http://2008.igem.org/wiki/index.php?title=Special:Contributions/Jkm&feed=atom&limit=50&target=Jkm&year=&month=2008.igem.org - User contributions [en]2024-03-29T05:51:44ZFrom 2008.igem.orgMediaWiki 1.16.5http://2008.igem.org/IGEM_PublicityIGEM Publicity2009-02-02T19:04:19Z<p>Jkm: </p>
<hr />
<div><div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [[Press_Kit | iGEM Press Kit]])</div><br />
<br />
<div><br />
<span style="color:#1e90ff; font-size:135%">'''blogs </span><span style="color:#3cb371; font-size:135%">covering iGEM 2009'''</span><br />
* [http://www.the-scientist.com/blog/browse/blogger/33/ <span style="color:red; font-size: 130%">'''The Scientist'''</span>] scroll down for several blog posts about iGEM and the Jamboree written by Alla Katsnelson.<br />
* [http://igemkuleuven.wordpress.com/ <span style="font-size:130%">'''KULeuven''']</span> blogs about their iGEM experience.<br />
* iGEM 2008 judge, [http://synthesis.typepad.com/synthesis/2008/11/igem-2008.html <span style="color:#e25500; font-size:130%">'''Rob Carlson''']</span>, shares his thoughts about iGEM and the successes of this year's teams.<br />
* [http://blog.ginkgobioworks.com/2008/11/09/team-slovenia-igem-grand-prize-winners-again/ <span style="color:#000; font-size:130%; font-weight:bold;">'''Ginkgo</span><span style="color:#77bc35; font-size:130%;">bioworks</span>'''], with several of its members serving as judges this year, blog about the iGEM 2008 Jamboree.<br />
* Eric Bland writes about iGEM on his [http://blogs.discovery.com/news_interior_design/2008/11/igem-2008.html<span style="color:#000; font-size:130%; font-weight:bold">Interior</span> <span style="color:#21528a; font-size:140%">Design</span>] blog, as part of '''Discovery Channel News'''.<br />
* [http://bostonist.com/2008/11/12/beaker_hill_synthetic_biology_igem.php <span style="color:#323232; font-weight: bold; font-size: 135%;">boston</span><span style="color:#323232; font-size: 135%;">ist</span>] talks about the Jamboree in a [http://bostonist.com/tags/igem three part series] on iGEM.<br />
* The UC Berkeley team blogs about their iGEM experience on their [http://blogs.coe.berkeley.edu/igem/about/ <span style="color:#323232; font-weight: bold; font-size: 135%;">Berkeley Engineering</span>] blog.<br />
</div><br />
<br />
<br />
<div><br />
====<font size=4><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 Scientist''': ''Brick by Brick'' Feb 1, 2009. [http://www.the-scientist.com/article/display/55378/ The Scientist]<br />
*'''The NewsHour with Jim Lehrer''': ''Students, Scientists Build Biological Machines'' Dec. 30, 2008 [http://www.pbs.org/newshour/science/technology/index.html The NewsHour] [http://www-tc.pbs.org/newshour/rss/media/2008/12/30/20081230_biology.mp3 ''(Download Audio)'']<br />
*'''National Geographic News''': ''New Biological "Machines" Fight Disease, Produce Power'' [http://news.nationalgeographic.com/news/2008/11/081112-synthetic-biology.html National Geographic News]<br />
*[http://radar.oreilly.com/archives/2007/12/stories-we-want-1.html '''O'Reilly Radar: Looking Forward to iGEM in 2008''']<br />
*[http://www.sciencefriday.com/program/archives/200802291 '''Science Friday: Anticipating Synthetic Biology] ([http://podcastdownload.npr.org/anon.npr-podcasts/podcast/510221/87816117/npr_87816117.mp3 mp3 here)]<br />
<br />
====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
*'''Hawaii''': "Bioengineers bring home awards from iGEM", [http://media.www.kaleo.org/media/storage/paper872/news/2008/12/04/News/Bioengineers.Bring.Home.Awards.From.Igem-3568981.shtml Ka Leo O Hawaii (December 4th, 2008)]<br />
<br />
* '''Alberta NINT & University of Alberta''' "U of A students bring home hardware from iGEM competition", [http://www.expressnews.ualberta.ca/article.cfm?id=9758 University of Alberta Express News (November 13, 2008)]<br />
<br />
* '''Alberta NINT''' "iGEM Jamboree Attracts 7 Alberta Student Teams", [http://www.innovationanthology.com/programs.php?id=195 Innovation Anthology (November 6, 2008)] [http://www.innovationanthology.com/uploads/Innovation%20Anthology%20182.mp3 (mp3 here)]<br />
<br />
* '''Alberta NINT & University of Alberta''' "Success of Butanerds fires up competitive spirit in synthetic biology", [http://www.expressnews.ualberta.ca/article.cfm?id=9605 University of Alberta Express News (September 18, 2008)] <br />
* '''Guelph''' "Student Project Aims to Prevent Blindness in Children Using Bacteria", [http://www.uoguelph.ca/news/2008/11/post_151.html University of Guelph Press Release (November 13, 2008)] <br />
* '''Guelph''' "Student Project Aims to Prevent Blindness in Children Using Bacteria", [http://news.guelphmercury.com/News/BreakingNews/article/403883 The Guelph Mercury (November 13, 2008)] <br />
* '''Guelph''' "Guelph Team wins metal at MIT iGEM", [http://www.plant.uoguelph.ca/news/archive/fall2008.html Plant Agriculture News (November 13, 2008)] <br />
*'''Guelph''' "Student Project Aims to Prevent Blindness in Children Using Bacteria", [http://www.exchangemagazine.com/morningpost/2008/week46/Friday/111416.htm Exchange Morning Post (November 14,2008)] <br />
*'''Guelph''' "Researchers at the University of Guelph Make Bacteria with Unusual Properties", [http://www.youtube.com/watch?v=6AWAuOkJm1c 1460 CJOY AM (November 14, 2008)]<br />
<br />
*'''Guelph''' "Guelph team wins at international science competition at MIT - project has potential to solve problem of vitamin A deficiency", [http://theontarion.ca/viewarticle.php?id_pag=2067 The Ontarion; The University of Guelph's Independant Student Newspaper (November 20, 2008)]<br />
* '''KULeuven''': ''"Synthetische biologie: Open source biologie" ("Synthetic biology: Open source biology")'', [https://static.igem.org/mediawiki/2008/a/ac/22-23_memo7_synthetischebiologie.pdf C<sub>2</sub>W Life Sciences (July 12 2008) & MeMo (September 11 2008)] (MeMo is a magazine of the [http://www.kvcv.be/ Koninklijke Vlaamse Chemische Vereniging])<br />
* '''KULeuven''': ''"Studenten K.U.Leuven met ontwerp intelligente bacterie Dr. Coli in MIT-wedstrijd synthetische biologie" ("Students of K.U.Leuven with design of intelligent bacterium Dr. Coli in MIT-competition synthetic biology")'', [http://dagkrant.kuleuven.be/?q=node/5098 Dagkrant K.U.Leuven (September 1 2008)]<br />
*'''MIT+Caltech''': ''Engineering Edible Bacteria'', [http://www.technologyreview.com/printer_friendly_article.aspx?id=21654 MIT Tech Review] (with video)<br />
* '''Rice''':''"Better beer: college team creating anti-cancer brew",'' [http://www.media.rice.edu/media/NewsBot.asp?MODE=VIEW&ID=11642 Rice University News and Media (October 17th, 2008)]<br />
* '''Rice''':''"'BioBeer' with wine's cancer fighting qualities",'' [http://www.dailyindia.com/show/277686.php Daily India (October 17th, 2008)]<br />
* '''Rice''':''"College students making anti-cancer beer,'' [http://www.upi.com/Health_News/2008/10/17/College_students_making_anti-cancer_beer/UPI-33191224299305/ United Press International (October 17th, 2008)]<br />
* '''Rice''':''"Drink beer to avoid cancer…",'' [http://blogs.zdnet.com/emergingtech/?p=1067 ZDNet (October 17th, 2008)]<br />
* '''Rice''':''"Coming soon, ”BioBeer” with wine’’s cancer fighting qualities",'' [http://www.thaindian.com/newsportal/india-news/coming-soon-biobeer-with-wines-cancer-fighting-qualities_100108232.html Thaindian.com/ANI wire (October 17th, 2008)]<br />
* '''Rice''':''"College Team Create Anticancer 'BioBeer' For Entry In Synthetic Biology's IGEM Contest",'' [http://www.medicalnewstoday.com/articles/125887.php Medical News Today (October 17th, 2008)]<br />
* '''Rice''':''"Rice Biology Students: Making Beer Better",'' [http://blogs.houstonpress.com/hairballs/2008/10/biobeer_genetic_resveratrol.php Houston Press (October 17th, 2008)]<br />
* '''Rice''':''"Can drinking beer help cure cancer?",'' [http://mobile.itwire.com/content/view/21268/1066/ IT Wire (October 17th, 2008)]<br />
* '''Rice''':''"Creating anticancer beer",'' [http://current.com/items/89430237_creating_anticancer_beer Current.com (October 20th, 2008)]<br />
* '''Rice''':''"It's Oktober: I'll Drink To That!",'' [http://www.popsci.com/rachel-durfee/article/2008-10/its-oktober-ill-drink?page= Popular Science, (October 21st, 2008)]<br />
* '''Rice''':''"University researchers developing cancer-fighting beer",'' [http://www.computerworld.com/action/article.do?command=printArticleBasic&taxonomyName=Development&articleId=9117656&taxonomyId=11 Computerworld, (October 21st, 2008)]<br />
* '''Rice''':''"Free Beer in an Unfree World",'' [http://reason.com/blog/show/129611.html Reason.com (October 22nd, 2008)]<br />
* '''Rice''':''"Researchers Developing Cancer-Fighting Beer",'' [http://science.slashdot.org/article.pl?sid=08/10/22/2230223&from=rss Slashdot (October 22nd, 2008)]<br />
* '''Rice''':''"Cold, Frosty and Healthy ...",'' [http://www.foxnews.com/story/0,2933,443798,00.html Fox News (October 24th, 2008)]<br />
* '''Rice''':''"Snart kan öl bekämpa cancer",'' [http://www.metro.se/se/article/2008/10/24/15/4736-68/index.xml Metro Teknik (October 24th, 2008)]<br />
* '''Rice''':''"'Fancy a swift twother?'",'' [http://www.thepublican.com/story.asp?sectioncode=7&storycode=61591&c=1 The Publican (October 24th, 2008)]<br />
* '''Rice''':''"Breakthroughs, tips and trends: Beer therapy",'' [http://www.timesonline.co.uk/tol/life_and_style/health/article5007974.ece Times of London (October 25th, 2008)]<br />
* '''Rice''':''"BioBeer Fights Cancer And Gets You Drunk",'' [http://www.javno.com/en/world/clanak.php?id=196507 Javno (Croatia) (October 25th, 2008)]<br />
* '''Rice''':''"A beer that battles cancer?",'' [http://www.odt.co.nz/lifestyle/health/28866/a-beer-battles-cancer Otago Daily Times (New Zealand) (October 25th, 2008)]<br />
* '''Rice''':''"Geek Week: Geek-o-naut returns home, researchers go for the foam",'' [http://weblog.infoworld.com/robertxcringely/archives/2008/10/geek_week_geeko.html InfoWorld (October 27th, 2008)]<br />
* '''Rice''':''"Rice University Students Aim For Creating A Beer Healthier Than Red Wine",'' [http://www.allheadlinenews.com/articles/7012806080 All Headline News (October 27th, 2008)]<br />
* '''Rice''':''"University researchers developing cancer-fighting beer",'' [http://www.techworld.com.au/article/264626/university_researchers_developing_cancer-fighting_beer TechWorld Australia (This Computerworld story also appears in CIO Magazine, Australia) (October 28th, 2008)]<br />
* '''Rice''':''"Beer Drinkers Rejoice: Cancer-Fighting Beer in Development",'' [http://www.nationalledger.com/artman/publish/article_272623475.shtml The National Ledger (October 28th, 2008)]<br />
* '''Rice''':''"Anti-cancer beer under development",'' [http://www.cosmosmagazine.com/news/2278/anti-cancer-beer-under-development COSMOS magazine (October 29th, 2008)]<br />
* '''Rice''':''"Beer taps wine's health benefit",'' [http://www.signonsandiego.com/uniontrib/20081028/news_lz1c28lafee.html San Diego Union-Tribune (October 29th, 2008)]<br />
* '''TU Delft''': ''"Studenten pimpen bacteriën" ("Students pimp bacteria")'', [http://www.tudelft.nl/live/pagina.jsp?id=23acdd35-ef09-4bfc-bee6-19e0f974913f&lang=nl TU Delft news (June 25 2008)]<br />
* '''TU Delft''': ''"Synthetische biologie: Open source biologie" ("Synthetic biology: Open source biology")'', [http://www.biw.kuleuven.be/persberichten/fiches/c2wLS-080712.pdf C2W Life Sciences (July 12 2008)]<br />
* '''TU Delft''': ''"Creatief met genetische lego - studenten bouwen thermometerbacteriën in internationale competitie" ("Being creative with genetic lego - Students build thermometer bacteria in an international competition")'', NRC newspaper (August 21 2008)<br />
* '''TU Delft''': "Studenten maken een thermometerbacterie" ("Students engineer a thermometer bacterium"), [http://www.delta.tudelft.nl/nl/archief/artikel/studenten-maken-een-thermometerbacterie/18466 ''TU Delta (September 25 2008)'']</div>Jkmhttp://2008.igem.org/IGEM_PublicityIGEM Publicity2008-11-12T23:59:13Z<p>Jkm: </p>
<hr />
<div><div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [[Press_Kit | iGEM Press Kit]])</div><br />
<br />
<div><br />
<span style="color:#1e90ff; font-size:135%">'''blogs </span><span style="color:#3cb371; font-size:135%">covering iGEM'''</span><br />
* [http://www.the-scientist.com/blog/browse/blogger/33/ <span style="color:red; font-size: 130%">'''The Scientist'''</span>] scroll down for several blog posts about iGEM and the Jamboree written by Alla Katsnelson.<br />
* [http://igemkuleuven.wordpress.com/ <span style="font-size:130%">'''KULeuven''']</span> blogs about their iGEM experience.<br />
* iGEM 2008 judge, [http://synthesis.typepad.com/synthesis/2008/11/igem-2008.html <span style="color:#e25500; font-size:130%">'''Rob Carlson''']</span>, shares his thoughts about iGEM and the successes of this year's teams.<br />
* [http://blog.ginkgobioworks.com/2008/11/09/team-slovenia-igem-grand-prize-winners-again/ <span style="color:#000; font-size:130%; font-weight:bold;">'''Ginkgo</span><span style="color:#77bc35; font-size:130%;">bioworks</span>'''], with several of its members serving as judges this year, blog about the iGEM 2008 Jamboree.<br />
* Eric Bland writes about iGEM on his [http://blogs.discovery.com/news_interior_design/2008/11/igem-2008.html<span style="color:#000; font-size:130%; font-weight:bold">Interior</span> <span style="color:#21528a; font-size:140%">Design</span>] blog, as part of '''Discovery Channel News'''.<br />
* [http://bostonist.com/2008/11/12/beaker_hill_synthetic_biology_igem.php <span style="color:#323232; font-weight: bold; font-size: 135%;">boston</span><span style="color:#323232; font-size: 135%;">ist</span>] talks about iGEM and the Jamboree.<br />
</div><br />
<br />
<br />
<div><br />
====<font size=4><font color=dodgerblue>'''news articles</font><font color=mediumseagreen> about iGEM'''</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 />
*'''Finalists''': ''New Biological "Machines" Fight Disease, Produce Power'' [http://news.nationalgeographic.com/news/pf/17802599.html National Geographic]<br />
*'''MIT+Caltech''': ''Engineering Edible Bacteria'', [http://www.technologyreview.com/printer_friendly_article.aspx?id=21654 MIT Tech Review] (with video)<br />
*[http://radar.oreilly.com/archives/2007/12/stories-we-want-1.html '''O'Reilly Radar: Looking Forward to iGEM in 2008''']<br />
*[http://www.sciencefriday.com/program/archives/200802291 '''Science Friday: Anticipating Synthetic Biology] ([http://podcastdownload.npr.org/anon.npr-podcasts/podcast/510221/87816117/npr_87816117.mp3 mp3 here)]<br />
* '''TU Delft''': ''"Studenten pimpen bacteriën" ("Students pimp bacteria")'', [http://www.tudelft.nl/live/pagina.jsp?id=23acdd35-ef09-4bfc-bee6-19e0f974913f&lang=nl TU Delft news (June 25 2008)]<br />
* '''TU Delft''': ''"Synthetische biologie: Open source biologie" ("Synthetic biology: Open source biology")'', [http://www.biw.kuleuven.be/persberichten/fiches/c2wLS-080712.pdf C2W Life Sciences (July 12 2008)]<br />
* '''TU Delft''': ''"Creatief met genetische lego - studenten bouwen thermometerbacteriën in internationale competitie" ("Being creative with genetic lego - Students build thermometer bacteria in an international competition")'', NRC newspaper (August 21 2008)<br />
* '''TU Delft''': "Studenten maken een thermometerbacterie" ("Students engineer a thermometer bacterium"), [http://www.delta.tudelft.nl/nl/archief/artikel/studenten-maken-een-thermometerbacterie/18466 ''TU Delta (September 25 2008)''] <br />
<br />
* '''KULeuven''': ''"Synthetische biologie: Open source biologie" ("Synthetic biology: Open source biology")'', [https://static.igem.org/mediawiki/2008/a/ac/22-23_memo7_synthetischebiologie.pdf C<sub>2</sub>W Life Sciences (July 12 2008) & MeMo (September 11 2008)] (MeMo is a magazine of the [http://www.kvcv.be/ Koninklijke Vlaamse Chemische Vereniging])<br />
* '''KULeuven''': ''"Studenten K.U.Leuven met ontwerp intelligente bacterie Dr. Coli in MIT-wedstrijd synthetische biologie" ("Students of K.U.Leuven with design of intelligent bacterium Dr. Coli in MIT-competition synthetic biology")'', [http://dagkrant.kuleuven.be/?q=node/5098 Dagkrant K.U.Leuven (September 1 2008)]<br />
* '''Rice''':''"Better beer: college team creating anti-cancer brew",'' [http://www.media.rice.edu/media/NewsBot.asp?MODE=VIEW&ID=11642 Rice University News and Media (October 17th, 2008)]<br />
* '''Rice''':''"'BioBeer' with wine's cancer fighting qualities",'' [http://www.dailyindia.com/show/277686.php Daily India (October 17th, 2008)]<br />
* '''Rice''':''"College students making anti-cancer beer,'' [http://www.upi.com/Health_News/2008/10/17/College_students_making_anti-cancer_beer/UPI-33191224299305/ United Press International (October 17th, 2008)]<br />
* '''Rice''':''"Drink beer to avoid cancer…",'' [http://blogs.zdnet.com/emergingtech/?p=1067 ZDNet (October 17th, 2008)]<br />
* '''Rice''':''"Coming soon, ”BioBeer” with wine’’s cancer fighting qualities",'' [http://www.thaindian.com/newsportal/india-news/coming-soon-biobeer-with-wines-cancer-fighting-qualities_100108232.html Thaindian.com/ANI wire (October 17th, 2008)]<br />
* '''Rice''':''"College Team Create Anticancer 'BioBeer' For Entry In Synthetic Biology's IGEM Contest",'' [http://www.medicalnewstoday.com/articles/125887.php Medical News Today (October 17th, 2008)]<br />
* '''Rice''':''"Rice Biology Students: Making Beer Better",'' [http://blogs.houstonpress.com/hairballs/2008/10/biobeer_genetic_resveratrol.php Houston Press (October 17th, 2008)]<br />
* '''Rice''':''"Can drinking beer help cure cancer?",'' [http://mobile.itwire.com/content/view/21268/1066/ IT Wire (October 17th, 2008)]<br />
* '''Rice''':''"Creating anticancer beer",'' [http://current.com/items/89430237_creating_anticancer_beer Current.com (October 20th, 2008)]<br />
* '''Rice''':''"It's Oktober: I'll Drink To That!",'' [http://www.popsci.com/rachel-durfee/article/2008-10/its-oktober-ill-drink?page= Popular Science, (October 21st, 2008)]<br />
* '''Rice''':''"University researchers developing cancer-fighting beer",'' [http://www.computerworld.com/action/article.do?command=printArticleBasic&taxonomyName=Development&articleId=9117656&taxonomyId=11 Computerworld, (October 21st, 2008)]<br />
* '''Rice''':''"Free Beer in an Unfree World",'' [http://reason.com/blog/show/129611.html Reason.com (October 22nd, 2008)]<br />
* '''Rice''':''"Researchers Developing Cancer-Fighting Beer",'' [http://science.slashdot.org/article.pl?sid=08/10/22/2230223&from=rss Slashdot (October 22nd, 2008)]<br />
* '''Rice''':''"Cold, Frosty and Healthy ...",'' [http://www.foxnews.com/story/0,2933,443798,00.html Fox News (October 24th, 2008)]<br />
* '''Rice''':''"Snart kan öl bekämpa cancer",'' [http://www.metro.se/se/article/2008/10/24/15/4736-68/index.xml Metro Teknik (October 24th, 2008)]<br />
* '''Rice''':''"'Fancy a swift twother?'",'' [http://www.thepublican.com/story.asp?sectioncode=7&storycode=61591&c=1 The Publican (October 24th, 2008)]<br />
* '''Rice''':''"Breakthroughs, tips and trends: Beer therapy",'' [http://www.timesonline.co.uk/tol/life_and_style/health/article5007974.ece Times of London (October 25th, 2008)]<br />
* '''Rice''':''"BioBeer Fights Cancer And Gets You Drunk",'' [http://www.javno.com/en/world/clanak.php?id=196507 Javno (Croatia) (October 25th, 2008)]<br />
* '''Rice''':''"A beer that battles cancer?",'' [http://www.odt.co.nz/lifestyle/health/28866/a-beer-battles-cancer Otago Daily Times (New Zealand) (October 25th, 2008)]<br />
* '''Rice''':''"Geek Week: Geek-o-naut returns home, researchers go for the foam",'' [http://weblog.infoworld.com/robertxcringely/archives/2008/10/geek_week_geeko.html InfoWorld (October 27th, 2008)]<br />
* '''Rice''':''"Rice University Students Aim For Creating A Beer Healthier Than Red Wine",'' [http://www.allheadlinenews.com/articles/7012806080 All Headline News (October 27th, 2008)]<br />
* '''Rice''':''"University researchers developing cancer-fighting beer",'' [http://www.techworld.com.au/article/264626/university_researchers_developing_cancer-fighting_beer TechWorld Australia (This Computerworld story also appears in CIO Magazine, Australia) (October 28th, 2008)]<br />
* '''Rice''':''"Beer Drinkers Rejoice: Cancer-Fighting Beer in Development",'' [http://www.nationalledger.com/artman/publish/article_272623475.shtml The National Ledger (October 28th, 2008)]<br />
* '''Rice''':''"Anti-cancer beer under development",'' [http://www.cosmosmagazine.com/news/2278/anti-cancer-beer-under-development COSMOS magazine (October 29th, 2008)]<br />
* '''Rice''':''"Beer taps wine's health benefit",'' [http://www.signonsandiego.com/uniontrib/20081028/news_lz1c28lafee.html San Diego Union-Tribune (October 29th, 2008)]<br />
<br />
</div></div>Jkmhttp://2008.igem.org/IGEM_PublicityIGEM Publicity2008-11-12T23:59:02Z<p>Jkm: </p>
<hr />
<div><div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [[Press_Kit | iGEM Press Kit]])</div><br />
<br />
<div><br />
<span style="color:#1e90ff; font-size:135%">'''blogs </span><span style="color:#3cb371; font-size:135%">covering iGEM'''</span><br />
* [http://www.the-scientist.com/blog/browse/blogger/33/ <span style="color:red; font-size: 130%">'''The Scientist'''</span>] scroll down for several blog posts about iGEM and the Jamboree written by Alla Katsnelson.<br />
* [http://igemkuleuven.wordpress.com/ <span style="font-size:130%">'''KULeuven''']</span> blogs about their iGEM experience.<br />
* iGEM 2008 judge, [http://synthesis.typepad.com/synthesis/2008/11/igem-2008.html <span style="color:#e25500; font-size:130%">'''Rob Carlson''']</span>, shares his thoughts about iGEM and the successes of this year's teams.<br />
* [http://blog.ginkgobioworks.com/2008/11/09/team-slovenia-igem-grand-prize-winners-again/ <span style="color:#000; font-size:130%; font-weight:bold;">'''Ginkgo</span><span style="color:#77bc35; font-size:130%;">bioworks</span>'''], with several of its members serving as judges this year, blog about the iGEM 2008 Jamboree.<br />
* Eric Bland writes about iGEM on his [http://blogs.discovery.com/news_interior_design/2008/11/igem-2008.html<span style="color:#000; font-size:130%; font-weight:bold">Interior</span> <span style="color:#21528a; font-size:140%">Design</span>] blog, as part of '''Discovery Channel News'''.<br />
* [http://bostonist.com/2008/11/12/beaker_hill_synthetic_biology_igem.php <span style="color:#323232; font-weight: bold; font-size: 135%;">boston</span><span style="color:#323232; font-size: 135%;">ist</span>] talks about iGEM and the Jamboree.<br />
</div><br />
<br />
<br />
<div><br />
====<font size=4><font color=dodgerblue>'''news articles</font><font color=mediumseagreen> about iGEM'''</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 />
*'''Finalists''': ''New Biological "Machines" Fight Disease, Produce Power'' [http://news.nationalgeographic.com/news/pf/17802599.html National Geographic]]<br />
*'''MIT+Caltech''': ''Engineering Edible Bacteria'', [http://www.technologyreview.com/printer_friendly_article.aspx?id=21654 MIT Tech Review] (with video)<br />
*[http://radar.oreilly.com/archives/2007/12/stories-we-want-1.html '''O'Reilly Radar: Looking Forward to iGEM in 2008''']<br />
*[http://www.sciencefriday.com/program/archives/200802291 '''Science Friday: Anticipating Synthetic Biology] ([http://podcastdownload.npr.org/anon.npr-podcasts/podcast/510221/87816117/npr_87816117.mp3 mp3 here)]<br />
* '''TU Delft''': ''"Studenten pimpen bacteriën" ("Students pimp bacteria")'', [http://www.tudelft.nl/live/pagina.jsp?id=23acdd35-ef09-4bfc-bee6-19e0f974913f&lang=nl TU Delft news (June 25 2008)]<br />
* '''TU Delft''': ''"Synthetische biologie: Open source biologie" ("Synthetic biology: Open source biology")'', [http://www.biw.kuleuven.be/persberichten/fiches/c2wLS-080712.pdf C2W Life Sciences (July 12 2008)]<br />
* '''TU Delft''': ''"Creatief met genetische lego - studenten bouwen thermometerbacteriën in internationale competitie" ("Being creative with genetic lego - Students build thermometer bacteria in an international competition")'', NRC newspaper (August 21 2008)<br />
* '''TU Delft''': "Studenten maken een thermometerbacterie" ("Students engineer a thermometer bacterium"), [http://www.delta.tudelft.nl/nl/archief/artikel/studenten-maken-een-thermometerbacterie/18466 ''TU Delta (September 25 2008)''] <br />
<br />
* '''KULeuven''': ''"Synthetische biologie: Open source biologie" ("Synthetic biology: Open source biology")'', [https://static.igem.org/mediawiki/2008/a/ac/22-23_memo7_synthetischebiologie.pdf C<sub>2</sub>W Life Sciences (July 12 2008) & MeMo (September 11 2008)] (MeMo is a magazine of the [http://www.kvcv.be/ Koninklijke Vlaamse Chemische Vereniging])<br />
* '''KULeuven''': ''"Studenten K.U.Leuven met ontwerp intelligente bacterie Dr. Coli in MIT-wedstrijd synthetische biologie" ("Students of K.U.Leuven with design of intelligent bacterium Dr. Coli in MIT-competition synthetic biology")'', [http://dagkrant.kuleuven.be/?q=node/5098 Dagkrant K.U.Leuven (September 1 2008)]<br />
* '''Rice''':''"Better beer: college team creating anti-cancer brew",'' [http://www.media.rice.edu/media/NewsBot.asp?MODE=VIEW&ID=11642 Rice University News and Media (October 17th, 2008)]<br />
* '''Rice''':''"'BioBeer' with wine's cancer fighting qualities",'' [http://www.dailyindia.com/show/277686.php Daily India (October 17th, 2008)]<br />
* '''Rice''':''"College students making anti-cancer beer,'' [http://www.upi.com/Health_News/2008/10/17/College_students_making_anti-cancer_beer/UPI-33191224299305/ United Press International (October 17th, 2008)]<br />
* '''Rice''':''"Drink beer to avoid cancer…",'' [http://blogs.zdnet.com/emergingtech/?p=1067 ZDNet (October 17th, 2008)]<br />
* '''Rice''':''"Coming soon, ”BioBeer” with wine’’s cancer fighting qualities",'' [http://www.thaindian.com/newsportal/india-news/coming-soon-biobeer-with-wines-cancer-fighting-qualities_100108232.html Thaindian.com/ANI wire (October 17th, 2008)]<br />
* '''Rice''':''"College Team Create Anticancer 'BioBeer' For Entry In Synthetic Biology's IGEM Contest",'' [http://www.medicalnewstoday.com/articles/125887.php Medical News Today (October 17th, 2008)]<br />
* '''Rice''':''"Rice Biology Students: Making Beer Better",'' [http://blogs.houstonpress.com/hairballs/2008/10/biobeer_genetic_resveratrol.php Houston Press (October 17th, 2008)]<br />
* '''Rice''':''"Can drinking beer help cure cancer?",'' [http://mobile.itwire.com/content/view/21268/1066/ IT Wire (October 17th, 2008)]<br />
* '''Rice''':''"Creating anticancer beer",'' [http://current.com/items/89430237_creating_anticancer_beer Current.com (October 20th, 2008)]<br />
* '''Rice''':''"It's Oktober: I'll Drink To That!",'' [http://www.popsci.com/rachel-durfee/article/2008-10/its-oktober-ill-drink?page= Popular Science, (October 21st, 2008)]<br />
* '''Rice''':''"University researchers developing cancer-fighting beer",'' [http://www.computerworld.com/action/article.do?command=printArticleBasic&taxonomyName=Development&articleId=9117656&taxonomyId=11 Computerworld, (October 21st, 2008)]<br />
* '''Rice''':''"Free Beer in an Unfree World",'' [http://reason.com/blog/show/129611.html Reason.com (October 22nd, 2008)]<br />
* '''Rice''':''"Researchers Developing Cancer-Fighting Beer",'' [http://science.slashdot.org/article.pl?sid=08/10/22/2230223&from=rss Slashdot (October 22nd, 2008)]<br />
* '''Rice''':''"Cold, Frosty and Healthy ...",'' [http://www.foxnews.com/story/0,2933,443798,00.html Fox News (October 24th, 2008)]<br />
* '''Rice''':''"Snart kan öl bekämpa cancer",'' [http://www.metro.se/se/article/2008/10/24/15/4736-68/index.xml Metro Teknik (October 24th, 2008)]<br />
* '''Rice''':''"'Fancy a swift twother?'",'' [http://www.thepublican.com/story.asp?sectioncode=7&storycode=61591&c=1 The Publican (October 24th, 2008)]<br />
* '''Rice''':''"Breakthroughs, tips and trends: Beer therapy",'' [http://www.timesonline.co.uk/tol/life_and_style/health/article5007974.ece Times of London (October 25th, 2008)]<br />
* '''Rice''':''"BioBeer Fights Cancer And Gets You Drunk",'' [http://www.javno.com/en/world/clanak.php?id=196507 Javno (Croatia) (October 25th, 2008)]<br />
* '''Rice''':''"A beer that battles cancer?",'' [http://www.odt.co.nz/lifestyle/health/28866/a-beer-battles-cancer Otago Daily Times (New Zealand) (October 25th, 2008)]<br />
* '''Rice''':''"Geek Week: Geek-o-naut returns home, researchers go for the foam",'' [http://weblog.infoworld.com/robertxcringely/archives/2008/10/geek_week_geeko.html InfoWorld (October 27th, 2008)]<br />
* '''Rice''':''"Rice University Students Aim For Creating A Beer Healthier Than Red Wine",'' [http://www.allheadlinenews.com/articles/7012806080 All Headline News (October 27th, 2008)]<br />
* '''Rice''':''"University researchers developing cancer-fighting beer",'' [http://www.techworld.com.au/article/264626/university_researchers_developing_cancer-fighting_beer TechWorld Australia (This Computerworld story also appears in CIO Magazine, Australia) (October 28th, 2008)]<br />
* '''Rice''':''"Beer Drinkers Rejoice: Cancer-Fighting Beer in Development",'' [http://www.nationalledger.com/artman/publish/article_272623475.shtml The National Ledger (October 28th, 2008)]<br />
* '''Rice''':''"Anti-cancer beer under development",'' [http://www.cosmosmagazine.com/news/2278/anti-cancer-beer-under-development COSMOS magazine (October 29th, 2008)]<br />
* '''Rice''':''"Beer taps wine's health benefit",'' [http://www.signonsandiego.com/uniontrib/20081028/news_lz1c28lafee.html San Diego Union-Tribune (October 29th, 2008)]<br />
<br />
</div></div>Jkmhttp://2008.igem.org/IGEM_PublicityIGEM Publicity2008-11-11T22:10:28Z<p>Jkm: </p>
<hr />
<div><div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [[Press_Kit | iGEM Press Kit]])</div><br />
<br />
<div><br />
<span style="color:#1e90ff; font-size:135%">'''blogs </span><span style="color:#3cb371; font-size:135%">covering iGEM'''</span><br />
* [http://www.the-scientist.com/blog/browse/blogger/33/ <span style="color:red; font-size: 130%">'''The Scientist'''</span>] scroll down for several blog posts about iGEM and the Jamboree written by Alla Katsnelson.<br />
* [http://igemkuleuven.wordpress.com/ <span style="font-size:130%">'''KULeuven''']</span> blogs about their iGEM experience.<br />
* iGEM 2008 judge, [http://synthesis.typepad.com/synthesis/2008/11/igem-2008.html <span style="color:#e25500; font-size:130%">'''Rob Carlson''']</span>, shares his thoughts about iGEM and the successes of this year's teams.<br />
* [http://blog.ginkgobioworks.com/2008/11/09/team-slovenia-igem-grand-prize-winners-again/ <span style="color:#000; font-size:130%; font-weight:bold;">'''Ginkgo</span><span style="color:#77bc35; font-size:130%;">bioworks</span>'''], with several of its members serving as judges this year, blog about the iGEM 2008 Jamboree.<br />
* Eric Bland writes about iGEM on his [http://blogs.discovery.com/news_interior_design/2008/11/igem-2008.html<span style="color:#000; font-size:130%; font-weight:bold">Interior</span> <span style="color:#21528a; font-size:140%">Design</span>] blog, as part of '''Discovery Channel News'''.<br />
</div><br />
<br />
<br />
<div><br />
====<font size=4><font color=dodgerblue>'''news articles</font><font color=mediumseagreen> about iGEM'''</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 />
*'''MIT+Caltech''': ''Engineering Edible Bacteria'', [http://www.technologyreview.com/printer_friendly_article.aspx?id=21654 MIT Tech Review]<br />
*[http://radar.oreilly.com/archives/2007/12/stories-we-want-1.html '''O'Reilly Radar: Looking Forward to iGEM in 2008''']<br />
*[http://www.sciencefriday.com/program/archives/200802291 '''Science Friday: Anticipating Synthetic Biology] ([http://podcastdownload.npr.org/anon.npr-podcasts/podcast/510221/87816117/npr_87816117.mp3 mp3 here)]<br />
* '''TU Delft''': ''"Studenten pimpen bacteriën" ("Students pimp bacteria")'', [http://www.tudelft.nl/live/pagina.jsp?id=23acdd35-ef09-4bfc-bee6-19e0f974913f&lang=nl TU Delft news (June 25 2008)]<br />
* '''TU Delft''': ''"Synthetische biologie: Open source biologie" ("Synthetic biology: Open source biology")'', [http://www.biw.kuleuven.be/persberichten/fiches/c2wLS-080712.pdf C2W Life Sciences (July 12 2008)]<br />
* '''TU Delft''': ''"Creatief met genetische lego - studenten bouwen thermometerbacteriën in internationale competitie" ("Being creative with genetic lego - Students build thermometer bacteria in an international competition")'', NRC newspaper (August 21 2008)<br />
* '''TU Delft''': "Studenten maken een thermometerbacterie" ("Students engineer a thermometer bacterium"), [http://www.delta.tudelft.nl/nl/archief/artikel/studenten-maken-een-thermometerbacterie/18466 ''TU Delta (September 25 2008)''] <br />
<br />
* '''KULeuven''': ''"Synthetische biologie: Open source biologie" ("Synthetic biology: Open source biology")'', [https://static.igem.org/mediawiki/2008/a/ac/22-23_memo7_synthetischebiologie.pdf C<sub>2</sub>W Life Sciences (July 12 2008) & MeMo (September 11 2008)] (MeMo is a magazine of the [http://www.kvcv.be/ Koninklijke Vlaamse Chemische Vereniging])<br />
* '''KULeuven''': ''"Studenten K.U.Leuven met ontwerp intelligente bacterie Dr. Coli in MIT-wedstrijd synthetische biologie" ("Students of K.U.Leuven with design of intelligent bacterium Dr. Coli in MIT-competition synthetic biology")'', [http://dagkrant.kuleuven.be/?q=node/5098 Dagkrant K.U.Leuven (September 1 2008)]<br />
* '''Rice''':''"Better beer: college team creating anti-cancer brew",'' [http://www.media.rice.edu/media/NewsBot.asp?MODE=VIEW&ID=11642 Rice University News and Media (October 17th, 2008)]<br />
* '''Rice''':''"'BioBeer' with wine's cancer fighting qualities",'' [http://www.dailyindia.com/show/277686.php Daily India (October 17th, 2008)]<br />
* '''Rice''':''"College students making anti-cancer beer,'' [http://www.upi.com/Health_News/2008/10/17/College_students_making_anti-cancer_beer/UPI-33191224299305/ United Press International (October 17th, 2008)]<br />
* '''Rice''':''"Drink beer to avoid cancer…",'' [http://blogs.zdnet.com/emergingtech/?p=1067 ZDNet (October 17th, 2008)]<br />
* '''Rice''':''"Coming soon, ”BioBeer” with wine’’s cancer fighting qualities",'' [http://www.thaindian.com/newsportal/india-news/coming-soon-biobeer-with-wines-cancer-fighting-qualities_100108232.html Thaindian.com/ANI wire (October 17th, 2008)]<br />
* '''Rice''':''"College Team Create Anticancer 'BioBeer' For Entry In Synthetic Biology's IGEM Contest",'' [http://www.medicalnewstoday.com/articles/125887.php Medical News Today (October 17th, 2008)]<br />
* '''Rice''':''"Rice Biology Students: Making Beer Better",'' [http://blogs.houstonpress.com/hairballs/2008/10/biobeer_genetic_resveratrol.php Houston Press (October 17th, 2008)]<br />
* '''Rice''':''"Can drinking beer help cure cancer?",'' [http://mobile.itwire.com/content/view/21268/1066/ IT Wire (October 17th, 2008)]<br />
* '''Rice''':''"Creating anticancer beer",'' [http://current.com/items/89430237_creating_anticancer_beer Current.com (October 20th, 2008)]<br />
* '''Rice''':''"It's Oktober: I'll Drink To That!",'' [http://www.popsci.com/rachel-durfee/article/2008-10/its-oktober-ill-drink?page= Popular Science, (October 21st, 2008)]<br />
* '''Rice''':''"University researchers developing cancer-fighting beer",'' [http://www.computerworld.com/action/article.do?command=printArticleBasic&taxonomyName=Development&articleId=9117656&taxonomyId=11 Computerworld, (October 21st, 2008)]<br />
* '''Rice''':''"Free Beer in an Unfree World",'' [http://reason.com/blog/show/129611.html Reason.com (October 22nd, 2008)]<br />
* '''Rice''':''"Researchers Developing Cancer-Fighting Beer",'' [http://science.slashdot.org/article.pl?sid=08/10/22/2230223&from=rss Slashdot (October 22nd, 2008)]<br />
* '''Rice''':''"Cold, Frosty and Healthy ...",'' [http://www.foxnews.com/story/0,2933,443798,00.html Fox News (October 24th, 2008)]<br />
* '''Rice''':''"Snart kan öl bekämpa cancer",'' [http://www.metro.se/se/article/2008/10/24/15/4736-68/index.xml Metro Teknik (October 24th, 2008)]<br />
* '''Rice''':''"'Fancy a swift twother?'",'' [http://www.thepublican.com/story.asp?sectioncode=7&storycode=61591&c=1 The Publican (October 24th, 2008)]<br />
* '''Rice''':''"Breakthroughs, tips and trends: Beer therapy",'' [http://www.timesonline.co.uk/tol/life_and_style/health/article5007974.ece Times of London (October 25th, 2008)]<br />
* '''Rice''':''"BioBeer Fights Cancer And Gets You Drunk",'' [http://www.javno.com/en/world/clanak.php?id=196507 Javno (Croatia) (October 25th, 2008)]<br />
* '''Rice''':''"A beer that battles cancer?",'' [http://www.odt.co.nz/lifestyle/health/28866/a-beer-battles-cancer Otago Daily Times (New Zealand) (October 25th, 2008)]<br />
* '''Rice''':''"Geek Week: Geek-o-naut returns home, researchers go for the foam",'' [http://weblog.infoworld.com/robertxcringely/archives/2008/10/geek_week_geeko.html InfoWorld (October 27th, 2008)]<br />
* '''Rice''':''"Rice University Students Aim For Creating A Beer Healthier Than Red Wine",'' [http://www.allheadlinenews.com/articles/7012806080 All Headline News (October 27th, 2008)]<br />
* '''Rice''':''"University researchers developing cancer-fighting beer",'' [http://www.techworld.com.au/article/264626/university_researchers_developing_cancer-fighting_beer TechWorld Australia (This Computerworld story also appears in CIO Magazine, Australia) (October 28th, 2008)]<br />
* '''Rice''':''"Beer Drinkers Rejoice: Cancer-Fighting Beer in Development",'' [http://www.nationalledger.com/artman/publish/article_272623475.shtml The National Ledger (October 28th, 2008)]<br />
* '''Rice''':''"Anti-cancer beer under development",'' [http://www.cosmosmagazine.com/news/2278/anti-cancer-beer-under-development COSMOS magazine (October 29th, 2008)]<br />
* '''Rice''':''"Beer taps wine's health benefit",'' [http://www.signonsandiego.com/uniontrib/20081028/news_lz1c28lafee.html San Diego Union-Tribune (October 29th, 2008)]<br />
<br />
</div></div>Jkmhttp://2008.igem.org/Team:Caltech/Protocols/Coculture_Inhibition_AssayTeam:Caltech/Protocols/Coculture Inhibition Assay2008-10-30T01:06:21Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Coculture Inhibition Assay</font></div><br />
__NOTOC__<br />
<br><br />
<br />
===Strains===<br />
* The engineered strain was JI377 transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K137076 K137076] (spxB) in pSB1A2 (AmpR). <br />
* The target strain was JI377 transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 B0015] (a transcriptional terminator) in pSB1AK3 (AmpR KanR). <br />
* The negative control was JI377 transformed with a modified pUC18 vector (AmpR) containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K137017 galactose oxidase]. (Kindly provided by Professor Arnold at Caltech.) <br />
<br />
===Protocol===<br />
# Grow overnight cultures of each strain in LB + Amp.<br />
# In the morning, back dilute cultures 1:100 into SOC + IPTG + Amp and grow to an OD600 of ~0.8. <br />
# To begin the assay, inoculate the target strain into 2.5 ml cultures of the engineered or control strains in amounts of (A) 1:1,000 (B) 1:10,000 and (C) 1:100,000. <br />
# Immediately serially dilute aliquots of the cocultures and plate to single colonies on LB+Kan plates for CFU counting. <br />
#*Co-culture "A" should be plated at dilutions of 1:100, 1:1,000, and 1:10,000. <br />
#*Co-culture "B" should be plated at dilutions of 1:1,000 1:10,000 and 1:100,000. <br />
#*Co-culture "C" should be plated at dilutions of 1:10,000 1:100,000 and 1:1,000,000.<br />
# Induce the co-cultures to produce hydrogen peroxide by bringing them to 10 nM AHL.<br />
# Incubate co-cultures for 6hrs and then plate to single colonies as before. <br />
# After incubation at 37C, count the CFU of each plate.<br />
<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/Protocols/TiteringTeam:Caltech/Protocols/Titering2008-10-30T01:05:51Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Titering Bacteriophage</font></div><br />
__NOTOC__<br />
<br><br />
#Grow fresh overnight cultures of the bacteria of interest (i.e. LE392, D1210, etc.) [[Image:Phage_titer.jpg|right|frame|An example plate. Dark spots are cleared zones in a lawn of bacteria.]]<br />
#Incubate the cultures until they reach an OD600 of roughly 0.1 <br />
