and sequencing with VF2. We used this part in combination with several genes for building our biobricks e.g. <partinfo>BBa_K190061</partinfo>.<br />
|}<br />
<br />
'''<partinfo>BBa_B0014</partinfo> Double terminator (B0012-B0011)''' <br> Double terminator consisting of BBa_B0012 and BBa_B0011 <br><br />
<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> This parts creates gas vesicles inside the cell wich enables it to float. It consist of 6064 base pairs. It's backbone is <partinfo>BBa_J61035</partinfo> <br><br />
<br />
'''<partinfo>BBa_P1010</partinfo> ccdB cell death gene'''<br />
<br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 3</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
P1010 is used when putting BioBrick parts into BioBrick plasmids. The part to be inserted and the plasmid are cut with BioBrick enzymes and mixed. The mixture will include both the original uncut or religated plasmid and the desired structure. However, because of CcdB, all of the cells containing the original plasmid die and the surviving colonies are the desired result. The ccdB cell death gene worked as expected killing our ''E. coli'' TOP10 cells, and keeping our ''E. coli'' DB3 cells alive. After noticing the inconsistent sequencing result for the pSB2K3 plasmid with ccdB cell death gene, we decided to choose a different pSB2K3 plasmid with random part to continue with our assemblies. This to minimize the chance of unwanted surprises in the end.<br />
|}<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> Constitutive promoter family member (high expression)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> <br />
<br />
'''<partinfo>J23101</partinfo> Constitutive promoter family member (high expression, reference)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> <br />
<br />
'''<partinfo>J23106</partinfo> Constitutive promoter family member (medium expression)''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> <br />
<br />
'''<partinfo>J23109</partinfo> Constitutive promoter family member (low expression)''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> <br />
<br />
'''<partinfo>R0010</partinfo> Promoter (lacI regulated)''' <br> This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first binds the CAP protein, which is generally present in E.coli and is asocciated with cell health and availability of glucose. The second binds LacI protein. <br> <br />
<br />
'''<partinfo>I0500</partinfo> pBad/araC''' <br> This part is <br> <br />
<br />
===Vectors===<br />
'''<partinfo>pSB1AC3</partinfo> High copy BioBrick assembly plasmid'''<br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
pSB1AC3 is a high copy number plasmid carrying ampicillin and chloramphenicol resistance. Together with pSB1A2 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. The transformations with pSB1AC3 (containing different biobricks of own design) into ''E. coli'' TOP10 cells, growth on both antibiotics, and gel analysis (undigested and digested with the EcoRI and PstI) worked as expected. The high (copy) number of plasmids per cell make it an easy to work with plasmid, ideal for cloning and assembly work.<br />
|}<br />
<br />
'''<partinfo>pSB1A2</partinfo> pSB1A2 (Replaced by pSB1A3 in registry) ''' <br> pSB1A2 is a high copy number plasmid carrying ampicillin resistance. Together with pSB1AC3 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br><br />
<br />
'''<partinfo>pSB2K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br><br />
<br />
'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br><br />
<br />
'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a rsistance against Ampicillin <br><br />
<br />
'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br></div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-17T17:09:41Z<p>Verhoeven1981: /* Vectors */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Used Parts==<br />
{{todo}}Maybe some general information of parts used from the registry.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===Miscellaneous===<br />
'''<partinfo>BBa_B0034</partinfo> RBS ((Elowitz 1999) -- defines RBS efficiency)''' <br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ligation of a part behind the RBS succeded, and was confirmed by gel (correct vector size) and sequencing with VF2. We used this part in combination with several genes for building our biobricks e.g. <partinfo>BBa_K190061</partinfo>.<br />
|}<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_B0034:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_B0014</partinfo> Double terminator (B0012-B0011)''' <br> Double terminator consisting of BBa_B0012 and BBa_B0011 <br><br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_B0014:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> This parts creates gas vesicles inside the cell wich enables it to float. It consist of 6064 base pairs. It's backbone is <partinfo>BBa_J61035</partinfo> <br><br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_I750016:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_P1010</partinfo> ccdB cell death gene''' <br> P1010 is used when putting BioBrick parts into BioBrick plasmids. The part to be inserted and the plasmid are cut with BioBrick enzymes and mixed. The mixture will include both the original uncut or religated plasmid and the desired structure. However, because of CcdB, all of the cells containing the original plasmid die and the surviving colonies are the desired result. <br><br />
<br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 3</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ccdB cell death gene worked as expected killing our ''E. coli'' TOP10 cells, and keeping our ''E. coli'' DB3 cells alive. After noticing the inconsistent sequencing result for the pSB2K3 plasmid with ccdB cell death gene, we decided to choose a different pSB2K3 plasmid with random part to continue with our assemblies. This to minimize the chance of unwanted surprises in the end.<br />
|}<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190028:Design| More Information].'''<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> Constitutive promoter family member (high expression)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[http://partsregistry.org/Part:BBa_J23100:Design| More Information]'''<br />
<br />
'''<partinfo>J23101</partinfo> Constitutive promoter family member (high expression, reference)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[http://partsregistry.org/Part:BBa_J23101:Design| More Information]'''<br />
<br />
'''<partinfo>J23106</partinfo> Constitutive promoter family member (medium expression)''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[http://partsregistry.org/Part:BBa_J23106:Design| More Information]'''<br />
<br />
'''<partinfo>J23109</partinfo> Constitutive promoter family member (low expression)''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> '''[http://partsregistry.org/Part:BBa_J23109:Design| More Information]'''<br />
<br />
'''<partinfo>R0010</partinfo> Promoter (lacI regulated)''' <br> This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first binds the CAP protein, which is generally present in E.coli and is asocciated with cell health and availability of glucose. The second binds LacI protein. <br> '''[http://partsregistry.org/Part:BBa_R0010:Design| More information]'''<br />
<br />
'''<partinfo>I0500</partinfo> pBad/araC''' <br> This part is <br> '''[http://partsregistry.org/Part:BBa_I0500:Design| More information]'''<br />
<br />
===Vectors===<br />
'''<partinfo>pSB1AC3</partinfo> High copy BioBrick assembly plasmid''' <br> pSB1AC3 is a high copy number plasmid carrying ampicillin and chloramphenicol resistance. Together with pSB1A2 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br><br />
<br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The transformations with pSB1AC3 (containing different biobricks of own design) into ''E. coli'' TOP10 cells, growth on both antibiotics, and gel analysis (undigested and digested with the EcoRI and PstI) worked as expected. The high (copy) number of plasmids per cell make it an easy to work with plasmid, ideal for cloning and assembly work.<br />
|}<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190028:Design| More Information].'''<br />
<br />
--<br />
<br />
'''<partinfo>pSB1A2</partinfo> pSB1A2 (Replaced by pSB1A3 in registry) ''' <br> pSB1A2 is a high copy number plasmid carrying ampicillin resistance. Together with pSB1AC3 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br>'''[http://partsregistry.org/Part:pSB1A2:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>pSB2K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[http://partsregistry.org/Part:pSB2K3:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[http://partsregistry.org/Part:pSB3K3:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a rsistance against Ampicillin <br>'''[http://partsregistry.org/Part:J61002:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61035|More information]]'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-17T15:29:07Z<p>Verhoeven1981: /* Miscellaneous */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Used Parts==<br />
{{todo}}Maybe some general information of parts used from the registry.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===Miscellaneous===<br />
'''<partinfo>BBa_B0034</partinfo> RBS ((Elowitz 1999) -- defines RBS efficiency)''' <br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ligation of a part behind the RBS succeded, and was confirmed by gel (correct vector size) and sequencing with VF2. We used this part in combination with several genes for building our biobricks e.g. <partinfo>BBa_K190061</partinfo>.<br />
|}<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_B0034:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_B0014</partinfo> Double terminator (B0012-B0011)''' <br> Double terminator consisting of BBa_B0012 and BBa_B0011 <br><br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_B0014:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> This parts creates gas vesicles inside the cell wich enables it to float. It consist of 6064 base pairs. It's backbone is <partinfo>BBa_J61035</partinfo> <br><br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_I750016:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_P1010</partinfo> ccdB cell death gene''' <br> P1010 is used when putting BioBrick parts into BioBrick plasmids. The part to be inserted and the plasmid are cut with BioBrick enzymes and mixed. The mixture will include both the original uncut or religated plasmid and the desired structure. However, because of CcdB, all of the cells containing the original plasmid die and the surviving colonies are the desired result. <br><br />
<br />
{|<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 3</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ccdB cell death gene worked as expected killing our ''E. coli'' TOP10 cells, and keeping our ''E. coli'' DB3 cells alive. After noticing the inconsistent sequencing result for the pSB2K3 plasmid with ccdB cell death gene, we decided to choose a different pSB2K3 plasmid with random part to continue with our assemblies. This to minimize the chance of unwanted surprises in the end.<br />
