http://2009.igem.org/wiki/index.php?title=Special:Contributions/110hs&feed=atom&limit=50&target=110hs&year=&month=2009.igem.org - User contributions [en]2024-03-28T08:47:36ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/IGEM_PublicityIGEM Publicity2010-06-21T08:35:50Z<p>110hs: /* team specific */</p>
<hr />
<div>__NOTOC__<br />
[https://2008.igem.org/IGEM_Publicity iGEM 2008 Publicity] | [https://2007.igem.org/Media iGEM 2007 Publicity] | [https://2006.igem.org/IGEM_News iGEM 2006 Publicity]<br />
<br />
<div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [[Press_Kit | iGEM Press Kit]])</div><br />
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<span style="color:#1e90ff; font-size:175%">'''blogs </span><span style="color:#3cb371; font-size:175%">covering iGEM 2009'''</span><br />
* The [http://igem.sdu.dk/ <span style="color:#453221; font-size: 130%">'''SDU-Denmark'''</span>] team blog about their iGEM experience.<br />
* A blog about the iGEM project of the [http://aboutgmos.org/iGEM <span style="color:#453221; font-size: 130%">'''Uppsala-Sweden'''</span>] team.<br />
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<br />
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<br />
====<font size=5><font color=dodgerblue>'''video/radio</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
*'''Slovenia, Heidelberg, SDU-Denmark''': [http://www.dradio.de/dlf/sendungen/forschak/1062608/ Die Biobastler von Boston], Nov 2, 2009, Deutschlandfunk radio (in German).<br />
*'''Cambridge, Imperial''': [http://www.sciencefriday.com/program/archives/200911063 Synthetic Biology Competition], Nov 6, 2009, NPR Science Friday (in English).<br />
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<br />
====<font size=5><font color=dodgerblue>'''news articles</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
<br />
<br />
If you would like to share an article that was written about iGEM or your iGEM team, please link to it on this page. If you have multiple articles featuring your team, link to them all individually!<br />
<br />
Post the name of your team, the title of your article, where it was featured, and provide a link to it. <br />
<br />
''Example'':<br> <br />
'''Team Example''': ''Title of article'', Nature, [link]<br />
<br />
====<font size=4><font color=dodgerblue>'''general'''</font></font>====<br />
*'''The Economist''': ''Biohacking: Hacking goes squishy'' Sep 3, 2009 [http://www.economist.com/search/displaystory.cfm?story_id=14299634 The Economist]<br />
*'''The Scientist''': ''Brick by Brick'' Feb 1, 2009. [http://www.the-scientist.com/article/display/55378/ The Scientist]<br />
*'''Technology Review''': ''A Genetically Engineered Rainbow of Bacteria'' Nov 03, 2009. [http://www.technologyreview.com/blog/editors/24351/ Technology Review]<br />
*'''Discovery News''': '' Bright Bacteria Wins Synthetic Biology Competition'' Nov 6, 2009. [http://blogs.discoverychannel.co.uk/discovery-news/2009/11/bright-bacteria-wins-synthetic-biology-competition.html Discovery News]<br />
*'''Wired''': '' Building new life forms at the iGEM Jamboree'' Nov 9, 2009. [http://www.wired.co.uk/news/archive/2009-11/09/building-new-life-forms-at-the-igem-jamboree.aspx Wired]<br />
*'''Nature Biotechnology''': ''Building outside of the box: iGEM and the BioBricks Foundation'' [http://www.nature.com/nbt/journal/v27/n12/full/nbt1209-1099.html Nature Biotechnology 27, 1099 - 1102 (2009)]<br />
*'''Nature Biotechnology''': ''About the cover'' [http://www.nature.com/nbt/journal/v27/n12/covers/index.html Nature Biotechnology 27(12), cover (2009)]<br />
*'''Repubblica''': ''Il biotech alla guerra del tumore'' (Italian) [http://ricerca.repubblica.it/repubblica/archivio/repubblica/2009/02/16/il-biotech-alla-guerra-del-tumore.html February 16, 2009]<br />
*'''Nature''': ''Five hard truths for synthetic biology'', [http://www.nature.com/news/2010/100120/full/463288a.html ''Nature'' 463, 288-290 (2010)]<br />
<br />
====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
*'''[[Team:IBB_Pune]]''': "<i>Bio-champs in the making</i>" (English), [http://www.punemirror.in/index.aspx?Page=article&sectname=News%20-%20City&sectid=2&contentid=200909142009091423331646a131eacc#ftr2 (Monday, September 14, 2009 at 11:33:25 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Ganiti Prakriyanmadhe Jeevanuncha Vaapar! (Use of Bacteria in mathematical devices)</i>" (Marathi), [http://beta.esakal.com/2009/09/09220618/pune-use-of-bacterias-in-maths.html(Wednesday, September 09th, 2009 AT 10:09 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Synthetic Biologiteel Bharari ("Advances in Synthetic Biology")</i>" (Marathi), [https://2009.igem.org/Team:IBB_Pune/press (Wednesday 7 October 2009)]<br />
<br />
*'''[[Team:Groningen]]''': "<i>Students from Groningen in competition for best bacteria</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*'''[[Team:Groningen]]''': "<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Team iGEM Groningen has been awarded a Gold Medal</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen iGEM 2009 participants</i>" ([http://www.rug.nl/corporate/nieuws/archief/archief2009/persberichten/174_09?lang=en English]/[http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Dutch]), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09?lang=en Press release University Groningen (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>With a pacmanbacterium in de finals</i>" (Dutch), [http://www.uk.rug.nl/archief/jaargang39/11/12e.php UK (Paper of the Groningen University) (November 12, 2009)]<br />
*'''[[Team:Groningen]]''' & '''[[Team:TUDelft]]''': "<i>iGEM Gold for Dutch</i>" (Dutch), [http://www.bionieuws.nl/artikel.php?id=4918&zoek=iGEM Bionieuws (November 14, 2009)]<br />
*'''[[Team:Paris]]''' : "<i>Un gène éthique qui vaut de l'or</i>" Le Monde, (French), [http://www.lemonde.fr/planete/article/2009/11/27/un-gene-ethique-qui-vaut-de-l-or_1272940_3244.html (November 27, 2009)]<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Un concours organisé par le M.I.T : en route pour Boston ! </i>" (French), [http://www.supbiotech.fr/2009/09/boston-supbiotech-igem.html (September 08, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Les étudiants de la biotech interpellent la biologie synthétique</i>" (French), [http://www.vivagora.org/spip.php?breve210 (October 09, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>Un equipo de la UAB, en el concurso de biología sintética del MIT</i>" (Spanish), [http://www.uab.es/servlet/Satellite?cid=1096481466568&pagename=UABDivulga%2FPage%2FTemplatePageDetallArticleInvestigar&param1=1253860327385 (September 23, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>UAB to participate in the synthetic biology competition at MIT</i>" (English), [http://www.uab.es/servlet/Satellite/latest-news/news-detail/uab-to-participate-in-the-synthetic-biology-competition-at-mit-1096476786473.html?noticiaid=1253657853358 (September 23, 2009)]<br />
<br />
*'''[[Team:UNIPV-Pavia]]''': <i>"Questo è il profumo dell'amore" - L'arte della Biopoesia al MIT</i>, Repubblica (Italian), [http://www.repubblica.it/2009/11/sezioni/scienze/bio-ingegneria/bio-ingegneria/bio-ingegneria.html (September, 2009)]<br />
<br />
*'''[[Team:UNIPV-Pavia]]''': "<i>Five hard truths for synthetic biology</i>", Nature, [http://www.nature.com/news/2010/100120/full/463288a.html online January 20, 2010; ''Nature'' 463, 288-290 (2010)]<br />
<br />
*'''[[Team:UNIPV-Pavia]]''': "<i>Con i «bricogeni» nasce la vita sintetica - Già realtà la produzione di farmaci e di bioetanolo </i>", Corriere (Italian), [http://archiviostorico.corriere.it/2010/febbraio/21/Con_bricogeni_nasce_vita_sintetica_co_9_100221109.shtml (February 21, 2010)]<br />
<br />
*'''[[Team:Valencia]]''': "<i>TheValencia team is awarded the Synthetic Standard Prize</i>" (Spanish), [http://www.elpais.com/articulo/sociedad/Levaduras/funcionan/pixeles/elpepuespval/20091103elpepusoc_9/Tes]<br />
<br />
*'''[[Team:Cambridge]]''': "<i>University of Cambridge team wins iGEM synthetic biology competition </i>" (English), [http://www.biotechniques.com/news/University-of-Cambridge-team-wins-iGEM-synthetic-biology-competition/biotechniques-180278.html (November 5 2009)]<br />
<br />
*'''[[Team:Virginia]]''': ''[http://www.washingtonpost.com/wp-dyn/content/article/2009/10/22/AR2009102204628.html New works of science nonfiction]'', The Washington Post (October 23, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Estafette voor bacterien'',(Dutch) Algemeen Dagblad (November 6, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette'', (Dutch) [http://noorderlicht.vpro.nl/themasites/mediaplayer/index.jsp?media=42722491&refernr=42560747&portalnr=3626936&hostname=noorderlicht&mediatype=audio&portalid=noorderlicht# Podcast van Radio 1, VPRO Noorderlicht, Annemieke Smit] (November 9, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette wint goud en award'', (Dutch) [http://www.tudelft.nl/live/pagina.jsp?id=ee44be90-289e-4efe-8a12-4636f1c86b28&lang=nl TNW Today Editie 2] (November 5, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette wint goud en award'', (Dutch) [http://www.tudelft.nl/live/pagina.jsp?id=ee44be90-289e-4efe-8a12-4636f1c86b28&lang=nl TUDelft Nieuws] (November 4, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterial relay race wins gold and award'', [http://www.tudelft.nl/live/pagina.jsp?id=26f61b2e-5a3f-40be-b737-a460b08bced7&lang=en Delft University of Technology Press release] (November 4, 2009)<br />
<br />
*'''[[Team:TorontoMaRSDiscovery]]''': ''A gem of a genetics competition'', [http://www.news.utoronto.ca/science-and-technology/a-gem-of-a-genetics-competition.html University of Toronto eBulletin] (November 23, 2009)<br />
<br />
*'''[[Team:Edinburgh]]''':"<i>Synthetic biology against land mines</i>" (Dutch), [http://www.c2w.nl/synthetische-biologie-tegen-landmijnen.72122.lynkx C<sub>2</sub>W Life Sciences (November 17 2009)]<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Pretty Fly (for a Small Guy)</i>" (English), [http://www.scienceline.org/2009/11/14/blog-bleicher-genetically-engineered-machines/ Scienceline Magazine] (Nov 14, 2009)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Gabriel See builds robot for MIT engineering Competition</i>" (English), [http://sammamishreview.com/2009/11/25/gabriel-see-builds-robot-for-mit-engineering-competition Sammamish Review] (Nov 25, 2009)<br />
<br />
*'''[[Team:Washington]] and [[Team:Washington-Software]]''':"<i>Gold, silver medals in UW teams in the International Genetically Engineered Machine Competition</i>" (English), [http://uwnews.org/uweek/article.aspx?id=53790 University Week] (Nov 19, 2009)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Seven Grader Scores in College Research Competition</i>" (English), [http://www.lwsd.org/News/News-from-the-Schools/Pages/12-07-2009-News-from-the-Schools.aspx News from Schools] (Dec 7, 2009)<br />
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*'''[[Team:Washington-Software]]''':"<i>iGEM genius</i>" (English), [http://scienceblogs.com/oscillator/2010/01/igem_geniuses.php Oscillator] (Jan 24, 2010)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Hidden gems in iGEM</i>" (English), [http://blog.ginkgobioworks.com/2009/11/03/hidden-gems-of-igem/ Gingkoo, Ginko Bioworks] (Nov 3, 2009)</div>110hshttp://2009.igem.org/IGEM_PublicityIGEM Publicity2010-06-21T08:32:00Z<p>110hs: /* general */</p>
<hr />
<div>__NOTOC__<br />
[https://2008.igem.org/IGEM_Publicity iGEM 2008 Publicity] | [http://parts.mit.edu/igem07/index.php/Media iGEM 2007 Publicity] | [http://parts.mit.edu/wiki/index.php/IGEM_News iGEM 2006 Publicity]<br />
<br />
<div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [[Press_Kit | iGEM Press Kit]])</div><br />
<br />
<html><br />
<iframe frameborder="0" width="728" height="90" marginwidth="0" marginheight="0" style="margin:0px 0px 25px 100px;"<br />
src="http://www.google.com/uds/modules/elements/newsshow/iframe.html?q=iGEM&rsz=large&format=728x90"><br />
</iframe><br />
</html><br />
<br />
<br />
<span style="color:#1e90ff; font-size:175%">'''blogs </span><span style="color:#3cb371; font-size:175%">covering iGEM 2009'''</span><br />
* The [http://igem.sdu.dk/ <span style="color:#453221; font-size: 130%">'''SDU-Denmark'''</span>] team blog about their iGEM experience.<br />
* A blog about the iGEM project of the [http://aboutgmos.org/iGEM <span style="color:#453221; font-size: 130%">'''Uppsala-Sweden'''</span>] team.<br />
<br />
<br />
<br />
<br />
====<font size=5><font color=dodgerblue>'''video/radio</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
*'''Slovenia, Heidelberg, SDU-Denmark''': [http://www.dradio.de/dlf/sendungen/forschak/1062608/ Die Biobastler von Boston], Nov 2, 2009, Deutschlandfunk radio (in German).<br />
*'''Cambridge, Imperial''': [http://www.sciencefriday.com/program/archives/200911063 Synthetic Biology Competition], Nov 6, 2009, NPR Science Friday (in English).<br />
<br />
<br />
<br />
<br />
====<font size=5><font color=dodgerblue>'''news articles</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
<br />
<br />
If you would like to share an article that was written about iGEM or your iGEM team, please link to it on this page. If you have multiple articles featuring your team, link to them all individually!<br />
<br />
Post the name of your team, the title of your article, where it was featured, and provide a link to it. <br />
<br />
''Example'':<br> <br />
'''Team Example''': ''Title of article'', Nature, [link]<br />
<br />
====<font size=4><font color=dodgerblue>'''general'''</font></font>====<br />
*'''The Economist''': ''Biohacking: Hacking goes squishy'' Sep 3, 2009 [http://www.economist.com/search/displaystory.cfm?story_id=14299634 The Economist]<br />
*'''The Scientist''': ''Brick by Brick'' Feb 1, 2009. [http://www.the-scientist.com/article/display/55378/ The Scientist]<br />
*'''Technology Review''': ''A Genetically Engineered Rainbow of Bacteria'' Nov 03, 2009. [http://www.technologyreview.com/blog/editors/24351/ Technology Review]<br />
*'''Discovery News''': '' Bright Bacteria Wins Synthetic Biology Competition'' Nov 6, 2009. [http://blogs.discoverychannel.co.uk/discovery-news/2009/11/bright-bacteria-wins-synthetic-biology-competition.html Discovery News]<br />
*'''Wired''': '' Building new life forms at the iGEM Jamboree'' Nov 9, 2009. [http://www.wired.co.uk/news/archive/2009-11/09/building-new-life-forms-at-the-igem-jamboree.aspx Wired]<br />
*'''Nature Biotechnology''': ''Building outside of the box: iGEM and the BioBricks Foundation'' [http://www.nature.com/nbt/journal/v27/n12/full/nbt1209-1099.html Nature Biotechnology 27, 1099 - 1102 (2009)]<br />
*'''Nature Biotechnology''': ''About the cover'' [http://www.nature.com/nbt/journal/v27/n12/covers/index.html Nature Biotechnology 27(12), cover (2009)]<br />
*'''Repubblica''': ''Il biotech alla guerra del tumore'' (Italian) [http://ricerca.repubblica.it/repubblica/archivio/repubblica/2009/02/16/il-biotech-alla-guerra-del-tumore.html February 16, 2009]<br />
*'''Nature''': ''Five hard truths for synthetic biology'', [http://www.nature.com/news/2010/100120/full/463288a.html ''Nature'' 463, 288-290 (2010)]<br />
<br />
====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
*'''[[Team:IBB_Pune]]''': "<i>Bio-champs in the making</i>" (English), [http://www.punemirror.in/index.aspx?Page=article&sectname=News%20-%20City&sectid=2&contentid=200909142009091423331646a131eacc#ftr2 (Monday, September 14, 2009 at 11:33:25 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Ganiti Prakriyanmadhe Jeevanuncha Vaapar! (Use of Bacteria in mathematical devices)</i>" (Marathi), [http://beta.esakal.com/2009/09/09220618/pune-use-of-bacterias-in-maths.html(Wednesday, September 09th, 2009 AT 10:09 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Synthetic Biologiteel Bharari ("Advances in Synthetic Biology")</i>" (Marathi), [https://2009.igem.org/Team:IBB_Pune/press (Wednesday 7 October 2009)]<br />
<br />
*'''[[Team:Groningen]]''': "<i>Students from Groningen in competition for best bacteria</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*'''[[Team:Groningen]]''': "<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Team iGEM Groningen has been awarded a Gold Medal</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen iGEM 2009 participants</i>" ([http://www.rug.nl/corporate/nieuws/archief/archief2009/persberichten/174_09?lang=en English]/[http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Dutch]), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09?lang=en Press release University Groningen (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>With a pacmanbacterium in de finals</i>" (Dutch), [http://www.uk.rug.nl/archief/jaargang39/11/12e.php UK (Paper of the Groningen University) (November 12, 2009)]<br />
*'''[[Team:Groningen]]''' & '''[[Team:TUDelft]]''': "<i>iGEM Gold for Dutch</i>" (Dutch), [http://www.bionieuws.nl/artikel.php?id=4918&zoek=iGEM Bionieuws (November 14, 2009)]<br />
*'''[[Team:Paris]]''' : "<i>Un gène éthique qui vaut de l'or</i>" Le Monde, (French), [http://www.lemonde.fr/planete/article/2009/11/27/un-gene-ethique-qui-vaut-de-l-or_1272940_3244.html (November 27, 2009)]<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Un concours organisé par le M.I.T : en route pour Boston ! </i>" (French), [http://www.supbiotech.fr/2009/09/boston-supbiotech-igem.html (September 08, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Les étudiants de la biotech interpellent la biologie synthétique</i>" (French), [http://www.vivagora.org/spip.php?breve210 (October 09, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>Un equipo de la UAB, en el concurso de biología sintética del MIT</i>" (Spanish), [http://www.uab.es/servlet/Satellite?cid=1096481466568&pagename=UABDivulga%2FPage%2FTemplatePageDetallArticleInvestigar&param1=1253860327385 (September 23, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>UAB to participate in the synthetic biology competition at MIT</i>" (English), [http://www.uab.es/servlet/Satellite/latest-news/news-detail/uab-to-participate-in-the-synthetic-biology-competition-at-mit-1096476786473.html?noticiaid=1253657853358 (September 23, 2009)]<br />
<br />
*'''[[Team:UNIPV-Pavia]]''': <i>"Questo è il profumo dell'amore" - L'arte della Biopoesia al MIT</i>, Repubblica (Italian), [http://www.repubblica.it/2009/11/sezioni/scienze/bio-ingegneria/bio-ingegneria/bio-ingegneria.html (September, 2009)]<br />
<br />
*'''[[Team:UNIPV-Pavia]]''': "<i>Con i «bricogeni» nasce la vita sintetica - Già realtà la produzione di farmaci e di bioetanolo </i>", Corriere (Italian), [http://archiviostorico.corriere.it/2010/febbraio/21/Con_bricogeni_nasce_vita_sintetica_co_9_100221109.shtml (February 21, 2010)]<br />
<br />
*'''[[Team:Valencia]]''': "<i>TheValencia team is awarded the Synthetic Standard Prize</i>" (Spanish), [http://www.elpais.com/articulo/sociedad/Levaduras/funcionan/pixeles/elpepuespval/20091103elpepusoc_9/Tes]<br />
<br />
*'''[[Team:Cambridge]]''': "<i>University of Cambridge team wins iGEM synthetic biology competition </i>" (English), [http://www.biotechniques.com/news/University-of-Cambridge-team-wins-iGEM-synthetic-biology-competition/biotechniques-180278.html (November 5 2009)]<br />
<br />
*'''[[Team:Virginia]]''': ''[http://www.washingtonpost.com/wp-dyn/content/article/2009/10/22/AR2009102204628.html New works of science nonfiction]'', The Washington Post (October 23, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Estafette voor bacterien'',(Dutch) Algemeen Dagblad (November 6, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette'', (Dutch) [http://noorderlicht.vpro.nl/themasites/mediaplayer/index.jsp?media=42722491&refernr=42560747&portalnr=3626936&hostname=noorderlicht&mediatype=audio&portalid=noorderlicht# Podcast van Radio 1, VPRO Noorderlicht, Annemieke Smit] (November 9, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette wint goud en award'', (Dutch) [http://www.tudelft.nl/live/pagina.jsp?id=ee44be90-289e-4efe-8a12-4636f1c86b28&lang=nl TNW Today Editie 2] (November 5, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette wint goud en award'', (Dutch) [http://www.tudelft.nl/live/pagina.jsp?id=ee44be90-289e-4efe-8a12-4636f1c86b28&lang=nl TUDelft Nieuws] (November 4, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterial relay race wins gold and award'', [http://www.tudelft.nl/live/pagina.jsp?id=26f61b2e-5a3f-40be-b737-a460b08bced7&lang=en Delft University of Technology Press release] (November 4, 2009)<br />
<br />
*'''[[Team:TorontoMaRSDiscovery]]''': ''A gem of a genetics competition'', [http://www.news.utoronto.ca/science-and-technology/a-gem-of-a-genetics-competition.html University of Toronto eBulletin] (November 23, 2009)<br />
<br />
*'''[[Team:Edinburgh]]''':"<i>Synthetic biology against land mines</i>" (Dutch), [http://www.c2w.nl/synthetische-biologie-tegen-landmijnen.72122.lynkx C<sub>2</sub>W Life Sciences (November 17 2009)]<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Pretty Fly (for a Small Guy)</i>" (English), [http://www.scienceline.org/2009/11/14/blog-bleicher-genetically-engineered-machines/ Scienceline Magazine] (Nov 14, 2009)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Gabriel See builds robot for MIT engineering Competition</i>" (English), [http://sammamishreview.com/2009/11/25/gabriel-see-builds-robot-for-mit-engineering-competition Sammamish Review] (Nov 25, 2009)<br />
<br />
*'''[[Team:Washington]] and [[Team:Washington-Software]]''':"<i>Gold, silver medals in UW teams in the International Genetically Engineered Machine Competition</i>" (English), [http://uwnews.org/uweek/article.aspx?id=53790 University Week] (Nov 19, 2009)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Seven Grader Scores in College Research Competition</i>" (English), [http://www.lwsd.org/News/News-from-the-Schools/Pages/12-07-2009-News-from-the-Schools.aspx News from Schools] (Dec 7, 2009)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>iGEM genius</i>" (English), [http://scienceblogs.com/oscillator/2010/01/igem_geniuses.php Oscillator] (Jan 24, 2010)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Hidden gems in iGEM</i>" (English), [http://blog.ginkgobioworks.com/2009/11/03/hidden-gems-of-igem/ Gingkoo, Ginko Bioworks] (Nov 3, 2009)</div>110hshttp://2009.igem.org/IGEM_PublicityIGEM Publicity2010-06-21T08:26:54Z<p>110hs: /* general */</p>
<hr />
<div>__NOTOC__<br />
[https://2008.igem.org/IGEM_Publicity iGEM 2008 Publicity] | [http://parts.mit.edu/igem07/index.php/Media iGEM 2007 Publicity] | [http://parts.mit.edu/wiki/index.php/IGEM_News iGEM 2006 Publicity]<br />
<br />
<div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [[Press_Kit | iGEM Press Kit]])</div><br />
<br />
<html><br />
<iframe frameborder="0" width="728" height="90" marginwidth="0" marginheight="0" style="margin:0px 0px 25px 100px;"<br />
src="http://www.google.com/uds/modules/elements/newsshow/iframe.html?q=iGEM&rsz=large&format=728x90"><br />
</iframe><br />
</html><br />
<br />
<br />
<span style="color:#1e90ff; font-size:175%">'''blogs </span><span style="color:#3cb371; font-size:175%">covering iGEM 2009'''</span><br />
* The [http://igem.sdu.dk/ <span style="color:#453221; font-size: 130%">'''SDU-Denmark'''</span>] team blog about their iGEM experience.<br />
* A blog about the iGEM project of the [http://aboutgmos.org/iGEM <span style="color:#453221; font-size: 130%">'''Uppsala-Sweden'''</span>] team.<br />
<br />
<br />
<br />
<br />
====<font size=5><font color=dodgerblue>'''video/radio</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
*'''Slovenia, Heidelberg, SDU-Denmark''': [http://www.dradio.de/dlf/sendungen/forschak/1062608/ Die Biobastler von Boston], Nov 2, 2009, Deutschlandfunk radio (in German).<br />
*'''Cambridge, Imperial''': [http://www.sciencefriday.com/program/archives/200911063 Synthetic Biology Competition], Nov 6, 2009, NPR Science Friday (in English).<br />
<br />
<br />
<br />
<br />
====<font size=5><font color=dodgerblue>'''news articles</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
<br />
<br />
If you would like to share an article that was written about iGEM or your iGEM team, please link to it on this page. If you have multiple articles featuring your team, link to them all individually!<br />
<br />
Post the name of your team, the title of your article, where it was featured, and provide a link to it. <br />
<br />
''Example'':<br> <br />
'''Team Example''': ''Title of article'', Nature, [link]<br />
<br />
====<font size=4><font color=dodgerblue>'''general'''</font></font>====<br />
*'''The Economist''': ''Biohacking: Hacking goes squishy'' Sep 3, 2009 [http://www.economist.com/search/displaystory.cfm?story_id=14299634 The Economist]<br />
*'''The Scientist''': ''Brick by Brick'' Feb 1, 2009. [http://www.the-scientist.com/article/display/55378/ The Scientist]<br />
*'''Technology Review''': ''A Genetically Engineered Rainbow of Bacteria'' Nov 03, 2009. [http://www.technologyreview.com/blog/editors/24351/ Technology Review]<br />
*'''Discovery News''': '' Bright Bacteria Wins Synthetic Biology Competition'' Nov 6, 2009. [http://blogs.discoverychannel.co.uk/discovery-news/2009/11/bright-bacteria-wins-synthetic-biology-competition.html Discovery News]<br />
*'''Wired''': '' Building new life forms at the iGEM Jamboree'' Nov 9, 2009. [http://www.wired.co.uk/news/archive/2009-11/09/building-new-life-forms-at-the-igem-jamboree.aspx Wired]<br />
*'''Nature Biotechnology''': ''Building outside of the box: iGEM and the BioBricks Foundation'' [http://www.nature.com/nbt/journal/v27/n12/full/nbt1209-1099.html Nature Biotechnology 27, 1099 - 1102 (2009)]<br />
*'''Nature Biotechnology''': ''About the cover'' [http://www.nature.com/nbt/journal/v27/n12/covers/index.html Nature Biotechnology 27(12), cover (2009)]<br />
*'''Repubblica''': ''Il biotech alla guerra del tumore'' (Italian) [http://ricerca.repubblica.it/repubblica/archivio/repubblica/2009/02/16/il-biotech-alla-guerra-del-tumore.html February 16, 2009]<br />
<br />
====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
*'''[[Team:IBB_Pune]]''': "<i>Bio-champs in the making</i>" (English), [http://www.punemirror.in/index.aspx?Page=article&sectname=News%20-%20City&sectid=2&contentid=200909142009091423331646a131eacc#ftr2 (Monday, September 14, 2009 at 11:33:25 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Ganiti Prakriyanmadhe Jeevanuncha Vaapar! (Use of Bacteria in mathematical devices)</i>" (Marathi), [http://beta.esakal.com/2009/09/09220618/pune-use-of-bacterias-in-maths.html(Wednesday, September 09th, 2009 AT 10:09 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Synthetic Biologiteel Bharari ("Advances in Synthetic Biology")</i>" (Marathi), [https://2009.igem.org/Team:IBB_Pune/press (Wednesday 7 October 2009)]<br />
<br />
*'''[[Team:Groningen]]''': "<i>Students from Groningen in competition for best bacteria</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*'''[[Team:Groningen]]''': "<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Team iGEM Groningen has been awarded a Gold Medal</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen iGEM 2009 participants</i>" ([http://www.rug.nl/corporate/nieuws/archief/archief2009/persberichten/174_09?lang=en English]/[http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Dutch]), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09?lang=en Press release University Groningen (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>With a pacmanbacterium in de finals</i>" (Dutch), [http://www.uk.rug.nl/archief/jaargang39/11/12e.php UK (Paper of the Groningen University) (November 12, 2009)]<br />
*'''[[Team:Groningen]]''' & '''[[Team:TUDelft]]''': "<i>iGEM Gold for Dutch</i>" (Dutch), [http://www.bionieuws.nl/artikel.php?id=4918&zoek=iGEM Bionieuws (November 14, 2009)]<br />
*'''[[Team:Paris]]''' : "<i>Un gène éthique qui vaut de l'or</i>" Le Monde, (French), [http://www.lemonde.fr/planete/article/2009/11/27/un-gene-ethique-qui-vaut-de-l-or_1272940_3244.html (November 27, 2009)]<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Un concours organisé par le M.I.T : en route pour Boston ! </i>" (French), [http://www.supbiotech.fr/2009/09/boston-supbiotech-igem.html (September 08, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Les étudiants de la biotech interpellent la biologie synthétique</i>" (French), [http://www.vivagora.org/spip.php?breve210 (October 09, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>Un equipo de la UAB, en el concurso de biología sintética del MIT</i>" (Spanish), [http://www.uab.es/servlet/Satellite?cid=1096481466568&pagename=UABDivulga%2FPage%2FTemplatePageDetallArticleInvestigar&param1=1253860327385 (September 23, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>UAB to participate in the synthetic biology competition at MIT</i>" (English), [http://www.uab.es/servlet/Satellite/latest-news/news-detail/uab-to-participate-in-the-synthetic-biology-competition-at-mit-1096476786473.html?noticiaid=1253657853358 (September 23, 2009)]<br />
<br />
*'''[[Team:UNIPV-Pavia]]''': <i>"Questo è il profumo dell'amore" - L'arte della Biopoesia al MIT</i>, Repubblica (Italian), [http://www.repubblica.it/2009/11/sezioni/scienze/bio-ingegneria/bio-ingegneria/bio-ingegneria.html (September, 2009)]<br />
<br />
*'''[[Team:UNIPV-Pavia]]''': "<i>Con i «bricogeni» nasce la vita sintetica - Già realtà la produzione di farmaci e di bioetanolo </i>", Corriere (Italian), [http://archiviostorico.corriere.it/2010/febbraio/21/Con_bricogeni_nasce_vita_sintetica_co_9_100221109.shtml (February 21, 2010)]<br />
<br />
*'''[[Team:Valencia]]''': "<i>TheValencia team is awarded the Synthetic Standard Prize</i>" (Spanish), [http://www.elpais.com/articulo/sociedad/Levaduras/funcionan/pixeles/elpepuespval/20091103elpepusoc_9/Tes]<br />
<br />
*'''[[Team:Cambridge]]''': "<i>University of Cambridge team wins iGEM synthetic biology competition </i>" (English), [http://www.biotechniques.com/news/University-of-Cambridge-team-wins-iGEM-synthetic-biology-competition/biotechniques-180278.html (November 5 2009)]<br />
<br />
*'''[[Team:Virginia]]''': ''[http://www.washingtonpost.com/wp-dyn/content/article/2009/10/22/AR2009102204628.html New works of science nonfiction]'', The Washington Post (October 23, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Estafette voor bacterien'',(Dutch) Algemeen Dagblad (November 6, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette'', (Dutch) [http://noorderlicht.vpro.nl/themasites/mediaplayer/index.jsp?media=42722491&refernr=42560747&portalnr=3626936&hostname=noorderlicht&mediatype=audio&portalid=noorderlicht# Podcast van Radio 1, VPRO Noorderlicht, Annemieke Smit] (November 9, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette wint goud en award'', (Dutch) [http://www.tudelft.nl/live/pagina.jsp?id=ee44be90-289e-4efe-8a12-4636f1c86b28&lang=nl TNW Today Editie 2] (November 5, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette wint goud en award'', (Dutch) [http://www.tudelft.nl/live/pagina.jsp?id=ee44be90-289e-4efe-8a12-4636f1c86b28&lang=nl TUDelft Nieuws] (November 4, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterial relay race wins gold and award'', [http://www.tudelft.nl/live/pagina.jsp?id=26f61b2e-5a3f-40be-b737-a460b08bced7&lang=en Delft University of Technology Press release] (November 4, 2009)<br />
<br />
*'''[[Team:TorontoMaRSDiscovery]]''': ''A gem of a genetics competition'', [http://www.news.utoronto.ca/science-and-technology/a-gem-of-a-genetics-competition.html University of Toronto eBulletin] (November 23, 2009)<br />
<br />
*'''[[Team:Edinburgh]]''':"<i>Synthetic biology against land mines</i>" (Dutch), [http://www.c2w.nl/synthetische-biologie-tegen-landmijnen.72122.lynkx C<sub>2</sub>W Life Sciences (November 17 2009)]<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Pretty Fly (for a Small Guy)</i>" (English), [http://www.scienceline.org/2009/11/14/blog-bleicher-genetically-engineered-machines/ Scienceline Magazine] (Nov 14, 2009)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Gabriel See builds robot for MIT engineering Competition</i>" (English), [http://sammamishreview.com/2009/11/25/gabriel-see-builds-robot-for-mit-engineering-competition Sammamish Review] (Nov 25, 2009)<br />
<br />
*'''[[Team:Washington]] and [[Team:Washington-Software]]''':"<i>Gold, silver medals in UW teams in the International Genetically Engineered Machine Competition</i>" (English), [http://uwnews.org/uweek/article.aspx?id=53790 University Week] (Nov 19, 2009)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Seven Grader Scores in College Research Competition</i>" (English), [http://www.lwsd.org/News/News-from-the-Schools/Pages/12-07-2009-News-from-the-Schools.aspx News from Schools] (Dec 7, 2009)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>iGEM genius</i>" (English), [http://scienceblogs.com/oscillator/2010/01/igem_geniuses.php Oscillator] (Jan 24, 2010)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Hidden gems in iGEM</i>" (English), [http://blog.ginkgobioworks.com/2009/11/03/hidden-gems-of-igem/ Gingkoo, Ginko Bioworks] (Nov 3, 2009)</div>110hshttp://2009.igem.org/IGEM_PublicityIGEM Publicity2010-06-21T08:21:36Z<p>110hs: /* team specific */</p>
<hr />
<div>__NOTOC__<br />
[https://2008.igem.org/IGEM_Publicity iGEM 2008 Publicity] | [http://parts.mit.edu/igem07/index.php/Media iGEM 2007 Publicity] | [http://parts.mit.edu/wiki/index.php/IGEM_News iGEM 2006 Publicity]<br />
<br />
<div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [[Press_Kit | iGEM Press Kit]])</div><br />
<br />
<html><br />
<iframe frameborder="0" width="728" height="90" marginwidth="0" marginheight="0" style="margin:0px 0px 25px 100px;"<br />
src="http://www.google.com/uds/modules/elements/newsshow/iframe.html?q=iGEM&rsz=large&format=728x90"><br />
</iframe><br />
</html><br />
<br />
<br />
<span style="color:#1e90ff; font-size:175%">'''blogs </span><span style="color:#3cb371; font-size:175%">covering iGEM 2009'''</span><br />
* The [http://igem.sdu.dk/ <span style="color:#453221; font-size: 130%">'''SDU-Denmark'''</span>] team blog about their iGEM experience.<br />
* A blog about the iGEM project of the [http://aboutgmos.org/iGEM <span style="color:#453221; font-size: 130%">'''Uppsala-Sweden'''</span>] team.<br />
<br />
<br />
<br />
<br />
====<font size=5><font color=dodgerblue>'''video/radio</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
*'''Slovenia, Heidelberg, SDU-Denmark''': [http://www.dradio.de/dlf/sendungen/forschak/1062608/ Die Biobastler von Boston], Nov 2, 2009, Deutschlandfunk radio (in German).<br />
*'''Cambridge, Imperial''': [http://www.sciencefriday.com/program/archives/200911063 Synthetic Biology Competition], Nov 6, 2009, NPR Science Friday (in English).<br />
<br />
<br />
<br />
<br />
====<font size=5><font color=dodgerblue>'''news articles</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
<br />
<br />
If you would like to share an article that was written about iGEM or your iGEM team, please link to it on this page. If you have multiple articles featuring your team, link to them all individually!<br />
<br />
Post the name of your team, the title of your article, where it was featured, and provide a link to it. <br />
<br />
''Example'':<br> <br />
'''Team Example''': ''Title of article'', Nature, [link]<br />
<br />
====<font size=4><font color=dodgerblue>'''general'''</font></font>====<br />
*'''The Economist''': ''Biohacking: Hacking goes squishy'' Sep 3, 2009 [http://www.economist.com/search/displaystory.cfm?story_id=14299634 The Economist]<br />
*'''The Scientist''': ''Brick by Brick'' Feb 1, 2009. [http://www.the-scientist.com/article/display/55378/ The Scientist]<br />
*'''Technology Review''': ''A Genetically Engineered Rainbow of Bacteria'' Nov 03, 2009. [http://www.technologyreview.com/blog/editors/24351/ Technology Review]<br />
*'''Discovery News''': '' Bright Bacteria Wins Synthetic Biology Competition'' Nov 6, 2009. [http://blogs.discoverychannel.co.uk/discovery-news/2009/11/bright-bacteria-wins-synthetic-biology-competition.html Discovery News]<br />
*'''Wired''': '' Building new life forms at the iGEM Jamboree'' Nov 9, 2009. [http://www.wired.co.uk/news/archive/2009-11/09/building-new-life-forms-at-the-igem-jamboree.aspx Wired]<br />
*'''Nature Biotechnology''': ''Building outside of the box: iGEM and the BioBricks Foundation'' [http://www.nature.com/nbt/journal/v27/n12/full/nbt1209-1099.html Nature Biotechnology 27, 1099 - 1102 (2009)]<br />
*'''Nature Biotechnology''': ''About the cover'' [http://www.nature.com/nbt/journal/v27/n12/covers/index.html Nature Biotechnology 27(12), cover (2009)]<br />
<br />
<br />
====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
*'''[[Team:IBB_Pune]]''': "<i>Bio-champs in the making</i>" (English), [http://www.punemirror.in/index.aspx?Page=article&sectname=News%20-%20City&sectid=2&contentid=200909142009091423331646a131eacc#ftr2 (Monday, September 14, 2009 at 11:33:25 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Ganiti Prakriyanmadhe Jeevanuncha Vaapar! (Use of Bacteria in mathematical devices)</i>" (Marathi), [http://beta.esakal.com/2009/09/09220618/pune-use-of-bacterias-in-maths.html(Wednesday, September 09th, 2009 AT 10:09 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Synthetic Biologiteel Bharari ("Advances in Synthetic Biology")</i>" (Marathi), [https://2009.igem.org/Team:IBB_Pune/press (Wednesday 7 October 2009)]<br />
<br />
*'''[[Team:Groningen]]''': "<i>Students from Groningen in competition for best bacteria</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*'''[[Team:Groningen]]''': "<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Team iGEM Groningen has been awarded a Gold Medal</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen iGEM 2009 participants</i>" ([http://www.rug.nl/corporate/nieuws/archief/archief2009/persberichten/174_09?lang=en English]/[http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Dutch]), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09?lang=en Press release University Groningen (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>With a pacmanbacterium in de finals</i>" (Dutch), [http://www.uk.rug.nl/archief/jaargang39/11/12e.php UK (Paper of the Groningen University) (November 12, 2009)]<br />
*'''[[Team:Groningen]]''' & '''[[Team:TUDelft]]''': "<i>iGEM Gold for Dutch</i>" (Dutch), [http://www.bionieuws.nl/artikel.php?id=4918&zoek=iGEM Bionieuws (November 14, 2009)]<br />
*'''[[Team:Paris]]''' : "<i>Un gène éthique qui vaut de l'or</i>" Le Monde, (French), [http://www.lemonde.fr/planete/article/2009/11/27/un-gene-ethique-qui-vaut-de-l-or_1272940_3244.html (November 27, 2009)]<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Un concours organisé par le M.I.T : en route pour Boston ! </i>" (French), [http://www.supbiotech.fr/2009/09/boston-supbiotech-igem.html (September 08, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Les étudiants de la biotech interpellent la biologie synthétique</i>" (French), [http://www.vivagora.org/spip.php?breve210 (October 09, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>Un equipo de la UAB, en el concurso de biología sintética del MIT</i>" (Spanish), [http://www.uab.es/servlet/Satellite?cid=1096481466568&pagename=UABDivulga%2FPage%2FTemplatePageDetallArticleInvestigar&param1=1253860327385 (September 23, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>UAB to participate in the synthetic biology competition at MIT</i>" (English), [http://www.uab.es/servlet/Satellite/latest-news/news-detail/uab-to-participate-in-the-synthetic-biology-competition-at-mit-1096476786473.html?noticiaid=1253657853358 (September 23, 2009)]<br />
<br />
*'''[[Team:UNIPV-Pavia]]''': <i>"Questo è il profumo dell'amore" - L'arte della Biopoesia al MIT</i>, Repubblica (Italian), [http://www.repubblica.it/2009/11/sezioni/scienze/bio-ingegneria/bio-ingegneria/bio-ingegneria.html (September, 2009)]<br />
<br />
*'''[[Team:UNIPV-Pavia]]''': "<i>Con i «bricogeni» nasce la vita sintetica - Già realtà la produzione di farmaci e di bioetanolo </i>", Corriere (Italian), [http://archiviostorico.corriere.it/2010/febbraio/21/Con_bricogeni_nasce_vita_sintetica_co_9_100221109.shtml (February 21, 2010)]<br />
<br />
