http://2009.igem.org/wiki/index.php?title=Special:Contributions/Gcromar&feed=atom&limit=50&target=Gcromar&year=&month=2009.igem.org - User contributions [en]2024-03-29T01:53:56ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/IGEM_PublicityIGEM Publicity2009-11-26T20:08:17Z<p>Gcromar: /* team specific */</p>
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[https://2008.igem.org/IGEM_Publicity iGEM 2008 Publicity] | [http://parts.mit.edu/igem07/index.php/Media iGEM 2007 Publicity] | [http://parts.mit.edu/wiki/index.php/IGEM_News iGEM 2006 Publicity]<br />
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<div style="color:#aaa; padding-bottom:20px;">(Members of the press, please see the [[Press_Kit | iGEM Press Kit]])</div><br />
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<span style="color:#1e90ff; font-size:175%">'''blogs </span><span style="color:#3cb371; font-size:175%">covering iGEM 2009'''</span><br />
* The [http://igem.sdu.dk/ <span style="color:#453221; font-size: 130%">'''SDU-Denmark'''</span>] team blog about their iGEM experience.<br />
* A blog about the iGEM project of the [http://aboutgmos.org/iGEM <span style="color:#453221; font-size: 130%">'''Uppsala-Sweden'''</span>] team.<br />
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====<font size=5><font color=dodgerblue>'''video/radio</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
*'''Slovenia, Heidelberg, SDU-Denmark''': [http://www.dradio.de/dlf/sendungen/forschak/1062608/ Die Biobastler von Boston], Nov 2, 2009, Deutschlandfunk radio (in German).<br />
*'''Cambridge, Imperial''': [http://www.sciencefriday.com/program/archives/200911063 Synthetic Biology Competition], Nov 6, 2009, NPR Science Friday (in English).<br />
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====<font size=5><font color=dodgerblue>'''news articles</font><font color=mediumseagreen> about iGEM 2009'''</font></font>====<br />
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<br />
If you would like to share an article that was written about iGEM or your iGEM team, please link to it on this page. If you have multiple articles featuring your team, link to them all individually!<br />
<br />
Post the name of your team, the title of your article, where it was featured, and provide a link to it. <br />
<br />
''Example'':<br> <br />
'''Team Example''': ''Title of article'', Nature, [link]<br />
<br />
====<font size=4><font color=dodgerblue>'''general'''</font></font>====<br />
*'''The Economist''': ''Biohacking: Hacking goes squishy'' Sep 3, 2009 [http://www.economist.com/search/displaystory.cfm?story_id=14299634 The Economist]<br />
*'''The Scientist''': ''Brick by Brick'' Feb 1, 2009. [http://www.the-scientist.com/article/display/55378/ The Scientist]<br />
*'''Technology Review''': ''A Genetically Engineered Rainbow of Bacteria'' Nov 03, 2009. [http://www.technologyreview.com/blog/editors/24351/ Technology Review]<br />
*'''Discovery News''': '' Bright Bacteria Wins Synthetic Biology Competition'' Nov 6, 2009. [http://blogs.discoverychannel.co.uk/discovery-news/2009/11/bright-bacteria-wins-synthetic-biology-competition.html Discovery News]<br />
*'''Wired''': '' Building new life forms at the iGEM Jamboree'' Nov 9, 2009. [http://www.wired.co.uk/news/archive/2009-11/09/building-new-life-forms-at-the-igem-jamboree.aspx Wired]<br />
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====<font size=4><font color=dodgerblue>'''team specific'''</font></font>====<br />
<br />
*'''[[Team:IBB_Pune]]''': "<i>Bio-champs in the making</i>" (English), [http://www.punemirror.in/index.aspx?Page=article&sectname=News%20-%20City&sectid=2&contentid=200909142009091423331646a131eacc#ftr2 (Monday, September 14, 2009 at 11:33:25 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Ganiti Prakriyanmadhe Jeevanuncha Vaapar! (Use of Bacteria in mathematical devices)</i>" (Marathi), [http://beta.esakal.com/2009/09/09220618/pune-use-of-bacterias-in-maths.html(Wednesday, September 09th, 2009 AT 10:09 PM)]<br />
*'''[[Team:IBB_Pune]]''': "<i>Synthetic Biologiteel Bharari ("Advances in Synthetic Biology")</i>" (Marathi), [https://2009.igem.org/Team:IBB_Pune/press (Wednesday 7 October 2009)]<br />
<br />
*'''[[Team:Groningen]]''': "<i>Students from Groningen in competition for best bacteria</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/groningse-studenten-synthetische-biologie-maken-bacterie City of Talent (August 20, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Talent of the Purest Water</i>" (Dutch, advertisement) [http://www.volkskrant.nl/vk-online/VK/20091024___/1_003/ad6.html Volkskrant, (October 24 2009)]<br />
*'''[[Team:Groningen]]''': "<i>RUG-team in finals synthetic biology</i>" (Dutch), [http://www.uk.rug.nl/cms-basis/nieuws.php?subaction=showfull&id=1257343172&archive=&start_from=&ucat=1 UK (Paper of the Groningen University) (November 4, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Team iGEM Groningen has been awarded a Gold Medal</i>" ([http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille English]/[http://www.cityoftalent.nl/nl/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille Dutch]), [http://www.cityoftalent.nl/en/content/nieuws/nieuws/team-igem-groningen-wint-gouden-medaille City of Talent (November 10, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen iGEM 2009 participants</i>" ([http://www.rug.nl/corporate/nieuws/archief/archief2009/persberichten/174_09?lang=en English]/[http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09 Dutch]), [http://www.rug.nl/Corporate/nieuws/archief/archief2009/persberichten/174_09?lang=en Press release University Groningen (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Performance of world class RUG in Boston</i>" (Dutch), [http://www.groningergezinsbode.nl/profile/redactiegroningergezinsbode/article73205.ece/wereldprestatie_rug_in_boston Groninger Gezinsbode (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Groningen students finalists gentech-competition</i>" (Dutch), [http://www.studned.nl/1084/wetenschap/groningse-studenten-finalisten-gentech-wedstrijd StudNed.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>International price for Groningen students</i>" (Dutch), [http://www.rtvnoord.nl/nieuws/indexwm.asp?actie=totaalbericht&pid=86497 RTV-Noord (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.noorderlink.nl/nieuws/goud-en-een-finale-plaats-voor-groningse-deelnemers-igem-2009 NoorderLink (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>Gold and a place in the finals for Groningen contestants iGEM 2009</i>" (Dutch), [http://www.headlinez.nl/?nr=2295334 HeadLinez.nl (November 11, 2009)]<br />
*'''[[Team:Groningen]]''': "<i>With a pacmanbacterium in de finals</i>" (Dutch), [http://www.uk.rug.nl/archief/jaargang39/11/12e.php UK (Paper of the Groningen University) (November 12, 2009)]<br />
*'''[[Team:Groningen]]''' & '''[[Team:TUDelft]]''': "<i>iGEM Gold for Dutch</i>" (Dutch), [http://www.bionieuws.nl/artikel.php?id=4918&zoek=iGEM Bionieuws (November 14, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Un concours organisé par le M.I.T : en route pour Boston ! </i>" (French), [http://www.supbiotech.fr/2009/09/boston-supbiotech-igem.html (September 08, 2009)]<br />
<br />
*'''[[Team:SupBiotech-Paris]]''': "<i>Les étudiants de la biotech interpellent la biologie synthétique</i>" (French), [http://www.vivagora.org/spip.php?breve210 (October 09, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>Un equipo de la UAB, en el concurso de biología sintética del MIT</i>" (Spanish), [http://www.uab.es/servlet/Satellite?cid=1096481466568&pagename=UABDivulga%2FPage%2FTemplatePageDetallArticleInvestigar&param1=1253860327385 (September 23, 2009)]<br />
<br />
*'''[[Team:UAB-Barcelona]]''': "<i>UAB to participate in the synthetic biology competition at MIT</i>" (English), [http://www.uab.es/servlet/Satellite/latest-news/news-detail/uab-to-participate-in-the-synthetic-biology-competition-at-mit-1096476786473.html?noticiaid=1253657853358 (September 23, 2009)]<br />
<br />
*'''[[Team:Valencia]]''': "<i>TheValencia team is awarded the Synthetic Standard Prize</i>" (Spanish), [http://www.elpais.com/articulo/sociedad/Levaduras/funcionan/pixeles/elpepuespval/20091103elpepusoc_9/Tes]<br />
<br />
*'''[[Team:Cambridge]]''': "<i>University of Cambridge team wins iGEM synthetic biology competition </i>" (English), [http://www.biotechniques.com/news/University-of-Cambridge-team-wins-iGEM-synthetic-biology-competition/biotechniques-180278.html (November 5 2009)]<br />
<br />
*'''[[Team:Virginia]]''': ''[http://www.washingtonpost.com/wp-dyn/content/article/2009/10/22/AR2009102204628.html New works of science nonfiction]'', The Washington Post (October 23, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Estafette voor bacterien'',(Dutch) Algemeen Dagblad (November 6, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette'', (Dutch) [http://noorderlicht.vpro.nl/themasites/mediaplayer/index.jsp?media=42722491&refernr=42560747&portalnr=3626936&hostname=noorderlicht&mediatype=audio&portalid=noorderlicht# Podcast van Radio 1, VPRO Noorderlicht, Annemieke Smit] (November 9, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette wint goud en award'', (Dutch) [http://www.tudelft.nl/live/pagina.jsp?id=ee44be90-289e-4efe-8a12-4636f1c86b28&lang=nl TNW Today Editie 2] (November 5, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterie-estafette wint goud en award'', (Dutch) [http://www.tudelft.nl/live/pagina.jsp?id=ee44be90-289e-4efe-8a12-4636f1c86b28&lang=nl TUDelft Nieuws] (November 4, 2009)<br />
<br />
*'''[[Team:TUDelft]]''': ''Bacterial relay race wins gold and award'', [http://www.tudelft.nl/live/pagina.jsp?id=26f61b2e-5a3f-40be-b737-a460b08bced7&lang=en Delft University of Technology Press release] (November 4, 2009)<br />
<br />
*'''[[Team:TorontoMaRSDiscovery]]''': ''A gem of a genetics competition'', [http://www.news.utoronto.ca/science-and-technology/a-gem-of-a-genetics-competition.html University of Toronto eBulletin] (November 23, 2009)</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/NotebookTeam:TorontoMaRSDiscovery/Notebook2009-10-22T03:05:02Z<p>Gcromar: /* Summary of Results */</p>
<hr />
<div>[[image:To_igem_wiki_banner.jpg|965px]]<br />
{| style="color:white;background-color:#99CCFF;" height:100px cellpadding="2" cellspacing="0" border="0" width="100%" align="center" class="menu"<br />
!align="center"|[[Team:TorontoMaRSDiscovery|Home]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Team|The Team]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Project|The Project]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Parts|BioBricks]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Modeling|Modelling]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Safety|Safety]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Notebook|Notebook]]<br />
|}<br />
<br><br />
<br />
==Monthly Notebook==<br />
<ul><br />
<li>[https://2009.igem.org/Team:TorontoMaRSDiscovery/Notebook/April April]<br />
<li>[https://2009.igem.org/Team:TorontoMaRSDiscovery/Notebook/May May]<br />
<li>[https://2009.igem.org/Team:TorontoMaRSDiscovery/Notebook/June June]<br />
<li>[https://2009.igem.org/Team:TorontoMaRSDiscovery/Notebook/July July]<br />
<li>[https://2009.igem.org/Team:TorontoMaRSDiscovery/Notebook/Augst August]<br />
<li>[https://2009.igem.org/Team:TorontoMaRSDiscovery/Notebook/September September]<br />
<li>[https://2009.igem.org/Team:TorontoMaRSDiscovery/Notebook/October October]<br />
</ul><br />
<br />
==Summary of Results==<br />
<br />
{| border="1" cellpadding="5" cellspacing="0"<br />
|'''Part Type'''<br />
|'''Arbitrary Name'''<br />
|'''Registry Code'''<br />
|'''Construct Used For'''<br />
|'''Status'''<br />
|'''Transformants Stocked?'''<br />
|'''Antibiotic Resistant, Backbone Plasmid Visualized?'''<br />
|'''Part Visualized?'''<br />
|'''Sequenced?'''<br />
|----<br />
|rowspan="2"|<br />
'''New Parts'''<br />
|Encapsulin (Enc)<br />
|K192000<br />
|Encapsulin<br />
|Confirmed<br />
|Yes<br />
|Yes<br />
|Yes<br />
|Yes<br />
|----<br />
|eCFPtgt<br />
|K192001<br />
|CFP target protein<br />
|Synthesized by Mr Gene<br />
|No<br />
|No<br />
|No<br />
|Yes<br />
|----<br />
|rowspan="13"|<br />
'''Registry Parts'''<br />
|1<br />
|JJ23100<br />
|Control, Encapsulin<br />
|Confirmed<br />
|Yes<br />
|Yes<br />
|Too small<br />
|No<br />
|----<br />
|2<br />
|B0034<br />
|Control, Encapsulin<br />
|Confirmed<br />
|Yes<br />
|Yes<br />
|Too small<br />
|No<br />
|----<br />
|3<br />
|C0040<br />
|Control<br />
|Confirmed<br />
|Yes<br />
|Yes<br />
|Yes<br />
|No<br />
|----<br />
|4<br />
|C0012<br />
|Control<br />
|Confirmed<br />
|Yes<br />
|Yes<br />
|Yes<br />
|No<br />
|----<br />
|5<br />
|B0015<br />
|Control, Encapsulin<br />
|Confirmed<br />
|Yes<br />
|Yes<br />
|Yes<br />
|No<br />
|----<br />
|C (Amp + C)<br />
|pSB1AC3<br />
|Assembly<br />
|Confirmed<br />
|Yes<br />
|Yes<br />
|Yes<br />
|No<br />
|----<br />
|C (C only)<br />
|pSB1C3<br />
|Assembly<br />
|Confirmed<br />
|Yes<br />
|Yes<br />
|Yes<br />
|No<br />
|----<br />
|K (ccdb)<br />
|pSB1AK3<br />
|Assembly<br />
|Confirmed<br />
|Yes<br />
|Yes<br />
|Yes<br />
|No<br />
|----<br />
|K (RFP)<br />
|pSB1K3<br />
|Assembly<br />
|Confirmed<br />
|Yes<br />
|Yes<br />
|Yes<br />
|No<br />
|----<br />
|Tet (ccdb)<br />
|pSB1AT3<br />
|Assembly<br />
|Confirmed<br />
|Yes<br />
|Yes<br />
|Yes<br />
|No<br />
|----<br />
|Tet (RFP)<br />
|pSB1T3<br />
|Assembly<br />
|Confirmed<br />
|Yes<br />
|Yes<br />
|Yes<br />
|No<br />
|----<br />
|7<br />
|J13002<br />
|Encapsulin<br />
|Transfected<br />
|Yes<br />
|No<br />
|No<br />
|No<br />
|----<br />
|Amp<br />
|pSB1A3<br />
|Assembly<br />
|Transfected<br />
|Yes<br />
|No<br />
|No<br />
|No<br />
|-<br />
|rowspan="7"|<br />
'''Assembled Parts'''<br />
|----<br />
|Enc in C (Amp+C)<br />
|<br />
|Submission, Encapsulin<br />
|Confirmed<br />
|Yes<br />
|Yes<br />
|Yes<br />
|Yes<br />
|----<br />
|1+2 in C (Amp+C)<br />
|<br />
|Control, Encapsulin<br />
|Confirmed<br />
|Yes<br />
|Yes<br />
|Too small<br />
|No<br />
|----<br />
|3+2 in C (Amp+C)<br />
|<br />
|Control<br />
|Confirmed<br />
|Yes<br />
|Yes<br />
|Yes<br />
|No<br />
|----<br />
|4+5 in Tet (ccdb)<br />
|<br />
|Control<br />
|Confirmed<br />
|Yes<br />
|Yes<br />
|Yes<br />
|No<br />
|----<br />
|1+2+3+2 in K (RFP)<br />
|<br />
|Control<br />
|Transfected<br />
|Yes<br />
|No<br />
|No<br />
|No<br />
|----<br />
|1+2+Enc in K<br />
|<br />
|Encapsulin<br />
|Confirmed<br />
|Yes<br />
|Yes<br />
|Yes<br />
|In Progress<br />
|----<br />
|}</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/BioinformaticsTeam:TorontoMaRSDiscovery/Bioinformatics2009-10-22T03:03:26Z<p>Gcromar: /* Future Work */</p>
<hr />
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{| style="color:white;background-color:#99CCFF;" height:100px cellpadding="2" cellspacing="0" border="0" width="100%" align="center" class="menu"<br />
!align="center"|[[Team:TorontoMaRSDiscovery|Home]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Team|The Team]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Project|The Project]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Parts|BioBricks]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Modeling|Modelling]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Safety|Safety]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Notebook|Notebook]]<br />
|}<br />
<br><br />
<p style="font-size:18pt;">The diversity of bacterial microcompartments in nature and their potential for biotechnological applications</p><br />
<br />
==Summary==<br />
<br />
Spatial segregation is widely believed to be a defining organizational feature of eukaryotic cells: proteins, nucleic acids and small molecules are contained within and often actively transported between the many membrane-bound, subcellular organelles such as mitochondria, lysosomes, Golgi apparatus, etc. However, it has recently been found that a number of bacteria conditionally express proteinaceous microcompartments. These polyhedral organelles are usually 100-150 nm in cross section [1] and consist of proteinaceous outer shells, reminiscent of viral capsids, surrounding a core of enzymes[2]. It is thought that microcompartments allow bacteria to sequester specific metabolic enzymes and their substrates to enhance enzymatic efficiency (enzyme channeling) and protect cells from the toxic effects of certain intermediates. While several examples of these compartments have been reported their diversity has not been fully explored. We wish to design new microcompartments with modified properties or novel enzymatic activities, which could result in potentially useful applications in biotechnology. With this goal in mind, we attempted to address the following two questions using a bioinformatics approach (1) What enzymes will benefit the most from enzymatic channeling? and (2) Are there any other alternative microcompartments that could be explored for enzymatic channeling engineering?<br />
<br />
==Background==<br />
<br />
Most of our knowledge on bacterial microcompartments has been derived from the three well-studied microcompartment systems in nature.<br />
<br />
[[image:TMDT_Nature.jpg|right|thumb|Figure 1: The carbon concentrating mechanism in carboxysome.]]<br />
#Carboxysomes<br />
#*First reported in 1956, carboxysomes were the first bacteria microcompartments to be discovered. They are often present in cyanobacteria and other chemoautotrophic bacteria [3]. They are known to play a key role in enhancing autotrophic carbon fixation in the Calvin cycle. The shell of the carboxysome encodes the enzymes carbonic anhydrase (CA) and ribulose bis-phosphate carboxylase monooxygenase (RuBisCO). CA converts bicarbonate ions into carbon dioxide, which is then converted into 3-phosphoglycerate (3-PGA) by RuBisCO. Carboxysome not only allows for co-localization of CA and RuBisCO, but also acts as a diffusion barrier to retain carbon dioxide in the immediate vicinity of RuBisCO and thus catalyzes the conversion [2]. See Figure 1.<br />
#''Pdu'' microcompartment<br />
#*In 1994, homologues of carboxysome shell proteins were reported in ''S. enterica''. They are amongst a cluster of genes that are involved in coenzyme B12-dependent metabolism of 1,2 propanediol [4]. The gene cluster was later termed the pdu operon and the microcompartment formed was later termed propanediol utilization microcompartment. The proposed fuction of the pdu microcompartment is to encapsulate the enzymes that are necessary for cell to degrade propanediol and most importantly, to protect the cell from the toxic effects of propionaldehyde, an intermediate formed during the process [5].<br />
#''Eut'' microcompartment<br />
#*Later, similar structures were also found in ''E.coli'' and ''S. enterica'' when they were grown using ethanolamine as energy source. These structures were named ethanolamine utilization microcompartment (encoded by eut operon) and they often display very high genetic similarity with pdu microcompartment. Eut microcompartments contain enzymes involved in the degradation of ethanolamine and protect cell from acetaldehyde [6-8].<br />
#*''Generally speaking, both pdu and eut microcompartments are less uniform in size and more irregular in shape than carboxysomes[2].'' <br />
<br />
These compartments are generally unsuitable for our purposes because a) Their shell proteins consist of several different types whose functions are not well characterized and b) It is not known how the associated enzymes are targeted to the microcompartment. However, several other putative microcompartments have been inferred by sequence similarity. These include <br />
*A microcompartment was suggested to be involved in the oxidation of ethanol by ''Clostridium kluyveri''. [9].<br />
*A microcompartment associated with a puryvate-formate lyase homolog is proposed to be involved in the production of ethanol from pyruvate [10].<br />
*A putative microcompartment in ''Rhodopirellula baltica'' is proposed to be associated with a lactate dehydrogenase homologue [1].<br />
*A putative microcompartment in ''Carboxydothermus hydrogenoformans'' is associated with an isochorismatase-family protein [1].<br />
*A putative microcompartment Solibacter usitatus can be associated with a dihydrdipicolinate synthase homologue [1].<br />
<br />
The observation that diverse microcompartment structures are composed of proteins with homologous sequences led to the identification of a protein domain in the shell of all polyhedral bacterial microcompartments. This conserved protein domain is known as the Bacterial Microcompartment (BMC) domain ([http://pfam.sanger.ac.uk/family?acc=PF00936 Pfam00936]). It is approximately 84 amino acids long and can be either found as a part of a large protein or in tandem copies within the same operon. This domain is found to be present in 189 bacterial species to date. [2]<br />
<br />
Microcompartments typically promote the catalysis of a particular metabolic reaction by sequestering or co-localizing functionally related enzymes. The product of one enzyme will be delivered to the next enzyme at high concentration since it cannot readily diffuse. This greatly enhances enzyme efficiency. Microcompartments can also occlude toxic intermediates that cannot be degraded by normal bacterial machinery. This was shown to be true in ''pdu'' and ''eut'' microcompartments. The degradation pathway of 1,2-propanediol and ethanolamine both proceed through aldehyde intermediates (propionaldehyde and acetaldehyde respectively). The toxicity of these aldehyde intermediates was later shown in growth assays in which cells accumulating large amounts of propionaldehyde underwent growth arrest due to propionaldehyde toxicity [5, 11]. Therefore, it is reasonable to propose that bacteria use microcompartments to mitigate the toxicity of aldehyde by encapsulating these poisonous intermediates.<br />
<br />
==Methods/Results==<br />
===a) Search for enzyme pairs===<br />
[[image:TMDT_Pathway.jpg|right|thumb|Figure 2: Complete pathway map obtained from KEGG database with the first enzyme colored in red:.]]<br />
In order to identify candidate enzyme pairs likely to display an observable benefit from enzyme channeling we made the following assumptions:<br />
#Enzyme pairs with specific thermodynamic properties will likely benefit. Specifically, for two sequential reactions the second one will be thermodynamically more favorable than the first reaction. Consequently, the products from the first reaction be consumed at a faster rate than being generated, hence driving the reaction to completion. <br />
#*Predictions based on free energy values: [[Media:Scored.xls|Scored.xls]]<br />
#Enzymes involved in reactions with toxic intermediates will likely benefit from enzymatic channeling since the toxin will be less likely to interact with cellular components.<br />
#*Enzyme pairs that possess a common metabolite that is known to be toxic: [[Media:ToxicPairs.xls|ToxicPairs.xls]]<br />
#Enzymes that were identified as gene fusion products in other organisms will likely benefit from enzyme channeling since fusion of multi-functional enzymes is an extreme example of colocalization.<br />
#*Enzyme pairs predicted to form multifunctional enzymes by the Prolinks Rosetta Stone analysis: [[Media:RosettaEnzymePairs.xls|RosettaEnzymePairs.xls]]<br />
#*Enzyme pairs annotated to form multifunctional enzymes in SwissProt database: [[Media:SwissProtPairs.xls|SwissProtPairs.xls]]<br />
These results provided us with a combined list that was further refined by removing enzymes that are not immediately adjacent to each other in their biochemical pathway based on the KEGG database (Figure 2). In addition, enzymes with a molecular weight well beyond the microcompartment capacity (100 kDa) were also eliminated. The remaining enzyme pairs meeting these criteria were considered candidates for enzyme channeling and have been summarized in the following table:<br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0"<br />
| align="center" |'''Enzyme1 EC'''<br />
| align="center" |'''Enzyme1 Size (kDa)'''<br />
| align="center" |'''Enzyme2 EC'''<br />
| align="center" |'''Enzyme2 Size (kDa)'''<br />
| align="center" |'''Source'''<br />
| align="center" |'''Pathway'''<br />
| align="center" |'''Major Product'''<br />
| align="center" |'''Applications'''<br />
|-<br />
| 2.1.3.2||32-125||6.3.4.4||47-96||Rosette||Alanine and aspartate metabolism||adenylosuccinate||<br />
|-<br />
| 1.1.1.1||25-57||1.2.1.10||96.7-520||Swiss Prot||Butanoate metabolism ||Butanoyl-coA||<br />
|-<br />
| 1.1.1.95||35-250||2.6.1.52||35-96||Rosette||Glycine, serine and threonine metabolism ||Phosphoserine||<br />
|-<br />
| 2.3.3.9||52-81||4.1.3.1||14-65||Swiss Prot &Rosette||Glyoxylate and dicarboxylate metabolism||Isocitrate||<br />
|-<br />
| 1.5.1.15||32-34||3.5.4.9||30-45||Swiss Prot||One carbon pool by folate||Formyl-THF||<br />
|-<br />
| 1.5.1.5||30-150||3.5.4.9||30-45||Swiss Prot||One carbon pool by folate||Formyl-THF||<br />
|-<br />
| 1.1.1.57||||5.3.1.12||||Rosette||Pentose and glucuronate interconversions||D-Glucuronate||<br />
|-<br />
| 2.2.1.1||100-141||5.1.3.1||45-200||Rosette||Pentose phosphate pathway||D-ribolose-5P||<br />
|-<br />
| 2.2.1.2||35-75||5.3.1.9||27-67||Rosette||Pentose phosphate pathway||a-glucose-6P||<br />
|-<br />
| 1.3.1.13||52-210||4.2.1.51||12-137||Rosette||Phenylalanine, tyrosine and tryptophan biosynthesis||Phenyl-pyruvate||<br />
|-<br />
| 2.3.1.8||35-71||2.7.2.15||43||Rosette||Propanoate metabolism||Propanoate||Food preservatives<br />
|-<br />
| 2.3.1.8||35-70||2.7.2.1||12 to 66||Rosette||Propanoate metabolism/Pyruvate metabolism||Propanoate/acetate||Energy<br />
|-<br />
| 2.4.2.10||39-140||4.1.1.23||14-64||Swiss Prot||Pyrimidine metabolism||UMP||<br />
|-<br />
| 2.4.2.1||45-86||2.4.2.3||32-160||Rosette||Pyrimidine metabolism||Uridine||<br />
|-<br />
| 2.4.1.15||45-630||3.1.3.12||25-973||Rosette||Starch and sucrose metabolism||a,a trehalose||biotechnology application<br />
|-<br />
| 1.2.1.41||41-189||2.7.2.11||47-354||Swiss Prot &Rosette||Urea cycle and metabolism of amino groups||Glutamate||Flavor enhancer, Plant growth<br />
|}<br />
<br />
===b) Alternative microcompartments===<br />
<br />
Upon further examination, most of our known microcompartments tend to consist of a large number of protein subunits with very complex structures. Because of their complicated regulatory patterns and stoichiometry, these structures are not ideal to be used in enzymatic channeling engineering.<br />
<br />
*Carboxysome: 80-150 nm in cross section. Composed of several thousands polypeptide of 10-15 different types. Contain as many as 250 RuBisCO per carboxysome molecule[12, 13].<br />
<br />
*''Pdu'' Microcomparment: 100-150 nm in cross section. Composed of about 18000 individual polypeptides of about 14-18 different types [1].<br />
<br />
*''Eut'' Microcomparment: Similar to pdu microcompartment in terms of both size and protein composition.<br />
<br />
<br />
One of the putative microcompartment systems in ''Clostridium kluyveri'' associated with the oxidation of ethanol, however, shows promise as an alternative microcompartment for use in our system because:<br />
<br />
#The gene cluster involved contains only seven genes: including two genes for a pair of nearly identical acetaldehyde dehydrogenases, three genes for highly similar ethanol dehydrogenases, and two genes for microcompartment proteins, which are orthologs of ethanolamine using genes (eutML) of ''Salmonella typhimurium'' [9].<br />
#From cell extracts of ''C. kluyveri'' a macromolecular complex of ethanol dehydrogenase and acetaldehyde dehydrogenase can be purified by differential manganese sulfate precipitation, indicating the presence of a functional and intact microcompartment enclosing these two enzymes[9].<br />