|}<br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190028:Design| More Information].'''<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> Constitutive promoter family member (high expression)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[http://partsregistry.org/Part:BBa_J23100:Design| More Information]'''<br />
<br />
'''<partinfo>J23101</partinfo> Constitutive promoter family member (high expression, reference)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[http://partsregistry.org/Part:BBa_J23101:Design| More Information]'''<br />
<br />
'''<partinfo>J23106</partinfo> Constitutive promoter family member (medium expression)''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[http://partsregistry.org/Part:BBa_J23106:Design| More Information]'''<br />
<br />
'''<partinfo>J23109</partinfo> Constitutive promoter family member (low expression)''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> '''[http://partsregistry.org/Part:BBa_J23109:Design| More Information]'''<br />
<br />
'''<partinfo>R0010</partinfo> Promoter (lacI regulated)''' <br> This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first binds the CAP protein, which is generally present in E.coli and is asocciated with cell health and availability of glucose. The second binds LacI protein. <br> '''[http://partsregistry.org/Part:BBa_R0010:Design| More information]'''<br />
<br />
'''<partinfo>I0500</partinfo> pBad/araC''' <br> This part is <br> '''[http://partsregistry.org/Part:BBa_I0500:Design| More information]'''<br />
<br />
===Vectors===<br />
'''<partinfo>pSB1AC3</partinfo> High copy BioBrick assembly plasmid''' <br> pSB1AC3 is a high copy number plasmid carrying ampicillin and chloramphenicol resistance. Together with pSB1A2 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br>'''[http://partsregistry.org/Part:pSB1AC3:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>pSB1A2</partinfo> pSB1A2 (Replaced by pSB1A3 in registry) ''' <br> pSB1A2 is a high copy number plasmid carrying ampicillin resistance. Together with pSB1AC3 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br>'''[http://partsregistry.org/Part:pSB1A2:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>pSB2K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[http://partsregistry.org/Part:pSB2K3:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[http://partsregistry.org/Part:pSB3K3:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a rsistance against Ampicillin <br>'''[http://partsregistry.org/Part:J61002:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61035|More information]]'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-17T15:10:56Z<p>Verhoeven1981: /* Miscellaneous */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Used Parts==<br />
{{todo}}Maybe some general information of parts used from the registry.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===Miscellaneous===<br />
'''<partinfo>BBa_B0034</partinfo> RBS ((Elowitz 1999) -- defines RBS efficiency)''' <br />
{|<br />
|-<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ligation of a part behind the RBS succeded, and was confirmed by gel (correct vector size) and sequencing with VF2. We used this part in combination with several genes for building our biobricks e.g. <partinfo>BBa_K190061</partinfo>.<br />
|}<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_B0034:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_B0014</partinfo> Double terminator (B0012-B0011)''' <br> Double terminator consisting of BBa_B0012 and BBa_B0011 <br><br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_B0014:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> This parts creates gas vesicles inside the cell wich enables it to float. It consist of 6064 base pairs. It's backbone is <partinfo>BBa_J61035</partinfo> <br><br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_I750016:Design| More Information].'''<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> Constitutive promoter family member (high expression)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[http://partsregistry.org/Part:BBa_J23100:Design| More Information]'''<br />
<br />
'''<partinfo>J23101</partinfo> Constitutive promoter family member (high expression, reference)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[http://partsregistry.org/Part:BBa_J23101:Design| More Information]'''<br />
<br />
'''<partinfo>J23106</partinfo> Constitutive promoter family member (medium expression)''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[http://partsregistry.org/Part:BBa_J23106:Design| More Information]'''<br />
<br />
'''<partinfo>J23109</partinfo> Constitutive promoter family member (low expression)''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> '''[http://partsregistry.org/Part:BBa_J23109:Design| More Information]'''<br />
<br />
'''<partinfo>R0010</partinfo> Promoter (lacI regulated)''' <br> This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first binds the CAP protein, which is generally present in E.coli and is asocciated with cell health and availability of glucose. The second binds LacI protein. <br> '''[http://partsregistry.org/Part:BBa_R0010:Design| More information]'''<br />
<br />
'''<partinfo>I0500</partinfo> pBad/araC''' <br> This part is <br> '''[http://partsregistry.org/Part:BBa_I0500:Design| More information]'''<br />
<br />
===Vectors===<br />
'''<partinfo>pSB1AC3</partinfo> High copy BioBrick assembly plasmid''' <br> pSB1AC3 is a high copy number plasmid carrying ampicillin and chloramphenicol resistance. Together with pSB1A2 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br>'''[http://partsregistry.org/Part:pSB1AC3:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>pSB1A2</partinfo> pSB1A2 (Replaced by pSB1A3 in registry) ''' <br> pSB1A2 is a high copy number plasmid carrying ampicillin resistance. Together with pSB1AC3 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br>'''[http://partsregistry.org/Part:pSB1A2:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>pSB2K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[http://partsregistry.org/Part:pSB2K3:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[http://partsregistry.org/Part:pSB3K3:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a rsistance against Ampicillin <br>'''[http://partsregistry.org/Part:J61002:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61035|More information]]'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Submitted_PartsTeam:Groningen/Parts/Submitted Parts2009-10-17T15:04:00Z<p>Verhoeven1981: /* Devices */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Submitted Parts==<br />
{{todo}}Maybe some general information about our submitted parts.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
===Importers===<br />
<br />
'''<partinfo>BBa_K190028</partinfo> GlpF''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190028:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190061</partinfo> GlpF (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190061:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190062</partinfo> GlpF regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190062:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190063</partinfo> GlpF regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190063:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190018</partinfo> HmtA (contains PstI sites)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190018:Design| More Information].'''<br />
<br />
===Acummulators===<br />
<br />
'''<partinfo>BBa_K190019</partinfo> fMT''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190019:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190031</partinfo> fMT regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190031:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190032</partinfo> fMT regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190032:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190027</partinfo> MBP-ArsR (fusion protein)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190027:Design| More Information].'''<br />
<br />
'''(<partinfo>BBa_K190071</partinfo> MBP-ArsR (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190071:Design| More Information].'''<br />
<br />
'''(<partinfo>BBa_K190072</partinfo> MBP-ArsR regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190072:Design| More Information].'''<br />
<br />
===Promotors===<br />
<br />
'''<partinfo>BBa_K190015</partinfo> Arsenic Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190015:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190023</partinfo> Arsenic Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190023:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190016</partinfo> Zinc Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190016:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190022</partinfo> Zinc Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190022:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190017</partinfo> Copper Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190017:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190024</partinfo> Copper Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190024:Design| More Information].'''<br />
<br />
===GVP Constructs===<br />
<br />
'''(<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> GVP consists of 6064 base pairs. It's Backbone is <partinfo>BBa_J61035</partinfo> With a resistance against Ampicillin/Gentamycin <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_I750016:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190025</partinfo> GVP regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190025:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190026</partinfo> GVP regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190026:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190033</partinfo> GVP regulated by pArsR''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190033:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190034</partinfo> GVP regulated by pZntR''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190034:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190035</partinfo> GVP regulated by pCueO''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190035:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190036</partinfo> GVP regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190036:Design| More Information].'''<br />
<br />
===Devices===<br />
<br />
'''<partinfo>BBa_K190038</partinfo> Arsenic Accumulation Device (IPTG induced)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190038:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190039</partinfo> Arsenic Accumulation Device (constitutive)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190039:Design| More Information].'''<br />
<br />