#* More specifically, when the culture is swirled, cloudiness is observed<br />
#Do NOT titer with bacterial cultures that have just been diluted from a culture past OD600 of 0.8 (complications will occur)<br />
#Place agar/agarose plates in the 42 C oven and ready the 48 C water bath<br />
#Prepare fresh 10 mM MgSO4 solution from the 1 M MgSO4 stock solution (100 ul MgSO4 in 10 mL sterile water works conveniently)<br />
#In 1.5 mL eppendorf tubes, add 1 mL of the 10 mM MgSO4 solution.<br />
#Prepare phage dilutions by adding appropriate amount of phage into MgSO4 solution (i.e. 1000-fold dilution is 1 ul phage in 1 ml MgSO4)<br />
#After addition of phage to solution, invert tube at least 12 times for sufficient mixing<br />
#For further dilutions, can simply dilute made phage dilutions (i.e. 1,000,000-fold can be obtained from taking 1 ul 1000-fold diluted phage and adding to another 1 mL of 10 mM MgSO4<br />
#Aliquot 0.1 mL cell solution (i.e. LE392, D1210, etc.) into new 1.5 mL eppendorf tubes<br />
#To begin infection, add appropriate amount of diluted phage to cell solution (10 ul usually works well for starters) and start the timer<br />
#During this 30 minute infection period, label the plates and place back in the 42 C oven<br />
#~3 minutes before the end of infection, can take 3 mL aliquots of the top medium in 13 mL falcon tubes or 5 mL round-bottom tubes and microwave till complete liquification has occurred.<br />
#*Note that top agar is the same composition as is used to pour the plates, only with half the agar (so ~7g/L agar rather than 15 g/L).<br />
#Right before the end of infection, take out the plates and place on the bench with the cover on top. Have a flame close-by to prevent contamination. Also, set the pippette to the right volume of infected cell solution and have sterile pipette non-filter tips ready <br />
#Very quickly with pipette in hand, pippette out infected cell solution, add to top medium, invert the tube at least 2 times, and pour the mixed contents onto the corresponding plate. <br />
#Rock the plate gently to allow the top medium to uniformly cover the plate and use the sterile tips to poke any bubbles.<br />
#Let the plate cool on the bench for at least 5 minutes (10 minutes is usually enough) with the cover completely on the plate.<br />
#Place the plate in the 37 C incubator.<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/ProtocolsTeam:Caltech/Protocols2008-10-30T01:04:32Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Protocols</font></div><br />
<br><br />
__NOTOC__<br />
*Oxidative Burst<br />
**[[Team:Caltech/Protocols/H2O2 Production Assay|H<sub>2</sub>O<sub>2</sub> production assay]]<br />
**[[Team:Caltech/Protocols/MIC Assay|Minimum inhibitory concentration (MIC) assay]]<br />
**[[Team:Caltech/Protocols/Coculture Inhibition Assay|Coculture inhibition assay]]<br />
**[[Team:Caltech/Protocols/Measuring H2O2|Measuring H<sub>2</sub>O<sub>2</sub>]]<br />
*Phage Pathogen Defense<br />
**[[Team:Caltech/Protocols/rcsA Lysogen Induction|rcsA lysogen induction]]<br />
**[[Team:Caltech/Protocols/Titering|Titering]]<br />
*Lactose Intolerance<br />
**[[Team:Caltech/Protocols/LacZ Assay|LacZ assay]]<br />
*Vitamin Production<br />
**[[Team:Caltech/Protocols/Folate_assay|Folate microbiological assay protocol]] <br />
**[[Team:Caltech/Protocols/PABA_HPLC_assay| para-Aminobenzoic Acid (pABA) HPLC protocol]]<br />
*Asynchronous Random State Generator<br />
**[[Team:Caltech/Protocols/Flow cytometry|Flow cytometry]]<br />
***[[Team:Caltech/Protocols/BioAssay_Buffer|Bioassay Buffer]]<br />
**[[Team:Caltech/Protocols/FimE|FimE inversion assay]]<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/Protocols/Coculture_Inhibition_AssayTeam:Caltech/Protocols/Coculture Inhibition Assay2008-10-30T00:27:03Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Coculture Inhibition Assay</font></div><br />
__NOTOC__<br />
<br><br />
<br />
===Strains===<br />
* The engineered strain was JI377 transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K137076 K137076] (spxB) in pSB1A2 (AmpR). <br />
* The target strain was JI377 transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 B0015] (a transcriptional terminator) in pSB1AK3 (AmpR KanR). <br />
* The negative control was JI377 transformed with a modified pUC18 vector (AmpR) containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K137017 galactose oxidase]. (Kindly provided by Professor Arnold at Caltech.) <br />
<br />
===Protocol===<br />
# Grow overnight cultures of each strain in LB + Amp.<br />
# In the morning, back dilute cultures 1:100 into SOC + IPTG + Amp and grow to an OD600 of ~0.8. <br />
# To begin the assay, inoculate the target strain into 2.5 ml cultures of the engineered or control strains in amounts of (A) 1:1,000 (B) 1:10,000 and (C) 1:100,000. <br />
# Immediately serially dilute cocultures and plate to single colonies on LB+Kan plates for CFU counting. <br />
#*Co-culture "A" should be plated at dilutions of 1:100, 1:1,000, and 1:10,000. <br />
#*Co-culture "B" should be plated at dilutions of 1:1,000 1:10,000 and 1:100,000. <br />
#*Co-culture "C" should be plated at dilutions of 1:10,000 1:100,000 and 1:1,000,000.<br />
# Induce the co-cultures to produce hydrogen peroxide by bringing them to 10 nM AHL.<br />
# Incubate co-cultures for 6hrs and then plate to single colonies as before. <br />
# After incubation at 37C, count the CFU of each plate.<br />
<br />
}}</div>Jkmhttp://2008.igem.org/File:Digestive_system_diagram_en.svg.pngFile:Digestive system diagram en.svg.png2008-10-29T23:54:29Z<p>Jkm: uploaded a new version of "Image:Digestive system diagram en.svg.png"</p>
<hr />
<div>From [http://en.wikipedia.org/wiki/Image:Digestive_system_diagram_en.svg]</div>Jkmhttp://2008.igem.org/File:Packshot_mutaflor.jpgFile:Packshot mutaflor.jpg2008-10-29T23:52:04Z<p>Jkm: uploaded a new version of "Image:Packshot mutaflor.jpg"</p>
<hr />
<div>[http://www.ardeypharm.de/pdfs/presse/packshot_mutaflor.jpg], Copyright Ardeypharm GmbH.</div>Jkmhttp://2008.igem.org/File:Digestive_system_diagram_en.svg.pngFile:Digestive system diagram en.svg.png2008-10-29T23:51:44Z<p>Jkm: uploaded a new version of "Image:Digestive system diagram en.svg.png"</p>
<hr />
<div>From [http://en.wikipedia.org/wiki/Image:Digestive_system_diagram_en.svg]</div>Jkmhttp://2008.igem.org/File:Gut_flora_color.pngFile:Gut flora color.png2008-10-29T23:51:29Z<p>Jkm: uploaded a new version of "Image:Gut flora color.png"</p>
<hr />
<div></div>Jkmhttp://2008.igem.org/File:Gut_flora_color.pngFile:Gut flora color.png2008-10-29T23:50:18Z<p>Jkm: uploaded a new version of "Image:Gut flora color.png"</p>
<hr />
<div></div>Jkmhttp://2008.igem.org/Team:Caltech/ProjectTeam:Caltech/Project2008-10-29T20:49:15Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
__NOTOC__<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Subprojects</font></div><br />
<div style="font-size:10pt;"><br />
<br />
<font face="verdana" style="color:#BB4400">Note: Click on the subproject title or picture for a detailed description of the subproject</font></div><br />
<br><br />
<br />
==[[Team:Caltech/Project/Oxidative Burst|<font face="verdana" style="color:#BB4400">Oxidative Burst</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}{{navimg|xsize=200|ysize=272|image=neutrophil-shigella.jpg|link=Team:Caltech/Project/Oxidative_Burst}}<br />
{{!}}<br />
Specialized white blood cells called neutrophils defend us from illness by killing bacteria with a potent concoction of degradative enzymes and oxidizing agents, including hydrogen peroxide. However, pathogens of the human large intestine are able to cause serious illness while being sheltered from neutrophils. We engineered a strain of ''Escherichia coli'' that is able to mimic a neutrophil by producing cytotoxic amounts of hydrogen peroxide in a controlled, inducible manner. Our engineered ''E. coli'' use the transcriptional activator LuxR to detect the presence of acyl-homoserine lactones, quorum sensing signaling molecules secreted by invading pathogens. LuxR activates production of the pyruvate oxidase of ''Streptococcus pneumoniae'', which produces large amounts of hydrogen peroxide by oxidizing pyruvate. The engineered ''E. coli'' is capable of killing certain strains of antibiotic resistant ''E. coli'' within six hours. When translated into a probiotic strain such as Nissle 1917, this system has the potential to be an effective means of combating enteric pathogens.<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Phage Pathogen Defense|<font face="verdana" style="color:#BB4400">Phage Pathogen Defense</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}<br />
Bacterial food poisoning is a prevalent problem around the world. In the year 2006, the United States had more than 325,000 hospitalizations from food borne illnesses. This subproject focuses on the prevention of food poisioning by the release of pathogen-specific bacteriophage. A model system for ‘manufacturing’ phage was built around ''Escherichia coli'' bacteriophage λ lysogens. We began with a strain of ''E. coli'' which does not express the receptor, LamB, necessary for λ infection. Next we expressed the receptor in the cell, allowing infection and lysogeny. Then the receptor was removed, again rendering the cell non-susceptible. Finally, a plasmid was constructed to control the induction of λ by regulating the expression of ''rcsA'' and ''cI''. When fully implemented, this system could be used to treat food poisoning due to pathogenic ''E. coli''. The system can also be modified to target other pathogenic species, such as ''Salmonella'', by replacing λ phage with other temperate bacteriophages and using similar methods to incorporate the non-native phage into ''E. coli''. <br />
{{!}}{{navimg|xsize=220|ysize=258|image=Phage.jpg|link=Team:Caltech/Project/Phage_Pathogen_Defense}}<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Lactose intolerance|<font face="verdana" style="color:#BB4400">Lactose Intolerance</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}{{navimg|xsize=250|ysize=187|image=Milk.jpg|link=Team:Caltech/Project/Lactose_intolerance}}<br />
{{!}}<br />
Approximately 75% of adults worldwide suffer from lactose intolerance, the inability to metabolize lactose in the small intestine. We propose to treat lactose intolerance by engineering a strain of ''Escherichia coli'' that can reside in the large intestine. The engineered strain will sense lactose and subsequently release ß-galactosidase to convert lactose into glucose and galactose, both of which can be reabsorbed by the host. To treat lactose intolerance, our engineered bacterial strain will contain two plasmids: one with constitutive expression of a mutant lactose permease and ß-galactosidase, and the second with lactose-inducible expression of the λ phage lysis cassette. The mutant lactose permease allows the cells to import lactose under all conditions. When the cells uptake enough lactose, the second plasmid will induce cell lysis through activation of the λ phage lysis cassette, resulting in cell lysis and release of ß-galactosidase into the large intestine. Data covering the construction and characterization of these plasmid constructs is [[Team:Caltech/Project/Lactose_intolerance|<font style="color:#BB4400">discussed</font>]].<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Vitamins|<font face="verdana" style="color:#BB4400">Vitamin Production</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}<br />
Folate, a term which encompasses the various forms of the vitamin B9, is an essential vitamin involved in everyday cell functions such as DNA replication. Unable to naturally produce folate, humans must obtain it from vegetables or folate-supplements. In regions with little or no access to these foods, folate deficiencies can cause serious birth defects. One possible solution to alleviate the effects of folate deficiency is to engineer a strain of gut microbes to produce bioavailable folate directly in the colon. We tested a total of four heterologous genes, two from the folate biosynthesis gene cluster and two from the paraaminobenzoic acid (pABA) synthesis pathway. Using standardized genetic sequences, folate biosynthesis genes extracted from the ''Lactoccocus lactis'' genome were cloned into Biobricks plasmids, transformed into ''Escherichia coli'' and overexpressed. We measured the effects of overexpression<br />
in terms of total folate and paraaminobenzoic acid levels. PABA, an intermediate in folate synthesis, was detected using [[Team:Caltech/Protocols/PABA_HPLC_assay|<font style="color:#BB4400">high performance liquid chromatography</font>]] (HPLC). Folate detection was achieved via a [[Team:Caltech/Protocols/Folate_assay|<font style="color:#BB4400">microbiological assay</font>]]. A measurable increase in folate production in ''E. coli'' provides proof-of-concept for both the feasibility of engineering overproduction of folate in ''E. coli'', as well as using standardized genetic components to do so.<br />
{{!}}{{navimg|xsize=193|ysize=288|image=Folate_foods.jpg|link=Team:Caltech/Project/Vitamins}}<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Population Variation|<font face="verdana" style="color:#BB4400">Population Variation</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}{{navimg|xsize=220|ysize=231|image=Differentiation.jpg|link=Team:Caltech/Project/Population_Variation}}<br />
{{!}}<br />
As more complicated and interconnected biological circuits are built, there is an increasing need for the simple integration of multiple functions into a single bacterial cell line. However, some of these functions may be incompatible or may kill the cell, such that each cell can only express a single function at any time or must be regenerated if the functionality is fatal. We aim to combine multiple mutually exclusive and potentially fatal functions into a single bacterial cell line that, as a population, exhibits the entire set of functions. <br />
<br />
In particular, we want only one of the four subprojects described above to be turned on in any given cell, or else the cell may be overburdened by our constructs. At the same time, we want our bacterial population to have the capability to exhibit all four functions. In addition, three of the four subprojects result in the death of the host cell through different methods of self-induced lysis. Therefore, we need a system that is able to combine all subprojects into one coherent system and that allows for self-renewal of the population.<br />
<br />
We propose a system in which a single bacterial strain encodes all four functions in its genome and can access each function independently through probabalistic molecular events. The engineered bacterial cells start in an undifferentiated state and randomly differentiate into one of three possible final states. To build this system, we designed two genetic devices. One relies on DNA polymerase slippage upon replication of a long stretch of short nucleotide repeats, a phenomenon termed slipped-strand mispairing (SSM). The other device uses the recombinase protein FimE to flip DNA segments. The two devices can form a system that permits the novel introduction of random multi-state differentiation into bacterial cells. Our experimental results demonstrate that short nucleotide repeats can be used as a stochastic switch and that FimE activity depends on the length of the segment being flipped. <br />
{{!}}}<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/Project/Lactose_intoleranceTeam:Caltech/Project/Lactose intolerance2008-10-29T01:42:45Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Curing Lactose Intolerance</font></div><br />
<br />
==Introduction==<br />
The gut flora of our digestive tract contains microorganisms that perform various useful functions for their hosts. Examples of such functions include growth inhibition of harmful microorganisms<sup>1</sup>, defense against the causes of many forms of Inflammatory Bowel Disease<sup>2</sup>, and the fermentation of carbohydrates and other molecules the human body cannot normally digest. Some bacterial strains, including the E. coli strain ‘Nissle 1917’, can persist in the gut of mice for months. Engineering these bacteria provide a new platform to treat various human diseases. <br>Lactose intolerance is characterized by the inability to break down lactose in the small intestine. The undigested lactose instead passes to the large intestine, leading to two negative processes: osmotic imbalance and bacterial fermentation. High lactose levels raise the osmolarity of the colon, causing diarrhea. In addition, gut microbes metabolize the lactose into methane gas, causing abdominal pain. Both problems must be addressed in order to fully treat lactose intolerance. If we simply have our strain metabolize lactose, another strain will further ferment the byproducts, resulting in the same side effects. <br><br />
[[image:LysisCaltech.JPG|thumb|200px|left|In frame 1, Beta-Gal is being produced at a high constitutive level at all times. When lactose enters the colon in frame 2, the cell uptakes lactose via mutated LacY which in turn induces lysis genes lysing the cell, and releasing Beta-Gal in the colon cleaving lactose. The host will then uptake glucose and galactose.]]<br />
<br>Instead, we have our engineered two plasmids which allow the host to uptake lactose, clearing the sugar from the colon. To do this, we have engineered the ‘Nissle 1917’ strain to release ß-galactosidase, an enzyme that cleaves lactose into glucose and galactose. Since protein secretion is difficult in E. coli, our cells were engineered to lyse in order to release ß-galactosidase. However, lysis must occur only when lactose is present in the gut. If lysis were to occur when lactose was absent, this would kill all our engineered cells at any given moment. The cells also express a lactose permease, allowing the strain to sense lactose in all conditions. <br><br><br><Br><Br><Br><br />
<br />
<br />
==System Design==<br />
===Lactose Regulator===<br />
[[image:Lactose_regulator.JPG|thumb|200px|left|Figure 1. A mutated LacY allows the uptake of lactose while LacZ produces large amounts of ß-galactosidase. The plasmid contains a strong constitutive promoter regulating both genes and a ColE1 replication origin.]]<br />
The first plasmid consists of a synthetic lactose operon under strong constitutive expression (Fig. 1). Our synthetic lactose operon encodes the ß-galactosidase LacZ and a lactose permease LacY. LacY is a membrane protein that actively transports lactose into the cell. Since most membrane proteins are toxic when overexpressed, we optimized our system to express appropriate levels of LacY without killing the cell. In the end, we want to express as much ß-galactosidase as we can to cleave as much lactose present in the large intestine. <br>The human gut is an unpredictable environment, and we wish our engineered cells to behave reliably despite this variability. E. coli are unable to uptake lactose in the presence of glucose, a phenomenon known as carbon catabolite repression. Catabolite repression is mediated by Enzyme IIA Glucose (IIAGluc), which inhibits the uptake of lactose in the presence of glucose by binding to LacY<sup>3</sup>. Previous research has identified various LacY mutations that prevent this inhibition and achieve increased uptake of lactose in the presence of glucose<sup>4</sup>.<br />
<br />
{{clear}}<br />
<br />
===Lactose-Induced Lysis===<br />
[[image:Lactose-inducible_lysis.JPG|thumb|200px|right|Figure 2. The lysis cassette plasmid acts as a lactose sensor. Intracellular lactose accumulation induces overexpression of the lysis cassette. Lactose inhibits binding of the LacI repressor to the promoter and P1 high copy origin of replication. A mutated holin gene in the lysis cassette allows faster lysis times.]]<br />
The second plasmid contains lactose inducible expression of λ phage lysis genes under a lactose inducible promoter (Fig. 2). To release ß-galactosidase, the cells will lyse when enough lactose is present in the cell to induce the expression of the lysis genes. It is important that the lactose-inducible promoter is tightly regulated since leaky expression will cause spontaneous lysis. To accomplish tight expression, specific lac promoters will keep our system from lysing<sup>5</sup>. In addition, the plasmid copy number remains low copy until induced with lactose, when the plasmid copy number increases to high copy. <br>Using wild type λ phage lysis genes, lysis occurs 40-45 minutes after induction by lactose. Decreasing the lag time will reduce the extent of lactose fermentation and therefore produce fewer deleterious effects. Previous research has uncovered mutations that shorten the lysis time to approximately 10-15 minutes<sup>6</sup>, and these mutations will be incorporated into our final construct.<br />
<br />
{{clear}}<br />
<br />
==Results==<br />
===Lactose Regulator===<br />
[[IMAGE:Promoter_strength.JPG|thumb|300px|left|Figure 3. Varying the expression levels of LacY with different promoters and ribosome binding sites. Cells died at our two highest expression levels, but survived on the other four. The construct with the weakest RBS was selected since we could express LacZ in the same operon with a strong promoter. This combination allows our cell to express high levels of LacZ and non-toxic levels of LacY.]]<br />
In the first plasmid, it was discovered that LacY is toxic when overexpressed. To determine safe expression levels, constructs were built with varying levels of LacY expression. Six plasmids were built from combinations of three different promoters and two different ribosome binding sites (Fig. 3). The cells appeared to have died when expressing the strongest and medium strength promoters along with the strongest ribosome binding site. Our information was based off transformants, and the plates with no transformants were classified as dead cells due to high LacY expression. Based on these results, we decided to express our synthetic lac operon by combining the strongest constitutive promoter with the weakest RBS for LacY and the strongest RBS for LacZ, preventing LacY toxicity while expressing LacZ in large quantities. <br>As mentioned earlier, our cells had to inhibit carbon catabolite repression to ensure uptake of lactose under any condition. Various mutations, including the insertion of two histidyl residues between amino acids S194 and A195 (ref 4) in the LacY gene, prevent the inhibition by IIAGluc. These insertions have been made and will be tested in future studies. <br />
<br />
{{clear}}<br />
<br />
[[image:Figure_1_beta_assay.JPG|thumb|300px|right|Figure 4. ß-galactosidase activity of plasmid #1, with and without the strong constitutive promoter. Error bars on the data with a promoter show the standard deviation of two measurements.]]The ß-galactosidase assay was performed on the synthetic network containing the lactose operon (Fig. 4). The first assay was performed on a strain containing the plasmid lacking the strong constitutive promoter. This assay was performed to simulate the eventual system regulation. We saw significant levels of ß-galactosidase, a surprising result since the plasmid lacked a promoter. These levels are likely due to spurious transcription amplified by our strong ribosome binding site and high copy plasmid. We then repeated the assay with the strong constitutive promoter to observe the dynamic range of LacZ expression in this system. Under these conditions, the system shows a 8-fold dynamic range, from 2000 MU without a promoter to 16000 MU with a promoter. This plasmid is still being characterized in combination with the lysis plasmid, and results will be available at the Jamboree.<br />
{{clear}}<br />
<br />
===Lactose-Induced Lysis===<br />
Our second plasmid is still being constructed and characterized. Further results will be available at the Jamboree. <br />
<br />
==Discussion==<br />
Our final system was not completely finished; however, future construction will be minimal. The synthetic lac operon plasmid was completely constructed with the insertion of two histidyl residues in their appropriate locations. Our lactose-induced lysis plasmid was one cloning step away from being completed. The promoter and flanking terminator were cloned in parallel with the lysis cassette and a terminator. The last cloning step would be to ligate the two together. In addition, the two separate point mutations have not been made, and the final step in our cloning would be to add those mutations.<br />
<br />
After completing the construction phase, we would characterize our system. Characterization will be completed on a construct containing our lactose inducible promoters and GFP. This would show our promoters can maintain tight expression of GFP from uninduced to induced. We would combine this construct with our first plasmid containing the mutated LacY, to show we can achieve induction by lactose even in the presence of glucose. To show the effect our lysis plasmid, it would be necessary to achieve lysis with lactose. In addition, we want to show that we can achieve lysis with lactose in the presence of glucose. Finally, the final assay we want to perform on our system would show our cells able to uptake lactose in the presence of glucose, and lyse soon after. Once the cells lyse, the cell lysis should contain ß-galactosidase at levels close to what we received from our ß-galactosidase assay on plasmid 1. Once characterization is completed, our construct would be moved to the ‘Nissle 1917’ strain where further characterization and modifications would take place.<br />
<br />
<br />
==Methods==<br />
Methods can be found [[Team:Caltech/Protocols|<font style="color:#BB4400">here</font>]]<br />
<br />
==Parts==<br />
{{{!}} border="1"<br />
! Registry Number !! Plasmid !! Part !! Status <br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_K137002 BBa_K137002]<br />
{{!}} pSB1A2 {{!}}{{!}} LacY {{!}}{{!}} Constructed <br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_K137126 BBa_K137126]<br />
{{!}} pSB1A2 {{!}}{{!}} Lysis Cassette {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S03970 BBa_S03970]<br />
{{!}} pSB1A2 {{!}}{{!}} B0031 + LacY {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S03971 BBa_S03971]<br />
{{!}} pSB1A2 {{!}}{{!}} B0033 + LacY {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S03973 BBa_S03973]<br />
{{!}} pSB1AK3 {{!}}{{!}} B0034-LacZ + B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04107 BBa_S04107]<br />
{{!}} pSB1AK3 {{!}}{{!}} B0031-LacY + B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04108 BBa_S04108]<br />
{{!}} pSB1AK3 {{!}}{{!}} B0033-LacY + B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04109 BBa_S04109]<br />
{{!}} J61002 {{!}}{{!}} J23113 + B0031-LacY-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04122 BBa_S04122]<br />
{{!}} J61002 {{!}}{{!}} J23106 + B0031-LacY-B0015 {{!}}{{!}} Unavailable<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04123 BBa_S04123]<br />
{{!}} J61002 {{!}}{{!}} J23100 + B0031-LacY-B0015 {{!}}{{!}} Unavailable<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04110 BBa_S04110]<br />
{{!}} J61002 {{!}}{{!}} J23113 + B0033-LacY-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04111 BBa_S04111]<br />
{{!}} J61002 {{!}}{{!}} J23106 + B0033-LacY-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04112 BBa_S04112]<br />
{{!}} J61002 {{!}}{{!}} J23100 + B0033-LacY-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04041 BBa_S04041]<br />
{{!}} pSB1A2 {{!}}{{!}} B0033 + LacY +H {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04022 BBa_S04022]<br />
{{!}} pSB1A2 {{!}}{{!}} B0033 + LacY +2H {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04113 BBa_S04113]<br />
{{!}} pSB1AK3 {{!}}{{!}} B0033-LacY +H + B0034-LacZ-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04054 BBa_S04054]<br />
{{!}} pSB1AK3 {{!}}{{!}} B0033-LacY +2H + B0034-LacZ-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04055 BBa_S04055]<br />
{{!}} J61002 {{!}}{{!}} Final synthetic LacYZ operon {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_K137125 BBa_K137125]<br />
{{!}} pSB1A2 {{!}}{{!}} LacI Repressed Promoter B4 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04114 BBa_S04114]<br />
{{!}} pSB2K3 {{!}}{{!}} Lysis + B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04105 BBa_S04105]<br />
{{!}} pSB2K3 {{!}}{{!}} B0034-LacI + B0015-B4 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04106 BBa_S04106]<br />
{{!}} pSB2K3 {{!}}{{!}} J23100 + B0034-LacI-B0015-B4 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_K137131 BBa_K137131]<br />
{{!}} pSB2K3 {{!}}{{!}} J23100-B0034-LacI-B0015-B4 + Lysis-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_K137132 BBa_K137132]<br />
{{!}} pSB2K3 {{!}}{{!}} J23100-B0034-LacI-B0015-B4 + B0034-GFP-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
{{!}}}<br />
<br />
<br />
==References==<br />
# Guarner F and Malagelada JR. 2003. '''Gut flora in health and disease.''' ''The Lancet'', Volume 361, Issue 9356, 8 February 2003, Pages 512-519. <br />
# Sears CL. 2005. '''A dynamic partnership: Celebrating our gut flora.''' ''Anaerobe'', Volume 11, Issue 5, Pages 247-251.<br />
# Deutscher, J., Francke, C. and Postma, P.W. 2006. '''How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bactertia.''' ''Microbial and Molecular Biology Reviews.'' 2006: 939-1031<br />
# Hoischen, C., Levin, J., Pitaknarongphorn, S., Reizer, J., and Saier , M. H. Jr. 1996. '''Involvement of the central loop of the lactose permease of Escherichia coli in its allosteris regulation by the glucose-specific enzyme IIA of the phosphoenolpyruvate-dependent phosphotransferase system.''' ''J. Bacteriol.'' 178: 6082-6086<br />
# Cox RS III, Surette MG, Elowitz MB. '''Programming gene expression with combinatorial promoters.''' ''Mol. Syst. Biol.'' 2007;3:145.<br />
# Gründling, A., M. D. Manson, and R. Young. 2001. '''Holins kill without warning.''' ''Proc. Natl. Acad. Sci.'' USA 98:9348-9352.<br />
<br />
<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/BiosafetyTeam:Caltech/Biosafety2008-10-29T01:40:04Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Biosafety Concerns</font></div><br />
<br><br />
__NOTOC__<br />
==General Concerns==<br />
There are risks in probiotic engineering that may prevent or delay our system from being implemented in humans. One common risk is increasing the chance of bacterial sepsis for those who are immune deficient. Other risk factors include premature infants, CVC (central venous catheter), cardiac vascular disease, and diarrhea<sup>1</sup>. We are also unsure of the effects that probiotics have on host metabolic activities. However, the unmodified Nissle 1917 strain has been shown to be safe for long term use<sup>2</sup>.<br />
<br />
The engineered probiotic will persist in the gut longer if antibiotics are used to suppress other gut flora<sup>3</sup>. However, such resistance may pass over into pathogenic strains and cause future problems for the host. The concern of introducing foreign genes into the gut is described below for each individual project.<br />
<br />
==Subproject Concerns==<br />
===Oxidative Burst===<br />
*'''Short Term:''' There are no safety concerns beyond that of typical ''E. coli'' lab strains. Considering that 260 mM H<sub>2</sub>O<sub>2</sub> is applied directly to the skin to disinfect cuts and scrapes, the 800 uM H<sub>2</sub>O<sub>2</sub> produced by the engineered strain should not be a health concern to anyone working in the lab.<br />
*'''Long Term:''' Production of hydrogen peroxide is not a normal occurrence in the large intestine, and its effects would need to be investigated before the engineered strain could be used to fight infection. Some concerns of hydrogen peroxide production in the intestine would be if:<br />
**it irritates the bowel<sup>4</sup>.<br />
**it kills off significant amounts of the native gut flora.<br />
**it significantly damages gut epithelial cells.<br />
**the ability to produce peroxide can be transmitted to other gut flora.<br />
<br />
<br />
===Phage Pathogen Defense===<br />
* Phage therapy is generally not harmful to the host due to phage specificity to bacterial hosts. However, there could be potentially several other complications associated with phage therapy:<br />
** Often times, there are pathogenic and benign strains of the same species of bacteria, and when targeting a species of pathogen, the associated benign strains could be destroyed as well.<br />
** Bacterial lysis leads to a release of endotoxins within the host, this could lead to a variety of side effects including fever and toxic shock[http://en.wikipedia.org/wiki/Herxheimer_reaction]<br />
** Phage lysogeny can often induce pathogenesis in otherwise harmless species by carrying virulence genes in the phage genome (''Vibrio cholerae'' is a classic example). We can avoid this problem by switching to phage variants which are only lysogenic in our engineered host.<br />
<br />
===Lactose Intolerance===<br />
* '''Synthetic LacY/Z Plasmid:''' There are no known safety concerns regarding this plasmid. The only foreign enzyme not present in humans that is expressed is β-Galactosidase, which simply acts as a lactase. Several lactase preparations are formally 'Generally Recognized as Safe' by the FDA[http://www.cfsan.fda.gov/~rdb/opa-g132.html] and we expect no difficulties in finding a suitable lactase.<br />
* '''Lysis Cassette Plasmid:''' We expect little effect of this plasmid, as there would be a strong selective pressure against lateral transmission. However, bacterial lysis can lead to undesirable side effects as described above.<br />
<br />
===Vitamin Production===<br />
* '''Overexpression of Folate in the Gut:''' While an excess of folate (Vitamin B9) in the colon would need further testing, the risk of toxicity from overdose is very low, since folate is water-soluble[http://en.wikipedia.org/wiki/Folate]. The tolerable upper intake level for folate is around 1 mg, and many cereals contain 100% of the recommended daily dose[http://www.cdc.gov/ncbddd/folicacid/cereals.htm]. <br />
* '''Folate biosynthesis plasmids:''' While these plasmids are constitutively high copy, these plasmids produce intermediates for folate, which again, has a very low risk of toxicity. <br />
* '''pABA biosynthesis plasmids''' para-aminobenzoic acid (pABA) is also an intermediate for folate that is commonly used in sunscreen to absorb UV radiation. There are no known serious toxic effects of pABA overdose, though there has also not been extensive testing[http://www.sciencelab.com/xMSDS-4_Aminobenzoic_acid-9922876]. <br />
<br />
===Population Variation===<br />
* The current constructs are regulatory in nature and present few safety concerns aside from those involved in the treatment subprojects. Minor concerns are discussed below:<br />
** In the simple FimE constructs, when the promoter starts in the configuration pointing upstream, DNA upstream of the constructs may be transcribed by the cell.<br />
** In the final design, the terminator that sits in the population variation generator may not be 100% efficient. Thus, the efficiency of the terminator should be tested before genes that are hazardous when co-expressed are placed into the system.<br />
** The engineered FimE system may interfere with natural fimbriae expression in Nissle 1917 or other gut flora. Since fimbriae are important for intestinal colonization, this could effect the intestinal composition.<br />
<br />
==References==<br />
# Boyle RJ, Robins-Browne RM, Tang ML. Probiotic use in clinical practice: what are the risks? Am J Clin Nutr 2006;83:1256-64.<br />
# Westendorf AM, Gunzer F, Deppenmeier S, Tapadar D, Hunger JK, Schmidt MA, Buer J, and Bruder D. '''Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules'''. ''FEMS Immunol Med Microbiol'' 2005 Mar 1; 43(3) 373-84.<br />
# Rao S, Hu S, McHugh L, Lueders K, Henry K, Zhao Q, Fekete RA, Kar S, Adhya S, and Hamer DH. '''Toward a live microbial microbicide for HIV: commensal bacteria secreting an HIV fusion inhibitor peptide'''. ''Proc Natl Acad Sci U S A'' 2005 Aug 23; 102(34) 11993-8.<br />
# Ackermann M, Stecher B, Freed NE, Songhet P, Hardt WD, and Doebeli M. '''Self-destructive cooperation mediated by phenotypic noise'''. ''Nature'' 2008 Aug 21; 454(7207) 987-90. <br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/ProjectTeam:Caltech/Project2008-10-29T01:34:20Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
__NOTOC__<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Subprojects</font></div><br />
<div style="font-size:10pt;"><br />
<br />
<font face="verdana" style="color:#BB4400">Note: Click on the subproject title or picture for a detailed description of the subproject</font></div><br />
<br><br />
<br />
==[[Team:Caltech/Project/Oxidative Burst|<font face="verdana" style="color:#BB4400">Oxidative Burst</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}{{navimg|xsize=200|ysize=272|image=neutrophil-shigella.jpg|link=Team:Caltech/Project/Oxidative_Burst}}<br />
{{!}}<br />
Specialized white blood cells called neutrophils defend us from illness by killing bacteria with a potent concoction of degradative enzymes and oxidizing agents, including hydrogen peroxide. However, pathogens of the human large intestine are able to cause serious illness while being sheltered from neutrophils. We engineered a strain of ''Escherichia coli'' that is able to mimic a neutrophil by producing cytotoxic amounts of hydrogen peroxide in a controlled, inducible manner. Our engineered ''E. coli'' use the transcriptional activator LuxR to detect the presence of acyl-homoserine lactones, quorum sensing signaling molecules secreted by invading pathogens. LuxR activates production of the pyruvate oxidase of ''Streptococcus pneumoniae'', which produces large amounts of hydrogen peroxide by oxidizing pyruvate. The engineered ''E. coli'' is capable of killing certain strains of antibiotic resistant ''E. coli'' within six hours. When translated into a probiotic strain such as Nissle 1917, this system has the potential to be an effective means of combating enteric pathogens.<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Phage Pathogen Defense|<font face="verdana" style="color:#BB4400">Phage Pathogen Defense</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}<br />
Another aspect of bacterial pathogen defense for our probiotic is to produce bacteriophages that would rapidly infect and wipe out pathogens. Our method uses the bacteriophage λ, a temperate phage which targets ''Escherichia coli''. λ infects ''E. coli'' through the LamB receptor, where absence of this receptor prevents λ infection. We have taken advantage of this property of bacteriophage λ to engineer an ''E. coli'' strain that is resistant to the phage, but harbors a phage in its genome and can specifically trigger phage release to destroy susceptible pathogenic ''E. coli''. This system is useful in itself, targeting pathogenic ''E. coli'', and as a model system for the introduction of non-natural phage into ''E. coli'' such as the ''Salmonella'' phage P22.<br />
{{!}}{{navimg|xsize=220|ysize=258|image=Phage.jpg|link=Team:Caltech/Project/Phage_Pathogen_Defense}}<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Lactose intolerance|<font face="verdana" style="color:#BB4400">Lactose Intolerance</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}{{navimg|xsize=250|ysize=187|image=Milk.jpg|link=Team:Caltech/Project/Lactose_intolerance}}<br />
{{!}}<br />
Approximately 75% of adults worldwide suffer from lactose intolerance, the inability to metabolize lactose in the small intestine. We propose to treat lactose intolerance by engineering a strain of ''Escherichia coli'' that can reside in the large intestine. The engineered strain will sense lactose and subsequently release ß-galactosidase to convert lactose into glucose and galactose, both of which can be reabsorbed by the host. To treat lactose intolerance, our engineered bacterial strain will contain two plasmids: one with constitutive expression of a mutant lactose permease and ß-galactosidase, and the second with lactose-inducible expression of the λ phage lysis cassette. The mutant lactose permease allows the cells to import lactose under all conditions. When the cells uptake enough lactose, the second plasmid will induce cell lysis through activation of the λ phage lysis cassette, resulting in cell lysis and release of ß-galactosidase into the large intestine. Data covering the construction and characterization of these plasmid constructs is [[Team:Caltech/Project/Lactose_intolerance|<font style="color:#BB4400">discussed</font>]].<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Vitamins|<font face="verdana" style="color:#BB4400">Vitamin Production</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}<br />