<br />
In order to co-opt this stucture for our own use it is necessary to understand how the associated enzymes are targeted to the compartment so that alternative enzymes of our choosing can be substituted. To search for a consensus targeting sequence we obtained the ''C. kluyveri'' compartment sequence from NCBI and performed a position specific iterative Blast search for related sequences among bacteria. We used the following two query sequences: <br />
<br />
{| border="1" cellspacing="0" width="50%"<br />
|-<br />
|'''CKL_1072'''<br />
|-<br />
|>gi|153953697|ref|YP_001394462.1| microcompartment protein [Clostridium kluyveri DSM 555]<br />
|-<br />
|MGQEALGMIETKGLVGAIEAADSMVKAANVALIGYEKIGSGLVTVMVRGDVGAVKAATDAGAASAKRVGE<br />
VISVHVIPRPHTDVEKILPNIG<br />
|}<br />
<br />
<br />
{| border="1" cellspacing="0" width="50%"<br />
|-<br />
|'''CKL_1073'''<br />
|-<br />
|>gi|153953698|ref|YP_001394463.1| microcompartment protein [Clostridium kluyveri DSM 555]<br />
|-<br />
|MNNELIEKVLGEVRKSLDLKNFDQEKLNKVVESTTEKLSDSKKEEAIKEAKPDVKVAEESKQAVVEQKAN<br />
DVKTAPTMTEFVGTAGGDTVGLVIANVDSLLHKHLGLDNTCRSIGIISARVGAPAQMMAADEAVKGTNTE<br />
VATIELPRDTKGGAGHGIFIVLKAADVSDARRAVEIALKQTDKYLGNVYLCDAGHLEVQYTARASLIFEK<br />
AFGAPSGQAFGIMHAAPAGVGMIVADTALKTADVKLITYGSPTNGVLSYTNEILITISGDSGAVLQSLTA<br />
ARKAGLSILRSMGQDPVSMSKPTF<br />
|}<br />
<br />
PSI-BLAST constructs and modifies the Position Specific Scoring Matrix constantly and uses it during the next round of search. This iterative searching strategy results in increased sensitivity. The top results were saved after 5 iterations. Download the [[Media:1072_3 BLAST.pdf|BLAST results]] and [[Media:taxonomy_report.txt|taxonomy report]].A [[Media:MUSCLE_alignment.pdf|multiple alignment]] of these sequences was constructed using CLCBio Genomic Workbench to assess the level of conservation between species and identify consensus sequence. <br />
<br />
Since microcompartment proteins are known to be found adjacent to their corresponding enzymes, we searched the top hits for adjacent genes 5’ upstream and 3’ downstream of the hit gene. We then attempted to apply multiple sequence alignment to these sequences in an effort to identify conserved motifs corresponding to a potential targeting sequence similar to the method employed by Suter ''et al''. A targeting sequence, if found, would allow translocation of enzymes into the microcompartment. Due to the incomplete annotation of certain genomes, the alignments did not yield a statistically significant targeting motif. We are attempting further analyses using different reading frames to refine the protein candidates used during the multiple alignment.<br />
<br />
==Conclusions==<br />
We have short-listed a number of enzymes which might benefit from channeling according to several properties such as free energy, toxicity and the occurrence of multi-functional homologues. In addition, while concluding that several classic examples of bacterial microcompartment are unsuitable for experimental manipulation, we have conducted a sequence-based search for additional protein family members corresponding to the ''Clostridium kluyveri'' microcompartment and searched the corresponding upstream and downstream genes for conserved motifs in an effort to find the targetting sequence for these enzymes. The ''Clostridium kluyveri'' system remains under consideration as a potentially viable alternative to the encapsulin-based nanocompartment. However, its suitability will depend on the results of further efforts to identify a targeting sequence for this compartment.<br />
<br />
==Future Work==<br />
In order to find the targeting sequence for the ''Clostridium kluyveri'' microcompartment, it will be necessary to fine tune the enzyme queries and potentially check out other alternative reading frames. In addition, enzymes will be selected from those short-listed above to proceed with a proof of concept experiment and potentially commercially viable application.<br />
<br />
==References==<br />
1.Cheng, S., ''et al''., Bacterial microcompartments: their properties and paradoxes. Bioessays, 2008. 30(11-12): p. 1084-95.<br />
<br />
2.Yeates, T.O., ''et al''., Protein-based organelles in bacteria: carboxysomes and related microcompartments. Nat Rev Microbiol, 2008. 6(9): p. 681-91.<br />
<br />
3.Shively, J.M., Inclusion bodies of prokaryotes. Annu Rev Microbiol, 1974. 28(0): p. 167-87.<br />
<br />
4.Chen, P., D.I. Andersson, and J.R. Roth, The control region of the pdu/cob regulon in Salmonella typhimurium. J <br />
Bacteriol, 1994. 176(17): p. 5474-82.<br />
<br />
5.Havemann, G.D., E.M. Sampson, and T.A. Bobik, PduA is a shell protein of polyhedral organelles involved in coenzyme B(12)-dependent degradation of 1,2-propanediol in Salmonella enterica serovar typhimurium LT2. J Bacteriol, 2002. 184(5): p. 1253-61.<br />
<br />
6.Brinsmade, S.R., T. Paldon, and J.C. Escalante-Semerena, Minimal functions and physiological conditions required for growth of salmonella enterica on ethanolamine in the absence of the metabolosome. J Bacteriol, 2005. 187(23): p. 8039-46.<br />
<br />
7.Penrod, J.T. and J.R. Roth, Conserving a volatile metabolite: a role for carboxysome-like organelles in Salmonella enterica. J Bacteriol, 2006. 188(8): p. 2865-74.<br />
<br />
8.Stojiljkovic, I., A.J. Baumler, and F. Heffron, Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster. J Bacteriol, 1995. 177(5): p. 1357-66.<br />
<br />
9.Seedorf, H., ''et al''., The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features. Proc Natl Acad Sci U S A, 2008. 105(6): p. 2128-33.<br />
<br />
10.Wackett, L.P., ''et al''., Genomic and biochemical studies demonstrating the absence of an alkane-producing phenotype in Vibrio furnissii M1. Appl Environ Microbiol, 2007. 73(22): p. 7192-8.<br />
<br />
11.Sampson, E.M. and T.A. Bobik, Microcompartments for B12-dependent 1,2-propanediol degradation provide protection from DNA and cellular damage by a reactive metabolic intermediate. J Bacteriol, 2008. 190(8): p. 2966-71.<br />
<br />
12.Yeates, T.O., ''et al''., Self-assembly in the carboxysome: a viral capsid-like protein shell in bacterial cells. Biochem Soc Trans, 2007. 35(Pt 3): p. 508-11.<br />
<br />
13.Tanaka, S., ''et al''., Atomic-level models of the bacterial carboxysome shell. Science, 2008. 319(5866): p. 1083-6.<br />
<br />
14.Sutter, M. ''et al''. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/BioinformaticsTeam:TorontoMaRSDiscovery/Bioinformatics2009-10-22T03:03:01Z<p>Gcromar: /* References */</p>
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<p style="font-size:18pt;">The diversity of bacterial microcompartments in nature and their potential for biotechnological applications</p><br />
<br />
==Summary==<br />
<br />
Spatial segregation is widely believed to be a defining organizational feature of eukaryotic cells: proteins, nucleic acids and small molecules are contained within and often actively transported between the many membrane-bound, subcellular organelles such as mitochondria, lysosomes, Golgi apparatus, etc. However, it has recently been found that a number of bacteria conditionally express proteinaceous microcompartments. These polyhedral organelles are usually 100-150 nm in cross section [1] and consist of proteinaceous outer shells, reminiscent of viral capsids, surrounding a core of enzymes[2]. It is thought that microcompartments allow bacteria to sequester specific metabolic enzymes and their substrates to enhance enzymatic efficiency (enzyme channeling) and protect cells from the toxic effects of certain intermediates. While several examples of these compartments have been reported their diversity has not been fully explored. We wish to design new microcompartments with modified properties or novel enzymatic activities, which could result in potentially useful applications in biotechnology. With this goal in mind, we attempted to address the following two questions using a bioinformatics approach (1) What enzymes will benefit the most from enzymatic channeling? and (2) Are there any other alternative microcompartments that could be explored for enzymatic channeling engineering?<br />
<br />
==Background==<br />
<br />
Most of our knowledge on bacterial microcompartments has been derived from the three well-studied microcompartment systems in nature.<br />
<br />
[[image:TMDT_Nature.jpg|right|thumb|Figure 1: The carbon concentrating mechanism in carboxysome.]]<br />
#Carboxysomes<br />
#*First reported in 1956, carboxysomes were the first bacteria microcompartments to be discovered. They are often present in cyanobacteria and other chemoautotrophic bacteria [3]. They are known to play a key role in enhancing autotrophic carbon fixation in the Calvin cycle. The shell of the carboxysome encodes the enzymes carbonic anhydrase (CA) and ribulose bis-phosphate carboxylase monooxygenase (RuBisCO). CA converts bicarbonate ions into carbon dioxide, which is then converted into 3-phosphoglycerate (3-PGA) by RuBisCO. Carboxysome not only allows for co-localization of CA and RuBisCO, but also acts as a diffusion barrier to retain carbon dioxide in the immediate vicinity of RuBisCO and thus catalyzes the conversion [2]. See Figure 1.<br />
#''Pdu'' microcompartment<br />
#*In 1994, homologues of carboxysome shell proteins were reported in ''S. enterica''. They are amongst a cluster of genes that are involved in coenzyme B12-dependent metabolism of 1,2 propanediol [4]. The gene cluster was later termed the pdu operon and the microcompartment formed was later termed propanediol utilization microcompartment. The proposed fuction of the pdu microcompartment is to encapsulate the enzymes that are necessary for cell to degrade propanediol and most importantly, to protect the cell from the toxic effects of propionaldehyde, an intermediate formed during the process [5].<br />
#''Eut'' microcompartment<br />
#*Later, similar structures were also found in ''E.coli'' and ''S. enterica'' when they were grown using ethanolamine as energy source. These structures were named ethanolamine utilization microcompartment (encoded by eut operon) and they often display very high genetic similarity with pdu microcompartment. Eut microcompartments contain enzymes involved in the degradation of ethanolamine and protect cell from acetaldehyde [6-8].<br />
#*''Generally speaking, both pdu and eut microcompartments are less uniform in size and more irregular in shape than carboxysomes[2].'' <br />
<br />
These compartments are generally unsuitable for our purposes because a) Their shell proteins consist of several different types whose functions are not well characterized and b) It is not known how the associated enzymes are targeted to the microcompartment. However, several other putative microcompartments have been inferred by sequence similarity. These include <br />
*A microcompartment was suggested to be involved in the oxidation of ethanol by ''Clostridium kluyveri''. [9].<br />
*A microcompartment associated with a puryvate-formate lyase homolog is proposed to be involved in the production of ethanol from pyruvate [10].<br />
*A putative microcompartment in ''Rhodopirellula baltica'' is proposed to be associated with a lactate dehydrogenase homologue [1].<br />
*A putative microcompartment in ''Carboxydothermus hydrogenoformans'' is associated with an isochorismatase-family protein [1].<br />
*A putative microcompartment Solibacter usitatus can be associated with a dihydrdipicolinate synthase homologue [1].<br />
<br />
The observation that diverse microcompartment structures are composed of proteins with homologous sequences led to the identification of a protein domain in the shell of all polyhedral bacterial microcompartments. This conserved protein domain is known as the Bacterial Microcompartment (BMC) domain ([http://pfam.sanger.ac.uk/family?acc=PF00936 Pfam00936]). It is approximately 84 amino acids long and can be either found as a part of a large protein or in tandem copies within the same operon. This domain is found to be present in 189 bacterial species to date. [2]<br />
<br />
Microcompartments typically promote the catalysis of a particular metabolic reaction by sequestering or co-localizing functionally related enzymes. The product of one enzyme will be delivered to the next enzyme at high concentration since it cannot readily diffuse. This greatly enhances enzyme efficiency. Microcompartments can also occlude toxic intermediates that cannot be degraded by normal bacterial machinery. This was shown to be true in ''pdu'' and ''eut'' microcompartments. The degradation pathway of 1,2-propanediol and ethanolamine both proceed through aldehyde intermediates (propionaldehyde and acetaldehyde respectively). The toxicity of these aldehyde intermediates was later shown in growth assays in which cells accumulating large amounts of propionaldehyde underwent growth arrest due to propionaldehyde toxicity [5, 11]. Therefore, it is reasonable to propose that bacteria use microcompartments to mitigate the toxicity of aldehyde by encapsulating these poisonous intermediates.<br />
<br />
==Methods/Results==<br />
===a) Search for enzyme pairs===<br />
[[image:TMDT_Pathway.jpg|right|thumb|Figure 2: Complete pathway map obtained from KEGG database with the first enzyme colored in red:.]]<br />
In order to identify candidate enzyme pairs likely to display an observable benefit from enzyme channeling we made the following assumptions:<br />
#Enzyme pairs with specific thermodynamic properties will likely benefit. Specifically, for two sequential reactions the second one will be thermodynamically more favorable than the first reaction. Consequently, the products from the first reaction be consumed at a faster rate than being generated, hence driving the reaction to completion. <br />
#*Predictions based on free energy values: [[Media:Scored.xls|Scored.xls]]<br />
#Enzymes involved in reactions with toxic intermediates will likely benefit from enzymatic channeling since the toxin will be less likely to interact with cellular components.<br />
#*Enzyme pairs that possess a common metabolite that is known to be toxic: [[Media:ToxicPairs.xls|ToxicPairs.xls]]<br />
#Enzymes that were identified as gene fusion products in other organisms will likely benefit from enzyme channeling since fusion of multi-functional enzymes is an extreme example of colocalization.<br />
#*Enzyme pairs predicted to form multifunctional enzymes by the Prolinks Rosetta Stone analysis: [[Media:RosettaEnzymePairs.xls|RosettaEnzymePairs.xls]]<br />
#*Enzyme pairs annotated to form multifunctional enzymes in SwissProt database: [[Media:SwissProtPairs.xls|SwissProtPairs.xls]]<br />
These results provided us with a combined list that was further refined by removing enzymes that are not immediately adjacent to each other in their biochemical pathway based on the KEGG database (Figure 2). In addition, enzymes with a molecular weight well beyond the microcompartment capacity (100 kDa) were also eliminated. The remaining enzyme pairs meeting these criteria were considered candidates for enzyme channeling and have been summarized in the following table:<br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0"<br />
| align="center" |'''Enzyme1 EC'''<br />
| align="center" |'''Enzyme1 Size (kDa)'''<br />
| align="center" |'''Enzyme2 EC'''<br />
| align="center" |'''Enzyme2 Size (kDa)'''<br />
| align="center" |'''Source'''<br />
| align="center" |'''Pathway'''<br />
| align="center" |'''Major Product'''<br />
| align="center" |'''Applications'''<br />
|-<br />
| 2.1.3.2||32-125||6.3.4.4||47-96||Rosette||Alanine and aspartate metabolism||adenylosuccinate||<br />
|-<br />
| 1.1.1.1||25-57||1.2.1.10||96.7-520||Swiss Prot||Butanoate metabolism ||Butanoyl-coA||<br />
|-<br />
| 1.1.1.95||35-250||2.6.1.52||35-96||Rosette||Glycine, serine and threonine metabolism ||Phosphoserine||<br />
|-<br />
| 2.3.3.9||52-81||4.1.3.1||14-65||Swiss Prot &Rosette||Glyoxylate and dicarboxylate metabolism||Isocitrate||<br />
|-<br />
| 1.5.1.15||32-34||3.5.4.9||30-45||Swiss Prot||One carbon pool by folate||Formyl-THF||<br />
|-<br />
| 1.5.1.5||30-150||3.5.4.9||30-45||Swiss Prot||One carbon pool by folate||Formyl-THF||<br />
|-<br />
| 1.1.1.57||||5.3.1.12||||Rosette||Pentose and glucuronate interconversions||D-Glucuronate||<br />
|-<br />
| 2.2.1.1||100-141||5.1.3.1||45-200||Rosette||Pentose phosphate pathway||D-ribolose-5P||<br />
|-<br />
| 2.2.1.2||35-75||5.3.1.9||27-67||Rosette||Pentose phosphate pathway||a-glucose-6P||<br />
|-<br />
| 1.3.1.13||52-210||4.2.1.51||12-137||Rosette||Phenylalanine, tyrosine and tryptophan biosynthesis||Phenyl-pyruvate||<br />
|-<br />
| 2.3.1.8||35-71||2.7.2.15||43||Rosette||Propanoate metabolism||Propanoate||Food preservatives<br />
|-<br />
| 2.3.1.8||35-70||2.7.2.1||12 to 66||Rosette||Propanoate metabolism/Pyruvate metabolism||Propanoate/acetate||Energy<br />
|-<br />
| 2.4.2.10||39-140||4.1.1.23||14-64||Swiss Prot||Pyrimidine metabolism||UMP||<br />
|-<br />
| 2.4.2.1||45-86||2.4.2.3||32-160||Rosette||Pyrimidine metabolism||Uridine||<br />
|-<br />
| 2.4.1.15||45-630||3.1.3.12||25-973||Rosette||Starch and sucrose metabolism||a,a trehalose||biotechnology application<br />
|-<br />
| 1.2.1.41||41-189||2.7.2.11||47-354||Swiss Prot &Rosette||Urea cycle and metabolism of amino groups||Glutamate||Flavor enhancer, Plant growth<br />
|}<br />
<br />
===b) Alternative microcompartments===<br />
<br />
Upon further examination, most of our known microcompartments tend to consist of a large number of protein subunits with very complex structures. Because of their complicated regulatory patterns and stoichiometry, these structures are not ideal to be used in enzymatic channeling engineering.<br />
<br />
*Carboxysome: 80-150 nm in cross section. Composed of several thousands polypeptide of 10-15 different types. Contain as many as 250 RuBisCO per carboxysome molecule[12, 13].<br />
<br />
*''Pdu'' Microcomparment: 100-150 nm in cross section. Composed of about 18000 individual polypeptides of about 14-18 different types [1].<br />
<br />
*''Eut'' Microcomparment: Similar to pdu microcompartment in terms of both size and protein composition.<br />
<br />
<br />
One of the putative microcompartment systems in ''Clostridium kluyveri'' associated with the oxidation of ethanol, however, shows promise as an alternative microcompartment for use in our system because:<br />
<br />
#The gene cluster involved contains only seven genes: including two genes for a pair of nearly identical acetaldehyde dehydrogenases, three genes for highly similar ethanol dehydrogenases, and two genes for microcompartment proteins, which are orthologs of ethanolamine using genes (eutML) of ''Salmonella typhimurium'' [9].<br />
#From cell extracts of ''C. kluyveri'' a macromolecular complex of ethanol dehydrogenase and acetaldehyde dehydrogenase can be purified by differential manganese sulfate precipitation, indicating the presence of a functional and intact microcompartment enclosing these two enzymes[9].<br />
<br />
In order to co-opt this stucture for our own use it is necessary to understand how the associated enzymes are targeted to the compartment so that alternative enzymes of our choosing can be substituted. To search for a consensus targeting sequence we obtained the ''C. kluyveri'' compartment sequence from NCBI and performed a position specific iterative Blast search for related sequences among bacteria. We used the following two query sequences: <br />
<br />
{| border="1" cellspacing="0" width="50%"<br />
|-<br />
|'''CKL_1072'''<br />
|-<br />
|>gi|153953697|ref|YP_001394462.1| microcompartment protein [Clostridium kluyveri DSM 555]<br />
|-<br />
|MGQEALGMIETKGLVGAIEAADSMVKAANVALIGYEKIGSGLVTVMVRGDVGAVKAATDAGAASAKRVGE<br />
VISVHVIPRPHTDVEKILPNIG<br />
|}<br />
<br />
<br />
{| border="1" cellspacing="0" width="50%"<br />
|-<br />
|'''CKL_1073'''<br />
|-<br />
|>gi|153953698|ref|YP_001394463.1| microcompartment protein [Clostridium kluyveri DSM 555]<br />
|-<br />
|MNNELIEKVLGEVRKSLDLKNFDQEKLNKVVESTTEKLSDSKKEEAIKEAKPDVKVAEESKQAVVEQKAN<br />
DVKTAPTMTEFVGTAGGDTVGLVIANVDSLLHKHLGLDNTCRSIGIISARVGAPAQMMAADEAVKGTNTE<br />
VATIELPRDTKGGAGHGIFIVLKAADVSDARRAVEIALKQTDKYLGNVYLCDAGHLEVQYTARASLIFEK<br />
AFGAPSGQAFGIMHAAPAGVGMIVADTALKTADVKLITYGSPTNGVLSYTNEILITISGDSGAVLQSLTA<br />
ARKAGLSILRSMGQDPVSMSKPTF<br />
|}<br />
<br />
PSI-BLAST constructs and modifies the Position Specific Scoring Matrix constantly and uses it during the next round of search. This iterative searching strategy results in increased sensitivity. The top results were saved after 5 iterations. Download the [[Media:1072_3 BLAST.pdf|BLAST results]] and [[Media:taxonomy_report.txt|taxonomy report]].A [[Media:MUSCLE_alignment.pdf|multiple alignment]] of these sequences was constructed using CLCBio Genomic Workbench to assess the level of conservation between species and identify consensus sequence. <br />
<br />
Since microcompartment proteins are known to be found adjacent to their corresponding enzymes, we searched the top hits for adjacent genes 5’ upstream and 3’ downstream of the hit gene. We then attempted to apply multiple sequence alignment to these sequences in an effort to identify conserved motifs corresponding to a potential targeting sequence similar to the method employed by Suter ''et al''. A targeting sequence, if found, would allow translocation of enzymes into the microcompartment. Due to the incomplete annotation of certain genomes, the alignments did not yield a statistically significant targeting motif. We are attempting further analyses using different reading frames to refine the protein candidates used during the multiple alignment.<br />
<br />
==Conclusions==<br />
We have short-listed a number of enzymes which might benefit from channeling according to several properties such as free energy, toxicity and the occurrence of multi-functional homologues. In addition, while concluding that several classic examples of bacterial microcompartment are unsuitable for experimental manipulation, we have conducted a sequence-based search for additional protein family members corresponding to the ''Clostridium kluyveri'' microcompartment and searched the corresponding upstream and downstream genes for conserved motifs in an effort to find the targetting sequence for these enzymes. The ''Clostridium kluyveri'' system remains under consideration as a potentially viable alternative to the encapsulin-based nanocompartment. However, its suitability will depend on the results of further efforts to identify a targeting sequence for this compartment.<br />
<br />
==Future Work==<br />
In order to find the targeting sequence for the Clostridium kluyveri microcompartment, it will be necessary to fine tune the enzyme queries and potentially check out other alternative reading frames. In addition, enzymes will be selected from those short-listed above to proceed with a proof of concept experiment and potentially commercially viable application.<br />
<br />
==References==<br />
1.Cheng, S., ''et al''., Bacterial microcompartments: their properties and paradoxes. Bioessays, 2008. 30(11-12): p. 1084-95.<br />
<br />
2.Yeates, T.O., ''et al''., Protein-based organelles in bacteria: carboxysomes and related microcompartments. Nat Rev Microbiol, 2008. 6(9): p. 681-91.<br />
<br />
3.Shively, J.M., Inclusion bodies of prokaryotes. Annu Rev Microbiol, 1974. 28(0): p. 167-87.<br />
<br />
4.Chen, P., D.I. Andersson, and J.R. Roth, The control region of the pdu/cob regulon in Salmonella typhimurium. J <br />
Bacteriol, 1994. 176(17): p. 5474-82.<br />
<br />
5.Havemann, G.D., E.M. Sampson, and T.A. Bobik, PduA is a shell protein of polyhedral organelles involved in coenzyme B(12)-dependent degradation of 1,2-propanediol in Salmonella enterica serovar typhimurium LT2. J Bacteriol, 2002. 184(5): p. 1253-61.<br />
<br />
6.Brinsmade, S.R., T. Paldon, and J.C. Escalante-Semerena, Minimal functions and physiological conditions required for growth of salmonella enterica on ethanolamine in the absence of the metabolosome. J Bacteriol, 2005. 187(23): p. 8039-46.<br />
<br />
7.Penrod, J.T. and J.R. Roth, Conserving a volatile metabolite: a role for carboxysome-like organelles in Salmonella enterica. J Bacteriol, 2006. 188(8): p. 2865-74.<br />
<br />
8.Stojiljkovic, I., A.J. Baumler, and F. Heffron, Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster. J Bacteriol, 1995. 177(5): p. 1357-66.<br />
<br />
9.Seedorf, H., ''et al''., The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features. Proc Natl Acad Sci U S A, 2008. 105(6): p. 2128-33.<br />
<br />
10.Wackett, L.P., ''et al''., Genomic and biochemical studies demonstrating the absence of an alkane-producing phenotype in Vibrio furnissii M1. Appl Environ Microbiol, 2007. 73(22): p. 7192-8.<br />
<br />
11.Sampson, E.M. and T.A. Bobik, Microcompartments for B12-dependent 1,2-propanediol degradation provide protection from DNA and cellular damage by a reactive metabolic intermediate. J Bacteriol, 2008. 190(8): p. 2966-71.<br />
<br />
12.Yeates, T.O., ''et al''., Self-assembly in the carboxysome: a viral capsid-like protein shell in bacterial cells. Biochem Soc Trans, 2007. 35(Pt 3): p. 508-11.<br />
<br />
13.Tanaka, S., ''et al''., Atomic-level models of the bacterial carboxysome shell. Science, 2008. 319(5866): p. 1083-6.<br />
<br />
14.Sutter, M. ''et al''. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/BioinformaticsTeam:TorontoMaRSDiscovery/Bioinformatics2009-10-22T03:02:08Z<p>Gcromar: /* References */</p>
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<br><br />
<p style="font-size:18pt;">The diversity of bacterial microcompartments in nature and their potential for biotechnological applications</p><br />
<br />
==Summary==<br />
<br />
Spatial segregation is widely believed to be a defining organizational feature of eukaryotic cells: proteins, nucleic acids and small molecules are contained within and often actively transported between the many membrane-bound, subcellular organelles such as mitochondria, lysosomes, Golgi apparatus, etc. However, it has recently been found that a number of bacteria conditionally express proteinaceous microcompartments. These polyhedral organelles are usually 100-150 nm in cross section [1] and consist of proteinaceous outer shells, reminiscent of viral capsids, surrounding a core of enzymes[2]. It is thought that microcompartments allow bacteria to sequester specific metabolic enzymes and their substrates to enhance enzymatic efficiency (enzyme channeling) and protect cells from the toxic effects of certain intermediates. While several examples of these compartments have been reported their diversity has not been fully explored. We wish to design new microcompartments with modified properties or novel enzymatic activities, which could result in potentially useful applications in biotechnology. With this goal in mind, we attempted to address the following two questions using a bioinformatics approach (1) What enzymes will benefit the most from enzymatic channeling? and (2) Are there any other alternative microcompartments that could be explored for enzymatic channeling engineering?<br />
<br />
==Background==<br />
<br />
Most of our knowledge on bacterial microcompartments has been derived from the three well-studied microcompartment systems in nature.<br />
<br />
[[image:TMDT_Nature.jpg|right|thumb|Figure 1: The carbon concentrating mechanism in carboxysome.]]<br />
#Carboxysomes<br />