*'''[[Team:Valencia]]''': "<i>TheValencia team is awarded the Synthetic Standard Prize</i>" (Spanish), [http://www.elpais.com/articulo/sociedad/Levaduras/funcionan/pixeles/elpepuespval/20091103elpepusoc_9/Tes]<br />
<br />
*'''[[Team:Cambridge]]''': "<i>University of Cambridge team wins iGEM synthetic biology competition </i>" (English), [http://www.biotechniques.com/news/University-of-Cambridge-team-wins-iGEM-synthetic-biology-competition/biotechniques-180278.html (November 5 2009)]<br />
<br />
*'''[[Team:Virginia]]''': ''[http://www.washingtonpost.com/wp-dyn/content/article/2009/10/22/AR2009102204628.html New works of science nonfiction]'', The Washington Post (October 23, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Estafette voor bacterien'',(Dutch) Algemeen Dagblad (November 6, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette'', (Dutch) [http://noorderlicht.vpro.nl/themasites/mediaplayer/index.jsp?media=42722491&refernr=42560747&portalnr=3626936&hostname=noorderlicht&mediatype=audio&portalid=noorderlicht# Podcast van Radio 1, VPRO Noorderlicht, Annemieke Smit] (November 9, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette wint goud en award'', (Dutch) [http://www.tudelft.nl/live/pagina.jsp?id=ee44be90-289e-4efe-8a12-4636f1c86b28&lang=nl TNW Today Editie 2] (November 5, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette wint goud en award'', (Dutch) [http://www.tudelft.nl/live/pagina.jsp?id=ee44be90-289e-4efe-8a12-4636f1c86b28&lang=nl TUDelft Nieuws] (November 4, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterial relay race wins gold and award'', [http://www.tudelft.nl/live/pagina.jsp?id=26f61b2e-5a3f-40be-b737-a460b08bced7&lang=en Delft University of Technology Press release] (November 4, 2009)<br />
<br />
*'''[[Team:TorontoMaRSDiscovery]]''': ''A gem of a genetics competition'', [http://www.news.utoronto.ca/science-and-technology/a-gem-of-a-genetics-competition.html University of Toronto eBulletin] (November 23, 2009)<br />
<br />
*'''[[Team:Edinburgh]]''':"<i>Synthetic biology against land mines</i>" (Dutch), [http://www.c2w.nl/synthetische-biologie-tegen-landmijnen.72122.lynkx C<sub>2</sub>W Life Sciences (November 17 2009)]<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Pretty Fly (for a Small Guy)</i>" (English), [http://www.scienceline.org/2009/11/14/blog-bleicher-genetically-engineered-machines/ Scienceline Magazine] (Nov 14, 2009)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Gabriel See builds robot for MIT engineering Competition</i>" (English), [http://sammamishreview.com/2009/11/25/gabriel-see-builds-robot-for-mit-engineering-competition Sammamish Review] (Nov 25, 2009)<br />
<br />
*'''[[Team:Washington]] and [[Team:Washington-Software]]''':"<i>Gold, silver medals in UW teams in the International Genetically Engineered Machine Competition</i>" (English), [http://uwnews.org/uweek/article.aspx?id=53790 University Week] (Nov 19, 2009)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Seven Grader Scores in College Research Competition</i>" (English), [http://www.lwsd.org/News/News-from-the-Schools/Pages/12-07-2009-News-from-the-Schools.aspx News from Schools] (Dec 7, 2009)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>iGEM genius</i>" (English), [http://scienceblogs.com/oscillator/2010/01/igem_geniuses.php Oscillator] (Jan 24, 2010)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Hidden gems in iGEM</i>" (English), [http://blog.ginkgobioworks.com/2009/11/03/hidden-gems-of-igem/ Gingkoo, Ginko Bioworks] (Nov 3, 2009)</div>110hshttp://2009.igem.org/IGEM_PublicityIGEM Publicity2010-06-21T08:18:07Z<p>110hs: /* team specific */</p>
<hr />
<div>__NOTOC__<br />
[https://2008.igem.org/IGEM_Publicity iGEM 2008 Publicity] | [http://parts.mit.edu/igem07/index.php/Media iGEM 2007 Publicity] | [http://parts.mit.edu/wiki/index.php/IGEM_News iGEM 2006 Publicity]<br />
<br />
<div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [[Press_Kit | iGEM Press Kit]])</div><br />
<br />
<html><br />
<iframe frameborder="0" width="728" height="90" marginwidth="0" marginheight="0" style="margin:0px 0px 25px 100px;"<br />
src="http://www.google.com/uds/modules/elements/newsshow/iframe.html?q=iGEM&rsz=large&format=728x90"><br />
</iframe><br />
</html><br />
<br />
<br />
<span style="color:#1e90ff; font-size:175%">'''blogs </span><span style="color:#3cb371; font-size:175%">covering iGEM 2009'''</span><br />
* The [http://igem.sdu.dk/ <span style="color:#453221; font-size: 130%">'''SDU-Denmark'''</span>] team blog about their iGEM experience.<br />
* A blog about the iGEM project of the [http://aboutgmos.org/iGEM <span style="color:#453221; font-size: 130%">'''Uppsala-Sweden'''</span>] team.<br />
<br />
<br />
<br />
<br />
====<font size=5><font color=dodgerblue>'''video/radio</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
*'''Slovenia, Heidelberg, SDU-Denmark''': [http://www.dradio.de/dlf/sendungen/forschak/1062608/ Die Biobastler von Boston], Nov 2, 2009, Deutschlandfunk radio (in German).<br />
*'''Cambridge, Imperial''': [http://www.sciencefriday.com/program/archives/200911063 Synthetic Biology Competition], Nov 6, 2009, NPR Science Friday (in English).<br />
<br />
<br />
<br />
<br />
====<font size=5><font color=dodgerblue>'''news articles</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
<br />
<br />
If you would like to share an article that was written about iGEM or your iGEM team, please link to it on this page. If you have multiple articles featuring your team, link to them all individually!<br />
<br />
Post the name of your team, the title of your article, where it was featured, and provide a link to it. <br />
<br />
''Example'':<br> <br />
'''Team Example''': ''Title of article'', Nature, [link]<br />
<br />
====<font size=4><font color=dodgerblue>'''general'''</font></font>====<br />
*'''The Economist''': ''Biohacking: Hacking goes squishy'' Sep 3, 2009 [http://www.economist.com/search/displaystory.cfm?story_id=14299634 The Economist]<br />
*'''The Scientist''': ''Brick by Brick'' Feb 1, 2009. [http://www.the-scientist.com/article/display/55378/ The Scientist]<br />
*'''Technology Review''': ''A Genetically Engineered Rainbow of Bacteria'' Nov 03, 2009. [http://www.technologyreview.com/blog/editors/24351/ Technology Review]<br />
*'''Discovery News''': '' Bright Bacteria Wins Synthetic Biology Competition'' Nov 6, 2009. [http://blogs.discoverychannel.co.uk/discovery-news/2009/11/bright-bacteria-wins-synthetic-biology-competition.html Discovery News]<br />
*'''Wired''': '' Building new life forms at the iGEM Jamboree'' Nov 9, 2009. [http://www.wired.co.uk/news/archive/2009-11/09/building-new-life-forms-at-the-igem-jamboree.aspx Wired]<br />
*'''Nature Biotechnology''': ''Building outside of the box: iGEM and the BioBricks Foundation'' [http://www.nature.com/nbt/journal/v27/n12/full/nbt1209-1099.html Nature Biotechnology 27, 1099 - 1102 (2009)]<br />
*'''Nature Biotechnology''': ''About the cover'' [http://www.nature.com/nbt/journal/v27/n12/covers/index.html Nature Biotechnology 27(12), cover (2009)]<br />
<br />
<br />
====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
*'''[[Team:IBB_Pune]]''': "<i>Bio-champs in the making</i>" (English), [http://www.punemirror.in/index.aspx?Page=article&sectname=News%20-%20City&sectid=2&contentid=200909142009091423331646a131eacc#ftr2 (Monday, September 14, 2009 at 11:33:25 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Ganiti Prakriyanmadhe Jeevanuncha Vaapar! (Use of Bacteria in mathematical devices)</i>" (Marathi), [http://beta.esakal.com/2009/09/09220618/pune-use-of-bacterias-in-maths.html(Wednesday, September 09th, 2009 AT 10:09 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Synthetic Biologiteel Bharari ("Advances in Synthetic Biology")</i>" (Marathi), [https://2009.igem.org/Team:IBB_Pune/press (Wednesday 7 October 2009)]<br />
<br />
*'''[[Team:Groningen]]''': "<i>Students from Groningen in competition for best bacteria</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*'''[[Team:Groningen]]''': "<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Team iGEM Groningen has been awarded a Gold Medal</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen iGEM 2009 participants</i>" ([http://www.rug.nl/corporate/nieuws/archief/archief2009/persberichten/174_09?lang=en English]/[http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Dutch]), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09?lang=en Press release University Groningen (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>With a pacmanbacterium in de finals</i>" (Dutch), [http://www.uk.rug.nl/archief/jaargang39/11/12e.php UK (Paper of the Groningen University) (November 12, 2009)]<br />
*'''[[Team:Groningen]]''' & '''[[Team:TUDelft]]''': "<i>iGEM Gold for Dutch</i>" (Dutch), [http://www.bionieuws.nl/artikel.php?id=4918&zoek=iGEM Bionieuws (November 14, 2009)]<br />
*'''[[Team:Paris]]''' : "<i>Un gène éthique qui vaut de l'or</i>" Le Monde, (French), [http://www.lemonde.fr/planete/article/2009/11/27/un-gene-ethique-qui-vaut-de-l-or_1272940_3244.html (November 27, 2009)]<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Un concours organisé par le M.I.T : en route pour Boston ! </i>" (French), [http://www.supbiotech.fr/2009/09/boston-supbiotech-igem.html (September 08, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Les étudiants de la biotech interpellent la biologie synthétique</i>" (French), [http://www.vivagora.org/spip.php?breve210 (October 09, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>Un equipo de la UAB, en el concurso de biología sintética del MIT</i>" (Spanish), [http://www.uab.es/servlet/Satellite?cid=1096481466568&pagename=UABDivulga%2FPage%2FTemplatePageDetallArticleInvestigar&param1=1253860327385 (September 23, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>UAB to participate in the synthetic biology competition at MIT</i>" (English), [http://www.uab.es/servlet/Satellite/latest-news/news-detail/uab-to-participate-in-the-synthetic-biology-competition-at-mit-1096476786473.html?noticiaid=1253657853358 (September 23, 2009)]<br />
<br />
*'''[[Team:UNIPV-Pavia]]''': <i>"Questo è il profumo dell'amore" - L'arte della Biopoesia al MIT</i> (Italian), [http://www.repubblica.it/2009/11/sezioni/scienze/bio-ingegneria/bio-ingegneria/bio-ingegneria.html (September, 2009)]<br />
<br />
*'''[[Team:UNIPV-Pavia]]''': "<i>Con i «bricogeni» nasce la vita sintetica - Già realtà la produzione di farmaci e di bioetanolo </i>" (Italian), [http://archiviostorico.corriere.it/2010/febbraio/21/Con_bricogeni_nasce_vita_sintetica_co_9_100221109.shtml (February 21, 2010)]<br />
<br />
*'''[[Team:Valencia]]''': "<i>TheValencia team is awarded the Synthetic Standard Prize</i>" (Spanish), [http://www.elpais.com/articulo/sociedad/Levaduras/funcionan/pixeles/elpepuespval/20091103elpepusoc_9/Tes]<br />
<br />
*'''[[Team:Cambridge]]''': "<i>University of Cambridge team wins iGEM synthetic biology competition </i>" (English), [http://www.biotechniques.com/news/University-of-Cambridge-team-wins-iGEM-synthetic-biology-competition/biotechniques-180278.html (November 5 2009)]<br />
<br />
*'''[[Team:Virginia]]''': ''[http://www.washingtonpost.com/wp-dyn/content/article/2009/10/22/AR2009102204628.html New works of science nonfiction]'', The Washington Post (October 23, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Estafette voor bacterien'',(Dutch) Algemeen Dagblad (November 6, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette'', (Dutch) [http://noorderlicht.vpro.nl/themasites/mediaplayer/index.jsp?media=42722491&refernr=42560747&portalnr=3626936&hostname=noorderlicht&mediatype=audio&portalid=noorderlicht# Podcast van Radio 1, VPRO Noorderlicht, Annemieke Smit] (November 9, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette wint goud en award'', (Dutch) [http://www.tudelft.nl/live/pagina.jsp?id=ee44be90-289e-4efe-8a12-4636f1c86b28&lang=nl TNW Today Editie 2] (November 5, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette wint goud en award'', (Dutch) [http://www.tudelft.nl/live/pagina.jsp?id=ee44be90-289e-4efe-8a12-4636f1c86b28&lang=nl TUDelft Nieuws] (November 4, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterial relay race wins gold and award'', [http://www.tudelft.nl/live/pagina.jsp?id=26f61b2e-5a3f-40be-b737-a460b08bced7&lang=en Delft University of Technology Press release] (November 4, 2009)<br />
<br />
*'''[[Team:TorontoMaRSDiscovery]]''': ''A gem of a genetics competition'', [http://www.news.utoronto.ca/science-and-technology/a-gem-of-a-genetics-competition.html University of Toronto eBulletin] (November 23, 2009)<br />
<br />
*'''[[Team:Edinburgh]]''':"<i>Synthetic biology against land mines</i>" (Dutch), [http://www.c2w.nl/synthetische-biologie-tegen-landmijnen.72122.lynkx C<sub>2</sub>W Life Sciences (November 17 2009)]<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Pretty Fly (for a Small Guy)</i>" (English), [http://www.scienceline.org/2009/11/14/blog-bleicher-genetically-engineered-machines/ Scienceline Magazine] (Nov 14, 2009)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Gabriel See builds robot for MIT engineering Competition</i>" (English), [http://sammamishreview.com/2009/11/25/gabriel-see-builds-robot-for-mit-engineering-competition Sammamish Review] (Nov 25, 2009)<br />
<br />
*'''[[Team:Washington]] and [[Team:Washington-Software]]''':"<i>Gold, silver medals in UW teams in the International Genetically Engineered Machine Competition</i>" (English), [http://uwnews.org/uweek/article.aspx?id=53790 University Week] (Nov 19, 2009)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Seven Grader Scores in College Research Competition</i>" (English), [http://www.lwsd.org/News/News-from-the-Schools/Pages/12-07-2009-News-from-the-Schools.aspx News from Schools] (Dec 7, 2009)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>iGEM genius</i>" (English), [http://scienceblogs.com/oscillator/2010/01/igem_geniuses.php Oscillator] (Jan 24, 2010)<br />
<br />
*'''[[Team:Washington-Software]]''':"<i>Hidden gems in iGEM</i>" (English), [http://blog.ginkgobioworks.com/2009/11/03/hidden-gems-of-igem/ Gingkoo, Ginko Bioworks] (Nov 3, 2009)</div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/SponsorsTeam:UNIPV-Pavia/Sponsors2010-06-19T14:30:57Z<p>110hs: </p>
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<br/><b><font size="6">Institutional Sponsors</font></b><br/><br/><hr/><br />
</td><br />
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<td width="33%"><br />
<a href="http://www.labmedinfo.org/" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2009/8/8e/Unipv_Lmi_logo.gif" width="220px" height="110px"/><br />
</a><br />
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<a href="http://cit.unipv.it/cit/" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2008/5/5d/Cit_logo.jpg"/><br />
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<b><big><u>Platinum</u>:</big></b><br />
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<a href="http://www.siram.it" target="_blank"><br />
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<img src="https://static.igem.org/mediawiki/2009/d/d8/Iuss_logo.gif" width="225px" height="188px"/><br />
</a><br />
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<hr/><br />
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<td><br />
<b><big><u>Gold</u>:</big></b><br />
</td><br />
<td><br />
<a href="http://digilander.libero.it/fondazionecosta/index.htm" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2009/3/34/Fondazione_Costa.gif"/><br />
</a><br />
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<td><br />
<a href="http://www.tecan.com/" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2009/c/cf/LogoTecan.gif" width="220px" height="80px"/><br />
</a><br />
</td><br />
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<b><big><u>Silver</u>:</big></b><br />
</td><br />
<td><br />
<a href="http://www.bmr-genomics.it/" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2009/f/fd/Logo_BMR.png"/><br />
</a><br />
</td><br />
<td/><br />
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<b><big><u>Bronze</u>:</big></b><br />
</td><br />
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<img src="https://static.igem.org/mediawiki/2009/2/22/CLU.jpg" width="100%"/><br />
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<td><br />
<big><b>SPIGA S.R.L.</b></big><br />
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</table></div>110hshttp://2009.igem.org/Template:UNIPV-Pavia/MenuTemplate:UNIPV-Pavia/Menu2010-05-21T20:35:57Z<p>110hs: </p>
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[[Image:EthanolPVanimation.gif]]</div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Project/SolutionTeam:UNIPV-Pavia/Project/Solution2009-10-22T00:07:09Z<p>110hs: /* SYSTEM OVERVIEW */</p>
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<font face="Pristina" size="50" ><b>Solution</b></font><br />
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<br />
Our aim is to merge and optimize the main features of naturally occurring lactose-cleaving and ethanol-producing microorganisms to build up an engineered biological system that can metabolize lactose and can ferment it in ethanol with high yield. Here we provide an overview of the main enzymes and biochemical pathways involved in these two processes.<br />
<br />
==MICROBIAL SOURCES OF LACTOSE==<br />
The complete lactose to ethanol transformation pathway is summarized in the following figure.<br />
<br />
{|align="center"<br />
|[[Image:Lactose_pathway.JPG|thumb|500px|Lactose to ethanol transformation pathway]]<br />
|}<br />
<br />
The first task of conversion is provided by β-D-Galactosidase. It can be obtained from a wide variety of sources such as microorganisms, plants and animals; however, according to its source, its properties differ markedly.<br />
<br />
{|align="center"<br />
|[[Image:B-galactosidase.jpg|thumb|500px|β-galactosidase]]<br />
|}<br />
<br />
The products of lactose hydrolysis may inhibit β-galactosidase action. Galactose is often a competitive inhibitor but glucose is usually ineffective, except at higher concentrations when it is usually non-competitive. Complete hydrolysis is therefore very difficult to achieve unless high concentrations of the enzyme are used. Yeast enzymes are generally inhibited by galactose (competitively) and glucose (non-competitively). Aspergillus niger enzyme is strongly inhibited by galactose; however, enzyme from A. oryzae is less subject to galactose inhibition.<br />
<br />
<br><br />
<font size="3"><b>Fungal enzymes</b></font><br />
<br />
These enzymes have a pH optimum in the acidic range 2.5–5.4, which makes them suitable for processing of acid whey and its ultrafiltration permeate. These have a relatively high temperature optimum and are typically used at temperatures up to 50°C, where they are reasonably stable. The production and purification of β-D-galactosidase from different fungal sources have been carried out using a variety of purification techniques. Fungal sources are: Aspergillus niger, Beauveria bassiana, Aspergillus fonsecaeus, Rhizomucor sp., Penicillium chrysogenum, Aspergillus carbonarius.<br />
<br />
<br><br />
<font size="3"><b>Yeast enzymes</b></font><br />
<br />
Yeast has been considered an important source of β-D-galactosidase from an industrial viewpoint. Yeast sources are: Kluyveromyces fragilis, Kluyveromyces lactis, Saccharomyces lactis, Saccharomyces anamensis, Kluyveromyces marxianus, recombinant yeast Saccharomyces cerevisiae.<br />
<br />
<br><br />
<font size="3"><b>Bacterial enzymes</b></font><br />
<br />
A large number of bacteria produce β-D-galactosidase, but relatively few bacterial species are regarded as safe sources. However, Streptococcus thermophilus and Bacillus stearothermophilus can be considered as potential bacterial sources. The former is eminently suitable as a source organism from the safety viewpoint because it is used as a starter culture in yoghurt and some cheeses.<br />
Other bacterial sources are: Escherichia coli, Propionibacterium shermanii, Streptococcus salivarius, Pseudoalteromonas sp., S. thermophilus, Thermoanaerobacter, Bacillus coagulans, Lactobacillus delbrueckii subsp. Bulgaricus.<br />
<br />
<br><br />
<font size="3"><b>Beta Galactosidase in <i>E. coli</i> (β-gal / gene name: LacZ)</b></font><br />
<br />
It is a tetrameric hydrolytic enzyme, coded by the LacZ gene and able to catalyse the hydrolysis of a disaccharide (β-galacotisedes) into two monosaccharides: it is, then, related to the reaction that injects new glucose into glycolysis.<br />
<br />
The <i>E. coli</i> isoform is 464K-Da weight and 1021 amino acid long. Each monomer is composed by five domains, the third of which hosts the active site. The protein can be split into two peptides, which are unable to catalyse reaction when separated. <br />
<br />
This peculiarity gives β.galactosidase a primary importance in synthetic and molecular biology, for it is used as a reporter gene, by fusing protein into the β-gal chain, disrupting the first peptide’s sequence, in a test known as “blue white screening”. A well known galactose homologue, X-Gal, turns blue when hydrolysed by this enzyme and shows whether colonies have incorporated the fusion genes or not.<br />
<br />
The active site is associated to Glu-537, that is the main actor of a nucleophil substitution between the carboxyl group of the side chain of Glu and the ketonic oxygen that links the two mono-saccharides. Result of this reaction is the substitution of an alcoholic group to the ketonic group and the following liberation of the other monosaccharide. Thus β-gal is the gateway for glucose into the glycolytic process.<br />
<br />
<br><br />
<br />
==ENGINEERING FERMENTATION USING Z. MOBILIS METABOLISM==<br />
<br />
<br><br />
<font size="3"><b><i>Zymomonas mobilis</i></b></font><br />
<br />
<i>Zymomonas mobilis</i> is a Gram-negative bacteria of the soil that has several appealing features for its use in the industrial production of ethanol. <i>Zymomonas</i> is the only known microorganism capable of oxidizing glucose anaerobically, via the Entner-Doudoroff pathway, as opposed to the classical glicolytic pathway.<br />
<br />
Unfortunately <i>Zymomonas</i> can only catabolize simple sugars as glucose, fructose and sucrose. Since our project relies on lactose catabolism, we decided to insert two <i>Zymomonas</i> key fermenting enzymes: the Pyruvate Decarboxylase (pdc) and the Alcohol Dehydrogenase II (adhB) in <i>E. coli</i>, which is, instead, capable of fermenting lactose.<br />
<br />
<i>E. coli</i> does not possess a native Pyruvate Decarboxylase: although, it uses Pyruvate Formate Lyase. This enzyme has an unbalanced consumption of NADH, and the microorganism balances it by producing acetic acid and succinic acid rather than ethanol. The insertion of <i>Zymomonas</i> pdc gene should determine the production of ethanol as only final product of the fermentation pathway.<br />
<br />
Although <i>E. coli</i> strains have Alcohol Dehydrogenase, the activity of this native enzyme is insufficient to achieve this high yield of ethanol, while <i>Zymomonas</i> adhB enzyme has a higher efficiency.<br />
<br />
<br><br />
<font size="3"><b>Pyruvate decarboxylase (PDC)</b></font><br />
<br />
Pyruvate decarboxylase is a homotetrameric enzyme (EC 4.1.1.1) that catalyses the decarboxylation of pyruvic acid to acetaldehyde and carbon dioxide. It is also called 2-oxo-acid carboxylase, alpha-ketoacid carboxylase, and pyruvic decarboxylase. In anaerobic conditions, this enzyme is part of an alternative pathway for fermentation,known as Entner-Dourdoff pathway, which is characterized by the bypass of Acetyl-CoA formation, and determines an irreversible loss of a CO<sub>2</sub> molecule, in order to produce acetaldehyde. Pyruvate decarboxylase starts this process by converting pyruvate into acetaldehyde and carbon dioxide. To do this, two thiamine pyrophosphate (TPP) and two magnesium ions are required as cofactors. This enzyme should not be mistaken for the unrelated enzyme pyruvate dehydrogenase, an oxidoreductase (EC 1.2.4.1), that catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA.<br />
<br />
<br><br />
<font size="3"><b>Alcohol dehydrogenase (ADH)</b></font><br />
<br />
They are a class of enzymes that catalyse the conversion between alcohols and aldehydes or ketons, in order to decrease the toxicity of alcoholic products.<br />
Though, in the glycolytic process it is used in the opposite direction, by converting aldehydes into alcohols. All the reactions involved are reduction or oxidations, with the contribution of nicotinamide dinucleotide (NAD), as a cofactor.<br />
<br />
ADH2 (gene name adhB) catalyses the crucial step into ethanol conversion: acetaldehyde comes from the last steps of glycolysis and the bacteria converts it into an ethanol molecule in order to gain a NAD<sup>+</sup>, as a reducing power, when in anaerobic, or microaerobial environment.<br />
<br />
Structurally, the alcohol dehydrogenase family is composed by multimeric proteins, about 300 amino acids long (ADH2 of <i>Z.mobilis</i> is by 383 amino acids and 40,145 KDa weight). The ratio between isoforms of each monomer may vary. Moreover, each unit has two zinc binding domains, where a zinc ion is the core of the active site: it settles an hydrogen bond with NAD, helped by Cys-146, Cys-174, and His-67; while the lateral chains of Phe-319, Ala-317, His-51, Ile-269 and Val-292 bind aldehyde. <br />
<br />
Deprotonation of NAD is catalysed by Ser-48, which is deprotonated by His-51. When NAD<sup>+</sup> loses its positive charge, it can reduce acetaldehyde to ethanol and become NADH.<br />
<br />
We selected <i>Zymomonas mobilis</i> as the source of the adhB transgene for implementing the conversion efficiency in the glucose-to-ethanol process, because it has been found that this microbiota follows a special glucose degradation pathway (Entner-Dourdoff pathway), that hass as only final byproducts, two ethanol molecules by each glucose, while two ATP molecules are produced.<br />
<br />
Moreover,<i> Z.mobilis</i> has another adh gene (adhA), which does not seem an isoform even if it accomplishes the same function. In addition, adhB seems more efficient in ethanol conversion than its homologue.<br />
<br />
==SYSTEM OVERVIEW==<br />
<br />
Our purpose is to build up a biological system to convert lactose into ethanol. We chose <i>E. coli</i> bacterium as the system’s chassis and we have planned to engineer an efficient lactose-ethanol conversion system using three main enzymes: beta-galactosidase, pyruvate decarboxylase and alcohol dehydrogenase II.<br />
The engineered gene network we want to build up and characterize quantitatively is shown in figure.<br />
<br />
{|align="center"<br />
|[[Image:Full_gene_network.jpg|thumb|500px|Full gene network]]<br />
|}<br />
<br />
The full characterization of this network will allow to know the efficiency and the feasibility of the entire system in cheese whey valorisation, as well as the estimation of B.O.D.5/C.O.D. parameters before and after the treatment of the waste with our engineered system.<br />
<br />
<br><br />
<font size="3"><b>Lactose conversion in glucose through beta-galactosidase</b></font><br />
<br />
<i>E. coli</i> has a well known gene system to grow in a lactose-rich and glucose-poor environment, whose main enzyme is the beta-galactosidase, which performs the conversion of lactose in glucose and galactose. The latter is then metabolized by other enzymes and is converted in glucose too.<br />
<br />
Although <i>E. coli</i> already has this system in nature, the lactose metabolism may be improved in several ways:<br />
*overexpressing the native E. coli beta-galactosidase;<br />
*engineering the expression of a more efficient heterologous beta-galactosidase;<br />
*engineering a glucose-independent lactose transport inside the cell;<br />
*limiting the metabolic burden of the strain by controlling the expression of beta-galactosidase with a lactose-inducible system just like in lac operon, but without the dependency of glucose.<br />
<br />
In this project, we want to overexpress the native E. coli enzyme under the control of a glucose-independent lactose-inducible strong promoter. We hope that this system will be able to convert lactose in glucose rapidly and without glucose inhibition. The genetic parts which can perform these functions are shown and described below.<br />
<br />
<br><br />
<font size="3"><b>Glucose-independent lactose/IPTG inducible system</b></font><br />
<br />
{|align="center"<br />
|[[Image:IPTG_inducible_system.jpg|thumb|500px|Glucose-independent lactose/IPTG inducile system]]<br />
|}<br />
<br />
BBa_R0011 is an artificial hybrid promoter derived from the lambda phage Pλ repressible strong promoter. In this hybrid promoter sequence, the cI binding sites have been replaced with lacO1 binding sites, so lacI repressor can bind and repress transcription in this promoter. Transcription can be switched on by adding lactose or IPTG, which bind lacI and repress its activity.<br />
The main advantage of using this promoter instead of the native <i>E. coli</i> lac promoter (BBa_R0010) is that the native one has a CAP binding site. So, when glucose is present in the environment the transcription is inhibited, even if lactose is present in the medium. The behaviour of the native promoter does not fit our project specifications because we want to engineer a strain which can metabolize lactose until it is over, so we used R0011 hybrid promoter.<br />
During the summer, we used BBa_J23118 constitutive promoter from UC Berkeley promoter collection to produce constantly lacI regulator, so that the lac promoter is in the “off” state until lactose or IPTG are added to the culture. Anyway, other constitutive promoters can be used for lacI production, in order to optimize the expression of beta-gal in real processes.<br />
This inducible system will be built up, characterized and submitted to the Registry as a lactose/IPTG input -> PoPS output device, which can be generalized by assembling the desired protein generator downstream.<br />
<br />
Used BioBrick parts:<br />
*<partinfo>BBa_J23118</partinfo> - <partinfo>BBa_B0034</partinfo> - <partinfo>BBa_C0012</partinfo> - <partinfo>BBa_B0015</partinfo>: pre-assembled BioBrick from Bologna iGEM 2009 team<br />
*<partinfo>BBa_R0011</partinfo>: Hybrid lacI repressible promoter<br />
<br />
<br><br />
<font size="3"><b>Beta-galactosidase protein generator</b></font><br />
<br />
{|align="center"<br />
|[[Image:Protein_generator.jpg|thumb|500px|Beta-galactosidase protein generator]]<br />
|}<br />
<br />
The gene which encodes for beta galactosidase in <i>E. coli</i> is called lacZ and it is present in the Registry as BBa_I732005. We used it in association with a strong RBS to build up a beta-galactosidase protein generator which can ensure an efficient overexpression of the enzyme (the protein generator was actually present in the Registry as BBa_I732019, but its sequence analysis gave bad results in 2008 Distribution, so we re-built it).<br />
As written above, beta-galactosidase performs the conversion of lactose in glucose and galactose; on the other hand, galactose is metabolized by the cell and converted in glucose through another pathway. So, the expression of this protein generator is the only needed step for the conversion of lactose in glucose, which is essential for the ethanol fermentation we plan to engineer in our project.<br />
<br />
In this artificial protein generator, lacZ gene can be replaced with any other beta-galactosidase genes taken from the genome of other organisms in order to compare their activity and to choose the most efficient one.<br />
<br />
Used BioBrick parts:<br />
*<partinfo>BBa_I732017</partinfo> - <partinfo>pSB1AK3</partinfo>: lacZ transcriptional unit<br />
*<partinfo>BBa_B0015</partinfo> - <partinfo>pSB1AK3</partinfo>: Double terminator<br />
<br />
<u>Future work</u><br />
<br />
Other ways to improve lactose metabolism in <i>E. coli</i> may be to:<br />
*use a more efficient heterologous beta-galactosidase;<br />
*use an engineered lactose permease to engineer a glucose-independent lactose transport, e.g. the lacY gene proposed by Caltech 2008 iGEM Team<br />
<br />
<br><br />
<font size="3"><b>Ethanol production from glucose: engineering a high efficiency fermentation in <i>E. coli</i></b></font><br />
<br />
{|align="center"<br />
|[[Image:Pyruvate.jpg|thumb|500px|Pyruvate decarboxylase and alcohol dehydrogenase generator]]<br />
|}<br />
<br />
In this project, two genes from the fermentative bacterium <i>Zymomonas mobilis</i> are used to build up an ethanol-producing operon in <i>E. coli</i>. Genes pdc and adhB, which encode for pyruvate decarboxylase and alcohol dehydrogenase respectively, will be assembled to engineer an ethanol producing system.<br />
<br />
DNA synthesis technology and the Registry of Standard Biological Parts will be used to optimize the characteristics of the operon, in particular:<br />
*the codon usage;<br />
*the ethanol yield;<br />
*the metabolic burden of the strain bearing an expressed operon.<br />
<br />
The codon usage has been optimized for <i>E. coli</i>, through DNA de novo synthesis, in order to have a high efficiency translation of pdc and adhB genes. The ethanol yield and the metabolic burden will be calibrated using different PoPS inputs.<br />
This optimized operon can be considered as the final actuator for our project: it converts glucose, the intermediate product, in ethanol, which can be used as a biofuel directly or can be converted in different biofuels, e.g. biodiesel through a transesterification process.<br />
The final version of the actuator should be controlled by a constitutive promoter in order to ensure an efficient and continuous ethanol production without any additional cost for inducer molecules. However, the strength of the promoter has to be calibrated and the assembly of the operon with a high number of promoter BioBricks can be avoided using well characterized inducible promoters. These inducible systems can be used as user-controlled knobs for gene expression.<br />
In order to compare the strengths of different promoters, a standard measurement system has been used: the Relative Promoter Units (RPUs), proposed by Kelly J. et al. (2008). The characterization of these systems would significantly contribute to Synthetic Biology community, because it would provide well-characterized and re-usable parts to the Registry. In particular, we have focused our attention on IPTG, aTc and 3OC6HSL inducible systems.<br />
<br />
Used BioBrick parts:<br />
*pdc (<partinfo>BBa_K173016</partinfo>): codon-optimized coding sequence from Mr Gene de novo synthesis service<br />
*adhB (<partinfo>BBa_K173017</partinfo>): codon-optimized coding sequence from Mr Gene de novo synthesis service<br />
*<partinfo>BBa_B0030</partinfo> - pSB1A2: RBS with efficiency 0.6<br />
*<partinfo>B0015</partinfo> - pSB1AK3: Double terminator<br />
*A promoter to be chosen<br />
<br />
<u>Future work</u><br />
*can this optimized operon be used to convert other sugars in ethanol?<br />
*can Synthetic Biology principles optimize the process of transesterification of ethanol in biodiesel?<br />
<br />
<br><br />
<font size="3"><b>Constitutive promoters and Inducible systems</b></font><br />
<br />
The final part containing pdc and adhB II has to be controlled by the best promoter, i.e. the promoter with transcriptional strength that ensures a yield as close as possible to the theoretical value.<br />
We want to build up a collection of well characterized promoters both constitutive and inducible, to use to optimize the operon yield, as well as every other device.<br />
In order to characterize inducible system behaviour, an important part of our project will be dedicated to perform quantitative experiments of these constructs.<br />
<br />
The systems we want to use are <br />
*aTc inducible device<br />
This device is based on the expression of tetR, a molecule which has the ability of inhibit the activity of ptet promoter. In presence of aTc, tetR is inactivated and ptet is free to express.<br />
We want to test different promoters to regulate the expression of the repressor molecule, so we plan to assemble several promoters of Anderson Promoters Collection. <br />
<br />
*3OC6HSL inducible device<br />
This device gives PoPS as output and can be induced with 3OC6-HSL autoinducer molecule: it binds luxR protein (encoded by <partinfo>BBa_C0062</partinfo>), which is constitutively expressed by tetR promoter (<partinfo>BBa_R0040</partinfo>). LuxR-HSL complex can work as a transcriptional activator for lux promoter (<partinfo>BBa_R0062</partinfo>).<br />
<br />
*IPTG/lactose inducible device<br />
The hybrid lac promoter (<partinfo>BBa_R0011</partinfo>) has been designed taking the Plambda promoter (<partinfo>BBa_R0051</partinfo>) and substituting its cI (<partinfo>BBa_C0051</partinfo>) binding sites with two lacI binding sites.<br />
This promoter can be repressed by lacI (<partinfo>BBa_C0012</partinfo>), which can be repressed by lactose or IPTG, providing a lactose/IPTG inducible system. Differently from wild type lac promoter, this part does not have any CAP binding sites, so its behaviour is glucose-independent.<br />
<br />
<br><br />
We plan to use these devices as knobs to calibrate promoter strenght, to determine best promoter for our operon. <br />
Our approach can be generalized to optimize the regulation of any device. <br />
We plan to characterize these device using RPU method.<br />
<br />
</td><br />
</tr><br />
</table></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1SepTeam:UNIPV-Pavia/Notebook/Week1Sep2009-10-21T23:42:01Z<p>110hs: /* September, 6th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<html><a name="week_start"></a></html><br />
= Week from August 31st, to September 6th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Aug#week_start"><br />
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</td><br />
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Next Week</a><br />
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<br />
== <html><font class="dayw_style">August, 31st</font></html> ==<br />
<br />
*We inoculated:<br />
**10 colonies from B5new2 plate in 4 ml of LB + Kan for screening;<br />
**B8-5 (8 ul from glycerol stock) in 4 ml of LB + Kan (for purification process);<br />
<br />
*We sent A15-2 purified DNA (stored at -20°C) to BMR Genomics for sequencing.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 1st</font></html> ==<br />
*We received sequencing results for:<br />
**A16-4: sequence ok!<br />
**A14-3: sequence had a deletion in C0012, as we expected because of A11-2 sequencing results, previously received;<br />
**B8-5: sequence wrong in VF2 and good in VR;<br />
**A17: chromatogram was good, but ligation failed (R0011 was not in the insert).<br />
<br />
*COMMENTS after sequencing results:<br />
**now we have a new aTc sensor (A16) to test together with A9;<br />
**B8 has to be repeated or purified, we will try both the approaches;<br />
**A14 has to be repeated using A11-3, which has a consistent sequencing result;<br />
**A17 is going to be ligated today (this time using gel extraction).<br />
<br />
<br />
<br />
<br />
*Glycerol stocks for the 10 B5new2 grown cultures.<br />
<br />
*Miniprep for B5new2 (10 samples) and for B8-5.<br />
<br />
*Digestion for:<br />
**B5new2 (10 samples) for screening (E-P cut);<br />
**B8-5 for purification from gel (E-P cut);<br />
**R0011 (stored at -20°C) for A17new ligation (S-P cut)<br />
**E0240 (stored at -20°C) for A17new ligation (X-P cut)<br />
<br />
*Medium-size gel for B5new2(E-P); small-size gel for B8-5(E-P), R0011(S-P) and E0240(X-P).<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B5new2_digestion_screening.jpg|thumb|500px|left|Digestion screening for B5new2.]]<br />
|}<br />
</font><br />
<br />
*Medium gel results: B5new2 clones all showed the correct length for ligated plasmid! we decided to keep B5new2-3 to continue the assemblies and B5new2-9 as a backup copy. Both plasmids were sent to BMR Genomics for sequencing.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B8_5_actual.jpg|thumb|500px|left|B8-5 construct was actually a wrong plasmid: the 4Kbp fragment was non digested DNA. Sequencing results will tell us what there is inside the cloning site.]]<br />