Folate, a term which encompasses the various forms of the vitamin B9, is an essential vitamin involved in everyday cell functions such as DNA replication. Unable to naturally produce folate, humans must obtain it from vegetables or folate-supplements. In regions with little or no access to these foods, folate deficiencies can cause serious birth defects. One possible solution to alleviate the effects of folate deficiency is to engineer a strain of gut microbes to produce bioavailable folate directly in the colon. We tested a total of four heterologous genes, two from the folate biosynthesis gene cluster and two from the paraaminobenzoic acid (pABA) synthesis pathway. Using standardized genetic sequences, folate biosynthesis genes extracted from the ''Lactoccocus lactis'' genome were cloned into Biobricks plasmids, transformed into ''Escherichia coli'' and overexpressed. We measured the effects of overexpression<br />
in terms of total folate and paraaminobenzoic acid levels. PABA, an intermediate in folate synthesis, was detected using [[Team:Caltech/Protocols/PABA_HPLC_assay|<font style="color:#BB4400">high performance liquid chromatography</font>]] (HPLC). Folate detection was achieved via a [[Team:Caltech/Protocols/Folate_assay|<font style="color:#BB4400">microbiological assay</font>]]. A measurable increase in folate production in ''E. coli'' provides proof-of-concept for both the feasibility of engineering overproduction of folate in ''E. coli'', as well as using standardized genetic components to do so.<br />
{{!}}{{navimg|xsize=193|ysize=288|image=Folate_foods.jpg|link=Team:Caltech/Project/Vitamins}}<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Population Variation|<font face="verdana" style="color:#BB4400">Population Variation</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}{{navimg|xsize=220|ysize=231|image=Differentiation.jpg|link=Team:Caltech/Project/Population_Variation}}<br />
{{!}}<br />
As more complicated and interconnected biological circuits are built, there is an increasing need for the simple integration of multiple functions into a single bacterial cell line. However, some of these functions may be incompatible or may kill the cell, such that each cell can only express a single function at any time or must be regenerated if the functionality is fatal. We aim to combine multiple mutually exclusive and potentially fatal functions into a single bacterial cell line that, as a population, exhibits the entire set of functions. <br />
<br />
In particular, we want only one of the four subprojects described above to be turned on in any given cell, or else the cell may be overburdened by our constructs. At the same time, we want our bacterial population to have the capability to exhibit all four functions. In addition, three of the four subprojects result in the death of the host cell through different methods of self-induced lysis. Therefore, we need a system that is able to combine all subprojects into one coherent system and that allows for self-renewal of the population.<br />
<br />
We propose a system in which a single bacterial strain encodes all four functions in its genome and can access each function independently through probabalistic molecular events. The engineered bacterial cells start in an undifferentiated state and randomly differentiate into one of three possible final states. To build this system, we designed two genetic devices. One relies on DNA polymerase slippage upon replication of a long stretch of short nucleotide repeats, a phenomenon termed slipped-strand mispairing (SSM). The other device uses the recombinase protein FimE to flip DNA segments. The two devices can form a system that permits the novel introduction of random multi-state differentiation into bacterial cells. Our experimental results demonstrate that short nucleotide repeats can be used as a stochastic switch and that FimE activity depends on the length of the segment being flipped. <br />
{{!}}}<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/ProjectTeam:Caltech/Project2008-10-29T01:33:54Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
__NOTOC__<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Subprojects</font></div><br />
<div style="font-size:10pt;"><br />
<br />
<font face="verdana" style="color:#BB4400">Note: Click on the subproject title or picture for a detailed description of the subproject</font></div><br />
<br><br />
<br />
==[[Team:Caltech/Project/Oxidative Burst|<font face="verdana" style="color:#BB4400">Oxidative Burst</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}{{navimg|xsize=200|ysize=272|image=neutrophil-shigella.jpg|link=Team:Caltech/Project/Oxidative_Burst}}<br />
{{!}}<br />
Specialized white blood cells called neutrophils defend us from illness by killing bacteria with a potent concoction of degradative enzymes and oxidizing agents, including hydrogen peroxide. However, pathogens of the human large intestine are able to cause serious illness while being sheltered from neutrophils. We engineered a strain of ''Escherichia coli'' that is able to mimic a neutrophil by producing cytotoxic amounts of hydrogen peroxide in a controlled, inducible manner. Our engineered ''E. coli'' use the transcriptional activator LuxR to detect the presence of acyl-homoserine lactones, quorum sensing signaling molecules secreted by invading pathogens. LuxR activates production of the pyruvate oxidase of ''Streptococcus pneumoniae'', which produces large amounts of hydrogen peroxide by oxidizing pyruvate. The engineered ''E. coli'' is capable of killing certain strains of antibiotic resistant ''E. coli'' within six hours. When translated into a probiotic strain such as Nissle 1917, this system has the potential to be an effective means of combating enteric pathogens.<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Phage Pathogen Defense|<font face="verdana" style="color:#BB4400">Phage Pathogen Defense</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}<br />
Another aspect of bacterial pathogen defense for our probiotic is to produce bacteriophages that would rapidly infect and wipe out pathogens. Our method uses the bacteriophage λ, a temperate phage which targets ''Escherichia coli''. λ infects ''E. coli'' through the lamB receptor, where absence of this receptor prevents λ infection. We have taken advantage of this property of bacteriophage λ to engineer an ''E. coli'' strain that is resistant to the phage, but harbors a phage in its genome and can specifically trigger phage release to destroy susceptible pathogenic ''E. coli''. This system is useful in itself, targeting pathogenic ''E. coli'', and as a model system for the introduction of non-natural phage into ''E. coli'' such as the ''Salmonella'' phage P22.<br />
{{!}}{{navimg|xsize=220|ysize=258|image=Phage.jpg|link=Team:Caltech/Project/Phage_Pathogen_Defense}}<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Lactose intolerance|<font face="verdana" style="color:#BB4400">Lactose Intolerance</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}{{navimg|xsize=250|ysize=187|image=Milk.jpg|link=Team:Caltech/Project/Lactose_intolerance}}<br />
{{!}}<br />
Approximately 75% of adults worldwide suffer from lactose intolerance, the inability to metabolize lactose in the small intestine. We propose to treat lactose intolerance by engineering a strain of ''Escherichia coli'' that can reside in the large intestine. The engineered strain will sense lactose and subsequently release ß-galactosidase to convert lactose into glucose and galactose, both of which can be reabsorbed by the host. To treat lactose intolerance, our engineered bacterial strain will contain two plasmids: one with constitutive expression of a mutant lactose permease and ß-galactosidase, and the second with lactose-inducible expression of the λ phage lysis cassette. The mutant lactose permease allows the cells to import lactose under all conditions. When the cells uptake enough lactose, the second plasmid will induce cell lysis through activation of the λ phage lysis cassette, resulting in cell lysis and release of ß-galactosidase into the large intestine. Data covering the construction and characterization of these plasmid constructs is [[Team:Caltech/Project/Lactose_intolerance|<font style="color:#BB4400">discussed</font>]].<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Vitamins|<font face="verdana" style="color:#BB4400">Vitamin Production</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}<br />
Folate, a term which encompasses the various forms of the vitamin B9, is an essential vitamin involved in everyday cell functions such as DNA replication. Unable to naturally produce folate, humans must obtain it from vegetables or folate-supplements. In regions with little or no access to these foods, folate deficiencies can cause serious birth defects. One possible solution to alleviate the effects of folate deficiency is to engineer a strain of gut microbes to produce bioavailable folate directly in the colon. We tested a total of four heterologous genes, two from the folate biosynthesis gene cluster and two from the paraaminobenzoic acid (pABA) synthesis pathway. Using standardized genetic sequences, folate biosynthesis genes extracted from the ''Lactoccocus lactis'' genome were cloned into Biobricks plasmids, transformed into ''Escherichia coli'' and overexpressed. We measured the effects of overexpression<br />
in terms of total folate and paraaminobenzoic acid levels. PABA, an intermediate in folate synthesis, was detected using [[Team:Caltech/Protocols/PABA_HPLC_assay|<font style="color:#BB4400">high performance liquid chromatography</font>]] (HPLC). Folate detection was achieved via a [[Team:Caltech/Protocols/Folate_assay|<font style="color:#BB4400">microbiological assay</font>]]. A measurable increase in folate production in ''E. coli'' provides proof-of-concept for both the feasibility of engineering overproduction of folate in ''E. coli'', as well as using standardized genetic components to do so.<br />
{{!}}{{navimg|xsize=193|ysize=288|image=Folate_foods.jpg|link=Team:Caltech/Project/Vitamins}}<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Population Variation|<font face="verdana" style="color:#BB4400">Population Variation</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}{{navimg|xsize=220|ysize=231|image=Differentiation.jpg|link=Team:Caltech/Project/Population_Variation}}<br />
{{!}}<br />
As more complicated and interconnected biological circuits are built, there is an increasing need for the simple integration of multiple functions into a single bacterial cell line. However, some of these functions may be incompatible or may kill the cell, such that each cell can only express a single function at any time or must be regenerated if the functionality is fatal. We aim to combine multiple mutually exclusive and potentially fatal functions into a single bacterial cell line that, as a population, exhibits the entire set of functions. <br />
<br />
In particular, we want only one of the four subprojects described above to be turned on in any given cell, or else the cell may be overburdened by our constructs. At the same time, we want our bacterial population to have the capability to exhibit all four functions. In addition, three of the four subprojects result in the death of the host cell through different methods of self-induced lysis. Therefore, we need a system that is able to combine all subprojects into one coherent system and that allows for self-renewal of the population.<br />
<br />
We propose a system in which a single bacterial strain encodes all four functions in its genome and can access each function independently through probabalistic molecular events. The engineered bacterial cells start in an undifferentiated state and randomly differentiate into one of three possible final states. To build this system, we designed two genetic devices. One relies on DNA polymerase slippage upon replication of a long stretch of short nucleotide repeats, a phenomenon termed slipped-strand mispairing (SSM). The other device uses the recombinase protein FimE to flip DNA segments. The two devices can form a system that permits the novel introduction of random multi-state differentiation into bacterial cells. Our experimental results demonstrate that short nucleotide repeats can be used as a stochastic switch and that FimE activity depends on the length of the segment being flipped. <br />
{{!}}}<br />
}}</div>Jkmhttp://2008.igem.org/Template:Caltech_iGEM_08Template:Caltech iGEM 082008-10-29T01:31:43Z<p>Jkm: </p>
<hr />
<div><html><br />
<style type="text/css"><br />
body {background: #FFFFFF}<br />
</style><br />
</html><br />
{|<br />
|-valign="top"<br />
|{{navimg|xsize=960|ysize=36|image=Caltech_header_top.jpg|link=Team:Caltech}}<br />
|}<br> <br />
<div style="font-size:24pt;"><br />
<font face="verdana" style="color:#BB4400"><center>iGEM 2008<br />
</center></font></div><br />
<br><br />
{|<br />
|-valign="top"<br />
|{{navimg|xsize=960|ysize=48|image=Caltech_header_bottom.jpg|link=Team:Caltech}}<br />
|}<br />
<div style="color: #ffffff; background-color: #ffffff; width: 900px"><br />
</div><br />
<br />
<div><br />
{| cellspacing="0"<br />
|-<br />
|style="background-color: #ffffff" width="75px" valign="top"|<br />
<br><center>[[Team:Caltech| <font face="verdana" style="color:#BB4400"> '''Home''' </font>]] <br><br><br />
[[Team:Caltech/Members | <font face="verdana" style="color:#BB4400"> '''People''' </font>]] <br><br><br />
[[Team:Caltech/Project | <font face="verdana" style="color:#BB4400"> '''Project Details''' </font>]] <br><br><br />
[[Team:Caltech/Protocols | <font face="verdana" style="color:#BB4400"> '''Protocols''' </font>]] <br><br><br />
[[Team:Caltech/Parts | <font face="verdana" style="color:#BB4400"> '''Completed Systems''' </font>]] <br><br><br />
[[Team:Caltech/Biosafety | <font face="verdana" style="color:#BB4400"> '''Biosafety''' </font>]] <br><br><br />
[[Team:Caltech/Acknowledgments | <font face="verdana" style="color:#BB4400"> '''Support''' </font>]] <br><br><br />
<br />
<br />
|width="880" valign="top" style="padding: 10px; border: 5px solid #FFFFFF; color: #000; background-color: white" | <br />
{{{Content}}}<br />
[[Image:Caltech_logo.gif|right]]<br />
{{Clear}}<br />
[[Image:Caltech_footer.jpg|left]]<br />
|}</div>Jkmhttp://2008.igem.org/Team:Caltech/AcknowledgmentsTeam:Caltech/Acknowledgments2008-10-28T19:13:33Z<p>Jkm: New page: {{Caltech_iGEM_08| Content=<div style="font-size:18pt;"> <font face="verdana" style="color:#CC3300">Acknowledgments</font></div> <br> __NOTOC__ ==Funding== *Major funding was provided by ...</p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Acknowledgments</font></div><br />
<br><br />
__NOTOC__<br />
<br />
==Funding==<br />
*Major funding was provided by the Howard Hughes Medical Institute<br />
*Fellowship support was provided by Amgen Scholars (Victoria Hsiao and Doug Tischer), Caltech SURF (Fei Chen, Allen Lin, and Robert Ovadia), and the NSF (Josh Michener)<br />
<br />
==Materials==<br />
*Strain JI377 was provided by Professor Jim Imlay<br />
*The phage λcm was a gift from Dr. Sankar Adhya<br />
*Dr. Sidney Cox provided the lactose-inducible promoter B4<br />
}}</div>Jkmhttp://2008.igem.org/Template:Caltech_iGEM_08Template:Caltech iGEM 082008-10-28T19:10:06Z<p>Jkm: </p>
<hr />
<div><html><br />
<style type="text/css"><br />
body {background: #FFFFFF}<br />
</style><br />
</html><br />
{|<br />
|-valign="top"<br />
|{{navimg|xsize=960|ysize=36|image=Caltech_header_top.jpg|link=Team:Caltech}}<br />
|}<br> <br />
<div style="font-size:24pt;"><br />
<font face="verdana" style="color:#BB4400"><center>iGEM 2008<br />
</center></font></div><br />
<br><br />
{|<br />
|-valign="top"<br />
|{{navimg|xsize=960|ysize=48|image=Caltech_header_bottom.jpg|link=Team:Caltech}}<br />
|}<br />
<div style="color: #ffffff; background-color: #ffffff; width: 900px"><br />
</div><br />
<br />
<div><br />
{| cellspacing="0"<br />
|-<br />
|style="background-color: #ffffff" width="75px" valign="top"|<br />
<br><center>[[Team:Caltech| <font face="verdana" style="color:#BB4400"> '''Home''' </font>]] <br><br><br />
[[Team:Caltech/Members | <font face="verdana" style="color:#BB4400"> '''People''' </font>]] <br><br><br />
[[Team:Caltech/Project | <font face="verdana" style="color:#BB4400"> '''Project Details''' </font>]] <br><br><br />
[[Team:Caltech/Protocols | <font face="verdana" style="color:#BB4400"> '''Protocols''' </font>]] <br><br><br />
[[Team:Caltech/Parts | <font face="verdana" style="color:#BB4400"> '''Completed Systems''' </font>]] <br><br><br />
[[Team:Caltech/Biosafety | <font face="verdana" style="color:#BB4400"> '''Biosafety''' </font>]] <br><br><br />
[[Team:Caltech/Acknowledgments | <font face="verdana" style="color:#BB4400"> '''Acknowledgments''' </font>]] <br><br><br />
<br />
<br />
|width="880" valign="top" style="padding: 10px; border: 5px solid #FFFFFF; color: #000; background-color: white" | <br />
{{{Content}}}<br />
[[Image:Caltech_logo.gif|right]]<br />
{{Clear}}<br />
[[Image:Caltech_footer.jpg|left]]<br />
|}</div>Jkmhttp://2008.igem.org/Team:Caltech/BiosafetyTeam:Caltech/Biosafety2008-10-28T18:14:18Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Biosafety Concerns</font></div><br />
<br><br />
__NOTOC__<br />
==General Concerns==<br />
There are risks in probiotic engineering that may prevent or delay our system from being implemented in humans. One common risk is increasing the chance of bacterial sepsis for those who are immune deficient. Other risk factors include premature infants, CVC (central venous catheter), cardiac vascular disease, and diarrhea<sup>1</sup>. We are also unsure of the effects that probiotics have on host metabolic activities. However, the unmodified Nissle 1917 strain has been shown to be safe for long term use<sup>2</sup>.<br />
<br />
The engineered probiotic will persist in the gut longer if antibiotics are used to suppress other gut flora<sup>3</sup>. However, such resistance may pass over into pathogenic strains and cause future problems for the host. The concern of introducing foreign genes into the gut is described below for each individual project.<br />
<br />
==Subproject Concerns==<br />
===Oxidative Burst===<br />
*'''Short Term:''' There are no safety concerns beyond that of typical ''E. coli'' lab strains. Considering that 260 mM H<sub>2</sub>O<sub>2</sub> is applied directly to the skin to disinfect cuts and scrapes, the 800 uM H<sub>2</sub>O<sub>2</sub> produced by the engineered strain should not be a health concern to anyone working in the lab.<br />
*'''Long Term:''' Production of hydrogen peroxide is not a normal occurrence in the large intestine, and its effects would need to be investigated before the engineered strain could be used to fight infection. Some concerns of hydrogen peroxide production in the intestine would be if:<br />
**it irritates the bowel<sup>4</sup>.<br />
**it kills off significant amounts of the native gut flora.<br />
**it significantly damages gut epithelial cells.<br />
**the ability to produce peroxide can be transmitted to other gut flora.<br />
<br />
<br />
===Phage Pathogen Defense===<br />
<br />
===Lactose Intolerance===<br />
* '''Synthetic LacY/Z Plasmid''' There are no known safety concerns regarding this plasmid. The only foreign enzyme not present in humans that is expressed is β-Galactosidase, which simply acts as a lactase. Several lactase preparations are formally 'Generally Recognized as Safe' by the FDA[http://www.cfsan.fda.gov/~rdb/opa-g132.html] and we expect no difficulties in finding a suitable lactase.<br />
* '''Lysis Cassette Plasmid''' This plasmid may cause problems in the gut, if passed over from our strain to another strain. If passed over to a pathogenic strain, this may of course help the host in lysing that strain when lactose is present, but if passed over to a helpful strain, this may wipe out a great amount of our gut flora.<br />
<br />
===Vitamin Production===<br />
<br />
===Population Variation===<br />
* The current constructs are regulatory in nature and present few safety concerns aside from those involved in the treatment subprojects. Minor concerns are discussed below:<br />
* In the simple FimE constructs, when the promoter starts in the configuration pointing upstream, DNA upstream of the constructs may be transcribed by the cell.<br />
* In the final design, the terminator that sits in the population variation generator may not be 100% efficient. Thus, the efficiency of the terminator should be tested before genes that are hazardous when co-expressed are placed into the system.<br />
* The engineered FimE system may interfere with natural fimbriae expression in Nissle 1917 or other gut flora. Since fimbriae are important for intestinal colonization, this could effect the intestinal composition.<br />
<br />
==References==<br />
# Boyle RJ, Robins-Browne RM, Tang ML. Probiotic use in clinical practice: what are the risks? Am J Clin Nutr 2006;83:1256-64.<br />
# Westendorf AM, Gunzer F, Deppenmeier S, Tapadar D, Hunger JK, Schmidt MA, Buer J, and Bruder D. '''Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules'''. ''FEMS Immunol Med Microbiol'' 2005 Mar 1; 43(3) 373-84.<br />
# Rao S, Hu S, McHugh L, Lueders K, Henry K, Zhao Q, Fekete RA, Kar S, Adhya S, and Hamer DH. '''Toward a live microbial microbicide for HIV: commensal bacteria secreting an HIV fusion inhibitor peptide'''. ''Proc Natl Acad Sci U S A'' 2005 Aug 23; 102(34) 11993-8.<br />
# Ackermann M, Stecher B, Freed NE, Songhet P, Hardt WD, and Doebeli M. '''Self-destructive cooperation mediated by phenotypic noise'''. ''Nature'' 2008 Aug 21; 454(7207) 987-90. <br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/ProjectTeam:Caltech/Project2008-10-27T20:27:24Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
__NOTOC__<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Subprojects</font></div><br />
<div style="font-size:10pt;"><br />
<br />
<font face="verdana" style="color:#BB4400">Note: Click on the subproject title or picture for a detailed description of the subproject</font></div><br />
<br><br />
<br />
==[[Team:Caltech/Project/Oxidative Burst|<font face="verdana" style="color:#BB4400">Oxidative Burst</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}{{navimg|xsize=200|ysize=272|image=neutrophil-shigella.jpg|link=Team:Caltech/Project/Oxidative_Burst}}<br />
{{!}}<br />
Specialized white blood cells called neutrophils defend us from illness by killing bacteria with a potent concoction of degradative enzymes and oxidizing agents, including hydrogen peroxide. However, pathogens of the human large intestine are able to cause serious illness while being sheltered from neutrophils. We engineered a strain of ''Escherichia coli'' that is able to mimic a neutrophil by producing cytotoxic amounts of hydrogen peroxide in a controlled, inducible manner. Our engineered ''E. coli'' use the transcriptional activator LuxR to detect the presence of acyl-homoserine lactones, quorum sensing signaling molecules secreted by invading pathogens. LuxR activates production of the pyruvate oxidase of ''Streptococcus pneumoniae'', which produces large amounts of hydrogen peroxide by oxidizing pyruvate. The engineered ''E. coli'' is capable of killing certain strains of antibiotic resistant ''E. coli'' within six hours. When translated into a probiotic strain such as Nissle 1917, this system has the potential to be an effective means of combating enteric pathogens.<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Phage Pathogen Defense|<font face="verdana" style="color:#BB4400">Phage Pathogen Defense</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}<br />
Another aspect of bacterial pathogen defense for our probiotic is to produce bacteriophages that would rapidly infect and wipe out pathogens. Our method uses the bacteriophage λ, a temperate phage which targets ''Escherichia coli''. λ infects ''E. coli'' through the lamB receptor, where absence of this receptor prevents λ infection. We have taken advantage of this property of bacteriophage λ to engineer an ''E. coli'' strain that is resistant to the phage, but harbors a phage in its genome and releases it to destroy susceptible pathogenic ''E. coli''. <br />
{{!}}{{navimg|xsize=220|ysize=258|image=Phage.jpg|link=Team:Caltech/Project/Phage_Pathogen_Defense}}<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Lactose intolerance|<font face="verdana" style="color:#BB4400">Lactose Intolerance</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}{{navimg|xsize=250|ysize=187|image=Milk.jpg|link=Team:Caltech/Project/Lactose_intolerance}}<br />
{{!}}<br />
Approximately 75% of adults worldwide suffer from lactose intolerance, the inability to metabolize lactose in the small intestine. We propose to treat lactose intolerance by engineering a strain of ''Escherichia coli'' that can reside in the large intestine. The engineered strain will sense lactose and subsequently release ß-galactosidase to convert lactose into glucose and galactose, both of which can be reabsorbed by the host. To treat lactose intolerance, our engineered bacterial strain will contain two plasmids: one with constitutive expression of a mutant lactose permease and ß-galactosidase, and the second with lactose-inducible expression of the λ phage lysis cassette. The mutant lactose permease allows the cells to import lactose under all conditions. When the cells uptake enough lactose, the second plasmid will induce cell lysis through activation of the λ phage lysis cassette, resulting in cell lysis and release of ß-galactosidase into the large intestine. Data covering the construction and characterization of these plasmid constructs is [[Team:Caltech/Project/Lactose_intolerance|<font style="color:#BB4400">discussed</font>]].<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Vitamins|<font face="verdana" style="color:#BB4400">Vitamin Production</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}<br />
Folate, a term which encompasses the various forms of the vitamin B9, is an essential vitamin involved in everyday cell functions such as DNA replication. Unable to naturally produce folate, humans must obtain it from vegetables or folate-supplements. In regions with little or no access to these foods, folate deficiencies can cause serious birth defects. One possible solution to alleviate the effects of folate deficiency is to engineer a strain of gut microbes to produce bioavailable folate directly in the colon. We tested a total of four heterologous genes, two from the folate biosynthesis gene cluster and two from the paraaminobenzoic acid (pABA) synthesis pathway. Using standardized genetic sequences, folate biosynthesis genes extracted from the ''Lactoccocus lactis'' genome were cloned into Biobricks plasmids, transformed into ''Escherichia coli'' and overexpressed. We measured the effects of overexpression<br />
in terms of total folate and paraaminobenzoic acid levels. PABA, an intermediate in folate synthesis, was detected using [[Team:Caltech/Protocols/PABA_HPLC_assay|<font style="color:#BB4400">high performance liquid chromatography</font>]] (HPLC). Folate detection was achieved via a [[Team:Caltech/Protocols/Folate_assay|<font style="color:#BB4400">microbiological assay</font>]]. A measurable increase in folate production in ''E. coli'' provides proof-of-concept for both the feasibility of engineering overproduction of folate in ''E. coli'', as well as using standardized genetic components to do so.<br />
{{!}}{{navimg|xsize=193|ysize=288|image=Folate_foods.jpg|link=Team:Caltech/Project/Vitamins}}<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Population Variation|<font face="verdana" style="color:#BB4400">Population Variation</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}{{navimg|xsize=220|ysize=231|image=Differentiation.jpg|link=Team:Caltech/Project/Population_Variation}}<br />
{{!}}<br />
As more complicated and interconnected biological circuits are built, there is an increasing need for the simple integration of multiple functions into a single bacterial cell line. However, some of these functions may be incompatible or may kill the cell, such that each cell can only express a single function at any time or must be regenerated if the functionality is fatal. We aim to combine multiple mutually exclusive and potentially fatal functions into a single bacterial cell line that, as a population, exhibits the entire set of functions. <br />
<br />
In particular, we want only one of the four subprojects described above to be turned on in any given cell, or else the cell may be overburdened by our constructs. At the same time, we want our bacterial population to have the capability to exhibit all four functions. In addition, three of the four subprojects result in the death of the host cell through different methods of self-induced lysis. Therefore, we need a system that is able to combine all subprojects into one coherent system and that allows for self-renewal of the population.<br />
<br />
We propose a system in which a single bacterial strain encodes all four functions in its genome and can access each function independently through probabalistic molecular events. The engineered bacterial cells start in an undifferentiated state and randomly differentiate into one of three possible final states. To build this system, we designed two genetic devices. One relies on DNA polymerase slippage upon replication of a long stretch of short nucleotide repeats, a phenomenon termed slipped-strand mispairing (SSM). The other device uses the recombinase protein FimE to flip DNA segments. The two devices can form a system that permits the novel introduction of random multi-state differentiation into bacterial cells. Our experimental results demonstrate that short nucleotide repeats can be used as a stochastic switch and that FimE activity depends on the length of the segment being flipped. <br />
{{!}}}<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/BiosafetyTeam:Caltech/Biosafety2008-10-25T17:05:16Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Biosafety Concerns</font></div><br />
<br><br />
__NOTOC__<br />
==General Concerns==<br />
<br />
==Subproject Concerns==<br />
===Oxidative Burst===<br />
*'''Short Term:''' There are no safety concerns beyond that of typical ''E. coli'' lab strains. Considering that 260 mM H<sub>2</sub>O<sub>2</sub> is applied directly to the skin to disinfect cuts and scrapes, the 800 uM H<sub>2</sub>O<sub>2</sub> produced by the engineered strain should not be a health concern to anyone working in the lab.<br />
*'''Long Term:''' Production of hydrogen peroxide is not a normal occurance in the large instestine, and its effects would need to be investigated before the engineered strain could be used to fight infection. Some concerns of hydrogen peroxide production in the intestine would be if:<br />
**it irritates the bowel<sup>1</sup>.<br />
**it kills off significant amounts of the native gut flora.<br />
**it significantly damages gut epithelial cells.<br />
**the ability to produce peroxide can be transmitted to other gut flora.<br />
<br />
<br />
===Phage Pathogen Defense===<br />
<br />
===Lactose Intolerance===<br />
<br />
===Vitamin Production===<br />
<br />
===Population Variation===<br />
<br />
==References==<br />
# Ackermann M, Stecher B, Freed NE, Songhet P, Hardt WD, and Doebeli M. '''Self-destructive cooperation mediated by phenotypic noise'''. ''Nature'' 2008 Aug 21; 454(7207) 987-90. <br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/BiosafetyTeam:Caltech/Biosafety2008-10-25T17:01:30Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Biosafety Concerns</font></div><br />
<br><br />
__NOTOC__<br />
==General Concerns==<br />
<br />
==Subproject Concerns==<br />
===Oxidative Burst===<br />
*'''Short Term:''' There are no safety concerns beyond that of typical ''E. coli'' lab strains. Considering that 260 mM H<sub>2</sub>O<sub>2</sub> is applied directly to the skin to disinfect cuts and scrapes, the 800 uM H<sub>2</sub>O<sub>2</sub> produced by the engineered strain should not be a health concern to anyone working in the lab.<br />
*'''Long Term:''' Production of hydrogen peroxide is not a normal occurance in the large instestine, and its effects would need to be investigated before the engineered strain could be used to fight infection. Some concerns of hydrogen peroxide production in the intestine would be if:<br />
**it irritates the bowel.<br />
**it kills off significant amounts of the native gut flora.<br />
**it significantly damages gut epithelial cells.<br />
**the ability to produce peroxide can be transmitted to other gut flora.<br />
<br />
<br />
===Phage Pathogen Defense===<br />
<br />
===Lactose Intolerance===<br />
<br />
===Vitamin Production===<br />
<br />
===Population Variation===<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/Project/Phage_Pathogen_DefenseTeam:Caltech/Project/Phage Pathogen Defense2008-10-25T17:00:16Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Phage Pathogen Defense</font></div><br />
<br><br />
__NOTOC__<br />
<br />
==Introduction==<br />
[[Image:PathogenicEcoli.jpg|thumb|left|Pathogenic ''E. coli''.]]<br />
There are 10<sup>14</sup> bacterial cells that naturally reside in the human gut, an order of magnitude greater than all the cells in the body. Humans enjoy a mutualistic relationship with intestinal microbiota, wherein the microorganisms perform a host of useful functions, such as processing unused energy substrates, training the immune system, and inhibiting growth of harmful bacterial species [1]. However, not all bacteria populations within the human intestine are benign. There are many pathogenic bacteria which cause a wide variety of diseases when present in the human gut. As one example, cholera is one of the most widespread and damaging of such infectious microorganisms, with the World Health Organization reporting 132,000 cases in 2006 [2]. But cholera is just one of many examples. In the United States alone, there were more than 325,000 hospitalizations from food-borne illnesses in the year of 2006 [3]. <br />
<br />
There are many medical treatments for food-borne illnesses, the most commonly prescribed being antibiotics. Unfortunately, antibiotics are indiscriminant and lead to rapid depletion of benign bacterial populations within the intestine. Due to this undesireable side effect, dietary supplements have been popularized which aim to introduce beneficial bacteria back into the gut after antibiotic treatment; these substances have been termed ‘probiotics’. Natural probiotics have two main advantages: they provide useful functions for the host and they competitively inhibit growth of pathogenic bacteria.<br />
<br />
Natural probiotics do not convey any more advantages than bacteria in a healthy human intestine. Modern synthetic biology techniques should allow us to create an engineered probiotic that goes beyond the limitations of natural probiotics. The viability of such engineered probiotics within humans has already been established [4]. As part of a collaborative effort by the Caltech iGEM team to engineer a novel probiotic with improved medical applications, this project focuses on engineering a pathogen defense system within ''E. coli''. <br />
<br />
There are many ways for the engineered probiotic to combat pathogens in the large intestine; we chose to use bacteriophages. Bacteriophages present a novel and effective agent for eliminating pathogenic bacteria. Two important factors contribute to a bacteriophage’s effectiveness: bacteriophages are highly infectious and thus highly efficient at destroying targeted populations, and bacteriophages are specific to their hosts. The latter factor is readily illustrated by the coliphage λ, which possesses a mode of infection dependent on an ''E. coli'' specific surface protein, LamB [5]. Benign, non-''E. coli'' intestinal bacteria lack this surface receptor and therefore are immune to destruction. <br />
<br />
===System Design===<br />
<br />
The engineered probiotic acts as a delivery vehicle for phage into the large intestine. There are four important design goals for such as system: (1) the system releases phage into the large intestine, (2) the production of phage is regulated with a high dynamic range between a distinct on and off state, (3) the system can target a wide range of pathogenic bacteria, and (4) the system integrates well within the existing iGEM probiotic. <br />
<br />
An obvious delivery mechanism would be the adaptation of natural lysogens, a phase of the phage life cycle in which the phage integrates its DNA into the genome of the host organism. Lysogens lie dormant until disturbed by some external stimuli, but can be stimulated to enter the lytic cycle and release phage. There is one major limitation to this method: phage released will be specific to the lysogenic hosts, ''E. coli''. We can avoid this limitation by incorporating as a lysogen a phage that is not normally infectious to ''E. coli'', such as the P22 phage from ''Salmonella''. This phage could potentially target pathogenic bacteria besides ''E. coli''. <br />
<br />
To demonstrate the feasibility of this approach, a simple model system was developed using λ phage and JW3996-1, a strain of ''E. coli'' immune to λ infection due to a ''lamB'' deletion [5]. In this model, the host is immune to infection unless LamB is expressed from a plasmid. After infection and selection for lysogens, the ''lamB'' plasmid is counterselected against to regain immunity. Extending this model to a more realistic situation, we envision using a phage that targets a pathogenic bacterial species, constitutively expressing the phage receptor within ''E. coli'' to create lysogens, and inducing the lysogens to infect non-''E. coli'' pathogenic bacteria. <br />
<br />
We require one other plasmid which controls the induction of the lysogens to release phage into the environment. Control of this aspect of the system is vital for integration with the overall iGEM project. The induction of λ phage lysogens has been explored since 1950 [6], and serves as a model system for the study of other temperate phages. The most famous induction mechanism is ultraviolet radiation (UV). In UV induction of λ lysogens, UV induced DNA damage activates the cellular SOS mechanism, which activates the expression of RecA, a protein involved in SOS response. RecA, in turn, cleaves the λ repressor and leads to phage activation. In this study, activation of the ''recA'' pathway was not readily feasible, so an alternative effector was used to elicit induction. A secondary plasmid expressing the ''E. coli'' regulatory gene, ''rcsA'', is used as the trigger for λ phage induction. ''rcsA'' is one of several regulatory genes which provide a ''recA'' independent pathway for λ induction [7]. Overexpression of RcsA leads to λ induction, and, furthermore, RcsA appears to directly compete with the λ repressor cI [6]. In this project, ''rcsA'' is used to trigger phage production. The effectiveness and dynamic range of ''rcsA'' induction is explored.<br />
<br />
===Constructs===<br />
<br />
[[Image:lambteta.png|thumb|right|300px|Figure 1 - Constructs involved in expression of the ''lamB'' receptor and the TetA cassette ]]<br />
All constructs were made with standard assembly methods. ''lamB'' and ''rcsA'' were cloned out of DH10B genomic DNA. Two families of plasmids were constructed for this project. The first consists of plasmids that express the ''lamB'' receptor and the tetracycline resistance cassette. The level of ''lamB'' expression required for λ phage infection is unknown, so the strength of the promoter/ribosome binding site (RBS) combination upstream of ''lamB'' was varied between the four constructs of this family.<br />