#*First reported in 1956, carboxysomes were the first bacteria microcompartments to be discovered. They are often present in cyanobacteria and other chemoautotrophic bacteria [3]. They are known to play a key role in enhancing autotrophic carbon fixation in the Calvin cycle. The shell of the carboxysome encodes the enzymes carbonic anhydrase (CA) and ribulose bis-phosphate carboxylase monooxygenase (RuBisCO). CA converts bicarbonate ions into carbon dioxide, which is then converted into 3-phosphoglycerate (3-PGA) by RuBisCO. Carboxysome not only allows for co-localization of CA and RuBisCO, but also acts as a diffusion barrier to retain carbon dioxide in the immediate vicinity of RuBisCO and thus catalyzes the conversion [2]. See Figure 1.<br />
#''Pdu'' microcompartment<br />
#*In 1994, homologues of carboxysome shell proteins were reported in ''S. enterica''. They are amongst a cluster of genes that are involved in coenzyme B12-dependent metabolism of 1,2 propanediol [4]. The gene cluster was later termed the pdu operon and the microcompartment formed was later termed propanediol utilization microcompartment. The proposed fuction of the pdu microcompartment is to encapsulate the enzymes that are necessary for cell to degrade propanediol and most importantly, to protect the cell from the toxic effects of propionaldehyde, an intermediate formed during the process [5].<br />
#''Eut'' microcompartment<br />
#*Later, similar structures were also found in ''E.coli'' and ''S. enterica'' when they were grown using ethanolamine as energy source. These structures were named ethanolamine utilization microcompartment (encoded by eut operon) and they often display very high genetic similarity with pdu microcompartment. Eut microcompartments contain enzymes involved in the degradation of ethanolamine and protect cell from acetaldehyde [6-8].<br />
#*''Generally speaking, both pdu and eut microcompartments are less uniform in size and more irregular in shape than carboxysomes[2].'' <br />
<br />
These compartments are generally unsuitable for our purposes because a) Their shell proteins consist of several different types whose functions are not well characterized and b) It is not known how the associated enzymes are targeted to the microcompartment. However, several other putative microcompartments have been inferred by sequence similarity. These include <br />
*A microcompartment was suggested to be involved in the oxidation of ethanol by ''Clostridium kluyveri''. [9].<br />
*A microcompartment associated with a puryvate-formate lyase homolog is proposed to be involved in the production of ethanol from pyruvate [10].<br />
*A putative microcompartment in ''Rhodopirellula baltica'' is proposed to be associated with a lactate dehydrogenase homologue [1].<br />
*A putative microcompartment in ''Carboxydothermus hydrogenoformans'' is associated with an isochorismatase-family protein [1].<br />
*A putative microcompartment Solibacter usitatus can be associated with a dihydrdipicolinate synthase homologue [1].<br />
<br />
The observation that diverse microcompartment structures are composed of proteins with homologous sequences led to the identification of a protein domain in the shell of all polyhedral bacterial microcompartments. This conserved protein domain is known as the Bacterial Microcompartment (BMC) domain ([http://pfam.sanger.ac.uk/family?acc=PF00936 Pfam00936]). It is approximately 84 amino acids long and can be either found as a part of a large protein or in tandem copies within the same operon. This domain is found to be present in 189 bacterial species to date. [2]<br />
<br />
Microcompartments typically promote the catalysis of a particular metabolic reaction by sequestering or co-localizing functionally related enzymes. The product of one enzyme will be delivered to the next enzyme at high concentration since it cannot readily diffuse. This greatly enhances enzyme efficiency. Microcompartments can also occlude toxic intermediates that cannot be degraded by normal bacterial machinery. This was shown to be true in ''pdu'' and ''eut'' microcompartments. The degradation pathway of 1,2-propanediol and ethanolamine both proceed through aldehyde intermediates (propionaldehyde and acetaldehyde respectively). The toxicity of these aldehyde intermediates was later shown in growth assays in which cells accumulating large amounts of propionaldehyde underwent growth arrest due to propionaldehyde toxicity [5, 11]. Therefore, it is reasonable to propose that bacteria use microcompartments to mitigate the toxicity of aldehyde by encapsulating these poisonous intermediates.<br />
<br />
==Methods/Results==<br />
===a) Search for enzyme pairs===<br />
[[image:TMDT_Pathway.jpg|right|thumb|Figure 2: Complete pathway map obtained from KEGG database with the first enzyme colored in red:.]]<br />
In order to identify candidate enzyme pairs likely to display an observable benefit from enzyme channeling we made the following assumptions:<br />
#Enzyme pairs with specific thermodynamic properties will likely benefit. Specifically, for two sequential reactions the second one will be thermodynamically more favorable than the first reaction. Consequently, the products from the first reaction be consumed at a faster rate than being generated, hence driving the reaction to completion. <br />
#*Predictions based on free energy values: [[Media:Scored.xls|Scored.xls]]<br />
#Enzymes involved in reactions with toxic intermediates will likely benefit from enzymatic channeling since the toxin will be less likely to interact with cellular components.<br />
#*Enzyme pairs that possess a common metabolite that is known to be toxic: [[Media:ToxicPairs.xls|ToxicPairs.xls]]<br />
#Enzymes that were identified as gene fusion products in other organisms will likely benefit from enzyme channeling since fusion of multi-functional enzymes is an extreme example of colocalization.<br />
#*Enzyme pairs predicted to form multifunctional enzymes by the Prolinks Rosetta Stone analysis: [[Media:RosettaEnzymePairs.xls|RosettaEnzymePairs.xls]]<br />
#*Enzyme pairs annotated to form multifunctional enzymes in SwissProt database: [[Media:SwissProtPairs.xls|SwissProtPairs.xls]]<br />
These results provided us with a combined list that was further refined by removing enzymes that are not immediately adjacent to each other in their biochemical pathway based on the KEGG database (Figure 2). In addition, enzymes with a molecular weight well beyond the microcompartment capacity (100 kDa) were also eliminated. The remaining enzyme pairs meeting these criteria were considered candidates for enzyme channeling and have been summarized in the following table:<br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0"<br />
| align="center" |'''Enzyme1 EC'''<br />
| align="center" |'''Enzyme1 Size (kDa)'''<br />
| align="center" |'''Enzyme2 EC'''<br />
| align="center" |'''Enzyme2 Size (kDa)'''<br />
| align="center" |'''Source'''<br />
| align="center" |'''Pathway'''<br />
| align="center" |'''Major Product'''<br />
| align="center" |'''Applications'''<br />
|-<br />
| 2.1.3.2||32-125||6.3.4.4||47-96||Rosette||Alanine and aspartate metabolism||adenylosuccinate||<br />
|-<br />
| 1.1.1.1||25-57||1.2.1.10||96.7-520||Swiss Prot||Butanoate metabolism ||Butanoyl-coA||<br />
|-<br />
| 1.1.1.95||35-250||2.6.1.52||35-96||Rosette||Glycine, serine and threonine metabolism ||Phosphoserine||<br />
|-<br />
| 2.3.3.9||52-81||4.1.3.1||14-65||Swiss Prot &Rosette||Glyoxylate and dicarboxylate metabolism||Isocitrate||<br />
|-<br />
| 1.5.1.15||32-34||3.5.4.9||30-45||Swiss Prot||One carbon pool by folate||Formyl-THF||<br />
|-<br />
| 1.5.1.5||30-150||3.5.4.9||30-45||Swiss Prot||One carbon pool by folate||Formyl-THF||<br />
|-<br />
| 1.1.1.57||||5.3.1.12||||Rosette||Pentose and glucuronate interconversions||D-Glucuronate||<br />
|-<br />
| 2.2.1.1||100-141||5.1.3.1||45-200||Rosette||Pentose phosphate pathway||D-ribolose-5P||<br />
|-<br />
| 2.2.1.2||35-75||5.3.1.9||27-67||Rosette||Pentose phosphate pathway||a-glucose-6P||<br />
|-<br />
| 1.3.1.13||52-210||4.2.1.51||12-137||Rosette||Phenylalanine, tyrosine and tryptophan biosynthesis||Phenyl-pyruvate||<br />
|-<br />
| 2.3.1.8||35-71||2.7.2.15||43||Rosette||Propanoate metabolism||Propanoate||Food preservatives<br />
|-<br />
| 2.3.1.8||35-70||2.7.2.1||12 to 66||Rosette||Propanoate metabolism/Pyruvate metabolism||Propanoate/acetate||Energy<br />
|-<br />
| 2.4.2.10||39-140||4.1.1.23||14-64||Swiss Prot||Pyrimidine metabolism||UMP||<br />
|-<br />
| 2.4.2.1||45-86||2.4.2.3||32-160||Rosette||Pyrimidine metabolism||Uridine||<br />
|-<br />
| 2.4.1.15||45-630||3.1.3.12||25-973||Rosette||Starch and sucrose metabolism||a,a trehalose||biotechnology application<br />
|-<br />
| 1.2.1.41||41-189||2.7.2.11||47-354||Swiss Prot &Rosette||Urea cycle and metabolism of amino groups||Glutamate||Flavor enhancer, Plant growth<br />
|}<br />
<br />
===b) Alternative microcompartments===<br />
<br />
Upon further examination, most of our known microcompartments tend to consist of a large number of protein subunits with very complex structures. Because of their complicated regulatory patterns and stoichiometry, these structures are not ideal to be used in enzymatic channeling engineering.<br />
<br />
*Carboxysome: 80-150 nm in cross section. Composed of several thousands polypeptide of 10-15 different types. Contain as many as 250 RuBisCO per carboxysome molecule[12, 13].<br />
<br />
*''Pdu'' Microcomparment: 100-150 nm in cross section. Composed of about 18000 individual polypeptides of about 14-18 different types [1].<br />
<br />
*''Eut'' Microcomparment: Similar to pdu microcompartment in terms of both size and protein composition.<br />
<br />
<br />
One of the putative microcompartment systems in ''Clostridium kluyveri'' associated with the oxidation of ethanol, however, shows promise as an alternative microcompartment for use in our system because:<br />
<br />
#The gene cluster involved contains only seven genes: including two genes for a pair of nearly identical acetaldehyde dehydrogenases, three genes for highly similar ethanol dehydrogenases, and two genes for microcompartment proteins, which are orthologs of ethanolamine using genes (eutML) of ''Salmonella typhimurium'' [9].<br />
#From cell extracts of ''C. kluyveri'' a macromolecular complex of ethanol dehydrogenase and acetaldehyde dehydrogenase can be purified by differential manganese sulfate precipitation, indicating the presence of a functional and intact microcompartment enclosing these two enzymes[9].<br />
<br />
In order to co-opt this stucture for our own use it is necessary to understand how the associated enzymes are targeted to the compartment so that alternative enzymes of our choosing can be substituted. To search for a consensus targeting sequence we obtained the ''C. kluyveri'' compartment sequence from NCBI and performed a position specific iterative Blast search for related sequences among bacteria. We used the following two query sequences: <br />
<br />
{| border="1" cellspacing="0" width="50%"<br />
|-<br />
|'''CKL_1072'''<br />
|-<br />
|>gi|153953697|ref|YP_001394462.1| microcompartment protein [Clostridium kluyveri DSM 555]<br />
|-<br />
|MGQEALGMIETKGLVGAIEAADSMVKAANVALIGYEKIGSGLVTVMVRGDVGAVKAATDAGAASAKRVGE<br />
VISVHVIPRPHTDVEKILPNIG<br />
|}<br />
<br />
<br />
{| border="1" cellspacing="0" width="50%"<br />
|-<br />
|'''CKL_1073'''<br />
|-<br />
|>gi|153953698|ref|YP_001394463.1| microcompartment protein [Clostridium kluyveri DSM 555]<br />
|-<br />
|MNNELIEKVLGEVRKSLDLKNFDQEKLNKVVESTTEKLSDSKKEEAIKEAKPDVKVAEESKQAVVEQKAN<br />
DVKTAPTMTEFVGTAGGDTVGLVIANVDSLLHKHLGLDNTCRSIGIISARVGAPAQMMAADEAVKGTNTE<br />
VATIELPRDTKGGAGHGIFIVLKAADVSDARRAVEIALKQTDKYLGNVYLCDAGHLEVQYTARASLIFEK<br />
AFGAPSGQAFGIMHAAPAGVGMIVADTALKTADVKLITYGSPTNGVLSYTNEILITISGDSGAVLQSLTA<br />
ARKAGLSILRSMGQDPVSMSKPTF<br />
|}<br />
<br />
PSI-BLAST constructs and modifies the Position Specific Scoring Matrix constantly and uses it during the next round of search. This iterative searching strategy results in increased sensitivity. The top results were saved after 5 iterations. Download the [[Media:1072_3 BLAST.pdf|BLAST results]] and [[Media:taxonomy_report.txt|taxonomy report]].A [[Media:MUSCLE_alignment.pdf|multiple alignment]] of these sequences was constructed using CLCBio Genomic Workbench to assess the level of conservation between species and identify consensus sequence. <br />
<br />
Since microcompartment proteins are known to be found adjacent to their corresponding enzymes, we searched the top hits for adjacent genes 5’ upstream and 3’ downstream of the hit gene. We then attempted to apply multiple sequence alignment to these sequences in an effort to identify conserved motifs corresponding to a potential targeting sequence similar to the method employed by Suter ''et al''. A targeting sequence, if found, would allow translocation of enzymes into the microcompartment. Due to the incomplete annotation of certain genomes, the alignments did not yield a statistically significant targeting motif. We are attempting further analyses using different reading frames to refine the protein candidates used during the multiple alignment.<br />
<br />
==Conclusions==<br />
We have short-listed a number of enzymes which might benefit from channeling according to several properties such as free energy, toxicity and the occurrence of multi-functional homologues. In addition, while concluding that several classic examples of bacterial microcompartment are unsuitable for experimental manipulation, we have conducted a sequence-based search for additional protein family members corresponding to the ''Clostridium kluyveri'' microcompartment and searched the corresponding upstream and downstream genes for conserved motifs in an effort to find the targetting sequence for these enzymes. The ''Clostridium kluyveri'' system remains under consideration as a potentially viable alternative to the encapsulin-based nanocompartment. However, its suitability will depend on the results of further efforts to identify a targeting sequence for this compartment.<br />
<br />
==Future Work==<br />
In order to find the targeting sequence for the Clostridium kluyveri microcompartment, it will be necessary to fine tune the enzyme queries and potentially check out other alternative reading frames. In addition, enzymes will be selected from those short-listed above to proceed with a proof of concept experiment and potentially commercially viable application.<br />
<br />
==References==<br />
1.Cheng, S., ''et al''., Bacterial microcompartments: their properties and paradoxes. Bioessays, 2008. 30(11-12): p. 1084-95.<br />
<br />
2.Yeates, T.O., ''et al''., Protein-based organelles in bacteria: carboxysomes and related microcompartments. Nat Rev Microbiol, 2008. 6(9): p. 681-91.<br />
<br />
3.Shively, J.M., Inclusion bodies of prokaryotes. Annu Rev Microbiol, 1974. 28(0): p. 167-87.<br />
<br />
4.Chen, P., D.I. Andersson, and J.R. Roth, The control region of the pdu/cob regulon in Salmonella typhimurium. J <br />
Bacteriol, 1994. 176(17): p. 5474-82.<br />
<br />
5.Havemann, G.D., E.M. Sampson, and T.A. Bobik, PduA is a shell protein of polyhedral organelles involved in coenzyme B(12)-dependent degradation of 1,2-propanediol in Salmonella enterica serovar typhimurium LT2. J Bacteriol, 2002. 184(5): p. 1253-61.<br />
<br />
6.Brinsmade, S.R., T. Paldon, and J.C. Escalante-Semerena, Minimal functions and physiological conditions required for growth of salmonella enterica on ethanolamine in the absence of the metabolosome. J Bacteriol, 2005. 187(23): p. 8039-46.<br />
<br />
7.Penrod, J.T. and J.R. Roth, Conserving a volatile metabolite: a role for carboxysome-like organelles in Salmonella enterica. J Bacteriol, 2006. 188(8): p. 2865-74.<br />
<br />
8.Stojiljkovic, I., A.J. Baumler, and F. Heffron, Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster. J Bacteriol, 1995. 177(5): p. 1357-66.<br />
<br />
9.Seedorf, H., ''et al''., The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features. Proc Natl Acad Sci U S A, 2008. 105(6): p. 2128-33.<br />
<br />
10.Wackett, L.P., ''et al''., Genomic and biochemical studies demonstrating the absence of an alkane-producing phenotype in Vibrio furnissii M1. Appl Environ Microbiol, 2007. 73(22): p. 7192-8.<br />
<br />
11.Sampson, E.M. and T.A. Bobik, Microcompartments for B12-dependent 1,2-propanediol degradation provide protection from DNA and cellular damage by a reactive metabolic intermediate. J Bacteriol, 2008. 190(8): p. 2966-71.<br />
<br />
12.Yeates, T.O., ''et al''., Self-assembly in the carboxysome: a viral capsid-like protein shell in bacterial cells. Biochem Soc Trans, 2007. 35(Pt 3): p. 508-11.<br />
<br />
13.Tanaka, S., ''et al''., Atomic-level models of the bacterial carboxysome shell. Science, 2008. 319(5866): p. 1083-6.<br />
<br />
14.Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/BioinformaticsTeam:TorontoMaRSDiscovery/Bioinformatics2009-10-22T03:00:03Z<p>Gcromar: /* Background */</p>
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<br><br />
<p style="font-size:18pt;">The diversity of bacterial microcompartments in nature and their potential for biotechnological applications</p><br />
<br />
==Summary==<br />
<br />
Spatial segregation is widely believed to be a defining organizational feature of eukaryotic cells: proteins, nucleic acids and small molecules are contained within and often actively transported between the many membrane-bound, subcellular organelles such as mitochondria, lysosomes, Golgi apparatus, etc. However, it has recently been found that a number of bacteria conditionally express proteinaceous microcompartments. These polyhedral organelles are usually 100-150 nm in cross section [1] and consist of proteinaceous outer shells, reminiscent of viral capsids, surrounding a core of enzymes[2]. It is thought that microcompartments allow bacteria to sequester specific metabolic enzymes and their substrates to enhance enzymatic efficiency (enzyme channeling) and protect cells from the toxic effects of certain intermediates. While several examples of these compartments have been reported their diversity has not been fully explored. We wish to design new microcompartments with modified properties or novel enzymatic activities, which could result in potentially useful applications in biotechnology. With this goal in mind, we attempted to address the following two questions using a bioinformatics approach (1) What enzymes will benefit the most from enzymatic channeling? and (2) Are there any other alternative microcompartments that could be explored for enzymatic channeling engineering?<br />
<br />
==Background==<br />
<br />
Most of our knowledge on bacterial microcompartments has been derived from the three well-studied microcompartment systems in nature.<br />
<br />
[[image:TMDT_Nature.jpg|right|thumb|Figure 1: The carbon concentrating mechanism in carboxysome.]]<br />
#Carboxysomes<br />
#*First reported in 1956, carboxysomes were the first bacteria microcompartments to be discovered. They are often present in cyanobacteria and other chemoautotrophic bacteria [3]. They are known to play a key role in enhancing autotrophic carbon fixation in the Calvin cycle. The shell of the carboxysome encodes the enzymes carbonic anhydrase (CA) and ribulose bis-phosphate carboxylase monooxygenase (RuBisCO). CA converts bicarbonate ions into carbon dioxide, which is then converted into 3-phosphoglycerate (3-PGA) by RuBisCO. Carboxysome not only allows for co-localization of CA and RuBisCO, but also acts as a diffusion barrier to retain carbon dioxide in the immediate vicinity of RuBisCO and thus catalyzes the conversion [2]. See Figure 1.<br />
#''Pdu'' microcompartment<br />
#*In 1994, homologues of carboxysome shell proteins were reported in ''S. enterica''. They are amongst a cluster of genes that are involved in coenzyme B12-dependent metabolism of 1,2 propanediol [4]. The gene cluster was later termed the pdu operon and the microcompartment formed was later termed propanediol utilization microcompartment. The proposed fuction of the pdu microcompartment is to encapsulate the enzymes that are necessary for cell to degrade propanediol and most importantly, to protect the cell from the toxic effects of propionaldehyde, an intermediate formed during the process [5].<br />
#''Eut'' microcompartment<br />
#*Later, similar structures were also found in ''E.coli'' and ''S. enterica'' when they were grown using ethanolamine as energy source. These structures were named ethanolamine utilization microcompartment (encoded by eut operon) and they often display very high genetic similarity with pdu microcompartment. Eut microcompartments contain enzymes involved in the degradation of ethanolamine and protect cell from acetaldehyde [6-8].<br />
#*''Generally speaking, both pdu and eut microcompartments are less uniform in size and more irregular in shape than carboxysomes[2].'' <br />
<br />
These compartments are generally unsuitable for our purposes because a) Their shell proteins consist of several different types whose functions are not well characterized and b) It is not known how the associated enzymes are targeted to the microcompartment. However, several other putative microcompartments have been inferred by sequence similarity. These include <br />
*A microcompartment was suggested to be involved in the oxidation of ethanol by ''Clostridium kluyveri''. [9].<br />
*A microcompartment associated with a puryvate-formate lyase homolog is proposed to be involved in the production of ethanol from pyruvate [10].<br />
*A putative microcompartment in ''Rhodopirellula baltica'' is proposed to be associated with a lactate dehydrogenase homologue [1].<br />
*A putative microcompartment in ''Carboxydothermus hydrogenoformans'' is associated with an isochorismatase-family protein [1].<br />
*A putative microcompartment Solibacter usitatus can be associated with a dihydrdipicolinate synthase homologue [1].<br />
<br />
The observation that diverse microcompartment structures are composed of proteins with homologous sequences led to the identification of a protein domain in the shell of all polyhedral bacterial microcompartments. This conserved protein domain is known as the Bacterial Microcompartment (BMC) domain ([http://pfam.sanger.ac.uk/family?acc=PF00936 Pfam00936]). It is approximately 84 amino acids long and can be either found as a part of a large protein or in tandem copies within the same operon. This domain is found to be present in 189 bacterial species to date. [2]<br />
<br />
Microcompartments typically promote the catalysis of a particular metabolic reaction by sequestering or co-localizing functionally related enzymes. The product of one enzyme will be delivered to the next enzyme at high concentration since it cannot readily diffuse. This greatly enhances enzyme efficiency. Microcompartments can also occlude toxic intermediates that cannot be degraded by normal bacterial machinery. This was shown to be true in ''pdu'' and ''eut'' microcompartments. The degradation pathway of 1,2-propanediol and ethanolamine both proceed through aldehyde intermediates (propionaldehyde and acetaldehyde respectively). The toxicity of these aldehyde intermediates was later shown in growth assays in which cells accumulating large amounts of propionaldehyde underwent growth arrest due to propionaldehyde toxicity [5, 11]. Therefore, it is reasonable to propose that bacteria use microcompartments to mitigate the toxicity of aldehyde by encapsulating these poisonous intermediates.<br />
<br />
==Methods/Results==<br />
===a) Search for enzyme pairs===<br />
[[image:TMDT_Pathway.jpg|right|thumb|Figure 2: Complete pathway map obtained from KEGG database with the first enzyme colored in red:.]]<br />
In order to identify candidate enzyme pairs likely to display an observable benefit from enzyme channeling we made the following assumptions:<br />
#Enzyme pairs with specific thermodynamic properties will likely benefit. Specifically, for two sequential reactions the second one will be thermodynamically more favorable than the first reaction. Consequently, the products from the first reaction be consumed at a faster rate than being generated, hence driving the reaction to completion. <br />
#*Predictions based on free energy values: [[Media:Scored.xls|Scored.xls]]<br />
#Enzymes involved in reactions with toxic intermediates will likely benefit from enzymatic channeling since the toxin will be less likely to interact with cellular components.<br />
#*Enzyme pairs that possess a common metabolite that is known to be toxic: [[Media:ToxicPairs.xls|ToxicPairs.xls]]<br />
#Enzymes that were identified as gene fusion products in other organisms will likely benefit from enzyme channeling since fusion of multi-functional enzymes is an extreme example of colocalization.<br />
#*Enzyme pairs predicted to form multifunctional enzymes by the Prolinks Rosetta Stone analysis: [[Media:RosettaEnzymePairs.xls|RosettaEnzymePairs.xls]]<br />
#*Enzyme pairs annotated to form multifunctional enzymes in SwissProt database: [[Media:SwissProtPairs.xls|SwissProtPairs.xls]]<br />
These results provided us with a combined list that was further refined by removing enzymes that are not immediately adjacent to each other in their biochemical pathway based on the KEGG database (Figure 2). In addition, enzymes with a molecular weight well beyond the microcompartment capacity (100 kDa) were also eliminated. The remaining enzyme pairs meeting these criteria were considered candidates for enzyme channeling and have been summarized in the following table:<br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0"<br />
| align="center" |'''Enzyme1 EC'''<br />
| align="center" |'''Enzyme1 Size (kDa)'''<br />
| align="center" |'''Enzyme2 EC'''<br />
| align="center" |'''Enzyme2 Size (kDa)'''<br />
| align="center" |'''Source'''<br />
| align="center" |'''Pathway'''<br />
| align="center" |'''Major Product'''<br />
| align="center" |'''Applications'''<br />
|-<br />
| 2.1.3.2||32-125||6.3.4.4||47-96||Rosette||Alanine and aspartate metabolism||adenylosuccinate||<br />
|-<br />
| 1.1.1.1||25-57||1.2.1.10||96.7-520||Swiss Prot||Butanoate metabolism ||Butanoyl-coA||<br />
|-<br />
| 1.1.1.95||35-250||2.6.1.52||35-96||Rosette||Glycine, serine and threonine metabolism ||Phosphoserine||<br />
|-<br />
| 2.3.3.9||52-81||4.1.3.1||14-65||Swiss Prot &Rosette||Glyoxylate and dicarboxylate metabolism||Isocitrate||<br />
|-<br />
| 1.5.1.15||32-34||3.5.4.9||30-45||Swiss Prot||One carbon pool by folate||Formyl-THF||<br />
|-<br />
| 1.5.1.5||30-150||3.5.4.9||30-45||Swiss Prot||One carbon pool by folate||Formyl-THF||<br />
|-<br />
| 1.1.1.57||||5.3.1.12||||Rosette||Pentose and glucuronate interconversions||D-Glucuronate||<br />
|-<br />
| 2.2.1.1||100-141||5.1.3.1||45-200||Rosette||Pentose phosphate pathway||D-ribolose-5P||<br />
|-<br />
| 2.2.1.2||35-75||5.3.1.9||27-67||Rosette||Pentose phosphate pathway||a-glucose-6P||<br />
|-<br />
| 1.3.1.13||52-210||4.2.1.51||12-137||Rosette||Phenylalanine, tyrosine and tryptophan biosynthesis||Phenyl-pyruvate||<br />
|-<br />
| 2.3.1.8||35-71||2.7.2.15||43||Rosette||Propanoate metabolism||Propanoate||Food preservatives<br />
|-<br />
| 2.3.1.8||35-70||2.7.2.1||12 to 66||Rosette||Propanoate metabolism/Pyruvate metabolism||Propanoate/acetate||Energy<br />
|-<br />
| 2.4.2.10||39-140||4.1.1.23||14-64||Swiss Prot||Pyrimidine metabolism||UMP||<br />
|-<br />
| 2.4.2.1||45-86||2.4.2.3||32-160||Rosette||Pyrimidine metabolism||Uridine||<br />
|-<br />
| 2.4.1.15||45-630||3.1.3.12||25-973||Rosette||Starch and sucrose metabolism||a,a trehalose||biotechnology application<br />
|-<br />
| 1.2.1.41||41-189||2.7.2.11||47-354||Swiss Prot &Rosette||Urea cycle and metabolism of amino groups||Glutamate||Flavor enhancer, Plant growth<br />
|}<br />
<br />
===b) Alternative microcompartments===<br />
<br />