'''((<partinfo>BBa_K190073</partinfo> Arsenic Accumulation Device (IPTG induced, able to regulate pArsR)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190073:Design| More Information].'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Submitted_PartsTeam:Groningen/Parts/Submitted Parts2009-10-17T15:02:29Z<p>Verhoeven1981: /* GVP Constructs */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Submitted Parts==<br />
{{todo}}Maybe some general information about our submitted parts.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
===Importers===<br />
<br />
'''<partinfo>BBa_K190028</partinfo> GlpF''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190028:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190061</partinfo> GlpF (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190061:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190062</partinfo> GlpF regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190062:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190063</partinfo> GlpF regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190063:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190018</partinfo> HmtA (contains PstI sites)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190018:Design| More Information].'''<br />
<br />
===Acummulators===<br />
<br />
'''<partinfo>BBa_K190019</partinfo> fMT''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190019:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190031</partinfo> fMT regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190031:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190032</partinfo> fMT regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190032:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190027</partinfo> MBP-ArsR (fusion protein)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190027:Design| More Information].'''<br />
<br />
'''(<partinfo>BBa_K190071</partinfo> MBP-ArsR (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190071:Design| More Information].'''<br />
<br />
'''(<partinfo>BBa_K190072</partinfo> MBP-ArsR regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190072:Design| More Information].'''<br />
<br />
===Promotors===<br />
<br />
'''<partinfo>BBa_K190015</partinfo> Arsenic Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190015:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190023</partinfo> Arsenic Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190023:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190016</partinfo> Zinc Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190016:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190022</partinfo> Zinc Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190022:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190017</partinfo> Copper Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190017:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190024</partinfo> Copper Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190024:Design| More Information].'''<br />
<br />
===GVP Constructs===<br />
<br />
'''(<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> GVP consists of 6064 base pairs. It's Backbone is <partinfo>BBa_J61035</partinfo> With a resistance against Ampicillin/Gentamycin <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_I750016:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190025</partinfo> GVP regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190025:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190026</partinfo> GVP regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190026:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190033</partinfo> GVP regulated by pArsR''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190033:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190034</partinfo> GVP regulated by pZntR''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190034:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190035</partinfo> GVP regulated by pCueO''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190035:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190036</partinfo> GVP regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190036:Design| More Information].'''<br />
<br />
===Devices===<br />
<br />
'''<partinfo>BBa_K190038</partinfo> Arsenic Accumulation Device (IPTG induced)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190039</partinfo> Arsenic Accumulation Device (constitutive)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''((<partinfo>BBa_K190073</partinfo> Arsenic Accumulation Device (IPTG induced, able to regulate pArsR)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]))'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Submitted_PartsTeam:Groningen/Parts/Submitted Parts2009-10-17T14:59:40Z<p>Verhoeven1981: /* Promotors */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Submitted Parts==<br />
{{todo}}Maybe some general information about our submitted parts.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
===Importers===<br />
<br />
'''<partinfo>BBa_K190028</partinfo> GlpF''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190028:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190061</partinfo> GlpF (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190061:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190062</partinfo> GlpF regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190062:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190063</partinfo> GlpF regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190063:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190018</partinfo> HmtA (contains PstI sites)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190018:Design| More Information].'''<br />
<br />
===Acummulators===<br />
<br />
'''<partinfo>BBa_K190019</partinfo> fMT''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190019:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190031</partinfo> fMT regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190031:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190032</partinfo> fMT regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190032:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190027</partinfo> MBP-ArsR (fusion protein)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190027:Design| More Information].'''<br />
<br />
'''(<partinfo>BBa_K190071</partinfo> MBP-ArsR (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190071:Design| More Information].'''<br />
<br />
'''(<partinfo>BBa_K190072</partinfo> MBP-ArsR regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190072:Design| More Information].'''<br />
<br />
===Promotors===<br />
<br />
'''<partinfo>BBa_K190015</partinfo> Arsenic Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190015:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190023</partinfo> Arsenic Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190023:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190016</partinfo> Zinc Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190016:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190022</partinfo> Zinc Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190022:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190017</partinfo> Copper Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190017:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190024</partinfo> Copper Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190024:Design| More Information].'''<br />
<br />
===GVP Constructs===<br />
<br />
'''(<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> GVP consists of 6064 base pairs. It's Backbone is <partinfo>BBa_J61035</partinfo> With a resistance against Ampicillin/Gentamycin '''[[Team Groningen:Dummielink|More information]])'''<br />
<br />
'''<partinfo>BBa_K190025</partinfo> GVP regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190026</partinfo> GVP regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190033</partinfo> GVP regulated by pArsR''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190034</partinfo> GVP regulated by pZntR''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190035</partinfo> GVP regulated by pCueO''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190036</partinfo> GVP regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Devices===<br />
<br />
'''<partinfo>BBa_K190038</partinfo> Arsenic Accumulation Device (IPTG induced)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190039</partinfo> Arsenic Accumulation Device (constitutive)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''((<partinfo>BBa_K190073</partinfo> Arsenic Accumulation Device (IPTG induced, able to regulate pArsR)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]))'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Submitted_PartsTeam:Groningen/Parts/Submitted Parts2009-10-17T14:57:37Z<p>Verhoeven1981: /* Acummulators */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Submitted Parts==<br />
{{todo}}Maybe some general information about our submitted parts.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
===Importers===<br />
<br />
'''<partinfo>BBa_K190028</partinfo> GlpF''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190028:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190061</partinfo> GlpF (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190061:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190062</partinfo> GlpF regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190062:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190063</partinfo> GlpF regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190063:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190018</partinfo> HmtA (contains PstI sites)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190018:Design| More Information].'''<br />
<br />
===Acummulators===<br />
<br />
'''<partinfo>BBa_K190019</partinfo> fMT''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190019:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190031</partinfo> fMT regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190031:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190032</partinfo> fMT regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190032:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190027</partinfo> MBP-ArsR (fusion protein)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190027:Design| More Information].'''<br />
<br />
'''(<partinfo>BBa_K190071</partinfo> MBP-ArsR (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190071:Design| More Information].'''<br />
<br />
'''(<partinfo>BBa_K190072</partinfo> MBP-ArsR regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190072:Design| More Information].'''<br />
<br />
===Promotors===<br />
<br />
'''<partinfo>BBa_K190015</partinfo> Arsenic Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190023</partinfo> Arsenic Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190016</partinfo> Zinc Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190022</partinfo> Zinc Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190017</partinfo> Copper Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190024</partinfo> Copper Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===GVP Constructs===<br />