{{clear}}<br />
[[Image:rcsa.png|thumb|left|300px|Figure 2 - Constructs for characterization of rcsA induction ]]<br />
<br />
The second family consists of constructs used to characterize ''rcsA'' induction of λ phage. ''rcsA'' was placed downstream of an acyl-homoserine lactone (AHL) inducible promoter, LuxR. This promoter was extensively characterized by Doug for the [[Team:Caltech/Project/Population_Variation|<font style="color:#BB4400">oxidative burst project</font>]]. Using flow cytometry data, the peak response for this promoter was determined to be 10 nM AHL. Plasmids with three different RBSs in front of ''rcsA'' were constructed to characterize the response of induction. Furthermore, a promoterless ''rcsA'' construct was built as a negative control, and a construct with constitutively active λ repressor ''cI'' driven by J23106 was also assembled.<br />
<br />
{{Clear}}<br />
<br />
==Results==<br />
<br />
===''lamB''<sup>-</sup> Infection===<br />
''lamB'' <sup>-</sup> cells were successfully infected by λ phage while constitutively expressing the ''lamB'' receptor. Furthermore, the level of infection directly correlated with the level of ''lamB'' expression , suggesting that phage infection was limited by the presence of ''lamB'' receptors. Next, lysogens were selected using chloramphenicol resistance. The presence of the ''lamB''/tetA construct within the lysogens was confirmed with colony PCR. This plasmid was successfully removed via tetracycline counterselection [10]. This procedure successfully established a lysogenic strain lacking the ''lamB'' receptor and thus immune to phage infection. <br />
<br />
<gallery widths="375px" heights="250px" ><br />
Image:lamb.png|Figure 3 - λ Infection of Constitutively Active lamB Constructs. Constitutively active ''lamB'' (Table 1) was used to augment the ''lamB''- phenotype in JW3996-1 cells. Cells were titered with a known high concentration stock of phage 1x10<sup>10</sup> phage/mL. The efficiency of infection of ''lamB'' constructs can be seen through the observed phage concentration after titering. Zero plaques were observed for the J23113+33 construct.<br />
Image:LamBExpression.png|Figure 4 - ''lamB'' Infection vs ''lamB'' expression level. Infection levels for the constructs in Figure 3 plotted against the expression levels recorded in the registry for the Promoter/RBSs used.<br />
</gallery><br />
<br />
===''rcsA'' Induction===<br />
<br />
<br />
Overexpression of ''rcsA'' led to induction of λ lysogens. ''rcsA'' was placed downstream of an AHL inducible promoter and overexpressed through induction with 10 nM AHL. Following induction, phage concentration in the supernatant was recorded through titering. This concentration was compared to the phage concentration in the supernatant without ''rcsA'' overexpression, given by a construct where ''rcsA'' is not driven by a promoter. The data show a 40 fold increase in phage concentration between the promoter-less ''rcsA'' construct and the strongest AHL induced ''rcsA'' construct (Figure 5, columns 1 and 4). The promoter-less ''rcsA'' construct should accurately represent background phage levels for the lysogen absent of any ''rcsA'' expression. <br />
Further work was done to characterize the response of the AHL inducible switch under three different RBSs. The data show induction efficiency is directly correlated with strength of ''rcsA'' overexpression (Figure 5, columns 2-4). In each of the constructs, there is a 3 to 5 fold increase in phage levels after induction with 10 nM AHL. This increase does not accurately reflect the dynamic range of the AHL inducible promoter, because it does not take into account the background phage levels without the promoter. In the weaker RBSs (B0032 and B0033), the phage concentration from the uninduced supernatant is comparable to the background level from the promoter-less construct (Figure 5, columns 1-3). Thus, in these inducible promoter systems, there is high dynamic range and negligible background compared to basal phage concentrations. However, in the strongest RBS (B0034), the uninduced phage level is an order of magnitude higher than the promoter-less background level. This high basal level expression suggests that the higher expression levels due to the strong RBS is leading to leaky ''rcsA'' activation of the phage lytic cycle. <br />
<br />
[[Image:Rcsainduction.png|600px|thumb|center|Figure 5 - ''rscA'' induction of phage production. In this figure, each construct was infected with λ phage and lysogens were selected. Lysogens were grown to OD600=0.1 and ''rcsA'' was induced with 10 nM of AHL. 1.5 hours after induction, the supernatant was used to titer wildtype stock of ''E. coli''. Plaques were counted to calculate the phage concentration inside the supernatant after induction. For more information see [[Team:Caltech/Protocols/rcsA_Lysogen_Induction|<font style="color:#BB4400">induction protocol.</font>]]]]<br />
{{Clear}}<br />
<br />
===''cI'' reduces ''rcsA'' induction===<br />
<br />
The high level of background phage induction in the strongest AHL inducible ''rcsA'' switch was not optimal. If the high basal phage levels could be reduced, then the dynamic range of the inducible switch can be greatly increased. cI, the λ repressor, has been shown to directly inhibit UV induction of lysogens [13], and RcsA seems to competitively interact with cI in λ induction [7]. Both of these properties suggest that increased concentration of the λ repressor should lead to lower phage induction levels. ''cI'' was constitutively expressed downstream of the strongest AHL inducible switch. As expected, expression of cI reduces the phage release and ''rcsA'' induction in both the off-state as well as the induced state. Phage concentration was reduced by more than 1.5x106 pfu/mL in both control and induced states (Figure 5, Columns 4 and 5). In Figure 5, for the construct expressing ''cI'', there is an 8 fold increase in phage concentration between induced and uninduced states, more than double the dynamic range of the construct without ''cI'' expression. <br />
<br />
==Conclusion==<br />
We have successfully developed a model system in which a bacteriophage can be integrated as a lysogen within an ''E. coli'' host that is not able to be infected by that phage. We successfully demonstrated this model with bacteriophage λ and the JW3996-1 strain of ''E. coli'' which is immune to λ infection. A system for inducing the release of phage was constructed around the ''E. coli'' regulatory protein RcsA. Work was done to optimize a switch using an AHL inducible promoter for ''rcsA'' induction. RcsA overexpression led to significant phage induction, producing approximately 1x10<sup>8</sup> plaque forming units (pfu)/mL. This value is 4 orders of magnitude greater than the phage induction reported by Rozanov et al. However, direct comparisons cannot be readily made between the data, because the method of overexpression of ''rcsA'' differs between the two papers, and the level at which ''rcsA'' was overexpressed in Rozanov’s study is unknown. The background phage concentration in uninduced lysogens was also much higher than reported in literature [7]. At present, there is no clear cause for this increase in basal induction levels within the λ lysogens. Phage induction via ''rcsA'' can be reduced through lambda repressor overexpression. The mechanism by which RcsA activates the lytic cycle of the dormant prophage is largely unknown; however, there is evidence that RscA acts through inactivation of the phage repressor, ''cI'' [7]. Therefore, increased production of the repressor ''cI'' led to a decrease in ''rcsA'' mediated phage induction. The addition of constitutively active ''cI'' repressor led to the construction of an ''rcsA'' phage induction system with a high dynamic range and relatively low background. <br />
<br />
Future work should be focused on broadening the host range of the system. Thus, a key aspect needs to be adapting other temperate phages to the model system. A significant extension of the results presented here would be the incorporation and production of a non-''E. coli'' bacteriophage by ''E. coli''. A promising candidate for this is the Salmonella phage P22; P22 has been shown to be viably produced in ''E. coli'' when the prophage is expressed [14]. Incorporation of the P22 prophage should be feasible following the framework of the current model. Furthermore, co-culture experiments with the current constructs and wild-type ''E. coli'' can be used to validate the current system's effectiveness at eliminating pathogens. <br />
<br />
==References==<br />
# Sears CL. 2005. "'''A dynamic partnership: Celebrating our gut flora.'''" Anaerobe, Volume 11, Issue 5, Pages 247-251.<br />
# "'''Cholera Statistics 2006.'''" World Health Organization. 4 July 2008 <http://www.who.int/wer/2006/wer8131.pdf>. <br />
# "'''Food Borne Illness FAQ.'''" World Health Organization. 5 July 2008 <http://www.who.int/mediacentre/factsheets/fs237/en/>. <br />
# Westendorf AM, Gunzer F, Deppenmeier S, Tapadar D, Hunger JK, Schmidt MA, Buer J, and Bruder D. “'''Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules.'''” FEMS Immunol Med Microbiol 2005 Mar 1; 43(3) 373-84. <br />
# Clément JM, Hofnung M. “'''Gene sequence of the lambda receptor, an outer membrane protein of E. coli K12.'''” Cell. 1981 Dec; 27(3 Pt 2):507–514.<br />
# Lwoff, A., L. Siminovitch, and N. Kjeldgaard. 1950. “'''Induction de la production de bacteriophages chez une bacterie lysogene.'''” Ann. Inst. Pasteur (Paris) 79:815-859.<br />
# Rozanov, Dmitry V., Richard D'ari, and Sergey P. Sineoky. "'''RecA-Independent Pathways of Lambdoid Prophage Induction in Escherichia coli.'''" Journal of Bacteriology 180 (1998): 6306-315.<br />
# “'''Biobricks Assembly'''” OpenWetware <http://openwetware.org/wiki/BioBricks_construction_tutorial><br />
# Sambrook, Joe, and David Russell. "'''Molecular Cloning.'''" New York: Cold Spring Harbor Laboratory P, 2000.<br />
# Maloy, Stanley R., and William D. Nunn. "'''Selection for Loss of Tetracycline Resistance by Escherichia coli.'''" Journal of Bacteriology 45 (1985): 1110-112.<br />
# Grayson, Paul. "'''Titering.'''" California Institute of Technology. <http://www.rpgroup.caltech.edu/wiki/index.php?title=titering>.<br />
# Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H. “'''Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.'''” Mol Syst Biol. 2006;2:2006.0008<br />
# John Baluch, Raquel Sussman. “'''Correlation Between UV Dose Requirement for Lambda Bacteriophage Induction and Lambda Repressor Concentration'''” Journal of Virology, June 1978, P. 595-602<br />
# Botstein, David and Ira Herskowitz. "'''Properties of hybrids between Salmonella phage P22 and coliphage lambda.'''" Nature. 1974 Oct 18;251(5476):584-9.<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/BiosafetyTeam:Caltech/Biosafety2008-10-24T17:38:21Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Biosafety Concerns</font></div><br />
<br><br />
__NOTOC__<br />
==General Concerns==<br />
<br />
==Subproject Concerns==<br />
===Oxidative Burst===<br />
<br />
===Phage Pathogen Defense===<br />
<br />
===Lactose Intolerance===<br />
<br />
===Vitamin Production===<br />
<br />
===Population Variation===<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/BiosafetyTeam:Caltech/Biosafety2008-10-24T17:36:48Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Biosafety Concerns</font></div><br />
<br><br />
__NOTOC__<br />
===General Concerns===<br />
<br />
===Oxidative Burst===<br />
<br />
===Phage Pathogen Defense===<br />
<br />
===Lactose Intolerance===<br />
<br />
===Vitamin Production===<br />
<br />
===Population Variation===<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/BiosafetyTeam:Caltech/Biosafety2008-10-24T15:44:13Z<p>Jkm: New page: {{Caltech_iGEM_08| Content=<div style="font-size:18pt;"> <font face="verdana" style="color:#CC3300">Biosafety Concerns</font></div> <br> __NOTOC__ ===Oxidative Burst=== ===Phage Pathogen ...</p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Biosafety Concerns</font></div><br />
<br><br />
__NOTOC__<br />
===Oxidative Burst===<br />
<br />
===Phage Pathogen Defense===<br />
<br />
===Lactose Intolerance===<br />
<br />
===Vitamin Production===<br />
<br />
===Population Variation===<br />
}}</div>Jkmhttp://2008.igem.org/Template:Caltech_iGEM_08Template:Caltech iGEM 082008-10-24T15:40:09Z<p>Jkm: </p>
<hr />
<div><html><br />
<style type="text/css"><br />
body {background: #FFFFFF}<br />
</style><br />
</html><br />
{|<br />
|-valign="top"<br />
|{{navimg|xsize=960|ysize=36|image=Caltech_header_top.jpg|link=Team:Caltech}}<br />
|}<br> <br />
<div style="font-size:24pt;"><br />
<font face="verdana" style="color:#BB4400"><center>iGEM 2008<br />
</center></font></div><br />
<br><br />
{|<br />
|-valign="top"<br />
|{{navimg|xsize=960|ysize=48|image=Caltech_header_bottom.jpg|link=Team:Caltech}}<br />
|}<br />
<div style="color: #ffffff; background-color: #ffffff; width: 900px"><br />
</div><br />
<br />
<div><br />
{| cellspacing="0"<br />
|-<br />
|style="background-color: #ffffff" width="75px" valign="top"|<br />
<br><center>[[Team:Caltech| <font face="verdana" style="color:#BB4400"> '''Home''' </font>]] <br><br><br />
[[Team:Caltech/Members | <font face="verdana" style="color:#BB4400"> '''People''' </font>]] <br><br><br />
[[Team:Caltech/Project | <font face="verdana" style="color:#BB4400"> '''Project Details''' </font>]] <br><br><br />
[[Team:Caltech/Protocols | <font face="verdana" style="color:#BB4400"> '''Protocols''' </font>]] <br><br><br />
[[Team:Caltech/Parts | <font face="verdana" style="color:#BB4400"> '''Completed Systems''' </font>]] <br><br><br />
[[Team:Caltech/Biosafety | <font face="verdana" style="color:#BB4400"> '''Biosafety''' </font>]] <br><br><br />
<br />
<br />
|width="880" valign="top" style="padding: 10px; border: 5px solid #FFFFFF; color: #000; background-color: white" | <br />
{{{Content}}}<br />
[[Image:Caltech_logo.gif|right]]<br />
{{Clear}}<br />
[[Image:Caltech_footer.jpg|left]]<br />
|}</div>Jkmhttp://2008.igem.org/Team:Caltech/Protocols/rcsA_Lysogen_InductionTeam:Caltech/Protocols/rcsA Lysogen Induction2008-10-24T05:17:15Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">rcsA Lysogen Induction</font></div><br />
__NOTOC__<br />
<br><br />
<br />
#Grow fresh overnight cultures of lysogens of bacteria carrying the rcsA construct.<br />
#Grow fresh overnight cultures wildtype E. coli. [[Image:Induction.png|right|thumb|400px|Diagram of the induction process.]]<br />
#Make a 1:1000 dilution of lysogen culture and incubate the cultures until they reach an OD600 of 0.1 <br />
#* More specifically, when the culture is swirled, cloudiness is observed. Check OD on plate reader as it is important to keep OD constant between trials and experiments.<br />
#At this time also make a 1:1000 dilution of the wildtype E. coli culture. <br />
#Add AHL to bring the concentration within the Lysogen culture to 10 nM.<br />
#Resume incubation at 37 degrees C for 1.5 hours.<br />
#At 1.5 hours, add 5% v/v formaldehyde to the lysogen culture and vortex vigorously.<br />
#Spin down cells at 5000xg for 5 minutes.<br />
#Remove an aliquot of the supernatant. <br />
#Follow the phage [[Team:Caltech/Protocols/Titering|<font style="color:#BB4400">titering</font>]] protocols using the supernatant as the phage solution, and titer against the culture of wildtype E. coli.<br />
#Count plaques the following day to estimate the concentration of phage in supernatant. <br />
<br />
<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/Project/Phage_Pathogen_DefenseTeam:Caltech/Project/Phage Pathogen Defense2008-10-23T19:59:36Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Phage Pathogen Defense</font></div><br />
<br><br />
__NOTOC__<br />
<br />
==Introduction==<br />
[[Image:PathogenicEcoli.jpg|thumb|left|Pathogenic ''E. coli''.]]<br />
There are 10<sup>14</sup> bacterial cells that naturally reside in the human gut, an order of magnitude greater than all the cells in the body. Humans enjoy a mutualistic relationship with intestinal microbiota, wherein the microorganisms perform a host of useful functions, such as processing unused energy substrates, training the immune system, and inhibiting growth of harmful bacterial species [1]. However, not all bacteria populations within the human intestine are benign. There are many pathogenic bacteria which cause a wide variety of diseases when present in the human gut. As one example, cholera is one of the most widespread and damaging of such infectious microorganisms, with the World Health Organization reporting 132,000 cases in 2006 [2]. But cholera is just one of many examples. In the United States alone, there were more than 325,000 hospitalizations from food-borne illnesses in the year of 2006 [3] <br />
<br />
There are many medical treatments for food-borne illnesses, the most commonly prescribed being antibiotics. Unfortunately, antibiotics are indiscriminant and lead to rapid depletion of benign bacterial populations within the intestine. Due to this fact, dietary supplements have been popularized which aim to introduce beneficial bacteria back into the gut after antibiotic treatment; these substances have been termed ‘probiotics’. Natural probiotics have two main advantages: they provide useful functions for the host and they competitively inhibit growth of pathogenic bacteria.<br />
<br />
Natural probiotics do not convey any more advantages than bacteria in a healthy human intestine. Modern synthetic biology techniques should allow us to create an engineered probiotic that goes beyond the limitations of natural probiotics. The viability of such engineered probiotics within humans has already been established [4]. As part of a collaborative effort by the Caltech iGEM team to create a novel probiotic with improved medical applications, this project focuses on engineering a pathogen defense system within ''E. coli''. <br />
<br />
There are many ways for the engineered probiotic to combat pathogens in the large intestine; we chose to use bacteriophages. Bacteriophages present a novel and effective agent for eliminating pathogenic bacteria. Two important factors contribute to a bacteriophage’s effectiveness: bacteriophages are highly infectious and thus highly efficient at destroying targeted populations, and bacteriophages are specific to their hosts. The latter factor is readily illustrated by the coliphage λ, which possesses a mode of infection dependent on an ''E. coli'' specific surface protein, LamB[5]. Benign, non-''E. coli'' intestinal bacteria lack this surface receptor and therefore are immune to destruction. <br />
<br />
===System Design===<br />
<br />
The engineered probiotic acts as a delivery vehicle for phage into the large intestine. There are four important design goals for such as system: (1) the system releases phage into the large intestine, (2) the production of phage is regulated with a high dynamic range between a distinct on and off state, (3) the system can target a wide range of pathogenic bacteria, and (4) the system integrates well within the existing iGEM probiotic. <br />
<br />
An obvious delivery mechanism would be the adaptation of natural lysogens, a phase of the phage life cycle in which the phage integrates its DNA with the host organism. Lysogens lie dormant until disturbed by some external stimuli, but can be stimulated to enter the lytic cycle and release phage. There is one major limitation to this method: phage released will be specific to the lysogenic hosts, ''E. coli''. We can avoid this limitation by incorporating as a lysogen a phage which is not normally infectious to ''E. coli'', such as the P22 phage from ''Salmonella''. This phage could potentially target pathogenic bacteria besides ''E. coli''. <br />
<br />
To demonstrate the feasibility of this approach, a simple model system was developed using λ phage and JW3996-1, a strain of ''E. coli'' immune to λ infection due to a ''lamB'' deletion[5]. In this model, the host is immune to infection unless LamB is expressed from a plasmid. After infection and selection for lysogens, the ''lamB'' plasmid is counterselected against to regain immunity. Extending this model to a more realistic situation, we envision using a phage which targets a pathogenic bacterial species, constitutively expressing the phage receptor within ''E. coli'' to create lysogens, and inducing the lysogens to infect non-''E. coli'' pathogenic bacteria. <br />
<br />
We require one other plasmid which controls the induction of the lysogens to release phage into the environment. Control of this aspect of the system is vital for integration with the overall iGEM project. The induction of λ phage lysogens has been explored since 1950[6], and serves as a model system for the study of other temperate phages. The most famous induction mechanism is ultraviolet radiation (UV). In UV induction of λ lysogens, UV induced DNA damage activates the cellular SOS mechanism, which activates the expression of RecA, a protein involved in SOS response. RecA, in turn, cleaves the λ repressor and leads to phage activation. In this study, activation of the ''recA'' pathway was not readily feasible, so an alternative effector was used to elicit induction. A secondary plasmid expressing the ''E. coli'' regulatory gene, ''rcsA'', is used as the trigger for λ phage induction. ''rcsA'' is one of several regulatory genes which provide a ''recA'' independent pathway for λ induction[7]. Overexpression of RcsA leads to λ induction, and, furthermore, RcsA appears to directly compete with the λ repressor cI. [6] In this project, ''rcsA'' is used to trigger phage production. The effectiveness and dynamic range of ''rcsA'' induction is explored.<br />
<br />
===Constructs===<br />
<br />
[[Image:lambteta.png|thumb|right|300px|Figure 1 - Constructs involved in expression of the ''lamB'' receptor and the TetA cassette ]]<br />
All constructs were made with standard assembly methods. ''lamB'' and ''rcsA'' were cloned out of DH10B genomic DNA. Two families of plasmids were created for this project. The first consists of plasmids which express the ''lamB'' receptor and the tetracycline resistance cassette. The level of ''lamB'' expression required for λ phage infection as unknown, so the strength of the promoter/RBS combination upstream of ''lamB'' was varied between the four constructs of this family.<br />
{{clear}}<br />
[[Image:rcsa.png|thumb|left|300px|Figure 2 - Constructs for characterization of rcsA induction ]]<br />
<br />
The second family consists of constructs used to characterize ''rcsA'' induction of λ phage. ''rcsA'' was placed downstream of an acyl-homoserine lactone (AHL) inducible promoter, LuxR. This promoter was extensively characterized by Doug for the [[Team:Caltech/Project/Population_Variation|<font style="color:#BB4400">oxidative burst project</font>]]. Using flow cytometry data, the peak response for this promoter was determined to be 10 nM AHL. Constructs with 3 different RBS's in front of ''rcsA'' were created to characterize the response of induction. Furthermore, a promoterless ''rcsA'' construct was created as a negative control, and a construct with constitutively active λ repressor ''cI'' driven by J23106 was also assembled.<br />
<br />
{{Clear}}<br />
<br />
==Results==<br />
<br />
===''lamB''<sup>-</sup> Infection===<br />
''lamB'' <sup>-</sup> cells were successfully infected by λ phage while constitutively expressing the ''lamB'' receptor. Furthermore, the level of infection directly correlated with the level of ''lamB'' expression , suggesting that phage infection was limited by the presence of ''lamB'' receptors. Next, lysogens were selected using chloramphenicol resistance. The presence of the ''lamB''/tetA construct within the lysogens was confirmed with colony PCR. This plasmid was successfully removed via tetracycline counterselection[10]. This procedure successfully established a lysogenic strain lacking the ''lamB'' receptor and thus immune to phage infection. <br />
<br />
<gallery widths="375px" heights="250px" ><br />
Image:lamb.png|Figure 3 - λ Infection of Constitutively Active lamB Constructs. Constitutively active ''lamB'' (Table 1) was used to augment the ''lamB''- phenotype in JW3996-1 cells. Cells were titered with a known high concentration stock of phage 1x10<sup>10</sup> phage/mL. The efficiency of infection of ''lamB'' constructs can be seen through the observed phage concentration after titering. Zero plaques were observed for the J23113+33 construct.<br />
Image:LamBExpression.png|Figure 4 - ''lamB'' Infection vs ''lamB'' expression level. Infection levels for the constructs in Figure 3 plotted against the expression levels recorded in the registry for the Promoter/RBSs used.<br />
</gallery><br />
<br />
===''rcsA'' Induction===<br />
<br />
<br />
Overexpression of ''rcsA'' led to induction of λ lysogens. ''rcsA'' was placed downstream of an AHL inducible promoter and overexpressed through induction with 10 nM AHL. Following induction, phage concentration in the supernatant was recorded through titering. This concentration was compared to the phage concentration in the supernatant without ''rcsA'' overexpression, given by a construct where ''rcsA'' is not driven by a promoter. The data show a 40 fold increase in phage concentration between the promoter-less ''rcsA'' construct and the strongest AHL induced ''rcsA'' construct (Figure 5, columns 1 and 4). The promoter-less ''rcsA'' construct should accurately represent background phage levels for the lysogen absent of any ''rcsA'' expression. <br />
Further work was done to characterize the response of the AHL inducible switch under three different ribosome binding sites (RBSs). The data show induction efficiency is directly correlated with strength of ''rcsA'' overexpression (Figure 5, columns 2-4). In each of the constructs, there is a 3 to 5 fold increase in phage levels after induction with 10nm AHL. This increase does not accurately reflect the dynamic range of the AHL inducible promoter, because it does not take into account the background phage levels without the promoter. In the weaker RBSs (B0032 and B0033), the phage concentration from the uninduced supernatant is comparable to the background level from the promoter-less construct (Figure 5, columns 1-3). Thus, in these inducible promoter systems, there is high dynamic range and negligible background compared to basal phage concentrations.. However, in the strongest RBS (B0034), the uninduced phage level is an order of magnitude higher than the promoter-less background level. This high basal level expression suggests that the higher expression levels due to the strong RBS is leading to leaky ''rcsA'' activation of the phage lytic cycle. <br />
<br />
[[Image:Rcsainduction.png|600px|thumb|center|Figure 5 - ''rscA'' induction of phage production. In this figure, each construct was infected with λ phage and lysogens were selected. Lysogens were grown to OD600=0.1 and ''rcsA'' was induced with 10 nM of AHL. 1.5 hours after induction, the supernatant was used to titer wildtype stock of ''E. coli''. Plaques were counted to calculate the phage concentration inside the supernatant after induction. For more information see [[Team:Caltech/Protocols/rcsA_Lysogen_Induction|<font style="color:#BB4400">induction protocol.</font>]]]]<br />
{{Clear}}<br />
<br />
===''cI'' reduces ''rcsA'' induction===<br />
<br />
The high level of background phage induction in the strongest AHL inducible ''rcsA'' switch was not optimal. If the high basal phage levels could be reduced, then the dynamic range of the inducible switch can be greatly increased. cI, the λ repressor, has been shown to directly inhibit UV induction of lysogens [13], and RcsA seems competitively interact with cI in λ induction [7]. Both of these reasons suggest that increased concentration of the λ repressor should lead to lower phage induction levels. ''cI'' was constitutively expressed downstream of the strongest AHL inducible switch. As expected, expression of cI reduces the phage release and ''rcsA'' induction in both the off-state as well as the induced state. Phage concentration was reduced by more than 1.5x106 pfu/mL in both control and induced states (Figure 5, Columns 4 and 5). In Figure 5, for the construct expressing ''cI'', there is an 8 fold increase in phage concentration between induced and un-induced states, more than double the dynamic range of the construct without ''cI'' expression. <br />
<br />
==Conclusion==<br />
We have successfully developed a model system in which bacteriophages not able to infect ''E. coli'' can be integrated as a lysogen within an ''E. coli'' host. We successfully demonstrated this model with bacteriophage λ and the JW3996-1 strain of ''E. coli'' which is immune to λ infection. A system for inducing the release of phage was constructed around the ''E. coli'' regulatory protein RcsA. Work was done to optimize a switch using an AHL inducible promoter for ''rcsA'' induction. RcsA overexpression led to significant phage induction, producing approximately 1x10<sup>8</sup> plaque forming units (pfu)/mL. This value is 4 orders of magnitude greater than the phage induction reported by Rozanov et al. However, direct comparisons cannot be readily made between the data, because the method of overexpression of ''rcsA'' differs between the two papers, and the level at which ''rcsA'' was overexpressed in Rozanov’s study is unknown. The background phage concentration in uninduced lysogens was also much higher than reported in literature [7]. At present, there is no clear cause for this increase in basal induction levels within the λ lysogens. Phage induction via ''rcsA'' can be reduced through lambda repressor overexpression. The mechanism by which RcsA activates the lytic cycle of the dormant prophage is largely unknown; however, there is evidence that RscA acts through inactivation of the phage repressor, ''cI''. [7] Therefore, increased production of the repressor ''cI'' led to a decrease in ''rcsA'' mediated phage induction. The addition of constitutively active ''cI'' repressor led to the construction of an ''rcsA'' phage induction system with a high dynamic range and relatively low background. <br />
<br />
Future work should be focused on broadening the host range of the system. Thus, a key aspect needs to be adapting other temperate phages the model system. A significant result would be the incorporation and production of a non-''E. coli'' bacteriophage by ''E. coli''. A promising candidate for this is the Salmonella phage P22; P22 has been shown to be viably produced in ''E. coli'' when the prophage is expressed[14]. Incorporation of the P22 prophage should be feasible following the framework of the current model. Furthermore, co-culture experiments with the current constructs and wild-type ''E. coli'' could be used to validate the current system's effectiveness at eliminating pathogens. <br />
<br />
==References==<br />
# Sears CL. 2005. "'''A dynamic partnership: Celebrating our gut flora.'''" Anaerobe, Volume 11, Issue 5, Pages 247-251.<br />
# "'''Cholera Statistics 2006.'''" World Health Organization. 4 July 2008 <http://www.who.int/wer/2006/wer8131.pdf>. <br />
# "'''Food Borne Illness FAQ.'''" World Health Organization. 5 July 2008 <http://www.who.int/mediacentre/factsheets/fs237/en/>. <br />
# Westendorf AM, Gunzer F, Deppenmeier S, Tapadar D, Hunger JK, Schmidt MA, Buer J, and Bruder D. “'''Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules.'''” FEMS Immunol Med Microbiol 2005 Mar 1; 43(3) 373-84. <br />
# Clément JM, Hofnung M. “'''Gene sequence of the lambda receptor, an outer membrane protein of E. coli K12.'''” Cell. 1981 Dec; 27(3 Pt 2):507–514.<br />
# Lwoff, A., L. Siminovitch, and N. Kjeldgaard. 1950. “'''Induction de la production de bacteriophages chez une bacterie lysogene.'''” Ann. Inst. Pasteur (Paris) 79:815-859.<br />
# Rozanov, Dmitry V., Richard D'ari, and Sergey P. Sineoky. "'''RecA-Independent Pathways of Lambdoid Prophage Induction in Escherichia coli.'''" Journal of Bacteriology 180 (1998): 6306-315.<br />
# “'''Biobricks Assembly'''” OpenWetware <http://openwetware.org/wiki/BioBricks_construction_tutorial><br />
# Sambrook, Joe, and David Russell. "'''Molecular Cloning.'''" New York: Cold Spring Harbor Laboratory P, 2000.<br />
# Maloy, Stanley R., and William D. Nunn. "'''Selection for Loss of Tetracycline Resistance by Escherichia coli.'''" Journal of Bacteriology 45 (1985): 1110-112.<br />
# Grayson, Paul. "'''Titering.'''" California Institute of Technology. <http://www.rpgroup.caltech.edu/wiki/index.php?title=titering>.<br />
# Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H. “'''Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.'''” Mol Syst Biol. 2006;2:2006.0008<br />
# John Baluch, Raquel Sussman. “'''Correlation Between UV Dose Requirement for Lambda Bacteriophage Induction and Lambda Repressor Concentration'''” Journal of Virology, June 1978, P. 595-602<br />
# Botstein, David and Ira Herskowitz. "'''Properties of hybrids between Salmonella phage P22 and coliphage lambda.'''" Nature. 1974 Oct 18;251(5476):584-9.<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/Project/Lactose_intoleranceTeam:Caltech/Project/Lactose intolerance2008-10-23T19:43:17Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Curing Lactose Intolerance</font></div><br />
<br />
==Introduction==<br />
The gut flora of our digestive tract contains microorganisms that perform various useful functions for their hosts. Examples of such functions include growth inhibition of harmful microorganisms<sup>1</sup>, defense against the causes of many forms of Inflammatory Bowel Disease<sup>2</sup>, and the fermentation of carbohydrates and other molecules the human body cannot normally digest. Some bacterial strains, including the E. coli strain ‘Nissle 1917’, can persist in the gut of mice for months. Engineering these bacteria provide a new platform to treat various human diseases. <br>Lactose intolerance is characterized by the inability to break down lactose in the small intestine. The undigested lactose instead passes to the large intestine, leading to two negative processes: osmotic imbalance and bacterial fermentation. High lactose levels raise the osmolarity of the colon, causing diarrhea. In addition, gut microbes metabolize the lactose into methane gas, causing abdominal pain. Both problems must be addressed in order to fully treat lactose intolerance. If we simply have our strain metabolize lactose, another strain will further ferment the byproducts, resulting in the same side effects. <br><br />
[[image:LysisCaltech.JPG|thumb|200px|left|In frame 1, Beta-Gal is being produced at a high constitutive level at all times. When lactose enters the colon in frame 2, the cell uptakes lactose via mutated LacY which in turn induces lysis genes lysing the cell, and releasing Beta-Gal in the colon cleaving lactose. The host will then uptake glucose and galactose.]]<br />
<br>Instead, we have our engineered two plasmids which allow the host to uptake lactose, clearing the sugar from the colon. To do this, we have engineered the ‘Nissle 1917’ strain to release ß-galactosidase, an enzyme that cleaves lactose into glucose and galactose. Since protein secretion is difficult in E. coli, our cells were engineered to lyse in order to release ß-galactosidase. However, lysis must occur only when lactose is present in the gut. If lysis were to occur when lactose was absent, this would kill all our engineered cells at any given moment. The cells also express a lactose permease, allowing the strain to sense lactose in all conditions. <br><br><br><Br><Br><Br><br />
<br />
<br />
==System Design==<br />
===Lactose Regulator===<br />
[[image:Lactose_regulator.JPG|thumb|200px|left|Figure 1. A mutated LacY allows the uptake of lactose while LacZ produces large amounts of ß-galactosidase. The plasmid contains a strong constitutive promoter regulating both genes and a ColE1 replication origin.]]<br />
The first plasmid consists of a synthetic lactose operon under strong constitutive expression (Fig. 1). Our synthetic lactose operon encodes the ß-galactosidase LacZ and a lactose permease LacY. LacY is a membrane protein that actively transports lactose into the cell. Since most membrane proteins are toxic when overexpressed, we optimized our system to express appropriate levels of LacY without killing the cell. In the end, we want to express as much ß-galactosidase as we can to cleave as much lactose present in the large intestine. <br>The human gut is an unpredictable environment, and we wish our engineered cells to behave reliably despite this variability. E. coli are unable to uptake lactose in the presence of glucose, a phenomenon known as carbon catabolite repression. Catabolite repression is mediated by Enzyme IIA Glucose (IIAGluc), which inhibits the uptake of lactose in the presence of glucose by binding to LacY<sup>3</sup>. Previous research has identified various LacY mutations that prevent this inhibition and achieve increased uptake of lactose in the presence of glucose<sup>4</sup>.<br />
<br />
{{clear}}<br />
<br />
===Lactose-Induced Lysis===<br />
[[image:Lactose-inducible_lysis.JPG|thumb|200px|right|Figure 2. The lysis cassette plasmid acts as a lactose sensor. Intracellular lactose accumulation induces overexpression of the lysis cassette. Lactose inhibits binding of the LacI repressor to the promoter and P1 high copy origin of replication. A mutated holin gene in the lysis cassette allows faster lysis times.]]<br />
The second plasmid contains lactose inducible expression of λ phage lysis genes under a lactose inducible promoter (Fig. 2). To release ß-galactosidase, the cells will lyse when enough lactose is present in the cell to induce the expression of the lysis genes. It is important that the lactose-inducible promoter is tightly regulated since leaky expression will cause spontaneous lysis. To accomplish tight expression, specific lac promoters will keep our system from lysing<sup>5</sup>. In addition, the plasmid copy number remains low copy until induced with lactose, when the plasmid copy number increases to high copy. <br>Using wild type λ phage lysis genes, lysis occurs 40-45 minutes after induction by lactose. Decreasing the lag time will reduce the extent of lactose fermentation and therefore produce fewer deleterious effects. Previous research has uncovered mutations that shorten the lysis time to approximately 10-15 minutes<sup>6</sup>, and these mutations will be incorporated into our final construct.<br />
<br />
{{clear}}<br />
<br />
==Results==<br />
===Lactose Regulator===<br />
[[IMAGE:Promoter_strength.JPG|thumb|300px|left|Figure 3. Varying the expression levels of LacY with different promoters and ribosome binding sites. Cells died at our two highest expression levels, but survived on the other four. The construct with the weakest RBS was selected since we could express LacZ in the same operon with a strong promoter. This combination allows our cell to express high levels of LacZ and non-toxic levels of LacY.]]<br />
In the first plasmid, it was discovered that LacY is toxic when overexpressed. To determine safe expression levels, constructs were built with varying levels of LacY expression. Six plasmids were built from combinations of three different promoters and two different ribosome binding sites (Fig. 3). The cells appeared to have died when expressing the strongest and medium strength promoters along with the strongest ribosome binding site. Our information was based off transformants, and the plates with no transformants were classified as dead cells due to high LacY expression. Based on these results, we decided to express our synthetic lac operon by combining the strongest constitutive promoter with the weakest RBS for LacY and the strongest RBS for LacZ, preventing LacY toxicity while expressing LacZ in large quantities. <br>As mentioned earlier, our cells had to inhibit carbon catabolite repression to ensure uptake of lactose under any condition. Various mutations, including the insertion of two histidyl residues between amino acids S194 and A195 (ref 4) in the LacY gene, prevent the inhibition by IIAGluc. These insertions have been made and will be tested in future studies. <br />