Upon further examination, most of our known microcompartments tend to consist of a large number of protein subunits with very complex structures. Because of their complicated regulatory patterns and stoichiometry, these structures are not ideal to be used in enzymatic channeling engineering.<br />
<br />
*Carboxysome: 80-150 nm in cross section. Composed of several thousands polypeptide of 10-15 different types. Contain as many as 250 RuBisCO per carboxysome molecule[12, 13].<br />
<br />
*''Pdu'' Microcomparment: 100-150 nm in cross section. Composed of about 18000 individual polypeptides of about 14-18 different types [1].<br />
<br />
*''Eut'' Microcomparment: Similar to pdu microcompartment in terms of both size and protein composition.<br />
<br />
<br />
One of the putative microcompartment systems in ''Clostridium kluyveri'' associated with the oxidation of ethanol, however, shows promise as an alternative microcompartment for use in our system because:<br />
<br />
#The gene cluster involved contains only seven genes: including two genes for a pair of nearly identical acetaldehyde dehydrogenases, three genes for highly similar ethanol dehydrogenases, and two genes for microcompartment proteins, which are orthologs of ethanolamine using genes (eutML) of ''Salmonella typhimurium'' [9].<br />
#From cell extracts of ''C. kluyveri'' a macromolecular complex of ethanol dehydrogenase and acetaldehyde dehydrogenase can be purified by differential manganese sulfate precipitation, indicating the presence of a functional and intact microcompartment enclosing these two enzymes[9].<br />
<br />
In order to co-opt this stucture for our own use it is necessary to understand how the associated enzymes are targeted to the compartment so that alternative enzymes of our choosing can be substituted. To search for a consensus targeting sequence we obtained the ''C. kluyveri'' compartment sequence from NCBI and performed a position specific iterative Blast search for related sequences among bacteria. We used the following two query sequences: <br />
<br />
{| border="1" cellspacing="0" width="50%"<br />
|-<br />
|'''CKL_1072'''<br />
|-<br />
|>gi|153953697|ref|YP_001394462.1| microcompartment protein [Clostridium kluyveri DSM 555]<br />
|-<br />
|MGQEALGMIETKGLVGAIEAADSMVKAANVALIGYEKIGSGLVTVMVRGDVGAVKAATDAGAASAKRVGE<br />
VISVHVIPRPHTDVEKILPNIG<br />
|}<br />
<br />
<br />
{| border="1" cellspacing="0" width="50%"<br />
|-<br />
|'''CKL_1073'''<br />
|-<br />
|>gi|153953698|ref|YP_001394463.1| microcompartment protein [Clostridium kluyveri DSM 555]<br />
|-<br />
|MNNELIEKVLGEVRKSLDLKNFDQEKLNKVVESTTEKLSDSKKEEAIKEAKPDVKVAEESKQAVVEQKAN<br />
DVKTAPTMTEFVGTAGGDTVGLVIANVDSLLHKHLGLDNTCRSIGIISARVGAPAQMMAADEAVKGTNTE<br />
VATIELPRDTKGGAGHGIFIVLKAADVSDARRAVEIALKQTDKYLGNVYLCDAGHLEVQYTARASLIFEK<br />
AFGAPSGQAFGIMHAAPAGVGMIVADTALKTADVKLITYGSPTNGVLSYTNEILITISGDSGAVLQSLTA<br />
ARKAGLSILRSMGQDPVSMSKPTF<br />
|}<br />
<br />
PSI-BLAST constructs and modifies the Position Specific Scoring Matrix constantly and uses it during the next round of search. This iterative searching strategy results in increased sensitivity. The top results were saved after 5 iterations. Download the [[Media:1072_3 BLAST.pdf|BLAST results]] and [[Media:taxonomy_report.txt|taxonomy report]].A [[Media:MUSCLE_alignment.pdf|multiple alignment]] of these sequences was constructed using CLCBio Genomic Workbench to assess the level of conservation between species and identify consensus sequence. <br />
<br />
Since microcompartment proteins are known to be found adjacent to their corresponding enzymes, we searched the top hits for adjacent genes 5’ upstream and 3’ downstream of the hit gene. We then attempted to apply multiple sequence alignment to these sequences in an effort to identify conserved motifs corresponding to a potential targeting sequence similar to the method employed by Suter ''et al''. A targeting sequence, if found, would allow translocation of enzymes into the microcompartment. Due to the incomplete annotation of certain genomes, the alignments did not yield a statistically significant targeting motif. We are attempting further analyses using different reading frames to refine the protein candidates used during the multiple alignment.<br />
<br />
==Conclusions==<br />
We have short-listed a number of enzymes which might benefit from channeling according to several properties such as free energy, toxicity and the occurrence of multi-functional homologues. In addition, while concluding that several classic examples of bacterial microcompartment are unsuitable for experimental manipulation, we have conducted a sequence-based search for additional protein family members corresponding to the ''Clostridium kluyveri'' microcompartment and searched the corresponding upstream and downstream genes for conserved motifs in an effort to find the targetting sequence for these enzymes. The ''Clostridium kluyveri'' system remains under consideration as a potentially viable alternative to the encapsulin-based nanocompartment. However, its suitability will depend on the results of further efforts to identify a targeting sequence for this compartment.<br />
<br />
==Future Work==<br />
In order to find the targeting sequence for the Clostridium kluyveri microcompartment, it will be necessary to fine tune the enzyme queries and potentially check out other alternative reading frames. In addition, enzymes will be selected from those short-listed above to proceed with a proof of concept experiment and potentially commercially viable application.<br />
<br />
==References==<br />
1.Cheng, S., et al., Bacterial microcompartments: their properties and paradoxes. Bioessays, 2008. 30(11-12): p. 1084-95.<br />
<br />
2.Yeates, T.O., et al., Protein-based organelles in bacteria: carboxysomes and related microcompartments. Nat Rev Microbiol, 2008. 6(9): p. 681-91.<br />
<br />
3.Shively, J.M., Inclusion bodies of prokaryotes. Annu Rev Microbiol, 1974. 28(0): p. 167-87.<br />
<br />
4.Chen, P., D.I. Andersson, and J.R. Roth, The control region of the pdu/cob regulon in Salmonella typhimurium. J <br />
Bacteriol, 1994. 176(17): p. 5474-82.<br />
<br />
5.Havemann, G.D., E.M. Sampson, and T.A. Bobik, PduA is a shell protein of polyhedral organelles involved in coenzyme B(12)-dependent degradation of 1,2-propanediol in Salmonella enterica serovar typhimurium LT2. J Bacteriol, 2002. 184(5): p. 1253-61.<br />
<br />
6.Brinsmade, S.R., T. Paldon, and J.C. Escalante-Semerena, Minimal functions and physiological conditions required for growth of salmonella enterica on ethanolamine in the absence of the metabolosome. J Bacteriol, 2005. 187(23): p. 8039-46.<br />
<br />
7.Penrod, J.T. and J.R. Roth, Conserving a volatile metabolite: a role for carboxysome-like organelles in Salmonella enterica. J Bacteriol, 2006. 188(8): p. 2865-74.<br />
<br />
8.Stojiljkovic, I., A.J. Baumler, and F. Heffron, Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster. J Bacteriol, 1995. 177(5): p. 1357-66.<br />
<br />
9.Seedorf, H., et al., The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features. Proc Natl Acad Sci U S A, 2008. 105(6): p. 2128-33.<br />
<br />
10.Wackett, L.P., et al., Genomic and biochemical studies demonstrating the absence of an alkane-producing phenotype in Vibrio furnissii M1. Appl Environ Microbiol, 2007. 73(22): p. 7192-8.<br />
<br />
11.Sampson, E.M. and T.A. Bobik, Microcompartments for B12-dependent 1,2-propanediol degradation provide protection from DNA and cellular damage by a reactive metabolic intermediate. J Bacteriol, 2008. 190(8): p. 2966-71.<br />
<br />
12.Yeates, T.O., et al., Self-assembly in the carboxysome: a viral capsid-like protein shell in bacterial cells. Biochem Soc Trans, 2007. 35(Pt 3): p. 508-11.<br />
<br />
13.Tanaka, S., et al., Atomic-level models of the bacterial carboxysome shell. Science, 2008. 319(5866): p. 1083-6.</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/BioinformaticsTeam:TorontoMaRSDiscovery/Bioinformatics2009-10-22T02:58:13Z<p>Gcromar: /* b) Alternative microcompartments */</p>
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<br><br />
<p style="font-size:18pt;">The diversity of bacterial microcompartments in nature and their potential for biotechnological applications</p><br />
<br />
==Summary==<br />
<br />
Spatial segregation is widely believed to be a defining organizational feature of eukaryotic cells: proteins, nucleic acids and small molecules are contained within and often actively transported between the many membrane-bound, subcellular organelles such as mitochondria, lysosomes, Golgi apparatus, etc. However, it has recently been found that a number of bacteria conditionally express proteinaceous microcompartments. These polyhedral organelles are usually 100-150 nm in cross section [1] and consist of proteinaceous outer shells, reminiscent of viral capsids, surrounding a core of enzymes[2]. It is thought that microcompartments allow bacteria to sequester specific metabolic enzymes and their substrates to enhance enzymatic efficiency (enzyme channeling) and protect cells from the toxic effects of certain intermediates. While several examples of these compartments have been reported their diversity has not been fully explored. We wish to design new microcompartments with modified properties or novel enzymatic activities, which could result in potentially useful applications in biotechnology. With this goal in mind, we attempted to address the following two questions using a bioinformatics approach (1) What enzymes will benefit the most from enzymatic channeling? and (2) Are there any other alternative microcompartments that could be explored for enzymatic channeling engineering?<br />
<br />
==Background==<br />
<br />
Most of our knowledge on bacterial microcompartments has been derived from the three well-studied microcompartment systems in nature.<br />
<br />
[[image:TMDT_Nature.jpg|right|thumb|Figure 1: The carbon concentrating mechanism in carboxysome.]]<br />
#Carboxysomes<br />
#*First reported in 1956, carboxysomes were the first bacteria microcompartments to be discovered. They are often present in cyanobacteria and other chemoautotrophic bacteria [3]. They are known to play a key role in enhancing autotrophic carbon fixation in the Calvin cycle. The shell of the carboxysome encodes the enzymes carbonic anhydrase (CA) and ribulose bis-phosphate carboxylase monooxygenase (RuBisCO). CA converts bicarbonate ions into carbon dioxide, which is then converted into 3-phosphoglycerate (3-PGA) by RuBisCO. Carboxysome not only allows for co-localization of CA and RuBisCO, but also acts as a diffusion barrier to retain carbon dioxide in the immediate vicinity of RuBisCO and thus catalyzes the conversion [2]. See Figure 1.<br />
#''Pdu'' microcompartment<br />
#*In 1994, homologues of carboxysome shell proteins were reported in S. enterica. They are amongst a cluster of genes that are involved in coenzyme B12-dependent metabolism of 1,2 propanediol [4]. The gene cluster was later termed the pdu operon and the microcompartment formed was later termed propanediol utilization microcompartment. The proposed fuction of the pdu microcompartment is to encapsulate the enzymes that are necessary for cell to degrade propanediol and most importantly, to protect the cell from the toxic effects of propionaldehyde, an intermediate formed during the process [5].<br />
#''Eut'' microcompartment<br />
#*Later, similar structures were also found in E.coli and S. enterica when they were grown using ethanolamine as energy source. These structures were named ethanolamine utilization microcompartment (encoded by eut operon) and they often display very high genetic similarity with pdu microcompartment. Eut microcompartments contain enzymes involved in the degradation of ethanolamine and protect cell from acetaldehyde [6-8].<br />
#*''Generally speaking, both pdu and eut microcompartments are less uniform in size and more irregular in shape than carboxysomes[2].'' <br />
<br />
These compartments are generally unsuitable for our purposes because a) Their shell proteins consist of several different types whose functions are not well characterized and b) It is not known how the associated enzymes are targeted to the microcompartment. However, several other putative microcompartments have been inferred by sequence similarity. These include <br />
*A microcompartment was suggested to be involved in the oxidation of ethanol by Clostridium kluyveri. [9].<br />
*A microcompartment associated with a puryvate-formate lyase homolog is proposed to be involved in the production of ethanol from pyruvate [10].<br />
*A putative microcompartment in Rhodopirellula baltica is proposed to be associated with a lactate dehydrogenase homologue [1].<br />
*A putative microcompartment in Carboxydothermus hydrogenoformans is associated with an isochorismatase-family protein [1].<br />
*A putative microcompartment Solibacter usitatus can be associated with a dihydrdipicolinate synthase homologue [1].<br />
<br />
The observation that diverse microcompartment structures are composed of proteins with homologous sequences led to the identification of a protein domain in the shell of all polyhedral bacterial microcompartments. This conserved protein domain is known as the Bacterial Microcompartment (BMC) domain ([http://pfam.sanger.ac.uk/family?acc=PF00936 Pfam00936]). It is approximately 84 amino acids long and can be either found as a part of a large protein or in tandem copies within the same operon. This domain is found to be present in 189 bacterial species to date. [2]<br />
<br />
Microcompartments typically promote the catalysis of a particular metabolic reaction by sequestering or co-localizing functionally related enzymes. The product of one enzyme will be delivered to the next enzyme at high concentration since it cannot readily diffuse. This greatly enhances enzyme efficiency. Microcompartments can also occlude toxic intermediates that cannot be degraded by normal bacterial machinery. This was shown to be true in ''pdu'' and ''eut'' microcompartments. The degradation pathway of 1,2-propanediol and ethanolamine both proceed through aldehyde intermediates (propionaldehyde and acetaldehyde respectively). The toxicity of these aldehyde intermediates was later shown in growth assays in which cells accumulating large amounts of propionaldehyde underwent growth arrest due to propionaldehyde toxicity [5, 11]. Therefore, it is reasonable to propose that bacteria use microcompartments to mitigate the toxicity of aldehyde by encapsulating these poisonous intermediates.<br />
<br />
==Methods/Results==<br />
===a) Search for enzyme pairs===<br />
[[image:TMDT_Pathway.jpg|right|thumb|Figure 2: Complete pathway map obtained from KEGG database with the first enzyme colored in red:.]]<br />
In order to identify candidate enzyme pairs likely to display an observable benefit from enzyme channeling we made the following assumptions:<br />
#Enzyme pairs with specific thermodynamic properties will likely benefit. Specifically, for two sequential reactions the second one will be thermodynamically more favorable than the first reaction. Consequently, the products from the first reaction be consumed at a faster rate than being generated, hence driving the reaction to completion. <br />
#*Predictions based on free energy values: [[Media:Scored.xls|Scored.xls]]<br />
#Enzymes involved in reactions with toxic intermediates will likely benefit from enzymatic channeling since the toxin will be less likely to interact with cellular components.<br />
#*Enzyme pairs that possess a common metabolite that is known to be toxic: [[Media:ToxicPairs.xls|ToxicPairs.xls]]<br />
#Enzymes that were identified as gene fusion products in other organisms will likely benefit from enzyme channeling since fusion of multi-functional enzymes is an extreme example of colocalization.<br />
#*Enzyme pairs predicted to form multifunctional enzymes by the Prolinks Rosetta Stone analysis: [[Media:RosettaEnzymePairs.xls|RosettaEnzymePairs.xls]]<br />
#*Enzyme pairs annotated to form multifunctional enzymes in SwissProt database: [[Media:SwissProtPairs.xls|SwissProtPairs.xls]]<br />
These results provided us with a combined list that was further refined by removing enzymes that are not immediately adjacent to each other in their biochemical pathway based on the KEGG database (Figure 2). In addition, enzymes with a molecular weight well beyond the microcompartment capacity (100 kDa) were also eliminated. The remaining enzyme pairs meeting these criteria were considered candidates for enzyme channeling and have been summarized in the following table:<br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0"<br />
| align="center" |'''Enzyme1 EC'''<br />
| align="center" |'''Enzyme1 Size (kDa)'''<br />
| align="center" |'''Enzyme2 EC'''<br />
| align="center" |'''Enzyme2 Size (kDa)'''<br />
| align="center" |'''Source'''<br />
| align="center" |'''Pathway'''<br />
| align="center" |'''Major Product'''<br />
| align="center" |'''Applications'''<br />
|-<br />
| 2.1.3.2||32-125||6.3.4.4||47-96||Rosette||Alanine and aspartate metabolism||adenylosuccinate||<br />
|-<br />
| 1.1.1.1||25-57||1.2.1.10||96.7-520||Swiss Prot||Butanoate metabolism ||Butanoyl-coA||<br />
|-<br />
| 1.1.1.95||35-250||2.6.1.52||35-96||Rosette||Glycine, serine and threonine metabolism ||Phosphoserine||<br />
|-<br />
| 2.3.3.9||52-81||4.1.3.1||14-65||Swiss Prot &Rosette||Glyoxylate and dicarboxylate metabolism||Isocitrate||<br />
|-<br />
| 1.5.1.15||32-34||3.5.4.9||30-45||Swiss Prot||One carbon pool by folate||Formyl-THF||<br />
|-<br />
| 1.5.1.5||30-150||3.5.4.9||30-45||Swiss Prot||One carbon pool by folate||Formyl-THF||<br />
|-<br />
| 1.1.1.57||||5.3.1.12||||Rosette||Pentose and glucuronate interconversions||D-Glucuronate||<br />
|-<br />
| 2.2.1.1||100-141||5.1.3.1||45-200||Rosette||Pentose phosphate pathway||D-ribolose-5P||<br />
|-<br />
| 2.2.1.2||35-75||5.3.1.9||27-67||Rosette||Pentose phosphate pathway||a-glucose-6P||<br />
|-<br />
| 1.3.1.13||52-210||4.2.1.51||12-137||Rosette||Phenylalanine, tyrosine and tryptophan biosynthesis||Phenyl-pyruvate||<br />
|-<br />
| 2.3.1.8||35-71||2.7.2.15||43||Rosette||Propanoate metabolism||Propanoate||Food preservatives<br />
|-<br />
| 2.3.1.8||35-70||2.7.2.1||12 to 66||Rosette||Propanoate metabolism/Pyruvate metabolism||Propanoate/acetate||Energy<br />
|-<br />
| 2.4.2.10||39-140||4.1.1.23||14-64||Swiss Prot||Pyrimidine metabolism||UMP||<br />
|-<br />
| 2.4.2.1||45-86||2.4.2.3||32-160||Rosette||Pyrimidine metabolism||Uridine||<br />
|-<br />
| 2.4.1.15||45-630||3.1.3.12||25-973||Rosette||Starch and sucrose metabolism||a,a trehalose||biotechnology application<br />
|-<br />
| 1.2.1.41||41-189||2.7.2.11||47-354||Swiss Prot &Rosette||Urea cycle and metabolism of amino groups||Glutamate||Flavor enhancer, Plant growth<br />
|}<br />
<br />
===b) Alternative microcompartments===<br />
<br />
Upon further examination, most of our known microcompartments tend to consist of a large number of protein subunits with very complex structures. Because of their complicated regulatory patterns and stoichiometry, these structures are not ideal to be used in enzymatic channeling engineering.<br />
<br />
*Carboxysome: 80-150 nm in cross section. Composed of several thousands polypeptide of 10-15 different types. Contain as many as 250 RuBisCO per carboxysome molecule[12, 13].<br />
<br />
*''Pdu'' Microcomparment: 100-150 nm in cross section. Composed of about 18000 individual polypeptides of about 14-18 different types [1].<br />
<br />
*''Eut'' Microcomparment: Similar to pdu microcompartment in terms of both size and protein composition.<br />
<br />
<br />
One of the putative microcompartment systems in ''Clostridium kluyveri'' associated with the oxidation of ethanol, however, shows promise as an alternative microcompartment for use in our system because:<br />
<br />
#The gene cluster involved contains only seven genes: including two genes for a pair of nearly identical acetaldehyde dehydrogenases, three genes for highly similar ethanol dehydrogenases, and two genes for microcompartment proteins, which are orthologs of ethanolamine using genes (eutML) of ''Salmonella typhimurium'' [9].<br />
#From cell extracts of ''C. kluyveri'' a macromolecular complex of ethanol dehydrogenase and acetaldehyde dehydrogenase can be purified by differential manganese sulfate precipitation, indicating the presence of a functional and intact microcompartment enclosing these two enzymes[9].<br />
<br />
In order to co-opt this stucture for our own use it is necessary to understand how the associated enzymes are targeted to the compartment so that alternative enzymes of our choosing can be substituted. To search for a consensus targeting sequence we obtained the ''C. kluyveri'' compartment sequence from NCBI and performed a position specific iterative Blast search for related sequences among bacteria. We used the following two query sequences: <br />
<br />
{| border="1" cellspacing="0" width="50%"<br />
|-<br />
|'''CKL_1072'''<br />
|-<br />
|>gi|153953697|ref|YP_001394462.1| microcompartment protein [Clostridium kluyveri DSM 555]<br />
|-<br />
|MGQEALGMIETKGLVGAIEAADSMVKAANVALIGYEKIGSGLVTVMVRGDVGAVKAATDAGAASAKRVGE<br />
VISVHVIPRPHTDVEKILPNIG<br />
|}<br />
<br />
<br />
{| border="1" cellspacing="0" width="50%"<br />
|-<br />
|'''CKL_1073'''<br />
|-<br />
|>gi|153953698|ref|YP_001394463.1| microcompartment protein [Clostridium kluyveri DSM 555]<br />
|-<br />
|MNNELIEKVLGEVRKSLDLKNFDQEKLNKVVESTTEKLSDSKKEEAIKEAKPDVKVAEESKQAVVEQKAN<br />
DVKTAPTMTEFVGTAGGDTVGLVIANVDSLLHKHLGLDNTCRSIGIISARVGAPAQMMAADEAVKGTNTE<br />
VATIELPRDTKGGAGHGIFIVLKAADVSDARRAVEIALKQTDKYLGNVYLCDAGHLEVQYTARASLIFEK<br />
AFGAPSGQAFGIMHAAPAGVGMIVADTALKTADVKLITYGSPTNGVLSYTNEILITISGDSGAVLQSLTA<br />
ARKAGLSILRSMGQDPVSMSKPTF<br />
|}<br />
<br />
PSI-BLAST constructs and modifies the Position Specific Scoring Matrix constantly and uses it during the next round of search. This iterative searching strategy results in increased sensitivity. The top results were saved after 5 iterations. Download the [[Media:1072_3 BLAST.pdf|BLAST results]] and [[Media:taxonomy_report.txt|taxonomy report]].A [[Media:MUSCLE_alignment.pdf|multiple alignment]] of these sequences was constructed using CLCBio Genomic Workbench to assess the level of conservation between species and identify consensus sequence. <br />
<br />
Since microcompartment proteins are known to be found adjacent to their corresponding enzymes, we searched the top hits for adjacent genes 5’ upstream and 3’ downstream of the hit gene. We then attempted to apply multiple sequence alignment to these sequences in an effort to identify conserved motifs corresponding to a potential targeting sequence similar to the method employed by Suter ''et al''. A targeting sequence, if found, would allow translocation of enzymes into the microcompartment. Due to the incomplete annotation of certain genomes, the alignments did not yield a statistically significant targeting motif. We are attempting further analyses using different reading frames to refine the protein candidates used during the multiple alignment.<br />
<br />
==Conclusions==<br />
We have short-listed a number of enzymes which might benefit from channeling according to several properties such as free energy, toxicity and the occurrence of multi-functional homologues. In addition, while concluding that several classic examples of bacterial microcompartment are unsuitable for experimental manipulation, we have conducted a sequence-based search for additional protein family members corresponding to the ''Clostridium kluyveri'' microcompartment and searched the corresponding upstream and downstream genes for conserved motifs in an effort to find the targetting sequence for these enzymes. The ''Clostridium kluyveri'' system remains under consideration as a potentially viable alternative to the encapsulin-based nanocompartment. However, its suitability will depend on the results of further efforts to identify a targeting sequence for this compartment.<br />
<br />
==Future Work==<br />
In order to find the targeting sequence for the Clostridium kluyveri microcompartment, it will be necessary to fine tune the enzyme queries and potentially check out other alternative reading frames. In addition, enzymes will be selected from those short-listed above to proceed with a proof of concept experiment and potentially commercially viable application.<br />
<br />
==References==<br />
1.Cheng, S., et al., Bacterial microcompartments: their properties and paradoxes. Bioessays, 2008. 30(11-12): p. 1084-95.<br />
<br />
2.Yeates, T.O., et al., Protein-based organelles in bacteria: carboxysomes and related microcompartments. Nat Rev Microbiol, 2008. 6(9): p. 681-91.<br />
<br />
3.Shively, J.M., Inclusion bodies of prokaryotes. Annu Rev Microbiol, 1974. 28(0): p. 167-87.<br />
<br />
4.Chen, P., D.I. Andersson, and J.R. Roth, The control region of the pdu/cob regulon in Salmonella typhimurium. J <br />
Bacteriol, 1994. 176(17): p. 5474-82.<br />
<br />
5.Havemann, G.D., E.M. Sampson, and T.A. Bobik, PduA is a shell protein of polyhedral organelles involved in coenzyme B(12)-dependent degradation of 1,2-propanediol in Salmonella enterica serovar typhimurium LT2. J Bacteriol, 2002. 184(5): p. 1253-61.<br />
<br />
6.Brinsmade, S.R., T. Paldon, and J.C. Escalante-Semerena, Minimal functions and physiological conditions required for growth of salmonella enterica on ethanolamine in the absence of the metabolosome. J Bacteriol, 2005. 187(23): p. 8039-46.<br />
<br />
7.Penrod, J.T. and J.R. Roth, Conserving a volatile metabolite: a role for carboxysome-like organelles in Salmonella enterica. J Bacteriol, 2006. 188(8): p. 2865-74.<br />
<br />
8.Stojiljkovic, I., A.J. Baumler, and F. Heffron, Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster. J Bacteriol, 1995. 177(5): p. 1357-66.<br />
<br />
9.Seedorf, H., et al., The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features. Proc Natl Acad Sci U S A, 2008. 105(6): p. 2128-33.<br />
<br />
10.Wackett, L.P., et al., Genomic and biochemical studies demonstrating the absence of an alkane-producing phenotype in Vibrio furnissii M1. Appl Environ Microbiol, 2007. 73(22): p. 7192-8.<br />
<br />
11.Sampson, E.M. and T.A. Bobik, Microcompartments for B12-dependent 1,2-propanediol degradation provide protection from DNA and cellular damage by a reactive metabolic intermediate. J Bacteriol, 2008. 190(8): p. 2966-71.<br />
<br />
12.Yeates, T.O., et al., Self-assembly in the carboxysome: a viral capsid-like protein shell in bacterial cells. Biochem Soc Trans, 2007. 35(Pt 3): p. 508-11.<br />
<br />
13.Tanaka, S., et al., Atomic-level models of the bacterial carboxysome shell. Science, 2008. 319(5866): p. 1083-6.</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/BioinformaticsTeam:TorontoMaRSDiscovery/Bioinformatics2009-10-22T02:57:34Z<p>Gcromar: /* Conclusions */</p>
<hr />
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<br><br />
<p style="font-size:18pt;">The diversity of bacterial microcompartments in nature and their potential for biotechnological applications</p><br />
<br />
==Summary==<br />
<br />