<br />
'''(<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> GVP consists of 6064 base pairs. It's Backbone is <partinfo>BBa_J61035</partinfo> With a resistance against Ampicillin/Gentamycin '''[[Team Groningen:Dummielink|More information]])'''<br />
<br />
'''<partinfo>BBa_K190025</partinfo> GVP regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190026</partinfo> GVP regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190033</partinfo> GVP regulated by pArsR''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190034</partinfo> GVP regulated by pZntR''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190035</partinfo> GVP regulated by pCueO''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190036</partinfo> GVP regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Devices===<br />
<br />
'''<partinfo>BBa_K190038</partinfo> Arsenic Accumulation Device (IPTG induced)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190039</partinfo> Arsenic Accumulation Device (constitutive)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''((<partinfo>BBa_K190073</partinfo> Arsenic Accumulation Device (IPTG induced, able to regulate pArsR)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]))'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Submitted_PartsTeam:Groningen/Parts/Submitted Parts2009-10-17T14:54:23Z<p>Verhoeven1981: /* Importers */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Submitted Parts==<br />
{{todo}}Maybe some general information about our submitted parts.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
===Importers===<br />
<br />
'''<partinfo>BBa_K190028</partinfo> GlpF''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190028:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190061</partinfo> GlpF (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190061:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190062</partinfo> GlpF regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190062:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190063</partinfo> GlpF regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190063:Design| More Information].'''<br />
<br />
'''<partinfo>BBa_K190018</partinfo> HmtA (contains PstI sites)''' <br> About 100 to 200 words chracterizing the Parts ... bla... <br />
<br />
'''Have a look at the part design page on the registry for [http://partsregistry.org/Part:BBa_K190018:Design| More Information].'''<br />
<br />
===Acummulators===<br />
<br />
'''<partinfo>BBa_K190019</partinfo> fMT''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190031</partinfo> fMT regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190032</partinfo> fMT regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190027</partinfo> MBP-ArsR (fusion protein)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''(<partinfo>BBa_K190071</partinfo> MBP-ArsR (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]])'''<br />
<br />
'''(<partinfo>BBa_K190072</partinfo> MBP-ArsR regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]])'''<br />
<br />
===Promotors===<br />
<br />
'''<partinfo>BBa_K190015</partinfo> Arsenic Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190023</partinfo> Arsenic Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190016</partinfo> Zinc Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190022</partinfo> Zinc Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190017</partinfo> Copper Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190024</partinfo> Copper Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===GVP Constructs===<br />
<br />
'''(<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> GVP consists of 6064 base pairs. It's Backbone is <partinfo>BBa_J61035</partinfo> With a resistance against Ampicillin/Gentamycin '''[[Team Groningen:Dummielink|More information]])'''<br />
<br />
'''<partinfo>BBa_K190025</partinfo> GVP regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190026</partinfo> GVP regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190033</partinfo> GVP regulated by pArsR''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190034</partinfo> GVP regulated by pZntR''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190035</partinfo> GVP regulated by pCueO''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190036</partinfo> GVP regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Devices===<br />
<br />
'''<partinfo>BBa_K190038</partinfo> Arsenic Accumulation Device (IPTG induced)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190039</partinfo> Arsenic Accumulation Device (constitutive)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''((<partinfo>BBa_K190073</partinfo> Arsenic Accumulation Device (IPTG induced, able to regulate pArsR)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]))'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-16T20:00:00Z<p>Verhoeven1981: /* Used Parts */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Used Parts==<br />
{{todo}}Maybe some general information of parts used from the registry.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===Miscellaneous===<br />
'''<partinfo>BBa_B0034</partinfo> RBS ((Elowitz 1999) -- defines RBS efficiency)''' <br />
{|width='80%' style='border:1px solid gray'<br />
|-<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ligation of a part behind the RBS succeded, and was confirmed by gel (correct vector size) and sequencing with VF2. We used this part in combination with several genes for building our biobricks e.g. <partinfo>BBa_K190061</partinfo>.<br />
|}<br />
'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_B0014</partinfo> Double terminator (B0012-B0011)''' <br> Double terminator consisting of BBa_B0012 and BBa_B0011 <br>'''[http://partsregistry.org/Part:BBa_B0014:Design| More Information]'''<br />
<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> This parts creates gas vesicles inside the cell wich enables it to float. It consist of 6064 base pairs. It's backbone is <partinfo>BBa_J61035</partinfo> <br>'''[[Team: Groningen/Modelling/Used Parts/GVP|More information]]'''<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> Constitutive promoter family member (high expression)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[http://partsregistry.org/Part:BBa_J23100:Design| More Information]'''<br />
<br />
'''<partinfo>J23101</partinfo> Constitutive promoter family member (high expression, reference)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[http://partsregistry.org/Part:BBa_J23101:Design| More Information]'''<br />
<br />
'''<partinfo>J23106</partinfo> Constitutive promoter family member (medium expression)''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[http://partsregistry.org/Part:BBa_J23106:Design| More Information]'''<br />
<br />
'''<partinfo>J23109</partinfo> Constitutive promoter family member (low expression)''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> '''[http://partsregistry.org/Part:BBa_J23109:Design| More Information]'''<br />
<br />
'''<partinfo>R0010</partinfo> Promoter (lacI regulated)''' <br> This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first binds the CAP protein, which is generally present in E.coli and is asocciated with cell health and availability of glucose. The second binds LacI protein. <br> '''[http://partsregistry.org/Part:BBa_R0010:Design| More information]'''<br />
<br />
'''<partinfo>I0500</partinfo> pBad/araC''' <br> This part is <br> '''[http://partsregistry.org/Part:BBa_I0500:Design| More information]'''<br />
<br />
===Vectors===<br />
'''<partinfo>pSB1AC3</partinfo> High copy BioBrick assembly plasmid''' <br> pSB1AC3 is a high copy number plasmid carrying ampicillin and chloramphenicol resistance. Together with pSB1A2 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br>'''[http://partsregistry.org/Part:pSB1AC3:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>pSB1A2</partinfo> pSB1A2 (Replaced by pSB1A3 in registry) ''' <br> pSB1A2 is a high copy number plasmid carrying ampicillin resistance. Together with pSB1AC3 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br>'''[http://partsregistry.org/Part:pSB1A2:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>pSB2K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[http://partsregistry.org/Part:pSB2K3:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[http://partsregistry.org/Part:pSB3K3:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a rsistance against Ampicillin <br>'''[http://partsregistry.org/Part:J61002:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61035|More information]]'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-16T19:49:29Z<p>Verhoeven1981: /* Vectors */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Used Parts==<br />
{{todo}}Maybe some general information of parts used from the registry.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===GVP Constructs===<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> This parts creates gas vesicles inside the cell wich enables it to float. It consist of 6064 base pairs. It's backbone is <partinfo>BBa_J61035</partinfo> <br>'''[[Team: Groningen/Modelling/Used Parts/GVP|More information]]'''<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Miscellaneous===<br />
'''<partinfo>BBa_B0034</partinfo> RBS ((Elowitz 1999) -- defines RBS efficiency)''' <br />
{|width='80%' style='border:1px solid gray'<br />
|-<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ligation of a part behind the RBS succeded, and was confirmed by gel (correct vector size) and sequencing with VF2. We used this part in combination with several genes for building our biobricks e.g. <partinfo>BBa_K190061</partinfo>.<br />
|}<br />
'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> Constitutive promoter family member (high expression)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[http://partsregistry.org/Part:BBa_J23100:Design| More Information]'''<br />
<br />
'''<partinfo>J23101</partinfo> Constitutive promoter family member (high expression, reference)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[http://partsregistry.org/Part:BBa_J23101:Design| More Information]'''<br />
<br />
'''<partinfo>J23106</partinfo> Constitutive promoter family member (medium expression)''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[http://partsregistry.org/Part:BBa_J23106:Design| More Information]'''<br />
<br />
'''<partinfo>J23109</partinfo> Constitutive promoter family member (low expression)''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> '''[http://partsregistry.org/Part:BBa_J23109:Design| More Information]'''<br />
<br />
'''<partinfo>R0010</partinfo> Promoter (lacI regulated)''' <br> This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first binds the CAP protein, which is generally present in E.coli and is asocciated with cell health and availability of glucose. The second binds LacI protein. <br> '''[http://partsregistry.org/Part:BBa_R0010:Design| More information]'''<br />
<br />