<br />
{{clear}}<br />
<br />
[[image:Figure_1_beta_assay.JPG|thumb|300px|right|Figure 4. ß-galactosidase activity of plasmid #1, with and without the strong constitutive promoter. Error bars on the data with a promoter show the standard deviation of two measurements.]]The ß-galactosidase assay was performed on the synthetic network containing the lactose operon (Fig. 4). The first assay was performed on a strain containing the plasmid lacking the strong constitutive promoter. This assay was performed to simulate the eventual system regulation. We saw significant levels of ß-galactosidase, a surprising result since the plasmid lacked a promoter. These levels are likely due to spurious transcription amplified by our strong ribosome binding site and high copy plasmid. We then repeated the assay with the strong constitutive promoter to observe the dynamic range of LacZ expression in this system. Under these conditions, the system shows a 8-fold dynamic range, from 2000 MU without a promoter to 16000 MU with a promoter.<br />
{{clear}}<br />
<br />
===Lactose-Induced Lysis===<br />
Our second plasmid was not finished, but necessary parts have been cloned for future construction. There remains one more cloning step to place the lysis cassette into the vector containing the promoters. The single base mutations in the lysis cassette were not made, and if time allowed, they would have been completed as the final step.<br />
<br />
==Discussion==<br />
Our final system was not completely finished; however, future construction will be minimal. The synthetic lac operon plasmid was completely constructed with the insertion of two histidyl residues in their appropriate locations. Our lactose-induced lysis plasmid was one cloning step away from being completed. The promoter and flanking terminator were cloned in parallel with the lysis cassette and a terminator. The last cloning step would be to ligate the two together. In addition, the two separate point mutations have not been made, and the final step in our cloning would be to add those mutations.<br>After completing the construction phase, we would characterize our system. Characterization will be completed on a construct containing our lactose inducible promoters and GFP. This would show our promoters can maintain tight expression of GFP from uninduced to induced. We would combine this construct with our first plasmid containing the mutated LacY, to show we can achieve induction by lactose even in the presence of glucose. To show the effect our lysis plasmid, it would be necessary to achieve lysis with lactose. In addition, we want to show that we can achieve lysis with lactose in the presence of glucose. Finally, the final assay we want to perform on our system would show our cells able to uptake lactose in the presence of glucose, and lyse soon after. Once the cells lyse, the cell lysis should contain ß-galactosidase at levels close to what we received from our ß-galactosidase assay on plasmid 1. Once characterization is completed, our construct would be moved to the ‘Nissle 1917’ strain where further characterization and modifications would take place.<br />
<br />
<br />
==Methods==<br />
Methods can be found [[Team:Caltech/Protocols|<font style="color:#BB4400">here</font>]]<br />
<br />
==Parts==<br />
{{{!}} border="1"<br />
! Registry Number !! Plasmid !! Part !! Status <br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_K137002 BBa_K137002]<br />
{{!}} pSB1A2 {{!}}{{!}} LacY {{!}}{{!}} Constructed <br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_K137126 BBa_K137126]<br />
{{!}} pSB1A2 {{!}}{{!}} Lysis Cassette {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S03970 BBa_S03970]<br />
{{!}} pSB1A2 {{!}}{{!}} B0031 + LacY {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S03971 BBa_S03971]<br />
{{!}} pSB1A2 {{!}}{{!}} B0033 + LacY {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S03973 BBa_S03973]<br />
{{!}} pSB1AK3 {{!}}{{!}} B0034-LacZ + B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04107 BBa_S04107]<br />
{{!}} pSB1AK3 {{!}}{{!}} B0031-LacY + B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04108 BBa_S04108]<br />
{{!}} pSB1AK3 {{!}}{{!}} B0033-LacY + B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04109 BBa_S04109]<br />
{{!}} J61002 {{!}}{{!}} J23113 + B0031-LacY-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04122 BBa_S04122]<br />
{{!}} J61002 {{!}}{{!}} J23106 + B0031-LacY-B0015 {{!}}{{!}} Unavailable<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04123 BBa_S04123]<br />
{{!}} J61002 {{!}}{{!}} J23100 + B0031-LacY-B0015 {{!}}{{!}} Unavailable<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04110 BBa_S04110]<br />
{{!}} J61002 {{!}}{{!}} J23113 + B0033-LacY-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04111 BBa_S04111]<br />
{{!}} J61002 {{!}}{{!}} J23106 + B0033-LacY-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04112 BBa_S04112]<br />
{{!}} J61002 {{!}}{{!}} J23100 + B0033-LacY-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04041 BBa_S04041]<br />
{{!}} pSB1A2 {{!}}{{!}} B0033 + LacY +H {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04022 BBa_S04022]<br />
{{!}} pSB1A2 {{!}}{{!}} B0033 + LacY +2H {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04113 BBa_S04113]<br />
{{!}} pSB1AK3 {{!}}{{!}} B0033-LacY +H + B0034-LacZ-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04054 BBa_S04054]<br />
{{!}} pSB1AK3 {{!}}{{!}} B0033-LacY +2H + B0034-LacZ-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04055 BBa_S04055]<br />
{{!}} J61002 {{!}}{{!}} Final synthetic LacYZ operon {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_K137125 BBa_K137125]<br />
{{!}} pSB1A2 {{!}}{{!}} LacI Repressed Promoter B4 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04114 BBa_S04114]<br />
{{!}} pSB2K3 {{!}}{{!}} Lysis + B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04105 BBa_S04105]<br />
{{!}} pSB2K3 {{!}}{{!}} B0034-LacI + B0015-B4 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04106 BBa_S04106]<br />
{{!}} pSB2K3 {{!}}{{!}} J23100 + B0034-LacI-B0015-B4 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_K137131 BBa_K137131]<br />
{{!}} pSB2K3 {{!}}{{!}} J23100-B0034-LacI-B0015-B4 + Lysis-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_K137132 BBa_K137132]<br />
{{!}} pSB2K3 {{!}}{{!}} J23100-B0034-LacI-B0015-B4 + B0034-GFP-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
{{!}}}<br />
<br />
<br />
==References==<br />
# Guarner F and Malagelada JR. 2003. '''Gut flora in health and disease.''' ''The Lancet'', Volume 361, Issue 9356, 8 February 2003, Pages 512-519. <br />
# Sears CL. 2005. '''A dynamic partnership: Celebrating our gut flora.''' ''Anaerobe'', Volume 11, Issue 5, Pages 247-251.<br />
# Deutscher, J., Francke, C. and Postma, P.W. 2006. '''How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bactertia.''' ''Microbial and Molecular Biology Reviews.'' 2006: 939-1031<br />
# Hoischen, C., Levin, J., Pitaknarongphorn, S., Reizer, J., and Saier , M. H. Jr. 1996. '''Involvement of the central loop of the lactose permease of Escherichia coli in its allosteris regulation by the glucose-specific enzyme IIA of the phosphoenolpyruvate-dependent phosphotransferase system.''' ''J. Bacteriol.'' 178: 6082-6086<br />
# Cox RS III, Surette MG, Elowitz MB. '''Programming gene expression with combinatorial promoters.''' ''Mol. Syst. Biol.'' 2007;3:145.<br />
# Gründling, A., M. D. Manson, and R. Young. 2001. '''Holins kill without warning.''' ''Proc. Natl. Acad. Sci.'' USA 98:9348-9352.<br />
<br />
<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/Project/Lactose_intoleranceTeam:Caltech/Project/Lactose intolerance2008-10-23T19:41:53Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Curing Lactose Intolerance</font></div><br />
<br />
==Introduction==<br />
The gut flora of our digestive tract contains microorganisms that perform various useful functions for their hosts. Examples of such functions include growth inhibition of harmful microorganisms<sup>1</sup>, defense against the causes of many forms of Inflammatory Bowel Disease<sup>2</sup>, and the fermentation of carbohydrates and other molecules the human body cannot normally digest. Some bacterial strains, including the E. coli strain ‘Nissle 1917’, can persist in the gut of mice for months. Engineering these bacteria provide a new platform to treat various human diseases. <br>Lactose intolerance is characterized by the inability to break down lactose in the small intestine. The undigested lactose instead passes to the large intestine, leading to two negative processes: osmotic imbalance and bacterial fermentation. High lactose levels raise the osmolarity of the colon, causing diarrhea. In addition, gut microbes metabolize the lactose into methane gas, causing abdominal pain. Both problems must be addressed in order to fully treat lactose intolerance. If we simply have our strain metabolize lactose, another strain will further ferment the byproducts, resulting in the same side effects. <br><br />
[[image:LysisCaltech.JPG|thumb|200px|left|In frame 1, Beta-Gal is being produced at a high constitutive level at all times. When lactose enters the colon in frame 2, the cell uptakes lactose via mutated LacY which in turn induces lysis genes lysing the cell, and releasing Beta-Gal in the colon cleaving lactose. The host will then uptake glucose and galactose.]]<br />
<br>Instead, we have our engineered two plasmids which allow the host to uptake lactose, clearing the sugar from the colon. To do this, we have engineered the ‘Nissle 1917’ strain to release ß-galactosidase, an enzyme that cleaves lactose into glucose and galactose. Since protein secretion is difficult in E. coli, our cells were engineered to lyse in order to release ß-galactosidase. However, lysis must occur only when lactose is present in the gut. If lysis were to occur when lactose was absent, this would kill all our engineered cells at any given moment. The cells also express a lactose permease, allowing the strain to sense lactose in all conditions. <br><br><br><Br><Br><Br><br />
<br />
<br />
==System Design==<br />
===Lactose Regulator===<br />
[[image:Lactose_regulator.JPG|thumb|200px|left|Figure 1. A mutated LacY allows the uptake of lactose while LacZ produces large amounts of ß-galactosidase. The plasmid contains a strong constitutive promoter regulating both genes and a ColE1 replication origin.]]<br />
The first plasmid consists of a synthetic lactose operon under strong constitutive expression (Fig. 1). Our synthetic lactose operon encodes the ß-galactosidase LacZ and a lactose permease LacY. LacY is a membrane protein that actively transports lactose into the cell. Since most membrane proteins are toxic when overexpressed, we optimized our system to express appropriate levels of LacY without killing the cell. In the end, we want to express as much ß-galactosidase as we can to cleave as much lactose present in the large intestine. <br>The human gut is an unpredictable environment, and we wish our engineered cells to behave reliably despite this variability. E. coli are unable to uptake lactose in the presence of glucose, a phenomenon known as carbon catabolite repression. Catabolite repression is mediated by Enzyme IIA Glucose (IIAGluc), which inhibits the uptake of lactose in the presence of glucose by binding to LacY<sup>3</sup>. Previous research has identified various LacY mutations that prevent this inhibition and achieve increased uptake of lactose in the presence of glucose<sup>4</sup>.<br />
<br />
{{clear}}<br />
<br />
===Lactose-Induced Lysis===<br />
[[image:Lactose-inducible_lysis.JPG|thumb|200px|right|Figure 2. The lysis cassette plasmid acts as a lactose sensor. Intracellular lactose accumulation induces overexpression of the lysis cassette. Lactose inhibits binding of the LacI repressor to the promoter and P1 high copy origin of replication. A mutated holin gene in the lysis cassette allows faster lysis times.]]<br />
The second plasmid contains lactose inducible expression of λ phage lysis genes under a lactose inducible promoter (Fig. 2). To release ß-galactosidase, the cells will lyse when enough lactose is present in the cell to induce the expression of the lysis genes. It is important that the lactose-inducible promoter is tightly regulated since leaky expression will cause spontaneous lysis. To accomplish tight expression, specific lac promoters will keep our system from lysing<sup>5</sup>. In addition, the plasmid copy number remains low copy until induced with lactose, when the plasmid copy number increases to high copy. <br>Using wild type λ phage lysis genes, lysis occurs 40-45 minutes after induction by lactose. Decreasing the lag time will reduce the extent of lactose fermentation and therefore produce fewer deleterious effects. Previous research has uncovered mutations that shorten the lysis time to approximately 10-15 minutes<sup>6</sup>, and these mutations will be incorporated into our final construct.<br />
<br />
{{clear}}<br />
<br />
==Results==<br />
===Lactose Regulator===<br />
[[IMAGE:Promoter_strength.JPG|thumb|300px|left|Figure 3. Varying the expression levels of LacY with different promoters and ribosome binding sites. Cells died at our two highest expression levels, but survived on the other four. The construct with the weakest RBS was selected since we could express LacZ in the same operon with a strong promoter. This combination allows our cell to express high levels of LacZ and non-toxic levels of LacY.]]<br />
In the first plasmid, it was discovered that LacY is toxic when overexpressed. To determine safe expression levels, constructs were built with varying levels of LacY expression. Six plasmids were built from combinations of three different promoters and two different ribosome binding sites (Fig. 3). The cells appeared to have died when expressing the strongest and medium strength promoters along with the strongest ribosome binding site. Our information was based off transformants, and the plates with no transformants were classified as dead cells due to high LacY expression. Based on these results, we decided to express our synthetic lac operon by combining the strongest constitutive promoter with the weakest RBS for LacY and the strongest RBS for LacZ, preventing LacY toxicity while expressing LacZ in large quantities. <br>As mentioned earlier, our cells had to inhibit carbon catabolite repression to ensure uptake of lactose under any condition. Various mutations, including the insertion of two histidyl residues between amino acids S194 and A195 (ref 4) in the LacY gene, prevent the inhibition by IIAGluc. These insertions have been made and will be tested in future studies. <br />
<br />
{{clear}}<br />
<br />
[[image:Figure_1_beta_assay.JPG|thumb|300px|right|Figure 4. ß-galactosidase activity of plasmid #1, with and without the strong constitutive promoter. Error bars on the data with a promoter show the standard deviation of two measurements.]]The ß-galactosidase assay was performed on the synthetic network containing the lactose operon (Fig. 4). The first assay was performed on a strain containing the plasmid lacking the strong constitutive promoter. This assay was performed to simulate the eventual system regulation. We saw significant levels of ß-galactosidase, a surprising result since the plasmid lacked a promoter. These levels are likely due to spurious transcription amplified by our strong ribosome binding site and high copy plasmid. We then repeated the assay with the strong constitutive promoter to observe the dynamic range of LacZ expression in this system. Under these conditions, the system shows a 8-fold dynamic range, from 2000 MU without a promoter to 16000 MU with a promoter.<br />
{{clear}}<br />
<br />
===Lactose-Induced Lysis===<br />
Our second plasmid was not finished, but necessary parts have been cloned for future construction. There remains one more cloning step to place the lysis cassette into the vector containing the promoters. The single base mutations in the lysis cassette were not made, and if time allowed, they would have been completed as the final step.<br />
<br />
==Discussion==<br />
Our final system was not completely finished; however, future construction will be minimal. The synthetic lac operon plasmid was completely constructed with the insertion of two histidyl residues in their appropriate locations. Our lactose-induced lysis plasmid was one cloning step away from being completed. The promoter and flanking terminator were cloned in parallel with the lysis cassette and a terminator. The last cloning step would be to ligate the two together. In addition, the two separate point mutations have not been made, and the final step in our cloning would be to add those mutations.<br>After completing the construction phase, we would characterize our system. Characterization will be completed on a construct containing our lactose inducible promoters and GFP. This would show our promoters can maintain tight expression of GFP from uninduced to induced. We would combine this construct with our first plasmid containing the mutated LacY, to show we can achieve induction by lactose even in the presence of glucose. To show the effect our lysis plasmid, it would be necessary to achieve lysis with lactose. In addition, we want to show that we can achieve lysis with lactose in the presence of glucose. Finally, the final assay we want to perform on our system would show our cells able to uptake lactose in the presence of glucose, and lyse soon after. Once the cells lyse, the cell lysis should contain ß-galactosidase at levels close to what we received from our ß-galactosidase assay on plasmid 1. Once characterization is completed, our construct would be moved to the ‘Nissle 1917’ strain where further characterization and modifications would take place.<br />
<br />
<br />
==Methods==<br />
Methods can be found [[Team:Caltech/Protocols|<font style="color:#BB4400">here</font>]]<br />
<br />
==Parts==<br />
{{{!}} border="1"<br />
! Registry Number !! Plasmid !! Part !! Status <br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_K137002 BBa_K137002]<br />
{{!}} pSB1A2 {{!}}{{!}} LacY {{!}}{{!}} Constructed <br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_K137126 BBa_K137126]<br />
{{!}} pSB1A2 {{!}}{{!}} Lysis Cassette {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S03970 BBa_S03970]<br />
{{!}} pSB1A2 {{!}}{{!}} B0031 + LacY {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S03971 BBa_S03971]<br />
{{!}} pSB1A2 {{!}}{{!}} B0033 + LacY {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S03973 BBa_S03973]<br />
{{!}} pSB1AK3 {{!}}{{!}} B0034-LacZ + B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04107 BBa_S04107]<br />
{{!}} pSB1AK3 {{!}}{{!}} B0031-LacY + B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04108 BBa_S04108]<br />
{{!}} pSB1AK3 {{!}}{{!}} B0033-LacY + B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04109 BBa_S04109]<br />
{{!}} J61002 {{!}}{{!}} J23113 + B0031-LacY-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04122 BBa_S04122]<br />
{{!}} J61002 {{!}}{{!}} J23106 + B0031-LacY-B0015 {{!}}{{!}} Unavailable<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04123 BBa_S04123]<br />
{{!}} J61002 {{!}}{{!}} J23100 + B0031-LacY-B0015 {{!}}{{!}} Unavailable<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04110 BBa_S04110]<br />
{{!}} J61002 {{!}}{{!}} J23113 + B0033-LacY-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04111 BBa_S04111]<br />
{{!}} J61002 {{!}}{{!}} J23106 + B0033-LacY-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04112 BBa_S04112]<br />
{{!}} J61002 {{!}}{{!}} J23100 + B0033-LacY-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04041 BBa_S04041]<br />
{{!}} pSB1A2 {{!}}{{!}} B0033 + LacY +H {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04022 BBa_S04022]<br />
{{!}} pSB1A2 {{!}}{{!}} B0033 + LacY +2H {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04113 BBa_S04113]<br />
{{!}} pSB1AK3 {{!}}{{!}} B0033-LacY +H + B0034-LacZ-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04054 BBa_S04054]<br />
{{!}} pSB1AK3 {{!}}{{!}} B0033-LacY +2H + B0034-LacZ-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04055 BBa_S04055]<br />
{{!}} J61002 {{!}}{{!}} J23100 + B0033-LacY SDM +2H-B0034-LacZ-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_K137125 BBa_K137125]<br />
{{!}} pSB1A2 {{!}}{{!}} LacI Repressed Promoter B4 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04114 BBa_S04114]<br />
{{!}} pSB2K3 {{!}}{{!}} Lysis + B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04116 BBa_S04116]<br />
{{!}} pSB2K3 {{!}}{{!}} B0015 + B4 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04105 BBa_S04105]<br />
{{!}} pSB2K3 {{!}}{{!}} B0034-LacI + B0015-B4 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_S04106 BBa_S04106]<br />
{{!}} pSB2K3 {{!}}{{!}} J23100 + B0034-LacI-B0015-B4 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_K137131 BBa_K137131]<br />
{{!}} pSB2K3 {{!}}{{!}} J23100-B0034-LacI-B0015-B4 + Lysis-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
! [http://partsregistry.org/Part:BBa_K137132 BBa_K137132]<br />
{{!}} pSB2K3 {{!}}{{!}} J23100-B0034-LacI-B0015-B4 + B0034-GFP-B0015 {{!}}{{!}} Constructed<br />
{{!}}-<br />
{{!}}}<br />
<br />
<br />
==References==<br />
# Guarner F and Malagelada JR. 2003. '''Gut flora in health and disease.''' ''The Lancet'', Volume 361, Issue 9356, 8 February 2003, Pages 512-519. <br />
# Sears CL. 2005. '''A dynamic partnership: Celebrating our gut flora.''' ''Anaerobe'', Volume 11, Issue 5, Pages 247-251.<br />
# Deutscher, J., Francke, C. and Postma, P.W. 2006. '''How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bactertia.''' ''Microbial and Molecular Biology Reviews.'' 2006: 939-1031<br />
# Hoischen, C., Levin, J., Pitaknarongphorn, S., Reizer, J., and Saier , M. H. Jr. 1996. '''Involvement of the central loop of the lactose permease of Escherichia coli in its allosteris regulation by the glucose-specific enzyme IIA of the phosphoenolpyruvate-dependent phosphotransferase system.''' ''J. Bacteriol.'' 178: 6082-6086<br />
# Cox RS III, Surette MG, Elowitz MB. '''Programming gene expression with combinatorial promoters.''' ''Mol. Syst. Biol.'' 2007;3:145.<br />
# Gründling, A., M. D. Manson, and R. Young. 2001. '''Holins kill without warning.''' ''Proc. Natl. Acad. Sci.'' USA 98:9348-9352.<br />
<br />
<br />
}}</div>Jkmhttp://2008.igem.org/File:Rcsainduction.pngFile:Rcsainduction.png2008-10-23T18:44:27Z<p>Jkm: uploaded a new version of "Image:Rcsainduction.png"</p>
<hr />
<div>rcsA induction of lambda lysogens.</div>Jkmhttp://2008.igem.org/Team:Caltech/Project/Phage_Pathogen_DefenseTeam:Caltech/Project/Phage Pathogen Defense2008-10-23T18:43:00Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Phage Pathogen Defense</font></div><br />
<br><br />
__NOTOC__<br />
<br />
==Introduction==<br />
[[Image:PathogenicEcoli.jpg|thumb|left|Pathogenic ''E. coli''.]]<br />
There are 10<sup>14</sup> bacterial cells that naturally reside in the human gut, an order of magnitude greater than all the cells in the body. Humans enjoy a mutualistic relationship with intestinal microbiota, wherein the microorganisms perform a host of useful functions, such as processing unused energy substrates, training the immune system, and inhibiting growth of harmful bacterial species [1]. However, not all bacteria populations within the human intestine are benign. There are many pathogenic bacteria which cause a wide variety of diseases when present in the human gut. As one example, cholera is one of the most widespread and damaging of such infectious microorganisms, with the World Health Organization reporting 132,000 cases in 2006 [2]. But cholera is just one of many examples. In the United States alone, there were more than 325,000 hospitalizations from food-borne illnesses in the year of 2006 [3] <br />
<br />
There are many medical treatments for food-borne illnesses, the most commonly prescribed being antibiotics. Unfortunately, antibiotics are indiscriminant and lead to rapid depletion of benign bacterial populations within the intestine. Due to this fact, dietary supplements have been popularized which aim to introduce beneficial bacteria back into the gut after antibiotic treatment; these substances have been termed ‘probiotics’. Natural probiotics have two main advantages: they provide useful functions for the host and they competitively inhibit growth of pathogenic bacteria.<br />
<br />
Natural probiotics do not convey any more advantages than bacteria in a healthy human intestine. Modern synthetic biology techniques should allow us to create an engineered probiotic that goes beyond the limitations of natural probiotics. The viability of such engineered probiotics within humans has already been established [4]. As part of a collaborative effort by the Caltech iGEM team to create a novel probiotic with improved medical applications, this project focuses on engineering a pathogen defense system within ''E. coli''. <br />
<br />
There are many ways for the engineered probiotic to combat pathogens in the large intestine; we chose to use bacteriophages. Bacteriophages present a novel and effective agent for eliminating pathogenic bacteria. Two important factors contribute to a bacteriophage’s effectiveness: bacteriophages are highly infectious and thus highly efficient at destroying targeted populations, and bacteriophages are specific to their hosts. The latter factor is readily illustrated by the coliphage λ, which possesses a mode of infection dependent on an ''E. coli'' specific surface protein, LamB[5]. Benign, non-''E. coli'' intestinal bacteria lack this surface receptor and therefore are immune to destruction. <br />
<br />
===System Design===<br />
<br />
The engineered probiotic acts as a delivery vehicle for phage into the large intestine. There are four important design goals for such as system: (1) the system releases phage into the large intestine, (2) the production of phage is regulated with a high dynamic range between a distinct on and off state, (3) the system can target a wide range of pathogenic bacteria, and (4) the system integrates well within the existing iGEM probiotic. <br />
<br />
An obvious delivery mechanism would be the adaptation of natural lysogens, a phase of the phage life cycle in which the phage integrates its DNA with the host organism. Lysogens lie dormant until disturbed by some external stimuli, but can be stimulated to enter the lytic cycle and release phage. There is one major limitation to this method: phage released will be specific to the lysogenic hosts, ''E. coli''. We can avoid this limitation by incorporating as a lysogen a phage which is not normally infectious to ''E. coli'', such as the P22 phage from ''Salmonella''. This phage could potentially target pathogenic bacteria besides ''E. coli''. <br />
<br />
To demonstrate the feasibility of this approach, a simple model system was developed using λ phage and JW3996-1, a strain of ''E. coli'' immune to λ infection due to a ''lamB'' deletion[5]. In this model, the host is immune to infection unless LamB is expressed from a plasmid. After infection and selection for lysogens, the ''lamB'' plasmid is counterselected against to regain immunity. Extending this model to a more realistic situation, we envision using a phage which targets a pathogenic bacterial species, constitutively expressing the phage receptor within ''E. coli'' to create lysogens, and inducing the lysogens to infect non-''E. coli'' pathogenic bacteria. <br />
<br />
We require one other plasmid which controls the induction of the lysogens to release phage into the environment. Control of this aspect of the system is vital for integration with the overall iGEM project. The induction of λ phage lysogens has been explored since 1950[6], and serves as a model system for the study of other temperate phages. The most famous induction mechanism is ultraviolet radiation (UV). In UV induction of λ lysogens, UV induced DNA damage activates the cellular SOS mechanism, which activates the expression of RecA, a protein involved in SOS response. RecA, in turn, cleaves the λ repressor and leads to phage activation. In this study, activation of the ''recA'' pathway was not readily feasible, so an alternative effector was used to elicit induction. A secondary plasmid expressing the ''E. coli'' regulatory gene, ''rcsA'', is used as the trigger for λ phage induction. ''rcsA'' is one of several regulatory genes which provide a ''recA'' independent pathway for λ induction[7]. Overexpression of RcsA leads to λ induction, and, furthermore, RcsA appears to directly compete with the λ repressor cI. [6] In this project, ''rcsA'' is used to trigger phage production. The effectiveness and dynamic range of ''rcsA'' induction is explored.<br />
<br />
===Constructs===<br />
<br />
[[Image:lambteta.png|thumb|right|300px|Figure 1 - Constructs involved in expression of the ''lamB'' receptor and the TetA cassette ]]<br />
All constructs were made with standard assembly methods. ''lamB'' and ''rcsA'' were cloned out of DH10B genomic DNA. Two families of plasmids were created for this project. The first consists of plasmids which express the ''lamB'' receptor and the tetracycline resistance cassette. The level of ''lamB'' expression required for λ phage infection as unknown, so the strength of the promoter/RBS combination upstream of ''lamB'' was varied between the four constructs of this family.<br />
{{clear}}<br />
[[Image:rcsa.png|thumb|left|300px|Figure 2 - Constructs for characterization of rcsA induction ]]<br />
<br />
The second family consists of constructs used to characterize ''rcsA'' induction of λ phage. ''rcsA'' was placed downstream of an acyl-homoserine lactone (AHL) inducible promoter, LuxR. This promoter was extensively characterized by Doug for the [[Team:Caltech/Project/Population_Variation|<font style="color:#BB4400">oxidative burst project</font>]]. Using flow cytometry data, the peak response for this promoter was determined to be 10 nM AHL. Constructs with 3 different RBS's in front of ''rcsA'' were created to characterize the response of induction. Furthermore, a promoterless ''rcsA'' construct was created as a negative control, and a construct with constitutively active λ repressor ''cI'' driven by J23106 was also assembled.<br />
<br />
{{Clear}}<br />
<br />
==Results==<br />
<br />
===''lamB''<sup>-</sup> Infection===<br />
''lamB'' <sup>-</sup> cells were successfully infected by λ phage while constitutively expressing the ''lamB'' receptor. Furthermore, the level of infection directly correlated with the level of ''lamB'' expression , suggesting that phage infection was limited by the presence of ''lamB'' receptors. Next, lysogens were selected using chloramphenicol resistance. The presence of the ''lamB''/tetA construct within the lysogens was confirmed with colony PCR. This plasmid was successfully removed via tetracycline counterselection[10]. This procedure successfully established a lysogenic strain lacking the ''lamB'' receptor and thus immune to phage infection. <br />
<br />
<gallery widths="375px" heights="250px" ><br />
Image:lamb.png|Figure 3 - λ Infection of Constitutively Active lamB Constructs. Constitutively active ''lamB'' (Table 1) was used to augment the ''lamB''- phenotype in JW3996-1 cells. Cells were titered with a known high concentration stock of phage 1x10>sup>10</sup> phage/mL. The efficiency of infection of ''lamB'' constructs can be seen through the observed phage concentration after titering. Zero plaques were observed for the J23113+33 construct.<br />
Image:LamBExpression.png|Figure 4 - ''lamB'' Infection vs ''lamB'' expression level. Infection levels for the constructs in Figure 3 plotted against the expression levels recorded in the registry for the Promoter/RBSs used.<br />
</gallery><br />
<br />
===''rcsA'' Induction===<br />
<br />
<br />
Overexpression of ''rcsA'' led to induction of λ lysogens. ''rcsA'' was placed downstream of an AHL inducible promoter and overexpressed through induction with 10 nM AHL. Following induction, phage concentration in the supernatant was recorded through titering. This concentration was compared to the phage concentration in the supernatant without ''rcsA'' overexpression, given by a construct where ''rcsA'' is not driven by a promoter. The data show a 40 fold increase in phage concentration between the promoter-less ''rcsA'' construct and the strongest AHL induced ''rcsA'' construct (Figure 5, columns 1 and 4). The promoter-less ''rcsA'' construct should accurately represent background phage levels for the lysogen absent of any ''rcsA'' expression. <br />
Further work was done to characterize the response of the AHL inducible switch under three different ribosome binding sites (RBSs). The data show induction efficiency is directly correlated with strength of ''rcsA'' overexpression (Figure 5, columns 2-4). In each of the constructs, there is a 3 to 5 fold increase in phage levels after induction with 10nm AHL. This increase does not accurately reflect the dynamic range of the AHL inducible promoter, because it does not take into account the background phage levels without the promoter. In the weaker RBSs (B0032 and B0033), the phage concentration from the uninduced supernatant is comparable to the background level from the promoter-less construct (Figure 5, columns 1-3). Thus, in these inducible promoter systems, there is high dynamic range and negligible background compared to basal phage concentrations.. However, in the strongest RBS (B0034), the uninduced phage level is an order of magnitude higher than the promoter-less background level. This high basal level expression suggests that the higher expression levels due to the strong RBS is leading to leaky ''rcsA'' activation of the phage lytic cycle. <br />
<br />
[[Image:Rcsainduction.png|600px|thumb|center|Figure 5 - ''rscA'' induction of phage production. In this figure, each construct was infected with λ phage and lysogens were selected. Lysogens were grown to OD600=0.1 and ''rcsA'' was induced with 10 nM of AHL. 1.5 hours after induction, the supernatant was used to titer wildtype stock of ''E. coli''. Plaques were counted to calculate the phage concentration inside the supernatant after induction. For more information see [[Team:Caltech/Protocols/rcsA_Lysogen_Induction|<font style="color:#BB4400">induction protocol.</font>]]]]<br />
{{Clear}}<br />
<br />
===''cI'' reduces ''rcsA'' induction===<br />
<br />
The high level of background phage induction in the strongest AHL inducible ''rcsA'' switch was not optimal. If the high basal phage levels could be reduced, then the dynamic range of the inducible switch can be greatly increased. cI, the λ repressor, has been shown to directly inhibit UV induction of lysogens [13], and RcsA seems competitively interact with cI in λ induction [7]. Both of these reasons suggest that increased concentration of the λ repressor should lead to lower phage induction levels. ''cI'' was constitutively expressed downstream of the strongest AHL inducible switch. As expected, expression of cI reduces the phage release and ''rcsA'' induction in both the off-state as well as the induced state. Phage concentration was reduced by more than 1.5x106 pfu/mL in both control and induced states (Figure 5, Columns 4 and 5). In Figure 5, for the construct expressing ''cI'', there is an 8 fold increase in phage concentration between induced and un-induced states, more than double the dynamic range of the construct without ''cI'' expression. <br />
<br />
==Conclusion==<br />
We have successfully developed a model system in which bacteriophages not able to infect ''E. coli'' can be integrated as a lysogen within an ''E. coli'' host. We successfully demonstrated this model with bacteriophage λ and the JW3996-1 strain of ''E. coli'' which is immune to λ infection. A system for inducing the release of phage was constructed around the ''E. coli'' regulatory protein RcsA. Work was done to optimize a switch using an AHL inducible promoter for ''rcsA'' induction. RcsA overexpression led to significant phage induction, producing approximately 1x10<sup>8</sup> plaque forming units (pfu)/mL. This value is 4 orders of magnitude greater than the phage induction reported by Rozanov et al. However, direct comparisons cannot be readily made between the data, because the method of overexpression of ''rcsA'' differs between the two papers, and the level at which ''rcsA'' was overexpressed in Rozanov’s study is unknown. The background phage concentration in uninduced lysogens was also much higher than reported in literature [7]. At present, there is no clear cause for this increase in basal induction levels within the λ lysogens. Phage induction via ''rcsA'' can be reduced through lambda repressor overexpression. The mechanism by which RcsA activates the lytic cycle of the dormant prophage is largely unknown; however, there is evidence that RscA acts through inactivation of the phage repressor, ''cI''. [7] Therefore, increased production of the repressor ''cI'' led to a decrease in ''rcsA'' mediated phage induction. The addition of constitutively active ''cI'' repressor led to the construction of an ''rcsA'' phage induction system with a high dynamic range and relatively low background. <br />
<br />
Future work should be focused on broadening the host range of the system. Thus, a key aspect needs to be adapting other temperate phages the model system. A significant result would be the incorporation and production of a non-''E. coli'' bacteriophage by ''E. coli''. A promising candidate for this is the Salmonella phage P22; P22 has been shown to be viably produced in ''E. coli'' when the prophage is expressed[14]. Incorporation of the P22 prophage should be feasible following the framework of the current model. Furthermore, co-culture experiments with the current constructs and wild-type ''E. coli'' could be used to validate the current system's effectiveness at eliminating pathogens. <br />
<br />
==References==<br />
# Sears CL. 2005. "'''A dynamic partnership: Celebrating our gut flora.'''" Anaerobe, Volume 11, Issue 5, Pages 247-251.<br />
# "'''Cholera Statistics 2006.'''" World Health Organization. 4 July 2008 <http://www.who.int/wer/2006/wer8131.pdf>. <br />
# "'''Food Borne Illness FAQ.'''" World Health Organization. 5 July 2008 <http://www.who.int/mediacentre/factsheets/fs237/en/>. <br />
# Westendorf AM, Gunzer F, Deppenmeier S, Tapadar D, Hunger JK, Schmidt MA, Buer J, and Bruder D. “'''Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules.'''” FEMS Immunol Med Microbiol 2005 Mar 1; 43(3) 373-84. <br />
# Clément JM, Hofnung M. “'''Gene sequence of the lambda receptor, an outer membrane protein of E. coli K12.'''” Cell. 1981 Dec; 27(3 Pt 2):507–514.<br />
# Lwoff, A., L. Siminovitch, and N. Kjeldgaard. 1950. “'''Induction de la production de bacteriophages chez une bacterie lysogene.'''” Ann. Inst. Pasteur (Paris) 79:815-859.<br />
# Rozanov, Dmitry V., Richard D'ari, and Sergey P. Sineoky. "'''RecA-Independent Pathways of Lambdoid Prophage Induction in Escherichia coli.'''" Journal of Bacteriology 180 (1998): 6306-315.<br />
# “'''Biobricks Assembly'''” OpenWetware <http://openwetware.org/wiki/BioBricks_construction_tutorial><br />
# Sambrook, Joe, and David Russell. "'''Molecular Cloning.'''" New York: Cold Spring Harbor Laboratory P, 2000.<br />
# Maloy, Stanley R., and William D. Nunn. "'''Selection for Loss of Tetracycline Resistance by Escherichia coli.'''" Journal of Bacteriology 45 (1985): 1110-112.<br />
# Grayson, Paul. "'''Titering.'''" California Institute of Technology. <http://www.rpgroup.caltech.edu/wiki/index.php?title=titering>.<br />
# Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H. “'''Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.'''” Mol Syst Biol. 2006;2:2006.0008<br />