|}<br />
</font><br />
<br />
*Small gel results: R0011(S-P) and E0240(X-P) were cut and purified through gel-extraction, while B8-5 could not be cut because the bands were not correct: maybe the heaviest band noticed on August 24th was a non-completely digested fragment, not a correct-size insert. We decided to throw B8-5 glycerol stock away.<br />
<br />
*Ligation: A17new = R0011(S-P) + E0240(X-P) in pSB1A2<br />
<br />
*We incubated ligation reaction at 16°C overnight.<br />
<br />
<br />
<br />
*We inoculated:<br />
**B5new2-3<br />
**B6-3<br />
**A4<br />
**F2620MIT1<br />
**A11-3<br />
<br />
<br />
<br />
===== pH sensor =====<br />
*We streaked a single colony LB + Amp agar plate using (the right) K116002 glycerol stock. We incubated the plate at 37°C overnight.<br />
*Preparation of LBK (LB with potassium instead of sodium) at pH 5,5 - 6,6 - 7,5 - 8,5 for the first experiment we would have performed in the following days.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 2nd</font></html> ==<br />
<br />
*Miniprep for:<br />
**B5new2-3<br />
**B6-3<br />
**A11-3<br />
**A4<br />
**F2620MIT1<br />
**E0240 pellet (stored at -20°C)<br />
<br />
*Digestions:<br />
**B5new2-3(E-X)<br />
**B6-3(E-X)<br />
**A11-3(S-P)<br />
**A4(E-S)<br />
**F2620MIT1(E-S)<br />
**E0240(X-P)<br />
<br />
*Gel run, cut and band purification for all the samples.<br />
<br />
*Ligations:<br />
**B7new = A4(E-S) + B5new2-3(E-X) in pSB1AK3<br />
**B8new = A4(E-S) + B6-3(E-X) in pSB1AK3<br />
**B9 = F2620MIT1(E-S) + B5new2-3(E-X) in pSB1AK3<br />
**B10 = F2620MIT1(E-S) + B6-3(E-X) in pSB1AK3<br />
**A14L = A11-3(S-P) + E0240(X-P) in pSB1A2<br />
<br />
*We incubated the five reactions at 16°C overnight.<br />
<br />
<br />
<br />
<br />
*Transformation/plating for A17new ligation.<br />
<br />
<br />
<br />
===== pH sensor =====<br />
*We inoculated a single colony from K116002 streaked plate in 5 ml of LB + Amp.<br />
*Overnight incubation at 37°C, 220 rpm.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 3rd</font></html> ==<br />
<br />
*A17new overnight plate showed only one colony...easy screening for this plate! Colony PCR/electrophoresis for this sample. We stored this colony in 100ul of LB + Amp and incubated it at 37°C waiting for the end of the reaction.<br />
<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_pcr_A17new.jpg|thumb|500px|left|Colony PCR results for the only A17new colony on its plate.]]<br />
|}<br />
</font><br />
<br />
*Gel results: the blank was not so clean, but the only colony was positive:) We inoculated the colony in 5 ml of LB + Amp to grow an overnight culture (37°C, 220 rpm).<br />
<br />
<br />
<br />
*We transformed these ligations:<br />
**B7new 1:40 on Kan<br />
**B8new 1:40 on Kan<br />
**B9 1:40 on Kan<br />
**B10 1:40 on Kan<br />
**A14L 1:20 on Amp<br />
<br />
*We incubated B7new, B8new, B9 and B10 at 37°C in the morning. Then, five colonies for each plate were picked after about 11 hours. We inoculated these colonies in 5 ml of LB + Kan and incubated them overnight (37°C, 220 rpm). NOTE: B7new and B8new had smaller colonies than B9 and B10.<br />
<br />
*We incubated A14L plate overnight at 37°C.<br />
<br />
<br />
===== pH sensor =====<br />
*Miniprep for K116002 overnight culture. We incubated the plate (LB + Amp) with transformed bacteria overnight at 37°C. <br />
*We used 1 ul of this sample to transform TOP10 in order to have the pH sensor in the same strain as the reference promoter (i.e. A2).<br />
*We plated transformed bacteria and incubated overnight at 37°C.<br />
*We also sent purified DNA to BMR Genomics for sequencing.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 4th</font></html> ==<br />
<br />
*A14L plate showed colonies.<br />
<br />
*Colony PCR/electrophoresis for 6 colonies from A14L plate. Colonies were saved inoculating them in 1 ml of LB + Amp and incubating them at 37°C, 220 rpm waiting for the end of the reaction.<br />
<br />
*Gel results: all the colonies showed the amplicon with the right size, but even some extra band...we decided to stock all the colonies in glycerol. We also decided to keep A14L-1: we inoculated it in 5 ml of LB + Amp to grow an overnight culture to check the sequence.<br />
<br />
<br />
<br />
<br />
*We received sequencing results for B8-5again(VF2) and it was not correct.<br />
<br />
<br />
<br />
*Glycerol stock/miniprep for 21 cultures:<br />
**B7new X5 colonies<br />
**B8new X5 colonies<br />
**B9 X5 colonies<br />
**B10 X5 colonies<br />
**A17new<br />
<br />
*We sent A17new purified DNA to BMR Genomics for sequencing.<br />
<br />
*Screening (E-P digestion cut) for B7new, B8new, B9 and B10 samples.<br />
<br />
<br />
===== pH sensor =====<br />
*K116002(TOP10) plate showed a bacterial carpet (with some single colony).<br />
*We inoculated a single colony from K116002 (TOP10) plate in 1 ml of LB + Amp and incubated this inoculum for about 6 hours (37°C, 220 rpm).<br />
*Glycerol stock.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 5th</font></html> ==<br />
*Fermentation experiment setup<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 6th</font></html> ==<br />
*Fermentation experiment<br />
*Cloning<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Aug#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Aug#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
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</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1SepTeam:UNIPV-Pavia/Notebook/Week1Sep2009-10-21T23:41:47Z<p>110hs: /* September, 5th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<html><a name="week_start"></a></html><br />
= Week from August 31st, to September 6th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Aug#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Aug#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">August, 31st</font></html> ==<br />
<br />
*We inoculated:<br />
**10 colonies from B5new2 plate in 4 ml of LB + Kan for screening;<br />
**B8-5 (8 ul from glycerol stock) in 4 ml of LB + Kan (for purification process);<br />
<br />
*We sent A15-2 purified DNA (stored at -20°C) to BMR Genomics for sequencing.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 1st</font></html> ==<br />
*We received sequencing results for:<br />
**A16-4: sequence ok!<br />
**A14-3: sequence had a deletion in C0012, as we expected because of A11-2 sequencing results, previously received;<br />
**B8-5: sequence wrong in VF2 and good in VR;<br />
**A17: chromatogram was good, but ligation failed (R0011 was not in the insert).<br />
<br />
*COMMENTS after sequencing results:<br />
**now we have a new aTc sensor (A16) to test together with A9;<br />
**B8 has to be repeated or purified, we will try both the approaches;<br />
**A14 has to be repeated using A11-3, which has a consistent sequencing result;<br />
**A17 is going to be ligated today (this time using gel extraction).<br />
<br />
<br />
<br />
<br />
*Glycerol stocks for the 10 B5new2 grown cultures.<br />
<br />
*Miniprep for B5new2 (10 samples) and for B8-5.<br />
<br />
*Digestion for:<br />
**B5new2 (10 samples) for screening (E-P cut);<br />
**B8-5 for purification from gel (E-P cut);<br />
**R0011 (stored at -20°C) for A17new ligation (S-P cut)<br />
**E0240 (stored at -20°C) for A17new ligation (X-P cut)<br />
<br />
*Medium-size gel for B5new2(E-P); small-size gel for B8-5(E-P), R0011(S-P) and E0240(X-P).<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B5new2_digestion_screening.jpg|thumb|500px|left|Digestion screening for B5new2.]]<br />
|}<br />
</font><br />
<br />
*Medium gel results: B5new2 clones all showed the correct length for ligated plasmid! we decided to keep B5new2-3 to continue the assemblies and B5new2-9 as a backup copy. Both plasmids were sent to BMR Genomics for sequencing.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B8_5_actual.jpg|thumb|500px|left|B8-5 construct was actually a wrong plasmid: the 4Kbp fragment was non digested DNA. Sequencing results will tell us what there is inside the cloning site.]]<br />
|}<br />
</font><br />
<br />
*Small gel results: R0011(S-P) and E0240(X-P) were cut and purified through gel-extraction, while B8-5 could not be cut because the bands were not correct: maybe the heaviest band noticed on August 24th was a non-completely digested fragment, not a correct-size insert. We decided to throw B8-5 glycerol stock away.<br />
<br />
*Ligation: A17new = R0011(S-P) + E0240(X-P) in pSB1A2<br />
<br />
*We incubated ligation reaction at 16°C overnight.<br />
<br />
<br />
<br />
*We inoculated:<br />
**B5new2-3<br />
**B6-3<br />
**A4<br />
**F2620MIT1<br />
**A11-3<br />
<br />
<br />
<br />
===== pH sensor =====<br />
*We streaked a single colony LB + Amp agar plate using (the right) K116002 glycerol stock. We incubated the plate at 37°C overnight.<br />
*Preparation of LBK (LB with potassium instead of sodium) at pH 5,5 - 6,6 - 7,5 - 8,5 for the first experiment we would have performed in the following days.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 2nd</font></html> ==<br />
<br />
*Miniprep for:<br />
**B5new2-3<br />
**B6-3<br />
**A11-3<br />
**A4<br />
**F2620MIT1<br />
**E0240 pellet (stored at -20°C)<br />
<br />
*Digestions:<br />
**B5new2-3(E-X)<br />
**B6-3(E-X)<br />
**A11-3(S-P)<br />
**A4(E-S)<br />
**F2620MIT1(E-S)<br />
**E0240(X-P)<br />
<br />
*Gel run, cut and band purification for all the samples.<br />
<br />
*Ligations:<br />
**B7new = A4(E-S) + B5new2-3(E-X) in pSB1AK3<br />
**B8new = A4(E-S) + B6-3(E-X) in pSB1AK3<br />
**B9 = F2620MIT1(E-S) + B5new2-3(E-X) in pSB1AK3<br />
**B10 = F2620MIT1(E-S) + B6-3(E-X) in pSB1AK3<br />
**A14L = A11-3(S-P) + E0240(X-P) in pSB1A2<br />
<br />
*We incubated the five reactions at 16°C overnight.<br />
<br />
<br />
<br />
<br />
*Transformation/plating for A17new ligation.<br />
<br />
<br />
<br />
===== pH sensor =====<br />
*We inoculated a single colony from K116002 streaked plate in 5 ml of LB + Amp.<br />
*Overnight incubation at 37°C, 220 rpm.<br />
<br />
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<br />
== <html><font class="dayw_style">September, 3rd</font></html> ==<br />
<br />
*A17new overnight plate showed only one colony...easy screening for this plate! Colony PCR/electrophoresis for this sample. We stored this colony in 100ul of LB + Amp and incubated it at 37°C waiting for the end of the reaction.<br />
<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_pcr_A17new.jpg|thumb|500px|left|Colony PCR results for the only A17new colony on its plate.]]<br />
|}<br />
</font><br />
<br />
*Gel results: the blank was not so clean, but the only colony was positive:) We inoculated the colony in 5 ml of LB + Amp to grow an overnight culture (37°C, 220 rpm).<br />
<br />
<br />
<br />
*We transformed these ligations:<br />
**B7new 1:40 on Kan<br />
**B8new 1:40 on Kan<br />
**B9 1:40 on Kan<br />
**B10 1:40 on Kan<br />
**A14L 1:20 on Amp<br />
<br />
*We incubated B7new, B8new, B9 and B10 at 37°C in the morning. Then, five colonies for each plate were picked after about 11 hours. We inoculated these colonies in 5 ml of LB + Kan and incubated them overnight (37°C, 220 rpm). NOTE: B7new and B8new had smaller colonies than B9 and B10.<br />
<br />
*We incubated A14L plate overnight at 37°C.<br />
<br />
<br />
===== pH sensor =====<br />
*Miniprep for K116002 overnight culture. We incubated the plate (LB + Amp) with transformed bacteria overnight at 37°C. <br />
*We used 1 ul of this sample to transform TOP10 in order to have the pH sensor in the same strain as the reference promoter (i.e. A2).<br />
*We plated transformed bacteria and incubated overnight at 37°C.<br />
*We also sent purified DNA to BMR Genomics for sequencing.<br />
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<br />
== <html><font class="dayw_style">September, 4th</font></html> ==<br />
<br />
*A14L plate showed colonies.<br />
<br />
*Colony PCR/electrophoresis for 6 colonies from A14L plate. Colonies were saved inoculating them in 1 ml of LB + Amp and incubating them at 37°C, 220 rpm waiting for the end of the reaction.<br />
<br />
*Gel results: all the colonies showed the amplicon with the right size, but even some extra band...we decided to stock all the colonies in glycerol. We also decided to keep A14L-1: we inoculated it in 5 ml of LB + Amp to grow an overnight culture to check the sequence.<br />
<br />
<br />
<br />
<br />
*We received sequencing results for B8-5again(VF2) and it was not correct.<br />
<br />
<br />
<br />
*Glycerol stock/miniprep for 21 cultures:<br />
**B7new X5 colonies<br />
**B8new X5 colonies<br />
**B9 X5 colonies<br />
**B10 X5 colonies<br />
**A17new<br />
<br />
*We sent A17new purified DNA to BMR Genomics for sequencing.<br />
<br />
*Screening (E-P digestion cut) for B7new, B8new, B9 and B10 samples.<br />
<br />
<br />
===== pH sensor =====<br />
*K116002(TOP10) plate showed a bacterial carpet (with some single colony).<br />
*We inoculated a single colony from K116002 (TOP10) plate in 1 ml of LB + Amp and incubated this inoculum for about 6 hours (37°C, 220 rpm).<br />
*Glycerol stock.<br />
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== <html><font class="dayw_style">September, 5th</font></html> ==<br />
*Fermentation experiment setup<br />
*Wiki updating<br />
<br />
<br />
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<br />
== <html><font class="dayw_style">September, 6th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Aug#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Aug#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5SepTeam:UNIPV-Pavia/Notebook/Week5Sep2009-10-21T23:41:22Z<p>110hs: /* October, 3rd */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from September 28th, to October 4th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 28th</font></html> ==<br />
===== pH sensor =====<br />
''3rd experiment''<br />
*We put 50 ul into 5 ml LB NaCl 171 mM + Amp pH 6,6 falcon for each of the overnight cultures RBS33 - A2 - pNhaA and incubated them for four hours and a half.<br />
*OD measure of the three falcons with TECAN in order to make dilutions with a final volume of 3 ml and an OD equal to 0,02.<br />
*We diluted:<br />
**RBS into:<br />
*** LB NaCl 171 mM + Amp pH 5,5<br />
*** LB NaCl 171 mM + Amp pH 6,6<br />
*** LB NaCl 171 mM + Amp pH 7,5<br />
*** LB NaCl 171 mM + Amp pH 8,5<br />
**pNhaA into:<br />
*** LB NaCl 171 mM + Amp pH 5,5<br />
*** LB NaCl 171 mM + Amp pH 6,6<br />
*** LB NaCl 171 mM + Amp pH 7,5<br />
*** LB NaCl 171 mM + Amp pH 8,5<br />
**A2 into:<br />
*** LB NaCl 171 mM + Amp pH 5,5<br />
*** LB NaCl 171 mM + Amp pH 6,6<br />
*** LB NaCl 171 mM + Amp pH 7,5<br />
*** LB NaCl 171 mM + Amp pH 8,5<br />
*We started this 6 hours' experiment that performed absorbance and fluorescence measures (on three different wells) for each sample every 5 minutes.<br />
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== <html><font class="dayw_style">September, 29th</font></html> ==<br />
<br />
*A14 induction test using IPTG<br />
The part doesn't show any induction!! :-(<br />
To test what happened, we have inoculated A14 L-1 from glycerol stocks. tomorrow it will be diluted 1:100 in 2 falcons (5ml), one of wich will be induced 10nM.<br />
<br />
*Inoculum of T9002 from glycerol stocks in 2 falcon tubes, one grown anaerobically and the other areobically. They have been induced with 3OC6HSL 10nM and next morning the measure of GFP was about the half in the anaerobically grown culture respect to the aerobically grown one.<br />
<br />
*Preparation of 2 falcon tubes (50 ml) containing 35ml LB+Amp+2%glucose and infected from glycerol stocks with B9-2 and F2620MIT2. They have been incubated overnight at 37°C without shaking.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/IPTG A14 A17 induction test TEST 29-09-09.pdf" target="_blank">Download Protocol</a></html><br />
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<br />
== <html><font class="dayw_style">September, 30th</font></html> ==<br />
<br />
We watched B9 and F2620 grown overnight in anaerobic way. In B9 lots of foam has developed that we couldn't see in F2620. Is it for it fermented and made CO2? In the evening they have been pelletted and F2620's pellet was bigger than B9's one.<br />
<br />
LIGATIONS:<br />
*B7 = A4(E-S) + B5new2-3(E-X) in pSB1AK3<br />
*B9: F2620(E-S) + B5new2-3(X-P-ClaI) in pSB1A2<br />
*B12: J23118(S-P) + B5new2-3(E-X) in pSB1A2<br />
*B13: J23106(S-P) + B5new2-3(E-X) in pSB1A2 <br />
*A19: F2620(E-P) + 4C5(E-P) in pSB4C5<br />
<br />
Inoculum of A15-1, A15-3 F2620 from glycerol stocks for tomorrow lysis test. A15-3 has also been plated on LB+amp+agar2% to pick single colony for tomorrow test.<br />
<br />
A14 inoculated yesterday has been diluted 1:100 into 2 falcons and induced with 1 mM IPTG. Tomorrow we will see if the part works.<br />
<br />
*Inoculum of 15ul from glycerol stock of B9 and F2620 in 2 cultures each, one closed (anaerobic) and the other a little opened ("aerobic"). The cultures have been induced immediately with 3OC6HSL 10nM. <br />
They have been incubated overnight at 37°C without shaking.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/aTc induction test M9 TEST 30-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
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<br />
== <html><font class="dayw_style">October, 1st</font></html> ==<br />
<br />
*From the cultures incubated yesterday, F2620 grew, but B9 didn't. We have decided not to induce anymore and to exploit F2620 leakage to express pdc and adhB.<br />
<br />
Yesterday ligations have been inoculated in 1ml LB + AB from colonies and incubated at 37° 220rpm for about 6 hours. Then glycerol stocks were prepared and the remaining 250 ul of the cultures have been re-filled with 5 ml of LB + antibiotic and incubated overnight to prepare the screening. The cultures are:<br />
*B7-1 (Amp)<br />
*B7-2 (Amp)<br />
*B12-1 (Amp)<br />
*B12-2 (Amp)<br />
*B13-1 (Amp)<br />
*B13-2 (Amp)<br />
*B9new-1 (Amp)<br />
*B9new-2 (Amp)<br />
*B9new-3 (Amp)<br />
*B9new-4 (Amp)<br />
*B9new-5 (Amp)<br />
*A19-1 (Cm)<br />
*A19-2 (Cm)<br />
<br />
Dilution 1:100 of cultures:<br />
*A15-1<br />
*A15-3<br />
*F2620<br />
and inoculum of A15-3 colony picked from colony grown on plate infected yesterday.<br />
Cultures have been incubated for further 4 hours for lysis test at TECAN in the afternoon.<br />
*Preparation of 3OC6HSL at desired concentration, to be used for induction test in the afternoon.<br />
<br />
A14 L-1 induced and A14 L-1 NOT induced have been measured using TECAN: the fluorescence in green wasn't significantly different. It could mean that A14 has mutated, so we have prepared an inculum from glycerol stocks of <br />
*A11 <br />
*E0240<br />
to repeat ligation tomorrow.<br />
<br />
*Preparation of LB+Amp 10% glucose:<br />
LB + Amp has been prepared as always for 500ml, but just 400ml of water have been added.<br />
The further 100ml have been used to dilute 50g glucose and have been added after autoclave, filtering them.<br />
*Preparation of LB+Amp (500ml)<br />
<br />
TECAN test in the afternoon for A15 lysis.<br />
Glycerol stock for all the cultures incubated this morning.<br />
Inoculum of A8pg, A2, RBS32 for tomorrow induction test of A8pg construct with aTc.<br />
Inoculum of A15-3 for tomorrow lysis test (to repeat today's test because the results were not satisfying).<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/A14 tentative TEST 1_10_09.pdf" target="_blank">Download Protocol</a></html><br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/lysis A15 TEST 1-10-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
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<br />
== <html><font class="dayw_style">October, 2nd</font></html> ==<br />
<br />
Dilution of A15 and induction with 3OC6HSL to test the activity os this part (it didn't work yesterday!)<br />
<br />
In the morning we have miniprepped:<br />
<br />
*A11-3<br />
*GFP3<br />
<br />
to re-ligate A14<br />
<br />
We have also miniprepped:<br />
<br />
*B9 new-1<br />
*B9 new-2<br />
*B9 new-3<br />
*B9 new-4<br />
*B9 new-5<br />
*A19-1<br />
*A19-2<br />
<br />
incubated overnight at 37°C 220rpm. They have been refilled from yesterday cultures used for glycerol stocks.<br />
<br />
After mini-prep, the cultures have been digested:<br />
<br />
*A11-3 (S-P) for ligation<br />
*GFP3 (X-P) for ligation<br />
*B9 new-1 (E-P) for screening<br />
*B9 new-2 (E-P) for screening<br />
*B9 new-3 (E-P) for screening<br />
*B9 new-4 (E-P) for screening<br />
*B9 new-5 (E-P) for screening<br />
*A19-1 (E-P) for screening<br />
*A19-2 (E-P) for screening<br />
<br />
Gel has been filled as follows:<br />
B9new-1 - B9new-2 - B9new-3 - B9new-4 - B9new-5 - A19-1 - A19-2 - - Marker- - A11 - - GFP3 - - - <br />
<br />
<br />
<table align="center"><br />
<tr><br />
<td><br />
<font class='didascalia'><br />
[[Image:GEL021009.jpg|thumb|500px|left|Screening gel for B9new 1-5, A19-1 and A19-2. Gel extraction for A11-3 and E0240.]]<br />
</font><br />
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</table><br />
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</div><br />
<br />
== <html><font class="dayw_style">October, 3rd</font></html> ==<br />
*Cloning<br />
*Wiki updating<br />
<br />
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</div><br />
<br />
== <html><font class="dayw_style">October, 4th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3OctTeam:UNIPV-Pavia/Notebook/Week3Oct2009-10-21T23:40:33Z<p>110hs: /* October, 18th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from October 12th, to October 18th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
== <html><font class="dayw_style">October, 12th</font></html> ==<br />
*We repeated the digestion for F2620TOP10(S-P) and B5new2-3(X-P-ClaI).<br />
<br />
*Gel run/cut for the two plasmids: bands were good.<br />
<br />
*Gel extraction for:<br />
**F2620TOP10(S-P)<br />
**B5new2-3(X-P-ClaI)<br />
**B3(X-P)<br />
**B4(X-P)<br />
**A19-1(S-P)<br />
<br />
*Unfortunately we had a very low DNA yield for almost all of them: A19-1(S-P) and F2620(S-P) could not be used :'(<br />
<br />
*We decided not to repeat A19-1 extraction (and then the A21 re-cloning) and to assay our A21 construct for ethanol production.<br />
<br />
*F2620 DNA was over, so we inoculated 8 ul of F2620MIT1 glycerol stock in 7 ml of LB + Amp. We incubated this inoculum overnight (37°C, 220 rpm).<br />
<br />
*We inoculated 12 of our assemblies in 3 ml of LB + Amp in order to prepare the DNA to send to iGEM HQ:<br />
**A1<br />
**A2<br />
**A3<br />
**A4<br />
**A5<br />
**A6<br />
**A7<br />
**A8pg<br />
**A9pg<br />
**A11-3<br />
**A12-2<br />
**A15-3<br />
<br />
*We ordered a gas chromatography for a sample of the supernatants (taken the previous day).<br />
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<br />
== <html><font class="dayw_style">October, 13th</font></html> ==<br />
<br />
*Miniprep for the 12 inocula to be sent.<br />
<br />
*Miniprep, cut S-P, gel run/cut/purification for F2620MIT1. This time we had a good yield;)<br />
<br />
*Ligations:<br />
**B14 = F2620(S-P) + B3(X-P) in pSB1A2<br />
**B15 = F2620(S-P) + B4(X-P) in pSB1A2<br />
*We incubated them at 16°C overnight.<br />
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<br />
== <html><font class="dayw_style">October, 14th</font></html> ==<br />
<br />
*We transformed the two ligations (after 1:20 dilution) and incubated the plated bacteria overnight (37°C).<br />
<br />
*We inoculated all the remaining assemblies (12) in order to send purified DNA to iGEM HQ (3 ml of LB + Amp).<br />
<br />
*We inoculated 8 ul of B5new2-3, F2620TOP10 and B0015 glycerol stocks in 8 ml of LB + Amp + 2% glucose. We incubated them at 37°C, 220 rpm in the morning.<br />
<br />
*Team meeting<br />
<br />
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<br />
== <html><font class="dayw_style">October, 15th</font></html> ==<br />
<br />
*We diluted 25 ul of A14S-3 in 3 ml of LB + Amp and in 3 ml of LB + Amp + 1mM IPTG. We incubated the two cultures at 37°C, 220 rpm for 6 hours. Then we measured OD600 and fluorescence, but unfortunately their GFP levels were the same.<br />
<br />
*Miniprep for the remaining BioBricks.<br />
<br />
*pH measurement for B5new2-3, F2620TOP10 and B0015 24 hour cultures. We diluted 1:100 these three cultures in LB + Amp + 10% glucose and aliquoted them in the microplate reader (anaerobic).<br />
<br />
*We inoculated 4 colonies of B14 plate and 3 colonies of B15 plate in 5 ml of LB + Amp and incubated them at 37°C, 220 rpm overnight. Tomorrow they will be screened!<br />
<br />
*We received gas chromatography results.<br />
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<br />
== <html><font class="dayw_style">October, 16th</font></html> ==<br />
*Cloning<br />
*Fermentation experiment setup<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
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</div><br />
<br />
== <html><font class="dayw_style">October, 17th</font></html> ==<br />
*Fermentation experiment<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 18th</font></html> ==<br />
*Fermentation experiment<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3OctTeam:UNIPV-Pavia/Notebook/Week3Oct2009-10-21T23:40:24Z<p>110hs: /* October, 17th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from October 12th, to October 18th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
== <html><font class="dayw_style">October, 12th</font></html> ==<br />
*We repeated the digestion for F2620TOP10(S-P) and B5new2-3(X-P-ClaI).<br />
<br />
*Gel run/cut for the two plasmids: bands were good.<br />
<br />
*Gel extraction for:<br />
**F2620TOP10(S-P)<br />
**B5new2-3(X-P-ClaI)<br />
**B3(X-P)<br />
**B4(X-P)<br />
**A19-1(S-P)<br />
<br />
*Unfortunately we had a very low DNA yield for almost all of them: A19-1(S-P) and F2620(S-P) could not be used :'(<br />
<br />
*We decided not to repeat A19-1 extraction (and then the A21 re-cloning) and to assay our A21 construct for ethanol production.<br />
<br />
*F2620 DNA was over, so we inoculated 8 ul of F2620MIT1 glycerol stock in 7 ml of LB + Amp. We incubated this inoculum overnight (37°C, 220 rpm).<br />
<br />
*We inoculated 12 of our assemblies in 3 ml of LB + Amp in order to prepare the DNA to send to iGEM HQ:<br />
**A1<br />
**A2<br />
**A3<br />
**A4<br />
**A5<br />
**A6<br />
**A7<br />
**A8pg<br />
**A9pg<br />
**A11-3<br />
**A12-2<br />
**A15-3<br />
<br />
*We ordered a gas chromatography for a sample of the supernatants (taken the previous day).<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 13th</font></html> ==<br />
<br />
*Miniprep for the 12 inocula to be sent.<br />
<br />
*Miniprep, cut S-P, gel run/cut/purification for F2620MIT1. This time we had a good yield;)<br />
<br />
*Ligations:<br />
**B14 = F2620(S-P) + B3(X-P) in pSB1A2<br />
**B15 = F2620(S-P) + B4(X-P) in pSB1A2<br />
*We incubated them at 16°C overnight.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 14th</font></html> ==<br />
<br />
*We transformed the two ligations (after 1:20 dilution) and incubated the plated bacteria overnight (37°C).<br />
<br />
*We inoculated all the remaining assemblies (12) in order to send purified DNA to iGEM HQ (3 ml of LB + Amp).<br />
<br />
*We inoculated 8 ul of B5new2-3, F2620TOP10 and B0015 glycerol stocks in 8 ml of LB + Amp + 2% glucose. We incubated them at 37°C, 220 rpm in the morning.<br />
<br />
*Team meeting<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 15th</font></html> ==<br />
<br />
*We diluted 25 ul of A14S-3 in 3 ml of LB + Amp and in 3 ml of LB + Amp + 1mM IPTG. We incubated the two cultures at 37°C, 220 rpm for 6 hours. Then we measured OD600 and fluorescence, but unfortunately their GFP levels were the same.<br />
<br />
*Miniprep for the remaining BioBricks.<br />
<br />
*pH measurement for B5new2-3, F2620TOP10 and B0015 24 hour cultures. We diluted 1:100 these three cultures in LB + Amp + 10% glucose and aliquoted them in the microplate reader (anaerobic).<br />
<br />
*We inoculated 4 colonies of B14 plate and 3 colonies of B15 plate in 5 ml of LB + Amp and incubated them at 37°C, 220 rpm overnight. Tomorrow they will be screened!<br />
<br />
*We received gas chromatography results.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 16th</font></html> ==<br />
*Cloning<br />
*Fermentation experiment setup<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 17th</font></html> ==<br />
*Fermentation experiment<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 18th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3OctTeam:UNIPV-Pavia/Notebook/Week3Oct2009-10-21T23:40:09Z<p>110hs: /* October, 16th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from October 12th, to October 18th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
== <html><font class="dayw_style">October, 12th</font></html> ==<br />
*We repeated the digestion for F2620TOP10(S-P) and B5new2-3(X-P-ClaI).<br />
<br />
*Gel run/cut for the two plasmids: bands were good.<br />
<br />
*Gel extraction for:<br />
**F2620TOP10(S-P)<br />
**B5new2-3(X-P-ClaI)<br />
**B3(X-P)<br />
**B4(X-P)<br />
**A19-1(S-P)<br />
<br />
*Unfortunately we had a very low DNA yield for almost all of them: A19-1(S-P) and F2620(S-P) could not be used :'(<br />
<br />
*We decided not to repeat A19-1 extraction (and then the A21 re-cloning) and to assay our A21 construct for ethanol production.<br />
<br />
*F2620 DNA was over, so we inoculated 8 ul of F2620MIT1 glycerol stock in 7 ml of LB + Amp. We incubated this inoculum overnight (37°C, 220 rpm).<br />
<br />
*We inoculated 12 of our assemblies in 3 ml of LB + Amp in order to prepare the DNA to send to iGEM HQ:<br />
**A1<br />
**A2<br />
**A3<br />
**A4<br />
**A5<br />
**A6<br />
**A7<br />
**A8pg<br />
**A9pg<br />
**A11-3<br />
**A12-2<br />
**A15-3<br />
<br />
*We ordered a gas chromatography for a sample of the supernatants (taken the previous day).<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 13th</font></html> ==<br />
<br />
*Miniprep for the 12 inocula to be sent.<br />
<br />
*Miniprep, cut S-P, gel run/cut/purification for F2620MIT1. This time we had a good yield;)<br />
<br />
*Ligations:<br />
**B14 = F2620(S-P) + B3(X-P) in pSB1A2<br />
**B15 = F2620(S-P) + B4(X-P) in pSB1A2<br />
*We incubated them at 16°C overnight.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 14th</font></html> ==<br />
<br />
*We transformed the two ligations (after 1:20 dilution) and incubated the plated bacteria overnight (37°C).<br />
<br />
*We inoculated all the remaining assemblies (12) in order to send purified DNA to iGEM HQ (3 ml of LB + Amp).<br />
<br />
*We inoculated 8 ul of B5new2-3, F2620TOP10 and B0015 glycerol stocks in 8 ml of LB + Amp + 2% glucose. We incubated them at 37°C, 220 rpm in the morning.<br />
<br />
*Team meeting<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 15th</font></html> ==<br />
<br />
*We diluted 25 ul of A14S-3 in 3 ml of LB + Amp and in 3 ml of LB + Amp + 1mM IPTG. We incubated the two cultures at 37°C, 220 rpm for 6 hours. Then we measured OD600 and fluorescence, but unfortunately their GFP levels were the same.<br />
<br />
*Miniprep for the remaining BioBricks.<br />
<br />
*pH measurement for B5new2-3, F2620TOP10 and B0015 24 hour cultures. We diluted 1:100 these three cultures in LB + Amp + 10% glucose and aliquoted them in the microplate reader (anaerobic).<br />
<br />
*We inoculated 4 colonies of B14 plate and 3 colonies of B15 plate in 5 ml of LB + Amp and incubated them at 37°C, 220 rpm overnight. Tomorrow they will be screened!<br />
<br />
*We received gas chromatography results.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 16th</font></html> ==<br />
*Cloning<br />
*Fermentation experiment setup<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 17th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 18th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4SepTeam:UNIPV-Pavia/Notebook/Week4Sep2009-10-21T23:39:24Z<p>110hs: /* September, 26th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from September 21st, to September 27th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 21st</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 22nd</font></html> ==<br />
===== pH sensor =====<br />
*LB NaCl 70 mM pH 5,5 - 6,6 - 7,5 - 8,5 + Amp preparation.<br />
* Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:<br />
**RBS33<br />
**A2<br />
**pNhaA<br />
* Overnight incubation of these three cultures at 37°C, 220 rpm.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/T9002 TEST 22-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 23rd</font></html> ==<br />
===== pH sensor =====<br />
''2nd experiment''<br />
*We put 50 ul into 5 ml LB NaCl 70 mM + Amp pH 6,6 falcon for each of the overnight cultures RBS33 - A2 - pNhaA and incubated them for four hours and a half at 37°C, 220 rpm.<br />
*OD measure of the three falcons with TECAN in order to make dilutions with a final volume of 3 ml and an OD equal to 0,02.<br />
*We diluted:<br />
**RBS into:<br />
*** LB NaCl 70 mM + Amp pH 5,5<br />
*** LB NaCl 70 mM + Amp pH 6,6<br />
*** LB NaCl 70 mM + Amp pH 7,5<br />
*** LB NaCl 70 mM + Amp pH 8,5<br />
**pNhaA into:<br />
*** LB NaCl 70 mM + Amp pH 5,5<br />
*** LB NaCl 70 mM + Amp pH 6,6<br />
*** LB NaCl 70 mM + Amp pH 7,5<br />
*** LB NaCl 70 mM + Amp pH 8,5<br />
**A2 into:<br />
*** LB NaCl 70 mM + Amp pH 5,5<br />
*** LB NaCl 70 mM + Amp pH 6,6<br />
*** LB NaCl 70 mM + Amp pH 7,5<br />
*** LB NaCl 70 mM + Amp pH 8,5<br />
*We started this 6 hours' experiment that performed absorbance and fluorescence measures (on three different wells) for each sample every 5 minutes.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 24th</font></html> ==<br />
*Fermentation experiment setup<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 25th</font></html> ==<br />
===== pH sensor =====<br />
*Preparation of LB NaCl 151 mM at pH 5,5 - 6,6 - 7,5 - 8,5.<br />
*Preparation of LB NaCl 250 mM at pH 5,5 - 6,6 - 7,5 - 8,5.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 26th</font></html> ==<br />
*Fermentation experiment<br />
*Cloning<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 27th</font></html> ==<br />
===== pH sensor =====<br />
*Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:<br />
**RBS33<br />
**A2<br />
**pNhaA<br />
*Overnight incubation of these three cultures at 37°C, 220 rpm.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4SepTeam:UNIPV-Pavia/Notebook/Week4Sep2009-10-21T23:39:04Z<p>110hs: /* September, 24th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from September 21st, to September 27th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 21st</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 22nd</font></html> ==<br />
===== pH sensor =====<br />
*LB NaCl 70 mM pH 5,5 - 6,6 - 7,5 - 8,5 + Amp preparation.<br />
* Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:<br />
**RBS33<br />
**A2<br />
**pNhaA<br />
* Overnight incubation of these three cultures at 37°C, 220 rpm.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/T9002 TEST 22-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 23rd</font></html> ==<br />
===== pH sensor =====<br />
''2nd experiment''<br />
*We put 50 ul into 5 ml LB NaCl 70 mM + Amp pH 6,6 falcon for each of the overnight cultures RBS33 - A2 - pNhaA and incubated them for four hours and a half at 37°C, 220 rpm.<br />
*OD measure of the three falcons with TECAN in order to make dilutions with a final volume of 3 ml and an OD equal to 0,02.<br />
*We diluted:<br />
**RBS into:<br />
*** LB NaCl 70 mM + Amp pH 5,5<br />
*** LB NaCl 70 mM + Amp pH 6,6<br />
*** LB NaCl 70 mM + Amp pH 7,5<br />
*** LB NaCl 70 mM + Amp pH 8,5<br />
**pNhaA into:<br />
*** LB NaCl 70 mM + Amp pH 5,5<br />
*** LB NaCl 70 mM + Amp pH 6,6<br />
*** LB NaCl 70 mM + Amp pH 7,5<br />
*** LB NaCl 70 mM + Amp pH 8,5<br />
**A2 into:<br />
*** LB NaCl 70 mM + Amp pH 5,5<br />
*** LB NaCl 70 mM + Amp pH 6,6<br />
*** LB NaCl 70 mM + Amp pH 7,5<br />
*** LB NaCl 70 mM + Amp pH 8,5<br />
*We started this 6 hours' experiment that performed absorbance and fluorescence measures (on three different wells) for each sample every 5 minutes.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 24th</font></html> ==<br />
*Fermentation experiment setup<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 25th</font></html> ==<br />
===== pH sensor =====<br />
*Preparation of LB NaCl 151 mM at pH 5,5 - 6,6 - 7,5 - 8,5.<br />
*Preparation of LB NaCl 250 mM at pH 5,5 - 6,6 - 7,5 - 8,5.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 26th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 27th</font></html> ==<br />
===== pH sensor =====<br />
*Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:<br />
**RBS33<br />
**A2<br />
**pNhaA<br />
*Overnight incubation of these three cultures at 37°C, 220 rpm.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3SepTeam:UNIPV-Pavia/Notebook/Week3Sep2009-10-21T23:38:43Z<p>110hs: /* September, 20th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from September 14th, to September 20th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 14th</font></html> ==<br />
*Cloning<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 15th</font></html> ==<br />
*Fermentation experiment setup<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 16th</font></html> ==<br />
*Fermentation experiment<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 17th</font></html> ==<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/A1A2A7 TEST 17-09-09.pdf" target="_blank">Download Protocol</a></html><br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/Ethanol test TEST 17-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
===== pH sensor =====<br />
*Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:<br />
**RBS33<br />
**A2<br />
**K116002 (from now on it will be indicated only as pNhaA)<br />
*Overnight incubation of these three cultures at 37°C, 220 rpm.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 18th</font></html> ==<br />
<br />
===== pH sensor =====<br />
''1st experiment''<br />
*We put 50 ul into 5 ml LBK + Amp pH 6,6 falcon for each of the overnight cultures RBS33 - A2 - pNhaA and incubated them for four hours and a half at 37°C, 220 rpm.<br />
*OD measure of the three falcons with TECAN in order to make dilutions with a final volume of 3 ml and an OD equal to 0,02.<br />
*We diluted:<br />
**RBS into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
**pNhaA into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
**A2 into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
*We started this 6 hours' experiment that performed absorbance and fluorescence measures (on three different wells) for each sample every 5 minutes.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 19th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 20th</font></html> ==<br />
*Cloning<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3SepTeam:UNIPV-Pavia/Notebook/Week3Sep2009-10-21T23:38:16Z<p>110hs: /* September, 16th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from September 14th, to September 20th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 14th</font></html> ==<br />
*Cloning<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 15th</font></html> ==<br />
*Fermentation experiment setup<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 16th</font></html> ==<br />
*Fermentation experiment<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 17th</font></html> ==<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/A1A2A7 TEST 17-09-09.pdf" target="_blank">Download Protocol</a></html><br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/Ethanol test TEST 17-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
===== pH sensor =====<br />
*Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:<br />
**RBS33<br />
**A2<br />
**K116002 (from now on it will be indicated only as pNhaA)<br />
*Overnight incubation of these three cultures at 37°C, 220 rpm.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 18th</font></html> ==<br />