===Vectors===<br />
'''<partinfo>pSB1AC3</partinfo> High copy BioBrick assembly plasmid''' <br> pSB1AC3 is a high copy number plasmid carrying ampicillin and chloramphenicol resistance. Together with pSB1A2 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br>'''[http://partsregistry.org/Part:pSB1AC3:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>pSB1A2</partinfo> pSB1A2 (Replaced by pSB1A3 in registry) ''' <br> pSB1A2 is a high copy number plasmid carrying ampicillin resistance. Together with pSB1AC3 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br>'''[http://partsregistry.org/Part:pSB1A2:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>pSB2K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[http://partsregistry.org/Part:pSB2K3:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[http://partsregistry.org/Part:pSB3K3:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a rsistance against Ampicillin <br>'''[http://partsregistry.org/Part:J61002:Design| More Information]'''<br />
<br />
--<br />
<br />
'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61035|More information]]'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-16T19:26:50Z<p>Verhoeven1981: /* Used Parts */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Used Parts==<br />
{{todo}}Maybe some general information of parts used from the registry.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===GVP Constructs===<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> This parts creates gas vesicles inside the cell wich enables it to float. It consist of 6064 base pairs. It's backbone is <partinfo>BBa_J61035</partinfo> <br>'''[[Team: Groningen/Modelling/Used Parts/GVP|More information]]'''<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Miscellaneous===<br />
'''<partinfo>BBa_B0034</partinfo> RBS ((Elowitz 1999) -- defines RBS efficiency)''' <br />
{|width='80%' style='border:1px solid gray'<br />
|-<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ligation of a part behind the RBS succeded, and was confirmed by gel (correct vector size) and sequencing with VF2. We used this part in combination with several genes for building our biobricks e.g. <partinfo>BBa_K190061</partinfo>.<br />
|}<br />
'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> Constitutive promoter family member (high expression)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[http://partsregistry.org/Part:BBa_J23100:Design| More Information]'''<br />
<br />
'''<partinfo>J23101</partinfo> Constitutive promoter family member (high expression, reference)''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[http://partsregistry.org/Part:BBa_J23101:Design| More Information]'''<br />
<br />
'''<partinfo>J23106</partinfo> Constitutive promoter family member (medium expression)''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[http://partsregistry.org/Part:BBa_J23106:Design| More Information]'''<br />
<br />
'''<partinfo>J23109</partinfo> Constitutive promoter family member (low expression)''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> '''[http://partsregistry.org/Part:BBa_J23109:Design| More Information]'''<br />
<br />
'''<partinfo>R0010</partinfo> Promoter (lacI regulated)''' <br> This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first binds the CAP protein, which is generally present in E.coli and is asocciated with cell health and availability of glucose. The second binds LacI protein. <br> '''[http://partsregistry.org/Part:BBa_R0010:Design| More information]'''<br />
<br />
===Vectors===<br />
'''<partinfo>pSB1AC3</partinfo> High copy BioBrick assembly plasmid''' <br> pSB1AC3 is a high copy number plasmid carrying ampicillin and chloramphenicol resistance. Together with pSB1A2 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br>'''[http://partsregistry.org/Part:pSB1AC3:Design| More Information]'''<br />
<br />
'''<partinfo>pSB1A2</partinfo> pSB1A2 (Replaced by pSB1A3 in registry) ''' <br> pSB1A2 is a high copy number plasmid carrying ampicillin resistance. Together with pSB1AC3 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br>'''[http://partsregistry.org/Part:pSB1A2:Design| More Information]'''<br />
<br />
'''<partinfo>pSB2K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[http://partsregistry.org/Part:pSB2K3:Design| More Information]'''<br />
<br />
'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[http://partsregistry.org/Part:pSB3K3:Design| More Information]'''<br />
<br />
'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a rsistance against Ampicillin <br>'''[http://partsregistry.org/Part:J61002:Design| More Information]'''<br />
<br />
'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61035|More information]]'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-16T19:12:51Z<p>Verhoeven1981: /* Used Parts */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Used Parts==<br />
{{todo}}Maybe some general information of parts used from the registry.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===GVP Constructs===<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> This parts creates gas vesicles inside the cell wich enables it to float. It consist of 6064 base pairs. It's backbone is <partinfo>BBa_J61035</partinfo> <br>'''[[Team: Groningen/Modelling/Used Parts/GVP|More information]]'''<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Miscellaneous===<br />
'''<partinfo>BBa_B0034</partinfo> RBS ((Elowitz 1999) -- defines RBS efficiency)''' <br />
{|width='80%' style='border:1px solid gray'<br />
|-<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ligation of a part behind the RBS succeded, and was confirmed by gel (correct vector size) and sequencing with VF2. We used this part in combination with several genes for building our biobricks e.g. <partinfo>BBa_K190061</partinfo>.<br />
|}<br />
'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> pHigh High consituative promotor''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pHigh|More information]]'''<br />
<br />
'''<partinfo>J23101</partinfo> Reference High consituative promotor''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pHigh|More information]]'''<br />
<br />
'''<partinfo>J23106</partinfo> pMed Medium consituative promotor''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pMed|More information]]'''<br />
<br />
'''<partinfo>J23109</partinfo> pLow Low consituative promotor''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> '''[[Team:Groningen/Modelling/Used Parts/pLow|More information]]'''<br />
<br />
'''<partinfo>J23109</partinfo> pLow Low consituative promotor''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> '''[[Team:Groningen/Modelling/Used Parts/pLow|More information]]'''<br />
<br />
===Vectors===<br />
'''<partinfo>pSB1AC3</partinfo> High copy BioBrick assembly plasmid''' <br> pSB1AC3 is a high copy number plasmid carrying ampicillin and chloramphenicol resistance. Together with pSB1A2 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br>'''[http://partsregistry.org/Part:pSB1AC3:Design| More Information]'''<br />
<br />
'''<partinfo>pSB1A2</partinfo> pSB1A2 (Replaced by pSB1A3 in registry) ''' <br> pSB1A2 is a high copy number plasmid carrying ampicillin resistance. Together with pSB1AC3 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. <br>'''[http://partsregistry.org/Part:pSB1A2:Design| More Information]'''<br />
<br />
'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[[Team:Groningen/Modelling/Used Parts/pSB3K3|More information]]'''<br />
<br />
'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a rsistance against Ampicillin <br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61002|More information]]'''<br />
<br />
'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61035|More information]]'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-16T18:54:38Z<p>Verhoeven1981: /* Devices */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Used Parts==<br />
{{todo}}Maybe some general information of parts used from the registry.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===Importers===<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Miscellaneous===<br />
'''<partinfo>BBa_B0034</partinfo> RBS ((Elowitz 1999) -- defines RBS efficiency)''' <br />
{|width='80%' style='border:1px solid gray'<br />
|-<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ligation of a part behind the RBS succeded, and was confirmed by gel (correct vector size) and sequencing with VF2. We used this part in combination with several genes for building our biobricks e.g. <partinfo>BBa_K190061</partinfo>.<br />
|}<br />
'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> pHigh High consituative promotor''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pHigh|More information]]'''<br />
<br />
'''<partinfo>J23101</partinfo> Reference High consituative promotor''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pHigh|More information]]'''<br />
<br />
'''<partinfo>J23106</partinfo> pMed Medium consituative promotor''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pMed|More information]]'''<br />
<br />
'''<partinfo>J23109</partinfo> pLow Low consituative promotor''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> '''[[Team:Groningen/Modelling/Used Parts/pLow|More information]]'''<br />
<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===GVP Constructs===<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> This parts creates gas vesicles inside the cell wich enables it to float. It consist of 6064 base pairs. It's backbone is <partinfo>BBa_J61035</partinfo> <br>'''[[Team: Groningen/Modelling/Used Parts/GVP|More information]]'''<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Vectors===<br />
*'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[[Team:Groningen/Modelling/Used Parts/pSB3K3|More information]]'''<br />
*'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a rsistance against Ampicillin <br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61002|More information]]'''<br />
*'''<partinfo>pSB1AC3</partinfo> A high copy number base vector''' <br> A high copy number base vector which has a resistance against Ampicillin/Chloramphenicol <br>'''[[Team:Groningen/Modelling/Used Parts/pSB1AC3|More information]]'''<br />
*'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61035|More information]]'''</div>Verhoeven1981http://2009.igem.org/Template:Team:Groningen/HeaderTemplate:Team:Groningen/Header2009-10-16T18:53:28Z<p>Verhoeven1981: </p>
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!<div id="team">[[Team:Groningen/Team|The Team]]</div><br />
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<li class="contact"></html>[[Team:Groningen/Team#contact|contact]]<html></li><br />
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<li class="ethics"></html>[[Team:Groningen/Ethics|Ethics]]<html></li><br />
<li class="ourvision"></html>[[Team:Groningen/Vision|Vision]]<html></li><br />
<li class="communication"></html>[[Team:Groningen/Commucation|Communication]]<html></li><br />
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!<div id="project">[[Team:Groningen/Project|The Project]]</div><br />