# John Baluch, Raquel Sussman. “'''Correlation Between UV Dose Requirement for Lambda Bacteriophage Induction and Lambda Repressor Concentration'''” Journal of Virology, June 1978, P. 595-602<br />
# Botstein, David and Ira Herskowitz. "'''Properties of hybrids between Salmonella phage P22 and coliphage lambda.'''" Nature. 1974 Oct 18;251(5476):584-9.<br />
}}</div>Jkmhttp://2008.igem.org/File:Rcsainduction.pngFile:Rcsainduction.png2008-10-23T18:35:04Z<p>Jkm: uploaded a new version of "Image:Rcsainduction.png"</p>
<hr />
<div>rcsA induction of lambda lysogens.</div>Jkmhttp://2008.igem.org/Team:Caltech/PartsTeam:Caltech/Parts2008-10-22T01:21:06Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Completed systems</font></div><br />
<br><br />
__NOTOC__<br />
*Oxidative Burst<br />
**AHL Receiver + GFP[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137018]<br />
**AHL Receiver + tet Inverter + GFP[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137019]<br />
**AHL Inducible H<sub>2</sub>O<sub>2</sub> Production[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137076]<br />
**AHL Inducible expression of KatG[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137079]<br />
*Phage Pathogen Defense<br />
**AHL Receiver + rcsA[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137114]<br />
**AHL Receiver + rcsA + constitutive cI[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137117]<br />
**LamB/TetA expression plasmid[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137120]<br />
*Lactose Intolerance<br />
**Synthetic LacYZ operon[http://partsregistry.org/wiki/index.php?title=Part:BBa_S04055]<br />
*Vitamin Production<br />
**FolB Expression Construct[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137053]<br />
**FolKE Expression Construct[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137054]<br />
**PabA Expression Construct[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137055]<br />
*Asynchronous Random State Generator<br />
**GFP FimE switches, 150-850bp[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137057][http://partsregistry.org/wiki/index.php?title=Part:BBa_K137058][http://partsregistry.org/wiki/index.php?title=Part:BBa_K137059][http://partsregistry.org/wiki/index.php?title=Part:BBa_K137060][http://partsregistry.org/wiki/index.php?title=Part:BBa_K137061][http://partsregistry.org/wiki/index.php?title=Part:BBa_K137062]<br />
**TA Regulatory SSM[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137042][http://partsregistry.org/wiki/index.php?title=Part:BBa_K137043]<br />
**AGTC Coding SSM[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137040][http://partsregistry.org/wiki/index.php?title=Part:BBa_K137041]<br />
<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/Protocols/FimETeam:Caltech/Protocols/FimE2008-10-21T22:43:24Z<p>Jkm: New page: {{Caltech_iGEM_08| Content= <div style="font-size:18pt;"> <font face="verdana" style="color:#CC3300">FimE Inversion Assay</font></div> __NOTOC__ <br> Insert protocol here }}</p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">FimE Inversion Assay</font></div><br />
__NOTOC__<br />
<br><br />
<br />
Insert protocol here<br />
<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/ProtocolsTeam:Caltech/Protocols2008-10-21T22:43:13Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Protocols</font></div><br />
<br><br />
__NOTOC__<br />
*Oxidative Burst<br />
**[[Team:Caltech/Protocols/H2O2 Production Assay|H<sub>2</sub>O<sub>2</sub> production assay]]<br />
**[[Team:Caltech/Protocols/MIC Assay|Minimum inhibitory concentration (MIC) assay]]<br />
**[[Team:Caltech/Protocols/Coculture Inhibition Assay|Coculture inhibition assay]]<br />
**[[Team:Caltech/Protocols/Measuring H2O2|Measuring H<sub>2</sub>O<sub>2</sub>]]<br />
*Phage Pathogen Defense<br />
**[[Team:Caltech/Protocols/rcsA Lysogen Induction|rcsA lysogen induction]]<br />
**[[Team:Caltech/Protocols/Titering|Titering]]<br />
*Lactose Intolerance<br />
**[[Team:Caltech/Protocols/LacZ Assay|LacZ assay]]<br />
**[[Team:Caltech/Protocols/Lysis Assay|Lysis assay]]<br />
*Vitamin Production<br />
**[[Team:Caltech/Protocols/Folate_assay|Folate microbiological assay protocol]] <br />
**[[Team:Caltech/Protocols/PABA_HPLC_assay| para-Aminobenzoic Acid (pABA) HPLC protocol]]<br />
*Asynchronous Random State Generator<br />
**[[Team:Caltech/Protocols/Flow cytometry|Flow cytometry]]<br />
***[[Team:Caltech/Protocols/BioAssay_Buffer|Bioassay Buffer]]<br />
**[[Team:Caltech/Protocols/FimE|FimE inversion assay]]<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/Project/Lactose_intoleranceTeam:Caltech/Project/Lactose intolerance2008-10-21T18:28:54Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Curing Lactose Intolerance</font></div><br />
<br />
==Introduction==<br />
The gut flora of our digestive tract contains microorganisms that perform various useful functions for their hosts. Examples of such functions include growth inhibition of harmful microorganisms<sup>1</sup>, defense against the causes of many forms of Inflammatory Bowel Disease<sup>2</sup>, and the fermentation of carbohydrates and other molecules the human body cannot normally digest. Some bacterial strains, including the E. coli strain ‘Nissle 1917’, can persist in the gut of mice for months. Engineering these bacteria provide a new platform to treat various human diseases. <br>Lactose intolerance is characterized by the inability to break down lactose in the small intestine. The undigested lactose instead passes to the large intestine, leading to two negative processes: osmotic imbalance and bacterial fermentation. High lactose levels raise the osmolarity of the colon, causing diarrhea. In addition, gut microbes metabolize the lactose into methane gas, causing abdominal pain. Both problems must be addressed in order to fully treat lactose intolerance. If we simply have our strain metabolize lactose, another strain will further ferment the byproducts, resulting in the same side effects. <br>Instead, we have our engineered two plasmids which allow the host to uptake lactose, clearing the sugar from the colon. To do this, we have engineered the ‘Nissle 1917’ strain to release ß-galactosidase, an enzyme that cleaves lactose into glucose and galactose. Since protein secretion is difficult in E. coli, our cells were engineered to lyse in order to release ß-galactosidase. However, lysis must occur only when lactose is present in the gut. If lysis were to occur when lactose was absent, this would kill all our engineered cells at any given moment. The cells also express a lactose permease, allowing the strain to sense lactose in all conditions. <br> Figure showing before lysis, during lysis, and after lysis will be added soon.<br />
<br />
==System Design==<br />
===Lactose Regulator===<br />
[[image:Lactose_regulator.JPG|thumb|200px|left|Figure 1. A mutated LacY allows the uptake of lactose while LacZ produces large amounts of ß-galactosidase. The plasmid contains a strong constitutive promoter regulating both genes and a ColE1 replication origin.]]<br />
The first plasmid consists of a synthetic lactose operon under strong constitutive expression (Fig. 1). Our synthetic lactose operon encodes the ß-galactosidase LacZ and a lactose permease LacY. LacY is a membrane protein that actively transports lactose into the cell. Since most membrane proteins are toxic when overexpressed, we optimized our system to express appropriate levels of LacY without killing the cell. In the end, we want to express as much ß-galactosidase as we can to cleave as much lactose present in the large intestine. <br>The human gut is an unpredictable environment, and we wish our engineered cells to behave reliably despite this variability. E. coli are unable to uptake lactose in the presence of glucose, a phenomenon known as carbon catabolite repression. Catabolite repression is mediated by Enzyme IIA Glucose (IIAGluc), which inhibits the uptake of lactose in the presence of glucose by binding to LacY<sup>3</sup>. Previous research has identified various LacY mutations that prevent this inhibition and achieve increased uptake of lactose in the presence of glucose<sup>4</sup>.<br />
<br />
{{clear}}<br />
<br />
===Lactose-Induced Lysis===<br />
[[image:Lactose-inducible_lysis.JPG|thumb|200px|right|Figure 2. The lysis cassette plasmid acts as a lactose sensor. Intracellular lactose accumulation induces overexpression of the lysis cassette. Lactose inhibits binding of the LacI repressor to the promoter and P1 high copy origin of replication. A mutated holin gene in the lysis cassette allows faster lysis times.]]<br />
The second plasmid contains lactose inducible expression of λ phage lysis genes under a lactose inducible promoter (Fig. 2). To release ß-galactosidase, the cells will lyse when enough lactose is present in the cell to induce the expression of the lysis genes. It is important that the lactose-inducible promoter is tightly regulated since leaky expression will cause spontaneous lysis. To accomplish tight expression, specific lac promoters will keep our system from lysing<sup>5</sup>. In addition, the plasmid copy number remains low copy until induced with lactose, when the plasmid copy number increases to high copy. <br>Using wild type λ phage lysis genes, lysis occurs 40-45 minutes after induction by lactose. Decreasing the lag time will reduce the extent of lactose fermentation and therefore produce fewer deleterious effects. Previous research has uncovered mutations that shorten the lysis time to approximately 10-15 minutes<sup>6</sup>, and these mutations will be incorporated into our final construct.<br />
<br />
{{clear}}<br />
<br />
==Results==<br />
===Lactose Regulator===<br />
[[IMAGE:Promoter_strength.JPG|thumb|300px|left|Figure 3. Varying the expression levels of LacY with different promoters and ribosome binding sites. Cells died at our two highest expression levels, but survived on the other four. The construct with the weakest RBS was selected since we could express LacZ in the same operon with a strong promoter. This combination allows our cell to express high levels of LacZ and non-toxic levels of LacY.]]<br />
In the first plasmid, it was discovered that LacY is toxic when overexpressed. To determine safe expression levels, constructs were built with varying levels of LacY expression. Six plasmids were built from combinations of three different promoters and two different ribosome binding sites (Fig. 3). The cells appeared to have died when expressing the strongest and medium strength promoters along with the strongest ribosome binding site. Our information was based off transformants, and the plates with no transformants were classified as dead cells due to high LacY expression. Based on these results, we decided to express our synthetic lac operon by combining the strongest constitutive promoter with the weakest RBS for LacY and the strongest RBS for LacZ, preventing LacY toxicity while expressing LacZ in large quantities. <br>As mentioned earlier, our cells had to inhibit carbon catabolite repression to ensure uptake of lactose under any condition. Various mutations, including the insertion of two histidyl residues between amino acids S194 and A195 (ref 4) in the LacY gene, prevent the inhibition by IIAGluc. These insertions have been made and will be tested in future studies. <br />
<br />
{{clear}}<br />
<br />
[[image:Figure_1_beta_assay.JPG|thumb|300px|right|Figure 4. ß-galactosidase activity of plasmid #1, with and without the strong constitutive promoter. Error bars on the data with a promoter show the standard deviation of two measurements.]]The ß-galactosidase assay was performed on the synthetic network containing the lactose operon (Fig. 4). The first assay was performed on a strain containing the plasmid lacking the strong constitutive promoter. This assay was performed to simulate the eventual system regulation. We saw significant levels of ß-galactosidase, a surprising result since the plasmid lacked a promoter. These levels are likely due to spurious transcription amplified by our strong ribosome binding site and high copy plasmid. We then repeated the assay with the strong constitutive promoter to observe the dynamic range of LacZ expression in this system. <br />
{{clear}}<br />
<br />
===Lactose-Induced Lysis===<br />
Our second plasmid was not finished, but necessary parts have been cloned for future construction. There remains one more cloning step to place the lysis cassette into the vector containing the promoters. The single base mutations in the lysis cassette were not made, and if time allowed, they would have been completed as the final step.<br />
<br />
==Discussion==<br />
Our final system was not completely finished; however, future construction will be minimal. The synthetic lac operon plasmid was completely constructed with the insertion of two histidyl residues in their appropriate locations. Our lactose-induced lysis plasmid was one cloning step away from being completed. The promoter and flanking terminator were cloned in parallel with the lysis cassette and a terminator. The last cloning step would be to ligate the two together. In addition, the two separate point mutations have not been made, and the final step in our cloning would be to add those mutations.<br>After completing the construction phase, we would characterize our system. Characterization will be completed on a construct containing our lactose inducible promoters and GFP. This would show our promoters can maintain tight expression of GFP from uninduced to induced. We would combine this construct with our first plasmid containing the mutated LacY, to show we can achieve induction by lactose even in the presence of glucose. To show the effect our lysis plasmid, it would be necessary to achieve lysis with lactose. In addition, we want to show that we can achieve lysis with lactose in the presence of glucose. Finally, the final assay we want to perform on our system would show our cells able to uptake lactose in the presence of glucose, and lyse soon after. Once the cells lyse, the cell lysis should contain ß-galactosidase at levels close to what we received from our ß-galactosidase assay on plasmid 1. Once characterization is completed, our construct would be moved to the ‘Nissle 1917’ strain where further characterization and modifications would take place.<br />
<br />
<br />
==Methods==<br />
Methods can be found [[Team:Caltech/Protocols|<font style="color:#BB4400">here</font>]]<br />
<br />
==References==<br />
# Guarner F and Malagelada JR. 2003. '''Gut flora in health and disease.''' ''The Lancet'', Volume 361, Issue 9356, 8 February 2003, Pages 512-519. <br />
# Sears CL. 2005. '''A dynamic partnership: Celebrating our gut flora.''' ''Anaerobe'', Volume 11, Issue 5, Pages 247-251.<br />
# Deutscher, J., Francke, C. and Postma, P.W. 2006. '''How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bactertia.''' ''Microbial and Molecular Biology Reviews.'' 2006: 939-1031<br />
# Hoischen, C., Levin, J., Pitaknarongphorn, S., Reizer, J., and Saier , M. H. Jr. 1996. '''Involvement of the central loop of the lactose permease of Escherichia coli in its allosteris regulation by the glucose-specific enzyme IIA of the phosphoenolpyruvate-dependent phosphotransferase system.''' ''J. Bacteriol.'' 178: 6082-6086<br />
# Cox RS III, Surette MG, Elowitz MB. '''Programming gene expression with combinatorial promoters.''' ''Mol. Syst. Biol.'' 2007;3:145.<br />
# Gründling, A., M. D. Manson, and R. Young. 2001. '''Holins kill without warning.''' ''Proc. Natl. Acad. Sci.'' USA 98:9348-9352.<br />
<br />
<br />
}}</div>Jkmhttp://2008.igem.org/File:Promoter_strength.JPGFile:Promoter strength.JPG2008-10-21T18:27:52Z<p>Jkm: uploaded a new version of "Image:Promoter strength.JPG"</p>
<hr />
<div></div>Jkmhttp://2008.igem.org/Team:Caltech/PartsTeam:Caltech/Parts2008-10-21T18:07:39Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Completed systems</font></div><br />
<br><br />
__NOTOC__<br />
*Oxidative Burst<br />
**AHL Receiver + GFP[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137018]<br />
**AHL Receiver + tet Inverter + GFP[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137019]<br />
**AHL Inducible H<sub>2</sub>O<sub>2</sub> Production[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137076]<br />
**AHL Inducible expression of KatG[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137079]<br />
*Phage Pathogen Defense<br />
**AHL Receiver + rcsA[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137114]<br />
**AHL Receiver + rcsA + constitutive cI[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137117]<br />
*Lactose Intolerance<br />
**Synthetic LacYZ operon[http://partsregistry.org/wiki/index.php?title=Part:BBa_S04055]<br />
*Vitamin Production<br />
**FolB Expression Construct[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137053]<br />
**FolKE Expression Construct[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137054]<br />
**PabA Expression Construct[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137055]<br />
*Asynchronous Random State Generator<br />
**GFP FimE switches, 150-850bp[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137057][http://partsregistry.org/wiki/index.php?title=Part:BBa_K137058][http://partsregistry.org/wiki/index.php?title=Part:BBa_K137059][http://partsregistry.org/wiki/index.php?title=Part:BBa_K137060][http://partsregistry.org/wiki/index.php?title=Part:BBa_K137061][http://partsregistry.org/wiki/index.php?title=Part:BBa_K137062]<br />
**TA Regulatory SSM[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137042][http://partsregistry.org/wiki/index.php?title=Part:BBa_K137043]<br />
**AGTC Coding SSM[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137040][http://partsregistry.org/wiki/index.php?title=Part:BBa_K137041]<br />
<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/Project/Population_VariationTeam:Caltech/Project/Population Variation2008-10-21T18:01:53Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
<br />
<div style="font-size:18pt;line-height:1.1"> <br />
<font face="verdana" style="color:#CC3300">Population Variation:</font></div><div><br />
<div style="font-size:14pt;line-height:1.1"><font face="verdana" style="color:#CC3300">Construction of an Asynchronous Random State Generator</font></div><br />
__NOTOC__<br><br />
==Introduction==<br />
<br />
Biological circuits are commonly introduced into cells to produce non-native functions. Such functions include the production of antimalarial drugs in yeast cells<sup>1</sup>, the detection of light by bacterial cells<sup>2</sup>, and the enzymatic breakdown of bacterial biofilms by bacteriophages<sup>3</sup>. However, a problem arises when dealing with functions that inhibit the reproduction of cells. For example, the overproduction of certain compounds may burden cells with such a heavy load that they grow at a slower rate. Such production may involve lysing the cell to release compounds into solution. Once a cell is lysed, it can no longer produce progeny. If all the cells in a colony lyse, then the cell line may eventually die. Our solution is to introduce cellular differentiation. Two states can be created in a cell: one in which the cell simply grows and replicates, and another in which the fatal function is turned on. Cells in the former state can produce progeny, and the progeny that they produce can either be in either of the two states. Cells that differentiate into the latter state can die off without directly affecting the viability of the cell line. Thus, differentiation allows for the incorporation of fatal functions into cells.<br />
<br />
=== Random State ===<br />
Differentiation can also be used to combine multiple non-compatible functions into a single cell line. In this case, only one function will be turned on in each cell at any time, but the cell population will express the entire set of functions. Two functions may be non-compatible if they compete for the same resources inside a cell or if they signal through the same intracellular components. These two functions could be simply placed into separate cell lines, but if these two cell lines have different growth rates, one cell line would outcompete the other. Differentiation separates different functions into individual cells and allows the entire cell line to express multiple functions. In such a system, cells start off in an undifferentiated state. As undifferentiated cells replicate, their offspring can stay in the current state or switch into one of the mutually-exclusive states. As long as cells divide faster than they differentiate, the reproduction of undifferentiated cells maintains the undifferentiated population, which ensures that the entire cell line is kept alive. Thus, fatal and mutually-exclusive functions can be incorporated into cell lines by utilizing cellular differentiation.<br />
<br />
=== Random Time ===<br />
It is also important to have cells differentiate at random times. Having asynchrony ensures that a large portion of the population will not differentiate at once. Doing so might leave too few undifferentiated cells to maintain the population. It is hard to ensure that cells divide faster than they differentiate if there are bursts in time in which many cells differentiate into terminal states. In addition, if the environment the cells are in continually changes, cells may differentiate into a particular state that is incompatible with the current environment. For example, that particular function may be useless in the current environment, or performing that function in such an environment may lead to the cell’s death. As such, spreading out the time during which cells differentiate increases the chances that an individual cell committed to a state finds itself in a hospitable environment. Thus, both random state and random time are needed for differentiation.<br />
<br />
===System Outline===<br />
<br />
[[Image:ARSG.png| 400px | thumb| right| Figure 1: The probabilistic switch controls the production of a trigger that activates the random state generator.]]<br />
<br />
To implement cellular differentiation, we designed two BioBrick<sup>TM</sup> devices. The first device is a probabilistic switch that randomly activates the second device. The second device is a random state generator that, when triggered, determines the final state of the bacterial cell line. Combined, the two devices form a system that allows for the integration of multiple <i>de novo</i> functions into bacterial cell lines.<br />
{{Clear}}<br />
==Probabilistic Switch: Slipped Stand Mutations==<br />
<br />
To construct the first device, the probabilistic switch, we exploit the fact that DNA polymerase slips when replicating long stretches of short nucleotide repeats<sup>4,5</sup>. This phenomenon is called slipped-strand mispairing (SSM). We have created two families of such switches based on where the long repeat is integrated. The first family, called coding SSMs, places a repeat in between the start codon of a gene and the remaining coding region. To create an on-to-off switch, the gene is initially placed in-frame with the start codon, and thus the gene is correctly translated and expressed. When DNA polymerase slips and miscopies the stretch of nucleotide repeats, the coding sequence shifts out of frame with respect to the start codon, and the gene is no longer correctly translated<sup>6,7</sup>. In an off-to-on switch, the coding sequence is initially out of frame and can either go into the correct frame or stay out of frame when SSM occurs, depending on whether the stretch of repeats becomes a multiple of three. <br />
<br />
The second family, called regulatory SSMs, places the repeat between the -10 and -35 elements of a promoter. To create an on-to-off switch, the distance between the -10 and -35 elements is initially optimal for sigma factor binding. The sigma factor helps recruit RNA polymerase to the promoter and initiate transcription. When the polymerase slips and changes the number of nucleotide repeats, the distance between the -10 and -35 elements changes. Suboptimal spacing between promoter elements reduces sigma factor recognition, resulting in less frequent transcription events<sup>7,8</sup>. <br />
<br />
<gallery widths="375px" heights="250px" ><br />
Image:Types_of_SSMs.png|Figure 2: The two families of SSMs, shown with particular repeats. For the coding SSM (top), the three possible outcomes from DNA replication are shown; the switch is initially off, and has a chance of turning on with each replication. GFP is used to determine the state of the switch.<br />
Image:SSM_Example.png|Figure 3: Example of an coding SSM transition in which slippage causes the downstream GFP gene to go into frame, turning on the gene.<br />
</gallery><br />
<br><br />
<br />
===Results===<br />
<br />
To test the functionality of SSMs as genetic switches, we used the green fluorescent protein (GFP). By measuring the fluorescence of bacterial cells that contain our constructs, we could determine the expression of GFP in the on and off states. We constructed four classes of coding SSMs, and one class of regulatory SSMs. Each class contains one in-frame or ‘on’ variant, and one or more out-of-frame or ‘off’ variants. The fluorescence of these constructs was then measured for the population and at the single-cell level. We found that AGTC coding repeats and TA regulatory repeats display detectable GFP fluorescence in the ‘on’ state and significantly reduced fluorescence in the ‘off’ state. From flow cytometry data, a broad distribution in fluorescence was observed in cells with AGTC constructs covering three orders-of-magnitude. Since the constructs were placed in high copy plasmids, these distributions might reflect hundreds of individual GFP coding sequences shifting in and out of frame within each cell.<br />
<br><br><br />
<gallery widths="375px" heights="275px" align="center" perrow="2" cellspacing="0"><br />
Image:SSMs_Constructed.png|Table 1: In green are the four classes of coding SSMs, and in red is the class of regulatory SSM created. These repeats were placed into their respective constructs.<br />
Image:SSM_In_and_Out.png|Figure 4: The measured fluorescence of the coding AGTC construct and the regulatory TA construct on a plate reader. These two classes were found to have significant differences between the on and off state. Error bars represent the standard deviation of two samplings from the same culture.<br />
Image:SSM_Fluorescence_1.png|Figure 5: The fluorescence spectrum of individual cells containing the AGTC ‘in’ and ‘out’ coding SSM constructs. Spectrum of positive control is in green, negative control is in blue, AGTC ‘in’ construct is in brown, and AGTC ‘out’ is in red. Note that the ‘in’ repeat spectrum has a peak at a higher value than the ‘out’ repeat spectrum. In addition, note the broad distribution of the spectrum. The constructs were placed in a high copy plasmid (>100 copies per cell). Thus, each one of those plasmids has a chance of slipping, and the total number of GFP transcripts from all the plasmids in a cell determines the overall fluorescence of the cell. <br />
Image:SSM_Fluorescence_2.png|Figure 6: The fluorescence spectrum of individual cells containing the TA ‘on’ and ‘off’ regulatory SSM constructs. Spectrum of negative control is in red, TA ‘on’ is in blue, and TA ‘off’ is in green. The TA ‘on’ distribution lies to the right of the TA ‘off’ distribution, indicating that the TA ‘on’ construct is a better promoter than its counterpart. These distributions were taken at a higher PMT value than the distributions in the left image. The peaks in between 10<sup>0</sup> and 10<sup>1</sup> fluorescence may be debris the flow cytometer picked up. Our flow cytometer is commonly used with eukaryotic cells and has not yet been optimized for use with bacterial cells. The cells measured with the flow cytometer in this figure are the same ones measured with the plate reader.<br />
</gallery><br />
<br><br />
Sequencing results of long repeats suggest the occurrence of slipped-strand mispairing. If a slipped-strand mispairing occurs early on in a cell line’s history, then a large proportion of the population will have shifted to a construct with a differing number of repeats, and the resulting sequencing of the miniprep will show mixed products. If a slipped-strand mutation occurs later on in the colony’s lineage, then a smaller proportion of the population will have shifted, and the resulting sequencing will have clear peaks, indicating single nucleotides at those locations. <br><br><br />
[[Image:SSM_Sequencing.png| 600px | thumb| center|Figure 7: Sequencing results suggest slipped-strand mutation. Cells were transformed with just the coding SSM construct (AGTC)<sub>9</sub>. However, the sequencing trace for a colony transformed with (AGTC)<sub>9</sub> also shows the presence of (AGTC)<sub>8</sub>, suggesting that slippage at the AGTC repeat had occurred. The sequence for GFP after the start codon follows the AGTC repeats.]]<br><br />
===Future Work===<br />
In future work, we would use the SSM as a switch to turn on the gene that codes the trigger for the second device. To implement a coding SSM, we would place repeats after the start codon of the trigger gene. The trigger would initially be off and turned on when the polymerase slips. Or, to implement a regulatory SSM, we would place the trigger gene downstream of a SSM promoter. Then the gene would be transcribed when the promoter slips into the right length to bind to a transcriptional activator.<br />
<br />
We would also place coding and regulatory SSMs in front of LacZ, and then chromosomally integrating the construct into ''E. coli'' so that there is only one copy of the construct in a cell. Our current work utilizes a high copy number plasmid, and it is difficult to resolve individual slipped strand mutations and calculate slippage frequency in such cases. With LacZ, blue-white screening can then be conducted to determine the switching frequency of the SSMs. In addition, we plan to place the SSMs in front of the gene that codes for tetracycline resistance (tetA). We can then select for and against cells with tetracycline resistance. <br />
<br><br><br />
==Random State Generator: FimE Recombinase==<br />
<br />
[[Image:FimE_Simple.png| 400px | thumb| right|Figure 8: FimE flips the region flanked by IRL and IRL. A tetR promoter is placed between the two binding sites facing upstream. When the DNA segment is flipped, the promoter faces downstream, and GFP is produced. Once the in and out components of a binding site do not match, FimE no longer recognizes that site. The length of the recombination region (in red) was varied from 250 bp to 850 bp in increments of 100 or 200 bp.]]<br />
FimE is the trigger that will act on the random state generator. FimE is a recombinase protein that can flip DNA flanked by two binding sites. The random state generator consists of recombination regions flanked by FimE binding sites. FimE recognizes binding sites called IRR (inverted repeat right) and IRL (inverted repeat left). Each IRL and IRR consists of an inner and outer sequence, and fimE will only recognize a site that has matching inner and outer sequences. After FimE flips the DNA in between two correctly formed binding sites, the binding sites become distorted, and FimE can no longer act on the region. Taking advantage of this effect, one could place a promoter initially pointing upstream in between the two binding sites, and a gene downstream of the recombination region. Since the promoter is pointing in the opposite direction of the gene, the downstream gene will not be transcribed. However, once FimE flips the flanked recombination region, the promoter then points in the correct direction and can transcribe the downstream gene<sup>9</sup>.<br />
{{Clear}}<br />
===Results===<br />
[[Image:FimE_Vary_Length.png| 400px | thumb| left|Figure 9: The fluorescence of cells with constructs in Figure 6, with varying distances between the two FimE binding sites. A peak at around 250 bp is observed. Fluorescence is correlated to the proportion of plasmids that contain flipped promoter. Cells were measured on plate reader, and error bars show the standard deviation of experiments performed in triplicate.]]<br />
Given that we were placing a different DNA segment in between the two binding sites than is found in wild-type genes, we wanted to determine the range of lengths FimE will act on and how the length affects FimE activity. In addition, we want to determine if the ratio of determined states could be skewed by changing the lengths of different recombination sites. Thus, we tested how the length of the recombination site affects FimE’s ability to detect the two flanking binding sites. The fluorescence of cells with each construct was used as an indicator of FimE activity. We observed a relationship between GFP fluorescence and separation length (Figure 9). Maximum fluorescence was observed to occur at about a separation length of 250 bp. This distance may be a function of entropy and enthalpy. The chances that two DNA segments on the same strand come together statistically decrease as the two segments are separated by intervening sequences. In addition, two sites very close together are unlikely to come into contact with each other, as it is energetically unfavorable to bend DNA to such a great degree. These two opposing forces might explain the presence of an optimal separation length.<br />
{{Clear}}<br />
===Future Work===<br />
Utilizing the fact that FimE flips DNA and cannot recognize binding sites that it has distorted, we can design a system that uses two IRR and two IRL sites to produce a total of three possible states, depending on how FimE acts. Once FimE flips any region in this system, it cannot flip the DNA segment again. Hence, the state of the cell is established after FimE acts. This assumes that FimE is unable to flip adjacent IRL and IRR sites, as it is difficult to bend around such a short DNA segment. The system we propose is shown below. By varying the length of the segment containing the promoter and the length of the segment containing the terminator, we may be able to vary the ratio of determined states.<br><br><br />
[[Image:Pop_Var_Final.png| 600px | thumb| center|Figure 10: a) The device design for the population variation generator. Gene A and Gene B (in opposite orientation) are initially off. b) Depending which IRR and IRL sites FimE binds to, different DNA segments are flipped. The bracket indicates the region that was flipped in each case. In the first case, gene B is turned on. In the second case, gene A is turned on. In the third case, gene A and gene B remain off and stay off, since FimE no longer has a valid IRR and IRL to chose from (the middle two binding sites are too close for FimE to act upon). Terminators at the ends of gene A and B are not shown.]]<br />
==Conclusion==<br />
<br />
We have evidence that short nucleotides repeats can be placed in the promoter or behind a start codon and function as randomized genetic switches through slipped-strand mutation. We have also shown that FimE can flip a segment of non-native DNA, and have evidence that the probability that FimE flips the segment depends on the length between the two binding sites. In the future, selected SSMs can be integrated into a FimE expression construct; thus, FimE expression can be randomly switched on in cells. FimE can then interact with the random state generator. Linked together, the probabilistic switch and the random state generator allow for the asynchronous differentiation of cells into mutually exclusive and fatal states.<br />
<br />
Shown below is the final system design, incorporating the other subprojects in our iGEM team. Initially, ''fimE'' is out of frame. When (AGTC)<sub>10</sub> through SSM becomes (AGTC)<sub>9</sub>, ''fimE'' falls into frame, and the FimE trigger protein is expressed. It interacts with the population variation generator, either committing the cell into a state of [[Team:Caltech/Project/Oxidative_Burst|<font style="color:#BB4400">ROS production</font>]] or [[Team:Caltech/Project/Lactose_intolerance|<font style="color:#BB4400">curing lactose intolerance</font>]]. In the default, undifferentied state, cell will produce a basal level of [[Team:Caltech/Project/Vitamins|<font style="color:#BB4400">folate biosynthesis enzymes</font>]]. [[Team:Caltech/Project/Phage_Pathogen_Defense|<font style="color:#BB4400">Phage induction</font>]] will be controlled by another AGTC coding SSM. Thus, this population variation machinery links the [[Team:Caltech/Project|<font style="color:#BB4400">subprojects</font>]] produced by our team into one coherent system.<br />
<br><br><br />
[[Image:Pop_Var_Conclusion.png| 600px | thumb| center| The final system design, linking the subprojects in this iGEM together. The folate biosynthesis pathway will be continually expressed in all states, and the phage induction system will be controlled by a seperate coding SSM (not shown).]]<br />
<br><br><br />
==References==<br />
<br />
[1] Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, and Keasling JD. '''Production of the antimalarial drug precursor artemisinic acid in engineered yeast'''. Nature 2006 Apr 13; 440(7086) 940-3.<br />
<br />
[2] Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, Davidson EA, Scouras A, Ellington AD, Marcotte EM, and Voigt CA. '''Synthetic biology: engineering Escherichia coli to see light'''. Nature 2005 Nov 24; 438(7067) 441-2.<br />
<br />
[3] Lu TK and Collins JJ. '''Dispersing biofilms with engineered enzymatic bacteriophage'''. Proc Natl Acad Sci U S A 2007 Jul 3; 104(27) 11197-202.<br />
<br />
[4] Henderson IR, Owen P, and Nataro JP. '''Molecular switches -- the ON and OFF of bacterial phase variation'''. Mol Microbiol 1999 Sep; 33(5) 919-32.<br />
<br />
[5] Levinson G and Gutman GA. '''Slipped-strand mispairing: a major mechanism for DNA sequence evolution'''. Mol Biol Evol 1987 May; 4(3) 203-21.<br />
<br />
[6] Levinson G and Gutman GA. '''High frequencies of short frameshifts in poly-CA/TG tandem repeats borne by bacteriophage M13 in Escherichia coli K-12'''. Nucleic Acids Res 1987 Jul 10; 15(13) 5323-38.<br />
<br />
[7] Torres-Cruz J and van der Woude MW. '''Slipped-strand mispairing can function as a phase variation mechanism in Escherichia coli'''. J Bacteriol 2003 Dec; 185(23) 6990-4.<br />
<br />
[8] van Ham SM, van Alphen L, Mooi FR, and van Putten JP. '''Phase variation of H. influenzae fimbriae: transcriptional control of two divergent genes through a variable combined promoter region'''. Cell 1993 Jun 18; 73(6) 1187-96. <br />
<br />
[9] Ham TS, Lee SK, Keasling JD, and Arkin AP. '''A tightly regulated inducible expression system utilizing the fim inversion recombination switch'''. Biotechnol Bioeng 2006 May 5; 94(1) 1-4. <br />
}}</div>Jkmhttp://2008.igem.org/Template:Caltech_iGEM_08Template:Caltech iGEM 082008-10-21T16:37:38Z<p>Jkm: </p>
<hr />
<div><html><br />
<style type="text/css"><br />
body {background: #FFFFFF}<br />
</style><br />
</html><br />
{|<br />
|-valign="top"<br />
|{{navimg|xsize=960|ysize=36|image=Caltech_header_top.jpg|link=Team:Caltech}}<br />
|}<br />
<div style="font-size:24pt;"><br />
<font face="verdana" style="color:#BB4400"><center>iGEM 2008<br />
</center></font></div><br />
<br><br />
{|<br />
|-valign="top"<br />
|{{navimg|xsize=960|ysize=48|image=Caltech_header_bottom.jpg|link=Team:Caltech}}<br />
|}<br />
<div style="color: #ffffff; background-color: #ffffff; width: 900px"><br />
</div><br />
<br />
<div><br />
{| cellspacing="0"<br />
|-<br />
|style="background-color: #ffffff" width="75px" valign="top"|<br />
<br><center>[[Team:Caltech| <font face="verdana" style="color:#BB4400"> '''Home''' </font>]] <br><br><br />
[[Team:Caltech/Members | <font face="verdana" style="color:#BB4400"> '''People''' </font>]] <br><br><br />
[[Team:Caltech/Project | <font face="verdana" style="color:#BB4400"> '''Project Details''' </font>]] <br><br><br />
[[Team:Caltech/Protocols | <font face="verdana" style="color:#BB4400"> '''Protocols''' </font>]] <br><br><br />
[[Team:Caltech/Parts | <font face="verdana" style="color:#BB4400"> '''Completed Systems''' </font>]] <br><br><br />