<br />
===== pH sensor =====<br />
''1st experiment''<br />
*We put 50 ul into 5 ml LBK + Amp pH 6,6 falcon for each of the overnight cultures RBS33 - A2 - pNhaA and incubated them for four hours and a half at 37°C, 220 rpm.<br />
*OD measure of the three falcons with TECAN in order to make dilutions with a final volume of 3 ml and an OD equal to 0,02.<br />
*We diluted:<br />
**RBS into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
**pNhaA into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
**A2 into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
*We started this 6 hours' experiment that performed absorbance and fluorescence measures (on three different wells) for each sample every 5 minutes.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 19th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 20th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3SepTeam:UNIPV-Pavia/Notebook/Week3Sep2009-10-21T23:38:05Z<p>110hs: /* September, 15th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from September 14th, to September 20th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 14th</font></html> ==<br />
*Cloning<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 15th</font></html> ==<br />
*Fermentation experiment setup<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 16th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 17th</font></html> ==<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/A1A2A7 TEST 17-09-09.pdf" target="_blank">Download Protocol</a></html><br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/Ethanol test TEST 17-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
===== pH sensor =====<br />
*Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:<br />
**RBS33<br />
**A2<br />
**K116002 (from now on it will be indicated only as pNhaA)<br />
*Overnight incubation of these three cultures at 37°C, 220 rpm.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 18th</font></html> ==<br />
<br />
===== pH sensor =====<br />
''1st experiment''<br />
*We put 50 ul into 5 ml LBK + Amp pH 6,6 falcon for each of the overnight cultures RBS33 - A2 - pNhaA and incubated them for four hours and a half at 37°C, 220 rpm.<br />
*OD measure of the three falcons with TECAN in order to make dilutions with a final volume of 3 ml and an OD equal to 0,02.<br />
*We diluted:<br />
**RBS into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
**pNhaA into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
**A2 into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
*We started this 6 hours' experiment that performed absorbance and fluorescence measures (on three different wells) for each sample every 5 minutes.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 19th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 20th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3SepTeam:UNIPV-Pavia/Notebook/Week3Sep2009-10-21T23:37:54Z<p>110hs: /* September, 14th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from September 14th, to September 20th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 14th</font></html> ==<br />
*Cloning<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 15th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 16th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 17th</font></html> ==<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/A1A2A7 TEST 17-09-09.pdf" target="_blank">Download Protocol</a></html><br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/Ethanol test TEST 17-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
===== pH sensor =====<br />
*Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:<br />
**RBS33<br />
**A2<br />
**K116002 (from now on it will be indicated only as pNhaA)<br />
*Overnight incubation of these three cultures at 37°C, 220 rpm.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 18th</font></html> ==<br />
<br />
===== pH sensor =====<br />
''1st experiment''<br />
*We put 50 ul into 5 ml LBK + Amp pH 6,6 falcon for each of the overnight cultures RBS33 - A2 - pNhaA and incubated them for four hours and a half at 37°C, 220 rpm.<br />
*OD measure of the three falcons with TECAN in order to make dilutions with a final volume of 3 ml and an OD equal to 0,02.<br />
*We diluted:<br />
**RBS into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
**pNhaA into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
**A2 into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
*We started this 6 hours' experiment that performed absorbance and fluorescence measures (on three different wells) for each sample every 5 minutes.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 19th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 20th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2SepTeam:UNIPV-Pavia/Notebook/Week2Sep2009-10-21T23:37:30Z<p>110hs: /* September, 13th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from Semptember 7th, to September 13th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 7th</font></html> ==<br />
<br />
In the morning we have prepared culture of A2 to stock in glycerol and we have prepared LB medium.<br />
<br />
In the afternoon we have incubated in adequate medium:<br />
<br />
<br />
*B9 (10 ml LB+Amp)<br />
*B10 (10 ml LB+Amp)<br />
*T9002 (10 ml LB + Amp)<br />
for a qualitative test of ethanol production,<br />
<br />
<br />
*A9pg (5 ml LB + Amp)<br />
*A16 (5 ml LB + Amp)<br />
*A8 (5 ml LB + Amp)<br />
*A2 (5 ml LB + Amp)<br />
*RBS33 (5 ml LB + Amp)<br />
to perform an aTc induction test,<br />
<br />
<br />
*A15 (25 ml LB + Amp)<br />
*pSB3K3 (25 ml LB + Kan)<br />
*A14-1 L (5 ml LB + Amp)<br />
to miniprep tomorrow.<br />
<br />
<br />
These coltures have been incubated at 37°C, 220 rpm overnight.<br />
Tomorrow, we will miniprep A14-1 L and midiprep A15 and 3K3 (pSB3K3). We perform midiprep on pSB3K3 because it is a low copy number plasmid and on A15 because it is a lysis cassette, so for leackage only "few" cells survive.<br />
<br />
Glycerol stocks of A2-3 have been stored at -80°C, named A2-3 and A2-3 bis.The previous stock has been thrown away.<br />
<br />
'''Preparation of a qualitative test to assay the presence of ethanol'''<br />
<br />
We have also induced coltures B9 and B10 incubated yesterday morning at 37°C 220 rpm in flasks and grown overnight. In the morning the coltures have been diluited to reach the desired OD (0.03), then re-incubated for 4 hours and induced with 3OC6-HSL 10nM. The induced coltures have been incubated at 18.00 o'clock and will stay overnight. Flasks have been ermetically closed, to allow fermentation. This qualitative test is performed to assay the presence oh ethanol.<br />
<br />
Tomorrow we will use the "Ethanol assay kit" to test the presence of ethanol!!<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 8th</font></html> ==<br />
<br />
A first test with ethanol assay kit has been made using coltures B9 and B10 induced the evening before and using as blank the reaction mix of Kit without ethanol. In the evening, we have also made a measure of blank made by LB + mix.<br />
The test have been performed on the supernatant of the coltures, obtained by centrifugation for 20 minutes 13000rpm.<br />
<br />
* A14 L-1 have been miniprepped, with a yield of 33ng/ul<br />
*pSB3K3 and A15 have been midi-prepped and tomorrow will be digested E-P and ligated<br />
<br />
aTc induction test:<br />
*one of the coltures incubated the day before has been "accidentally" centrifuged, and has shown no growth in the next 3 hours, so the coltures have been re-inoculated, as the day before: <br />
*A9pg (5 ml LB + Amp)<br />
*A16 (5 ml LB + Amp)<br />
*A8 (5 ml LB + Amp)<br />
*A2 (5 ml LB + Amp)<br />
*RBS33 (5 ml LB + Amp)<br />
to perform a new aTc induction test, tomorrow.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/aTc induction test TEST 08-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 9th</font></html> ==<br />
<br />
Today we made aTc induction test:<br />
*coltures have been diluted 1:100 in the morning (50 ul of colture incubated overnight at 37° 220 rpm in 5 ml of new LB+Amp fresh medium) and incubated in the same conditions for further 4 hours<br />
*in the afternoon they have been diluted again to start from the desired O.D. of 0,02 using the usual phormula.<br />
*Implementation of induction test with aTc<br />
<br />
*Ligation of pSB3K3 with A15: digestion of both the BioBrick E-P, gel extraction and ligation overnight. <br />
* B9 B10 and F620 have been inoculated from glycerol stocks to be used tomorrow for thanol assay test.<br />
<br />
We have also received the resultes of A17 sequencing.<br />
In the afternoon we have made a qualitative ethanol assay test:<br />
coltures induced yesterday with 3OC6HSL 10nM have been centrifugate and supernatant has been diluted:<br />
*1:400<br />
*1:100<br />
*1:10<br />
<br />
The measure was not significant for the Ethanol Assay Kit doesn't work.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/aTc TEST 09-09-09.pdf" target="_blank">Download Protocol</a></html><br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/Ethanol Qualitative TEST 09-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 10th</font></html> ==<br />
The ligation of A15 and pSB3K3, named A19, has been tranformed in E. coli TOP10.<br />
The coltures B9, B10, F2620 have been diluted 1:100 and induced with 3OC6HSL 100nM to be tested, with glucose 10% added.<br />
<br />
Test aTc with good results in LB<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 11th</font></html> ==<br />
<br />
The lab has been closed for techinal problems, so we have just performed test at TECAN<br />
<br />
Ethanol test: ethanol assay kit doesn't work!!! We've tested coltures induced woth 3OC6HSL 10nM and with 10% glucose added. The measure has been made on the whole cultures, not centrifugated, for we couldn't have access to the lab.<br />
No result has been obtained for the enzyme of ethanol assay kit doesn't work!<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/PiatraEtanolo_11_09_09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
At 16.00, meeting of our team with representatives of Costa Foundation.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 12th</font></html> ==<br />
*Fermentation experiment setup<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 13th</font></html> ==<br />
*Fermentation experiment<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2SepTeam:UNIPV-Pavia/Notebook/Week2Sep2009-10-21T23:37:18Z<p>110hs: /* September, 12th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from Semptember 7th, to September 13th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 7th</font></html> ==<br />
<br />
In the morning we have prepared culture of A2 to stock in glycerol and we have prepared LB medium.<br />
<br />
In the afternoon we have incubated in adequate medium:<br />
<br />
<br />
*B9 (10 ml LB+Amp)<br />
*B10 (10 ml LB+Amp)<br />
*T9002 (10 ml LB + Amp)<br />
for a qualitative test of ethanol production,<br />
<br />
<br />
*A9pg (5 ml LB + Amp)<br />
*A16 (5 ml LB + Amp)<br />
*A8 (5 ml LB + Amp)<br />
*A2 (5 ml LB + Amp)<br />
*RBS33 (5 ml LB + Amp)<br />
to perform an aTc induction test,<br />
<br />
<br />
*A15 (25 ml LB + Amp)<br />
*pSB3K3 (25 ml LB + Kan)<br />
*A14-1 L (5 ml LB + Amp)<br />
to miniprep tomorrow.<br />
<br />
<br />
These coltures have been incubated at 37°C, 220 rpm overnight.<br />
Tomorrow, we will miniprep A14-1 L and midiprep A15 and 3K3 (pSB3K3). We perform midiprep on pSB3K3 because it is a low copy number plasmid and on A15 because it is a lysis cassette, so for leackage only "few" cells survive.<br />
<br />
Glycerol stocks of A2-3 have been stored at -80°C, named A2-3 and A2-3 bis.The previous stock has been thrown away.<br />
<br />
'''Preparation of a qualitative test to assay the presence of ethanol'''<br />
<br />
We have also induced coltures B9 and B10 incubated yesterday morning at 37°C 220 rpm in flasks and grown overnight. In the morning the coltures have been diluited to reach the desired OD (0.03), then re-incubated for 4 hours and induced with 3OC6-HSL 10nM. The induced coltures have been incubated at 18.00 o'clock and will stay overnight. Flasks have been ermetically closed, to allow fermentation. This qualitative test is performed to assay the presence oh ethanol.<br />
<br />
Tomorrow we will use the "Ethanol assay kit" to test the presence of ethanol!!<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 8th</font></html> ==<br />
<br />
A first test with ethanol assay kit has been made using coltures B9 and B10 induced the evening before and using as blank the reaction mix of Kit without ethanol. In the evening, we have also made a measure of blank made by LB + mix.<br />
The test have been performed on the supernatant of the coltures, obtained by centrifugation for 20 minutes 13000rpm.<br />
<br />
* A14 L-1 have been miniprepped, with a yield of 33ng/ul<br />
*pSB3K3 and A15 have been midi-prepped and tomorrow will be digested E-P and ligated<br />
<br />
aTc induction test:<br />
*one of the coltures incubated the day before has been "accidentally" centrifuged, and has shown no growth in the next 3 hours, so the coltures have been re-inoculated, as the day before: <br />
*A9pg (5 ml LB + Amp)<br />
*A16 (5 ml LB + Amp)<br />
*A8 (5 ml LB + Amp)<br />
*A2 (5 ml LB + Amp)<br />
*RBS33 (5 ml LB + Amp)<br />
to perform a new aTc induction test, tomorrow.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/aTc induction test TEST 08-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 9th</font></html> ==<br />
<br />
Today we made aTc induction test:<br />
*coltures have been diluted 1:100 in the morning (50 ul of colture incubated overnight at 37° 220 rpm in 5 ml of new LB+Amp fresh medium) and incubated in the same conditions for further 4 hours<br />
*in the afternoon they have been diluted again to start from the desired O.D. of 0,02 using the usual phormula.<br />
*Implementation of induction test with aTc<br />
<br />
*Ligation of pSB3K3 with A15: digestion of both the BioBrick E-P, gel extraction and ligation overnight. <br />
* B9 B10 and F620 have been inoculated from glycerol stocks to be used tomorrow for thanol assay test.<br />
<br />
We have also received the resultes of A17 sequencing.<br />
In the afternoon we have made a qualitative ethanol assay test:<br />
coltures induced yesterday with 3OC6HSL 10nM have been centrifugate and supernatant has been diluted:<br />
*1:400<br />
*1:100<br />
*1:10<br />
<br />
The measure was not significant for the Ethanol Assay Kit doesn't work.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/aTc TEST 09-09-09.pdf" target="_blank">Download Protocol</a></html><br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/Ethanol Qualitative TEST 09-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 10th</font></html> ==<br />
The ligation of A15 and pSB3K3, named A19, has been tranformed in E. coli TOP10.<br />
The coltures B9, B10, F2620 have been diluted 1:100 and induced with 3OC6HSL 100nM to be tested, with glucose 10% added.<br />
<br />
Test aTc with good results in LB<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 11th</font></html> ==<br />
<br />
The lab has been closed for techinal problems, so we have just performed test at TECAN<br />
<br />
Ethanol test: ethanol assay kit doesn't work!!! We've tested coltures induced woth 3OC6HSL 10nM and with 10% glucose added. The measure has been made on the whole cultures, not centrifugated, for we couldn't have access to the lab.<br />
No result has been obtained for the enzyme of ethanol assay kit doesn't work!<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/PiatraEtanolo_11_09_09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
At 16.00, meeting of our team with representatives of Costa Foundation.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 12th</font></html> ==<br />
*Fermentation experiment setup<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 13th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3OctTeam:UNIPV-Pavia/Notebook/Week3Oct2009-10-21T23:33:31Z<p>110hs: /* October, 18th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from October 12th, to October 18th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
== <html><font class="dayw_style">October, 12th</font></html> ==<br />
*We repeated the digestion for F2620TOP10(S-P) and B5new2-3(X-P-ClaI).<br />
<br />
*Gel run/cut for the two plasmids: bands were good.<br />
<br />
*Gel extraction for:<br />
**F2620TOP10(S-P)<br />
**B5new2-3(X-P-ClaI)<br />
**B3(X-P)<br />
**B4(X-P)<br />
**A19-1(S-P)<br />
<br />
*Unfortunately we had a very low DNA yield for almost all of them: A19-1(S-P) and F2620(S-P) could not be used :'(<br />
<br />
*We decided not to repeat A19-1 extraction (and then the A21 re-cloning) and to assay our A21 construct for ethanol production.<br />
<br />
*F2620 DNA was over, so we inoculated 8 ul of F2620MIT1 glycerol stock in 7 ml of LB + Amp. We incubated this inoculum overnight (37°C, 220 rpm).<br />
<br />
*We inoculated 12 of our assemblies in 3 ml of LB + Amp in order to prepare the DNA to send to iGEM HQ:<br />
**A1<br />
**A2<br />
**A3<br />
**A4<br />
**A5<br />
**A6<br />
**A7<br />
**A8pg<br />
**A9pg<br />
**A11-3<br />
**A12-2<br />
**A15-3<br />
<br />
*We ordered a gas chromatography for a sample of the supernatants (taken the previous day).<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 13th</font></html> ==<br />
<br />
*Miniprep for the 12 inocula to be sent.<br />
<br />
*Miniprep, cut S-P, gel run/cut/purification for F2620MIT1. This time we had a good yield;)<br />
<br />
*Ligations:<br />
**B14 = F2620(S-P) + B3(X-P) in pSB1A2<br />
**B15 = F2620(S-P) + B4(X-P) in pSB1A2<br />
*We incubated them at 16°C overnight.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 14th</font></html> ==<br />
<br />
*We transformed the two ligations (after 1:20 dilution) and incubated the plated bacteria overnight (37°C).<br />
<br />
*We inoculated all the remaining assemblies (12) in order to send purified DNA to iGEM HQ (3 ml of LB + Amp).<br />
<br />
*We inoculated 8 ul of B5new2-3, F2620TOP10 and B0015 glycerol stocks in 8 ml of LB + Amp + 2% glucose. We incubated them at 37°C, 220 rpm in the morning.<br />
<br />
*Team meeting<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 15th</font></html> ==<br />
<br />
*We diluted 25 ul of A14S-3 in 3 ml of LB + Amp and in 3 ml of LB + Amp + 1mM IPTG. We incubated the two cultures at 37°C, 220 rpm for 6 hours. Then we measured OD600 and fluorescence, but unfortunately their GFP levels were the same.<br />
<br />
*Miniprep for the remaining BioBricks.<br />
<br />
*pH measurement for B5new2-3, F2620TOP10 and B0015 24 hour cultures. We diluted 1:100 these three cultures in LB + Amp + 10% glucose and aliquoted them in the microplate reader (anaerobic).<br />
<br />
*We inoculated 4 colonies of B14 plate and 3 colonies of B15 plate in 5 ml of LB + Amp and incubated them at 37°C, 220 rpm overnight. Tomorrow they will be screened!<br />
<br />
*We received gas chromatography results.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 16th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 17th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 18th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start"><br />
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</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3OctTeam:UNIPV-Pavia/Notebook/Week3Oct2009-10-21T23:33:25Z<p>110hs: /* October, 17th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from October 12th, to October 18th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start"><br />
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</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
== <html><font class="dayw_style">October, 12th</font></html> ==<br />
*We repeated the digestion for F2620TOP10(S-P) and B5new2-3(X-P-ClaI).<br />
<br />
*Gel run/cut for the two plasmids: bands were good.<br />
<br />
*Gel extraction for:<br />
**F2620TOP10(S-P)<br />
**B5new2-3(X-P-ClaI)<br />
**B3(X-P)<br />
**B4(X-P)<br />
**A19-1(S-P)<br />
<br />
*Unfortunately we had a very low DNA yield for almost all of them: A19-1(S-P) and F2620(S-P) could not be used :'(<br />
<br />
*We decided not to repeat A19-1 extraction (and then the A21 re-cloning) and to assay our A21 construct for ethanol production.<br />
<br />
*F2620 DNA was over, so we inoculated 8 ul of F2620MIT1 glycerol stock in 7 ml of LB + Amp. We incubated this inoculum overnight (37°C, 220 rpm).<br />
<br />
*We inoculated 12 of our assemblies in 3 ml of LB + Amp in order to prepare the DNA to send to iGEM HQ:<br />
**A1<br />
**A2<br />
**A3<br />
**A4<br />
**A5<br />
**A6<br />
**A7<br />
**A8pg<br />
**A9pg<br />
**A11-3<br />
**A12-2<br />
**A15-3<br />
<br />
*We ordered a gas chromatography for a sample of the supernatants (taken the previous day).<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 13th</font></html> ==<br />
<br />
*Miniprep for the 12 inocula to be sent.<br />
<br />
*Miniprep, cut S-P, gel run/cut/purification for F2620MIT1. This time we had a good yield;)<br />
<br />
*Ligations:<br />
**B14 = F2620(S-P) + B3(X-P) in pSB1A2<br />
**B15 = F2620(S-P) + B4(X-P) in pSB1A2<br />
*We incubated them at 16°C overnight.<br />
<br />
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</div><br />
<br />
== <html><font class="dayw_style">October, 14th</font></html> ==<br />
<br />
*We transformed the two ligations (after 1:20 dilution) and incubated the plated bacteria overnight (37°C).<br />
<br />
*We inoculated all the remaining assemblies (12) in order to send purified DNA to iGEM HQ (3 ml of LB + Amp).<br />
<br />
*We inoculated 8 ul of B5new2-3, F2620TOP10 and B0015 glycerol stocks in 8 ml of LB + Amp + 2% glucose. We incubated them at 37°C, 220 rpm in the morning.<br />
<br />
*Team meeting<br />
<br />
<div align="right"><br />
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</div><br />
<br />
== <html><font class="dayw_style">October, 15th</font></html> ==<br />
<br />
*We diluted 25 ul of A14S-3 in 3 ml of LB + Amp and in 3 ml of LB + Amp + 1mM IPTG. We incubated the two cultures at 37°C, 220 rpm for 6 hours. Then we measured OD600 and fluorescence, but unfortunately their GFP levels were the same.<br />
<br />
*Miniprep for the remaining BioBricks.<br />
<br />
*pH measurement for B5new2-3, F2620TOP10 and B0015 24 hour cultures. We diluted 1:100 these three cultures in LB + Amp + 10% glucose and aliquoted them in the microplate reader (anaerobic).<br />
<br />
*We inoculated 4 colonies of B14 plate and 3 colonies of B15 plate in 5 ml of LB + Amp and incubated them at 37°C, 220 rpm overnight. Tomorrow they will be screened!<br />
<br />
*We received gas chromatography results.<br />
<br />
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</div><br />
<br />
== <html><font class="dayw_style">October, 16th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 17th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 18th</font></html> ==<br />
<br />
<br />
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[[#top|Top]]<br />
</div><br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
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</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3OctTeam:UNIPV-Pavia/Notebook/Week3Oct2009-10-21T23:33:17Z<p>110hs: /* October, 16th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from October 12th, to October 18th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start"><br />
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</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
== <html><font class="dayw_style">October, 12th</font></html> ==<br />
*We repeated the digestion for F2620TOP10(S-P) and B5new2-3(X-P-ClaI).<br />
<br />
*Gel run/cut for the two plasmids: bands were good.<br />
<br />
*Gel extraction for:<br />
**F2620TOP10(S-P)<br />
**B5new2-3(X-P-ClaI)<br />
**B3(X-P)<br />
**B4(X-P)<br />
**A19-1(S-P)<br />
<br />
*Unfortunately we had a very low DNA yield for almost all of them: A19-1(S-P) and F2620(S-P) could not be used :'(<br />
<br />
*We decided not to repeat A19-1 extraction (and then the A21 re-cloning) and to assay our A21 construct for ethanol production.<br />
<br />
*F2620 DNA was over, so we inoculated 8 ul of F2620MIT1 glycerol stock in 7 ml of LB + Amp. We incubated this inoculum overnight (37°C, 220 rpm).<br />
<br />
*We inoculated 12 of our assemblies in 3 ml of LB + Amp in order to prepare the DNA to send to iGEM HQ:<br />
**A1<br />
**A2<br />
**A3<br />
**A4<br />
**A5<br />
**A6<br />
**A7<br />
**A8pg<br />
**A9pg<br />
**A11-3<br />
**A12-2<br />
**A15-3<br />
<br />
*We ordered a gas chromatography for a sample of the supernatants (taken the previous day).<br />
<br />
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[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 13th</font></html> ==<br />
<br />
*Miniprep for the 12 inocula to be sent.<br />
<br />
*Miniprep, cut S-P, gel run/cut/purification for F2620MIT1. This time we had a good yield;)<br />
<br />
*Ligations:<br />
**B14 = F2620(S-P) + B3(X-P) in pSB1A2<br />
**B15 = F2620(S-P) + B4(X-P) in pSB1A2<br />
*We incubated them at 16°C overnight.<br />
<br />
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</div><br />
<br />
== <html><font class="dayw_style">October, 14th</font></html> ==<br />
<br />
*We transformed the two ligations (after 1:20 dilution) and incubated the plated bacteria overnight (37°C).<br />
<br />
*We inoculated all the remaining assemblies (12) in order to send purified DNA to iGEM HQ (3 ml of LB + Amp).<br />
<br />
*We inoculated 8 ul of B5new2-3, F2620TOP10 and B0015 glycerol stocks in 8 ml of LB + Amp + 2% glucose. We incubated them at 37°C, 220 rpm in the morning.<br />
<br />
*Team meeting<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 15th</font></html> ==<br />
<br />
*We diluted 25 ul of A14S-3 in 3 ml of LB + Amp and in 3 ml of LB + Amp + 1mM IPTG. We incubated the two cultures at 37°C, 220 rpm for 6 hours. Then we measured OD600 and fluorescence, but unfortunately their GFP levels were the same.<br />
<br />
*Miniprep for the remaining BioBricks.<br />
<br />
*pH measurement for B5new2-3, F2620TOP10 and B0015 24 hour cultures. We diluted 1:100 these three cultures in LB + Amp + 10% glucose and aliquoted them in the microplate reader (anaerobic).<br />
<br />
*We inoculated 4 colonies of B14 plate and 3 colonies of B15 plate in 5 ml of LB + Amp and incubated them at 37°C, 220 rpm overnight. Tomorrow they will be screened!<br />
<br />
*We received gas chromatography results.<br />
<br />
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[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 16th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 17th</font></html> ==<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<br />
== <html><font class="dayw_style">October, 18th</font></html> ==<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5SepTeam:UNIPV-Pavia/Notebook/Week5Sep2009-10-21T23:32:41Z<p>110hs: /* October, 4th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from September 28th, to October 4th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start"><br />
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</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 28th</font></html> ==<br />
===== pH sensor =====<br />
''3rd experiment''<br />
*We put 50 ul into 5 ml LB NaCl 171 mM + Amp pH 6,6 falcon for each of the overnight cultures RBS33 - A2 - pNhaA and incubated them for four hours and a half.<br />
*OD measure of the three falcons with TECAN in order to make dilutions with a final volume of 3 ml and an OD equal to 0,02.<br />
*We diluted:<br />
**RBS into:<br />
*** LB NaCl 171 mM + Amp pH 5,5<br />
*** LB NaCl 171 mM + Amp pH 6,6<br />
*** LB NaCl 171 mM + Amp pH 7,5<br />
*** LB NaCl 171 mM + Amp pH 8,5<br />
**pNhaA into:<br />
*** LB NaCl 171 mM + Amp pH 5,5<br />
*** LB NaCl 171 mM + Amp pH 6,6<br />
*** LB NaCl 171 mM + Amp pH 7,5<br />
*** LB NaCl 171 mM + Amp pH 8,5<br />
**A2 into:<br />
*** LB NaCl 171 mM + Amp pH 5,5<br />
*** LB NaCl 171 mM + Amp pH 6,6<br />
*** LB NaCl 171 mM + Amp pH 7,5<br />
*** LB NaCl 171 mM + Amp pH 8,5<br />
*We started this 6 hours' experiment that performed absorbance and fluorescence measures (on three different wells) for each sample every 5 minutes.<br />
<br />
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</div><br />
<br />
== <html><font class="dayw_style">September, 29th</font></html> ==<br />
<br />
*A14 induction test using IPTG<br />
The part doesn't show any induction!! :-(<br />
To test what happened, we have inoculated A14 L-1 from glycerol stocks. tomorrow it will be diluted 1:100 in 2 falcons (5ml), one of wich will be induced 10nM.<br />
<br />
*Inoculum of T9002 from glycerol stocks in 2 falcon tubes, one grown anaerobically and the other areobically. They have been induced with 3OC6HSL 10nM and next morning the measure of GFP was about the half in the anaerobically grown culture respect to the aerobically grown one.<br />
<br />
*Preparation of 2 falcon tubes (50 ml) containing 35ml LB+Amp+2%glucose and infected from glycerol stocks with B9-2 and F2620MIT2. They have been incubated overnight at 37°C without shaking.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/IPTG A14 A17 induction test TEST 29-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<div align="right"><br />
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</div><br />
<br />
== <html><font class="dayw_style">September, 30th</font></html> ==<br />
<br />
We watched B9 and F2620 grown overnight in anaerobic way. In B9 lots of foam has developed that we couldn't see in F2620. Is it for it fermented and made CO2? In the evening they have been pelletted and F2620's pellet was bigger than B9's one.<br />
<br />
LIGATIONS:<br />
*B7 = A4(E-S) + B5new2-3(E-X) in pSB1AK3<br />
*B9: F2620(E-S) + B5new2-3(X-P-ClaI) in pSB1A2<br />
*B12: J23118(S-P) + B5new2-3(E-X) in pSB1A2<br />
*B13: J23106(S-P) + B5new2-3(E-X) in pSB1A2 <br />
*A19: F2620(E-P) + 4C5(E-P) in pSB4C5<br />
<br />
Inoculum of A15-1, A15-3 F2620 from glycerol stocks for tomorrow lysis test. A15-3 has also been plated on LB+amp+agar2% to pick single colony for tomorrow test.<br />
<br />
A14 inoculated yesterday has been diluted 1:100 into 2 falcons and induced with 1 mM IPTG. Tomorrow we will see if the part works.<br />
<br />
*Inoculum of 15ul from glycerol stock of B9 and F2620 in 2 cultures each, one closed (anaerobic) and the other a little opened ("aerobic"). The cultures have been induced immediately with 3OC6HSL 10nM. <br />
They have been incubated overnight at 37°C without shaking.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/aTc induction test M9 TEST 30-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
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</div><br />
<br />
== <html><font class="dayw_style">October, 1st</font></html> ==<br />
<br />
*From the cultures incubated yesterday, F2620 grew, but B9 didn't. We have decided not to induce anymore and to exploit F2620 leakage to express pdc and adhB.<br />
<br />
Yesterday ligations have been inoculated in 1ml LB + AB from colonies and incubated at 37° 220rpm for about 6 hours. Then glycerol stocks were prepared and the remaining 250 ul of the cultures have been re-filled with 5 ml of LB + antibiotic and incubated overnight to prepare the screening. The cultures are:<br />
*B7-1 (Amp)<br />
*B7-2 (Amp)<br />
*B12-1 (Amp)<br />
*B12-2 (Amp)<br />
*B13-1 (Amp)<br />
*B13-2 (Amp)<br />
*B9new-1 (Amp)<br />
*B9new-2 (Amp)<br />
*B9new-3 (Amp)<br />
*B9new-4 (Amp)<br />
*B9new-5 (Amp)<br />
*A19-1 (Cm)<br />
*A19-2 (Cm)<br />
<br />
Dilution 1:100 of cultures:<br />
*A15-1<br />
*A15-3<br />
*F2620<br />
and inoculum of A15-3 colony picked from colony grown on plate infected yesterday.<br />
Cultures have been incubated for further 4 hours for lysis test at TECAN in the afternoon.<br />
*Preparation of 3OC6HSL at desired concentration, to be used for induction test in the afternoon.<br />
<br />
A14 L-1 induced and A14 L-1 NOT induced have been measured using TECAN: the fluorescence in green wasn't significantly different. It could mean that A14 has mutated, so we have prepared an inculum from glycerol stocks of <br />
*A11 <br />
*E0240<br />
to repeat ligation tomorrow.<br />
<br />
*Preparation of LB+Amp 10% glucose:<br />
LB + Amp has been prepared as always for 500ml, but just 400ml of water have been added.<br />
The further 100ml have been used to dilute 50g glucose and have been added after autoclave, filtering them.<br />
*Preparation of LB+Amp (500ml)<br />
<br />
TECAN test in the afternoon for A15 lysis.<br />
Glycerol stock for all the cultures incubated this morning.<br />
Inoculum of A8pg, A2, RBS32 for tomorrow induction test of A8pg construct with aTc.<br />
Inoculum of A15-3 for tomorrow lysis test (to repeat today's test because the results were not satisfying).<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/A14 tentative TEST 1_10_09.pdf" target="_blank">Download Protocol</a></html><br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/lysis A15 TEST 1-10-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
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</div><br />
<br />
== <html><font class="dayw_style">October, 2nd</font></html> ==<br />
<br />
Dilution of A15 and induction with 3OC6HSL to test the activity os this part (it didn't work yesterday!)<br />
<br />
In the morning we have miniprepped:<br />
<br />
*A11-3<br />
*GFP3<br />
<br />
to re-ligate A14<br />
<br />
We have also miniprepped:<br />
<br />
*B9 new-1<br />
*B9 new-2<br />
*B9 new-3<br />
*B9 new-4<br />
*B9 new-5<br />
*A19-1<br />
*A19-2<br />
<br />
incubated overnight at 37°C 220rpm. They have been refilled from yesterday cultures used for glycerol stocks.<br />
<br />
After mini-prep, the cultures have been digested:<br />
<br />
*A11-3 (S-P) for ligation<br />
*GFP3 (X-P) for ligation<br />
*B9 new-1 (E-P) for screening<br />
*B9 new-2 (E-P) for screening<br />
*B9 new-3 (E-P) for screening<br />
*B9 new-4 (E-P) for screening<br />
*B9 new-5 (E-P) for screening<br />
*A19-1 (E-P) for screening<br />
*A19-2 (E-P) for screening<br />
<br />
Gel has been filled as follows:<br />
B9new-1 - B9new-2 - B9new-3 - B9new-4 - B9new-5 - A19-1 - A19-2 - - Marker- - A11 - - GFP3 - - - <br />
<br />
<br />
<table align="center"><br />
<tr><br />
<td><br />
<font class='didascalia'><br />
[[Image:GEL021009.jpg|thumb|500px|left|Screening gel for B9new 1-5, A19-1 and A19-2. Gel extraction for A11-3 and E0240.]]<br />
</font><br />
</td><br />
</tr><br />
</table><br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 3rd</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 4th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5SepTeam:UNIPV-Pavia/Notebook/Week5Sep2009-10-21T23:32:31Z<p>110hs: /* October, 3rd */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from September 28th, to October 4th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 28th</font></html> ==<br />
===== pH sensor =====<br />
''3rd experiment''<br />
*We put 50 ul into 5 ml LB NaCl 171 mM + Amp pH 6,6 falcon for each of the overnight cultures RBS33 - A2 - pNhaA and incubated them for four hours and a half.<br />
*OD measure of the three falcons with TECAN in order to make dilutions with a final volume of 3 ml and an OD equal to 0,02.<br />
*We diluted:<br />
**RBS into:<br />
*** LB NaCl 171 mM + Amp pH 5,5<br />
*** LB NaCl 171 mM + Amp pH 6,6<br />
*** LB NaCl 171 mM + Amp pH 7,5<br />
*** LB NaCl 171 mM + Amp pH 8,5<br />
**pNhaA into:<br />
*** LB NaCl 171 mM + Amp pH 5,5<br />
*** LB NaCl 171 mM + Amp pH 6,6<br />
*** LB NaCl 171 mM + Amp pH 7,5<br />
*** LB NaCl 171 mM + Amp pH 8,5<br />
**A2 into:<br />
*** LB NaCl 171 mM + Amp pH 5,5<br />
*** LB NaCl 171 mM + Amp pH 6,6<br />
*** LB NaCl 171 mM + Amp pH 7,5<br />
*** LB NaCl 171 mM + Amp pH 8,5<br />
*We started this 6 hours' experiment that performed absorbance and fluorescence measures (on three different wells) for each sample every 5 minutes.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 29th</font></html> ==<br />
<br />
*A14 induction test using IPTG<br />
The part doesn't show any induction!! :-(<br />
To test what happened, we have inoculated A14 L-1 from glycerol stocks. tomorrow it will be diluted 1:100 in 2 falcons (5ml), one of wich will be induced 10nM.<br />
<br />
*Inoculum of T9002 from glycerol stocks in 2 falcon tubes, one grown anaerobically and the other areobically. They have been induced with 3OC6HSL 10nM and next morning the measure of GFP was about the half in the anaerobically grown culture respect to the aerobically grown one.<br />
<br />
*Preparation of 2 falcon tubes (50 ml) containing 35ml LB+Amp+2%glucose and infected from glycerol stocks with B9-2 and F2620MIT2. They have been incubated overnight at 37°C without shaking.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/IPTG A14 A17 induction test TEST 29-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 30th</font></html> ==<br />
<br />
We watched B9 and F2620 grown overnight in anaerobic way. In B9 lots of foam has developed that we couldn't see in F2620. Is it for it fermented and made CO2? In the evening they have been pelletted and F2620's pellet was bigger than B9's one.<br />
<br />
LIGATIONS:<br />
*B7 = A4(E-S) + B5new2-3(E-X) in pSB1AK3<br />
*B9: F2620(E-S) + B5new2-3(X-P-ClaI) in pSB1A2<br />
*B12: J23118(S-P) + B5new2-3(E-X) in pSB1A2<br />
*B13: J23106(S-P) + B5new2-3(E-X) in pSB1A2 <br />
*A19: F2620(E-P) + 4C5(E-P) in pSB4C5<br />
<br />
Inoculum of A15-1, A15-3 F2620 from glycerol stocks for tomorrow lysis test. A15-3 has also been plated on LB+amp+agar2% to pick single colony for tomorrow test.<br />
<br />
A14 inoculated yesterday has been diluted 1:100 into 2 falcons and induced with 1 mM IPTG. Tomorrow we will see if the part works.<br />
<br />
*Inoculum of 15ul from glycerol stock of B9 and F2620 in 2 cultures each, one closed (anaerobic) and the other a little opened ("aerobic"). The cultures have been induced immediately with 3OC6HSL 10nM. <br />
They have been incubated overnight at 37°C without shaking.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/aTc induction test M9 TEST 30-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 1st</font></html> ==<br />
<br />
*From the cultures incubated yesterday, F2620 grew, but B9 didn't. We have decided not to induce anymore and to exploit F2620 leakage to express pdc and adhB.<br />
<br />
Yesterday ligations have been inoculated in 1ml LB + AB from colonies and incubated at 37° 220rpm for about 6 hours. Then glycerol stocks were prepared and the remaining 250 ul of the cultures have been re-filled with 5 ml of LB + antibiotic and incubated overnight to prepare the screening. The cultures are:<br />
*B7-1 (Amp)<br />
*B7-2 (Amp)<br />
*B12-1 (Amp)<br />
*B12-2 (Amp)<br />
*B13-1 (Amp)<br />
*B13-2 (Amp)<br />
*B9new-1 (Amp)<br />
*B9new-2 (Amp)<br />
*B9new-3 (Amp)<br />
*B9new-4 (Amp)<br />
*B9new-5 (Amp)<br />
*A19-1 (Cm)<br />
*A19-2 (Cm)<br />
<br />
Dilution 1:100 of cultures:<br />
*A15-1<br />
*A15-3<br />
*F2620<br />
and inoculum of A15-3 colony picked from colony grown on plate infected yesterday.<br />
Cultures have been incubated for further 4 hours for lysis test at TECAN in the afternoon.<br />
*Preparation of 3OC6HSL at desired concentration, to be used for induction test in the afternoon.<br />
<br />
A14 L-1 induced and A14 L-1 NOT induced have been measured using TECAN: the fluorescence in green wasn't significantly different. It could mean that A14 has mutated, so we have prepared an inculum from glycerol stocks of <br />
*A11 <br />
*E0240<br />
to repeat ligation tomorrow.<br />
<br />
*Preparation of LB+Amp 10% glucose:<br />
LB + Amp has been prepared as always for 500ml, but just 400ml of water have been added.<br />
The further 100ml have been used to dilute 50g glucose and have been added after autoclave, filtering them.<br />
*Preparation of LB+Amp (500ml)<br />
<br />
TECAN test in the afternoon for A15 lysis.<br />