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<li class="transport"></html>[[Team:Groningen/Project/Transport|Transport]]<html></li><br />
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<li class="promoter"></html>[[Team:Groningen/Project/Promoters|Metal-sensitive Promoters]]<html></li><br />
<li class="vesicles"></html>[[Team:Groningen/Project/Vesicle|Gas Vesicles]]<html></li><br />
</ul></html></div><br />
<!--For the moment we'll try to integrate this with the "The Project page"--><br />
<!--!align="center"|[[Team:Groningen/Parts|Parts]]--><br />
!<div id="modelling">[[Team:Groningen/Modelling|Modelling]]</div><br />
<div class="navigation imageMap"><html><ul style="left:-12px; width:134px; height:50px;"><br />
<li class="detailedmodel"></html>[[Team:Groningen/Modelling/Arsenic|Detailed model]]<html></li><br />
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<!--<div class="navigation imageMap"><html><ul style="left:-67px; width:170px; height:50px;"><br />
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</ul></html></div><br />
!<div id="BioBricks">[[Team:Groningen/Parts|Biobricks]]</div><br />
<div class="navigation imageMap"><html><ul style="left:-11px; width:121px; height:70px;"><br />
<li class="ourregistry"></html>[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Groningen Our registry]<html></li><br />
<li class="submittedbiobricks"></html>[[Team:Groningen/Parts/Submitted_Parts|Submitted Biobricks]]<html></li><br />
<li class="usedbiobricks"></html>[[Team:Groningen/Parts/Used_Parts|Used Biobricks]]<html></li><br />
</ul></html></div><br />
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<div id="glossary">[[Team:Groningen/Glossary|Glossary]]</div><br />
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__NOTOC__</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Submitted_PartsTeam:Groningen/Parts/Submitted Parts2009-10-16T12:29:59Z<p>Verhoeven1981: /* Devices */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Submitted Parts==<br />
{{todo}}Maybe some general information about our submitted parts.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
===Importers===<br />
<br />
'''<partinfo>BBa_K190028</partinfo> GlpF''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190061</partinfo> GlpF (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190062</partinfo> GlpF regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190063</partinfo> GlpF regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190018</partinfo> HmtA (contains PstI sites)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Acummulators===<br />
<br />
'''<partinfo>BBa_K190019</partinfo> fMT''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190031</partinfo> fMT regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190032</partinfo> fMT regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190027</partinfo> MBP-ArsR (fusion protein)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''(<partinfo>BBa_K190071</partinfo> MBP-ArsR (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]])'''<br />
<br />
'''(<partinfo>BBa_K190072</partinfo> MBP-ArsR regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]])'''<br />
<br />
===Promotors===<br />
<br />
'''<partinfo>BBa_K190015</partinfo> Arsenic Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190023</partinfo> Arsenic Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190016</partinfo> Zinc Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190022</partinfo> Zinc Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190017</partinfo> Copper Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190024</partinfo> Copper Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===GVP Constructs===<br />
<br />
'''(<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> GVP consists of 6064 base pairs. It's Backbone is <partinfo>BBa_J61035</partinfo> With a resistance against Ampicillin/Gentamycin '''[[Team Groningen:Dummielink|More information]])'''<br />
<br />
'''<partinfo>BBa_K190025</partinfo> GVP regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190026</partinfo> GVP regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190033</partinfo> GVP regulated by pArsR''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190034</partinfo> GVP regulated by pZntR''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190035</partinfo> GVP regulated by pCueO''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190036</partinfo> GVP regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Devices===<br />
<br />
'''<partinfo>BBa_K190038</partinfo> Arsenic Accumulation Device (IPTG induced)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190039</partinfo> Arsenic Accumulation Device (constitutive)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''((<partinfo>BBa_K190073</partinfo> Arsenic Accumulation Device (IPTG induced, able to regulate pArsR)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]))'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Submitted_PartsTeam:Groningen/Parts/Submitted Parts2009-10-16T12:29:18Z<p>Verhoeven1981: /* GVP Constructs */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Submitted Parts==<br />
{{todo}}Maybe some general information about our submitted parts.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
===Importers===<br />
<br />
'''<partinfo>BBa_K190028</partinfo> GlpF''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190061</partinfo> GlpF (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190062</partinfo> GlpF regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190063</partinfo> GlpF regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190018</partinfo> HmtA (contains PstI sites)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Acummulators===<br />
<br />
'''<partinfo>BBa_K190019</partinfo> fMT''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190031</partinfo> fMT regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190032</partinfo> fMT regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190027</partinfo> MBP-ArsR (fusion protein)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''(<partinfo>BBa_K190071</partinfo> MBP-ArsR (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]])'''<br />
<br />
'''(<partinfo>BBa_K190072</partinfo> MBP-ArsR regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]])'''<br />
<br />
===Promotors===<br />
<br />
'''<partinfo>BBa_K190015</partinfo> Arsenic Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190023</partinfo> Arsenic Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190016</partinfo> Zinc Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190022</partinfo> Zinc Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190017</partinfo> Copper Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190024</partinfo> Copper Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===GVP Constructs===<br />
<br />
'''(<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> GVP consists of 6064 base pairs. It's Backbone is <partinfo>BBa_J61035</partinfo> With a resistance against Ampicillin/Gentamycin '''[[Team Groningen:Dummielink|More information]])'''<br />
<br />
'''<partinfo>BBa_K190025</partinfo> GVP regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190026</partinfo> GVP regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190033</partinfo> GVP regulated by pArsR''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190034</partinfo> GVP regulated by pZntR''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190035</partinfo> GVP regulated by pCueO''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190036</partinfo> GVP regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Devices===<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190038</partinfo> Arsenic Accumulation Device (IPTG induced)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190039</partinfo> Arsenic Accumulation Device (constitutive)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190073</partinfo> Arsenic Accumulation Device (IPTG induced, able to regulate pArsR)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Submitted_PartsTeam:Groningen/Parts/Submitted Parts2009-10-16T12:28:30Z<p>Verhoeven1981: /* Promotors */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Submitted Parts==<br />
{{todo}}Maybe some general information about our submitted parts.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
===Importers===<br />
<br />
'''<partinfo>BBa_K190028</partinfo> GlpF''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190061</partinfo> GlpF (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190062</partinfo> GlpF regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190063</partinfo> GlpF regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190018</partinfo> HmtA (contains PstI sites)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Acummulators===<br />
<br />
'''<partinfo>BBa_K190019</partinfo> fMT''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190031</partinfo> fMT regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190032</partinfo> fMT regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190027</partinfo> MBP-ArsR (fusion protein)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''(<partinfo>BBa_K190071</partinfo> MBP-ArsR (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]])'''<br />
<br />
'''(<partinfo>BBa_K190072</partinfo> MBP-ArsR regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]])'''<br />
<br />
===Promotors===<br />
<br />
'''<partinfo>BBa_K190015</partinfo> Arsenic Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190023</partinfo> Arsenic Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190016</partinfo> Zinc Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190022</partinfo> Zinc Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190017</partinfo> Copper Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190024</partinfo> Copper Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===GVP Constructs===<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> GVP consists of 6064 base pairs. It's Backbone is <partinfo>BBa_J61035</partinfo> With a resistance against Ampicillin/Gentamycin '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190025</partinfo> GVP regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190026</partinfo> GVP regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190033</partinfo> GVP regulated by pArsR''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190034</partinfo> GVP regulated by pZntR''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190035</partinfo> GVP regulated by pCueO''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190036</partinfo> GVP regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Devices===<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190038</partinfo> Arsenic Accumulation Device (IPTG induced)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190039</partinfo> Arsenic Accumulation Device (constitutive)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190073</partinfo> Arsenic Accumulation Device (IPTG induced, able to regulate pArsR)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Submitted_PartsTeam:Groningen/Parts/Submitted Parts2009-10-16T12:27:34Z<p>Verhoeven1981: /* Acummulators */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Submitted Parts==<br />