<br />
<br />
|width="880" valign="top" style="padding: 10px; border: 5px solid #FFFFFF; color: #000; background-color: white" | <br />
{{{Content}}}<br />
[[Image:Caltech_logo.gif|right]]<br />
{{Clear}}<br />
[[Image:Caltech_footer.jpg|left]]<br />
|}</div>Jkmhttp://2008.igem.org/Team:Caltech/Project/Population_VariationTeam:Caltech/Project/Population Variation2008-10-21T16:30:25Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
<br />
<div style="font-size:18pt;line-height:1.1"> <br />
<font face="verdana" style="color:#CC3300">Population Variation:</font></div><div><br />
<div style="font-size:14pt;line-height:1.1"><font face="verdana" style="color:#CC3300">Construction of an Asynchronous Random State Generator</font></div><br />
__NOTOC__<br><br />
==Introduction==<br />
<br />
Biological circuits are commonly introduced into cells to produce non-native functions. Such functions include the production of antimalarial drugs in yeast cells<sup>1</sup>, the detection of light by bacterial cells<sup>2</sup>, and the enzymatic breakdown of bacterial biofilms by bacteriophages<sup>3</sup>. However, a problem arises when dealing with functions that inhibit the reproduction of cells. For example, the overproduction of certain compounds may burden cells with such a heavy load that they grow at a slower rate. Such production may involve lysing the cell to release compounds into solution. Once a cell is lysed, it can no longer produce progeny. If all the cells in a colony lyse, then the cell line may eventually die. Our solution is to introduce cellular differentiation. Two states can be created in a cell: one in which the cell simply grows and replicates, and another in which the fatal function is turned on. Cells in the former state can produce progeny, and the progeny that they produce can either be in either of the two states. Cells that differentiate into the latter state can die off without directly affecting the viability of the cell line. Thus, differentiation allows for the incorporation of fatal functions into cells.<br />
<br />
=== Random State ===<br />
Differentiation can also be used to combine multiple non-compatible functions into a single cell line. In this case, only one function will be turned on in each cell at any time, but the cell population will express the entire set of functions. Two functions may be non-compatible if they compete for the same resources inside a cell or if they signal through the same intracellular components. These two functions could be simply placed into separate cell lines, but if these two cell lines have different growth rates, one cell line would outcompete the other. Differentiation separates different functions into individual cells and allows the entire cell line to express multiple functions. In such a system, cells start off in an undifferentiated state. As undifferentiated cells replicate, their offspring can stay in the current state or switch into one of the mutually-exclusive states. As long as cells divide faster than they differentiate, the reproduction of undifferentiated cells maintains the undifferentiated population, which ensures that the entire cell line is kept alive. Thus, fatal and mutually-exclusive functions can be incorporated into cell lines by utilizing cellular differentiation.<br />
<br />
=== Random Time ===<br />
It is also important to have cells differentiate at random times. Having asynchrony ensures that a large portion of the population will not differentiate at once. Doing so might leave too few undifferentiated cells to maintain the population. It is hard to ensure that cells divide faster than they differentiate if there are bursts in time in which many cells differentiate into terminal states. In addition, if the environment the cells are in continually changes, cells may differentiate into a particular state that is incompatible with the current environment. For example, that particular function may be useless in the current environment, or performing that function in such an environment may lead to the cell’s death. As such, spreading out the time during which cells differentiate increases the chances that an individual cell committed to a state finds itself in a hospitable environment. Thus, both random state and random time are needed for differentiation.<br />
<br />
===System Outline===<br />
<br />
[[Image:ARSG.png| 400px | thumb| right| The probabilistic switch controls the production of a trigger that activates the random state generator.]]<br />
<br />
To implement cellular differentiation, we designed two BioBrick<sup>TM</sup> devices. The first device is a probabilistic switch that randomly activates the second device. The second device is a random state generator that, when triggered, determines the final state of the bacterial cell line. Combined, the two devices form a system that allows for the integration of multiple <i>de novo</i> functions into bacterial cell lines.<br />
{{Clear}}<br />
==Probabilistic Switch: Slipped Stand Mutations==<br />
<br />
To construct the first device, the probabilistic switch, we exploit the fact that DNA polymerase slips when replicating long stretches of short nucleotide repeats<sup>4,5</sup>. This phenomenon is called slipped-strand mispairing (SSM). We have created two families of such switches based on where the long repeat is integrated. The first family, called coding SSMs, places a repeat in between the start codon of a gene and the remaining coding region. To create an on-to-off switch, the gene is initially placed in-frame with the start codon, and thus the gene is correctly translated and expressed. When DNA polymerase slips and miscopies the stretch of nucleotide repeats, the coding sequence shifts out of frame with respect to the start codon, and the gene is no longer correctly translated<sup>6,7</sup>. In an off-to-on switch, the coding sequence is initially out of frame and can either go into the correct frame or stay out of frame when SSM occurs, depending on whether the stretch of repeats becomes a multiple of three. <br />
<br />
The second family, called regulatory SSMs, places the repeat between the -10 and -35 elements of a promoter. To create an on-to-off switch, the distance between the -10 and -35 elements is initially optimal for sigma factor binding. The sigma factor helps recruit RNA polymerase to the promoter and initiate transcription. When the polymerase slips and changes the number of nucleotide repeats, the distance between the -10 and -35 elements changes. Suboptimal spacing between promoter elements reduces sigma factor recognition, resulting in less frequent transcription events<sup>7,8</sup>. <br />
<br />
<gallery widths="375px" heights="250px" ><br />
Image:Types_of_SSMs.png|The two families of SSMs, shown with particular repeats. For the coding SSM (top), the three possible outcomes from DNA replication are shown; the switch is initially off, and has a chance of turning on with each replication. GFP is used to determine the state of the switch.<br />
Image:SSM_Example.png|Example of an coding SSM transition in which slippage causes the downstream GFP gene to go into frame, turning on the gene.<br />
</gallery><br />
<br><br />
<br />
===Results===<br />
<br />
To test the functionality of SSMs as genetic switches, we used the green fluorescent protein (GFP). By measuring the fluorescence of bacterial cells that contain our constructs, we could determine the expression of GFP in the on and off states. We constructed four classes of coding SSMs, and one class of regulatory SSMs. Each class contains one in-frame or ‘on’ variant, and one or more out-of-frame or ‘off’ variants. The fluorescence of these constructs was then measured for the population and at the single-cell level. We found that AGTC coding repeats and TA regulatory repeats display detectable GFP fluorescence in the ‘on’ state and significantly reduced fluorescence in the ‘off’ state. From flow cytometry data, a broad distribution in fluorescence was observed in cells with AGTC constructs covering three orders-of-magnitude. Since the constructs were placed in high copy plasmids, these distributions might reflect hundreds of individual GFP coding sequences shifting in and out of frame within each cell.<br />
<br><br><br />
<gallery widths="375px" heights="275px" align="center" perrow="2" cellspacing="0"><br />
Image:SSMs_Constructed.png|In green are the four classes of coding SSMs, and in red is the class of regulatory SSM created. These repeats were placed into their respective constructs.<br />
Image:SSM_In_and_Out.png|The measured fluorescence of the coding AGTC construct and the regulatory TA construct on a plate reader. These two classes were found to have significant differences between the on and off state. Error bars represent the standard deviation of two samplings from the same culture.<br />
Image:SSM_Fluorescence_1.png|The fluorescence spectrum of individual cells containing the AGTC ‘in’ and ‘out’ coding SSM constructs. Spectrum of positive control is in green, negative control is in blue, AGTC ‘in’ construct is in brown, and AGTC ‘out’ is in red. Note that the ‘in’ repeat spectrum has a peak at a higher value than the ‘out’ repeat spectrum. In addition, note the broad distribution of the spectrum. The constructs were placed in a high copy plasmid (>100 copies per cell). Thus, each one of those plasmids has a chance of slipping, and the total number of GFP transcripts from all the plasmids in a cell determines the overall fluorescence of the cell. <br />
Image:SSM_Fluorescence_2.png|The fluorescence spectrum of individual cells containing the TA ‘on’ and ‘off’ regulatory SSM constructs. Spectrum of negative control is in red, TA ‘on’ is in blue, and TA ‘off’ is in green. The TA ‘on’ distribution lies to the right of the TA ‘off’ distribution, indicating that the TA ‘on’ construct is a better promoter than its counterpart. These distributions were taken at a higher PMT value than the distributions in the left image. The peaks in between 10<sup>0</sup> and 10<sup>1</sup> fluorescence may be debris the flow cytometer picked up. Our flow cytometer is commonly used with eukaryotic cells and has not yet been optimized for use with bacterial cells. The cells measured with the flow cytometer in this figure are the same ones measured with the plate reader.<br />
</gallery><br />
<br><br />
Sequencing results of long repeats suggest the occurrence of slipped-strand mispairing. If a slipped-strand mispairing occurs early on in a cell line’s history, then a large proportion of the population will have shifted to a construct with a differing number of repeats, and the resulting sequencing of the miniprep will show mixed products. If a slipped-strand mutation occurs later on in the colony’s lineage, then a smaller proportion of the population will have shifted, and the resulting sequencing will have clear peaks, indicating single nucleotides at those locations. <br><br><br />
[[Image:SSM_Sequencing.png| 600px | thumb| center| Sequencing results suggest slipped-strand mutation. Cells were transformed with just the coding SSM construct (AGTC)<sub>9</sub>. However, the sequencing trace for a colony transformed with (AGTC)<sub>9</sub> also shows the presence of (AGTC)<sub>8</sub>, suggesting that slippage at the AGTC repeat had occurred. The sequence for GFP after the start codon follows the AGTC repeats.]]<br><br />
===Future Work===<br />
In future work, we would use the SSM as a switch to turn on the gene that codes the trigger for the second device. To implement a coding SSM, we would place repeats after the start codon of the trigger gene. The trigger would initially be off and turned on when the polymerase slips. Or, to implement a regulatory SSM, we would place the trigger gene downstream of a SSM promoter. Then the gene would be transcribed when the promoter slips into the right length to bind to a transcriptional activator.<br />
<br />
We would also place coding and regulatory SSMs in front of LacZ, and then chromosomally integrating the construct into ''E. coli'' so that there is only one copy of the construct in a cell. Our current work utilizes a high copy number plasmid, and it is difficult to resolve individual slipped strand mutations and calculate slippage frequency in such cases. With LacZ, blue-white screening can then be conducted to determine the switching frequency of the SSMs. In addition, we plan to place the SSMs in front of the gene that codes for tetracycline resistance (tetA). We can then select for and against cells with tetracycline resistance. <br />
<br><br><br />
==Random State Generator: FimE Recombinase==<br />
<br />
[[Image:FimE_Simple.png| 400px | thumb| right| FimE flips the region flanked by IRL and IRL. A tetR promoter is placed between the two binding sites facing upstream. When the DNA segment is flipped, the promoter faces downstream, and GFP is produced. Once the in and out components of a binding site do not match, FimE no longer recognizes that site. The length of the recombination region (in red) was varied from 250 bp to 850 bp in increments of 100 or 200 bp.]]<br />
FimE is the trigger that will act on the random state generator. FimE is a recombinase protein that can flip DNA flanked by two binding sites. The random state generator consists of recombination regions flanked by FimE binding sites. FimE recognizes binding sites called IRR (inverted repeat right) and IRL (inverted repeat left). Each IRL and IRR consists of an inner and outer sequence, and fimE will only recognize a site that has matching inner and outer sequences. After FimE flips the DNA in between two correctly formed binding sites, the binding sites become distorted, and FimE can no longer act on the region. Taking advantage of this effect, one could place a promoter initially pointing upstream in between the two binding sites, and a gene downstream of the recombination region. Since the promoter is pointing in the opposite direction of the gene, the downstream gene will not be transcribed. However, once FimE flips the flanked recombination region, the promoter then points in the correct direction and can transcribe the downstream gene<sup>9</sup>.<br />
{{Clear}}<br />
===Results===<br />
[[Image:FimE_Vary_Length.png| 400px | thumb| left| The fluorescence of cells with constructs in Figure 6, with varying distances between the two FimE binding sites. A peak at around 250 bp is observed. Fluorescence is correlated to the proportion of plasmids that contain flipped promoter. Cells were measured on plate reader, and error bars show the standard deviation of experiments performed in triplicate.]]<br />
Given that we were placing a different DNA segment in between the two binding sites than is found in wild-type genes, we wanted to determine the range of lengths FimE will act on and how the length affects FimE activity. In addition, we want to determine if the ratio of determined states could be skewed by changing the lengths of different recombination sites. Thus, we tested how the length of the recombination site affects FimE’s ability to detect the two flanking binding sites. The fluorescence of cells with each construct was used as an indicator of FimE activity. We observed a relationship between GFP fluorescence and separation length (Figure 7). Maximum fluorescence was observed to occur at about a separation length of 250 bp. This distance may be a function of entropy and enthalpy. The chances that two DNA segments on the same strand come together statistically decrease as the two segments are separated by intervening sequences. In addition, two sites very close together are unlikely to come into contact with each other, as it is energetically unfavorable to bend DNA to such a great degree. These two opposing forces might explain the presence of an optimal separation length.<br />
{{Clear}}<br />
===Future Work===<br />
Utilizing the fact that FimE flips DNA and cannot recognize binding sites that it has distorted, we can design a system that uses two IRR and two IRL sites to produce a total of three possible states, depending on how FimE acts. Once FimE flips any region in this system, it cannot flip the DNA segment again. Hence, the state of the cell is established after FimE acts. This assumes that FimE is unable to flip adjacent IRL and IRR sites, as it is difficult to bend around such a short DNA segment. The system we propose is shown below. By varying the length of the segment containing the promoter and the length of the segment containing the terminator, we may be able to vary the ratio of determined states.<br><br><br />
[[Image:Pop_Var_Final.png| 600px | thumb| center|a) The device design for the population variation generator. Gene A and Gene B (in opposite orientation) are initially off. b) Depending which IRR and IRL sites FimE binds to, different DNA segments are flipped. The bracket indicates the region that was flipped in each case. In the first case, gene B is turned on. In the second case, gene A is turned on. In the third case, gene A and gene B remain off and stay off, since FimE no longer has a valid IRR and IRL to chose from (the middle two binding sites are too close for FimE to act upon). Terminators at the ends of gene A and B are not shown.]]<br />
==Conclusion==<br />
<br />
We have evidence that short nucleotides repeats can be placed in the promoter or behind a start codon and function as randomized genetic switches through slipped-strand mutation. We have also shown that FimE can flip a segment of non-native DNA, and have evidence that the probability that FimE flips the segment depends on the length between the two binding sites. In the future, selected SSMs can be integrated into a FimE expression construct; thus, FimE expression can be randomly switched on in cells. FimE can then interact with the random state generator. Linked together, the probabilistic switch and the random state generator allow for the asynchronous differentiation of cells into mutually exclusive and fatal states.<br />
<br />
Shown below is the final system design, incorporating the other subprojects in our iGEM team. Initially, ''fimE'' is out of frame. When (AGTC)<sub>10</sub> through SSM becomes (AGTC)<sub>9</sub>, ''fimE'' falls into frame, and the FimE trigger protein is expressed. It interacts with the population variation generator, either committing the cell into a state of [[Team:Caltech/Project/Oxidative_Burst|<font style="color:#BB4400">ROS production</font>]] or [[Team:Caltech/Project/Lactose_intolerance|<font style="color:#BB4400">curing lactose intolerance</font>]]. In the default, undifferentied state, cell will produce a basal level of [[Team:Caltech/Project/Vitamins|<font style="color:#BB4400">folate biosynthesis enzymes</font>]]. [[Team:Caltech/Project/Phage_Pathogen_Defense|<font style="color:#BB4400">Phage induction</font>]] will be controlled by another AGTC coding SSM. Thus, this population variation machinery links the [[Team:Caltech/Project|<font style="color:#BB4400">subprojects</font>]] produced by our team into one coherent system.<br />
<br><br><br />
[[Image:Pop_Var_Conclusion.png| 600px | thumb| center| The final system design, linking the subprojects in this iGEM together. The folate biosynthesis pathway will be continually expressed in all states, and the phage induction system will be controlled by a seperate coding SSM (not shown).]]<br />
<br><br><br />
==References==<br />
<br />
[1] Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, and Keasling JD. '''Production of the antimalarial drug precursor artemisinic acid in engineered yeast'''. Nature 2006 Apr 13; 440(7086) 940-3.<br />
<br />
[2] Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, Davidson EA, Scouras A, Ellington AD, Marcotte EM, and Voigt CA. '''Synthetic biology: engineering Escherichia coli to see light'''. Nature 2005 Nov 24; 438(7067) 441-2.<br />
<br />
[3] Lu TK and Collins JJ. '''Dispersing biofilms with engineered enzymatic bacteriophage'''. Proc Natl Acad Sci U S A 2007 Jul 3; 104(27) 11197-202.<br />
<br />
[4] Henderson IR, Owen P, and Nataro JP. '''Molecular switches -- the ON and OFF of bacterial phase variation'''. Mol Microbiol 1999 Sep; 33(5) 919-32.<br />
<br />
[5] Levinson G and Gutman GA. '''Slipped-strand mispairing: a major mechanism for DNA sequence evolution'''. Mol Biol Evol 1987 May; 4(3) 203-21.<br />
<br />
[6] Levinson G and Gutman GA. '''High frequencies of short frameshifts in poly-CA/TG tandem repeats borne by bacteriophage M13 in Escherichia coli K-12'''. Nucleic Acids Res 1987 Jul 10; 15(13) 5323-38.<br />
<br />
[7] Torres-Cruz J and van der Woude MW. '''Slipped-strand mispairing can function as a phase variation mechanism in Escherichia coli'''. J Bacteriol 2003 Dec; 185(23) 6990-4.<br />
<br />
[8] van Ham SM, van Alphen L, Mooi FR, and van Putten JP. '''Phase variation of H. influenzae fimbriae: transcriptional control of two divergent genes through a variable combined promoter region'''. Cell 1993 Jun 18; 73(6) 1187-96. <br />
<br />
[9] Ham TS, Lee SK, Keasling JD, and Arkin AP. '''A tightly regulated inducible expression system utilizing the fim inversion recombination switch'''. Biotechnol Bioeng 2006 May 5; 94(1) 1-4. <br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/ProjectTeam:Caltech/Project2008-10-21T16:19:20Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
__NOTOC__<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Subprojects</font></div><br />
<div style="font-size:10pt;"><br />
<br />
<font face="verdana" style="color:#BB4400">Note: Click on the subproject title or picture for a detailed description of the subproject</font></div><br />
<br><br />
<br />
==[[Team:Caltech/Project/Oxidative Burst|<font face="verdana" style="color:#BB4400">Oxidative Burst</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}{{navimg|xsize=200|ysize=272|image=neutrophil-shigella.jpg|link=Team:Caltech/Project/Oxidative_Burst}}<br />
{{!}}<br />
Specialized white blood cells called neutrophils defend us from illness by killing bacteria with a potent concoction of degradative enzymes and oxidizing agents, including hydrogen peroxide. However, pathogens of the human large intestine are able to cause serious illness while being sheltered from neutrophils. We engineered a strain of ''Escherichia coli'' that is able to mimic a neutrophil by producing cytotoxic amounts of hydrogen peroxide in a controlled, inducible manner. Our engineered ''E. coli'' use the transcriptional activator LuxR to detect the presence of acyl-homoserine lactones, quorum sensing signaling molecules secreted by invading pathogens. LuxR activates production of the pyruvate oxidase of ''Streptococcus pneumoniae'', which produces large amounts of hydrogen peroxide by oxidizing pyruvate. The engineered ''E. coli'' is capable of killing certain strains of antibiotic resistant ''E. coli'' within six hours. When translated into a probiotic strain such as Nissle 1917, this system has the potential to be an effective means of combating enteric pathogens.<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Phage Pathogen Defense|<font face="verdana" style="color:#BB4400">Phage Pathogen Defense</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}<br />
Another aspect of bacterial pathogen defense for our probiotic is to produce bacteriophages, which would rapidly infect and wipe out all of the pathogens. There are basically two methods to approach phage production, differentiated by the type of phage used. The first uses the bacteriophage λ, which targets E. Coli. The other is exploring the use of a temperate bacteriophage from B. Subtilis, however this method, if successful, can be adapted to temperate bacteriophages of any bacterial strain.<br />
<br />
Bacteriophage λ is a temperate phage with an E. Coli. host, λ infects E. Coli through the lamB receptor, and absence of this receptor prevents λ infection. We will takes advantage of this aspect of bacteriophage λ to create E. Coli which are resistant to the phage, but release the phage to destroy susceptible pathogenic E. Coli. <br />
<br />
The second approach is more versatile, and can target more strains of pathogenic bacteria. The goal is to create a phasmid out of the genome of a temperate bacteriophage. A phasmid combines a E. Coli plasmid Origin of Replication with a linear phage genome, circularizing it. This allows the phasmid to pass on as a plasmid within E. Coli, however when transferred to its native host, the phage phage is induced. <br />
{{!}}{{navimg|xsize=220|ysize=258|image=Phage.jpg|link=Team:Caltech/Project/Phage_Pathogen_Defense}}<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Lactose intolerance|<font face="verdana" style="color:#BB4400">Lactose Intolerance</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}{{navimg|xsize=250|ysize=187|image=Milk.jpg|link=Team:Caltech/Project/Lactose_intolerance}}<br />
{{!}}<br />
Approximately 75% of adults worldwide suffer from lactose intolerance, the inability to metabolize lactose in the small intestine. We propose to treat lactose intolerance by engineering a strain of ''Escherichia coli'' that can reside in the large intestine. The engineered strain will sense lactose and subsequently release ß-galactosidase to convert lactose into glucose and galactose, both of which can be reabsorbed by the host. To treat lactose intolerance, our engineered bacterial strain will contain two plasmids: one with constitutive expression of a mutant lactose permease and ß-galactosidase, and the second with lactose-inducible expression of the λ phage lysis cassette. The mutant lactose permease allows the cells to import lactose under all conditions. When the cells uptake enough lactose, the second plasmid will induce cell lysis through activation of the λ phage lysis cassette, resulting in cell lysis and release of ß-galactosidase into the large intestine. Data covering the construction and characterization of these plasmid constructs is [[Team:Caltech/Project/Lactose_intolerance|<font style="color:#BB4400">discussed</font>]].<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Vitamins|<font face="verdana" style="color:#BB4400">Vitamin Production</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}<br />
Folate, a term which encompasses the various forms of the vitamin B9, is an essential vitamin involved in everyday cell functions such as DNA replication. Unable to naturally produce folate, humans must obtain it from vegetables or folate-supplements. In regions with little or no access to these foods, folate deficiencies can cause serious birth defects. One possible solution to alleviate the effects of folate deficiency is to engineer a strain of gut microbes to produce bioavailable folate directly in the colon. A total of four heterologous genes, two from the folate biosynthesis gene cluster and two from the paraaminobenzoic acid (pABA) synthesis pathway, were tested. Using standardized genetic sequences, folate biosynthesis genes extracted from the ''Lactoccocus lactis'' genome were cloned into Biobricks plasmids, transformed into ''Escherichia coli'' and overexpressed. The effects of overexpression<br />
were measured in terms of total folate and paraaminobenzoic acid levels. PABA, an intermediate in folate synthesis, was detected using [[Team:Caltech/Protocols/PABA_HPLC_assay|<font style="color:#BB4400">high performance liquid chromatography</font>]] (HPLC). Folate detection was achieved via a [[Team:Caltech/Protocols/Folate_assay|<font style="color:#BB4400">microbiological assay</font>]]. A measurable increase in folate production in ''E. coli'' would be a proof-of-concept for both the feasibility of engineering overproduction of folate in ''E. coli'' as well as using standardized genetic components to do so.<br />
{{!}}{{navimg|xsize=193|ysize=288|image=Folate_foods.jpg|link=Team:Caltech/Project/Vitamins}}<br />
{{!}}}<br />
<br />
==[[Team:Caltech/Project/Population Variation|<font face="verdana" style="color:#BB4400">Population Variation</font>]]==<br />
{{{!}}<br />
{{!}}-valign="top"<br />
{{!}}{{navimg|xsize=220|ysize=231|image=Differentiation.jpg|link=Team:Caltech/Project/Population_Variation}}<br />
{{!}}<br />
As more complicated and interconnected biological circuits are synthesized, there becomes the need for the simple integration of multiple functions into a single bacterial cell line. However, some of these functions may be incompatible or may kill the cell, such that each cell can only express a single function at any time and must be regenerated if the functionality is fatal. We aim to combine multiple mutually exclusive and potentially fatal functions into a single bacterial cell line that, as a population, expresses the entire set of functions. <br />
<br />
In particular, we want only one of the four subprojects described above to be turned on in any given cell, or else the cell may be overburdened by our constructs. At the same time, we want our bacterial population to have all four abilities. In addition, three of the four subprojects cause the death of the host cell through self-induced lysis. We need a system that is able to combine all subprojects into one coherent system.<br />
<br />
We propose a system in which bacterial cells initially start in an undifferentiated state and randomly differentiate into one of three possible final states. To build this system, we designed two genetic devices. One relies on DNA polymerase slippage upon replication of a long stretch of short nucleotide repeats, a phenomenon termed slipped-strand mispairing (SSM). The other device uses the recombinase protein FimE to flip DNA segments. The two devices can form a system that permits the novel introduction of random multi-state differentiation into bacterial cells. Here, we present evidence that short nucleotide repeats can be used as a stochastic switch and that FimE activity depends on the length of the segment being flipped. <br />
{{!}}}<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/Project/VitaminsTeam:Caltech/Project/Vitamins2008-10-20T18:48:07Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#BB4400">''In vivo'' Folate Production</font></div><br />
<br><br />
<br />
==Background Information on Folate==<br />
[[Image:THF_structure.jpg|thumb|left|The structure of tetrahydrofolate.]] <br />
<br />
Folate, the generic term for the various forms of Vitamin B9, is an essential vitamin involved in single-carbon transfer reactions, which are important for many pathways including amino acid synthesis. Folate deficiencies in women can result in birth defects such as neural tube defects and other spinal cord malformations. As important as folate is, humans are unable to produce folate, and so must obtain it from eating foods such as green leafy vegetables or folate-fortified cereals. An engineered strain of bacteria that would constantly release folate into the gut would reduce the need to fortify breads and cereals with folate, as well as reduce folate-related birth defects in regions with little access to folate-containing foods. In addition to all the reasons stated above, folate is an ideal vitamin to be produced in the gut because, unlike many other vitamins, it has been shown to be absorbed in physiologically relevant quantities in the large intestine<sup>1, 2</sup>. <br />
<br />
Structurally, folate consists of three main parts: pteridine (derived from GTP), p-aminobenzoic acid (PABA, derived from chorismate), and a poly-glutamyl tail (derived from linking glutamate). <br />
<br />
==Folate Biosynthesis Pathway==<br />
<br />
[[Image:folate_gene_cluster.jpg |frame|center|50px|The folate gene cluster from ''L.lactis''. Black arrows represent genes which have been tested in metabolic engineering studies, shaded arrows represent genes involved in folate biosynthesis, and white arrows represent genes not involved in folate synthesis<sup>3</sup>.]]<br />
<br />
Although folate is naturally produced in ''E. coli'', the folate biosynthesis pathway in the bacteria ''Lactococcus lactis'' has been more heavily characterized and studied. In previous studies, this folate gene cluster has been successfully transformed into the folate-consuming bacteria ''L. gasseri'', turning the bacteria into folate-producers<sup>4</sup>. Using the folate gene cluster from ''L. lactis'' also offers the additional benefit of removing the operon from its natural regulatory context. There are six major enzymatic activities involved in folate synthesis, which, in ''L. lactis'', are contained in five genes: ''folB'', ''folKE'', ''folP'', ''folC'', and ''folA''<sup>1</sup>. The first four, which we have chosen to focus on, are located in a gene cluster approximately 4.4kb long. We have chosen not to test overexpression of ''folA'' because it encodes for an enzyme which turns one form of folate (dihydrofolate) into another form of folate (tetrahydrofolate). Since our assay would detect both types of folate as part of the total folate production, ''folA'' was not a prime target for overexpression of folate. <br />
<br />
[[Image:Llactis_folate_synthesis.jpg|thumb|left|The folate biosynthesis pathway from ''L.lactis''<sup>3</sup>.]] <br />
Our strategy is to clone the entire folate operon from the ''L.lactis'' genome and to transform the entire operon into ''E.coli''. However, because we are unsure of whether or not the ribosomal binding sites (RBS) within the ''L.lactis'' operon would work in ''E.coli'', we are also going to unpack the operon by cloning each of the four genes individually, placing them behind ''E.coli'' RBSs, and then recombine them into a single empty BioBricks™ plasmid. In addition to the main folate operon, we will also be experimenting with overexpression of the para-aminobenzoic acid (pABA) synthesis pathway from chorismate. Wegkamp ''et al.'' have shown that in order to increase overall total levels of folate, both the pABA synthesis genes and certain folate production genes need to be overexpressed<sup>5</sup>. The pABA pathway involves three enzymatic activities, ''pabA'', ''pabB'', and ''pabC'' – though in ''L.lactis'', ''pabB'' is actually a bifunctional enzyme containing the activities for both ''pabB'' and ''pabC''<sup>5</sup>.<br />
{{clear}}<br />
==System Design==<br />
[[Image:systemdesign_folate.png|thumb|right|Final folate biosynthesis plasmid]]<br />
[[Image:systemdesign_paba.png|thumb|right|Final PABA biosynthesis plasmid]]<br />
<br />
The overall system design for testing folate production in ''E. coli'' is to construct several plasmids: one for each individual gene, one for the folate biosynthesis pathway, and one for the PABA synthesis pathway. Each gene, or gene cluster, would be cloned in an inducible-copy plasmid, which would be low copy by default, but can be switched to high copy via the addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG)to the media. This will allow us to test overexpression of each plasmid separately. In addition, each plasmid contains a constitutive promoter, to ensure the constant production of folate. <br />
{{clear}}<br />
==Folate Detection Methods==<br />
[[Image:Lrham.jpg| thumb| left| Image of ''Lactobacillus rhamnosus.'' Itself a probiotic strain, ''L. rhamnosus'' is commonly used in yogurt [http://www.yaklasansaat.com/resimler/2008haberresimleri/haber_subat_resim/subat19_lactobacillus_rhamnosus.jpg][http://en.wikipedia.org/wiki/Lactobacillus_rhamnosus].]]<br />
We measured folate production, and thus the relative success of our engineering efforts, via a microbiological assay involving the folate-dependent strain ''Lactobacillus rhamnosus'' (ATCC 7469)<sup>6</sup>. The assay uses growth of the folate-dependent strain ''L. rhamnosus'' as an indicator of folate concentrations in the sample. The assay media specifically lacks folate, so folate becomes the limiting factor in the growth of ''L. rhamnosus'', with the only possible source being the bacterial lysates of the engineered ''E. coli''. In order to quantify the relative growth of the folate-dependent strain ''L. rhamnosus'', a standard growth curve must first be characterized using known quantities of folic acid in assay media. Once the standard curve has been established, then experimental growth levels, as quantified by spectrophotometry, can be interpolated. Protocols are based off of BD Biosciences Folate Assay Medium datasheet<sup>7</sup>. Growth was measured by taking the OD at 546nm.<br />
<br />
Further details are available on the [[Team:Caltech/Protocols/Folate assay|<font style="color:#BB4400">folate microbiological assay protocol</font>]] page.<br />
{{clear}}<br />
==pABA Detection Methods==<br />
[[Image:PABA-chem.png|thumb|left|para-Aminobenzoic Acid [http://en.wikipedia.org/wiki/4-Aminobenzoic_acid]]]<br />
para-Aminobenzoic Acid (PABA) can be detected using high performance liquid chromatography (HPLC)<sup>8</sup>. Using a 14 minute protocol, we were able to detect PABA peaks coming off a C18 column at 4.9 min. A standard curve was made by running a series of 1:3 PABA dilutions starting at a 10μg/ml concentration. The PABA for the standard curve was spiked into wild type ''E. coli'' cell lysate, which by itself did not show any detectable PABA peaks. Samples were run using the same HPLC protocol as the standards, and both lysate and supernatant were tested for PABA peaks. <br />
<br />
Again, details on the PABA detection method can be found on the [[Team:Caltech/Protocols/PABA HPLC assay|<font style="color:#BB4400">para-aminobenzoic acid (pABA) HPLC protocol</font>]] page.<br />
<br />
==Results==<br />
<br />
===Modifications to System Design===<br />
[[Image:Target_constructs.png | thumb | right | Target constructs for folate biosynthesis and pABA synthesis gene overexpression. We were able to successfully clone ''folB'' in an inducible-copy plasmid and ''folKE'' and ''pabA'', in high-copy plasmids. Unfortunately, we were unable to complete the ''folBKE'', ''pabB'', or ''pabA + pabB'' constructs.]]<br />
We were able to successfully extract and clone the following three genes: ''folB'', ''folKE'', and ''pabA''. We aimed to create individual constructs of each gene in the IPTG inducible-copy plasmid pSB2K3, as well as constructs with the two folate genes combined and with the two PABA synthesis genes combined. However, we had issues cloning our genes into the pSB2K3 + B0015 terminator construct, and so switched to completing the constructs in the high copy plasmid pSB1AK3 + B0015 terminator.<br />
<br />
Our final constructs, shown on the right, are ''folB'' in pSB2K3, and ''folKE'' and ''pabA'' in pSB1AK3. We were unable to sequence confirm our ''folBKE'' and ''pabB'' constructs, and so unfortunately could not include their data. Previous studies on folate overexpression have shown that both folate synthesis and PABA synthesis genes need to be overexpressed simultaneously in order to increase total folate levels<sup>3</sup>. Therefore, tests on ''folB'' and ''folKE'' were done with and without the addition of PABA during the ''E. coli'' inoculation; detection of higher folate levels on addition of PABA would indicate PABA as a limiting factor for folate production.<br />
<br />
We were unable to use PCR to extract several of the target genes: the entire folate gene cluster, ''folP'', and ''folC''. After analyzing the sequencing results of our other folate biosynthesis genes, we realized that the genetic sequences had several point mutations which were not `stop' codons. This indicated that we had a homologous, but not identical, genomic DNA than the one that we has used for PCR primer design. We believe that this homologous sequence could have resulted in too many differences between the primer and the genomic DNA, resulting in little to no binding. This may explain why we were unable to successfully extract ''folP'', ''folC'', and subsequently, the entire folate gene cluster (since ''folP'' and ''folC'' are the last two genes in the cluster) from the genomic DNA of ''L. lactis''. The revised target constructs are shown . <br />
<br />
<br />
===Folate Assay Results for ''folB'' AND ''folKE''===<br />
<br />