Glycerol stock for all the cultures incubated this morning.<br />
Inoculum of A8pg, A2, RBS32 for tomorrow induction test of A8pg construct with aTc.<br />
Inoculum of A15-3 for tomorrow lysis test (to repeat today's test because the results were not satisfying).<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/A14 tentative TEST 1_10_09.pdf" target="_blank">Download Protocol</a></html><br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/lysis A15 TEST 1-10-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 2nd</font></html> ==<br />
<br />
Dilution of A15 and induction with 3OC6HSL to test the activity os this part (it didn't work yesterday!)<br />
<br />
In the morning we have miniprepped:<br />
<br />
*A11-3<br />
*GFP3<br />
<br />
to re-ligate A14<br />
<br />
We have also miniprepped:<br />
<br />
*B9 new-1<br />
*B9 new-2<br />
*B9 new-3<br />
*B9 new-4<br />
*B9 new-5<br />
*A19-1<br />
*A19-2<br />
<br />
incubated overnight at 37°C 220rpm. They have been refilled from yesterday cultures used for glycerol stocks.<br />
<br />
After mini-prep, the cultures have been digested:<br />
<br />
*A11-3 (S-P) for ligation<br />
*GFP3 (X-P) for ligation<br />
*B9 new-1 (E-P) for screening<br />
*B9 new-2 (E-P) for screening<br />
*B9 new-3 (E-P) for screening<br />
*B9 new-4 (E-P) for screening<br />
*B9 new-5 (E-P) for screening<br />
*A19-1 (E-P) for screening<br />
*A19-2 (E-P) for screening<br />
<br />
Gel has been filled as follows:<br />
B9new-1 - B9new-2 - B9new-3 - B9new-4 - B9new-5 - A19-1 - A19-2 - - Marker- - A11 - - GFP3 - - - <br />
<br />
<br />
<table align="center"><br />
<tr><br />
<td><br />
<font class='didascalia'><br />
[[Image:GEL021009.jpg|thumb|500px|left|Screening gel for B9new 1-5, A19-1 and A19-2. Gel extraction for A11-3 and E0240.]]<br />
</font><br />
</td><br />
</tr><br />
</table><br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 3rd</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">October, 4th</font></html> ==<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Oct#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4SepTeam:UNIPV-Pavia/Notebook/Week4Sep2009-10-21T23:32:04Z<p>110hs: /* September, 26th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from September 21st, to September 27th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 21st</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 22nd</font></html> ==<br />
===== pH sensor =====<br />
*LB NaCl 70 mM pH 5,5 - 6,6 - 7,5 - 8,5 + Amp preparation.<br />
* Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:<br />
**RBS33<br />
**A2<br />
**pNhaA<br />
* Overnight incubation of these three cultures at 37°C, 220 rpm.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/T9002 TEST 22-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 23rd</font></html> ==<br />
===== pH sensor =====<br />
''2nd experiment''<br />
*We put 50 ul into 5 ml LB NaCl 70 mM + Amp pH 6,6 falcon for each of the overnight cultures RBS33 - A2 - pNhaA and incubated them for four hours and a half at 37°C, 220 rpm.<br />
*OD measure of the three falcons with TECAN in order to make dilutions with a final volume of 3 ml and an OD equal to 0,02.<br />
*We diluted:<br />
**RBS into:<br />
*** LB NaCl 70 mM + Amp pH 5,5<br />
*** LB NaCl 70 mM + Amp pH 6,6<br />
*** LB NaCl 70 mM + Amp pH 7,5<br />
*** LB NaCl 70 mM + Amp pH 8,5<br />
**pNhaA into:<br />
*** LB NaCl 70 mM + Amp pH 5,5<br />
*** LB NaCl 70 mM + Amp pH 6,6<br />
*** LB NaCl 70 mM + Amp pH 7,5<br />
*** LB NaCl 70 mM + Amp pH 8,5<br />
**A2 into:<br />
*** LB NaCl 70 mM + Amp pH 5,5<br />
*** LB NaCl 70 mM + Amp pH 6,6<br />
*** LB NaCl 70 mM + Amp pH 7,5<br />
*** LB NaCl 70 mM + Amp pH 8,5<br />
*We started this 6 hours' experiment that performed absorbance and fluorescence measures (on three different wells) for each sample every 5 minutes.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 24th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 25th</font></html> ==<br />
===== pH sensor =====<br />
*Preparation of LB NaCl 151 mM at pH 5,5 - 6,6 - 7,5 - 8,5.<br />
*Preparation of LB NaCl 250 mM at pH 5,5 - 6,6 - 7,5 - 8,5.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 26th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 27th</font></html> ==<br />
===== pH sensor =====<br />
*Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:<br />
**RBS33<br />
**A2<br />
**pNhaA<br />
*Overnight incubation of these three cultures at 37°C, 220 rpm.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4SepTeam:UNIPV-Pavia/Notebook/Week4Sep2009-10-21T23:31:56Z<p>110hs: /* September, 24th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from September 21st, to September 27th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 21st</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 22nd</font></html> ==<br />
===== pH sensor =====<br />
*LB NaCl 70 mM pH 5,5 - 6,6 - 7,5 - 8,5 + Amp preparation.<br />
* Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:<br />
**RBS33<br />
**A2<br />
**pNhaA<br />
* Overnight incubation of these three cultures at 37°C, 220 rpm.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/T9002 TEST 22-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 23rd</font></html> ==<br />
===== pH sensor =====<br />
''2nd experiment''<br />
*We put 50 ul into 5 ml LB NaCl 70 mM + Amp pH 6,6 falcon for each of the overnight cultures RBS33 - A2 - pNhaA and incubated them for four hours and a half at 37°C, 220 rpm.<br />
*OD measure of the three falcons with TECAN in order to make dilutions with a final volume of 3 ml and an OD equal to 0,02.<br />
*We diluted:<br />
**RBS into:<br />
*** LB NaCl 70 mM + Amp pH 5,5<br />
*** LB NaCl 70 mM + Amp pH 6,6<br />
*** LB NaCl 70 mM + Amp pH 7,5<br />
*** LB NaCl 70 mM + Amp pH 8,5<br />
**pNhaA into:<br />
*** LB NaCl 70 mM + Amp pH 5,5<br />
*** LB NaCl 70 mM + Amp pH 6,6<br />
*** LB NaCl 70 mM + Amp pH 7,5<br />
*** LB NaCl 70 mM + Amp pH 8,5<br />
**A2 into:<br />
*** LB NaCl 70 mM + Amp pH 5,5<br />
*** LB NaCl 70 mM + Amp pH 6,6<br />
*** LB NaCl 70 mM + Amp pH 7,5<br />
*** LB NaCl 70 mM + Amp pH 8,5<br />
*We started this 6 hours' experiment that performed absorbance and fluorescence measures (on three different wells) for each sample every 5 minutes.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 24th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 25th</font></html> ==<br />
===== pH sensor =====<br />
*Preparation of LB NaCl 151 mM at pH 5,5 - 6,6 - 7,5 - 8,5.<br />
*Preparation of LB NaCl 250 mM at pH 5,5 - 6,6 - 7,5 - 8,5.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 26th</font></html> ==<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
== <html><font class="dayw_style">September, 27th</font></html> ==<br />
===== pH sensor =====<br />
*Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:<br />
**RBS33<br />
**A2<br />
**pNhaA<br />
*Overnight incubation of these three cultures at 37°C, 220 rpm.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4SepTeam:UNIPV-Pavia/Notebook/Week4Sep2009-10-21T23:31:43Z<p>110hs: /* September, 21st */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from September 21st, to September 27th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 21st</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 22nd</font></html> ==<br />
===== pH sensor =====<br />
*LB NaCl 70 mM pH 5,5 - 6,6 - 7,5 - 8,5 + Amp preparation.<br />
* Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:<br />
**RBS33<br />
**A2<br />
**pNhaA<br />
* Overnight incubation of these three cultures at 37°C, 220 rpm.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/T9002 TEST 22-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 23rd</font></html> ==<br />
===== pH sensor =====<br />
''2nd experiment''<br />
*We put 50 ul into 5 ml LB NaCl 70 mM + Amp pH 6,6 falcon for each of the overnight cultures RBS33 - A2 - pNhaA and incubated them for four hours and a half at 37°C, 220 rpm.<br />
*OD measure of the three falcons with TECAN in order to make dilutions with a final volume of 3 ml and an OD equal to 0,02.<br />
*We diluted:<br />
**RBS into:<br />
*** LB NaCl 70 mM + Amp pH 5,5<br />
*** LB NaCl 70 mM + Amp pH 6,6<br />
*** LB NaCl 70 mM + Amp pH 7,5<br />
*** LB NaCl 70 mM + Amp pH 8,5<br />
**pNhaA into:<br />
*** LB NaCl 70 mM + Amp pH 5,5<br />
*** LB NaCl 70 mM + Amp pH 6,6<br />
*** LB NaCl 70 mM + Amp pH 7,5<br />
*** LB NaCl 70 mM + Amp pH 8,5<br />
**A2 into:<br />
*** LB NaCl 70 mM + Amp pH 5,5<br />
*** LB NaCl 70 mM + Amp pH 6,6<br />
*** LB NaCl 70 mM + Amp pH 7,5<br />
*** LB NaCl 70 mM + Amp pH 8,5<br />
*We started this 6 hours' experiment that performed absorbance and fluorescence measures (on three different wells) for each sample every 5 minutes.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 24th</font></html> ==<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 25th</font></html> ==<br />
===== pH sensor =====<br />
*Preparation of LB NaCl 151 mM at pH 5,5 - 6,6 - 7,5 - 8,5.<br />
*Preparation of LB NaCl 250 mM at pH 5,5 - 6,6 - 7,5 - 8,5.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 26th</font></html> ==<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
== <html><font class="dayw_style">September, 27th</font></html> ==<br />
===== pH sensor =====<br />
*Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:<br />
**RBS33<br />
**A2<br />
**pNhaA<br />
*Overnight incubation of these three cultures at 37°C, 220 rpm.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3SepTeam:UNIPV-Pavia/Notebook/Week3Sep2009-10-21T23:31:30Z<p>110hs: /* September, 20th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from September 14th, to September 20th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 14th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 15th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 16th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 17th</font></html> ==<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/A1A2A7 TEST 17-09-09.pdf" target="_blank">Download Protocol</a></html><br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/Ethanol test TEST 17-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
===== pH sensor =====<br />
*Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:<br />
**RBS33<br />
**A2<br />
**K116002 (from now on it will be indicated only as pNhaA)<br />
*Overnight incubation of these three cultures at 37°C, 220 rpm.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 18th</font></html> ==<br />
<br />
===== pH sensor =====<br />
''1st experiment''<br />
*We put 50 ul into 5 ml LBK + Amp pH 6,6 falcon for each of the overnight cultures RBS33 - A2 - pNhaA and incubated them for four hours and a half at 37°C, 220 rpm.<br />
*OD measure of the three falcons with TECAN in order to make dilutions with a final volume of 3 ml and an OD equal to 0,02.<br />
*We diluted:<br />
**RBS into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
**pNhaA into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
**A2 into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
*We started this 6 hours' experiment that performed absorbance and fluorescence measures (on three different wells) for each sample every 5 minutes.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 19th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 20th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3SepTeam:UNIPV-Pavia/Notebook/Week3Sep2009-10-21T23:31:22Z<p>110hs: /* September, 19th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from September 14th, to September 20th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 14th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 15th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 16th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 17th</font></html> ==<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/A1A2A7 TEST 17-09-09.pdf" target="_blank">Download Protocol</a></html><br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/Ethanol test TEST 17-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
===== pH sensor =====<br />
*Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:<br />
**RBS33<br />
**A2<br />
**K116002 (from now on it will be indicated only as pNhaA)<br />
*Overnight incubation of these three cultures at 37°C, 220 rpm.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 18th</font></html> ==<br />
<br />
===== pH sensor =====<br />
''1st experiment''<br />
*We put 50 ul into 5 ml LBK + Amp pH 6,6 falcon for each of the overnight cultures RBS33 - A2 - pNhaA and incubated them for four hours and a half at 37°C, 220 rpm.<br />
*OD measure of the three falcons with TECAN in order to make dilutions with a final volume of 3 ml and an OD equal to 0,02.<br />
*We diluted:<br />
**RBS into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
**pNhaA into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
**A2 into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
*We started this 6 hours' experiment that performed absorbance and fluorescence measures (on three different wells) for each sample every 5 minutes.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 19th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 20th</font></html> ==<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3SepTeam:UNIPV-Pavia/Notebook/Week3Sep2009-10-21T23:31:11Z<p>110hs: /* Week from September 14th, to September 20th, 2009 */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from September 14th, to September 20th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 14th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 15th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 16th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 17th</font></html> ==<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/A1A2A7 TEST 17-09-09.pdf" target="_blank">Download Protocol</a></html><br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/Ethanol test TEST 17-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
===== pH sensor =====<br />
*Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:<br />
**RBS33<br />
**A2<br />
**K116002 (from now on it will be indicated only as pNhaA)<br />
*Overnight incubation of these three cultures at 37°C, 220 rpm.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 18th</font></html> ==<br />
<br />
===== pH sensor =====<br />
''1st experiment''<br />
*We put 50 ul into 5 ml LBK + Amp pH 6,6 falcon for each of the overnight cultures RBS33 - A2 - pNhaA and incubated them for four hours and a half at 37°C, 220 rpm.<br />
*OD measure of the three falcons with TECAN in order to make dilutions with a final volume of 3 ml and an OD equal to 0,02.<br />
*We diluted:<br />
**RBS into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
**pNhaA into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
**A2 into:<br />
*** LBK + Amp pH 5,5<br />
*** LBK + Amp pH 6,6<br />
*** LBK + Amp pH 7,5<br />
*** LBK + Amp pH 8,5<br />
*We started this 6 hours' experiment that performed absorbance and fluorescence measures (on three different wells) for each sample every 5 minutes.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 19th</font></html> ==<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 20th</font></html> ==<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
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</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2SepTeam:UNIPV-Pavia/Notebook/Week2Sep2009-10-21T23:30:37Z<p>110hs: /* September, 13th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from Semptember 7th, to September 13th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 7th</font></html> ==<br />
<br />
In the morning we have prepared culture of A2 to stock in glycerol and we have prepared LB medium.<br />
<br />
In the afternoon we have incubated in adequate medium:<br />
<br />
<br />
*B9 (10 ml LB+Amp)<br />
*B10 (10 ml LB+Amp)<br />
*T9002 (10 ml LB + Amp)<br />
for a qualitative test of ethanol production,<br />
<br />
<br />
*A9pg (5 ml LB + Amp)<br />
*A16 (5 ml LB + Amp)<br />
*A8 (5 ml LB + Amp)<br />
*A2 (5 ml LB + Amp)<br />
*RBS33 (5 ml LB + Amp)<br />
to perform an aTc induction test,<br />
<br />
<br />
*A15 (25 ml LB + Amp)<br />
*pSB3K3 (25 ml LB + Kan)<br />
*A14-1 L (5 ml LB + Amp)<br />
to miniprep tomorrow.<br />
<br />
<br />
These coltures have been incubated at 37°C, 220 rpm overnight.<br />
Tomorrow, we will miniprep A14-1 L and midiprep A15 and 3K3 (pSB3K3). We perform midiprep on pSB3K3 because it is a low copy number plasmid and on A15 because it is a lysis cassette, so for leackage only "few" cells survive.<br />
<br />
Glycerol stocks of A2-3 have been stored at -80°C, named A2-3 and A2-3 bis.The previous stock has been thrown away.<br />
<br />
'''Preparation of a qualitative test to assay the presence of ethanol'''<br />
<br />
We have also induced coltures B9 and B10 incubated yesterday morning at 37°C 220 rpm in flasks and grown overnight. In the morning the coltures have been diluited to reach the desired OD (0.03), then re-incubated for 4 hours and induced with 3OC6-HSL 10nM. The induced coltures have been incubated at 18.00 o'clock and will stay overnight. Flasks have been ermetically closed, to allow fermentation. This qualitative test is performed to assay the presence oh ethanol.<br />
<br />
Tomorrow we will use the "Ethanol assay kit" to test the presence of ethanol!!<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 8th</font></html> ==<br />
<br />
A first test with ethanol assay kit has been made using coltures B9 and B10 induced the evening before and using as blank the reaction mix of Kit without ethanol. In the evening, we have also made a measure of blank made by LB + mix.<br />
The test have been performed on the supernatant of the coltures, obtained by centrifugation for 20 minutes 13000rpm.<br />
<br />
* A14 L-1 have been miniprepped, with a yield of 33ng/ul<br />
*pSB3K3 and A15 have been midi-prepped and tomorrow will be digested E-P and ligated<br />
<br />
aTc induction test:<br />
*one of the coltures incubated the day before has been "accidentally" centrifuged, and has shown no growth in the next 3 hours, so the coltures have been re-inoculated, as the day before: <br />
*A9pg (5 ml LB + Amp)<br />
*A16 (5 ml LB + Amp)<br />
*A8 (5 ml LB + Amp)<br />
*A2 (5 ml LB + Amp)<br />
*RBS33 (5 ml LB + Amp)<br />
to perform a new aTc induction test, tomorrow.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/aTc induction test TEST 08-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 9th</font></html> ==<br />
<br />
Today we made aTc induction test:<br />
*coltures have been diluted 1:100 in the morning (50 ul of colture incubated overnight at 37° 220 rpm in 5 ml of new LB+Amp fresh medium) and incubated in the same conditions for further 4 hours<br />
*in the afternoon they have been diluted again to start from the desired O.D. of 0,02 using the usual phormula.<br />
*Implementation of induction test with aTc<br />
<br />
*Ligation of pSB3K3 with A15: digestion of both the BioBrick E-P, gel extraction and ligation overnight. <br />
* B9 B10 and F620 have been inoculated from glycerol stocks to be used tomorrow for thanol assay test.<br />
<br />
We have also received the resultes of A17 sequencing.<br />
In the afternoon we have made a qualitative ethanol assay test:<br />
coltures induced yesterday with 3OC6HSL 10nM have been centrifugate and supernatant has been diluted:<br />
*1:400<br />
*1:100<br />
*1:10<br />
<br />
The measure was not significant for the Ethanol Assay Kit doesn't work.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/aTc TEST 09-09-09.pdf" target="_blank">Download Protocol</a></html><br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/Ethanol Qualitative TEST 09-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 10th</font></html> ==<br />
The ligation of A15 and pSB3K3, named A19, has been tranformed in E. coli TOP10.<br />
The coltures B9, B10, F2620 have been diluted 1:100 and induced with 3OC6HSL 100nM to be tested, with glucose 10% added.<br />
<br />
Test aTc with good results in LB<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 11th</font></html> ==<br />
<br />
The lab has been closed for techinal problems, so we have just performed test at TECAN<br />
<br />
Ethanol test: ethanol assay kit doesn't work!!! We've tested coltures induced woth 3OC6HSL 10nM and with 10% glucose added. The measure has been made on the whole cultures, not centrifugated, for we couldn't have access to the lab.<br />
No result has been obtained for the enzyme of ethanol assay kit doesn't work!<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/PiatraEtanolo_11_09_09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
At 16.00, meeting of our team with representatives of Costa Foundation.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 12th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 13th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2SepTeam:UNIPV-Pavia/Notebook/Week2Sep2009-10-21T23:30:28Z<p>110hs: /* September, 12th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from Semptember 7th, to September 13th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">September, 7th</font></html> ==<br />
<br />
In the morning we have prepared culture of A2 to stock in glycerol and we have prepared LB medium.<br />
<br />
In the afternoon we have incubated in adequate medium:<br />
<br />
<br />
*B9 (10 ml LB+Amp)<br />
*B10 (10 ml LB+Amp)<br />
*T9002 (10 ml LB + Amp)<br />
for a qualitative test of ethanol production,<br />
<br />
<br />
*A9pg (5 ml LB + Amp)<br />
*A16 (5 ml LB + Amp)<br />
*A8 (5 ml LB + Amp)<br />
*A2 (5 ml LB + Amp)<br />
*RBS33 (5 ml LB + Amp)<br />
to perform an aTc induction test,<br />
<br />
<br />
*A15 (25 ml LB + Amp)<br />
*pSB3K3 (25 ml LB + Kan)<br />
*A14-1 L (5 ml LB + Amp)<br />
to miniprep tomorrow.<br />
<br />
<br />
These coltures have been incubated at 37°C, 220 rpm overnight.<br />
Tomorrow, we will miniprep A14-1 L and midiprep A15 and 3K3 (pSB3K3). We perform midiprep on pSB3K3 because it is a low copy number plasmid and on A15 because it is a lysis cassette, so for leackage only "few" cells survive.<br />
<br />
Glycerol stocks of A2-3 have been stored at -80°C, named A2-3 and A2-3 bis.The previous stock has been thrown away.<br />
<br />
'''Preparation of a qualitative test to assay the presence of ethanol'''<br />
<br />
We have also induced coltures B9 and B10 incubated yesterday morning at 37°C 220 rpm in flasks and grown overnight. In the morning the coltures have been diluited to reach the desired OD (0.03), then re-incubated for 4 hours and induced with 3OC6-HSL 10nM. The induced coltures have been incubated at 18.00 o'clock and will stay overnight. Flasks have been ermetically closed, to allow fermentation. This qualitative test is performed to assay the presence oh ethanol.<br />
<br />
Tomorrow we will use the "Ethanol assay kit" to test the presence of ethanol!!<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 8th</font></html> ==<br />
<br />
A first test with ethanol assay kit has been made using coltures B9 and B10 induced the evening before and using as blank the reaction mix of Kit without ethanol. In the evening, we have also made a measure of blank made by LB + mix.<br />
The test have been performed on the supernatant of the coltures, obtained by centrifugation for 20 minutes 13000rpm.<br />
<br />
* A14 L-1 have been miniprepped, with a yield of 33ng/ul<br />
*pSB3K3 and A15 have been midi-prepped and tomorrow will be digested E-P and ligated<br />
<br />
aTc induction test:<br />
*one of the coltures incubated the day before has been "accidentally" centrifuged, and has shown no growth in the next 3 hours, so the coltures have been re-inoculated, as the day before: <br />
*A9pg (5 ml LB + Amp)<br />
*A16 (5 ml LB + Amp)<br />
*A8 (5 ml LB + Amp)<br />
*A2 (5 ml LB + Amp)<br />
*RBS33 (5 ml LB + Amp)<br />
to perform a new aTc induction test, tomorrow.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/aTc induction test TEST 08-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 9th</font></html> ==<br />
<br />
Today we made aTc induction test:<br />
*coltures have been diluted 1:100 in the morning (50 ul of colture incubated overnight at 37° 220 rpm in 5 ml of new LB+Amp fresh medium) and incubated in the same conditions for further 4 hours<br />
*in the afternoon they have been diluted again to start from the desired O.D. of 0,02 using the usual phormula.<br />
*Implementation of induction test with aTc<br />
<br />
*Ligation of pSB3K3 with A15: digestion of both the BioBrick E-P, gel extraction and ligation overnight. <br />
* B9 B10 and F620 have been inoculated from glycerol stocks to be used tomorrow for thanol assay test.<br />
<br />
We have also received the resultes of A17 sequencing.<br />
In the afternoon we have made a qualitative ethanol assay test:<br />
coltures induced yesterday with 3OC6HSL 10nM have been centrifugate and supernatant has been diluted:<br />
*1:400<br />
*1:100<br />
*1:10<br />
<br />
The measure was not significant for the Ethanol Assay Kit doesn't work.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/aTc TEST 09-09-09.pdf" target="_blank">Download Protocol</a></html><br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/Ethanol Qualitative TEST 09-09-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 10th</font></html> ==<br />
The ligation of A15 and pSB3K3, named A19, has been tranformed in E. coli TOP10.<br />
The coltures B9, B10, F2620 have been diluted 1:100 and induced with 3OC6HSL 100nM to be tested, with glucose 10% added.<br />
<br />
Test aTc with good results in LB<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 11th</font></html> ==<br />
<br />
The lab has been closed for techinal problems, so we have just performed test at TECAN<br />
<br />
Ethanol test: ethanol assay kit doesn't work!!! We've tested coltures induced woth 3OC6HSL 10nM and with 10% glucose added. The measure has been made on the whole cultures, not centrifugated, for we couldn't have access to the lab.<br />
No result has been obtained for the enzyme of ethanol assay kit doesn't work!<br />
<br />
''Experiment with Tecan F200''<br />
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/PiatraEtanolo_11_09_09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
At 16.00, meeting of our team with representatives of Costa Foundation.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 12th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 13th</font></html> ==<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1SepTeam:UNIPV-Pavia/Notebook/Week1Sep2009-10-21T23:29:31Z<p>110hs: /* September, 6th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<html><a name="week_start"></a></html><br />
= Week from August 31st, to September 6th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Aug#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Aug#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">August, 31st</font></html> ==<br />
<br />
*We inoculated:<br />
**10 colonies from B5new2 plate in 4 ml of LB + Kan for screening;<br />
**B8-5 (8 ul from glycerol stock) in 4 ml of LB + Kan (for purification process);<br />
<br />
*We sent A15-2 purified DNA (stored at -20°C) to BMR Genomics for sequencing.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 1st</font></html> ==<br />
*We received sequencing results for:<br />
**A16-4: sequence ok!<br />
**A14-3: sequence had a deletion in C0012, as we expected because of A11-2 sequencing results, previously received;<br />
**B8-5: sequence wrong in VF2 and good in VR;<br />
**A17: chromatogram was good, but ligation failed (R0011 was not in the insert).<br />
<br />
*COMMENTS after sequencing results:<br />
**now we have a new aTc sensor (A16) to test together with A9;<br />
**B8 has to be repeated or purified, we will try both the approaches;<br />
**A14 has to be repeated using A11-3, which has a consistent sequencing result;<br />
**A17 is going to be ligated today (this time using gel extraction).<br />
<br />
<br />
<br />
<br />
*Glycerol stocks for the 10 B5new2 grown cultures.<br />
<br />
*Miniprep for B5new2 (10 samples) and for B8-5.<br />
<br />
*Digestion for:<br />
**B5new2 (10 samples) for screening (E-P cut);<br />
**B8-5 for purification from gel (E-P cut);<br />
**R0011 (stored at -20°C) for A17new ligation (S-P cut)<br />
**E0240 (stored at -20°C) for A17new ligation (X-P cut)<br />
<br />
*Medium-size gel for B5new2(E-P); small-size gel for B8-5(E-P), R0011(S-P) and E0240(X-P).<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B5new2_digestion_screening.jpg|thumb|500px|left|Digestion screening for B5new2.]]<br />
|}<br />
</font><br />
<br />
*Medium gel results: B5new2 clones all showed the correct length for ligated plasmid! we decided to keep B5new2-3 to continue the assemblies and B5new2-9 as a backup copy. Both plasmids were sent to BMR Genomics for sequencing.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B8_5_actual.jpg|thumb|500px|left|B8-5 construct was actually a wrong plasmid: the 4Kbp fragment was non digested DNA. Sequencing results will tell us what there is inside the cloning site.]]<br />
|}<br />
</font><br />
<br />
*Small gel results: R0011(S-P) and E0240(X-P) were cut and purified through gel-extraction, while B8-5 could not be cut because the bands were not correct: maybe the heaviest band noticed on August 24th was a non-completely digested fragment, not a correct-size insert. We decided to throw B8-5 glycerol stock away.<br />
<br />
*Ligation: A17new = R0011(S-P) + E0240(X-P) in pSB1A2<br />
<br />
*We incubated ligation reaction at 16°C overnight.<br />
<br />
<br />
<br />
*We inoculated:<br />
**B5new2-3<br />
**B6-3<br />
**A4<br />
**F2620MIT1<br />
**A11-3<br />
<br />
<br />
<br />
===== pH sensor =====<br />
*We streaked a single colony LB + Amp agar plate using (the right) K116002 glycerol stock. We incubated the plate at 37°C overnight.<br />
*Preparation of LBK (LB with potassium instead of sodium) at pH 5,5 - 6,6 - 7,5 - 8,5 for the first experiment we would have performed in the following days.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 2nd</font></html> ==<br />
<br />
*Miniprep for:<br />
**B5new2-3<br />
**B6-3<br />
**A11-3<br />
**A4<br />
**F2620MIT1<br />
**E0240 pellet (stored at -20°C)<br />
<br />
*Digestions:<br />
**B5new2-3(E-X)<br />
**B6-3(E-X)<br />
**A11-3(S-P)<br />
**A4(E-S)<br />
**F2620MIT1(E-S)<br />
**E0240(X-P)<br />
<br />
*Gel run, cut and band purification for all the samples.<br />
<br />
*Ligations:<br />
**B7new = A4(E-S) + B5new2-3(E-X) in pSB1AK3<br />
**B8new = A4(E-S) + B6-3(E-X) in pSB1AK3<br />
**B9 = F2620MIT1(E-S) + B5new2-3(E-X) in pSB1AK3<br />
**B10 = F2620MIT1(E-S) + B6-3(E-X) in pSB1AK3<br />
**A14L = A11-3(S-P) + E0240(X-P) in pSB1A2<br />
<br />
*We incubated the five reactions at 16°C overnight.<br />
<br />
<br />
<br />
<br />
*Transformation/plating for A17new ligation.<br />
<br />
<br />
<br />
===== pH sensor =====<br />
*We inoculated a single colony from K116002 streaked plate in 5 ml of LB + Amp.<br />
*Overnight incubation at 37°C, 220 rpm.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 3rd</font></html> ==<br />
<br />
*A17new overnight plate showed only one colony...easy screening for this plate! Colony PCR/electrophoresis for this sample. We stored this colony in 100ul of LB + Amp and incubated it at 37°C waiting for the end of the reaction.<br />
<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_pcr_A17new.jpg|thumb|500px|left|Colony PCR results for the only A17new colony on its plate.]]<br />
|}<br />
</font><br />
<br />
*Gel results: the blank was not so clean, but the only colony was positive:) We inoculated the colony in 5 ml of LB + Amp to grow an overnight culture (37°C, 220 rpm).<br />
<br />
<br />
<br />
*We transformed these ligations:<br />
**B7new 1:40 on Kan<br />
**B8new 1:40 on Kan<br />
**B9 1:40 on Kan<br />
**B10 1:40 on Kan<br />
**A14L 1:20 on Amp<br />
<br />
*We incubated B7new, B8new, B9 and B10 at 37°C in the morning. Then, five colonies for each plate were picked after about 11 hours. We inoculated these colonies in 5 ml of LB + Kan and incubated them overnight (37°C, 220 rpm). NOTE: B7new and B8new had smaller colonies than B9 and B10.<br />
<br />
*We incubated A14L plate overnight at 37°C.<br />
<br />
<br />
===== pH sensor =====<br />
*Miniprep for K116002 overnight culture. We incubated the plate (LB + Amp) with transformed bacteria overnight at 37°C. <br />
*We used 1 ul of this sample to transform TOP10 in order to have the pH sensor in the same strain as the reference promoter (i.e. A2).<br />
*We plated transformed bacteria and incubated overnight at 37°C.<br />
*We also sent purified DNA to BMR Genomics for sequencing.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 4th</font></html> ==<br />
<br />
*A14L plate showed colonies.<br />
<br />
*Colony PCR/electrophoresis for 6 colonies from A14L plate. Colonies were saved inoculating them in 1 ml of LB + Amp and incubating them at 37°C, 220 rpm waiting for the end of the reaction.<br />
<br />
*Gel results: all the colonies showed the amplicon with the right size, but even some extra band...we decided to stock all the colonies in glycerol. We also decided to keep A14L-1: we inoculated it in 5 ml of LB + Amp to grow an overnight culture to check the sequence.<br />
<br />
<br />
<br />
<br />
*We received sequencing results for B8-5again(VF2) and it was not correct.<br />
<br />
<br />
<br />
*Glycerol stock/miniprep for 21 cultures:<br />
**B7new X5 colonies<br />
**B8new X5 colonies<br />
**B9 X5 colonies<br />
**B10 X5 colonies<br />
**A17new<br />
<br />
*We sent A17new purified DNA to BMR Genomics for sequencing.<br />
<br />
*Screening (E-P digestion cut) for B7new, B8new, B9 and B10 samples.<br />
<br />
<br />
===== pH sensor =====<br />
*K116002(TOP10) plate showed a bacterial carpet (with some single colony).<br />
*We inoculated a single colony from K116002 (TOP10) plate in 1 ml of LB + Amp and incubated this inoculum for about 6 hours (37°C, 220 rpm).<br />
*Glycerol stock.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 5th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 6th</font></html> ==<br />
*Wiki updating<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Aug#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Aug#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1SepTeam:UNIPV-Pavia/Notebook/Week1Sep2009-10-21T23:29:20Z<p>110hs: /* September, 5th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<html><a name="week_start"></a></html><br />
= Week from August 31st, to September 6th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Aug#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Aug#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">August, 31st</font></html> ==<br />
<br />
*We inoculated:<br />
**10 colonies from B5new2 plate in 4 ml of LB + Kan for screening;<br />
**B8-5 (8 ul from glycerol stock) in 4 ml of LB + Kan (for purification process);<br />
<br />
*We sent A15-2 purified DNA (stored at -20°C) to BMR Genomics for sequencing.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 1st</font></html> ==<br />
*We received sequencing results for:<br />
**A16-4: sequence ok!<br />
**A14-3: sequence had a deletion in C0012, as we expected because of A11-2 sequencing results, previously received;<br />
**B8-5: sequence wrong in VF2 and good in VR;<br />
**A17: chromatogram was good, but ligation failed (R0011 was not in the insert).<br />
<br />
*COMMENTS after sequencing results:<br />
**now we have a new aTc sensor (A16) to test together with A9;<br />
**B8 has to be repeated or purified, we will try both the approaches;<br />
**A14 has to be repeated using A11-3, which has a consistent sequencing result;<br />
**A17 is going to be ligated today (this time using gel extraction).<br />
<br />
<br />
<br />
<br />
*Glycerol stocks for the 10 B5new2 grown cultures.<br />
<br />
*Miniprep for B5new2 (10 samples) and for B8-5.<br />
<br />
*Digestion for:<br />
**B5new2 (10 samples) for screening (E-P cut);<br />
**B8-5 for purification from gel (E-P cut);<br />
**R0011 (stored at -20°C) for A17new ligation (S-P cut)<br />
**E0240 (stored at -20°C) for A17new ligation (X-P cut)<br />
<br />
*Medium-size gel for B5new2(E-P); small-size gel for B8-5(E-P), R0011(S-P) and E0240(X-P).<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B5new2_digestion_screening.jpg|thumb|500px|left|Digestion screening for B5new2.]]<br />
|}<br />
</font><br />
<br />
*Medium gel results: B5new2 clones all showed the correct length for ligated plasmid! we decided to keep B5new2-3 to continue the assemblies and B5new2-9 as a backup copy. Both plasmids were sent to BMR Genomics for sequencing.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B8_5_actual.jpg|thumb|500px|left|B8-5 construct was actually a wrong plasmid: the 4Kbp fragment was non digested DNA. Sequencing results will tell us what there is inside the cloning site.]]<br />
|}<br />
</font><br />
<br />
*Small gel results: R0011(S-P) and E0240(X-P) were cut and purified through gel-extraction, while B8-5 could not be cut because the bands were not correct: maybe the heaviest band noticed on August 24th was a non-completely digested fragment, not a correct-size insert. We decided to throw B8-5 glycerol stock away.<br />
<br />
*Ligation: A17new = R0011(S-P) + E0240(X-P) in pSB1A2<br />
<br />
*We incubated ligation reaction at 16°C overnight.<br />
<br />
<br />
<br />
*We inoculated:<br />
**B5new2-3<br />
**B6-3<br />
**A4<br />
**F2620MIT1<br />
**A11-3<br />
<br />
<br />
<br />
===== pH sensor =====<br />
*We streaked a single colony LB + Amp agar plate using (the right) K116002 glycerol stock. We incubated the plate at 37°C overnight.<br />
*Preparation of LBK (LB with potassium instead of sodium) at pH 5,5 - 6,6 - 7,5 - 8,5 for the first experiment we would have performed in the following days.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 2nd</font></html> ==<br />
<br />
*Miniprep for:<br />
**B5new2-3<br />
**B6-3<br />
**A11-3<br />
**A4<br />
**F2620MIT1<br />
**E0240 pellet (stored at -20°C)<br />
<br />
*Digestions:<br />
**B5new2-3(E-X)<br />
**B6-3(E-X)<br />
**A11-3(S-P)<br />
**A4(E-S)<br />
**F2620MIT1(E-S)<br />
**E0240(X-P)<br />
<br />
*Gel run, cut and band purification for all the samples.<br />
<br />
*Ligations:<br />
**B7new = A4(E-S) + B5new2-3(E-X) in pSB1AK3<br />
**B8new = A4(E-S) + B6-3(E-X) in pSB1AK3<br />
**B9 = F2620MIT1(E-S) + B5new2-3(E-X) in pSB1AK3<br />
**B10 = F2620MIT1(E-S) + B6-3(E-X) in pSB1AK3<br />
**A14L = A11-3(S-P) + E0240(X-P) in pSB1A2<br />
<br />
*We incubated the five reactions at 16°C overnight.<br />
<br />
<br />
<br />
<br />
*Transformation/plating for A17new ligation.<br />
<br />
<br />
<br />
===== pH sensor =====<br />
*We inoculated a single colony from K116002 streaked plate in 5 ml of LB + Amp.<br />
*Overnight incubation at 37°C, 220 rpm.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 3rd</font></html> ==<br />
<br />
*A17new overnight plate showed only one colony...easy screening for this plate! Colony PCR/electrophoresis for this sample. We stored this colony in 100ul of LB + Amp and incubated it at 37°C waiting for the end of the reaction.<br />
<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_pcr_A17new.jpg|thumb|500px|left|Colony PCR results for the only A17new colony on its plate.]]<br />
|}<br />
</font><br />
<br />
*Gel results: the blank was not so clean, but the only colony was positive:) We inoculated the colony in 5 ml of LB + Amp to grow an overnight culture (37°C, 220 rpm).<br />
<br />
<br />
<br />
*We transformed these ligations:<br />
**B7new 1:40 on Kan<br />
**B8new 1:40 on Kan<br />
**B9 1:40 on Kan<br />
**B10 1:40 on Kan<br />
**A14L 1:20 on Amp<br />
<br />
*We incubated B7new, B8new, B9 and B10 at 37°C in the morning. Then, five colonies for each plate were picked after about 11 hours. We inoculated these colonies in 5 ml of LB + Kan and incubated them overnight (37°C, 220 rpm). NOTE: B7new and B8new had smaller colonies than B9 and B10.<br />
<br />
*We incubated A14L plate overnight at 37°C.<br />
<br />
<br />
===== pH sensor =====<br />