{{todo}}Maybe some general information about our submitted parts.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
===Importers===<br />
<br />
'''<partinfo>BBa_K190028</partinfo> GlpF''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190061</partinfo> GlpF (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190062</partinfo> GlpF regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190063</partinfo> GlpF regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190018</partinfo> HmtA (contains PstI sites)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Acummulators===<br />
<br />
'''<partinfo>BBa_K190019</partinfo> fMT''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190031</partinfo> fMT regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190032</partinfo> fMT regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190027</partinfo> MBP-ArsR (fusion protein)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''(<partinfo>BBa_K190071</partinfo> MBP-ArsR (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]])'''<br />
<br />
'''(<partinfo>BBa_K190072</partinfo> MBP-ArsR regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]])'''<br />
<br />
===Promotors===<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190015</partinfo> Arsenic Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190023</partinfo> Arsenic Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190016</partinfo> Zinc Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190022</partinfo> Zinc Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190017</partinfo> Copper Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190024</partinfo> Copper Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===GVP Constructs===<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> GVP consists of 6064 base pairs. It's Backbone is <partinfo>BBa_J61035</partinfo> With a resistance against Ampicillin/Gentamycin '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190025</partinfo> GVP regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190026</partinfo> GVP regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190033</partinfo> GVP regulated by pArsR''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190034</partinfo> GVP regulated by pZntR''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190035</partinfo> GVP regulated by pCueO''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190036</partinfo> GVP regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Devices===<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190038</partinfo> Arsenic Accumulation Device (IPTG induced)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190039</partinfo> Arsenic Accumulation Device (constitutive)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190073</partinfo> Arsenic Accumulation Device (IPTG induced, able to regulate pArsR)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Submitted_PartsTeam:Groningen/Parts/Submitted Parts2009-10-16T12:26:01Z<p>Verhoeven1981: /* Importers */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Submitted Parts==<br />
{{todo}}Maybe some general information about our submitted parts.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
===Importers===<br />
<br />
'''<partinfo>BBa_K190028</partinfo> GlpF''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190061</partinfo> GlpF (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190062</partinfo> GlpF regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190063</partinfo> GlpF regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190018</partinfo> HmtA (contains PstI sites)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Acummulators===<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190019</partinfo> fMT''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190031</partinfo> fMT regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190032</partinfo> fMT regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190027</partinfo> MBP-ArsR (fusion protein)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190071</partinfo> MBP-ArsR (with strong RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190072</partinfo> MBP-ArsR regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Promotors===<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190015</partinfo> Arsenic Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190023</partinfo> Arsenic Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190016</partinfo> Zinc Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190022</partinfo> Zinc Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190017</partinfo> Copper Promoter''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190024</partinfo> Copper Promoter (with wild type RBS)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===GVP Constructs===<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> GVP consists of 6064 base pairs. It's Backbone is <partinfo>BBa_J61035</partinfo> With a resistance against Ampicillin/Gentamycin '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190025</partinfo> GVP regulated by low constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190026</partinfo> GVP regulated by medium constitutive promotor''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190033</partinfo> GVP regulated by pArsR''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190034</partinfo> GVP regulated by pZntR''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190035</partinfo> GVP regulated by pCueO''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190036</partinfo> GVP regulated by pLacI''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Devices===<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190038</partinfo> Arsenic Accumulation Device (IPTG induced)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190039</partinfo> Arsenic Accumulation Device (constitutive)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
'''<partinfo>BBa_K190073</partinfo> Arsenic Accumulation Device (IPTG induced, able to regulate pArsR)''' <br> About 100 to 200 words chracterizing the Parts ... bla... '''[[Team Groningen:Dummielink|More information]]'''</div>Verhoeven1981http://2009.igem.org/Judging/Judging_formJudging/Judging form2009-10-10T15:20:02Z<p>Verhoeven1981: New page: Who knows what the form has to look like?</p>
<hr />
<div>Who knows what the form has to look like?</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-09T19:24:03Z<p>Verhoeven1981: /* Miscellaneous */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Used Parts==<br />
{{todo}}Maybe some general information of parts used from the registry.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===Importers===<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Miscellaneous===<br />
'''<partinfo>BBa_B0034</partinfo> RBS ((Elowitz 1999) -- defines RBS efficiency)''' <br />
{|width='80%' style='border:1px solid gray'<br />
|-<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ligation of a part behind the RBS succeded, and was confirmed by gel (correct vector size) and sequencing with VF2. We used this part in combination with several genes for building our biobricks e.g. <partinfo>BBa_K190061</partinfo>.<br />
|}<br />
'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> pHigh High consituative promotor''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pHigh|More information]]'''<br />
<br />
'''<partinfo>J23101</partinfo> Reference High consituative promotor''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pHigh|More information]]'''<br />
<br />
'''<partinfo>J23106</partinfo> pMed Medium consituative promotor''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pMed|More information]]'''<br />
<br />
'''<partinfo>J23109</partinfo> pLow Low consituative promotor''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> '''[[Team:Groningen/Modelling/Used Parts/pLow|More information]]'''<br />
<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===GVP Constructs===<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> This parts creates gas vesicles inside the cell wich enables it to float. It consist of 6064 base pairs. It's backbone is <partinfo>BBa_J61035</partinfo> <br>'''[[Team: Groningen/Modelling/Used Parts/GVP|More information]]'''<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Vectors===<br />
*'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[[Team:Groningen/Modelling/Used Parts/pSB3K3|More information]]'''<br />
*'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a rsistance against Ampicillin <br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61002|More information]]'''<br />
*'''<partinfo>pSB1AC3</partinfo> A high copy number base vector''' <br> A high copy number base vector which has a resistance against Ampicillin/Chloramphenicol <br>'''[[Team:Groningen/Modelling/Used Parts/pSB1AC3|More information]]'''<br />
*'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61035|More information]]'''<br />
<br />
===Devices===<br />
*'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-09T19:21:08Z<p>Verhoeven1981: /* Mis */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Used Parts==<br />
{{todo}}Maybe some general information of parts used from the registry.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===Importers===<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Miscellaneous===<br />
'''<partinfo>BBa_B0034</partinfo> RBS''' <br />
{|width='80%' style='border:1px solid gray'<br />
|-<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ligation of a part behind the RBS succeded, and was confirmed by gel (correct vector size) and sequencing with VF2. We used this part in combination with several genes for building our biobricks e.g. <partinfo>BBa_K190061</partinfo>.<br />
|}<br />
'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> pHigh High consituative promotor''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pHigh|More information]]'''<br />
<br />
'''<partinfo>J23101</partinfo> Reference High consituative promotor''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pHigh|More information]]'''<br />
<br />
'''<partinfo>J23106</partinfo> pMed Medium consituative promotor''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pMed|More information]]'''<br />
<br />
'''<partinfo>J23109</partinfo> pLow Low consituative promotor''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> '''[[Team:Groningen/Modelling/Used Parts/pLow|More information]]'''<br />
<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===GVP Constructs===<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> This parts creates gas vesicles inside the cell wich enables it to float. It consist of 6064 base pairs. It's backbone is <partinfo>BBa_J61035</partinfo> <br>'''[[Team: Groningen/Modelling/Used Parts/GVP|More information]]'''<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Vectors===<br />