Spatial segregation is widely believed to be a defining organizational feature of eukaryotic cells: proteins, nucleic acids and small molecules are contained within and often actively transported between the many membrane-bound, subcellular organelles such as mitochondria, lysosomes, Golgi apparatus, etc. However, it has recently been found that a number of bacteria conditionally express proteinaceous microcompartments. These polyhedral organelles are usually 100-150 nm in cross section [1] and consist of proteinaceous outer shells, reminiscent of viral capsids, surrounding a core of enzymes[2]. It is thought that microcompartments allow bacteria to sequester specific metabolic enzymes and their substrates to enhance enzymatic efficiency (enzyme channeling) and protect cells from the toxic effects of certain intermediates. While several examples of these compartments have been reported their diversity has not been fully explored. We wish to design new microcompartments with modified properties or novel enzymatic activities, which could result in potentially useful applications in biotechnology. With this goal in mind, we attempted to address the following two questions using a bioinformatics approach (1) What enzymes will benefit the most from enzymatic channeling? and (2) Are there any other alternative microcompartments that could be explored for enzymatic channeling engineering?<br />
<br />
==Background==<br />
<br />
Most of our knowledge on bacterial microcompartments has been derived from the three well-studied microcompartment systems in nature.<br />
<br />
[[image:TMDT_Nature.jpg|right|thumb|Figure 1: The carbon concentrating mechanism in carboxysome.]]<br />
#Carboxysomes<br />
#*First reported in 1956, carboxysomes were the first bacteria microcompartments to be discovered. They are often present in cyanobacteria and other chemoautotrophic bacteria [3]. They are known to play a key role in enhancing autotrophic carbon fixation in the Calvin cycle. The shell of the carboxysome encodes the enzymes carbonic anhydrase (CA) and ribulose bis-phosphate carboxylase monooxygenase (RuBisCO). CA converts bicarbonate ions into carbon dioxide, which is then converted into 3-phosphoglycerate (3-PGA) by RuBisCO. Carboxysome not only allows for co-localization of CA and RuBisCO, but also acts as a diffusion barrier to retain carbon dioxide in the immediate vicinity of RuBisCO and thus catalyzes the conversion [2]. See Figure 1.<br />
#''Pdu'' microcompartment<br />
#*In 1994, homologues of carboxysome shell proteins were reported in S. enterica. They are amongst a cluster of genes that are involved in coenzyme B12-dependent metabolism of 1,2 propanediol [4]. The gene cluster was later termed the pdu operon and the microcompartment formed was later termed propanediol utilization microcompartment. The proposed fuction of the pdu microcompartment is to encapsulate the enzymes that are necessary for cell to degrade propanediol and most importantly, to protect the cell from the toxic effects of propionaldehyde, an intermediate formed during the process [5].<br />
#''Eut'' microcompartment<br />
#*Later, similar structures were also found in E.coli and S. enterica when they were grown using ethanolamine as energy source. These structures were named ethanolamine utilization microcompartment (encoded by eut operon) and they often display very high genetic similarity with pdu microcompartment. Eut microcompartments contain enzymes involved in the degradation of ethanolamine and protect cell from acetaldehyde [6-8].<br />
#*''Generally speaking, both pdu and eut microcompartments are less uniform in size and more irregular in shape than carboxysomes[2].'' <br />
<br />
These compartments are generally unsuitable for our purposes because a) Their shell proteins consist of several different types whose functions are not well characterized and b) It is not known how the associated enzymes are targeted to the microcompartment. However, several other putative microcompartments have been inferred by sequence similarity. These include <br />
*A microcompartment was suggested to be involved in the oxidation of ethanol by Clostridium kluyveri. [9].<br />
*A microcompartment associated with a puryvate-formate lyase homolog is proposed to be involved in the production of ethanol from pyruvate [10].<br />
*A putative microcompartment in Rhodopirellula baltica is proposed to be associated with a lactate dehydrogenase homologue [1].<br />
*A putative microcompartment in Carboxydothermus hydrogenoformans is associated with an isochorismatase-family protein [1].<br />
*A putative microcompartment Solibacter usitatus can be associated with a dihydrdipicolinate synthase homologue [1].<br />
<br />
The observation that diverse microcompartment structures are composed of proteins with homologous sequences led to the identification of a protein domain in the shell of all polyhedral bacterial microcompartments. This conserved protein domain is known as the Bacterial Microcompartment (BMC) domain ([http://pfam.sanger.ac.uk/family?acc=PF00936 Pfam00936]). It is approximately 84 amino acids long and can be either found as a part of a large protein or in tandem copies within the same operon. This domain is found to be present in 189 bacterial species to date. [2]<br />
<br />
Microcompartments typically promote the catalysis of a particular metabolic reaction by sequestering or co-localizing functionally related enzymes. The product of one enzyme will be delivered to the next enzyme at high concentration since it cannot readily diffuse. This greatly enhances enzyme efficiency. Microcompartments can also occlude toxic intermediates that cannot be degraded by normal bacterial machinery. This was shown to be true in ''pdu'' and ''eut'' microcompartments. The degradation pathway of 1,2-propanediol and ethanolamine both proceed through aldehyde intermediates (propionaldehyde and acetaldehyde respectively). The toxicity of these aldehyde intermediates was later shown in growth assays in which cells accumulating large amounts of propionaldehyde underwent growth arrest due to propionaldehyde toxicity [5, 11]. Therefore, it is reasonable to propose that bacteria use microcompartments to mitigate the toxicity of aldehyde by encapsulating these poisonous intermediates.<br />
<br />
==Methods/Results==<br />
===a) Search for enzyme pairs===<br />
[[image:TMDT_Pathway.jpg|right|thumb|Figure 2: Complete pathway map obtained from KEGG database with the first enzyme colored in red:.]]<br />
In order to identify candidate enzyme pairs likely to display an observable benefit from enzyme channeling we made the following assumptions:<br />
#Enzyme pairs with specific thermodynamic properties will likely benefit. Specifically, for two sequential reactions the second one will be thermodynamically more favorable than the first reaction. Consequently, the products from the first reaction be consumed at a faster rate than being generated, hence driving the reaction to completion. <br />
#*Predictions based on free energy values: [[Media:Scored.xls|Scored.xls]]<br />
#Enzymes involved in reactions with toxic intermediates will likely benefit from enzymatic channeling since the toxin will be less likely to interact with cellular components.<br />
#*Enzyme pairs that possess a common metabolite that is known to be toxic: [[Media:ToxicPairs.xls|ToxicPairs.xls]]<br />
#Enzymes that were identified as gene fusion products in other organisms will likely benefit from enzyme channeling since fusion of multi-functional enzymes is an extreme example of colocalization.<br />
#*Enzyme pairs predicted to form multifunctional enzymes by the Prolinks Rosetta Stone analysis: [[Media:RosettaEnzymePairs.xls|RosettaEnzymePairs.xls]]<br />
#*Enzyme pairs annotated to form multifunctional enzymes in SwissProt database: [[Media:SwissProtPairs.xls|SwissProtPairs.xls]]<br />
These results provided us with a combined list that was further refined by removing enzymes that are not immediately adjacent to each other in their biochemical pathway based on the KEGG database (Figure 2). In addition, enzymes with a molecular weight well beyond the microcompartment capacity (100 kDa) were also eliminated. The remaining enzyme pairs meeting these criteria were considered candidates for enzyme channeling and have been summarized in the following table:<br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0"<br />
| align="center" |'''Enzyme1 EC'''<br />
| align="center" |'''Enzyme1 Size (kDa)'''<br />
| align="center" |'''Enzyme2 EC'''<br />
| align="center" |'''Enzyme2 Size (kDa)'''<br />
| align="center" |'''Source'''<br />
| align="center" |'''Pathway'''<br />
| align="center" |'''Major Product'''<br />
| align="center" |'''Applications'''<br />
|-<br />
| 2.1.3.2||32-125||6.3.4.4||47-96||Rosette||Alanine and aspartate metabolism||adenylosuccinate||<br />
|-<br />
| 1.1.1.1||25-57||1.2.1.10||96.7-520||Swiss Prot||Butanoate metabolism ||Butanoyl-coA||<br />
|-<br />
| 1.1.1.95||35-250||2.6.1.52||35-96||Rosette||Glycine, serine and threonine metabolism ||Phosphoserine||<br />
|-<br />
| 2.3.3.9||52-81||4.1.3.1||14-65||Swiss Prot &Rosette||Glyoxylate and dicarboxylate metabolism||Isocitrate||<br />
|-<br />
| 1.5.1.15||32-34||3.5.4.9||30-45||Swiss Prot||One carbon pool by folate||Formyl-THF||<br />
|-<br />
| 1.5.1.5||30-150||3.5.4.9||30-45||Swiss Prot||One carbon pool by folate||Formyl-THF||<br />
|-<br />
| 1.1.1.57||||5.3.1.12||||Rosette||Pentose and glucuronate interconversions||D-Glucuronate||<br />
|-<br />
| 2.2.1.1||100-141||5.1.3.1||45-200||Rosette||Pentose phosphate pathway||D-ribolose-5P||<br />
|-<br />
| 2.2.1.2||35-75||5.3.1.9||27-67||Rosette||Pentose phosphate pathway||a-glucose-6P||<br />
|-<br />
| 1.3.1.13||52-210||4.2.1.51||12-137||Rosette||Phenylalanine, tyrosine and tryptophan biosynthesis||Phenyl-pyruvate||<br />
|-<br />
| 2.3.1.8||35-71||2.7.2.15||43||Rosette||Propanoate metabolism||Propanoate||Food preservatives<br />
|-<br />
| 2.3.1.8||35-70||2.7.2.1||12 to 66||Rosette||Propanoate metabolism/Pyruvate metabolism||Propanoate/acetate||Energy<br />
|-<br />
| 2.4.2.10||39-140||4.1.1.23||14-64||Swiss Prot||Pyrimidine metabolism||UMP||<br />
|-<br />
| 2.4.2.1||45-86||2.4.2.3||32-160||Rosette||Pyrimidine metabolism||Uridine||<br />
|-<br />
| 2.4.1.15||45-630||3.1.3.12||25-973||Rosette||Starch and sucrose metabolism||a,a trehalose||biotechnology application<br />
|-<br />
| 1.2.1.41||41-189||2.7.2.11||47-354||Swiss Prot &Rosette||Urea cycle and metabolism of amino groups||Glutamate||Flavor enhancer, Plant growth<br />
|}<br />
<br />
===b) Alternative microcompartments===<br />
<br />
Upon further examination, most of our known microcompartments tend to consist of a large number of protein subunits with very complex structures. Because of their complicated regulatory patterns and stoichiometry, these structures are not ideal to be used in enzymatic channeling engineering.<br />
<br />
*Carboxysome: 80-150 nm in cross section. Composed of several thousands polypeptide of 10-15 different types. Contain as many as 250 RuBisCO per carboxysome molecule[12, 13].<br />
<br />
*''Pdu'' Microcomparment: 100-150 nm in cross section. Composed of about 18000 individual polypeptides of about 14-18 different types [1].<br />
<br />
*''Eut'' Microcomparment: Similar to pdu microcompartment in terms of both size and protein composition.<br />
<br />
<br />
One of the putative microcompartment systems in ''Clostridium kluyveri'' associated with the oxidation of ethanol, however, shows promise as an alternative microcompartment for use in our system because:<br />
<br />
#The gene cluster involved contains only seven genes: including two genes for a pair of nearly identical acetaldehyde dehydrogenases, three genes for highly similar ethanol dehydrogenases, and two genes for microcompartment proteins, which are orthologs of ethanolamine using genes (eutML) of ''Salmonella typhimurium'' [9].<br />
#From cell extracts of ''C. kluyveri'' a macromolecular complex of ethanol dehydrogenase and acetaldehyde dehydrogenase can be purified by differential manganese sulfate precipitation, indicating the presence of a functional and intact microcompartment enclosing these two enzymes[9].<br />
<br />
In order to co-opt this stucture for our own use it is necessary to understand how the associated enzymes are targeted to the compartment so that alternative enzymes of our choosing can be substituted. To search for a consensus targeting sequence we obtained the ''C. kluyveri'' compartment sequence from NCBI and performed a position specific iterative Blast search for related sequences among bacteria. We used the following two query sequences: <br />
<br />
{| border="1" cellspacing="0" width="50%"<br />
|-<br />
|'''CKL_1072'''<br />
|-<br />
|>gi|153953697|ref|YP_001394462.1| microcompartment protein [Clostridium kluyveri DSM 555]<br />
|-<br />
|MGQEALGMIETKGLVGAIEAADSMVKAANVALIGYEKIGSGLVTVMVRGDVGAVKAATDAGAASAKRVGE<br />
VISVHVIPRPHTDVEKILPNIG<br />
|}<br />
<br />
<br />
{| border="1" cellspacing="0" width="50%"<br />
|-<br />
|'''CKL_1073'''<br />
|-<br />
|>gi|153953698|ref|YP_001394463.1| microcompartment protein [Clostridium kluyveri DSM 555]<br />
|-<br />
|MNNELIEKVLGEVRKSLDLKNFDQEKLNKVVESTTEKLSDSKKEEAIKEAKPDVKVAEESKQAVVEQKAN<br />
DVKTAPTMTEFVGTAGGDTVGLVIANVDSLLHKHLGLDNTCRSIGIISARVGAPAQMMAADEAVKGTNTE<br />
VATIELPRDTKGGAGHGIFIVLKAADVSDARRAVEIALKQTDKYLGNVYLCDAGHLEVQYTARASLIFEK<br />
AFGAPSGQAFGIMHAAPAGVGMIVADTALKTADVKLITYGSPTNGVLSYTNEILITISGDSGAVLQSLTA<br />
ARKAGLSILRSMGQDPVSMSKPTF<br />
|}<br />
<br />
PSI-BLAST constructs and modifies the Position Specific Scoring Matrix constantly and uses it during the next round of search. This iterative searching strategy results in increased sensitivity. The top results were saved after 5 iterations. Download the [[Media:1072_3 BLAST.pdf|BLAST results]] and [[Media:taxonomy_report.txt|taxonomy report]].A [[Media:MUSCLE_alignment.pdf|multiple alignment]] of these sequences was constructed using CLCBio Genomic Workbench to assess the level of conservation between species and identify consensus sequence. <br />
<br />
Since microcompartment proteins are known to be found adjacent to their corresponding enzymes, we searched the top hits for adjacent genes 5’ upstream and 3’ downstream of the hit gene. We then attempted to apply multiple sequence alignment to these sequences in an effort to identify conserved motifs corresponding to a potential targeting sequence similar to the method employed by Suter et al. A targeting sequence, if found, would allow translocation of enzymes into the microcompartment. Due to the incomplete annotation of certain genomes, the alignments did not yield a statistically significant targeting motif. We are attempting further analyses using different reading frames to refine the protein candidates used during the multiple alignment.<br />
<br />
==Conclusions==<br />
We have short-listed a number of enzymes which might benefit from channeling according to several properties such as free energy, toxicity and the occurrence of multi-functional homologues. In addition, while concluding that several classic examples of bacterial microcompartment are unsuitable for experimental manipulation, we have conducted a sequence-based search for additional protein family members corresponding to the ''Clostridium kluyveri'' microcompartment and searched the corresponding upstream and downstream genes for conserved motifs in an effort to find the targetting sequence for these enzymes. The ''Clostridium kluyveri'' system remains under consideration as a potentially viable alternative to the encapsulin-based nanocompartment. However, its suitability will depend on the results of further efforts to identify a targeting sequence for this compartment.<br />
<br />
==Future Work==<br />
In order to find the targeting sequence for the Clostridium kluyveri microcompartment, it will be necessary to fine tune the enzyme queries and potentially check out other alternative reading frames. In addition, enzymes will be selected from those short-listed above to proceed with a proof of concept experiment and potentially commercially viable application.<br />
<br />
==References==<br />
1.Cheng, S., et al., Bacterial microcompartments: their properties and paradoxes. Bioessays, 2008. 30(11-12): p. 1084-95.<br />
<br />
2.Yeates, T.O., et al., Protein-based organelles in bacteria: carboxysomes and related microcompartments. Nat Rev Microbiol, 2008. 6(9): p. 681-91.<br />
<br />
3.Shively, J.M., Inclusion bodies of prokaryotes. Annu Rev Microbiol, 1974. 28(0): p. 167-87.<br />
<br />
4.Chen, P., D.I. Andersson, and J.R. Roth, The control region of the pdu/cob regulon in Salmonella typhimurium. J <br />
Bacteriol, 1994. 176(17): p. 5474-82.<br />
<br />
5.Havemann, G.D., E.M. Sampson, and T.A. Bobik, PduA is a shell protein of polyhedral organelles involved in coenzyme B(12)-dependent degradation of 1,2-propanediol in Salmonella enterica serovar typhimurium LT2. J Bacteriol, 2002. 184(5): p. 1253-61.<br />
<br />
6.Brinsmade, S.R., T. Paldon, and J.C. Escalante-Semerena, Minimal functions and physiological conditions required for growth of salmonella enterica on ethanolamine in the absence of the metabolosome. J Bacteriol, 2005. 187(23): p. 8039-46.<br />
<br />
7.Penrod, J.T. and J.R. Roth, Conserving a volatile metabolite: a role for carboxysome-like organelles in Salmonella enterica. J Bacteriol, 2006. 188(8): p. 2865-74.<br />
<br />
8.Stojiljkovic, I., A.J. Baumler, and F. Heffron, Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster. J Bacteriol, 1995. 177(5): p. 1357-66.<br />
<br />
9.Seedorf, H., et al., The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features. Proc Natl Acad Sci U S A, 2008. 105(6): p. 2128-33.<br />
<br />
10.Wackett, L.P., et al., Genomic and biochemical studies demonstrating the absence of an alkane-producing phenotype in Vibrio furnissii M1. Appl Environ Microbiol, 2007. 73(22): p. 7192-8.<br />
<br />
11.Sampson, E.M. and T.A. Bobik, Microcompartments for B12-dependent 1,2-propanediol degradation provide protection from DNA and cellular damage by a reactive metabolic intermediate. J Bacteriol, 2008. 190(8): p. 2966-71.<br />
<br />
12.Yeates, T.O., et al., Self-assembly in the carboxysome: a viral capsid-like protein shell in bacterial cells. Biochem Soc Trans, 2007. 35(Pt 3): p. 508-11.<br />
<br />
13.Tanaka, S., et al., Atomic-level models of the bacterial carboxysome shell. Science, 2008. 319(5866): p. 1083-6.</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/BioinformaticsTeam:TorontoMaRSDiscovery/Bioinformatics2009-10-22T02:53:22Z<p>Gcromar: /* b) Alternative microcompartments */</p>
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<p style="font-size:18pt;">The diversity of bacterial microcompartments in nature and their potential for biotechnological applications</p><br />
<br />
==Summary==<br />
<br />
Spatial segregation is widely believed to be a defining organizational feature of eukaryotic cells: proteins, nucleic acids and small molecules are contained within and often actively transported between the many membrane-bound, subcellular organelles such as mitochondria, lysosomes, Golgi apparatus, etc. However, it has recently been found that a number of bacteria conditionally express proteinaceous microcompartments. These polyhedral organelles are usually 100-150 nm in cross section [1] and consist of proteinaceous outer shells, reminiscent of viral capsids, surrounding a core of enzymes[2]. It is thought that microcompartments allow bacteria to sequester specific metabolic enzymes and their substrates to enhance enzymatic efficiency (enzyme channeling) and protect cells from the toxic effects of certain intermediates. While several examples of these compartments have been reported their diversity has not been fully explored. We wish to design new microcompartments with modified properties or novel enzymatic activities, which could result in potentially useful applications in biotechnology. With this goal in mind, we attempted to address the following two questions using a bioinformatics approach (1) What enzymes will benefit the most from enzymatic channeling? and (2) Are there any other alternative microcompartments that could be explored for enzymatic channeling engineering?<br />
<br />
==Background==<br />
<br />
Most of our knowledge on bacterial microcompartments has been derived from the three well-studied microcompartment systems in nature.<br />
<br />
[[image:TMDT_Nature.jpg|right|thumb|Figure 1: The carbon concentrating mechanism in carboxysome.]]<br />
#Carboxysomes<br />
#*First reported in 1956, carboxysomes were the first bacteria microcompartments to be discovered. They are often present in cyanobacteria and other chemoautotrophic bacteria [3]. They are known to play a key role in enhancing autotrophic carbon fixation in the Calvin cycle. The shell of the carboxysome encodes the enzymes carbonic anhydrase (CA) and ribulose bis-phosphate carboxylase monooxygenase (RuBisCO). CA converts bicarbonate ions into carbon dioxide, which is then converted into 3-phosphoglycerate (3-PGA) by RuBisCO. Carboxysome not only allows for co-localization of CA and RuBisCO, but also acts as a diffusion barrier to retain carbon dioxide in the immediate vicinity of RuBisCO and thus catalyzes the conversion [2]. See Figure 1.<br />
#''Pdu'' microcompartment<br />
#*In 1994, homologues of carboxysome shell proteins were reported in S. enterica. They are amongst a cluster of genes that are involved in coenzyme B12-dependent metabolism of 1,2 propanediol [4]. The gene cluster was later termed the pdu operon and the microcompartment formed was later termed propanediol utilization microcompartment. The proposed fuction of the pdu microcompartment is to encapsulate the enzymes that are necessary for cell to degrade propanediol and most importantly, to protect the cell from the toxic effects of propionaldehyde, an intermediate formed during the process [5].<br />
#''Eut'' microcompartment<br />
#*Later, similar structures were also found in E.coli and S. enterica when they were grown using ethanolamine as energy source. These structures were named ethanolamine utilization microcompartment (encoded by eut operon) and they often display very high genetic similarity with pdu microcompartment. Eut microcompartments contain enzymes involved in the degradation of ethanolamine and protect cell from acetaldehyde [6-8].<br />
#*''Generally speaking, both pdu and eut microcompartments are less uniform in size and more irregular in shape than carboxysomes[2].'' <br />
<br />
These compartments are generally unsuitable for our purposes because a) Their shell proteins consist of several different types whose functions are not well characterized and b) It is not known how the associated enzymes are targeted to the microcompartment. However, several other putative microcompartments have been inferred by sequence similarity. These include <br />
*A microcompartment was suggested to be involved in the oxidation of ethanol by Clostridium kluyveri. [9].<br />
*A microcompartment associated with a puryvate-formate lyase homolog is proposed to be involved in the production of ethanol from pyruvate [10].<br />
*A putative microcompartment in Rhodopirellula baltica is proposed to be associated with a lactate dehydrogenase homologue [1].<br />
*A putative microcompartment in Carboxydothermus hydrogenoformans is associated with an isochorismatase-family protein [1].<br />
*A putative microcompartment Solibacter usitatus can be associated with a dihydrdipicolinate synthase homologue [1].<br />
<br />
The observation that diverse microcompartment structures are composed of proteins with homologous sequences led to the identification of a protein domain in the shell of all polyhedral bacterial microcompartments. This conserved protein domain is known as the Bacterial Microcompartment (BMC) domain ([http://pfam.sanger.ac.uk/family?acc=PF00936 Pfam00936]). It is approximately 84 amino acids long and can be either found as a part of a large protein or in tandem copies within the same operon. This domain is found to be present in 189 bacterial species to date. [2]<br />
<br />
Microcompartments typically promote the catalysis of a particular metabolic reaction by sequestering or co-localizing functionally related enzymes. The product of one enzyme will be delivered to the next enzyme at high concentration since it cannot readily diffuse. This greatly enhances enzyme efficiency. Microcompartments can also occlude toxic intermediates that cannot be degraded by normal bacterial machinery. This was shown to be true in ''pdu'' and ''eut'' microcompartments. The degradation pathway of 1,2-propanediol and ethanolamine both proceed through aldehyde intermediates (propionaldehyde and acetaldehyde respectively). The toxicity of these aldehyde intermediates was later shown in growth assays in which cells accumulating large amounts of propionaldehyde underwent growth arrest due to propionaldehyde toxicity [5, 11]. Therefore, it is reasonable to propose that bacteria use microcompartments to mitigate the toxicity of aldehyde by encapsulating these poisonous intermediates.<br />
<br />
==Methods/Results==<br />
===a) Search for enzyme pairs===<br />
[[image:TMDT_Pathway.jpg|right|thumb|Figure 2: Complete pathway map obtained from KEGG database with the first enzyme colored in red:.]]<br />
In order to identify candidate enzyme pairs likely to display an observable benefit from enzyme channeling we made the following assumptions:<br />
#Enzyme pairs with specific thermodynamic properties will likely benefit. Specifically, for two sequential reactions the second one will be thermodynamically more favorable than the first reaction. Consequently, the products from the first reaction be consumed at a faster rate than being generated, hence driving the reaction to completion. <br />
#*Predictions based on free energy values: [[Media:Scored.xls|Scored.xls]]<br />
#Enzymes involved in reactions with toxic intermediates will likely benefit from enzymatic channeling since the toxin will be less likely to interact with cellular components.<br />
#*Enzyme pairs that possess a common metabolite that is known to be toxic: [[Media:ToxicPairs.xls|ToxicPairs.xls]]<br />
#Enzymes that were identified as gene fusion products in other organisms will likely benefit from enzyme channeling since fusion of multi-functional enzymes is an extreme example of colocalization.<br />
#*Enzyme pairs predicted to form multifunctional enzymes by the Prolinks Rosetta Stone analysis: [[Media:RosettaEnzymePairs.xls|RosettaEnzymePairs.xls]]<br />
#*Enzyme pairs annotated to form multifunctional enzymes in SwissProt database: [[Media:SwissProtPairs.xls|SwissProtPairs.xls]]<br />
These results provided us with a combined list that was further refined by removing enzymes that are not immediately adjacent to each other in their biochemical pathway based on the KEGG database (Figure 2). In addition, enzymes with a molecular weight well beyond the microcompartment capacity (100 kDa) were also eliminated. The remaining enzyme pairs meeting these criteria were considered candidates for enzyme channeling and have been summarized in the following table:<br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0"<br />
| align="center" |'''Enzyme1 EC'''<br />
| align="center" |'''Enzyme1 Size (kDa)'''<br />
| align="center" |'''Enzyme2 EC'''<br />
| align="center" |'''Enzyme2 Size (kDa)'''<br />
| align="center" |'''Source'''<br />
| align="center" |'''Pathway'''<br />
| align="center" |'''Major Product'''<br />
| align="center" |'''Applications'''<br />
|-<br />
| 2.1.3.2||32-125||6.3.4.4||47-96||Rosette||Alanine and aspartate metabolism||adenylosuccinate||<br />
|-<br />
| 1.1.1.1||25-57||1.2.1.10||96.7-520||Swiss Prot||Butanoate metabolism ||Butanoyl-coA||<br />
|-<br />
| 1.1.1.95||35-250||2.6.1.52||35-96||Rosette||Glycine, serine and threonine metabolism ||Phosphoserine||<br />
|-<br />
| 2.3.3.9||52-81||4.1.3.1||14-65||Swiss Prot &Rosette||Glyoxylate and dicarboxylate metabolism||Isocitrate||<br />
|-<br />
| 1.5.1.15||32-34||3.5.4.9||30-45||Swiss Prot||One carbon pool by folate||Formyl-THF||<br />
|-<br />
| 1.5.1.5||30-150||3.5.4.9||30-45||Swiss Prot||One carbon pool by folate||Formyl-THF||<br />
|-<br />