*Miniprep for K116002 overnight culture. We incubated the plate (LB + Amp) with transformed bacteria overnight at 37°C. <br />
*We used 1 ul of this sample to transform TOP10 in order to have the pH sensor in the same strain as the reference promoter (i.e. A2).<br />
*We plated transformed bacteria and incubated overnight at 37°C.<br />
*We also sent purified DNA to BMR Genomics for sequencing.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 4th</font></html> ==<br />
<br />
*A14L plate showed colonies.<br />
<br />
*Colony PCR/electrophoresis for 6 colonies from A14L plate. Colonies were saved inoculating them in 1 ml of LB + Amp and incubating them at 37°C, 220 rpm waiting for the end of the reaction.<br />
<br />
*Gel results: all the colonies showed the amplicon with the right size, but even some extra band...we decided to stock all the colonies in glycerol. We also decided to keep A14L-1: we inoculated it in 5 ml of LB + Amp to grow an overnight culture to check the sequence.<br />
<br />
<br />
<br />
<br />
*We received sequencing results for B8-5again(VF2) and it was not correct.<br />
<br />
<br />
<br />
*Glycerol stock/miniprep for 21 cultures:<br />
**B7new X5 colonies<br />
**B8new X5 colonies<br />
**B9 X5 colonies<br />
**B10 X5 colonies<br />
**A17new<br />
<br />
*We sent A17new purified DNA to BMR Genomics for sequencing.<br />
<br />
*Screening (E-P digestion cut) for B7new, B8new, B9 and B10 samples.<br />
<br />
<br />
===== pH sensor =====<br />
*K116002(TOP10) plate showed a bacterial carpet (with some single colony).<br />
*We inoculated a single colony from K116002 (TOP10) plate in 1 ml of LB + Amp and incubated this inoculum for about 6 hours (37°C, 220 rpm).<br />
*Glycerol stock.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 5th</font></html> ==<br />
*Wiki updating<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">September, 6th</font></html> ==<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Aug#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Aug#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4AugTeam:UNIPV-Pavia/Notebook/Week4Aug2009-10-21T23:28:03Z<p>110hs: /* August, 28th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from August 24th, to August 30th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Aug#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Aug#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">August, 24th</font></html> ==<br />
*We inoculated competent DH5alpha strain (gift from Bologna iGEM09 Team) in 4 ml of LB and incubated it overnight (37°C, 220 rpm).<br />
<br />
*Miniprep for B7 (2 samples), B8 (5 samples), A14-3, A16-4 and I714891 (bacterial pellets stored at -20°C).<br />
<br />
*Screening digestion for all the miniprepped samples:<br />
**B7(E-P)<br />
**B8(E-P)<br />
**A14-3(E-P)<br />
**A16-4(E-P)<br />
**I714891(E-P)<br />
<br />
*Gel run for the digested samples.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B7_B8_A14_A16_3K3.jpg|thumb|500px|left|Digestion screening.]]<br />
|}<br />
</font><br />
<br />
*Gel results:<br />
**B7 - both screened colonies were negative (~3200bp of vector and ~3200bp of insert).<br />
**B8 - only B8-5 SEEMED to show the expected length for ligated plasmid (~3200bp of vector and ~4200bp of insert), but it also shows a single unexpected band...All the other 4 samples were negative (~3200bp of vector and ~3200bp of insert).<br />
**A14-3 - showed the expected length for ligated plasmid (2079bp of vector and ~2200bp of insert).<br />
**A16-4 - showed the expected length for ligated plasmid (2079bp of vector and ~1700bp of insert).<br />
**I714891 - showed the expected length for ligated plasmid (~2700bp of vector and ~700bp of insert).<br />
<br />
*Comments: it seems that we have the final constitutively expressed ethanol producing operon (B8 = RBS-tetR-TT-Ptet-RBS-adhB-RBS-pdc-TT in pSB1AK3). Anyway, an unexpected band is present in this part and in its ancestor (i.e. B6). So, we decided to follow three ways:<br />
**sequence B8-5 and see if it is correct;<br />
**repeat the last two assemblies of the missing version of the ethanol producing operon: B5=B1+B4 and B7=A4+B5;<br />
**try to purify the expected bands from the gel through gel extraction.<br />
<br />
<br />
<br />
<br />
*We sent purified DNA of:<br />
**B8-5<br />
**A14-3<br />
**A16-4<br />
*to BMR Genomics for sequencing.<br />
<br />
<br />
<br />
*We inoculated 8 ul of these glycerol stocks:<br />
**R0011<br />
**E0240<br />
**A15-2<br />
**B1-13<br />
**B4-2<br />
*in 4 ml of LB + suitable antibiotic to grow overnight cultures (37°C, 220 rpm).<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">August, 25th</font></html> ==<br />
*DH5alpha competent cells preparation.<br />
*We streaked a single colony LB agar plate with DH5alpha overnight liquid culture, in order to store it (tomorrow a glycerol stock for it will be prepared).<br />
<br />
*Miniprep for:<br />
**R0011<br />
**E0240<br />
**A15-2<br />
**B1-13<br />
**B4-2<br />
<br />
*Digestion for:<br />
**R0011(S-P)<br />
**E0240(X-P)<br />
**A15-2(E-P)<br />
**B1-13(E-S)<br />
**B4-2(E-X)<br />
**I714891(E-P) (the purified plasmid was stored at -20°C)<br />
<br />
*Precipitation with sodium acetate for all the 6 samples.<br />
<br />
*Ligations:<br />
**B5new = B1(E-S) + B4(E-X) in pSB1AK3<br />
**A17 = R0011(S-P) + E0240(X-P) in pSB1AK3<br />
**A18 = A15-2(E-P) + I714891(E-P) in pSB3K3<br />
*We incubated the ligations at 16°C overnight.<br />
<br />
<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We prepared 25 ml of LB + Kan and M9 supplemented + Kan with 0%, 2.5%, 3.5% and 4.5% ethanol (weight/volume).<br />
<br />
*We inoculated 8 ul of B0015 glycerol stock in 5 ml of LB + Kan in the morning. We let it grow during the day (37°C, 220 rpm) and after about 9 hours we diluted the culture (10 ul of B0015 bacteria in 5 ml of LB + Kan and 10 ul of B0015 bacteria in 5 ml of M9 supplemented + Kan). We incubated the diluted cultures overnight (37°C, 220 rpm).<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">August, 26th</font></html> ==<br />
<br />
*We transformed B5new, A17 and A18 overnight ligations in TOP10. We plated transformed bacteria on LB agar plates + Kan (for B5new and A18) or + Amp (for A17). We incubated B5new plate at 37°C in the morning.<br />
<br />
*After about 10 hours we picked 8 colonies from B5new plate and infected 4 ml of LB + Kan to grow overnight cultures (37°C, 220 rpm) for screening.<br />
<br />
*A17 and A18 plates were incubated overnight at 37°C.<br />
<br />
*We also inoculated:<br />
**DH5alpha strain (4 ml of LB without antibiotic) from the single colony streaked plate;<br />
**E0240 (4 ml of LB + Amp).<br />
<br />
<br />
<br />
*We received sequencing results for:<br />
**A15-1 - sequence ok! (long part) (actually it is not consistent with the Registry, but it is consistent with iGEM 2009 QC, in which there are some problems with K112808, even if the part should work)<br />
**A15-3 - sequence ok! (long part) (actually it is not consistent with the Registry, but it is consistent with iGEM 2009 QC, in which there are some problems with K112808, even if the part should work)<br />
**A11-2 - deletion of 20bp in lacI gene...<br />
**A11-3 - sequence ok!<br />
**B5-3 - sequence ok! (long part)<br />
**B6-3 - sequence ok! (long part)<br />
**B3-5 - sequence ok!<br />
<br />
*COMMENTS:<br />
**we used A11-2 and A15-2 for A14 and A18 ligations...so A14 had to be repeated using A11-3, while we will send A15-2 to BMR for sequencing soon (we forgot to do it!).<br />
**B3 native stock sequence was confirmed! but we still don't know why we see two unexpected bands after B3 digestions...<br />
**B5-3 and B6-3 sequences were confirmed! but we still don't know why we see two unexpected bands after B5 and B6 digestions...<br />
<br />
<br />
''Preparation of experiment with Tecan F200 (for tomorrow!)''<br />
<br />
*We infected 5 ml of LB + Amp with 8 ul of B0030, A2 and T9002 glycerol stocks.<br />
<br />
*We incubated the inocula at 37°C, 220 rpm overnight.<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:2000 the LB and the M9 5 ml overnight cultures of B0015 in all the 50 ml falcon tubes containing 25 ml of LB + Kan and M9 supplemented + Kan with different ethanol concentrations. Before diluting the cultures, we took 3 ml of all the media to use them as blank.<br />
<br />
*We incubated the diluted cultures at 37°C, 220 rpm in the morning.<br />
<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">August, 27th</font></html> ==<br />
*80% glycerol preparation.<br />
<br />
*Glycerol stock and miniprep for the 8 cultures of B5new.<br />
<br />
*Glycerol stock for DH5alpha strain.<br />
<br />
*Digestion (E-P) for all the 8 DNA samples.<br />
<br />
*Gel run for the digested samples.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B5new_digestion_screening.jpg|thumb|500px|left|No positive colonies for B5new.]]<br />
|}<br />
</font><br />
<br />
*Gel results: no positive colony:'( We will repeat the ligation.<br />
<br />
*We infected 4 ml of LB + suitable antibiotic with 8 ul of B1 and B4 glycerol stocks (to repeat "B5" ligation). We incubated these two inocula at 37°C, 220 rpm overnight.<br />
<br />
<br />
*Colony PCR for A17 plate (6 colonies). Bacteria were inoculated in 100 ul of LB + Amp before putting them in the PCR vials, waiting for the end of the reaction.<br />
<br />
*Electrophoresis for PCR results.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_A17_wrong.jpg|thumb|500px|left|Colony PCR results on A17: colonies 1, 4, 5 and 6 seem to be correct!]]<br />
|}<br />
</font><br />
<br />
*Gel results: we chose A17-4 to grow an overnight culture in LB + Amp (37°C, 220 rpm).<br />
<br />
<br />
<br />
*We picked 7 colonies from A18 plate to inoculate 4 ml of LB + Kan. We incubated them at 37°C, 220 rpm overnight.<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200 (for tomorrow!)''<br />
<br />
*We infected 4 ml of M9 + Amp with 8 ul of A2 and B0030 glycerol stocks.<br />
<br />
*We incubated the inocula at 37°C, 220 rpm overnight.<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:100 the overnight cultures of B0030, A2 and T9002 in 5 ml of LB + Amp and in 5 ml of M9 supplemented + Amp.<br />
<br />
*We incubated the diluted cultures at 37°C, 220 rpm for 4 hours.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
<br />
* <html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/T9002 induction test TEST 27-08-09.pdf" target="_blank">Download Protocol</a></html><br />
* <html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/Kynetic Cycle LB-M9 27_08_09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">August, 28th</font></html> ==<br />
*We prepared a pellet for E0240, discarded supernatant (LB), dried the pellet and stored the dry pellet at -20°C, ready to be miniprepped.<br />
<br />
*Glycerol stocks for the 7 cultures of A18 and for A17-4.<br />
<br />
*We prepared a bacterial pellet for the 7 cultures of A18, that will be screened after having A15-2 sequencing results.<br />
<br />
*Miniprep for A17-4, B1-13 and B4-2.<br />
<br />
*Digestion for:<br />
**B1(E-S)<br />
**B4(E-X)<br />
<br />
*Gel run for B1(E-S) (1 ul for check) and for B4(E-X) (all the volume). Gel cut/purification for B4(E-X); precipitation with sodium acetate for B1(E-S).<br />
<br />
*Ligation: B5new2 = B1(E-S) + B4 (E-X) in pSB1AK3. We incubated the ligation overnight at 16°C.<br />
<br />
<br />
*We sent A17-4 purified DNA to BMR Genomics for sequencing.<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:10 the overnight cultures of B0030 and A2 in 5 ml of M9 supplemented + Amp.<br />
<br />
*We incubated the diluted cultures at 37°C, 220 rpm for 4 hours.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">August, 29th</font></html> ==<br />
<br />
*We inactivated T4 ligase (65°C for 10 min) and put the ligation at -20°C.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">August, 30th</font></html> ==<br />
<br />
*We transformed B5new2 ligation, stored at -20°C with a 1:60 dilution. We incubated the plated bacteria at 37°C overnight.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Aug#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Aug#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4AugTeam:UNIPV-Pavia/Notebook/Week4Aug2009-10-21T23:27:44Z<p>110hs: /* August, 27th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from August 24th, to August 30th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Aug#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Aug#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">August, 24th</font></html> ==<br />
*We inoculated competent DH5alpha strain (gift from Bologna iGEM09 Team) in 4 ml of LB and incubated it overnight (37°C, 220 rpm).<br />
<br />
*Miniprep for B7 (2 samples), B8 (5 samples), A14-3, A16-4 and I714891 (bacterial pellets stored at -20°C).<br />
<br />
*Screening digestion for all the miniprepped samples:<br />
**B7(E-P)<br />
**B8(E-P)<br />
**A14-3(E-P)<br />
**A16-4(E-P)<br />
**I714891(E-P)<br />
<br />
*Gel run for the digested samples.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B7_B8_A14_A16_3K3.jpg|thumb|500px|left|Digestion screening.]]<br />
|}<br />
</font><br />
<br />
*Gel results:<br />
**B7 - both screened colonies were negative (~3200bp of vector and ~3200bp of insert).<br />
**B8 - only B8-5 SEEMED to show the expected length for ligated plasmid (~3200bp of vector and ~4200bp of insert), but it also shows a single unexpected band...All the other 4 samples were negative (~3200bp of vector and ~3200bp of insert).<br />
**A14-3 - showed the expected length for ligated plasmid (2079bp of vector and ~2200bp of insert).<br />
**A16-4 - showed the expected length for ligated plasmid (2079bp of vector and ~1700bp of insert).<br />
**I714891 - showed the expected length for ligated plasmid (~2700bp of vector and ~700bp of insert).<br />
<br />
*Comments: it seems that we have the final constitutively expressed ethanol producing operon (B8 = RBS-tetR-TT-Ptet-RBS-adhB-RBS-pdc-TT in pSB1AK3). Anyway, an unexpected band is present in this part and in its ancestor (i.e. B6). So, we decided to follow three ways:<br />
**sequence B8-5 and see if it is correct;<br />
**repeat the last two assemblies of the missing version of the ethanol producing operon: B5=B1+B4 and B7=A4+B5;<br />
**try to purify the expected bands from the gel through gel extraction.<br />
<br />
<br />
<br />
<br />
*We sent purified DNA of:<br />
**B8-5<br />
**A14-3<br />
**A16-4<br />
*to BMR Genomics for sequencing.<br />
<br />
<br />
<br />
*We inoculated 8 ul of these glycerol stocks:<br />
**R0011<br />
**E0240<br />
**A15-2<br />
**B1-13<br />
**B4-2<br />
*in 4 ml of LB + suitable antibiotic to grow overnight cultures (37°C, 220 rpm).<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">August, 25th</font></html> ==<br />
*DH5alpha competent cells preparation.<br />
*We streaked a single colony LB agar plate with DH5alpha overnight liquid culture, in order to store it (tomorrow a glycerol stock for it will be prepared).<br />
<br />
*Miniprep for:<br />
**R0011<br />
**E0240<br />
**A15-2<br />
**B1-13<br />
**B4-2<br />
<br />
*Digestion for:<br />
**R0011(S-P)<br />
**E0240(X-P)<br />
**A15-2(E-P)<br />
**B1-13(E-S)<br />
**B4-2(E-X)<br />
**I714891(E-P) (the purified plasmid was stored at -20°C)<br />
<br />
*Precipitation with sodium acetate for all the 6 samples.<br />
<br />
*Ligations:<br />
**B5new = B1(E-S) + B4(E-X) in pSB1AK3<br />
**A17 = R0011(S-P) + E0240(X-P) in pSB1AK3<br />
**A18 = A15-2(E-P) + I714891(E-P) in pSB3K3<br />
*We incubated the ligations at 16°C overnight.<br />
<br />
<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We prepared 25 ml of LB + Kan and M9 supplemented + Kan with 0%, 2.5%, 3.5% and 4.5% ethanol (weight/volume).<br />
<br />
*We inoculated 8 ul of B0015 glycerol stock in 5 ml of LB + Kan in the morning. We let it grow during the day (37°C, 220 rpm) and after about 9 hours we diluted the culture (10 ul of B0015 bacteria in 5 ml of LB + Kan and 10 ul of B0015 bacteria in 5 ml of M9 supplemented + Kan). We incubated the diluted cultures overnight (37°C, 220 rpm).<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">August, 26th</font></html> ==<br />
<br />
*We transformed B5new, A17 and A18 overnight ligations in TOP10. We plated transformed bacteria on LB agar plates + Kan (for B5new and A18) or + Amp (for A17). We incubated B5new plate at 37°C in the morning.<br />
<br />
*After about 10 hours we picked 8 colonies from B5new plate and infected 4 ml of LB + Kan to grow overnight cultures (37°C, 220 rpm) for screening.<br />
<br />
*A17 and A18 plates were incubated overnight at 37°C.<br />
<br />
*We also inoculated:<br />
**DH5alpha strain (4 ml of LB without antibiotic) from the single colony streaked plate;<br />
**E0240 (4 ml of LB + Amp).<br />
<br />
<br />
<br />
*We received sequencing results for:<br />
**A15-1 - sequence ok! (long part) (actually it is not consistent with the Registry, but it is consistent with iGEM 2009 QC, in which there are some problems with K112808, even if the part should work)<br />
**A15-3 - sequence ok! (long part) (actually it is not consistent with the Registry, but it is consistent with iGEM 2009 QC, in which there are some problems with K112808, even if the part should work)<br />
**A11-2 - deletion of 20bp in lacI gene...<br />
**A11-3 - sequence ok!<br />
**B5-3 - sequence ok! (long part)<br />
**B6-3 - sequence ok! (long part)<br />
**B3-5 - sequence ok!<br />
<br />
*COMMENTS:<br />
**we used A11-2 and A15-2 for A14 and A18 ligations...so A14 had to be repeated using A11-3, while we will send A15-2 to BMR for sequencing soon (we forgot to do it!).<br />
**B3 native stock sequence was confirmed! but we still don't know why we see two unexpected bands after B3 digestions...<br />
**B5-3 and B6-3 sequences were confirmed! but we still don't know why we see two unexpected bands after B5 and B6 digestions...<br />
<br />
<br />
''Preparation of experiment with Tecan F200 (for tomorrow!)''<br />
<br />
*We infected 5 ml of LB + Amp with 8 ul of B0030, A2 and T9002 glycerol stocks.<br />
<br />
*We incubated the inocula at 37°C, 220 rpm overnight.<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:2000 the LB and the M9 5 ml overnight cultures of B0015 in all the 50 ml falcon tubes containing 25 ml of LB + Kan and M9 supplemented + Kan with different ethanol concentrations. Before diluting the cultures, we took 3 ml of all the media to use them as blank.<br />
<br />
*We incubated the diluted cultures at 37°C, 220 rpm in the morning.<br />
<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">August, 27th</font></html> ==<br />
*80% glycerol preparation.<br />
<br />
*Glycerol stock and miniprep for the 8 cultures of B5new.<br />
<br />
*Glycerol stock for DH5alpha strain.<br />
<br />
*Digestion (E-P) for all the 8 DNA samples.<br />
<br />
*Gel run for the digested samples.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B5new_digestion_screening.jpg|thumb|500px|left|No positive colonies for B5new.]]<br />
|}<br />
</font><br />
<br />
*Gel results: no positive colony:'( We will repeat the ligation.<br />
<br />
*We infected 4 ml of LB + suitable antibiotic with 8 ul of B1 and B4 glycerol stocks (to repeat "B5" ligation). We incubated these two inocula at 37°C, 220 rpm overnight.<br />
<br />
<br />
*Colony PCR for A17 plate (6 colonies). Bacteria were inoculated in 100 ul of LB + Amp before putting them in the PCR vials, waiting for the end of the reaction.<br />
<br />
*Electrophoresis for PCR results.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_A17_wrong.jpg|thumb|500px|left|Colony PCR results on A17: colonies 1, 4, 5 and 6 seem to be correct!]]<br />
|}<br />
</font><br />
<br />
*Gel results: we chose A17-4 to grow an overnight culture in LB + Amp (37°C, 220 rpm).<br />
<br />
<br />
<br />
*We picked 7 colonies from A18 plate to inoculate 4 ml of LB + Kan. We incubated them at 37°C, 220 rpm overnight.<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200 (for tomorrow!)''<br />
<br />
*We infected 4 ml of M9 + Amp with 8 ul of A2 and B0030 glycerol stocks.<br />
<br />
*We incubated the inocula at 37°C, 220 rpm overnight.<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:100 the overnight cultures of B0030, A2 and T9002 in 5 ml of LB + Amp and in 5 ml of M9 supplemented + Amp.<br />
<br />
*We incubated the diluted cultures at 37°C, 220 rpm for 4 hours.<br />
<br />
<br />
''Experiment with Tecan F200''<br />
<br />
* <html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/T9002 induction test TEST 27-08-09.pdf" target="_blank">Download Protocol</a></html><br />
* <html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/Kynetic Cycle LB-M9 27_08_09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">August, 28th</font></html> ==<br />
*We prepared a pellet for E0240, discarded supernatant (LB), dried the pellet and stored the dry pellet at -20°C, ready to be miniprepped.<br />
<br />
*Glycerol stocks for the 7 cultures of A18 and for A17-4.<br />
<br />
*We prepared a bacterial pellet for the 7 cultures of A18, that will be screened after having A15-2 sequencing results.<br />
<br />
*Miniprep for A17-4, B1-13 and B4-2.<br />
<br />
*Digestion for:<br />
**B1(E-S)<br />
**B4(E-X)<br />
<br />
*Gel run for B1(E-S) (1 ul for check) and for B4(E-X) (all the volume). Gel cut/purification for B4(E-X); precipitation with sodium acetate for B1(E-S).<br />
<br />
*Ligation: B5new2 = B1(E-S) + B4 (E-X) in pSB1AK3. We incubated the ligation overnight at 16°C.<br />
<br />
<br />
*We sent A17-4 purified DNA to BMR Genomics for sequencing.<br />
<br />
<br />
<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:10 the overnight cultures of B0030 and A2 in 5 ml of M9 supplemented + Amp.<br />
<br />
*We incubated the diluted cultures at 37°C, 220 rpm for 4 hours.<br />
<br />
<br />
<br />
<br />
<br />
''Experiment with Tecan F200''<br />
<br />
* <html><u>Description</u></html><br />
* <html><u>Purpose:</u></html><br />
* <html><u>Materials & Methods</u></html><br />
* <html><u>Protocol</u></html><br />
* <html><u>Results</u></html><br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">August, 29th</font></html> ==<br />
<br />
*We inactivated T4 ligase (65°C for 10 min) and put the ligation at -20°C.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">August, 30th</font></html> ==<br />
<br />
*We transformed B5new2 ligation, stored at -20°C with a 1:60 dilution. We incubated the plated bacteria at 37°C overnight.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Aug#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Aug#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4AugTeam:UNIPV-Pavia/Notebook/Week4Aug2009-10-21T23:27:21Z<p>110hs: /* August, 26th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from August 24th, to August 30th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Aug#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Aug#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">August, 24th</font></html> ==<br />
*We inoculated competent DH5alpha strain (gift from Bologna iGEM09 Team) in 4 ml of LB and incubated it overnight (37°C, 220 rpm).<br />
<br />
*Miniprep for B7 (2 samples), B8 (5 samples), A14-3, A16-4 and I714891 (bacterial pellets stored at -20°C).<br />
<br />
*Screening digestion for all the miniprepped samples:<br />
**B7(E-P)<br />
**B8(E-P)<br />
**A14-3(E-P)<br />
**A16-4(E-P)<br />
**I714891(E-P)<br />
<br />
*Gel run for the digested samples.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B7_B8_A14_A16_3K3.jpg|thumb|500px|left|Digestion screening.]]<br />
|}<br />
</font><br />
<br />
*Gel results:<br />
**B7 - both screened colonies were negative (~3200bp of vector and ~3200bp of insert).<br />
**B8 - only B8-5 SEEMED to show the expected length for ligated plasmid (~3200bp of vector and ~4200bp of insert), but it also shows a single unexpected band...All the other 4 samples were negative (~3200bp of vector and ~3200bp of insert).<br />
**A14-3 - showed the expected length for ligated plasmid (2079bp of vector and ~2200bp of insert).<br />
**A16-4 - showed the expected length for ligated plasmid (2079bp of vector and ~1700bp of insert).<br />
**I714891 - showed the expected length for ligated plasmid (~2700bp of vector and ~700bp of insert).<br />
<br />
*Comments: it seems that we have the final constitutively expressed ethanol producing operon (B8 = RBS-tetR-TT-Ptet-RBS-adhB-RBS-pdc-TT in pSB1AK3). Anyway, an unexpected band is present in this part and in its ancestor (i.e. B6). So, we decided to follow three ways:<br />
**sequence B8-5 and see if it is correct;<br />
**repeat the last two assemblies of the missing version of the ethanol producing operon: B5=B1+B4 and B7=A4+B5;<br />
**try to purify the expected bands from the gel through gel extraction.<br />
<br />
<br />
<br />
<br />
*We sent purified DNA of:<br />
**B8-5<br />
**A14-3<br />
**A16-4<br />
*to BMR Genomics for sequencing.<br />
<br />
<br />
<br />
*We inoculated 8 ul of these glycerol stocks:<br />
**R0011<br />
**E0240<br />
**A15-2<br />
**B1-13<br />
**B4-2<br />
*in 4 ml of LB + suitable antibiotic to grow overnight cultures (37°C, 220 rpm).<br />
<br />
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<br />
== <html><font class="dayw_style">August, 25th</font></html> ==<br />
*DH5alpha competent cells preparation.<br />
*We streaked a single colony LB agar plate with DH5alpha overnight liquid culture, in order to store it (tomorrow a glycerol stock for it will be prepared).<br />
<br />
*Miniprep for:<br />
**R0011<br />
**E0240<br />
**A15-2<br />
**B1-13<br />
**B4-2<br />
<br />
*Digestion for:<br />
**R0011(S-P)<br />
**E0240(X-P)<br />
**A15-2(E-P)<br />
**B1-13(E-S)<br />
**B4-2(E-X)<br />
**I714891(E-P) (the purified plasmid was stored at -20°C)<br />
<br />
*Precipitation with sodium acetate for all the 6 samples.<br />
<br />
*Ligations:<br />
**B5new = B1(E-S) + B4(E-X) in pSB1AK3<br />
**A17 = R0011(S-P) + E0240(X-P) in pSB1AK3<br />
**A18 = A15-2(E-P) + I714891(E-P) in pSB3K3<br />
*We incubated the ligations at 16°C overnight.<br />
<br />
<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We prepared 25 ml of LB + Kan and M9 supplemented + Kan with 0%, 2.5%, 3.5% and 4.5% ethanol (weight/volume).<br />
<br />
*We inoculated 8 ul of B0015 glycerol stock in 5 ml of LB + Kan in the morning. We let it grow during the day (37°C, 220 rpm) and after about 9 hours we diluted the culture (10 ul of B0015 bacteria in 5 ml of LB + Kan and 10 ul of B0015 bacteria in 5 ml of M9 supplemented + Kan). We incubated the diluted cultures overnight (37°C, 220 rpm).<br />
<br />
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<br />
== <html><font class="dayw_style">August, 26th</font></html> ==<br />
<br />
*We transformed B5new, A17 and A18 overnight ligations in TOP10. We plated transformed bacteria on LB agar plates + Kan (for B5new and A18) or + Amp (for A17). We incubated B5new plate at 37°C in the morning.<br />
<br />
*After about 10 hours we picked 8 colonies from B5new plate and infected 4 ml of LB + Kan to grow overnight cultures (37°C, 220 rpm) for screening.<br />
<br />
*A17 and A18 plates were incubated overnight at 37°C.<br />
<br />
*We also inoculated:<br />
**DH5alpha strain (4 ml of LB without antibiotic) from the single colony streaked plate;<br />
**E0240 (4 ml of LB + Amp).<br />
<br />
<br />
<br />
*We received sequencing results for:<br />
**A15-1 - sequence ok! (long part) (actually it is not consistent with the Registry, but it is consistent with iGEM 2009 QC, in which there are some problems with K112808, even if the part should work)<br />
**A15-3 - sequence ok! (long part) (actually it is not consistent with the Registry, but it is consistent with iGEM 2009 QC, in which there are some problems with K112808, even if the part should work)<br />
**A11-2 - deletion of 20bp in lacI gene...<br />
**A11-3 - sequence ok!<br />
**B5-3 - sequence ok! (long part)<br />
**B6-3 - sequence ok! (long part)<br />
**B3-5 - sequence ok!<br />
<br />
*COMMENTS:<br />
**we used A11-2 and A15-2 for A14 and A18 ligations...so A14 had to be repeated using A11-3, while we will send A15-2 to BMR for sequencing soon (we forgot to do it!).<br />
**B3 native stock sequence was confirmed! but we still don't know why we see two unexpected bands after B3 digestions...<br />
**B5-3 and B6-3 sequences were confirmed! but we still don't know why we see two unexpected bands after B5 and B6 digestions...<br />
<br />
<br />
''Preparation of experiment with Tecan F200 (for tomorrow!)''<br />
<br />
*We infected 5 ml of LB + Amp with 8 ul of B0030, A2 and T9002 glycerol stocks.<br />
<br />
*We incubated the inocula at 37°C, 220 rpm overnight.<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:2000 the LB and the M9 5 ml overnight cultures of B0015 in all the 50 ml falcon tubes containing 25 ml of LB + Kan and M9 supplemented + Kan with different ethanol concentrations. Before diluting the cultures, we took 3 ml of all the media to use them as blank.<br />
<br />
*We incubated the diluted cultures at 37°C, 220 rpm in the morning.<br />
<br />
<br />
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<br />
== <html><font class="dayw_style">August, 27th</font></html> ==<br />
*80% glycerol preparation.<br />
<br />
*Glycerol stock and miniprep for the 8 cultures of B5new.<br />
<br />
*Glycerol stock for DH5alpha strain.<br />
<br />
*Digestion (E-P) for all the 8 DNA samples.<br />
<br />
*Gel run for the digested samples.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B5new_digestion_screening.jpg|thumb|500px|left|No positive colonies for B5new.]]<br />
|}<br />
</font><br />
<br />
*Gel results: no positive colony:'( We will repeat the ligation.<br />
<br />
*We infected 4 ml of LB + suitable antibiotic with 8 ul of B1 and B4 glycerol stocks (to repeat "B5" ligation). We incubated these two inocula at 37°C, 220 rpm overnight.<br />
<br />
<br />
*Colony PCR for A17 plate (6 colonies). Bacteria were inoculated in 100 ul of LB + Amp before putting them in the PCR vials, waiting for the end of the reaction.<br />
<br />
*Electrophoresis for PCR results.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_A17_wrong.jpg|thumb|500px|left|Colony PCR results on A17: colonies 1, 4, 5 and 6 seem to be correct!]]<br />
|}<br />
</font><br />
<br />
*Gel results: we chose A17-4 to grow an overnight culture in LB + Amp (37°C, 220 rpm).<br />
<br />
<br />
<br />
*We picked 7 colonies from A18 plate to inoculate 4 ml of LB + Kan. We incubated them at 37°C, 220 rpm overnight.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200 (for tomorrow!)''<br />
<br />
*We infected 4 ml of M9 + Amp with 8 ul of A2 and B0030 glycerol stocks.<br />
<br />
*We incubated the inocula at 37°C, 220 rpm overnight.<br />
<br />
<br />
<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:100 the overnight cultures of B0030, A2 and T9002 in 5 ml of LB + Amp and in 5 ml of M9 supplemented + Amp.<br />
<br />
*We incubated the diluted cultures at 37°C, 220 rpm for 4 hours.<br />
<br />
<br />
<br />
<br />
<br />
''Experiment with Tecan F200''<br />
<br />
* <html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/T9002 induction test TEST 27-08-09.pdf" target="_blank">Download Protocol</a></html><br />
* <html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/Kynetic Cycle LB-M9 27_08_09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
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<br />
== <html><font class="dayw_style">August, 28th</font></html> ==<br />
*We prepared a pellet for E0240, discarded supernatant (LB), dried the pellet and stored the dry pellet at -20°C, ready to be miniprepped.<br />
<br />
*Glycerol stocks for the 7 cultures of A18 and for A17-4.<br />
<br />
*We prepared a bacterial pellet for the 7 cultures of A18, that will be screened after having A15-2 sequencing results.<br />
<br />
*Miniprep for A17-4, B1-13 and B4-2.<br />
<br />
*Digestion for:<br />
**B1(E-S)<br />
**B4(E-X)<br />
<br />
*Gel run for B1(E-S) (1 ul for check) and for B4(E-X) (all the volume). Gel cut/purification for B4(E-X); precipitation with sodium acetate for B1(E-S).<br />
<br />
*Ligation: B5new2 = B1(E-S) + B4 (E-X) in pSB1AK3. We incubated the ligation overnight at 16°C.<br />
<br />
<br />
*We sent A17-4 purified DNA to BMR Genomics for sequencing.<br />
<br />
<br />
<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:10 the overnight cultures of B0030 and A2 in 5 ml of M9 supplemented + Amp.<br />
<br />
*We incubated the diluted cultures at 37°C, 220 rpm for 4 hours.<br />
<br />
<br />
<br />
<br />
<br />
''Experiment with Tecan F200''<br />
<br />
* <html><u>Description</u></html><br />
* <html><u>Purpose:</u></html><br />
* <html><u>Materials & Methods</u></html><br />
* <html><u>Protocol</u></html><br />
* <html><u>Results</u></html><br />
<div align="right"><br />
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</div><br />
<br />
== <html><font class="dayw_style">August, 29th</font></html> ==<br />
<br />
*We inactivated T4 ligase (65°C for 10 min) and put the ligation at -20°C.<br />
<br />
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<br />
== <html><font class="dayw_style">August, 30th</font></html> ==<br />
<br />
*We transformed B5new2 ligation, stored at -20°C with a 1:60 dilution. We incubated the plated bacteria at 37°C overnight.<br />
<br />
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</div><br />
<br />
<br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Aug#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Aug#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Sep#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
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</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1AugTeam:UNIPV-Pavia/Notebook/Week1Aug2009-10-21T23:26:24Z<p>110hs: /* August, 5th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from August 3rd, to August 9th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Jul#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Jul#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Aug#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Aug#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">August, 3rd</font></html> ==<br />
<br />
*This week we planned to continue the assembly of the synthetic ethanol producing operon.<br />
<br />
<br />
*We received DH5alpha, XL1-Blue and DB3.1 competent cells from Bologna iGEM Team. Thank you!!<br />
<br />
<br />
<br />
*We performed 2 screenings for the 7 samples of B3 and for the 7 samples of B4:<br />
<br />
*SCREENING 1 - Ojective: check the presence of non-ligated plasmids. We planned not to digest with standard enzymes because the resulting fragments would result either too small or too big. Insert excision would generate a possible 129pb fragment, while vector linearization would generate possible 3Kb or 5Kb fragments. We used ApE to decide the best enzymes to cut. Digestion with XhoI-PstI (6 ul of DNA, final volume 20ul). There are two XhoI restriction sites on pSB1AK3 plasmid: after the cut, we expected two fragments of the same size (~1.1Kbp) and one fragment of ~3Kbp for B3 positive clones (in negative clones the heaviest band would be ~1.4Kbp), while we expected the same two fragments of ~1.1Kbp and a heavier fragment of ~2.5Kbp for B4 positive clones (in negative clones the heaviest band would be ~1.4Kbp).<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B3_B4_digestion_PstI_XhoI.jpg|thumb|500px|left|B3 and B4 digested samples (XhoI-PstI).]]<br />
|}<br />
</font><br />
<br />
*Gel results:<br />
**Only B3-1 and B3-5 showed the bands with expected length for the correct plasmid.<br />
**All B4 samples showed the bands with expected lengths!<br />
<br />
*SCREENING 2 - Ojective: check the presence of the right insert length for B3 samples, because there was the possibility that B1 vector (pSB1A2) ligated in B0015 vector (pSB1AK3) as an insert. If so, no XbaI site is present in the final plasmid, while correct ligations should show a ~1900bp fragment as an insert. Digestion with XbaI-PstI (2 ul of DNA, final volume 20ul). We decided to perform the reaction for all B3 samples, even if only two were good.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B3_digestion_X-P.jpg.jpg|thumb|300px|left|B3 digesed XbaI-PstI.]]<br />
|}<br />
</font><br />
<br />
*Gel results: B3-1 and B3-5 showed the expected bands for pSB1AK3 (~3200 bp) and B1-B0015 ligated insert (~1900bp).<br />
<br />
*We sent the following purified DNA samples to BMR Genomics for sequencing:<br />
**B3-1<br />
**B3-5<br />
**B4-2<br />
**B4-4<br />
*We planned not to perform ligations this week and to wait for BMR Genomics sequencing results, in order to be sure of what we were assembling.<br />
<br />
<br />
*LB agar plates + Kan preparation.<br />
<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We inoculated 10 ul of A1, A2, A7, J23100, J23101 and J23118 glycerol stocks in 5 ml of LB + Amp, while we used a single colony from B0030 native plate to infect 5 ml of LB + Amp.<br />
<br />
*We incubated these inocula overnight (37°C, 220 rpm).<br />
<br />
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<br />
== <html><font class="dayw_style">August, 4th</font></html> ==<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:1000 the overnight cultures of A1, A2, A7, J23100, J23101, J23118 and B0030.<br />
<br />
*We incubated the diluted cultures for 5 hours (37°C, 220 rpm).<br />
<br />
*We adjusted OD600 to 0.03 diluting the cultures in LB + Amp.<br />
<br />
<br />
<br />
''Experiment with Tecan F200''<br />
<br />
* <html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/A1 A2 A7 in LB TEST 04-08-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200 (for the following day!)''<br />
<br />
*We picked a single colony from B0015 native plate and infected 5 ml of LB + Kan.<br />
<br />
*We incubated the inoculum overnight (37°C, 220 rpm).<br />
<br />
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<br />
== <html><font class="dayw_style">August, 5th</font></html> ==<br />
<br />
*We received sequencing results for the first two ligation steps of ethanologenic operon and for iGEM stabs:<br />
**B1-13: sequence ok!<br />
**B2-5: sequence ok!<br />
**K116001: the stab actually contained K116002! moreover, sequence analysis showed an inconsistent prefix and an additional "c" at the end of nhaA promoter, but we think that the measurement system should work. We will call it with its real name ("K116002") on our freezer page.<br />
**K116002: the stab actually contained J33204!<br />
**K112405: sequence ok!<br />
**P0412: sequence ok!<br />
**I746902: sequence ok!<br />
**I746903: the RBS in the sequence is actually B0030 and not B0034 as documented on the Registry. Anyway, it does not corrupt the function of this brick and the rest of the sequence was ok!<br />
**K101017: very bad sequencing.<br />
**F2620MIT1: sequence ok!<br />
**F2620MIT2: sequence ok!<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:100 the overnight culture of B0015.<br />
<br />
*We incubated the diluted culture for 5 hours (37°C, 220 rpm).<br />
<br />
<br />
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<br />
== <html><font class="dayw_style">August, 6th</font></html> ==<br />
<br />
*We received Ethanol Assay Kit and Lactose Assay Kit from BioVision!<br />
<br />
<br />
*We received sequencing results for the second ligation step of ethanologenic operon:<br />
**B3-1: sequence ok, but the chromatogram quality was not so good...<br />
**B3-5: sequence ok!<br />
**B4-2: sequence ok!<br />
**B4-4: sequence ok!<br />
<br />
*We decided to use B3-5 and B4-2 for the following assemblies.<br />
<br />
*We infected 4 ml of LB + Amp with the following glycerol stocks:<br />
{|cellpadding="20"<br />
|B1-13 (X2)<br />
|B2-5 (X2)<br />
|B3-5 (X2)<br />
|-<br />
|B4-2 (X2)<br />
|F2620MIT1<br />
|K112808<br />
|-<br />
|BOL1<br />
|R0011<br />
|}<br />
*Tomorrow they will be miniprepped to prepare the assemblies of the following week!<br />
<br />
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<br />
== <html><font class="dayw_style">August, 7th</font></html> ==<br />