The experimental setup included running a standard curve with known amounts of folic acid (0-10ng) simultaneously with the samples, in order to have the same basis for comparison. The hope was that the standard curve growth OD values would correspond with known concentrations, and so sample concentrations could be interpolated based upon the standard. ''folKE'', which was cloned into a constitutive high-copy plasmid, was tested with and without the addition of 500 ng of PABA during inoculation. ''folB'', which is in an inducible copy plasmid, it was tested induced to high-copy as well as uninduced low-copy, with and without 500 ng of PABA during inoculation. We assayed both the supernatant and the cell lysate, though only the supernatant had measurable results. <br />
<br />
The growth data for the samples are very encouraging since the relative folate levels match what we would expect. We see that the addition of 500 ng of PABA during inoculation dramatically increases overall folate levels for ''folKE'' relative to the samples without PABA. Furthermore, adding PABA to the wild type controls did not affect growth at all, suggesting that the assay bacteria ''L.rhamnosus'' was not metabolizing the extra PABA. The extremely high OD values for all the samples were possibly the result of not completely washing out all of the culture media prior to adding the ''L.rhamnosus'' to the assay samples. <br />
<br />
<gallery widths="375px" heights="250px" ><br />
Image:08-19-08 Folate Assay Std Curve Avg.png|Test #1: Folate Standard Curve from 0-10ng. This standard curve is pretty much useless.<br />
Image:08-19-08 Folate Assay supernatant.png|Test #1: Relative folate levels for ''folKE'' compared to ''E. coli'' wild type. Here we see encouraging confirmation of all the expected trends - the addition of PABA during inoculation increases total folate levels but only in the samples with overexpression of our overexpressed folate constructs.<br />
</gallery><br />
When we repeated the assay in duplicate, this time on ''folB'' as well, we were able to observe the same trends in growth with and without the addition of PABA. Again, we see that for ''folKE'', the addition of PABA does increase total folate levels, though not as dramatically as before. The rampant growth of the wild type control is disconcerting, but wild type folate production again appears to be unaffected by the addition of PABA. Furthermore, folate levels for ''folKE'' are still higher than the control where ''L.rhamnosus'' was added to only media without supernatant.<br />
<gallery widths="375px" heights="250px" ><br />
Image:08-20-08 Folate Assay Std 16 hrs.png|Test #2: Folate Standard Assay.A beautiful linear range between .1 and 10 ng can be seen, but overall growth does not correspond to sample growth. Could we be saturating the standard?<br />
Image:08-20-08 Folate Assay supernatant 16 hrs folKE, BKE.png|Test #2: Folate Assay Results with ''folKE'' and ''folB''. Although the wild type growth is mystifying, the folate levels for ''folKE'' exhibit the same trends as before, where increased folate is observed with the addition of PABA. ''FolB'' was tested in low (no IPTG) and high (with IPTG) copy since it is in an inducible-copy plasmid. In the folB data, induced high copy without PABA shows higher growth than low copy without PABA, and the addition of PABA shows the highest growth of all. It is interesting to note that that the two +PABA levels for ''folB'' appear to be the same.<br />
</gallery><br />
<br />
[[Image:bottleneck.png|thumb|right|Increasing the flux of both pathways upstream of pABA integration into the pterin (derived from GTP) component of folate may have created a bottleneck. Unfortunately we were unable to test this hypothesis since we could not overexpress ''folP''<sup>3</sup>]]<br />
And what of ''folB''? Recall that ''folB'' was the only gene to be successfully cloned into an inducible-copy plasmid, and so it was tested both induced and uninduced. The folate levels in the induced sample (high-copy) are higher than in the uninduced (low-copy) sample, which is consistent with what we would expect. The addition of PABA to both induced and uninduced increases relative levels of folate, which is also consistent. However, it is interesting to note that folate levels for the +PABA samples are the same for induced and uninduced. Of course, given the small sample size, this could just be due to variability, but it could also suggest that folate production reached an upper limit. This explanation seems even more likely if we reconsider the folate biosynthesis pathway, and we see that ''folB'' and ''folKE'' are both located upstream of actual integration of PABA into the molecule, accomplished by ''folP''. It is very possible that we are increasing the flux of both pathways going into the ''folP'' junction, but as we were unable to overexpress ''folP'' as well, we may have created a bottleneck.<br />
{{clear}}<br />
<br />
===para-Aminobenzoic Acid (pABA) HPLC Assay Results for ''pabA''===<br />
<gallery widths="375px" heights="250px" ><br />
Image:pABA.png|HPLC peaks for the ''pabA'' sample. The blue trace is the control cell supernatant, which has no peak at 4.9 min, the elution time we found for PABA. The area for the ''pabA'' peak, shown in green, is 82.55.<br />
Image:pABA trendline.png| Standard Curve with linear trendline (y = 117.39x). Using this linear approximation, the concentration of pABA is 703 ng/mL for ''pabA'' overexpression. <br />
</gallery><br />
<br />
Using high performance liquid chromatography (HPLC) we were able to detect PABA peaks from overexpression of ''pabA'' compared to a control sample of wild type supernatant. Compared to the peak areas of the standard curve, we were able to use a linear approximation to determine the concentration of PABA to be 703 ng/mL for ''pabA'' overexpression. <br />
<br />
===Conclusions and Future Work===<br />
<br />
We have shown, through very preliminary tests, the feasibility of overproduction of folate in ''E. coli'' using genes originally derived from ''L. lactis''. Our data have confirmed previous studies in the necessity of overexpressing both folate and PABA synthesis genes, and we have shown that folate appears to be mostly present in the supernatant. Our data, again confirming previous results<sup>9</sup>, also suggest that overexpression of ''folKE'' is the most effective, definitely more effective than ''folB''.<br />
<br />
We have also shown that it is possible to overexpress para-aminobenzoic acid production in ''E. coli'' and that overexpression of ''pabA'' increases total levels of para-aminobenzoic acid (PABA).<br />
<br />
Further work on this project would include repeating the folate assay to generate more data, perfecting the assay in order to quantify folate levels with the standard curve, making the ''pabA'' + ''pabB'' and ''folB'' + ''folKE'' constructs, making a ''folB'' + ''folKE'' + ''pabA'' + ''pabB'' construct, and extracting and cloning ''folP'' from the ''L. lactis'' genome. <br />
<br />
==Relevant Parts==<br />
==Basic Parts==<br />
{{{!}} border="1"<br />
{{!}}+ Basic Parts (Extracted from ''L.lactis subspecies IL1403'' genome)<br />
! Part Name !! Registry # !! Description !! Cloned? !! Sequence confirmed? <br />
{{!}}-<br />
! Entire folate synthesis operon<br />
{{!}} [http://partsregistry.org/Part:BBa_K137002 BBa_K137002]{{!}}{{!}} Includes folB+folKE+folP+ylgG+folC {{!}}{{!}} NO {{!}}{{!}} NO<br />
{{!}}-<br />
! folB<br />
{{!}} [http://partsregistry.org/Part:BBa_K137009 BBa_K137009]{{!}}{{!}} dihydroneopterin aldolase {{!}}{{!}} YES{{!}}{{!}} YES<br />
{{!}}-<br />
! folKE<br />
{{!}} [http://partsregistry.org/Part:BBa_K137011 BBa_K137011]{{!}}{{!}} fusion gene: GTP cyclohydrolase & 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase {{!}}{{!}} YES{{!}}{{!}} YES<br />
{{!}}-<br />
! folP<br />
{{!}} [http://partsregistry.org/Part:BBa_K137012 BBa_K137012]{{!}}{{!}} Dihydropteroate synthase {{!}}{{!}} NO{{!}}{{!}} NO<br />
{{!}}-<br />
! folC<br />
{{!}} [http://partsregistry.org/Part:BBa_K137013 BBa_K137013]{{!}}{{!}} folate synthetase/polyglutamyl folate synthetase {{!}}{{!}} NO{{!}}{{!}} NO<br />
{{!}}-<br />
! pabA<br />
{{!}} [http://partsregistry.org/Part:BBa_K137005 BBa_K137005]{{!}}{{!}} para-aminobenzoate synthetase component II {{!}}{{!}} YES{{!}}{{!}} YES<br />
{{!}}-<br />
! pabB<br />
{{!}} [http://partsregistry.org/Part:BBa_K137006 BBa_K137006]{{!}}{{!}} para-aminobenzoate synthetase component I {{!}}{{!}} YES{{!}}{{!}} YES<br />
{{!}}-<br />
{{!}}}<br />
==Construction Intermediates==<br />
{{{!}} border="1"<br />
{{!}}+ Construction Intermediates: Adding B0034 (strong RBS) before each individual gene<br />
! Part Name !! Registry # !! Description !! Cloned? !! Sequence confirmed? <br />
{{!}}-<br />
! folB + B0034<br />
{{!}} [http://partsregistry.org/Part:BBa_S03957 BBa_S03957]{{!}}{{!}} RBS + dihydroneopterin aldolase {{!}}{{!}} YES{{!}}{{!}} YES<br />
{{!}}-<br />
! folKE + B0034<br />
{{!}} [http://partsregistry.org/Part:BBa_S03958 BBa_S03958]{{!}}{{!}} RBS + fusion gene: GTP cyclohydrolase & 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase {{!}}{{!}} YES{{!}}{{!}} YES<br />
{{!}}-<br />
! folP + B0034<br />
{{!}} [http://partsregistry.org/Part:BBa_S03959 BBa_S03959]{{!}}{{!}} RBS + Dihydropteroate synthase {{!}}{{!}} NO{{!}}{{!}} NO<br />
{{!}}-<br />
! folC + B0034<br />
{{!}} [http://partsregistry.org/Part:BBa_S03960 BBa_S03960]{{!}}{{!}} RBS + folate synthetase/polyglutamyl folate synthetase {{!}}{{!}} NO{{!}}{{!}} NO<br />
{{!}}-<br />
! pabA + B0034<br />
{{!}} [http://partsregistry.org/Part:BBa_S03976 BBa_S03976]{{!}}{{!}} RBS + para-aminobenzoate synthetase component II {{!}}{{!}} YES{{!}}{{!}} YES<br />
{{!}}-<br />
! pabB + B0034<br />
{{!}} [http://partsregistry.org/Part:BBa_S03977 BBa_S03977]{{!}}{{!}} RBS + para-aminobenzoate synthetase component I {{!}}{{!}} YES{{!}}{{!}} YES<br />
{{!}}-<br />
! B0034 + folB + B0034 + folKE <br />
{{!}} [http://partsregistry.org/Part:BBa_S03961 BBa_S03961]{{!}}{{!}} Combining folB (with RBS) + folKE (with RBS) {{!}}{{!}} YES{{!}}{{!}} YES<br />
{{!}}-<br />
{{!}}}<br />
<br />
{{{!}} border="1"<br />
{{!}}+ Construction Intermediates: Adding J23100 (constitutive promoter) to each gene (with RBS already)<br />
! Part Name !! Registry # !! Description !! Cloned? !! Sequence confirmed? <br />
{{!}}-<br />
! folB + J23100<br />
{{!}} [http://partsregistry.org/Part:BBa_S04032 BBa_S04032]{{!}}{{!}} Promoter(j23100) + RBS(b0034) + dihydroneopterin aldolase {{!}}{{!}} YES{{!}}{{!}} YES<br />
{{!}}-<br />
! folKE + J23100<br />
{{!}} [http://partsregistry.org/Part:BBa_S04033 BBa_S04033]{{!}}{{!}} Promoter + RBS + fusion gene: GTP cyclohydrolase & 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase {{!}}{{!}} YES{{!}}{{!}} YES<br />
{{!}}-<br />
! pabA + J23100<br />
{{!}} [http://partsregistry.org/Part:BBa_S04034 BBa_S04034]{{!}}{{!}} Promoter + RBS + para-aminobenzoate synthetase component II {{!}}{{!}} YES{{!}}{{!}} YES<br />
{{!}}-<br />
! folBKE + J23100<br />
{{!}} [http://partsregistry.org/Part:BBa_S04035 BBa_S04035]{{!}}{{!}} Promoter + folB (with RBS) + folKE (with RBS) {{!}}{{!}} YES {{!}}{{!}} YES<br />
{{!}}-<br />
! pabB + J23100<br />
{{!}} [http://partsregistry.org/Part:BBa_S04039 BBa_S04039]{{!}}{{!}} Promoter + pabB {{!}}{{!}} YES {{!}}{{!}} YES <br />
{{!}}-<br />
! pabB + B0015<br />
{{!}} [http://partsregistry.org/Part:BBa_S04038 BBa_S04038]{{!}}{{!}} pabB + b0015 terminator {{!}}{{!}} MAYBE {{!}}{{!}} NO<br />
{{!}}-<br />
{{!}}}<br />
===Composite Parts===<br />
{{{!}} border="1"<br />
{{!}}+ Composite Parts: Adding B0015 (double terminator) to complete constructs with promoter + RBS already<br />
! Part Name !! Registry # !! Description !! Cloned? !! Sequence confirmed? <br />
{{!}}-<br />
! folB + B0015<br />
{{!}} [http://partsregistry.org/Part:BBa_K137053 BBa_K137053]{{!}}{{!}} Promoter(j23100) + RBS(b0034) + folB (dihydroneopterin aldolase) + double terminator (b0015) {{!}}{{!}} YES {{!}}{{!}} YES<br />
{{!}}-<br />
! folKE + B0015<br />
{{!}} [http://partsregistry.org/Part:BBa_K137054 BBa_K137054]{{!}}{{!}} Promoter + RBS + folKE (fusion gene: GTP cyclohydrolase & 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase) + double terminator (b0015) {{!}}{{!}} YES {{!}}{{!}} YES<br />
{{!}}-<br />
! pabA + B0015<br />
{{!}} [http://partsregistry.org/Part:BBa_K137055 BBa_K137055]{{!}}{{!}} Promoter + RBS + pabA (para-aminobenzoate synthetase component II) {{!}}{{!}} YES {{!}}{{!}} YES<br />
{{!}}-<br />
! pabB + B0015<br />
{{!}} [http://partsregistry.org/Part:BBa_K137109 BBa_K137109]{{!}}{{!}} Promoter + RBS + pabB (para-aminobenzoate synthetase component I) {{!}}{{!}} YES {{!}}{{!}} NO<br />
{{!}}-<br />
! folBKE + B0015<br />
{{!}} [http://partsregistry.org/Part:BBa_K137056 BBa_K137056]{{!}}{{!}} Promoter + RBS + folB + folKE + b0015 {{!}}{{!}} YES {{!}}{{!}} NO<br />
{{!}}-<br />
{{!}}}<br />
<br />
==References==<br />
[1] Asrar FM and O’Connor DL. '''Bacterially synthesized folate and supplemental folic acid are absorbed across the large intestine of piglets.''' J Nutr Biochem 2005 Oct; 16(10) 587-93.<br />
<br />
[2] Camilo E, Zimmerman J, Mason JB, Golner B, Russell R, Selhub J, and Rosenberg IH. '''Folate synthesized by bacteria in the human upper small intestine is assimilated by the host.''' Gastroenterology 1996 Apr; 110(4) 991-8.<br />
<br />
[3] Sybesma W, Starrenburg M, Kleerebezem M, Mierau I, de Vos WM, and Hugenholtz J. '''Increased production of folate by metabolic engineering of Lactococcus lactis.''' Appl Environ Microbiol 2003 Jun; 69(6) 3069-76.<br />
<br />
[4] Wegkamp A, Starrenburg M, de Vos WM, Hugenholtz J, and Sybesma W. '''Transformation of folate-consuming Lactobacillus gasseri into a folate producer.''' Appl Environ Microbiol 2004 May;70(5) 3146-8.<br />
<br />
[5] Wegkamp A, van Oorschot W, de Vos WM, and Smid EJ. '''Characterization of the role of para-aminobenzoic acid biosynthesis in folate production by Lactococcus lactis.''' Appl Environ Microbiol 2007 Apr; 73(8) 2673-81.<br />
<br />
[6] Horne DW and Patterson D. '''Lactobacillus casei microbiological assay of folic acid derivatives in 96-well microtiter plates.''' Clin Chem 1988 Nov; 34(11) 2357-9.<br />
<br />
[7] BD Biosciences. '''Folic Acid Assay Medium.''' [http://www.bd.com/ds/technicalCenter/inserts/Folic_Acid_Assay_Medium.pdf]<br />
<br />
[8] Zhang GF, Mortier KA, Storozhenko S, Steene JVD, Straeten DVD, Lambert WE. '''Free and total para-aminobenzoic acid analysis in plants with high-performance liquid chromatography/tandem mass spectrometry.''' Rapid Communications in Mass Spectrometry: Volume 19, Issue 8 , Pages 963 - 969, 2005.<br />
<br />
[9] Sybesma W, Burgess C, Starrenburg M, van Sinderen D, and Hugenholtz J. '''Multivitamin production in Lactococcus lactis using metabolic engineering.''' Metab Eng 2004 Apr; 6(2) 109-15.<br />
}}</div>Jkmhttp://2008.igem.org/Team:CaltechTeam:Caltech2008-10-20T18:47:10Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
__NOTOC__<br />
==Engineering multi-functional probiotic bacteria==<br />
[[Image:Gut_flora_color.png|right|thumb|200px|Engineered gut flora]]<br />
The human gut houses a diverse collection of microorganisms, with important implications for the health and welfare of the host. We aim to engineer a member of this microbial community to provide innovative medical treatments. Our work focuses on four main areas: (1) pathogen defense, either by expression of [[Team:Caltech/Project/Phage_Pathogen_Defense|<font style="color:#BB4400">pathogen-specific bacteriophage</font>]] or by targeted bursts of [[Team:Caltech/Project/Oxidative_Burst|<font style="color:#BB4400">reactive oxygen species</font>]]; (2) prevention of birth defects by [[Team:Caltech/Project/Vitamins|<font style="color:#BB4400">folate over-expression</font>]] and delivery; (3) treatment of [[Team:Caltech/Project/Lactose_intolerance|<font style="color:#BB4400">lactose intolerance</font>]], by cleaving lactose to allow absorption in the large intestine; and (4) [[Team:Caltech/Project/Population_Variation|<font style="color:#BB4400">regulation</font>]] of these three treatment functions to produce renewable subpopulations specialized for each function. Our research demonstrates that synthetic biology techniques can be used to modify naturally occurring microbial communities for applications in biomedicine and biotechnology.<br />
{{clear}}<br />
<br />
==Why engineer gut microbes?==<br />
<br />
===The large intestine: an ideal bioreactor===<br />
[[Image:Digestive system diagram en.svg.png|thumb|200px|right|The human digestive tract]]<br />
The human intestinal track is a perfect environment for bacteria. It is a 37°C mobile incubator with a constant stream of food. While bacteria are present in all parts of the intestinal track downstream of the stomach, the majority of those bacteria reside in the large intestine. There are approximately 10<sup>12</sup> bacterial per mL in the large intestinal lumen, comprised of between 500-1000 different species of bacteria. Of these species, approximately 30 species compose 99% of all bacteria in the large intestine. Estimating there is roughly 100 mL of feces in the large intestine, all the bacteria in our gut outnumber all the cells in the human body 100 to 1<sup>1</sup>.<br />
{{clear}}<br />
<br />
===Probiotic bacteria and other natural examples===<br />
[[Image:Lacbr.jpg|thumb|left|Electron micrograph of ''Lactobacillus brevis'', a probiotic lactic acid bacterium]]<br />
Most of the bacteria in our gut have yet to be characterized because they are difficult to culture, owing to their sensitivity to oxygen. However, several species are known. Many bacterial laboratory strains are derived from the well-known ''Escherichia coli'' (a non-pathogenic type) which is normally present in the large intestine. Because of its use in research, ''E. coli'' is the most well characterized bacteria to date. Another bacteria, ''Bacteroides fragilis'', plays an important role in proper development of the immune system and in controlling intestinal inflammation. Specifically, ''B. fragilis'' produces a starch called polysacharride A. In mice that had been raised in a sterile environment since birth (the intestinal track is initially sterile at birth and requires outside sources of bacteria to populate it), the immune system had less than normal levels of CD4+ killer T cells, a necessary white blood cell to battle infections. However the CD4+ levels returned to normal when the mice were raised again in a sterile environment, except for the presence of ''B. fragilis''. Polysaccharide A alone, not the mere presence of Bacteroides fragilis, was responsible for the improvement, since mice raised with ''B. fragilis'' that could not produce polysaccharide A showed the same levels of CD4+ cell as the sterile mice<sup>2</sup>.<br />
{{clear}}<br />
Several bacterial species can cause disease in humans by infecting the gut. ''E. coli'' is commonly associated with food poisoning. ''Salmonela enterica'' is responsible for typhoid fever. ''Campylobacter jejuni'' and ''Shigella'' can cause bowl inflammation, diarrhea and dysentery. Cholera is caused by ''Vibrio cholerae'', which infected around 230,000 people and caused 6,300 deaths in 2006, according to the World Health Organization (WHO)[http://www.who.int/wer/2007/wer8231.pdf]. These pathogens typically cause illness in otherwise healthy people. There are other, more opportunistic bacteria, which infect people in hospital setting who are otherwise sick or undergoing treatment that puts them at greater risk for infection of their intestinal track. Paradoxically, the treatment of one pathogen with antibiotics can make that same patient more susceptible to infection of their gut by opportunistic pathogens. It is thought that the right balance of natural gut flora prevent these opportunistic pathogens from colonizing the colon<sup>3</sup>.<br />
<br />
As a further example, humans cannot produce Vitamin K, an important cofactor in blood clotting. Instead, the vitamin is provided by various species of the gut microbiota, which collectively produce more than 30 times the vitamin K recommended daily allowance<sup>4</sup>.<br />
<br />
<br />
===Nissle 1917: Probiotic, commercially available ''E. coli''===<br />
[[Image:packshot_mutaflor.jpg|thumb|right|Mutaflor - a commercially available preparation of Nissle 1917]]<br />
Nissle 1917 is a commercially available[http://www.ardeypharm.de/en/] non-pathogenic, probiotic strain of ''E. coli''. It has been successfully used to treat gastrointestinal disorders including colitis and intestinal bowel disease<sup>5</sup> and shows little immunostimulatory activity<sup>6</sup>. Engineered versions of the Nissle 1917 strain have been developed as anti-HIV<sup>7</sup> and anti-cholera<sup>8</sup> agents. The ability of Nissle 1917 to efficiently colonize the gut without provoking an inflammatory response makes it an ideal chassis for ''in situ'' applications in biomedicine and biotechnology.<br />
<br />
{{clear}}<br />
<br />
For more details, please see our [[Team:Caltech/Project|<font style="color:#BB4400">project</font>]] page.<br />
<br />
===References===<br />
# Hooper LV, Midtvedt T, and Gordon JI. '''How host-microbial interactions shape the nutrient environment of the mammalian intestine'''. ''Annu Rev Nutr'' 2002; 22 283-307.<br />
# Mazmanian SK, Liu CH, Tzianabos AO, and Kasper DL. '''An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system'''. ''Cell'' 2005 Jul 15; 122(1) 107-18.<br />
# Donskey CJ. '''The role of the intestinal tract as a reservoir and source for transmission of nosocomial pathogens'''. ''Clin Infect Dis'' 2004 Jul 15; 39(2) 219-26.<br />
# Suttie JW. '''The importance of menaquinones in human nutrition'''. ''Annu Rev Nutr'' 1995; 15 399-417.<br />
# Krammer HJ, Kamper H, von Bunau R, Zieseniss E, Stange C, Schlieger F, Clever I, and Schulze J. '''Probiotic drug therapy with E. coli strain Nissle 1917 (EcN): results of a prospective study of the records of 3,807 patients'''. ''Z Gastroenterol'' 2006 Aug; 44(8) 651-6.<br />
# Westendorf AM, Gunzer F, Deppenmeier S, Tapadar D, Hunger JK, Schmidt MA, Buer J, and Bruder D. '''Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules'''. ''FEMS Immunol Med Microbiol'' 2005 Mar 1; 43(3) 373-84.<br />
# Rao S, Hu S, McHugh L, Lueders K, Henry K, Zhao Q, Fekete RA, Kar S, Adhya S, and Hamer DH. '''Toward a live microbial microbicide for HIV: commensal bacteria secreting an HIV fusion inhibitor peptide'''. ''Proc Natl Acad Sci U S A'' 2005 Aug 23; 102(34) 11993-8.<br />
# Duan F and March JC. '''Interrupting Vibrio cholerae infection of human epithelial cells with engineered commensal bacterial signaling'''. ''Biotechnol Bioeng'' 2008 Sep 1; 101(1) 128-34.<br />
}}</div>Jkmhttp://2008.igem.org/Team:CaltechTeam:Caltech2008-10-20T18:46:30Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
__NOTOC__<br />
==Engineering multi-functional probiotic bacteria==<br />
[[Image:Gut_flora_color.png|right|thumb|200px|Engineered gut flora]]<br />
The human gut houses a diverse collection of microorganisms, with important implications for the health and welfare of the host. We aim to engineer a member of this microbial community to provide innovative medical treatments. Our work focuses on four main areas: (1) pathogen defense, either by expression of [[Team:Caltech/Project/Phage_Pathogen_Defense|<font style="color:#BB4400">pathogen-specific bacteriophage</font>]] or by targeted bursts of [[Team:Caltech/Project/Oxidative_Burst|<font style="color:#BB4400">reactive oxygen species</font>]]; (2) prevention of birth defects by [[Team:Caltech/Project/Vitamins|<font style="color:#BB4400">folate over-expression</font>]] and delivery; (3) treatment of [[Team:Caltech/Project/Lactose_intolerance|<font style="color:#BB4400">lactose intolerance</font>]], by cleaving lactose to allow absorption in the large intestine; and (4) [[Team:Caltech/Project/Population_Variation|<font style="color:#BB4400">regulation</font>]] of these three treatment functions to produce renewable subpopulations specialized for each function. Our research demonstrates that synthetic biology techniques can be used to modify naturally occurring microbial communities for applications in biomedicine and biotechnology.<br />
{{clear}}<br />
<br />
==Why engineer gut microbes?==<br />
<br />
===The large intestine: an ideal bioreactor===<br />
[[Image:Digestive system diagram en.svg.png|thumb|200px|right|The human digestive tract]]<br />
The human intestinal track is a perfect environment for bacteria. It is a 37°C mobile incubator with a constant stream of food. While bacteria are present in all parts of the intestinal track downstream of the stomach, the majority of those bacteria reside in the large intestine. There are approximately 10<sup>12</sup> bacterial per mL in the large intestinal lumen, comprised of between 500-1000 different species of bacteria. Of these species, approximately 30 species compose 99% of all bacteria in the large intestine. Estimating there is roughly 100 mL of feces in the large intestine, all the bacteria in our gut outnumber all the cells in the human body 100 to 1<sup>1</sup>.<br />
{{clear}}<br />
<br />
===Probiotic bacteria and other natural examples===<br />
[[Image:Lacbr.jpg|thumb|left|Electron micrograph of ''Lactobacillus brevis'', a probiotic lactic acid bacterium]]<br />
Most of the bacteria in our gut have yet to be characterized because they are difficult to culture, owing to their sensitivity to oxygen. However, several species are known. Many bacterial laboratory strains are derived from the well-known ''Escherichia coli'' (a non-pathogenic type) which is normally present in the large intestine. Because of its use in research, ''E. coli'' is the most well characterized bacteria to date. Another bacteria, ''Bacteroides fragilis'', plays an important role in proper development of the immune system and in controlling intestinal inflammation. Specifically, ''B. fragilis'' produces a starch called polysacharride A. In mice that had been raised in a sterile environment since birth (the intestinal track is initially sterile at birth and requires outside sources of bacteria to populate it), the immune system had less than normal levels of CD4+ killer T cells, a necessary white blood cell to battle infections. However the CD4+ levels returned to normal when the mice were raised again in a sterile environment, except for the presence of ''B. fragilis''. Polysaccharide A alone, not the mere presence of Bacteroides fragilis, was responsible for the improvement, since mice raised with ''B. fragilis'' that could not produce polysaccharide A showed the same levels of CD4+ cell as the sterile mice<sup>2</sup>.<br />
{{clear}}<br />
Several bacterial species can cause disease in humans by infecting the gut. ''E. coli'' is commonly associated with food poisoning. ''Salmonela enterica'' is responsible for typhoid fever. ''Campylobacter jejuni'' and ''Shigella'' can cause bowl inflammation, diarrhea and dysentery. Cholera is caused by ''Vibrio cholerae'', which infected around 230,000 people and caused 6,300 deaths in 2006, according to the World Health Organization (WHO)[http://www.who.int/wer/2007/wer8231.pdf]. These pathogens typically cause illness in otherwise healthy people. There are other, more opportunistic bacteria, which infect people in hospital setting who are otherwise sick or undergoing treatment that puts them at greater risk for infection of their intestinal track. Paradoxically, the treatment of one pathogen with antibiotics can make that same patient more susceptible to infection of their gut by opportunistic pathogens. It is thought that the right balance of natural gut flora prevent these opportunistic pathogens from colonizing the colon<sup>3</sup>.<br />
<br />
As a further example, humans cannot produce Vitamin K, an important cofactor in blood clotting. Instead, the vitamin is provided by various species of the gut microbiota, which collectively produce more than 30 times the vitamin K recommended daily allowance<sup>4</sup>.<br />
<br />
<br />
===Nissle 1917: Probiotic, commercially available ''E. coli''===<br />
[[Image:packshot_mutaflor.jpg|thumb|right|Mutaflor - a commercially available preparation of Nissle 1917]]<br />
Nissle 1917 is a commercially available[http://www.ardeypharm.de/en/] non-pathogenic, probiotic strain of ''E. coli''. It has been successfully used to treat gastrointestinal disorders including colitis and intestinal bowel disease<sup>5</sup> and shows little immunostimulatory activity<sup>6</sup>. Engineered versions of the Nissle 1917 strain have been developed as anti-HIV<sup>7</sup> and anti-cholera<sup>8</sup> agents. The ability of Nissle 1917 to efficiently colonize the gut without provoking an inflammatory response makes it an ideal chassis for ''in situ'' applications in biomedicine and biotechnology.<br />
<br />
{{clear}}<br />
<br />
For more details, please see our [[Team:Caltech/Project|project]] page.<br />
<br />
===References===<br />
# Hooper LV, Midtvedt T, and Gordon JI. '''How host-microbial interactions shape the nutrient environment of the mammalian intestine'''. ''Annu Rev Nutr'' 2002; 22 283-307.<br />
# Mazmanian SK, Liu CH, Tzianabos AO, and Kasper DL. '''An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system'''. ''Cell'' 2005 Jul 15; 122(1) 107-18.<br />
# Donskey CJ. '''The role of the intestinal tract as a reservoir and source for transmission of nosocomial pathogens'''. ''Clin Infect Dis'' 2004 Jul 15; 39(2) 219-26.<br />
# Suttie JW. '''The importance of menaquinones in human nutrition'''. ''Annu Rev Nutr'' 1995; 15 399-417.<br />
# Krammer HJ, Kamper H, von Bunau R, Zieseniss E, Stange C, Schlieger F, Clever I, and Schulze J. '''Probiotic drug therapy with E. coli strain Nissle 1917 (EcN): results of a prospective study of the records of 3,807 patients'''. ''Z Gastroenterol'' 2006 Aug; 44(8) 651-6.<br />
# Westendorf AM, Gunzer F, Deppenmeier S, Tapadar D, Hunger JK, Schmidt MA, Buer J, and Bruder D. '''Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules'''. ''FEMS Immunol Med Microbiol'' 2005 Mar 1; 43(3) 373-84.<br />
# Rao S, Hu S, McHugh L, Lueders K, Henry K, Zhao Q, Fekete RA, Kar S, Adhya S, and Hamer DH. '''Toward a live microbial microbicide for HIV: commensal bacteria secreting an HIV fusion inhibitor peptide'''. ''Proc Natl Acad Sci U S A'' 2005 Aug 23; 102(34) 11993-8.<br />
# Duan F and March JC. '''Interrupting Vibrio cholerae infection of human epithelial cells with engineered commensal bacterial signaling'''. ''Biotechnol Bioeng'' 2008 Sep 1; 101(1) 128-34.<br />
}}</div>Jkmhttp://2008.igem.org/Team:Caltech/Project/Lactose_intoleranceTeam:Caltech/Project/Lactose intolerance2008-10-20T16:20:26Z<p>Jkm: </p>
<hr />
<div>{{Caltech_iGEM_08|<br />
Content=<br />
<br />
<div style="font-size:18pt;"><br />
<font face="verdana" style="color:#CC3300">Curing Lactose Intolerance</font></div><br />
<br />
==Introduction==<br />
The gut flora of our digestive tract contains microorganisms that perform various useful functions for their hosts. Examples of such functions include growth inhibition of harmful microorganisms<sup>1</sup>, defense against the causes of many forms of Inflammatory Bowel Disease<sup>2</sup>, and the fermentation of carbohydrates and other molecules the human body cannot normally digest. Some bacterial strains, including the ''Escherichia coli'' strain ‘Nissle 1917’, can persist in the gut of mice for months<sup>3</sup>. Engineering these bacteria provide a new platform to treat various human diseases.<br />
Lactose intolerance is characterized by the inability to break down lactose in the small intestine. The undigested lactose instead passes to the large intestine, leading to two negative processes: osmotic imbalance and bacterial fermentation. High lactose levels raise the osmolarity of the colon, causing diarrhea. In addition, gut microbes metabolize the lactose into methane gas, causing abdominal pain. Both problems must be addressed in order to fully treat lactose intolerance. If we simply have our strain metabolize lactose, another strain will further ferment the byproducts, resulting in the same side effects. <br />
Instead, we have our engineered strain allow the host to uptake lactose, clearing the sugar from the colon. To do this, we have engineered the ‘Nissle 1917’ strain to release ß-galactosidase, an enzyme that cleaves lactose into glucose and galactose. Since protein secretion is difficult in ''E. coli'', our cells were engineered to lyse in order to release ß-galactosidase. However, lysis must occur only when lactose is present in the gut. Therefore, the cells also express a mutant lactose permease, allowing the strain to sense lactose in all conditions. <br />
<br />
==System Design==<br />
===Lactose Regulator===<br />
[[image:Lactose_regulator.JPG|thumb|200px|left|A mutated LacY allows the uptake of lactose while LacZ produces large amounts of ß-galactosidase. The plasmid contains a strong constitutive promoter regulating both genes and a ColE1 replication origin.]]<br />
The first plasmid consists of a synthetic lactose operon under strong constitutive expression. Our synthetic lactose operon encodes the ß-galactosidase LacZ and a mutant form of the lactose permease LacY. LacY is a membrane protein that actively transports lactose into the cell. Since most membrane proteins are toxic when overexpressed, we optimized our system to express appropriate levels of LacY without killing the cell. In the end, we want to express as much ß-galactosidase as we can to cleave as much lactose present in the large intestine. <br />
The human gut is an unpredictable environment, and we wish our engineered cells to behave reliably despite this variability. E. coli are unable to uptake lactose in the presence of glucose, a phenomenon known as carbon catabolite repression. Catabolite repression is mediated by Enzyme IIA Glucose (IIAGluc), which inhibits the uptake of lactose in the presence of glucose by binding to LacY<sup>4</sup>. Previous research has identified various LacY mutations that prevent this inhibition and achieve increased uptake of lactose in the presence of glucose<sup>5</sup>.<br />
{{clear}}<br />
<br />
===Lactose-Induced Lysis===<br />
[[image:Lactose-inducible_lysis.JPG|thumb|200px|right|The lysis cassette plasmid acts as a lactose sensor. Intracellular lactose accumulation induces overexpression of the lysis cassette. Lactose inhibits binding of the LacI repressor to the promoter and P1 high copy origin of replication. A mutated holin gene in the lysis cassette allows faster lysis times.]]<br />
The second plasmid contains lactose inducible expression of λ phage lysis genes. To release ß-galactosidase, the cells will lyse when enough lactose is present in the cell to induce the expression of the lysis genes. Absent any stimulus, the plasmid containing the lysis genes remains at low copy but switches to high copy when induced with lactose. In addition, the lysis genes are placed behind lactose-inducible promoters. It is important that the lactose-inducible promoter be tightly regulated, since leaky expression will cause spontaneous lysis<sup>6</sup>.<br />
Using wild type λ phage lysis genes, lysis occurs 40-45 minutes after induction by lactose. Decreasing the lag time will reduce the extent of lactose fermentation and therefore produce fewer deleterious effects. Previous research has uncovered mutations that shorten the lysis time to approximately 10-15 minutes<sup>7</sup>, and these mutations will be incorporated into our final construct.<br />
{{clear}}<br />
<br />
==Results==<br />
===Lactose Regulator===<br />
[[IMAGE:Promoter_strength.JPG|thumn|200px|left|Varying the expression levels of LacY with different promoters and ribosome binding sites. Cells died at our two highest expression levels, but survived on the other four. The construct with the weakest RBS was selected since we could express LacZ in the same operon with a strong promoter. This combination allows our cell to express high levels of LacZ and non-toxic levels of LacY.]]<br />
In the first plasmid, it was discovered that LacY is toxic when overexpressed. To determine safe expression levels, constructs were built with varying levels of LacY expression. Six plasmids were built from combinations of three different promoters and two different ribosome binding sites. The cells died when expressing the strongest and medium strength promoters along with the strongest ribosome binding site. We decided to express LacZ by combining the weakest ribosome binding site with the strongest constitutive promoter, preventing LacY toxicity while expressing LacZ in large quantities. <br />
As mentioned earlier, our cells had to overcome carbon catabolite repression. Various mutations, including the insertion of two histidyl residues between amino acids S194 and A195 in the LacY gene, prevent the inhibition by IIAGluc. These insertions have been made, and tests are currently underway.<br />
{{clear}}<br />
<br />
[[image:Figure_1_beta_assay.JPG|thumb|200px|right|ß-galactosidase activity of plasmid #1, with and without the strong constitutive promoter. Error bars on the data with a promoter show the standard deviation of two measurements.]]ß-galactosidase assays were used to quantify our data from our lactose regulator. The assay was performed twice on the synthetic network containing the lactose operon. The first assay was performed on a strain containing the plasmid lacking the strong constitutive promoter. This assay was performed to simulate the eventual system regulation. Our completed project will be under the expression of a promoter controlled by the FimE recombinase. The promoter will initially be pointing away from our synthetic lac operon and will be inverted to face the operon when the system is turned on. We saw significant levels of ß-galactosidase, a surprising result since the plasmid lacked a promoter. These levels are likely due to spurious transcription amplified by our strong ribosome binding site and high copy plasmid. We then repeated the assay one more time with the strong constitutive promoter to observe the dynamic range of LacZ expression in this system.<br />
{{clear}}<br />
<br />
===Lactose-Induced Lysis===<br />
Our second plasmid was not completely cloned; therefore we were unable to perform assays on our second plasmid. There remains one more cloning step to place the lysis cassette into the vector containing the promoters. The single base mutations in the lysis cassette were not made, and if time allowed, they would have been completed as the final step.<br />
<br />
==Discussion==<br />
Our final system was not completely finished; however, we have made progress. The lactose regulator plasmid was completely constructed with the insertion of two histidyl residues in their appropriate locations. Our lactose-induced lysis plasmid was one cloning step away from being completed. The promoter and flanking terminator were cloned in parallel with the lysis cassette and a terminator. The last cloning step would be to ligate the two together. In addition, the two separate point mutations have not been made, and the final step in our cloning would be to add those mutations. <br> After completing the construction phase, we would characterize our system, and then move our complete network into the ‘Nissle 1917’ strain. A construct containing our lactose inducible promoters and GFP would show our promoters can maintain tight expression of GFP from uninduced to induced. We would combine this construct with our first plasmid containing the mutated LacY, to show we can achieve induction by lactose even in the presence of glucose. To show the effect our second plasmid, it would be necessary to achieve lysis with lactose. In addition, we want to show that we can achieve lysis with lactose in the presence of glucose. Finally, the final assay we want to perform on our system would show our cells able to uptake lactose in the presence of glucose, and lyse soon after. Once the cells lyse, the cell lysis should contain ß-galactosidase at levels close to what we received from our ß-galactosidase assay on our lactose regulator. <br />
<br />
==Methods==<br />
Methods can be found [[Team:Caltech/Protocols|<font style="color:#BB4400">here</font>]]<br />
<br />
==References==<br />
<br />
<br />
}}</div>Jkm