| 1.1.1.57||||5.3.1.12||||Rosette||Pentose and glucuronate interconversions||D-Glucuronate||<br />
|-<br />
| 2.2.1.1||100-141||5.1.3.1||45-200||Rosette||Pentose phosphate pathway||D-ribolose-5P||<br />
|-<br />
| 2.2.1.2||35-75||5.3.1.9||27-67||Rosette||Pentose phosphate pathway||a-glucose-6P||<br />
|-<br />
| 1.3.1.13||52-210||4.2.1.51||12-137||Rosette||Phenylalanine, tyrosine and tryptophan biosynthesis||Phenyl-pyruvate||<br />
|-<br />
| 2.3.1.8||35-71||2.7.2.15||43||Rosette||Propanoate metabolism||Propanoate||Food preservatives<br />
|-<br />
| 2.3.1.8||35-70||2.7.2.1||12 to 66||Rosette||Propanoate metabolism/Pyruvate metabolism||Propanoate/acetate||Energy<br />
|-<br />
| 2.4.2.10||39-140||4.1.1.23||14-64||Swiss Prot||Pyrimidine metabolism||UMP||<br />
|-<br />
| 2.4.2.1||45-86||2.4.2.3||32-160||Rosette||Pyrimidine metabolism||Uridine||<br />
|-<br />
| 2.4.1.15||45-630||3.1.3.12||25-973||Rosette||Starch and sucrose metabolism||a,a trehalose||biotechnology application<br />
|-<br />
| 1.2.1.41||41-189||2.7.2.11||47-354||Swiss Prot &Rosette||Urea cycle and metabolism of amino groups||Glutamate||Flavor enhancer, Plant growth<br />
|}<br />
<br />
===b) Alternative microcompartments===<br />
<br />
Upon further examination, most of our known microcompartments tend to consist of a large number of protein subunits with very complex structures. Because of their complicated regulatory patterns and stoichiometry, these structures are not ideal to be used in enzymatic channeling engineering.<br />
<br />
*Carboxysome: 80-150 nm in cross section. Composed of several thousands polypeptide of 10-15 different types. Contain as many as 250 RuBisCO per carboxysome molecule[12, 13].<br />
<br />
*''Pdu'' Microcomparment: 100-150 nm in cross section. Composed of about 18000 individual polypeptides of about 14-18 different types [1].<br />
<br />
*''Eut'' Microcomparment: Similar to pdu microcompartment in terms of both size and protein composition.<br />
<br />
<br />
One of the putative microcompartment systems in ''Clostridium kluyveri'' associated with the oxidation of ethanol, however, shows promise as an alternative microcompartment for use in our system because:<br />
<br />
#The gene cluster involved contains only seven genes: including two genes for a pair of nearly identical acetaldehyde dehydrogenases, three genes for highly similar ethanol dehydrogenases, and two genes for microcompartment proteins, which are orthologs of ethanolamine using genes (eutML) of ''Salmonella typhimurium'' [9].<br />
#From cell extracts of ''C. kluyveri'' a macromolecular complex of ethanol dehydrogenase and acetaldehyde dehydrogenase can be purified by differential manganese sulfate precipitation, indicating the presence of a functional and intact microcompartment enclosing these two enzymes[9].<br />
<br />
In order to co-opt this stucture for our own use it is necessary to understand how the associated enzymes are targeted to the compartment so that alternative enzymes of our choosing can be substituted. To search for a consensus targeting sequence we obtained the ''C. kluyveri'' compartment sequence from NCBI and performed a position specific iterative Blast search for related sequences among bacteria. We used the following two query sequences: <br />
<br />
{| border="1" cellspacing="0" width="50%"<br />
|-<br />
|'''CKL_1072'''<br />
|-<br />
|>gi|153953697|ref|YP_001394462.1| microcompartment protein [Clostridium kluyveri DSM 555]<br />
|-<br />
|MGQEALGMIETKGLVGAIEAADSMVKAANVALIGYEKIGSGLVTVMVRGDVGAVKAATDAGAASAKRVGE<br />
VISVHVIPRPHTDVEKILPNIG<br />
|}<br />
<br />
<br />
{| border="1" cellspacing="0" width="50%"<br />
|-<br />
|'''CKL_1073'''<br />
|-<br />
|>gi|153953698|ref|YP_001394463.1| microcompartment protein [Clostridium kluyveri DSM 555]<br />
|-<br />
|MNNELIEKVLGEVRKSLDLKNFDQEKLNKVVESTTEKLSDSKKEEAIKEAKPDVKVAEESKQAVVEQKAN<br />
DVKTAPTMTEFVGTAGGDTVGLVIANVDSLLHKHLGLDNTCRSIGIISARVGAPAQMMAADEAVKGTNTE<br />
VATIELPRDTKGGAGHGIFIVLKAADVSDARRAVEIALKQTDKYLGNVYLCDAGHLEVQYTARASLIFEK<br />
AFGAPSGQAFGIMHAAPAGVGMIVADTALKTADVKLITYGSPTNGVLSYTNEILITISGDSGAVLQSLTA<br />
ARKAGLSILRSMGQDPVSMSKPTF<br />
|}<br />
<br />
PSI-BLAST constructs and modifies the Position Specific Scoring Matrix constantly and uses it during the next round of search. This iterative searching strategy results in increased sensitivity. The top results were saved after 5 iterations. Download the [[Media:1072_3 BLAST.pdf|BLAST results]] and [[Media:taxonomy_report.txt|taxonomy report]].A [[Media:MUSCLE_alignment.pdf|multiple alignment]] of these sequences was constructed using CLCBio Genomic Workbench to assess the level of conservation between species and identify consensus sequence. <br />
<br />
Since microcompartment proteins are known to be found adjacent to their corresponding enzymes, we searched the top hits for adjacent genes 5’ upstream and 3’ downstream of the hit gene. We then attempted to apply multiple sequence alignment to these sequences in an effort to identify conserved motifs corresponding to a potential targeting sequence similar to the method employed by Suter et al. A targeting sequence, if found, would allow translocation of enzymes into the microcompartment. Due to the incomplete annotation of certain genomes, the alignments did not yield a statistically significant targeting motif. We are attempting further analyses using different reading frames to refine the protein candidates used during the multiple alignment.<br />
<br />
==Conclusions==<br />
We have short-listed a number of enzymes which might benefit from channeling according to several properties such as free energy, toxicity and the occurrence of multi-functional homologues. In addition, while concluding that several classic examples of bacterial microcompartment are unsuitable for experimental manipulation, we have conducted a sequence-based search for additional protein family members corresponding to the Clostridium kluyveri microcompartment and searched the corresponding upstream and downstream genes for conserved motifs in an effort to find the targetting sequence for these enzymes. The Clostridium kluyveri system remains under consideration as a potentially viable alternative to the encapsulin-based nanocompartment. However, its suitability will depend on the results of further efforts to identify a targeting sequence for this compartment.<br />
<br />
==Future Work==<br />
In order to find the targeting sequence for the Clostridium kluyveri microcompartment, it will be necessary to fine tune the enzyme queries and potentially check out other alternative reading frames. In addition, enzymes will be selected from those short-listed above to proceed with a proof of concept experiment and potentially commercially viable application.<br />
<br />
==References==<br />
1.Cheng, S., et al., Bacterial microcompartments: their properties and paradoxes. Bioessays, 2008. 30(11-12): p. 1084-95.<br />
<br />
2.Yeates, T.O., et al., Protein-based organelles in bacteria: carboxysomes and related microcompartments. Nat Rev Microbiol, 2008. 6(9): p. 681-91.<br />
<br />
3.Shively, J.M., Inclusion bodies of prokaryotes. Annu Rev Microbiol, 1974. 28(0): p. 167-87.<br />
<br />
4.Chen, P., D.I. Andersson, and J.R. Roth, The control region of the pdu/cob regulon in Salmonella typhimurium. J <br />
Bacteriol, 1994. 176(17): p. 5474-82.<br />
<br />
5.Havemann, G.D., E.M. Sampson, and T.A. Bobik, PduA is a shell protein of polyhedral organelles involved in coenzyme B(12)-dependent degradation of 1,2-propanediol in Salmonella enterica serovar typhimurium LT2. J Bacteriol, 2002. 184(5): p. 1253-61.<br />
<br />
6.Brinsmade, S.R., T. Paldon, and J.C. Escalante-Semerena, Minimal functions and physiological conditions required for growth of salmonella enterica on ethanolamine in the absence of the metabolosome. J Bacteriol, 2005. 187(23): p. 8039-46.<br />
<br />
7.Penrod, J.T. and J.R. Roth, Conserving a volatile metabolite: a role for carboxysome-like organelles in Salmonella enterica. J Bacteriol, 2006. 188(8): p. 2865-74.<br />
<br />
8.Stojiljkovic, I., A.J. Baumler, and F. Heffron, Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster. J Bacteriol, 1995. 177(5): p. 1357-66.<br />
<br />
9.Seedorf, H., et al., The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features. Proc Natl Acad Sci U S A, 2008. 105(6): p. 2128-33.<br />
<br />
10.Wackett, L.P., et al., Genomic and biochemical studies demonstrating the absence of an alkane-producing phenotype in Vibrio furnissii M1. Appl Environ Microbiol, 2007. 73(22): p. 7192-8.<br />
<br />
11.Sampson, E.M. and T.A. Bobik, Microcompartments for B12-dependent 1,2-propanediol degradation provide protection from DNA and cellular damage by a reactive metabolic intermediate. J Bacteriol, 2008. 190(8): p. 2966-71.<br />
<br />
12.Yeates, T.O., et al., Self-assembly in the carboxysome: a viral capsid-like protein shell in bacterial cells. Biochem Soc Trans, 2007. 35(Pt 3): p. 508-11.<br />
<br />
13.Tanaka, S., et al., Atomic-level models of the bacterial carboxysome shell. Science, 2008. 319(5866): p. 1083-6.</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/BioinformaticsTeam:TorontoMaRSDiscovery/Bioinformatics2009-10-22T02:47:14Z<p>Gcromar: /* Conclusions */</p>
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<br><br />
<p style="font-size:18pt;">The diversity of bacterial microcompartments in nature and their potential for biotechnological applications</p><br />
<br />
==Summary==<br />
<br />
Spatial segregation is widely believed to be a defining organizational feature of eukaryotic cells: proteins, nucleic acids and small molecules are contained within and often actively transported between the many membrane-bound, subcellular organelles such as mitochondria, lysosomes, Golgi apparatus, etc. However, it has recently been found that a number of bacteria conditionally express proteinaceous microcompartments. These polyhedral organelles are usually 100-150 nm in cross section [1] and consist of proteinaceous outer shells, reminiscent of viral capsids, surrounding a core of enzymes[2]. It is thought that microcompartments allow bacteria to sequester specific metabolic enzymes and their substrates to enhance enzymatic efficiency (enzyme channeling) and protect cells from the toxic effects of certain intermediates. While several examples of these compartments have been reported their diversity has not been fully explored. We wish to design new microcompartments with modified properties or novel enzymatic activities, which could result in potentially useful applications in biotechnology. With this goal in mind, we attempted to address the following two questions using a bioinformatics approach (1) What enzymes will benefit the most from enzymatic channeling? and (2) Are there any other alternative microcompartments that could be explored for enzymatic channeling engineering?<br />
<br />
==Background==<br />
<br />
Most of our knowledge on bacterial microcompartments has been derived from the three well-studied microcompartment systems in nature.<br />
<br />
[[image:TMDT_Nature.jpg|right|thumb|Figure 1: The carbon concentrating mechanism in carboxysome.]]<br />
#Carboxysomes<br />
#*First reported in 1956, carboxysomes were the first bacteria microcompartments to be discovered. They are often present in cyanobacteria and other chemoautotrophic bacteria [3]. They are known to play a key role in enhancing autotrophic carbon fixation in the Calvin cycle. The shell of the carboxysome encodes the enzymes carbonic anhydrase (CA) and ribulose bis-phosphate carboxylase monooxygenase (RuBisCO). CA converts bicarbonate ions into carbon dioxide, which is then converted into 3-phosphoglycerate (3-PGA) by RuBisCO. Carboxysome not only allows for co-localization of CA and RuBisCO, but also acts as a diffusion barrier to retain carbon dioxide in the immediate vicinity of RuBisCO and thus catalyzes the conversion [2]. See Figure 1.<br />
#''Pdu'' microcompartment<br />
#*In 1994, homologues of carboxysome shell proteins were reported in S. enterica. They are amongst a cluster of genes that are involved in coenzyme B12-dependent metabolism of 1,2 propanediol [4]. The gene cluster was later termed the pdu operon and the microcompartment formed was later termed propanediol utilization microcompartment. The proposed fuction of the pdu microcompartment is to encapsulate the enzymes that are necessary for cell to degrade propanediol and most importantly, to protect the cell from the toxic effects of propionaldehyde, an intermediate formed during the process [5].<br />
#''Eut'' microcompartment<br />
#*Later, similar structures were also found in E.coli and S. enterica when they were grown using ethanolamine as energy source. These structures were named ethanolamine utilization microcompartment (encoded by eut operon) and they often display very high genetic similarity with pdu microcompartment. Eut microcompartments contain enzymes involved in the degradation of ethanolamine and protect cell from acetaldehyde [6-8].<br />
#*''Generally speaking, both pdu and eut microcompartments are less uniform in size and more irregular in shape than carboxysomes[2].'' <br />
<br />
These compartments are generally unsuitable for our purposes because a) Their shell proteins consist of several different types whose functions are not well characterized and b) It is not known how the associated enzymes are targeted to the microcompartment. However, several other putative microcompartments have been inferred by sequence similarity. These include <br />
*A microcompartment was suggested to be involved in the oxidation of ethanol by Clostridium kluyveri. [9].<br />
*A microcompartment associated with a puryvate-formate lyase homolog is proposed to be involved in the production of ethanol from pyruvate [10].<br />
*A putative microcompartment in Rhodopirellula baltica is proposed to be associated with a lactate dehydrogenase homologue [1].<br />
*A putative microcompartment in Carboxydothermus hydrogenoformans is associated with an isochorismatase-family protein [1].<br />
*A putative microcompartment Solibacter usitatus can be associated with a dihydrdipicolinate synthase homologue [1].<br />
<br />
The observation that diverse microcompartment structures are composed of proteins with homologous sequences led to the identification of a protein domain in the shell of all polyhedral bacterial microcompartments. This conserved protein domain is known as the Bacterial Microcompartment (BMC) domain ([http://pfam.sanger.ac.uk/family?acc=PF00936 Pfam00936]). It is approximately 84 amino acids long and can be either found as a part of a large protein or in tandem copies within the same operon. This domain is found to be present in 189 bacterial species to date. [2]<br />
<br />
Microcompartments typically promote the catalysis of a particular metabolic reaction by sequestering or co-localizing functionally related enzymes. The product of one enzyme will be delivered to the next enzyme at high concentration since it cannot readily diffuse. This greatly enhances enzyme efficiency. Microcompartments can also occlude toxic intermediates that cannot be degraded by normal bacterial machinery. This was shown to be true in ''pdu'' and ''eut'' microcompartments. The degradation pathway of 1,2-propanediol and ethanolamine both proceed through aldehyde intermediates (propionaldehyde and acetaldehyde respectively). The toxicity of these aldehyde intermediates was later shown in growth assays in which cells accumulating large amounts of propionaldehyde underwent growth arrest due to propionaldehyde toxicity [5, 11]. Therefore, it is reasonable to propose that bacteria use microcompartments to mitigate the toxicity of aldehyde by encapsulating these poisonous intermediates.<br />
<br />
==Methods/Results==<br />
===a) Search for enzyme pairs===<br />
[[image:TMDT_Pathway.jpg|right|thumb|Figure 2: Complete pathway map obtained from KEGG database with the first enzyme colored in red:.]]<br />
In order to identify candidate enzyme pairs likely to display an observable benefit from enzyme channeling we made the following assumptions:<br />
#Enzyme pairs with specific thermodynamic properties will likely benefit. Specifically, for two sequential reactions the second one will be thermodynamically more favorable than the first reaction. Consequently, the products from the first reaction be consumed at a faster rate than being generated, hence driving the reaction to completion. <br />
#*Predictions based on free energy values: [[Media:Scored.xls|Scored.xls]]<br />
#Enzymes involved in reactions with toxic intermediates will likely benefit from enzymatic channeling since the toxin will be less likely to interact with cellular components.<br />
#*Enzyme pairs that possess a common metabolite that is known to be toxic: [[Media:ToxicPairs.xls|ToxicPairs.xls]]<br />
#Enzymes that were identified as gene fusion products in other organisms will likely benefit from enzyme channeling since fusion of multi-functional enzymes is an extreme example of colocalization.<br />
#*Enzyme pairs predicted to form multifunctional enzymes by the Prolinks Rosetta Stone analysis: [[Media:RosettaEnzymePairs.xls|RosettaEnzymePairs.xls]]<br />
#*Enzyme pairs annotated to form multifunctional enzymes in SwissProt database: [[Media:SwissProtPairs.xls|SwissProtPairs.xls]]<br />
These results provided us with a combined list that was further refined by removing enzymes that are not immediately adjacent to each other in their biochemical pathway based on the KEGG database (Figure 2). In addition, enzymes with a molecular weight well beyond the microcompartment capacity (100 kDa) were also eliminated. The remaining enzyme pairs meeting these criteria were considered candidates for enzyme channeling and have been summarized in the following table:<br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0"<br />
| align="center" |'''Enzyme1 EC'''<br />
| align="center" |'''Enzyme1 Size (kDa)'''<br />
| align="center" |'''Enzyme2 EC'''<br />
| align="center" |'''Enzyme2 Size (kDa)'''<br />
| align="center" |'''Source'''<br />
| align="center" |'''Pathway'''<br />
| align="center" |'''Major Product'''<br />
| align="center" |'''Applications'''<br />
|-<br />
| 2.1.3.2||32-125||6.3.4.4||47-96||Rosette||Alanine and aspartate metabolism||adenylosuccinate||<br />
|-<br />
| 1.1.1.1||25-57||1.2.1.10||96.7-520||Swiss Prot||Butanoate metabolism ||Butanoyl-coA||<br />
|-<br />
| 1.1.1.95||35-250||2.6.1.52||35-96||Rosette||Glycine, serine and threonine metabolism ||Phosphoserine||<br />
|-<br />
| 2.3.3.9||52-81||4.1.3.1||14-65||Swiss Prot &Rosette||Glyoxylate and dicarboxylate metabolism||Isocitrate||<br />
|-<br />
| 1.5.1.15||32-34||3.5.4.9||30-45||Swiss Prot||One carbon pool by folate||Formyl-THF||<br />
|-<br />
| 1.5.1.5||30-150||3.5.4.9||30-45||Swiss Prot||One carbon pool by folate||Formyl-THF||<br />
|-<br />
| 1.1.1.57||||5.3.1.12||||Rosette||Pentose and glucuronate interconversions||D-Glucuronate||<br />
|-<br />
| 2.2.1.1||100-141||5.1.3.1||45-200||Rosette||Pentose phosphate pathway||D-ribolose-5P||<br />
|-<br />
| 2.2.1.2||35-75||5.3.1.9||27-67||Rosette||Pentose phosphate pathway||a-glucose-6P||<br />
|-<br />
| 1.3.1.13||52-210||4.2.1.51||12-137||Rosette||Phenylalanine, tyrosine and tryptophan biosynthesis||Phenyl-pyruvate||<br />
|-<br />
| 2.3.1.8||35-71||2.7.2.15||43||Rosette||Propanoate metabolism||Propanoate||Food preservatives<br />
|-<br />
| 2.3.1.8||35-70||2.7.2.1||12 to 66||Rosette||Propanoate metabolism/Pyruvate metabolism||Propanoate/acetate||Energy<br />
|-<br />
| 2.4.2.10||39-140||4.1.1.23||14-64||Swiss Prot||Pyrimidine metabolism||UMP||<br />
|-<br />
| 2.4.2.1||45-86||2.4.2.3||32-160||Rosette||Pyrimidine metabolism||Uridine||<br />
|-<br />
| 2.4.1.15||45-630||3.1.3.12||25-973||Rosette||Starch and sucrose metabolism||a,a trehalose||biotechnology application<br />
|-<br />
| 1.2.1.41||41-189||2.7.2.11||47-354||Swiss Prot &Rosette||Urea cycle and metabolism of amino groups||Glutamate||Flavor enhancer, Plant growth<br />
|}<br />
<br />
===b) Alternative microcompartments===<br />
<br />
Upon further examination, most of our known microcompartments tend to consist of a large number of protein subunits with very complex structures. Because of their complicated regulatory patterns and stoichiometry, these structures are not ideal to be used in enzymatic channeling engineering.<br />
<br />
*Carboxysome: 80-150 nm in cross section. Composed of several thousands polypeptide of 10-15 different types. Contain as many as 250 RuBisCO per carboxysome molecule[12, 13].<br />
<br />
*''Pdu'' Microcomparment: 100-150 nm in cross section. Composed of about 18000 individual polypeptides of about 14-18 different types [1].<br />
<br />
*''Eut'' Microcomparment: Similar to pdu microcompartment in terms of both size and protein composition.<br />
<br />
<br />
One of the putative microcompartment systems in ''Clostridium kluyveri'' associated with the oxidation of ethanol, however, shows promise as an alternative microcompartment for use in our system because:<br />
<br />
#The gene cluster involved contains only seven genes: including two genes for a pair of nearly identical acetaldehyde dehydrogenases, three genes for highly similar ethanol dehydrogenases, and two genes for microcompartment proteins, which are orthologs of ethanolamine using genes (eutML) of ''Salmonella typhimurium'' [9].<br />
#From cell extracts of ''C. kluyveri'' a macromolecular complex of ethanol dehydrogenase and acetaldehyde dehydrogenase can be purified by differential manganese sulfate precipitation, indicating the presence of a functional and intact microcompartment enclosing these two enzymes[9].<br />
<br />
In order to co-opt this stucture for our own use it is necessary to understand how the associated enzymes are targeted to the compartment so that alternative enzymes of our choosing can be substituted. To search for a consensus targeting sequence we obtained the ''C. kluyveri'' compartment sequence from NCBI and performed a position specific iterative Blast search for related sequences among bacteria. We used the following two query sequences: <br />
<br />
{| border="1" cellspacing="0" width="50%"<br />
|-<br />
|'''CKL_1072'''<br />
|-<br />
|>gi|153953697|ref|YP_001394462.1| microcompartment protein [Clostridium kluyveri DSM 555]<br />
|-<br />
|MGQEALGMIETKGLVGAIEAADSMVKAANVALIGYEKIGSGLVTVMVRGDVGAVKAATDAGAASAKRVGE<br />
VISVHVIPRPHTDVEKILPNIG<br />
|}<br />
<br />
<br />
{| border="1" cellspacing="0" width="50%"<br />
|-<br />
|'''CKL_1073'''<br />
|-<br />
|>gi|153953698|ref|YP_001394463.1| microcompartment protein [Clostridium kluyveri DSM 555]<br />
|-<br />
|MNNELIEKVLGEVRKSLDLKNFDQEKLNKVVESTTEKLSDSKKEEAIKEAKPDVKVAEESKQAVVEQKAN<br />
DVKTAPTMTEFVGTAGGDTVGLVIANVDSLLHKHLGLDNTCRSIGIISARVGAPAQMMAADEAVKGTNTE<br />
VATIELPRDTKGGAGHGIFIVLKAADVSDARRAVEIALKQTDKYLGNVYLCDAGHLEVQYTARASLIFEK<br />
AFGAPSGQAFGIMHAAPAGVGMIVADTALKTADVKLITYGSPTNGVLSYTNEILITISGDSGAVLQSLTA<br />
ARKAGLSILRSMGQDPVSMSKPTF<br />
|}<br />
<br />
PSI-BLAST constructs and modifies the Position Specific Scoring Matrix constantly and uses it during the next round of search. This iterative searching strategy results in increased sensitivity. The top results were saved after 5 iterations. Download the [[Media:1072_3 BLAST.pdf|BLAST results]] and [[Media:taxonomy_report.txt|taxonomy report]].A [[Media:MUSCLE_alignment.pdf|multiple alignment]] of these sequences was constructed using CLCBio Genomic Workbench to assess the level of conservation between species and identify consensus sequence. <br />
<br />
Since microcompartment proteins are known to be found adjacent to their corresponding enzymes, we searched the top hits for adjacent genes 5’ upstream and 3’ downstream of the hit gene. We then attempted to apply multiple alignment to these adjacent protein sequences in an effort to identify potential conserved motifs that might indicate a potential targeting sequence to translocate enzymes into the microcompartment. However, due to the incomplete annotation of certain genomes, the alignment did not yield any statistically important targeting sequences. Future analysis will attempt different reading frames and refine the protein candidates used during the multiple alignment.<br />
<br />
==Conclusions==<br />
We have short-listed a number of enzymes which might benefit from channeling according to several properties such as free energy, toxicity and the occurrence of multi-functional homologues. In addition, while concluding that several classic examples of bacterial microcompartment are unsuitable for experimental manipulation, we have conducted a sequence-based search for additional protein family members corresponding to the Clostridium kluyveri microcompartment and searched the corresponding upstream and downstream genes for conserved motifs in an effort to find the targetting sequence for these enzymes. The Clostridium kluyveri system remains under consideration as a potentially viable alternative to the encapsulin-based nanocompartment. However, its suitability will depend on the results of further efforts to identify a targeting sequence for this compartment.<br />
<br />
==Future Work==<br />
In order to find the targeting sequence for the Clostridium kluyveri microcompartment, it will be necessary to fine tune the enzyme queries and potentially check out other alternative reading frames. In addition, enzymes will be selected from those short-listed above to proceed with a proof of concept experiment and potentially commercially viable application.<br />
<br />
==References==<br />
1.Cheng, S., et al., Bacterial microcompartments: their properties and paradoxes. Bioessays, 2008. 30(11-12): p. 1084-95.<br />
<br />
2.Yeates, T.O., et al., Protein-based organelles in bacteria: carboxysomes and related microcompartments. Nat Rev Microbiol, 2008. 6(9): p. 681-91.<br />
<br />
3.Shively, J.M., Inclusion bodies of prokaryotes. Annu Rev Microbiol, 1974. 28(0): p. 167-87.<br />
<br />
4.Chen, P., D.I. Andersson, and J.R. Roth, The control region of the pdu/cob regulon in Salmonella typhimurium. J <br />
Bacteriol, 1994. 176(17): p. 5474-82.<br />
<br />
5.Havemann, G.D., E.M. Sampson, and T.A. Bobik, PduA is a shell protein of polyhedral organelles involved in coenzyme B(12)-dependent degradation of 1,2-propanediol in Salmonella enterica serovar typhimurium LT2. J Bacteriol, 2002. 184(5): p. 1253-61.<br />
<br />
6.Brinsmade, S.R., T. Paldon, and J.C. Escalante-Semerena, Minimal functions and physiological conditions required for growth of salmonella enterica on ethanolamine in the absence of the metabolosome. J Bacteriol, 2005. 187(23): p. 8039-46.<br />
<br />
7.Penrod, J.T. and J.R. Roth, Conserving a volatile metabolite: a role for carboxysome-like organelles in Salmonella enterica. J Bacteriol, 2006. 188(8): p. 2865-74.<br />
<br />
8.Stojiljkovic, I., A.J. Baumler, and F. Heffron, Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster. J Bacteriol, 1995. 177(5): p. 1357-66.<br />
<br />
9.Seedorf, H., et al., The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features. Proc Natl Acad Sci U S A, 2008. 105(6): p. 2128-33.<br />
<br />
10.Wackett, L.P., et al., Genomic and biochemical studies demonstrating the absence of an alkane-producing phenotype in Vibrio furnissii M1. Appl Environ Microbiol, 2007. 73(22): p. 7192-8.<br />
<br />
11.Sampson, E.M. and T.A. Bobik, Microcompartments for B12-dependent 1,2-propanediol degradation provide protection from DNA and cellular damage by a reactive metabolic intermediate. J Bacteriol, 2008. 190(8): p. 2966-71.<br />
<br />
12.Yeates, T.O., et al., Self-assembly in the carboxysome: a viral capsid-like protein shell in bacterial cells. Biochem Soc Trans, 2007. 35(Pt 3): p. 508-11.<br />
<br />