*'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[[Team:Groningen/Modelling/Used Parts/pSB3K3|More information]]'''<br />
*'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a rsistance against Ampicillin <br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61002|More information]]'''<br />
*'''<partinfo>pSB1AC3</partinfo> A high copy number base vector''' <br> A high copy number base vector which has a resistance against Ampicillin/Chloramphenicol <br>'''[[Team:Groningen/Modelling/Used Parts/pSB1AC3|More information]]'''<br />
*'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61035|More information]]'''<br />
<br />
===Devices===<br />
*'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-09T19:18:43Z<p>Verhoeven1981: /* Acummulators */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Used Parts==<br />
{{todo}}Maybe some general information of parts used from the registry.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===Importers===<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Mis===<br />
'''<partinfo>BBa_B0034</partinfo> RBS''' <br />
{|width='80%' style='border:1px solid gray'<br />
|-<br />
|width='10%'|<br />
<partinfo>BBa_K190028 AddReview 5</partinfo><br />
<I> iGEM Groningen 2009 </I><br />
|width='60%' valign='top'|<br />
The ligation of a part behind the RBS succeded, and was confirmed by gel (correct vector size) and sequencing with VF2. We used this part in combination with several genes for building our biobricks e.g. <partinfo>BBa_K190061</partinfo>.<br />
|}<br />
'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> pHigh High consituative promotor''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pHigh|More information]]'''<br />
<br />
'''<partinfo>J23101</partinfo> Reference High consituative promotor''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pHigh|More information]]'''<br />
<br />
'''<partinfo>J23106</partinfo> pMed Medium consituative promotor''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pMed|More information]]'''<br />
<br />
'''<partinfo>J23109</partinfo> pLow Low consituative promotor''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> '''[[Team:Groningen/Modelling/Used Parts/pLow|More information]]'''<br />
<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===GVP Constructs===<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> This parts creates gas vesicles inside the cell wich enables it to float. It consist of 6064 base pairs. It's backbone is <partinfo>BBa_J61035</partinfo> <br>'''[[Team: Groningen/Modelling/Used Parts/GVP|More information]]'''<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Vectors===<br />
*'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[[Team:Groningen/Modelling/Used Parts/pSB3K3|More information]]'''<br />
*'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a rsistance against Ampicillin <br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61002|More information]]'''<br />
*'''<partinfo>pSB1AC3</partinfo> A high copy number base vector''' <br> A high copy number base vector which has a resistance against Ampicillin/Chloramphenicol <br>'''[[Team:Groningen/Modelling/Used Parts/pSB1AC3|More information]]'''<br />
*'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61035|More information]]'''<br />
<br />
===Devices===<br />
*'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-09T19:01:49Z<p>Verhoeven1981: /* GVP Constructs */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Used Parts==<br />
{{todo}}Maybe some general information of parts used from the registry.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===Importers===<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Acummulators===<br />
*'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> pHigh High consituative promotor''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pHigh|More information]]'''<br />
<br />
'''<partinfo>J23101</partinfo> Reference High consituative promotor''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pHigh|More information]]'''<br />
<br />
'''<partinfo>J23106</partinfo> pMed Medium consituative promotor''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pMed|More information]]'''<br />
<br />
'''<partinfo>J23109</partinfo> pLow Low consituative promotor''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> '''[[Team:Groningen/Modelling/Used Parts/pLow|More information]]'''<br />
<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===GVP Constructs===<br />
'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> This parts creates gas vesicles inside the cell wich enables it to float. It consist of 6064 base pairs. It's backbone is <partinfo>BBa_J61035</partinfo> <br>'''[[Team: Groningen/Modelling/Used Parts/GVP|More information]]'''<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Vectors===<br />
*'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[[Team:Groningen/Modelling/Used Parts/pSB3K3|More information]]'''<br />
*'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a rsistance against Ampicillin <br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61002|More information]]'''<br />
*'''<partinfo>pSB1AC3</partinfo> A high copy number base vector''' <br> A high copy number base vector which has a resistance against Ampicillin/Chloramphenicol <br>'''[[Team:Groningen/Modelling/Used Parts/pSB1AC3|More information]]'''<br />
*'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61035|More information]]'''<br />
<br />
===Devices===<br />
*'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-09T19:01:11Z<p>Verhoeven1981: /* Importers */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Used Parts==<br />
{{todo}}Maybe some general information of parts used from the registry.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===Importers===<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Acummulators===<br />
*'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> pHigh High consituative promotor''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pHigh|More information]]'''<br />
<br />
'''<partinfo>J23101</partinfo> Reference High consituative promotor''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pHigh|More information]]'''<br />
<br />
'''<partinfo>J23106</partinfo> pMed Medium consituative promotor''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pMed|More information]]'''<br />
<br />
'''<partinfo>J23109</partinfo> pLow Low consituative promotor''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> '''[[Team:Groningen/Modelling/Used Parts/pLow|More information]]'''<br />
<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===GVP Constructs===<br />
*'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> This parts creates gas vesicles inside the cell wich enables it to float. It consist of 6064 base pairs. It's backbone is <partinfo>BBa_J61035</partinfo> <br>'''[[Team: Groningen/Modelling/Used Parts/GVP|More information]]'''<br />
*'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Vectors===<br />
*'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[[Team:Groningen/Modelling/Used Parts/pSB3K3|More information]]'''<br />
*'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a rsistance against Ampicillin <br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61002|More information]]'''<br />
*'''<partinfo>pSB1AC3</partinfo> A high copy number base vector''' <br> A high copy number base vector which has a resistance against Ampicillin/Chloramphenicol <br>'''[[Team:Groningen/Modelling/Used Parts/pSB1AC3|More information]]'''<br />
*'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61035|More information]]'''<br />
<br />
===Devices===<br />
*'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''</div>Verhoeven1981http://2009.igem.org/Team:Groningen/Parts/Used_PartsTeam:Groningen/Parts/Used Parts2009-10-09T19:00:27Z<p>Verhoeven1981: /* Promotors */</p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
==Used Parts==<br />
{{todo}}Maybe some general information of parts used from the registry.<br />
*To get to the partsregistry site of a particular part one can click on the '''name of the part'''<br />
*To see what our main findings where (no details or derivations) regarding a particular part one can click on '''more information''' <br />
<br />
===Importers===<br />
*'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Acummulators===<br />
*'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Promotors===<br />
'''<partinfo>J23100</partinfo> pHigh High consituative promotor''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pHigh|More information]]'''<br />
<br />
'''<partinfo>J23101</partinfo> Reference High consituative promotor''' <br> pHigh is a high consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pHigh|More information]]'''<br />
<br />
'''<partinfo>J23106</partinfo> pMed Medium consituative promotor''' <br> pMed is a medium consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo> <br> '''[[Team:Groningen/Modelling/Used Parts/pMed|More information]]'''<br />
<br />
'''<partinfo>J23109</partinfo> pLow Low consituative promotor''' <br> pLow is a Low consituative promotor. It consists of about 35 base pairs. It has been placed in the following vectors. <partinfo>pSB3K3</partinfo>, <partinfo>BBa_J61002</partinfo> and <partinfo>pSB1AC3</partinfo><br> '''[[Team:Groningen/Modelling/Used Parts/pLow|More information]]'''<br />
<br />
'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===GVP Constructs===<br />
*'''<partinfo>BBa_I750016</partinfo> GVP Gas Vesicle Proteins''' <br> This parts creates gas vesicles inside the cell wich enables it to float. It consist of 6064 base pairs. It's backbone is <partinfo>BBa_J61035</partinfo> <br>'''[[Team: Groningen/Modelling/Used Parts/GVP|More information]]'''<br />
*'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''<br />
<br />
===Vectors===<br />
*'''<partinfo>pSB3K3</partinfo> A low copy number base vector''' <br> A low copy number base vector which as a resistance against Kanamycin <br>'''[[Team:Groningen/Modelling/Used Parts/pSB3K3|More information]]'''<br />
*'''<partinfo>BBa_J61002</partinfo> A normal base vector''' <br> A normal base vector which has a rsistance against Ampicillin <br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61002|More information]]'''<br />
*'''<partinfo>pSB1AC3</partinfo> A high copy number base vector''' <br> A high copy number base vector which has a resistance against Ampicillin/Chloramphenicol <br>'''[[Team:Groningen/Modelling/Used Parts/pSB1AC3|More information]]'''<br />
*'''<partinfo>BBa_J61035</partinfo> Vector we get with GVP''' <br> It's the backbone of our GVP part is and it a resistance against Ampicillin/Gentamycin<br>'''[[Team:Groningen/Modelling/Used Parts/BBa_J61035|More information]]'''<br />
<br />
===Devices===<br />
*'''[[Team Groningen:Dummielink|Name Part]] Main title with discription''' <br> About 100 to 200 words chracterizing the Parts and our experience with it ... bla... <br>'''[[Team Groningen:Dummielink|More information]]'''</div>Verhoeven1981