*Miniprep for:<br />
{|cellpadding="20"<br />
|B1-13 (X2)<br />
|B2-5 (X2)<br />
|B3-5 (X2)<br />
|-<br />
|B4-2 (X2)<br />
|R0011<br />
|F2620MIT1<br />
|-<br />
|BOL1<br />
|K112808<br />
|}<br />
*We stored purified DNA at -20°C and next week we will perform digestion for these 12 samples, in order to: i) finish the assembly of the ethanologenic operon, ii) re-assemble A11, which sequence analysis showed a deletion and iii) build up an inducible lysis device.<br />
<br />
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</div><br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Jul#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Jul#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Aug#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Aug#week_start"><br />
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</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5JulTeam:UNIPV-Pavia/Notebook/Week5Jul2009-10-21T23:25:59Z<p>110hs: /* July, 31st */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from July 27th, to August 2nd, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Jul#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Jul#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Aug#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Aug#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">July, 27th</font></html> ==<br />
<br />
*Screening for the 11 miniprepped DNA samples for B1: digestion S-P for all.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_digestion_B1_S-P.jpg|thumb|500px|left|Insert here]]<br />
|}<br />
</font><br />
<br />
*Gel results:<br />
**Samples 1, 4, 10, 11, 14, 19 and 20 showed an extra band for the non ligated plasmid;<br />
**Samples 2, 9, 13 and 18 were pure! we decided to keep B1-13 (lane 8) to perform future ligations.<br />
<br />
<br />
<br />
*NOTE: we had a pure sample for B1 (i.e. B1-13)and three almost-pure sample for B2. Anyway, we decided to perform ligation reactions for these samples and the extra band of B2 will be eliminated during gel cut/purification. WE DECIDED TO KEEP B2-5. Next weeks we will think about purifying B2-5 itself.<br />
<br />
<br />
<br />
*We transformed 20 pg of B1-13 purified DNA (stored at -20°C) in TOP10 in order to prepare a glycerol stock for this construct. We incubated the plate at 37°C overnight.<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We infected 5 ml of LB + Amp with 10 ul of A14pg, A8pg and A9pg glycerol stocks.<br />
<br />
*We also infected 5 ml of LB + Amp with a single colony taken from B0030 native plate (stored at +4°C).<br />
<br />
*We incubated the inocula at 37°C, 220 rpm overnight.<br />
<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 28th</font></html> ==<br />
<br />
*We streaked LB agar plates + suitable antibiotic with iGEM stabs:<br />
{|cellpadding="20"<br />
|K116001<br />
|K116002<br />
|K112405<br />
|-<br />
|P0412<br />
|I746902<br />
|I746903<br />
|-<br />
|K101017<br />
|F2620MIT1<br />
|F2620MIT2<br />
|}<br />
<br />
*We incubated these "single colonies" plates at 37°C overnight.<br />
<br />
<br />
<br />
*We picked a single colony from B1-13 plate to infect 1 ml of LB + Amp and incubated this inoculum for 5 hours and 1/2.<br />
<br />
*We prepared a glycerol stock for B1-13.<br />
<br />
*We aliquoted the remaining 250 ul of B1-13 bacterial culture in two different falcon tubes and re-filled them with 5 ml of LB + Amp.<br />
<br />
*We also infected 5 ml of LB + Amp with 10 ul of B0015(X2) and B2-5(X2) glycerol stocks.<br />
<br />
*We incubated these six cultures at 37°C, 220 rpm overnight.<br />
<br />
<br />
<br />
<br />
*We received sequencing results for:<br />
**A12-2: sequence ok!<br />
**A12-3: sequence ok!<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:1000 the overnight cultures of A14pg, A8pg, A9pg and B0030.<br />
<br />
*We incubated the diluted cultures for 5 hours (37°C, 220 rpm).<br />
<br />
*After 5 hours, we adjusted the OD600 at 0.025.<br />
<br />
<br />
<br />
''Experiment with Tecan F200''<br />
<br />
* <html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/A14 Induction test 28-07-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 29th</font></html> ==<br />
<br />
*Miniprep for:<br />
**B1-13<br />
**B1-13bis<br />
**B2-5<br />
**B2-5bis<br />
**B0015<br />
**B0015bis<br />
<br />
*Digestion:<br />
**B1-13(E-S)<br />
**B1-13bis(E-S)<br />
**B2-5(E-S)<br />
**B2-5bis(E-S)<br />
**B0015(E-X)<br />
**B0015bis(E-X) - 500ng<br />
<br />
*Gel run/cul/purification for:<br />
**B1-13(E-S) - ONLY FOR CHECK, NOT PURIFIED<br />
**B1-13bis(E-S) - ONLY FOR CHECK, NOT PURIFIED<br />
**B2-5(E-S)<br />
**B2-5bis(E-S)<br />
**B0015(E-X)<br />
**B0015bis(E-X)<br />
<br />
*Precipitation with sodium acetate for:<br />
**B1-13(E-S)<br />
**B1-13bis(E-S)<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B1E-S_B2E-S_B0015E-X_cut.jpg|thumb|500px|left|All the bands were in the right place!]]<br />
|}<br />
</font><br />
<br />
*We had good yields for all, except from B0015bis(E-X). We will use the other vector (i.e. B0015(E-X)) for ligation.<br />
<br />
*Ligation:<br />
**B3 = B1(E-S) + B0015(E-X) in pSB1AK3 (50 ng of vector)<br />
**B4 = B2(E-S) + B0015(E-X) in pSB1AK3 (26 ng of vector)<br />
*keeping the sample that gave the higher yield.<br />
<br />
*We incubated the ligations at 16°C overnight.<br />
<br />
<br />
<br />
<br />
*All the streaked plates showed single colonies! we prepared an inoculum for all of them picking a single colony and infecting 1 ml of LB + suitable antibiotic. We incubated these inocula for 5 hours and 1/2.<br />
<br />
*Then, we prepared a glycerol stock for each of them and re-filled the remaining 250 ul of bacterial culture with 3 ml of LB + suitable antibiotic.<br />
<br />
*We incubated the cultures at 37°C, 220 rpm overnight.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 30th</font></html> ==<br />
<br />
*We transformed B3 (50 pg) and B4 (20 pg) ligations. We incubated the plates at 37°C overnight.<br />
<br />
<br />
<br />
*Miniprep for the overnight cultures of:<br />
{|cellpadding="20"<br />
|K116001<br />
|K116002<br />
|K112405<br />
|-<br />
|P0412<br />
|I746902<br />
|I746903<br />
|-<br />
|K101017<br />
|F2620MIT1<br />
|F2620MIT2<br />
|}<br />
<br />
*We sent purified DNA to BMR Genomics for sequencing (VF2 and VR for all of them, except from K112405, whose plasmid does not have VF2 annealing site).<br />
<br />
*We also sent purified DNA of B1-13 and B2-5 (stored at -20°C) to BMR Genomics for sequencing.<br />
<br />
<br />
<br />
<br />
*We received sequencing results for A11: lacI sequence showed a deletion...we will repeat A11 ligation from BOL1 and R0011 parts.<br />
<br />
<br />
''Preparation of experiment with Tecan F200 (for the following day!)''<br />
<br />
*We inoculated 10 ul of J23100 and J23100-E0240 parts form UNIPV iGEM 2008 stocks.<br />
<br />
*We incubated these two inocula at 37°C, 220 rpm overnight.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 31st</font></html> ==<br />
<br />
*B3 and B4 plates showed colonies! We picked 7 colonies for each plate and infected 1 ml of LB + Kan. We incubated these 14 inocula for 5 hours and 1/2. Then we prepared glycerol stocks and re-filled the remaining 250 ul of bacterial culture with 5 ml of LB + Kan.<br />
<br />
*We incubated these cultures at 37°C, 220 rpm overnight.<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:100 J23100 and J23100-E0240 parts form UNIPV iGEM 2008 stocks overnight cultures.<br />
<br />
*We incubated these two cultures at 37°C, 220 rpm for about 3 hours.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">August, 1st</font></html> ==<br />
<br />
*Miniprep for the 7 colonies of B3 and for the 7 colonies of B4. We stored purified DNA at -20°C and next Monday we will perform screening for these 14 samples.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Jul#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Jul#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Aug#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Aug#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5JulTeam:UNIPV-Pavia/Notebook/Week5Jul2009-10-21T23:25:44Z<p>110hs: /* July, 30th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from July 27th, to August 2nd, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Jul#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Jul#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Aug#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Aug#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">July, 27th</font></html> ==<br />
<br />
*Screening for the 11 miniprepped DNA samples for B1: digestion S-P for all.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_digestion_B1_S-P.jpg|thumb|500px|left|Insert here]]<br />
|}<br />
</font><br />
<br />
*Gel results:<br />
**Samples 1, 4, 10, 11, 14, 19 and 20 showed an extra band for the non ligated plasmid;<br />
**Samples 2, 9, 13 and 18 were pure! we decided to keep B1-13 (lane 8) to perform future ligations.<br />
<br />
<br />
<br />
*NOTE: we had a pure sample for B1 (i.e. B1-13)and three almost-pure sample for B2. Anyway, we decided to perform ligation reactions for these samples and the extra band of B2 will be eliminated during gel cut/purification. WE DECIDED TO KEEP B2-5. Next weeks we will think about purifying B2-5 itself.<br />
<br />
<br />
<br />
*We transformed 20 pg of B1-13 purified DNA (stored at -20°C) in TOP10 in order to prepare a glycerol stock for this construct. We incubated the plate at 37°C overnight.<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We infected 5 ml of LB + Amp with 10 ul of A14pg, A8pg and A9pg glycerol stocks.<br />
<br />
*We also infected 5 ml of LB + Amp with a single colony taken from B0030 native plate (stored at +4°C).<br />
<br />
*We incubated the inocula at 37°C, 220 rpm overnight.<br />
<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 28th</font></html> ==<br />
<br />
*We streaked LB agar plates + suitable antibiotic with iGEM stabs:<br />
{|cellpadding="20"<br />
|K116001<br />
|K116002<br />
|K112405<br />
|-<br />
|P0412<br />
|I746902<br />
|I746903<br />
|-<br />
|K101017<br />
|F2620MIT1<br />
|F2620MIT2<br />
|}<br />
<br />
*We incubated these "single colonies" plates at 37°C overnight.<br />
<br />
<br />
<br />
*We picked a single colony from B1-13 plate to infect 1 ml of LB + Amp and incubated this inoculum for 5 hours and 1/2.<br />
<br />
*We prepared a glycerol stock for B1-13.<br />
<br />
*We aliquoted the remaining 250 ul of B1-13 bacterial culture in two different falcon tubes and re-filled them with 5 ml of LB + Amp.<br />
<br />
*We also infected 5 ml of LB + Amp with 10 ul of B0015(X2) and B2-5(X2) glycerol stocks.<br />
<br />
*We incubated these six cultures at 37°C, 220 rpm overnight.<br />
<br />
<br />
<br />
<br />
*We received sequencing results for:<br />
**A12-2: sequence ok!<br />
**A12-3: sequence ok!<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:1000 the overnight cultures of A14pg, A8pg, A9pg and B0030.<br />
<br />
*We incubated the diluted cultures for 5 hours (37°C, 220 rpm).<br />
<br />
*After 5 hours, we adjusted the OD600 at 0.025.<br />
<br />
<br />
<br />
''Experiment with Tecan F200''<br />
<br />
* <html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/A14 Induction test 28-07-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 29th</font></html> ==<br />
<br />
*Miniprep for:<br />
**B1-13<br />
**B1-13bis<br />
**B2-5<br />
**B2-5bis<br />
**B0015<br />
**B0015bis<br />
<br />
*Digestion:<br />
**B1-13(E-S)<br />
**B1-13bis(E-S)<br />
**B2-5(E-S)<br />
**B2-5bis(E-S)<br />
**B0015(E-X)<br />
**B0015bis(E-X) - 500ng<br />
<br />
*Gel run/cul/purification for:<br />
**B1-13(E-S) - ONLY FOR CHECK, NOT PURIFIED<br />
**B1-13bis(E-S) - ONLY FOR CHECK, NOT PURIFIED<br />
**B2-5(E-S)<br />
**B2-5bis(E-S)<br />
**B0015(E-X)<br />
**B0015bis(E-X)<br />
<br />
*Precipitation with sodium acetate for:<br />
**B1-13(E-S)<br />
**B1-13bis(E-S)<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B1E-S_B2E-S_B0015E-X_cut.jpg|thumb|500px|left|All the bands were in the right place!]]<br />
|}<br />
</font><br />
<br />
*We had good yields for all, except from B0015bis(E-X). We will use the other vector (i.e. B0015(E-X)) for ligation.<br />
<br />
*Ligation:<br />
**B3 = B1(E-S) + B0015(E-X) in pSB1AK3 (50 ng of vector)<br />
**B4 = B2(E-S) + B0015(E-X) in pSB1AK3 (26 ng of vector)<br />
*keeping the sample that gave the higher yield.<br />
<br />
*We incubated the ligations at 16°C overnight.<br />
<br />
<br />
<br />
<br />
*All the streaked plates showed single colonies! we prepared an inoculum for all of them picking a single colony and infecting 1 ml of LB + suitable antibiotic. We incubated these inocula for 5 hours and 1/2.<br />
<br />
*Then, we prepared a glycerol stock for each of them and re-filled the remaining 250 ul of bacterial culture with 3 ml of LB + suitable antibiotic.<br />
<br />
*We incubated the cultures at 37°C, 220 rpm overnight.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 30th</font></html> ==<br />
<br />
*We transformed B3 (50 pg) and B4 (20 pg) ligations. We incubated the plates at 37°C overnight.<br />
<br />
<br />
<br />
*Miniprep for the overnight cultures of:<br />
{|cellpadding="20"<br />
|K116001<br />
|K116002<br />
|K112405<br />
|-<br />
|P0412<br />
|I746902<br />
|I746903<br />
|-<br />
|K101017<br />
|F2620MIT1<br />
|F2620MIT2<br />
|}<br />
<br />
*We sent purified DNA to BMR Genomics for sequencing (VF2 and VR for all of them, except from K112405, whose plasmid does not have VF2 annealing site).<br />
<br />
*We also sent purified DNA of B1-13 and B2-5 (stored at -20°C) to BMR Genomics for sequencing.<br />
<br />
<br />
<br />
<br />
*We received sequencing results for A11: lacI sequence showed a deletion...we will repeat A11 ligation from BOL1 and R0011 parts.<br />
<br />
<br />
''Preparation of experiment with Tecan F200 (for the following day!)''<br />
<br />
*We inoculated 10 ul of J23100 and J23100-E0240 parts form UNIPV iGEM 2008 stocks.<br />
<br />
*We incubated these two inocula at 37°C, 220 rpm overnight.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 31st</font></html> ==<br />
<br />
*B3 and B4 plates showed colonies! We picked 7 colonies for each plate and infected 1 ml of LB + Kan. We incubated these 14 inocula for 5 hours and 1/2. Then we prepared glycerol stocks and re-filled the remaining 250 ul of bacterial culture with 5 ml of LB + Kan.<br />
<br />
*We incubated these cultures at 37°C, 220 rpm overnight.<br />
<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:100 J23100 and J23100-E0240 parts form UNIPV iGEM 2008 stocks overnight cultures.<br />
<br />
*We incubated these two cultures at 37°C, 220 rpm for about 3 hours.<br />
<br />
<br />
<br />
<br />
<br />
''Experiment with Tecan F200''<br />
<br />
* <html><u>Description</u></html><br />
* <html><u>Purpose:</u></html><br />
* <html><u>Materials & Methods</u></html><br />
* <html><u>Protocol</u></html><br />
* <html><u>Results</u></html><br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<br />
<br />
== <html><font class="dayw_style">August, 1st</font></html> ==<br />
<br />
*Miniprep for the 7 colonies of B3 and for the 7 colonies of B4. We stored purified DNA at -20°C and next Monday we will perform screening for these 14 samples.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Jul#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Jul#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Aug#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Aug#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4JulTeam:UNIPV-Pavia/Notebook/Week4Jul2009-10-21T23:25:09Z<p>110hs: /* July, 20th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from July 20th, to July 26th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Jul#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Jul#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Jul#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Jul#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">July, 20th</font></html> ==<br />
<br />
*Overnight digestion (20 ul reaction volume) for:<br />
{|cellpadding="20"<br />
|MRGENE1(X-P)<br />
|MRGENE1-2(X-P)<br />
|MRGENE1-3(X-P)<br />
|-<br />
|MRGENE2(X-P)(X2)<br />
|MRGENE2-2(X-P)<br />
|MRGENE2-3(X-P)<br />
|-<br />
|B0030(S-P)(X3)<br />
|}<br />
*NOTE: reactions which involve excision(X-P) were performed on 3 ug of DNA, while S-P digestions were performed with 1 ug of DNA. We decided to have 3 replicates to be sure to have an acceptable yield.<br />
<br />
*We decided to perform gel run/cut/purification for MRGENE2 and for B0030, while we decided to perform DNA precipitation with sodium acetate for MRGENE1.<br />
<br />
<br />
<br />
<br />
*We transformed about 2.5 pg of A14-4 in TOP10 in order to filter the wanted plasmids. We plated the transformed bacteria and incubated the plates at 37°C overnight.<br />
<br />
<br />
<br />
<br />
*We infected 5 ml of LB + Amp with 10 ul of A12-2 and A12-3 glycerol stocks. Tomorrow they will be miniprepped and sent to sequence!<br />
<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We infected 5 ml of LB + Kan with a single colony taken from the native plate of B0015.<br />
<br />
*We incubated the inoculum for 6 hours (37°C, 220 rpm).<br />
<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 21st</font></html> ==<br />
<br />
*Gel run for:<br />
**MRGENE1(for check)<br />
**MRGENE2<br />
**MRGENE2-2<br />
**MRGENE2-3<br />
**B0030(X3)<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_Digestion_mrGene_B0030.jpg|thumb|500px|left|Digestion for MrGene1 (X-P, for check, lane 3), MrGene2 (X-P, for cut/purification, lanes 5, 7 and 9) and RBS30 (S-P, for cut/purification, lanes 11, 13 and 15)]]<br />
|}<br />
</font><br />
<br />
*We noticed unwanted bands in MRGENE1 run...So, we performed an analysis on Mr Gene plasmids and we found an unwanted PstI site in pMK-RQ that gives the noticed bands...<br />
<br />
*We decided to proceed to ligation and to perform a massive screening of the ligated inserts in the next days.<br />
<br />
*Gel purification for:<br />
**MRGENE2<br />
**MRGENE2-2<br />
**MRGENE2-3<br />
**B0030(X3)<br />
<br />
*DNA precipitation with sodium acetate for MRGENE1, MRGENE1-2, MRGENE1-3.<br />
<br />
*Results: after quantifications at Nanodrop, all the purified DNA samples had a good yield! let's proceed to ligation!<br />
<br />
*Ligation:<br />
**B1 = B0030(S-P) + MRGENE1(X-P)<br />
**B2 = B0030(S-P) + MRGENE2(X-P)<br />
<br />
*We incubated the ligations at 16°C overnight.<br />
<br />
<br />
*Miniprep for A12-2 and A12-3. We sent purified DNA to BMR Genomics for sequencing.<br />
<br />
<br />
<br />
<br />
*A14pg plate showed colonies! we picked 7 colonies and infected 5 ml of LB + Amp. We incubated the 7 inocula at 37°C, 220 rpm overnight.<br />
<br />
<br />
<br />
*We received sequencing results for F2620: sequence was not consistent, which confirmed the results of iGEM 2009 QC.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 22nd</font></html> ==<br />
<br />
*We received the following stabs from iGEM HQ:<br />
{|cellpadding="20"<br />
|K116001<br />
|K116002<br />
|K112405<br />
|-<br />
|P0412<br />
|I746902<br />
|I746903<br />
|-<br />
|K101017<br />
|}<br />
*while F2620 will be shipped soon.<br />
<br />
<br />
<br />
*We transformed B1 and B2 overnight ligations in TOP10. Plates were incubated at 37°C overnight.<br />
<br />
<br />
<br />
<br />
*Glycerol stocks and miniprep for the 7 overnight cultures of A14pg.<br />
<br />
*Digestion E-P for the miniprepped DNA.<br />
<br />
*Gel run for these 7 samples.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_A14pg_screening_EP.jpg|thumb|300px|left|Screening digestion for A14pg.]]<br />
|}<br />
</font><br />
<br />
*Gel results: there was the expected ligated insert band in all the colonies, but some of them also have the non ligated insert (very small amount) or the same unexpected band (~1100 bp). None of these colonies was pure...We will discuss this result in the next days. If someone have suggestions about this problem, please e-mail lorenzo.pasotti@unipv.it . Thank you!<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 23rd</font></html> ==<br />
<br />
*Colony PCR for B2. The screened colonies were inoculated in 1 ml of LB + Amp and incubated at 37°C, 220 rpm waiting for the end of the PCR.<br />
<br />
*We also linearized three samples from A14pg previously extracted DNA, cutting in PstI. We chose to perform this test to check if the unexpected extra band was due to double or single digestion.<br />
<br />
*Electrophoresis for B2 PCR results and for the linearized samples of A14pg.<br />
<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_B2pcr_A14pgPstI.jpg|thumb|500px|left|PCR results for B2 (lanes 3 to 12), digestion PstI for three samples of A14pg (lanes 13 to 15).]]<br />
|}<br />
</font><br />
<br />
*Gel results:<br />
**All the screened colonies from B2 had the insert with expected length (~1450 bp). Anyway, in this gel the 250 pb band in the ladder was lost and so we do not know if the ten colonies were pure or not. We decided to keep colonies 1, 2, 3, 5, 7 and 8 for further screening. We prepared a glycerol stock for them.<br />
**The first and the third A14pg DNA sample had the unexpected extra band (~1100 bp), while the second seemed not to have it...we still don't know what it is. Moreover, all the three samples had the unwanted band (difficult to notice, because it was near the brightest one). We decided to keep the second DNA sample and to test TOP10 bearing this plasmid.<br />
<br />
*We re-filled the remaining 250 ul of B2-1, B2-5 and B2-8 bacterial culture with 5 ml of LB + Amp to grow an overnight culture.<br />
<br />
*We also inoculated 21 colonies from B1 plate in 5 ml of LB + Amp. Tomorrow they will be miniprepped and screened! we chose such a high number of colonies because the ligation of B1 had the simultaneous presence of our wanted insert and other digestion products due to an extra PstI site in the vector provided by Mr Gene.<br />
<br />
*We incubated the 24 cultures at 37°C, 220 rpm overnight.<br />
<br />
<br />
<br />
*We sent A11-1 purified DNA (stored at -20°C) to BMR Genomics for sequencing.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 24th</font></html> ==<br />
<br />
*Miniprep for the 24 overnight cultures.<br />
<br />
*Digestion E-P for all the 21 B1 samples.<br />
<br />
*Digestion S-P for B2-1, B2-5, B2-8 samples.<br />
<br />
*Electrophoresis for digestion products (two medium-size 1% gels).<br />
<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_digestion_B1.jpg|thumb|500px|left|B1 digestion E-P (samples 1 to 12)]]<br />
|}<br />
</font><br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_digestion_B1_B2.jpg|thumb|500px|left|lanes 2 to 10: B1 digestion E-P (samples 13 to 21), lanes 11 to 13: B2 digestion S-P]]<br />
|}<br />
</font><br />
<br />
*Gel results:<br />
**B1 - positive colonies containing ''pdc'' gene: 1, 2, 4, 9, 10, 11, 13, 14, 18, 19, 20. They will be screened to check for non ligated plasmid presence.<br />
**B2 - all the three samples showed the expected length for plasmid+insert, but also the expected length for non ligated plasmid...<br />
<br />
<br />
<br />
*We received TWO stabs for F2620 from iGEM HQ.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Jul#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Jul#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Jul#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5Jul#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2JulTeam:UNIPV-Pavia/Notebook/Week2Jul2009-10-21T23:24:34Z<p>110hs: /* July, 9th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from July 6th, to July 12nd, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jul#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jul#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Jul#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Jul#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">July, 6th</font></html> ==<br />
<br />
*We planned to dedicate this week to the assembly of an IPTG/lactose->PoPS device in order to have a lactose sensor for our project, but also a useful inducible system for general purpose applications.<br />
<br />
<br />
*We already had R0011 and BOL1 purified plasmids stored at -20°C. Digestion for:<br />
{|cellpadding="20"<br />
|R0011(E-X)<br />
|BOL1(E-S)<br />
|}<br />
<br />
*Gel run/cut and band purification.<br />
<br />
*The purified DNA gave a good result at Nanodrop, but we decided to perform the entire work on BOL1 again because we were not sure that we had extracted a pure band.<br />
<br />
*We stored R0011(E-X) DNA at -20°C.<br />
<br />
*We infected 5 ml of LB + Amp with 8 ul of BOL1 glycerol stock to grow two identical overnight cultures (37°C, 220 rpm).<br />
<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We picked a colony of B0030 plate (stored at +4°C) and infected 5 ml of LB + Amp.<br />
<br />
*We also infected 5 ml of LB + Amp with 10 ul of A1 and J23100 glycerol stocks.<br />
<br />
*We incubated the inocula overnight at 37°C, 220 rpm.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 7th</font></html> ==<br />
<br />
*Miniprep for BOL1 (X2).<br />
<br />
*Digestion for BOL1(E-S)(X2).<br />
<br />
*Gel run/cut/purification: none of the two BOL1 samples gave a good results in DNA quantification...we decided to cut the miniprepped plasmids again, but this time through an overnight digestion (both plasmids E-S).<br />
<br />
*In case of failure, we also infected 5 ml of LB + Amp with 8 ul of BOL1 glycerol stock to grow an overnight culture at 37°C, 220 rpm.<br />
<br />
*We prepared 0.5 l of LB + Amp.<br />
<br />
*We received sequencing results for:<br />
**T9002 - long part, but SEQUENCE CONFIRMED! while MIT results were "INCONSISTENT".<br />
**A10 - results showed a good chromatogram, but K117000 had not been correctly ligated. We will repeat this ligation one of the next weeks.<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:100 the overnight cultures of A1, B0030 and J23100.<br />
<br />
*We incubated the diluted cultures for 5 hours (37°C, 220 rpm).<br />
<br />
<br />
<br />
<br />
<br />
''Experiment with Tecan F200''<br />
<br />
* <html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/culture dilutions TEST 07-07-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<br />
<br />
<br />
''Preparation of tomorrow's experiment with Tecan F200''<br />
<br />
*We picked a colony from B0030 native plate (stored ad +4°C) and infected 5 ml of LB + Amp. We incubated the inoculum at 37°C, 220 rpm overnight.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 8th</font></html> ==<br />
<br />
*Gel run/cut/band purification for BOL1(E-S) overnight digestion. Results: gel showed that the enzymes had cut very well and this time also the purification gave a good quantification at Nanodrop.<br />
<br />
*We took R0011(E-X) stored ad -20°C and we were ready to ligate;)<br />
<br />
*Ligation:<br />
**A11: BOL1(E-S) + R0011(E-X) in pSB1A2<br />
<br />
*We incubated the ligation overnight at 16°C.<br />
<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:100 the overnight culture of B0030.<br />
<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 9th</font></html> ==<br />
<br />
*We resuspended F2620 BioBrick from iGEM 2009 plates.<br />
<br />
*We transformed about 40 pg of the overnight ligation and 1 ul of the resuspended F2620 BioBrick. We plated transformed bacteria and incubated the two plates overnight at 37°C.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 10th</font></html> ==<br />
<br />
*A11 and F2620 overnight plates showed colonies!<br />
<br />
*Colony PCR for 6 colonies of A11 plate: the picked colonies were inoculated in 1 ml of LB + Amp and incubated at 37°C, 220 rpm waiting for PCR results.<br />
<br />
*We also picked a colony from F2620 to infect 1 ml of LB + Amp. We incubated this inoculum with the others.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_colonypcr_A11.jpg|thumb|300px|left|Colony PCR for A11 plate: all the colonies showed the correct plasmid. We kept the 1st, 3rd and 6th colonies.]]<br />
|}<br />
</font><br />
<br />
*Gel results: all the picked colonies showed the expected amplicon of a correctly ligated plasmid! We decided to prepare a glycerol stock for:<br />
**A11-1<br />
**A11-3<br />
**A11-6<br />
<br />
*Next week, only A11-1 will be used, but the other colonies will be used in case of failure.<br />
<br />
*We also prepared a glycerol stock for F2620 culture.<br />
<br />
<br />
<br />
*We contacted iGEM HQ to request these BioBricks:<br />
{|cellpadding="20"<br />
|K116001<br />
|K116002<br />
|K112405<br />
|-<br />
|P0412<br />
|I746902<br />
|I746903<br />
|-<br />
|K101017<br />
|F2620<br />
|}<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jul#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jul#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Jul#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Jul#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2JulTeam:UNIPV-Pavia/Notebook/Week2Jul2009-10-21T23:24:24Z<p>110hs: /* July, 8th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<br />
<html><a name="week_start"></a></html><br />
= Week from July 6th, to July 12nd, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jul#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jul#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Jul#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Jul#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">July, 6th</font></html> ==<br />
<br />
*We planned to dedicate this week to the assembly of an IPTG/lactose->PoPS device in order to have a lactose sensor for our project, but also a useful inducible system for general purpose applications.<br />
<br />
<br />
*We already had R0011 and BOL1 purified plasmids stored at -20°C. Digestion for:<br />
{|cellpadding="20"<br />
|R0011(E-X)<br />
|BOL1(E-S)<br />
|}<br />
<br />
*Gel run/cut and band purification.<br />
<br />
*The purified DNA gave a good result at Nanodrop, but we decided to perform the entire work on BOL1 again because we were not sure that we had extracted a pure band.<br />
<br />
*We stored R0011(E-X) DNA at -20°C.<br />
<br />
*We infected 5 ml of LB + Amp with 8 ul of BOL1 glycerol stock to grow two identical overnight cultures (37°C, 220 rpm).<br />
<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We picked a colony of B0030 plate (stored at +4°C) and infected 5 ml of LB + Amp.<br />
<br />
*We also infected 5 ml of LB + Amp with 10 ul of A1 and J23100 glycerol stocks.<br />
<br />
*We incubated the inocula overnight at 37°C, 220 rpm.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 7th</font></html> ==<br />
<br />
*Miniprep for BOL1 (X2).<br />
<br />
*Digestion for BOL1(E-S)(X2).<br />
<br />
*Gel run/cut/purification: none of the two BOL1 samples gave a good results in DNA quantification...we decided to cut the miniprepped plasmids again, but this time through an overnight digestion (both plasmids E-S).<br />
<br />
*In case of failure, we also infected 5 ml of LB + Amp with 8 ul of BOL1 glycerol stock to grow an overnight culture at 37°C, 220 rpm.<br />
<br />
*We prepared 0.5 l of LB + Amp.<br />
<br />
*We received sequencing results for:<br />
**T9002 - long part, but SEQUENCE CONFIRMED! while MIT results were "INCONSISTENT".<br />
**A10 - results showed a good chromatogram, but K117000 had not been correctly ligated. We will repeat this ligation one of the next weeks.<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:100 the overnight cultures of A1, B0030 and J23100.<br />
<br />
*We incubated the diluted cultures for 5 hours (37°C, 220 rpm).<br />
<br />
<br />
<br />
<br />
<br />
''Experiment with Tecan F200''<br />
<br />
* <html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/culture dilutions TEST 07-07-09.pdf" target="_blank">Download Protocol</a></html><br />
<br />
<br />
<br />
<br />
''Preparation of tomorrow's experiment with Tecan F200''<br />
<br />
*We picked a colony from B0030 native plate (stored ad +4°C) and infected 5 ml of LB + Amp. We incubated the inoculum at 37°C, 220 rpm overnight.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 8th</font></html> ==<br />
<br />
*Gel run/cut/band purification for BOL1(E-S) overnight digestion. Results: gel showed that the enzymes had cut very well and this time also the purification gave a good quantification at Nanodrop.<br />
<br />
*We took R0011(E-X) stored ad -20°C and we were ready to ligate;)<br />
<br />
*Ligation:<br />
**A11: BOL1(E-S) + R0011(E-X) in pSB1A2<br />
<br />
*We incubated the ligation overnight at 16°C.<br />
<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We diluted 1:100 the overnight culture of B0030.<br />
<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 9th</font></html> ==<br />
<br />
*We resuspended F2620 BioBrick from iGEM 2009 plates.<br />
<br />
*We transformed about 40 pg of the overnight ligation and 1 ul of the resuspended F2620 BioBrick. We plated transformed bacteria and incubated the two plates overnight at 37°C.<br />
<br />
<br />
<br />
<br />
''End of experiment with Tecan F200 (last measurement)''<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">July, 10th</font></html> ==<br />
<br />
*A11 and F2620 overnight plates showed colonies!<br />
<br />
*Colony PCR for 6 colonies of A11 plate: the picked colonies were inoculated in 1 ml of LB + Amp and incubated at 37°C, 220 rpm waiting for PCR results.<br />
<br />
*We also picked a colony from F2620 to infect 1 ml of LB + Amp. We incubated this inoculum with the others.<br />
<br />
<font class='didascalia'><br />
{|align="center"<br />
|[[Image:pv_colonypcr_A11.jpg|thumb|300px|left|Colony PCR for A11 plate: all the colonies showed the correct plasmid. We kept the 1st, 3rd and 6th colonies.]]<br />
|}<br />
</font><br />
<br />
*Gel results: all the picked colonies showed the expected amplicon of a correctly ligated plasmid! We decided to prepare a glycerol stock for:<br />
**A11-1<br />
**A11-3<br />
**A11-6<br />
<br />
*Next week, only A11-1 will be used, but the other colonies will be used in case of failure.<br />
<br />
*We also prepared a glycerol stock for F2620 culture.<br />
<br />
<br />
<br />
*We contacted iGEM HQ to request these BioBricks:<br />
{|cellpadding="20"<br />
|K116001<br />
|K116002<br />
|K112405<br />
|-<br />
|P0412<br />
|I746902<br />
|I746903<br />
|-<br />
|K101017<br />
|F2620<br />
|}<br />
<br />
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[[#top|Top]]<br />
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</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3JunTeam:UNIPV-Pavia/Notebook/Week3Jun2009-10-21T23:23:28Z<p>110hs: /* June, 21st */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<html><a name="week_start"></a></html><br />
= Week from June 15th, to June 21st, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Jun#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Jun#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Jun#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Jun#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">June, 15th</font></html> ==<br />
<br />
*This week we wanted to complete the assembly of an aTc sensor and to test if lacZ works. Sequence analysis cannot be efficiently performed on I732017 or on A3 because of lacZ coding sequence length (>3 Kb), so we decided to check lacZ integrity assembling a constitutive promoter upstream of A3 and testing the ligation on X-Gal plates.<br />
<br />
<br />
*We already had J23100, J23101, A3 and A4 at -20°C. Digestions:<br />
{|cellpadding="20"<br />
|J23100(S-P)<br />
|J23101(E-S)<br />
|-<br />
|A3(E-X)<br />
|A4(X-P)<br />
|}<br />
<br />
*We ran/cut a 1% gel for J23100(S-P), A3(E-X) and A4(X-P). All the desired bands had the expected sizes and had been purified from agarose gel. Unlikely we had a low yield for A4(X-P) extraction...<br />
<br />
*We ran a 2% gel to isolate J23101(E-S) insert, but unfortunately we could not see any band at the expected size of 35 bp...<br />
<br />
*We planned not to perform any ligation: unlucky day for them!:(<br />
*NOTE: we did not re-try to extract J23101(E-S) because it is too small.<br><br />
Our A4 ligation is composed by RBS-tetR-TT-Ptet , so it can be considered as a constitutive promoter (Ptet) with an additional "non functional" part (RBS-tetR-TT). So it can be easily used as an insert for assembly because of its 900 bp size, that allows it to be efficiently isolated and extracted from a gel!<br />
<br />
*For this reason, we infected 5 ml of LB + Amp with 15 ul of A4 glycerol stock (X4 falcon tubes). The following day we plan to cut A4(E-S) (X2) to use it as a constitutive promoter for A3(E-X), while we plan to cut A4(X-P) (X2) to ligate it to J23100 to constitute an aTc->PoPS inducible device. We performed all the inocula X2 because we wanted to be sure to have a sufficient yield for ligations.<br />
<br />
*We stored J23100(S-P) and A3(E-X) at -20°C.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">June, 16th</font></html> ==<br />
<br />
*Miniprep for A4 (X4 overnight cultures).<br />
<br />
*Digestions:<br />
{|cellpadding="20"<br />
|A4(E-S) (X2)<br />
|A4(X-P) (X2)<br />
|}<br />
<br />
*Gel run/cut; gel extraction.<br />
<br />
*Ligations:<br />
**A5 = A4(E-S) + A3(E-X) in pSB1AK3<br />
**A6 = J23100(S-P) + A4(X-P) in J61002 without RFP protein generator<br />
<br />
*We incubated the two ligation reactions overnight at 16°C.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">June, 17th</font></html> ==<br />
<br />
*We prepared three X-Gal plates (of LB + Amp).<br />
<br />
*We transformed the two ligations (1 ul) in TOP10 E. coli.<br />
<br />
*We plated A6 transformed bacteria + SOC in a non-X-Gal plate.<br />
*We plated A5 transformed bacteria + SOC in two X-Gal plates: we plated 100 ul in the first plate (called A5) and 200 ul in the second plate (called A5bis) in order to check for blue colonies the following day.<br />
<br />
*We also plated 20 ul of E0240 glycerol stock in the third prepared X-Gal plate, in order to have a negative control to check if TOP10 E. coli actually don't express beta-galactosidase.<br />
<br />
*Expected results:<br />
**Plates A5 and A5bis should show blue colonies;<br />
**Plate E0240 should remain colorless.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">June, 18th</font></html> ==<br />
<br />
*Plate results:<br />
**A5 showed blue colonies;<br />
**A5bis showed blue colonies;<br />
**E0240 did not show any blue colony.<br />
<br />
*So, we can say that TOP10 E. coli don't express any beta-galactosidase and our lacZ gene works!<br />
<br />
<table width=100%><br />
<tr><br />
<td><br />
<font class='didascalia'><br />
[[Image:pv_A5xgal.jpg|thumb|200px|left|Blue colonies for A5 X-Gal overnight plate]]<br />
</font><br />
</td><br />
<td><br />
<font class='didascalia'><br />
[[Image:pv_A5xgalbis.jpg|thumb|200px|left|Blue colonies for A5bis X-Gal overnight plate]]<br />
</font><br />
</td><br />
<td><br />
<font class='didascalia'><br />
[[Image:pv_e0240xgal.jpg|thumb|200px|left|Normal-coloured colonies for E0240 X-Gal overnight plate - negative control]]<br />
</font><br />
</td><br />
</tr><br />
</table><br />
<br />
*We picked a blue colony from A5bis plate and infected 5 ml of LB + Amp. We incubated the inoculum overnight at 37°C, 220 rpm.<br />
<br />
<br />
*We decided to perform the X-Gal test on A3, which is the promoterless RBS-lacZ-TT in pSB1AK3:<br />
**we prepared another X-Gal plate (LB + Amp),<br />
**we mixed 20 ul of A3 glycerol stock with 100 ul of SOC medium,<br />
**we plated the diluted A3 culture and incubated the plate overnight at 37°C.<br />
<br />
<br />
<br />
*Colony PCR for A6 plate: we screened six colonies. We let the picked colonies grow in 1 ml of LB + Amp, 37°C, 220 rpm, waiting for the screening results.<br />
<br />
*We had a problem with the thermal cycler, it gave an error after about 20 min of the reaction and we had to restart it. At the end of the reaction we noticed that the cap of A6-2 vial had been melted and almost all the reaction evaporated...What a beautiful PCR! isn't it? @_@(...)<br />