13.Tanaka, S., et al., Atomic-level models of the bacterial carboxysome shell. Science, 2008. 319(5866): p. 1083-6.</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-22T02:20:56Z<p>Gcromar: </p>
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
==Design==<br />
<br />
'''Considerations<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A pair of enzymes that are expected to benefit from the effects of channeling.<br />
<br />
Our search for a suitable platform to effect channeling led us to bacterial microcompartments. A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our search for enzymes is described on our [[Team:TorontoMaRSDiscovery/Bioinformatics|bioinformatics]] page. <br />
<br />
We are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford [7]. We want to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be [[Team:TorontoMaRSDiscovery/Modeling|modeled]] using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products. Here, we present our proof-of-concept system based on an encapsulin microcompartment and fluorescent (eCFP) 'probe'. <br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in ''E. coli''.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
'''Components<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Using two new parts (see below) and several existing BioBricks [5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). We include a 'control module' expressing two repressors under control of a constitutive promoter as well as the structural, microcompartment protein, encapsulin (<partinfo>BBa_K192000</partinfo>) and a fluorescent 'probe' (<partinfo>BBa_K192001</partinfo>) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6]. By varying the amounts of inducers aTc and IPTG in the system it is possible to vary the expression of encapsulin and the targeted fluorescent probe (eCFP). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach to visualize the microcompartments. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using the recombinant, fluorescent marker protein (eCFP) by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized (see [[Team:TorontoMaRSDiscovery/Modeling|model]]). By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.<br />
<br />
==Progress==<br />
We have made substantial progress in the construction of all components as detailed in our [[Team:TorontoMaRSDiscovery/Bioinformatics|notebook]]. Specifically, the encapsulin biobrick was completed, sequenced and contributed to the parts registry. Also, as the construction of the fluorescent probe was not trivial, we took advantage of the offer from Mr. Gene to have this part synthesized. We have not yet completed standard assembly of the control module and are awaiting confirmation of this assembly before transforming the synthesized probe, at which point this part will also be contributed. There has been some difficulty obtaining the encapsulin assembly and we are beginning to suspect that our attempts to obtain the construct (initially with a constitutive promoter) may be killing the cells due to the effects of over-expression of the protein.<br />
<br />
Our study of the natural biology of microcompartments across bacteria suggests that their occurrence is confined to a protective function, most commonly isolating toxic or reactive intermediates in certain redox reactions. While other microcompartments exist, the lack of a discernible targeting sequence makes them unsuitable for use in our system and encapsulin remains our best option at present. We have short-listed a series of enzyme pairs that we feel are amenable to manipulation by enzyme channeling based on thermodynamic and other properties for use in our future work.<br />
<br />
==Future Work==<br />
We continue to work on completing the assembly of our proof-of-concept system at which point we will proceed with the characterization of the system as planned. Modeling of candidate enzyme pairs for a downstream application will proceed using the results of our enzyme search. We anticipate that this will lead to several candidate applications. For these applications, we will construct recombinant enzymes fused to the targeting sequence. These enzymes will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to first test an alternative, readily assayable system regardless of commercial relevance, to meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7]. As a further experiment, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in dynamic pathway switching.<br />
<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-22T02:18:18Z<p>Gcromar: /* Future Work */</p>
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
==Design==<br />
<br />
'''Considerations<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A pair of enzymes that are expected to benefit from the effects of channeling.<br />
<br />
Our search for a suitable platform to effect channeling led us to bacterial microcompartments. A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our search for enzymes is described on our [[Team:TorontoMaRSDiscovery/Bioinformatics|bioinformatics]] page. <br />
<br />
We are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford [7]. We want to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be [[Team:TorontoMaRSDiscovery/Modeling|modeled]] using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products. Here, we present our proof-of-concept system based on an encapsulin microcompartment and fluorescent (eCFP) 'probe'. <br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in ''E. coli''.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
'''Components<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Using two new parts (see below) and several existing BioBricks [5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). We include a 'control module' expressing two repressors under control of a constitutive promoter as well as the structural, microcompartment protein, encapsulin (<partinfo>BBa_K192000</partinfo>) and a fluorescent 'probe' (<partinfo>BBa_K192001</partinfo>) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6]. By varying the amounts of inducers aTc and IPTG in the system it is possible to vary the expression of encapsulin and the targeted fluorescent probe (eCFP). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach to visualize the microcompartments. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using the recombinant, fluorescent marker protein (eCFP) by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized (see [[Team:TorontoMaRSDiscovery/Modeling|model]]). By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.<br />
<br />
==Progress==<br />
We have made substantial progress in the construction of all components as detailed in our [[Team:TorontoMaRSDiscovery/Bioinformatics|notebook]]. Specifically, the encapsulin biobrick was completed, sequenced and contributed to the parts registry. Also, as the construction of the fluorescent probe was not trivial, we took advantage of the offer from Mr. Gene to have this part synthesized. We have not yet completed standard assembly of the control module and are awaiting confirmation of this assembly before transforming the synthesized probe, at which point this part will also be contributed. There has been some difficulty obtaining the encapsulin assembly and we are beginning to suspect that our attempts to obtain the construct (initially with a constitutive promoter) may be killing the cells due to the effects of over-expression of the protein.<br />
<br />
Our study of the natural biology of microcompartments across bacteria suggests that their occurrence is confined to a protective function, most commonly isolating toxic or reactive intermediates in certain redox reactions. While other microcompartments exist, the lack of a discernible targeting sequence makes them unsuitable for use in our system and encapsulin remains our best option at present. We have short-listed a series of enzyme pairs that we feel are amenable to manipulation by enzyme channeling based on thermodynamic and other properties for use in our future work.<br />
<br />
==Future Work==<br />
We continue to work on completing the assembly of our proof-of-concept system at which point we will proceed with the characterization of the system as planned. Modeling of candidate enzyme pairs for a downstream application will proceed using the results of our enzyme search. We anticipate that this will lead to several candidate applications. For these applications, we will construct recombinant enzymes fused to the targeting sequence. These enzymes will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in dynamic pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-22T02:11:50Z<p>Gcromar: /* Design */</p>
<hr />
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<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
==Design==<br />
<br />
'''Considerations<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A pair of enzymes that are expected to benefit from the effects of channeling.<br />
<br />
Our search for a suitable platform to effect channeling led us to bacterial microcompartments. A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our search for enzymes is described on our [[Team:TorontoMaRSDiscovery/Bioinformatics|bioinformatics]] page. <br />
<br />
We are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford [7]. We want to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be [[Team:TorontoMaRSDiscovery/Modeling|modeled]] using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products. Here, we present our proof-of-concept system based on an encapsulin microcompartment and fluorescent (eCFP) 'probe'. <br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in ''E. coli''.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
'''Components<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Using two new parts (see below) and several existing BioBricks [5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). We include a 'control module' expressing two repressors under control of a constitutive promoter as well as the structural, microcompartment protein, encapsulin (<partinfo>BBa_K192000</partinfo>) and a fluorescent 'probe' (<partinfo>BBa_K192001</partinfo>) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6]. By varying the amounts of inducers aTc and IPTG in the system it is possible to vary the expression of encapsulin and the targeted fluorescent probe (eCFP). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach to visualize the microcompartments. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using the recombinant, fluorescent marker protein (eCFP) by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized (see [[Team:TorontoMaRSDiscovery/Modeling|model]]). By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.<br />
<br />
==Progress==<br />
We have made substantial progress in the construction of all components as detailed in our [[Team:TorontoMaRSDiscovery/Bioinformatics|notebook]]. Specifically, the encapsulin biobrick was completed, sequenced and contributed to the parts registry. Also, as the construction of the fluorescent probe was not trivial, we took advantage of the offer from Mr. Gene to have this part synthesized. We have not yet completed standard assembly of the control module and are awaiting confirmation of this assembly before transforming the synthesized probe, at which point this part will also be contributed. There has been some difficulty obtaining the encapsulin assembly and we are beginning to suspect that our attempts to obtain the construct (initially with a constitutive promoter) may be killing the cells due to the effects of over-expression of the protein.<br />
<br />
Our study of the natural biology of microcompartments across bacteria suggests that their occurrence is confined to a protective function, most commonly isolating toxic or reactive intermediates in certain redox reactions. While other microcompartments exist, the lack of a discernible targeting sequence makes them unsuitable for use in our system and encapsulin remains our best option at present. We have short-listed a series of enzyme pairs that we feel are amenable to manipulation by enzyme channeling based on thermodynamic and other properties for use in our future work.<br />
<br />
==Future Work==<br />
We continue to work on completing the assembly of our proof-of-concept system at which point we will proceed with the characterization of the system as planned. Modeling of candidate enzyme pairs for a downstream application will proceed using the results of our enzyme search. We anticipate that this will lead to several candidate applications <br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-22T02:09:11Z<p>Gcromar: /* Progress */</p>
<hr />
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
==Design==<br />
<br />
'''Considerations<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A pair of enzymes that are expected to benefit from the effects of channeling.<br />
<br />
Our search for a suitable platform to instigate channeling led us to bacterial microcompartments. A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our search for enzymes is described on our [[Team:TorontoMaRSDiscovery/Bioinformatics|bioinformatics]] page. <br />
<br />
We are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford [7]. We want to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be [[Team:TorontoMaRSDiscovery/Modeling|modeled]] using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products. Here, we present our proof-of-concept system based on an encapsulin microcompartment and fluorescent (eCFP) 'probe'. <br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in ''E. coli''.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
'''Components<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Using two new parts (see below) and several existing BioBricks [5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). We include a 'control module' expressing two repressors under control of a constitutive promoter as well as the structural, microcompartment protein, encapsulin (<partinfo>BBa_K192000</partinfo>) and a fluorescent 'probe' (<partinfo>BBa_K192001</partinfo>) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6]. By varying the amounts of inducers aTc and IPTG in the system it is possible to vary the expression of encapsulin and the targeted fluorescent probe (eCFP). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach to visualize the microcompartments. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using the recombinant, fluorescent marker protein (eCFP) by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized (see [[Team:TorontoMaRSDiscovery/Modeling|model]]). By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.<br />
<br />
==Progress==<br />
We have made substantial progress in the construction of all components as detailed in our [[Team:TorontoMaRSDiscovery/Bioinformatics|notebook]]. Specifically, the encapsulin biobrick was completed, sequenced and contributed to the parts registry. Also, as the construction of the fluorescent probe was not trivial, we took advantage of the offer from Mr. Gene to have this part synthesized. We have not yet completed standard assembly of the control module and are awaiting confirmation of this assembly before transforming the synthesized probe, at which point this part will also be contributed. There has been some difficulty obtaining the encapsulin assembly and we are beginning to suspect that our attempts to obtain the construct (initially with a constitutive promoter) may be killing the cells due to the effects of over-expression of the protein.<br />
<br />
Our study of the natural biology of microcompartments across bacteria suggests that their occurrence is confined to a protective function, most commonly isolating toxic or reactive intermediates in certain redox reactions. While other microcompartments exist, the lack of a discernible targeting sequence makes them unsuitable for use in our system and encapsulin remains our best option at present. We have short-listed a series of enzyme pairs that we feel are amenable to manipulation by enzyme channeling based on thermodynamic and other properties for use in our future work.<br />
<br />
==Future Work==<br />
We continue to work on completing the assembly of our proof-of-concept system at which point we will proceed with the characterization of the system as planned. Modeling of candidate enzyme pairs for a downstream application will proceed using the results of our enzyme search. We anticipate that this will lead to several candidate applications <br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-22T01:55:16Z<p>Gcromar: </p>
<hr />
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|}<br />
<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
==Design==<br />
<br />
'''Considerations<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A pair of enzymes that are expected to benefit from the effects of channeling.<br />
<br />
Our search for a suitable platform to instigate channeling led us to bacterial microcompartments. A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our search for enzymes is described on our [[Team:TorontoMaRSDiscovery/Bioinformatics|bioinformatics]] page. <br />
<br />
We are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford [7]. We want to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be [[Team:TorontoMaRSDiscovery/Modeling|modeled]] using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products. Here, we present our proof-of-concept system based on an encapsulin microcompartment and fluorescent (eCFP) 'probe'. <br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in ''E. coli''.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
'''Components<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Using two new parts (see below) and several existing BioBricks [5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). We include a 'control module' expressing two repressors under control of a constitutive promoter as well as the structural, microcompartment protein, encapsulin (<partinfo>BBa_K192000</partinfo>) and a fluorescent 'probe' (<partinfo>BBa_K192001</partinfo>) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6]. By varying the amounts of inducers aTc and IPTG in the system it is possible to vary the expression of encapsulin and the targeted fluorescent probe (eCFP). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach to visualize the microcompartments. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using the recombinant, fluorescent marker protein (eCFP) by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized (see [[Team:TorontoMaRSDiscovery/Modeling|model]]). By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.<br />
<br />
==Progress==<br />
We have made substantial progress in the construction of all components as detailed in our [[Team:TorontoMaRSDiscovery/Bioinformatics|notebook]]. Specifically, the encapsulin biobrick was completed, sequenced and contributed to the parts registry. Also, as the construction of the fluorescent probe was not trivial, we took advantage of the offer from Mr. Gene to have this part synthesized. We have not yet completed standard assembly of the control module and are awaiting confirmation of this assembly before transforming the synthesized probe, at which point this part will also be contributed. There has been some difficulty obtaining the encapsulin assembly and we are beginning to suspect that our attempts to obtain the construct (initially with a constitutive promoter) may be killing the cells due to the effects of over-expression of the protein.<br />
<br />
==Future Work==<br />
We continue to work on completing the assembly of our proof-of-concept system at which point we will proceed with the characterization of the system as planned. Modeling of candidate enzyme pairs for a downstream application will proceed using the results of our enzyme search. We anticipate that this will lead to several candidate applications <br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-22T01:33:30Z<p>Gcromar: /* Design */</p>
<hr />
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
==Design==<br />
<br />
'''Considerations<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A pair of enzymes that are expected to benefit from the effects of channeling.<br />
<br />
Our search for a suitable platform to instigate channeling led us to bacterial microcompartments. A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our search for enzymes is described on our [[Team:TorontoMaRSDiscovery/Bioinformatics|bioinformatics]] page. <br />
<br />
We are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford [7]. We want to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be [[Team:TorontoMaRSDiscovery/Modeling|modeled]] using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products. Here, we present our proof-of-concept system based on an encapsulin microcompartment and fluorescent (eCFP) 'probe'. <br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in ''E. coli''.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
'''Components<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Using two new parts (see below) and several existing BioBricks [5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). We include a 'control module' expressing two repressors under control of a constitutive promoter as well as the structural, microcompartment protein, encapsulin (<partinfo>BBa_K192000</partinfo>) and a fluorescent 'probe' (<partinfo>BBa_K192001</partinfo>) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6]. By varying the amounts of inducers aTc and IPTG in the system it is possible to vary the expression of encapsulin and the targeted fluorescent probe (eCFP). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach to visualize the microcompartments. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using the recombinant, fluorescent marker protein (eCFP) by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized (see model). By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-22T01:28:48Z<p>Gcromar: /* Design */</p>
<hr />
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
==Design==<br />
<br />
'''Considerations<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A pair of enzymes that are expected to benefit from the effects of channeling.<br />
<br />
Our search for a suitable platform to instigate channeling led us to bacterial microcompartments. A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our search for enzymes is described on our [[Team:TorontoMaRSDiscovery/Bioinformatics|bioinformatics]] page. <br />
<br />
We are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford [7]. We want to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be [[Team:TorontoMaRSDiscovery/Modeling|modeled]] using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products. Here, we present our proof-of-concept system based on an encapsulin microcompartment and fluorescent (eCFP) 'probe'. <br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in ''E. coli''.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
'''Components<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Using two new parts (see below) and several existing BioBricks [5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). We include a 'control module' expressing two repressors under control of a constitutive promoter as well as the structural, microcompartment protein, encapsulin (<partinfo>BBa_K192000</partinfo>) and a fluorescent 'probe' with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6](<partinfo>BBa_K192001</partinfo>). By varying the amounts of inducers aTc and IPTG in the system it is possible to vary the expression of encapsulin and the targeted fluorescent probe (eCFP). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach to visualize the microcompartments. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using the recombinant, fluorescent marker protein (eCFP) by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-22T01:20:36Z<p>Gcromar: /* Design */</p>
<hr />
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
==Design==<br />
<br />
'''Considerations<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A pair of enzymes that are expected to benefit from the effects of channeling.<br />
<br />
Our search for a suitable platform to instigate channeling led us to bacterial microcompartments. A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our search for enzymes is described on our [[Team:TorontoMaRSDiscovery/Bioinformatics|bioinformatics]] page. <br />
<br />
We are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford [7]. We want to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be [[Team:TorontoMaRSDiscovery/Modeling|modeled]] using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products. Here, we present our proof-of-concept system based on an encapsulin microcompartment and fluorescent (eCFP) 'probe'. <br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in ''E. coli''.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
'''Components<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Using two parts of our own design and several existing BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). We include a 'control module' expressing two repressors under control of a constitutive promoter as well as the structural microcompartment protein, encapsulin (<partinfo>BBa_K192000</partinfo>) and a fluorescent 'probe' (<partinfo>BBa_K192001</partinfo>). By varying the amounts of inducers aTc and IPTG it is possible to vary the expression of the microcompartment protein (encapsulin) and a targeted fluorescent probe (eCFP). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-22T01:10:09Z<p>Gcromar: /* Design */</p>
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<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
==Design==<br />
<br />
'''Considerations<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A pair of enzymes that are expected to benefit from the effects of channeling.<br />
<br />
Our search for a suitable platform to instigate channeling led us to bacterial microcompartments. A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our search for enzymes is described on our [[Team:TorontoMaRSDiscovery/Bioinformatics|bioinformatics]] page. <br />
<br />
We are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford [7]. We want to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be [[Team:TorontoMaRSDiscovery/Modeling|modeled]] using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products. Here, we present our proof-of-concept system based on an encapsulin microcompartment and fluorescent (eCFP) 'probe'. <br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in ''E. coli''.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
'''Components<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). We include a 'control module' expressing two repressors under control of a constitutive promoter. By varying the amounts of inducers aTc and IPTG it is possible to vary the expression of the microcompartment protein (encapsulin) and a targeted fluorescent probe (eCFP). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-22T01:06:54Z<p>Gcromar: /* Design */</p>
<hr />
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
==Design==<br />
<br />
'''Considerations<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A pair of enzymes that are expected to benefit from the effects of channeling.<br />
<br />
Our search for a suitable platform to instigate channeling led us to bacterial microcompartments. A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our search for enzymes is described on our [[Team:TorontoMaRSDiscovery/Bioinformatics|bioinformatics]] page. <br />