<br />
*Electrophoresis for the PCR vials.<br />
<br />
<table align=center><br />
<tr><br />
<td><br />
<font class='didascalia'><br />
[[Image:pv_colonypcr_A6.jpg|thumb|300px|left|Colony PCR: we chose the 4th screened colony (A6-4, lane 5). No amplicons for the almost evaporated reaction (A6-2, lane 8)]]<br />
</font><br />
</td><br />
</tr><br />
</table><br />
<br />
*Gel results: the almost evaporated reaction (A6-2) did not show any amplicon (lane 8). All the other reactions showed the amplicon with the expected size (~1100 bp).<br />
<br />
*We chose the 4th colony (A6-4): we filled A6-4 incubated culture with LB + Amp to reach a total volume of 5 ml and let it grow overnight at 37°C, 220 rpm.<br />
<br />
*We infected 5 ml of LB + Amp with 15 ul of these glycerol stocks (to prepare samples for sequencing):<br />
{|cellpadding="20"<br />
|K131009<br />
|A3<br />
|A4<br />
|}<br />
<br />
*So, in summary, we have 5 overnight cultures:<br />
**K131009 (from glycerol)<br />
**A3 (from glycerol)<br />
**A4 (from glycerol)<br />
**A5 (from A5bis overnight plate, colony randomly chosen)<br />
**A6 (from A6 overnight plate, after colony PCR)<br />
<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We picked two colonies from E0240 overnight plate and infected two falcon tubes containing 5 ml of LB + Amp. We incubated the inocula at 37°C, 220 rpm for 2 hours before the test.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">June, 19th</font></html> ==<br />
<br />
*We prepared glycerol stocks for A5 and A6.<br />
*A3 plates incubated yesterday showed blue colonies, even if LacZ was not preceded by any promoter in the plasmid.<br />
<font class='didascalia'><br />
[[Image:pv_A3xgal.jpg|thumb|300px|center|Blue colonies for A3 X-Gal overnight plate]]<br />
</font><br />
*This result is strange, but we noticed that Caltech iGEM 2008 team had a similar outcome, performing the beta-galactosidase assay on a similar BioBrick [https://2008.igem.org/Team:Caltech/Project/Lactose_intolerance https://2008.igem.org/Team:Caltech/Project/Lactose_intolerance]: they found significant levels of beta-galactosidase in a high copy number plasmid containing RBS-lacZ-TT. Caltech wrote that it may be caused by 'spurious transcription' amplified by a strong ribosome binding site and high copy plasmid (from their wiki).<br />
*Anyway, we now know that our lacZ sequence is correct and it will be put under the control of the desired promoter during the following weeks. Unfortunately, because of the spurious expression of lacZ, we won't be able to perform a blue/white colony screening after the ligations that involve lacZ gene. For this reason, we are not sure that the blue colony picked on June 18th from A5bis plate is correctly ligated! We decided to purify it and to sequence it anyway. (the plasmid contained in this colony will be called "A5?")<br />
*Miniprep for A3, A4, A5, A6 and K131009.<br />
*We sent the purified plasmids to BMR Genomics for sequencing.<br />
<br />
*We received a gift from Bologna iGEM team: J23118-B0034-C0012-B0015 in pSB1A2! (we will call it BOL1) Thank you very much!!!;)<br />
<br />
== <html><font class="dayw_style">June, 20th</font></html> ==<br />
<br />
Spring Workshop in London.<br />
<br />
== <html><font class="dayw_style">June, 21st</font></html> ==<br />
<br />
Spring Workshop in London<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Jun#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Jun#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Jun#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Jun#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
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<br />
</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3JunTeam:UNIPV-Pavia/Notebook/Week3Jun2009-10-21T23:23:16Z<p>110hs: /* June, 21st */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<html><a name="week_start"></a></html><br />
= Week from June 15th, to June 21st, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Jun#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Jun#week_start">Previous Week</a><br />
</td><br />
<td align="right" width="50%"> <br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Jun#week_start">Next Week</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Jun#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/><br />
</a><br />
</td><br />
</tr><br />
</table><br />
</html><br />
<br />
== <html><font class="dayw_style">June, 15th</font></html> ==<br />
<br />
*This week we wanted to complete the assembly of an aTc sensor and to test if lacZ works. Sequence analysis cannot be efficiently performed on I732017 or on A3 because of lacZ coding sequence length (>3 Kb), so we decided to check lacZ integrity assembling a constitutive promoter upstream of A3 and testing the ligation on X-Gal plates.<br />
<br />
<br />
*We already had J23100, J23101, A3 and A4 at -20°C. Digestions:<br />
{|cellpadding="20"<br />
|J23100(S-P)<br />
|J23101(E-S)<br />
|-<br />
|A3(E-X)<br />
|A4(X-P)<br />
|}<br />
<br />
*We ran/cut a 1% gel for J23100(S-P), A3(E-X) and A4(X-P). All the desired bands had the expected sizes and had been purified from agarose gel. Unlikely we had a low yield for A4(X-P) extraction...<br />
<br />
*We ran a 2% gel to isolate J23101(E-S) insert, but unfortunately we could not see any band at the expected size of 35 bp...<br />
<br />
*We planned not to perform any ligation: unlucky day for them!:(<br />
*NOTE: we did not re-try to extract J23101(E-S) because it is too small.<br><br />
Our A4 ligation is composed by RBS-tetR-TT-Ptet , so it can be considered as a constitutive promoter (Ptet) with an additional "non functional" part (RBS-tetR-TT). So it can be easily used as an insert for assembly because of its 900 bp size, that allows it to be efficiently isolated and extracted from a gel!<br />
<br />
*For this reason, we infected 5 ml of LB + Amp with 15 ul of A4 glycerol stock (X4 falcon tubes). The following day we plan to cut A4(E-S) (X2) to use it as a constitutive promoter for A3(E-X), while we plan to cut A4(X-P) (X2) to ligate it to J23100 to constitute an aTc->PoPS inducible device. We performed all the inocula X2 because we wanted to be sure to have a sufficient yield for ligations.<br />
<br />
*We stored J23100(S-P) and A3(E-X) at -20°C.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">June, 16th</font></html> ==<br />
<br />
*Miniprep for A4 (X4 overnight cultures).<br />
<br />
*Digestions:<br />
{|cellpadding="20"<br />
|A4(E-S) (X2)<br />
|A4(X-P) (X2)<br />
|}<br />
<br />
*Gel run/cut; gel extraction.<br />
<br />
*Ligations:<br />
**A5 = A4(E-S) + A3(E-X) in pSB1AK3<br />
**A6 = J23100(S-P) + A4(X-P) in J61002 without RFP protein generator<br />
<br />
*We incubated the two ligation reactions overnight at 16°C.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">June, 17th</font></html> ==<br />
<br />
*We prepared three X-Gal plates (of LB + Amp).<br />
<br />
*We transformed the two ligations (1 ul) in TOP10 E. coli.<br />
<br />
*We plated A6 transformed bacteria + SOC in a non-X-Gal plate.<br />
*We plated A5 transformed bacteria + SOC in two X-Gal plates: we plated 100 ul in the first plate (called A5) and 200 ul in the second plate (called A5bis) in order to check for blue colonies the following day.<br />
<br />
*We also plated 20 ul of E0240 glycerol stock in the third prepared X-Gal plate, in order to have a negative control to check if TOP10 E. coli actually don't express beta-galactosidase.<br />
<br />
*Expected results:<br />
**Plates A5 and A5bis should show blue colonies;<br />
**Plate E0240 should remain colorless.<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">June, 18th</font></html> ==<br />
<br />
*Plate results:<br />
**A5 showed blue colonies;<br />
**A5bis showed blue colonies;<br />
**E0240 did not show any blue colony.<br />
<br />
*So, we can say that TOP10 E. coli don't express any beta-galactosidase and our lacZ gene works!<br />
<br />
<table width=100%><br />
<tr><br />
<td><br />
<font class='didascalia'><br />
[[Image:pv_A5xgal.jpg|thumb|200px|left|Blue colonies for A5 X-Gal overnight plate]]<br />
</font><br />
</td><br />
<td><br />
<font class='didascalia'><br />
[[Image:pv_A5xgalbis.jpg|thumb|200px|left|Blue colonies for A5bis X-Gal overnight plate]]<br />
</font><br />
</td><br />
<td><br />
<font class='didascalia'><br />
[[Image:pv_e0240xgal.jpg|thumb|200px|left|Normal-coloured colonies for E0240 X-Gal overnight plate - negative control]]<br />
</font><br />
</td><br />
</tr><br />
</table><br />
<br />
*We picked a blue colony from A5bis plate and infected 5 ml of LB + Amp. We incubated the inoculum overnight at 37°C, 220 rpm.<br />
<br />
<br />
*We decided to perform the X-Gal test on A3, which is the promoterless RBS-lacZ-TT in pSB1AK3:<br />
**we prepared another X-Gal plate (LB + Amp),<br />
**we mixed 20 ul of A3 glycerol stock with 100 ul of SOC medium,<br />
**we plated the diluted A3 culture and incubated the plate overnight at 37°C.<br />
<br />
<br />
<br />
*Colony PCR for A6 plate: we screened six colonies. We let the picked colonies grow in 1 ml of LB + Amp, 37°C, 220 rpm, waiting for the screening results.<br />
<br />
*We had a problem with the thermal cycler, it gave an error after about 20 min of the reaction and we had to restart it. At the end of the reaction we noticed that the cap of A6-2 vial had been melted and almost all the reaction evaporated...What a beautiful PCR! isn't it? @_@(...)<br />
<br />
*Electrophoresis for the PCR vials.<br />
<br />
<table align=center><br />
<tr><br />
<td><br />
<font class='didascalia'><br />
[[Image:pv_colonypcr_A6.jpg|thumb|300px|left|Colony PCR: we chose the 4th screened colony (A6-4, lane 5). No amplicons for the almost evaporated reaction (A6-2, lane 8)]]<br />
</font><br />
</td><br />
</tr><br />
</table><br />
<br />
*Gel results: the almost evaporated reaction (A6-2) did not show any amplicon (lane 8). All the other reactions showed the amplicon with the expected size (~1100 bp).<br />
<br />
*We chose the 4th colony (A6-4): we filled A6-4 incubated culture with LB + Amp to reach a total volume of 5 ml and let it grow overnight at 37°C, 220 rpm.<br />
<br />
*We infected 5 ml of LB + Amp with 15 ul of these glycerol stocks (to prepare samples for sequencing):<br />
{|cellpadding="20"<br />
|K131009<br />
|A3<br />
|A4<br />
|}<br />
<br />
*So, in summary, we have 5 overnight cultures:<br />
**K131009 (from glycerol)<br />
**A3 (from glycerol)<br />
**A4 (from glycerol)<br />
**A5 (from A5bis overnight plate, colony randomly chosen)<br />
**A6 (from A6 overnight plate, after colony PCR)<br />
<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We picked two colonies from E0240 overnight plate and infected two falcon tubes containing 5 ml of LB + Amp. We incubated the inocula at 37°C, 220 rpm for 2 hours before the test.<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
== <html><font class="dayw_style">June, 19th</font></html> ==<br />
<br />
*We prepared glycerol stocks for A5 and A6.<br />
*A3 plates incubated yesterday showed blue colonies, even if LacZ was not preceded by any promoter in the plasmid.<br />
<font class='didascalia'><br />
[[Image:pv_A3xgal.jpg|thumb|300px|center|Blue colonies for A3 X-Gal overnight plate]]<br />
</font><br />
*This result is strange, but we noticed that Caltech iGEM 2008 team had a similar outcome, performing the beta-galactosidase assay on a similar BioBrick [https://2008.igem.org/Team:Caltech/Project/Lactose_intolerance https://2008.igem.org/Team:Caltech/Project/Lactose_intolerance]: they found significant levels of beta-galactosidase in a high copy number plasmid containing RBS-lacZ-TT. Caltech wrote that it may be caused by 'spurious transcription' amplified by a strong ribosome binding site and high copy plasmid (from their wiki).<br />
*Anyway, we now know that our lacZ sequence is correct and it will be put under the control of the desired promoter during the following weeks. Unfortunately, because of the spurious expression of lacZ, we won't be able to perform a blue/white colony screening after the ligations that involve lacZ gene. For this reason, we are not sure that the blue colony picked on June 18th from A5bis plate is correctly ligated! We decided to purify it and to sequence it anyway. (the plasmid contained in this colony will be called "A5?")<br />
*Miniprep for A3, A4, A5, A6 and K131009.<br />
*We sent the purified plasmids to BMR Genomics for sequencing.<br />
<br />
*We received a gift from Bologna iGEM team: J23118-B0034-C0012-B0015 in pSB1A2! (we will call it BOL1) Thank you very much!!!;)<br />
<br />
== <html><font class="dayw_style">June, 20th</font></html> ==<br />
<br />
Spring Workshop in London.<br />
<br />
== <html><font class="dayw_style">June, 21st</font></html> ==<br />
<br />
Spring Workshop in London<br />
<br />
<br />
<div align="right"><br />
[[#top|Top]]<br />
</div><br />
<br />
<html><br />
<hr/><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Jun#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
</a><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Jun#week_start">Previous Week</a><br />
</td><br />
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</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3JunTeam:UNIPV-Pavia/Notebook/Week3Jun2009-10-21T23:23:00Z<p>110hs: /* June, 21st */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<html><a name="week_start"></a></html><br />
= Week from June 15th, to June 21st, 2009 =<br />
<html><br />
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<br />
== <html><font class="dayw_style">June, 15th</font></html> ==<br />
<br />
*This week we wanted to complete the assembly of an aTc sensor and to test if lacZ works. Sequence analysis cannot be efficiently performed on I732017 or on A3 because of lacZ coding sequence length (>3 Kb), so we decided to check lacZ integrity assembling a constitutive promoter upstream of A3 and testing the ligation on X-Gal plates.<br />
<br />
<br />
*We already had J23100, J23101, A3 and A4 at -20°C. Digestions:<br />
{|cellpadding="20"<br />
|J23100(S-P)<br />
|J23101(E-S)<br />
|-<br />
|A3(E-X)<br />
|A4(X-P)<br />
|}<br />
<br />
*We ran/cut a 1% gel for J23100(S-P), A3(E-X) and A4(X-P). All the desired bands had the expected sizes and had been purified from agarose gel. Unlikely we had a low yield for A4(X-P) extraction...<br />
<br />
*We ran a 2% gel to isolate J23101(E-S) insert, but unfortunately we could not see any band at the expected size of 35 bp...<br />
<br />
*We planned not to perform any ligation: unlucky day for them!:(<br />
*NOTE: we did not re-try to extract J23101(E-S) because it is too small.<br><br />
Our A4 ligation is composed by RBS-tetR-TT-Ptet , so it can be considered as a constitutive promoter (Ptet) with an additional "non functional" part (RBS-tetR-TT). So it can be easily used as an insert for assembly because of its 900 bp size, that allows it to be efficiently isolated and extracted from a gel!<br />
<br />
*For this reason, we infected 5 ml of LB + Amp with 15 ul of A4 glycerol stock (X4 falcon tubes). The following day we plan to cut A4(E-S) (X2) to use it as a constitutive promoter for A3(E-X), while we plan to cut A4(X-P) (X2) to ligate it to J23100 to constitute an aTc->PoPS inducible device. We performed all the inocula X2 because we wanted to be sure to have a sufficient yield for ligations.<br />
<br />
*We stored J23100(S-P) and A3(E-X) at -20°C.<br />
<br />
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</div><br />
<br />
== <html><font class="dayw_style">June, 16th</font></html> ==<br />
<br />
*Miniprep for A4 (X4 overnight cultures).<br />
<br />
*Digestions:<br />
{|cellpadding="20"<br />
|A4(E-S) (X2)<br />
|A4(X-P) (X2)<br />
|}<br />
<br />
*Gel run/cut; gel extraction.<br />
<br />
*Ligations:<br />
**A5 = A4(E-S) + A3(E-X) in pSB1AK3<br />
**A6 = J23100(S-P) + A4(X-P) in J61002 without RFP protein generator<br />
<br />
*We incubated the two ligation reactions overnight at 16°C.<br />
<br />
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<br />
== <html><font class="dayw_style">June, 17th</font></html> ==<br />
<br />
*We prepared three X-Gal plates (of LB + Amp).<br />
<br />
*We transformed the two ligations (1 ul) in TOP10 E. coli.<br />
<br />
*We plated A6 transformed bacteria + SOC in a non-X-Gal plate.<br />
*We plated A5 transformed bacteria + SOC in two X-Gal plates: we plated 100 ul in the first plate (called A5) and 200 ul in the second plate (called A5bis) in order to check for blue colonies the following day.<br />
<br />
*We also plated 20 ul of E0240 glycerol stock in the third prepared X-Gal plate, in order to have a negative control to check if TOP10 E. coli actually don't express beta-galactosidase.<br />
<br />
*Expected results:<br />
**Plates A5 and A5bis should show blue colonies;<br />
**Plate E0240 should remain colorless.<br />
<br />
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<br />
== <html><font class="dayw_style">June, 18th</font></html> ==<br />
<br />
*Plate results:<br />
**A5 showed blue colonies;<br />
**A5bis showed blue colonies;<br />
**E0240 did not show any blue colony.<br />
<br />
*So, we can say that TOP10 E. coli don't express any beta-galactosidase and our lacZ gene works!<br />
<br />
<table width=100%><br />
<tr><br />
<td><br />
<font class='didascalia'><br />
[[Image:pv_A5xgal.jpg|thumb|200px|left|Blue colonies for A5 X-Gal overnight plate]]<br />
</font><br />
</td><br />
<td><br />
<font class='didascalia'><br />
[[Image:pv_A5xgalbis.jpg|thumb|200px|left|Blue colonies for A5bis X-Gal overnight plate]]<br />
</font><br />
</td><br />
<td><br />
<font class='didascalia'><br />
[[Image:pv_e0240xgal.jpg|thumb|200px|left|Normal-coloured colonies for E0240 X-Gal overnight plate - negative control]]<br />
</font><br />
</td><br />
</tr><br />
</table><br />
<br />
*We picked a blue colony from A5bis plate and infected 5 ml of LB + Amp. We incubated the inoculum overnight at 37°C, 220 rpm.<br />
<br />
<br />
*We decided to perform the X-Gal test on A3, which is the promoterless RBS-lacZ-TT in pSB1AK3:<br />
**we prepared another X-Gal plate (LB + Amp),<br />
**we mixed 20 ul of A3 glycerol stock with 100 ul of SOC medium,<br />
**we plated the diluted A3 culture and incubated the plate overnight at 37°C.<br />
<br />
<br />
<br />
*Colony PCR for A6 plate: we screened six colonies. We let the picked colonies grow in 1 ml of LB + Amp, 37°C, 220 rpm, waiting for the screening results.<br />
<br />
*We had a problem with the thermal cycler, it gave an error after about 20 min of the reaction and we had to restart it. At the end of the reaction we noticed that the cap of A6-2 vial had been melted and almost all the reaction evaporated...What a beautiful PCR! isn't it? @_@(...)<br />
<br />
*Electrophoresis for the PCR vials.<br />
<br />
<table align=center><br />
<tr><br />
<td><br />
<font class='didascalia'><br />
[[Image:pv_colonypcr_A6.jpg|thumb|300px|left|Colony PCR: we chose the 4th screened colony (A6-4, lane 5). No amplicons for the almost evaporated reaction (A6-2, lane 8)]]<br />
</font><br />
</td><br />
</tr><br />
</table><br />
<br />
*Gel results: the almost evaporated reaction (A6-2) did not show any amplicon (lane 8). All the other reactions showed the amplicon with the expected size (~1100 bp).<br />
<br />
*We chose the 4th colony (A6-4): we filled A6-4 incubated culture with LB + Amp to reach a total volume of 5 ml and let it grow overnight at 37°C, 220 rpm.<br />
<br />
*We infected 5 ml of LB + Amp with 15 ul of these glycerol stocks (to prepare samples for sequencing):<br />
{|cellpadding="20"<br />
|K131009<br />
|A3<br />
|A4<br />
|}<br />
<br />
*So, in summary, we have 5 overnight cultures:<br />
**K131009 (from glycerol)<br />
**A3 (from glycerol)<br />
**A4 (from glycerol)<br />
**A5 (from A5bis overnight plate, colony randomly chosen)<br />
**A6 (from A6 overnight plate, after colony PCR)<br />
<br />
<br />
<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We picked two colonies from E0240 overnight plate and infected two falcon tubes containing 5 ml of LB + Amp. We incubated the inocula at 37°C, 220 rpm for 2 hours before the test.<br />
<br />
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<br />
== <html><font class="dayw_style">June, 19th</font></html> ==<br />
<br />
*We prepared glycerol stocks for A5 and A6.<br />
*A3 plates incubated yesterday showed blue colonies, even if LacZ was not preceded by any promoter in the plasmid.<br />
<font class='didascalia'><br />
[[Image:pv_A3xgal.jpg|thumb|300px|center|Blue colonies for A3 X-Gal overnight plate]]<br />
</font><br />
*This result is strange, but we noticed that Caltech iGEM 2008 team had a similar outcome, performing the beta-galactosidase assay on a similar BioBrick [https://2008.igem.org/Team:Caltech/Project/Lactose_intolerance https://2008.igem.org/Team:Caltech/Project/Lactose_intolerance]: they found significant levels of beta-galactosidase in a high copy number plasmid containing RBS-lacZ-TT. Caltech wrote that it may be caused by 'spurious transcription' amplified by a strong ribosome binding site and high copy plasmid (from their wiki).<br />
*Anyway, we now know that our lacZ sequence is correct and it will be put under the control of the desired promoter during the following weeks. Unfortunately, because of the spurious expression of lacZ, we won't be able to perform a blue/white colony screening after the ligations that involve lacZ gene. For this reason, we are not sure that the blue colony picked on June 18th from A5bis plate is correctly ligated! We decided to purify it and to sequence it anyway. (the plasmid contained in this colony will be called "A5?")<br />
*Miniprep for A3, A4, A5, A6 and K131009.<br />
*We sent the purified plasmids to BMR Genomics for sequencing.<br />
<br />
*We received a gift from Bologna iGEM team: J23118-B0034-C0012-B0015 in pSB1A2! (we will call it BOL1) Thank you very much!!!;)<br />
<br />
== <html><font class="dayw_style">June, 20th</font></html> ==<br />
<br />
Spring Workshop in London.<br />
<br />
== <html><font class="dayw_style">June, 21st</font></html> ==<br />
<br />
Spring Workshop in London<br />
<br />
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</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2JunTeam:UNIPV-Pavia/Notebook/Week2Jun2009-10-21T23:22:35Z<p>110hs: /* June, 8th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<html><a name="week_start"></a></html><br />
= Week from June 8th, to June 14th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jun#week_start"><br />
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/><br />
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<br />
== <html><font class="dayw_style">June, 8th</font></html> ==<br />
<br />
*We infected 5 ml of LB + Amp with 15 ul of these glycerol stocks:<br />
<table width=100%><br />
<tr><td><br />
{|cellpadding="20"<br />
|J23100<br />
|J23101<br />
|E0240 (X2 cultures)<br />
|-<br />
|P0040<br />
|R0040<br />
|I732017<br />
|-<br />
|B0015<br />
|}<br />
</td><br />
</tr><br />
</table><br />
<br />
*We incubated the inocula overnight at 37°C, 220 rpm.<br />
<br />
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</div><br />
<br />
== <html><font class="dayw_style">June, 9th</font></html> ==<br />
<br />
*This is the first ligation day! cross your sticky fingers:)<br />
<br />
<table width=100%><br />
<tr><br />
<td><br />
*Miniprep for:<br />
{|cellpadding="20"<br />
|J23100<br />
|J23101<br />
|E0240 (X2)<br />
|-<br />
|P0040<br />
|R0040<br />
|I732017<br />
|-<br />
|B0015<br />
|}<br />
</td><br />
<td><br />
<font class='didascalia'><br />
[[Image:pv_letizia_1miniprep.jpg|thumb|300px|left|Letizia miniprepping...]]<br />
</font><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<table width=100%><br />
<tr><br />
<td><br />
<font class='didascalia'><br />
[[Image:pv_letizia_1gel.jpg|thumb|300px|left|...and preparing the gel run!]]<br />
</font><br />
</td><br />
<td><br />
*Digestions:<br />
{|cellpadding="20"<br />
|J23100(S-P)<br />
|J23101(S-P)<br />
|E0240(X-P) (X2)<br />
|-<br />
|P0040(E-S)<br />
|R0040(E-X)<br />
|I732017(E-S)<br />
|-<br />
|B0015(E-X)<br />
|}<br />
</td><br />
</tr><br />
</table><br />
<br />
<table width="100%"><br />
<tr><br />
<td><br />
*Gel run and band cut for the digested plasmids. We used two gels: a medium one (short run to separate 1-2 Kb fragments) for J23100(S-P), J23101(S-P), E0240(X-P) (X2), P0040(E-S) and R0040(E-X); a small one (long run to separate 2-3 Kb fragments) for I732017(E-S) and B0015(E-X).<br />
<br />
*All the bands in the two gels had the expected size! only R0040 seemed a bit contaminated, but the expected fragment was much brighter than the smear (see figure below). Complete digestion for J23100, J23101, I732017. Incomplete digestion for E0240 (both) and P0440.<br />
<br />
*Gel extraction.<br />
<br />
*Ligations:<br />
**A1 = J23100(S-P) + E0240(X-P)<br />
**A2 = J23101(S-P) + E0240(X-P)<br />
**A3 = I732017(E-S) + B0015(E-X)<br />
**A4 = P0440(E-S) + R0040(E-X)<br />
<br />
*We incubated the ligation reaction overnight at 16°C.<br />
</td><br />
<td><br />
<font class='didascalia'><br />
[[Image:pv_cut_gel.jpg|thumb|350px|left|Medium gel after bands cut]]<br />
</font><br />
</td><br />
</tr><br />
</table><br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We streaked a plate with E0240 glycerol stock to prepare the bacterial growth test at Tecan F200.<br />
<br />
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</div><br />
<br />
== <html><font class="dayw_style">June, 10th</font></html> ==<br />
<br />
*We transformed the four ligation reactions.<br />
<br />
*Quality control results performed by MIT on the extracted lysis BioBricks:<br />
**K124017 BioBrick sequence had been classified as "inconsistent" by MIT (and sequence looked completely wrong!). In the following days we will plan how to substitute this part.<br />
**K131009 had some problems in prefix (1 bp deletion, but does not corrupt restriction sites), suffix (1 bp deletion, but does not corrupt restriction sites) and celB gene (point mutation that changes the amino acid L->M). We decided to sequence this part in the following days.<br />
**K117000 was good.<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We picked a colony of the freshly streaked E0240 overnight plate and infected 5 ml of LB + Amp.<br />
<br />
*We incubated the inoculum overnight at 37°C, 220 rpm.<br />
<br />
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</div><br />
<br />
== <html><font class="dayw_style">June, 11th</font></html> ==<br />
<br />
*We received sequencing results for B0030 and J23100: both sequences were correct, confirming that we were working correctly!:)<br />
<br />
*All the four overnight plates showed many colonies! A1 and A2 plates showed only a couple of red colonies...maybe a contamination of the non completely cut plasmids carrying RFP downstream of the promoter...<br />
<br />
*Colony PCR for A1, A2 and A4. We decided not to perform it on A3 because the insert was very long (>3 Kb!) and in our opinion colony PCR was not the ideal way to screen the plate. We let the picked colonies grow in 1 ml of LB + Amp, 37°C, 220 rpm for 5 and 1/2 hours, waiting for the end of the PCR screening.<br />
<br />
*Gel run for the PCR reactions.<br />
<br />
<table align="center"><br />
<tr><br />
<td><br />
<font class='didascalia'><br />
[[Image:pv_colonypcr_A1_A2_A4.jpg|thumb|500px|left|Colony PCR: we had at least a positive colony for each of the three screened plates! we chose the third colony for each plate. ]]<br />
</font><br />
</td><br />
</tr><br />
</table><br />
<br />
*Gel results: A1-2, A2-1 and A4-1 reactions did not show any amplicon...Anyway, the positive reactions gave the expected lengths! We chose:<br />
**3rd colony for A1 (expected length = ~1100 bp)<br />
**3rd colony for A2 (expected length = ~1100 bp)<br />
**3rd colony for A4 (expected length = ~1100 bp)<br />
*NOTE: A4-2 showed a 290 bp band, but in the picture above it's quite hard to see it.<br />
<br />
*Glycerol stocks for the chosen colonies.<br />
<br />
*We re-filled the remaining 250 ul of the chosen colony of A4 with 5 ml of LB + Amp and incubated the falcon tube at 37°C, 220 rpm overnight.<br />
<br />
*We picked five colonies from A3 plate and infected 5 ml of LB + Amp. We incubated them overnight at 37°C, 220 rpm. Tomorrow we will perform miniprep and we will screen the colonies through digestion/electrophoresis.<br />
<br />
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<br />
== <html><font class="dayw_style">June, 12th</font></html> ==<br />
<br />
*Miniprep for the chosen colony of A4.<br />
<br />
*Glycerol stocks/miniprep for the five 5 ml overnight cultures of A3.<br />
<br />
*We linearized the purified plasmids digesting 1 ug of DNA with PstI in a total digestion reaction volume of 20 ul. We incubated the digestion at 37°C for 3 hours. We also applied this protocol for B0015 plasmid (taken from -20°C freezer) in order to have a negative control.<br />
<br />
*NOTE: we expected to find an insert length of 3.2 Kb (I732017+B0015) if the ligation had worked, while we expected an insert length of 129 bp (only B0015) if it had not worked. We decided not to check the length of the inserts because the scaffold vector was about 3.2 Kb long and we could not separate it from the insert without a long gel run...but after a long run, the 129 bp fragment might be lost!<br><br />
For this reason, we decided to perform a run for the entire linearized plasmids to discriminate a 6.4 Kb fragment from a 3.3 Kb fragment.<br />
<br />
*Gel run for the six digestions.<br />
<br />
<table align="center"><br />
<tr><br />
<td><br />
<font class='didascalia'><br />
[[Image:pv_digestion_A3_B0015.jpg|thumb|300px|left|Digestion screening results: A3-1 is the most pure plasmid.]]<br />
</font><br />
</td><br />
</tr><br />
</table><br />
<br />
*Gel results: A3-1, A3-2 and A3-3 show the expected size of a correctly ligated linearized plasmid...but they show an unexpected identical heavy band, especially A3-2 and A3-3. In addition, the brightest bands of A4 and A5 are two and show a completely wrong size, different from B0015 lane, which corresponds to the expected size of a non ligated plasmid. Even A4 and A5 show an unexpected identical heavy band...All the heavy bands had a constant shift from the brightest bands.<br><br />
Maybe they are nicked DNA, but if you read this notebook and you have a different explanation, please e-mail: lorenzo.pasotti@unipv.it ...thank you:)<br><br />
<br />
*Next reactions will be performed with A3-1, which is the most pure colony.<br />
<br />
*Meeting for wiki planning.<br />
<br />
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</html></div>110hshttp://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2JunTeam:UNIPV-Pavia/Notebook/Week2Jun2009-10-21T23:22:27Z<p>110hs: /* June, 11th */</p>
<hr />
<div>{{UNIPV-Pavia/Month}}<br />
__NOTOC__ <br />
<div><br />
<html><a name="week_start"></a></html><br />
= Week from June 8th, to June 14th, 2009 =<br />
<html><br />
<table width="100%"><br />
<tr><br />
<td align="left" width="50%"><br />
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jun#week_start"><br />
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== <html><font class="dayw_style">June, 8th</font></html> ==<br />
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*We infected 5 ml of LB + Amp with 15 ul of these glycerol stocks:<br />
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{|cellpadding="20"<br />
|J23100<br />
|J23101<br />
|E0240 (X2 cultures)<br />
|-<br />
|P0040<br />
|R0040<br />
|I732017<br />
|-<br />
|B0015<br />
|}<br />
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*We incubated the inocula overnight at 37°C, 220 rpm.<br />
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''Experiment with Tecan F200''<br />
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* <html><u>Description</u></html><br />
* <html><u>Purpose:</u></html><br />
* <html><u>Materials & Methods</u></html><br />
* <html><u>Protocol</u></html><br />
* <html><u>Results</u></html><br />
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== <html><font class="dayw_style">June, 9th</font></html> ==<br />
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*This is the first ligation day! cross your sticky fingers:)<br />
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*Miniprep for:<br />
{|cellpadding="20"<br />
|J23100<br />
|J23101<br />
|E0240 (X2)<br />
|-<br />
|P0040<br />
|R0040<br />
|I732017<br />
|-<br />
|B0015<br />
|}<br />
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<td><br />
<font class='didascalia'><br />
[[Image:pv_letizia_1miniprep.jpg|thumb|300px|left|Letizia miniprepping...]]<br />
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<font class='didascalia'><br />
[[Image:pv_letizia_1gel.jpg|thumb|300px|left|...and preparing the gel run!]]<br />
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*Digestions:<br />
{|cellpadding="20"<br />
|J23100(S-P)<br />
|J23101(S-P)<br />
|E0240(X-P) (X2)<br />
|-<br />
|P0040(E-S)<br />
|R0040(E-X)<br />
|I732017(E-S)<br />
|-<br />
|B0015(E-X)<br />
|}<br />
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*Gel run and band cut for the digested plasmids. We used two gels: a medium one (short run to separate 1-2 Kb fragments) for J23100(S-P), J23101(S-P), E0240(X-P) (X2), P0040(E-S) and R0040(E-X); a small one (long run to separate 2-3 Kb fragments) for I732017(E-S) and B0015(E-X).<br />
<br />
*All the bands in the two gels had the expected size! only R0040 seemed a bit contaminated, but the expected fragment was much brighter than the smear (see figure below). Complete digestion for J23100, J23101, I732017. Incomplete digestion for E0240 (both) and P0440.<br />
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*Gel extraction.<br />
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*Ligations:<br />
**A1 = J23100(S-P) + E0240(X-P)<br />
**A2 = J23101(S-P) + E0240(X-P)<br />
**A3 = I732017(E-S) + B0015(E-X)<br />
**A4 = P0440(E-S) + R0040(E-X)<br />
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*We incubated the ligation reaction overnight at 16°C.<br />
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<font class='didascalia'><br />
[[Image:pv_cut_gel.jpg|thumb|350px|left|Medium gel after bands cut]]<br />
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''Preparation of experiment with Tecan F200''<br />
<br />
*We streaked a plate with E0240 glycerol stock to prepare the bacterial growth test at Tecan F200.<br />
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== <html><font class="dayw_style">June, 10th</font></html> ==<br />
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*We transformed the four ligation reactions.<br />
<br />
*Quality control results performed by MIT on the extracted lysis BioBricks:<br />
**K124017 BioBrick sequence had been classified as "inconsistent" by MIT (and sequence looked completely wrong!). In the following days we will plan how to substitute this part.<br />
**K131009 had some problems in prefix (1 bp deletion, but does not corrupt restriction sites), suffix (1 bp deletion, but does not corrupt restriction sites) and celB gene (point mutation that changes the amino acid L->M). We decided to sequence this part in the following days.<br />
**K117000 was good.<br />
<br />
''Preparation of experiment with Tecan F200''<br />
<br />
*We picked a colony of the freshly streaked E0240 overnight plate and infected 5 ml of LB + Amp.<br />
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*We incubated the inoculum overnight at 37°C, 220 rpm.<br />
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== <html><font class="dayw_style">June, 11th</font></html> ==<br />
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*We received sequencing results for B0030 and J23100: both sequences were correct, confirming that we were working correctly!:)<br />
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*All the four overnight plates showed many colonies! A1 and A2 plates showed only a couple of red colonies...maybe a contamination of the non completely cut plasmids carrying RFP downstream of the promoter...<br />
<br />
*Colony PCR for A1, A2 and A4. We decided not to perform it on A3 because the insert was very long (>3 Kb!) and in our opinion colony PCR was not the ideal way to screen the plate. We let the picked colonies grow in 1 ml of LB + Amp, 37°C, 220 rpm for 5 and 1/2 hours, waiting for the end of the PCR screening.<br />
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*Gel run for the PCR reactions.<br />
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<font class='didascalia'><br />
[[Image:pv_colonypcr_A1_A2_A4.jpg|thumb|500px|left|Colony PCR: we had at least a positive colony for each of the three screened plates! we chose the third colony for each plate. ]]<br />
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*Gel results: A1-2, A2-1 and A4-1 reactions did not show any amplicon...Anyway, the positive reactions gave the expected lengths! We chose:<br />
**3rd colony for A1 (expected length = ~1100 bp)<br />
**3rd colony for A2 (expected length = ~1100 bp)<br />
**3rd colony for A4 (expected length = ~1100 bp)<br />
*NOTE: A4-2 showed a 290 bp band, but in the picture above it's quite hard to see it.<br />
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*Glycerol stocks for the chosen colonies.<br />
<br />
*We re-filled the remaining 250 ul of the chosen colony of A4 with 5 ml of LB + Amp and incubated the falcon tube at 37°C, 220 rpm overnight.<br />
<br />
*We picked five colonies from A3 plate and infected 5 ml of LB + Amp. We incubated them overnight at 37°C, 220 rpm. Tomorrow we will perform miniprep and we will screen the colonies through digestion/electrophoresis.<br />
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== <html><font class="dayw_style">June, 12th</font></html> ==<br />
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*Miniprep for the chosen colony of A4.<br />
<br />
*Glycerol stocks/miniprep for the five 5 ml overnight cultures of A3.<br />
<br />
*We linearized the purified plasmids digesting 1 ug of DNA with PstI in a total digestion reaction volume of 20 ul. We incubated the digestion at 37°C for 3 hours. We also applied this protocol for B0015 plasmid (taken from -20°C freezer) in order to have a negative control.<br />
<br />
*NOTE: we expected to find an insert length of 3.2 Kb (I732017+B0015) if the ligation had worked, while we expected an insert length of 129 bp (only B0015) if it had not worked. We decided not to check the length of the inserts because the scaffold vector was about 3.2 Kb long and we could not separate it from the insert without a long gel run...but after a long run, the 129 bp fragment might be lost!<br><br />
For this reason, we decided to perform a run for the entire linearized plasmids to discriminate a 6.4 Kb fragment from a 3.3 Kb fragment.<br />
<br />
*Gel run for the six digestions.<br />
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<font class='didascalia'><br />
[[Image:pv_digestion_A3_B0015.jpg|thumb|300px|left|Digestion screening results: A3-1 is the most pure plasmid.]]<br />
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*Gel results: A3-1, A3-2 and A3-3 show the expected size of a correctly ligated linearized plasmid...but they show an unexpected identical heavy band, especially A3-2 and A3-3. In addition, the brightest bands of A4 and A5 are two and show a completely wrong size, different from B0015 lane, which corresponds to the expected size of a non ligated plasmid. Even A4 and A5 show an unexpected identical heavy band...All the heavy bands had a constant shift from the brightest bands.<br><br />
Maybe they are nicked DNA, but if you read this notebook and you have a different explanation, please e-mail: lorenzo.pasotti@unipv.it ...thank you:)<br><br />
<br />
*Next reactions will be performed with A3-1, which is the most pure colony.<br />
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*Meeting for wiki planning.<br />
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