<br />
We are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford [7]. We want to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be [[Team:TorontoMaRSDiscovery/Modeling|modeled]] using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products. Here, we present our proof-of-concept system based on an encapsulin microcompartment and fluorescent (eCFP) 'probe'. <br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in ''E. coli''.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
'''Components<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.<br />
We include a 'control module' expressing two repressors under control of a constitutive promoter. Varying amounts of inducers aTc and IPTG it is possible to vary the expression of the microcompartment protein (encapsulin) and the targeted fluorescent probe (eCFP).<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-22T01:06:11Z<p>Gcromar: </p>
<hr />
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
==Design==<br />
<br />
'''Considerations<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A pair of enzymes that are expected to benefit from the effects of channeling.<br />
<br />
Our search for a suitable platform to instigate channeling led us to bacterial microcompartments. A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our search for enzymes is described on our [[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]] page. <br />
<br />
We are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford [7]. We want to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be [[Team:TorontoMaRSDiscovery/Modeling|modeled]] using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products. Here, we present our proof-of-concept system based on an encapsulin microcompartment and fluorescent (eCFP) 'probe'. <br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in ''E. coli''.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
'''Components<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To determine whether encapsulin correctly assembles in this system we plan to use a standard negative staining (transmission EM) approach. To further establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. This 'probe' carries an LVA tag which targets the protein for degradation. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the inducers aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments.<br />
We include a 'control module' expressing two repressors under control of a constitutive promoter. Varying amounts of inducers aTc and IPTG it is possible to vary the expression of the microcompartment protein (encapsulin) and the targeted fluorescent probe (eCFP).<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T23:15:34Z<p>Gcromar: </p>
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<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
==Design==<br />
<br />
'''Considerations<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A pair of enzymes that are expected to benefit from the effects of channeling.<br />
<br />
Our search for a suitable platform to instigate channeling led us to bacterial microcompartments. A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our search for enzymes is described on our [[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]] page. <br />
<br />
We are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford [7]. We want to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be [[Team:TorontoMaRSDiscovery/Modeling|modeled]] using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products. Here, we present our proof-of-concept system based on an encapsulin microcompartment and fluorescent (eCFP) 'probe'. <br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in ''E. coli''.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
'''Components<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T23:01:24Z<p>Gcromar: </p>
<hr />
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
==Design==<br />
<br />
'''Considerations<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A pair of enzymes that are expected to benefit from the effects of channeling.<br />
<br />
A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our search for enzymes is described on our [[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]] page. Here, we present our proof-of-concept system based on an encapsulin microcompartment and fluorescent (eCFP) 'probe'.<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in ''E. coli''.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
'''Components<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T22:55:51Z<p>Gcromar: </p>
<hr />
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
==Design==<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A pair of enzymes that are expected to benefit from the effects of channeling.<br />
<br />
A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our search for enzymes is described on our [[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]] page. Here, we present our proof-of-concept system based on an encapsulin microcompartment and fluorescent (eCFP) 'probe'.<br />
<br />
===Experimental Approach (Proof of concept)===<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T22:54:44Z<p>Gcromar: /* Design */</p>
<hr />
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
==Design==<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A pair of enzymes that are expected to benefit from the effects of channeling.<br />
<br />
A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our search for enzymes is described on our [[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]] page. Here, we present our proof-of-concept system based on an encapsulin microcompartment and fluorescent (eCFP) 'probe'.<br />
<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T22:44:57Z<p>Gcromar: </p>
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<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
==Design==<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A list of candidate enzyme pairs that might be expected to benefit from the effects of channeling.<br />
<br />
A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our analysis of enzymes is described in our [[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]] page. Here, we present our proof-of-concept system, based on encapsulin.<br />
<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T22:43:51Z<p>Gcromar: /* Design */</p>
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Design==<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A list of candidate enzyme pairs that might be expected to benefit from the effects of channeling.<br />
<br />
A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our analysis of enzymes is described in our [[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]] page. Here, we present our proof-of-concept system, based on encapsulin.<br />
<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T22:43:11Z<p>Gcromar: /* Background */</p>
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Design==<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T22:41:27Z<p>Gcromar: /* Background */</p>
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<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A list of candidate enzyme pairs that might be expected to benefit from the effects of channeling.<br />
<br />
A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments. Our ongoing search for alternative microcompartments as well as our analysis of enzymes is described in our [[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]] page. Here, we present our proof-of-concept system, based on encapsulin.<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Design==<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T22:37:12Z<p>Gcromar: /* Background */</p>
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A list of candidate enzyme pairs that might be expected to benefit from the effects of channeling.<br />
<br />
A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from other microcompartments (see [[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]] for further discussion).<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Design==<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T22:36:02Z<p>Gcromar: /* Design */</p>
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A list of candidate enzyme pairs that might be expected to benefit from the effects of channeling.<br />
<br />
A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from others reported (see [[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]] for a further discussion).<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Design==<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T22:35:41Z<p>Gcromar: /* Background */</p>
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A list of candidate enzyme pairs that might be expected to benefit from the effects of channeling.<br />
<br />
A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from others reported (see [[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]] for a further discussion).<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Design==<br />
<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T22:35:09Z<p>Gcromar: /* Background */</p>
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<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A list of candidate enzyme pairs that might be expected to benefit from the effects of channeling.<br />
<br />
A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from others reported (see [[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]] for a further discussion).<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Design==<br />
<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T22:32:07Z<p>Gcromar: </p>
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A list of candidate enzyme pairs that might be expected to benefit from the effects of channeling.<br />
<br />
A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from others reported (see [[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]] for a more detailed review).<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Design==<br />
<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T22:30:37Z<p>Gcromar: /* Background */</p>
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A list of candidate enzyme pairs that might be expected to benefit from the effects of channeling.<br />
<br />
A detailed structural analysis of encapsulin nano-compartments has recently been reported [6]. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from others reported (see bioinformatics for a more detailed review).<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Design==<br />
<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T22:29:19Z<p>Gcromar: /* Background */</p>
<hr />
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A list of candidate enzyme pairs that might be expected to benefit from the effects of channeling.<br />
<br />
A detailed structural analysis of encapsulin nano-compartments has recently been reported []. Of particular interest to us is the fact that a discernible targeting sequence is known; a fact that distinguishes this compartment from others reported (see bioinformatics for a more detailed review).<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Design==<br />
<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T22:19:31Z<p>Gcromar: /* Background */</p>
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<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A list of candidate enzyme pairs that might be expected to benefit from the effects of channeling.<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Design==<br />
<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T22:16:59Z<p>Gcromar: /* Background */</p>
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
From an engineering perspective a successful implementation requires:<br />
<br />
#The expression of a subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to this space<br />
#A list of candidate enzyme pairs that might be expected to benefit from the effects of channeling.<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Design==<br />
<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T22:10:20Z<p>Gcromar: /* Background */</p>
<hr />
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
[[image:tmdt_design2.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications such as the production therapeutic molecules and biofuels [3] and the degradation of toxic wastes[4].<br />
<br />
From an engineering perspective, a successful implementation requires:<br />
<br />
#A subcellular space for co-localizing enzymes (and intermediates) which would restrict free diffusion.<br />
#A method of targeting chosen enzymes to the above space<br />
#<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Design==<br />
<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T21:49:58Z<p>Gcromar: /* Phase-One */</p>
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<br><br />
<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
[[image:tmdt_biobrick.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications.<br />
<br />
Potential applications range from the production of valuable compounds such as therapeutic molecules and biofuels[3] to the degradation of toxic wastes[4].<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Design==<br />
<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Progress==<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T21:48:36Z<p>Gcromar: /* Background */</p>
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<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
[[image:tmdt_biobrick.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
Proteins operate as parts of integrated biochemical pathways and exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. In metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits:<br />
<br />
#Optimization of catalytic efficiency by decreasing transit time for intermediates<br />
#Relief of the effects of product inhibition<br />
#Protection from the creation of potentially toxic or unstable intermediates<br />
#Regulation of substrate flux through mediating pathway cross-talk<br />
<br />
<br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications.<br />
<br />
Potential applications range from the production of valuable compounds such as therapeutic molecules and biofuels[3] to the degradation of toxic wastes[4].<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Phase-One==<br />
<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T21:42:09Z<p>Gcromar: /* Phase-Two */</p>
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<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
[[image:tmdt_biobrick.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
It is well established that genes and their products do not operate in isolation but rather form parts of integrated biochemical pathways. There is increasing evidence that in many pathways, individual components exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. <br />
<br />
For metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Metabolic channeling is defined as the process in which the intermediate produced by one enzyme is transferred to the next enzyme without complete mixing in the bulk phase [2]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits: 1) Optimization of catalytic efficiency by decreasing transit time for intermediates; 2) Relief of the effects of product inhibition; 3) Protection from the creation of potentially toxic or unstable intermediates; and 4) Regulation of substrate flux through mediating pathway cross-talk [1]. Potential applications range from the production of valuable compounds such as therapeutic molecules and biofuels[3] to the degradation of toxic wastes[4]. <br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications.<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Phase-One==<br />
<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Future Work==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/ProjectTeam:TorontoMaRSDiscovery/Project2009-10-21T21:38:59Z<p>Gcromar: /* Background */</p>
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<br />
<p style="font-size:18pt;">Engineering bacterial micro-compartments to investigate metabolic channeling and its potential uses in biotechnological applications</p><br />
<br />
==Background==<br />
[[image:tmdt_biobrick.png|right|thumb|Figure 1: A three-component design for the controlled expression of encapsulin and targeted eCFP.]]<br />
It is well established that genes and their products do not operate in isolation but rather form parts of integrated biochemical pathways. There is increasing evidence that in many pathways, individual components exhibit varying degrees of spatial organization ranging from sub-cellular compartmentalization to the formation of discrete complexes. <br />
<br />
For metabolic processes, the co-localization of enzymatic components has been shown to promote the transfer of substrates between consecutive reactions in a process termed “channeling”[1]. Metabolic channeling is defined as the process in which the intermediate produced by one enzyme is transferred to the next enzyme without complete mixing in the bulk phase [2]. Channeling results in the efficient translocation of substrates between enzymes and has been proposed to result in the following benefits: 1) Optimization of catalytic efficiency by decreasing transit time for intermediates; 2) Relief of the effects of product inhibition; 3) Protection from the creation of potentially toxic or unstable intermediates; and 4) Regulation of substrate flux through mediating pathway cross-talk [1]. Potential applications range from the production of valuable compounds such as therapeutic molecules and biofuels[3] to the degradation of toxic wastes[4]. <br />
<br />
We have taken an interdisciplinary approach to systematically investigate how nature implements metabolic channeling and how this knowledge may be exploited for biotechnological applications.<br />
<br />
==Experimental Approach==<br />
<br />
'''Objectives<br />
<br />
#Design, construct and characterize a micro-compartment expression system in E. coli.<br />
#Demonstrate in vivo assembly of the expressed micro-compartments.<br />
#Target a fluorescent marker (eCFP) to the micro-compartment.<br />
#Identify and prioritize candidate enzyme pairs for channeling.<br />
#Apply channeling to selected enzyme pairs.<br />
<br />
==Phase-One==<br />
<br />
Using standard BioBrick parts[5] we have designed and are in the process of constructing an expression system capable of producing functional, ''T. maritima'' derived, encapsulin micro-compartments in ''E. coli'' (Figure 1). To establish that these compartments are functional, we will test our ability to target peptides to this compartment using a recombinant, fluorescent marker protein (eCFP) with a c-terminal extension corresponding to the conserved targeting sequence described by Sutter et al [6] followed by fluorescence microscopy. We hypothesize that encapsulation of eCFP will prolong its half-life and that by varying the amounts of the de-repressors aTc and IPTG in the expression system it will be possible to obtain a state where sufficient encapsulin is produced to enclose detectable amounts of eCFP and where background signal is minimized. By measuring the difference in fluorescence between bacteria expressing encapsulin + eCFP versus those expressing only eCFP at a given optical density we hope to obtain an estimate of the amount of eCFP being protected from degradation and therefore localized to the micro-compartments. <br />
<br />
Concurrently, we are evaluating enzymes for use in our system which are likely to be amenable to channeling as predicted by C. Sanford. Modeling and Computational Prediction of Metabolic Channeling [7]. Our bioinformatics team, which consists of undergraduate students as well as graduate student advisors who have modeling and metabolomics expertise, will attempt to identify associated pathways containing substrates and products that are 1) easily assayed (e.g. using colorimetric or spectrophotometric tests) and 2) potentially commercially relevant (i.e. produces a desirable product or breaks down an undesirable compound). Short-listed pathways will be modeled using SimBiology [8] and Cell++[9] (the latter was developed in our host laboratory) to predict the effect of channeling on pathway intermediates and products as an aid to selecting a Phase-Two target application.<br />
<br />
==Phase-Two==<br />
Using pairs of enzymes from pathways modeled in our earlier screens we will construct recombinants fused to the targeting sequence. These enzyme pairs will be co-expressed in the presence and absence of encapsulin and the effect on pathway intermediates and/or products will be assayed to determine the effects of channeling and to compare them with our modeled prediction. The nature of the assays will depend on the chosen system. As a further experiment, if possible, we would like to apply channeling to a branching pathway to evaluate the potential role of channeling in pathway switching.<br />
<br />
==Comments==<br />
#Size of compartments<br />
#:It is possible that the extremely small size (230-240 angstroms diameter with a pore size of approximately 5 angstroms) of the encapsulin micro-compartment could lead to problems in accommodating some enzymes as well as the passage of some metabolites. <br />
#Control of expression<br />
#:In our system, each micro-compartment consists of sixty encapsulin monomers. The targeting sequence we used corresponds to the T. maritima ferritin-like protein (flp) extension in which the encapsulated enzymes are thought to form a pentamer of dimers[6]. This implies a particular optimum ratio of enzymes (or eCFP) to encapsulin which thereby must be achieved via matching of protein expression.<br />
#Search for an assayable application<br />
#:It is possible that few commercially relevant pathways have products that are easily assayed by colorimetric or spectrophotometric means. If this turns out to be the case, we propose to test an alternative, readily assayable system regardless of commercial relevance, to first meet our scientific objectives. We note that there are several such pathways related to glycolysis that would meet this objective, including one previously modeled by C Sanford [7].<br />
<br />
==References==<br />
#Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol, 8 280-91 (2005).<br />
#Spivey, H.O. & Ovadi, J. Substrate channeling. Methods 19, 306-21 (1999).<br />
#Atsumi, S. & Liao, J.C. Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol (2008).<br />
#Villas-Boas, S.G. & Bruheim, P. The potential of metabolomics tools in bioremediation studies. Omics 11, 305-13 (2007).<br />
#Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-93 (2008).<br />
#Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).<br />
#Sanford, C. M.Sc. Thesis: Modeling and Computational Prediction of Metabolic Channeling (2009).<br />
#SimBiology 3.0 - Model, simulate, and analyze biological systems. Vol. 2009 (The MathWorks).<br />
#Sanford, C., Yip, M.L., White, C. & Parkinson, J. Cell++--simulating biochemical pathways. Bioinformatics 22, 2918-25 (2006).</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/SafetyTeam:TorontoMaRSDiscovery/Safety2009-10-21T19:57:11Z<p>Gcromar: /* iGEM Safety */</p>
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!align="center"|[[Team:TorontoMaRSDiscovery/Team|The Team]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Project|The Project]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Parts|BioBricks]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Modeling|Modeling]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Safety|Safety]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Notebook|Notebook]]<br />
|}<br />
<br><br />
=iGEM Safety=<br />
<br />
Members of our lab team were required to take biological and chemical safety training courses offered through the Department of Occupational Health and Safety at the Hospital for Sick Children in Toronto. Each safety seminar was divided into 2 parts, each part being 2 hours in length followed by a short exam. In addition, due to our proximity to patients and hospital staff each member of the team was required to present proof of immunizations and a recent TB test as well as attending a hospital orientation and sensitivity training. We were closely supervised by our instructors during further lab-specific training for the first 4-6 weeks of our summer work period. As we were involved in setting up a new lab this year, we created and actively maintained a binder of Material Safety Data Sheets (MSDS) as required by WHMIS (Workplace Hazardous Material Information System).<br />
<br />
====''Do any of our project ideas raise safety issues in terms of researcher safety, public safety, or environmental safety?''====<br />
None beyond the usual safety requirements as provided for in a level-2 laboratory (Canadian standard).<br />
<br />
====''Is there a local biosafety group, committee, or review board at our institution?''====<br />
Yes - The Research Training Centre at The Hospital for Sick Children.<br />
<br />
====''What does our local biosafety group think about your project?''====<br />
The Research Institute has approached us to profile our work in media promotions. We take this as a sign that they approve.<br />
<br />
====''Do any of the new BioBrick parts that we made this year raise any safety issues?''====<br />
No. Our key part is already native to bacteria. Our manipulations lay the groundwork for new technologies that may influence the cost of producing some medicines. This is generally seen as a positive.</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/SafetyTeam:TorontoMaRSDiscovery/Safety2009-10-21T19:56:49Z<p>Gcromar: /* iGEM Safety */</p>
<hr />
<div>[[image:To_igem_wiki_banner.jpg|965px]]<br />
{| style="color:white;background-color:#99CCFF;" height:100px cellpadding="2" cellspacing="0" border="0" width="100%" align="center" class="menu"<br />
!align="center"|[[Team:TorontoMaRSDiscovery|Home]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Team|The Team]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Project|The Project]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Parts|BioBricks]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Modeling|Modeling]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Safety|Safety]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Notebook|Notebook]]<br />
|}<br />
<br><br />
=iGEM Safety=<br />
Members of our lab team were required to take biological and chemical safety training courses offered through the Department of Occupational Health and Safety at the Hospital for Sick Children in Toronto. Each safety seminar was divided into 2 parts, each part being 2 hours in length followed by a short exam. In addition, due to our proximity to patients and hospital staff each member of the team was required to present proof of immunizations and a recent TB test as well as attending a hospital orientation and sensitivity training. We were closely supervised by our instructors during further lab-specific training for the first 4-6 weeks of our summer work period. As we were involved in setting up a new lab this year, we created and actively maintained a binder of Material Safety Data Sheets (MSDS) as required by WHMIS (Workplace Hazardous Material Information System).<br />
<br />
====''Do any of our project ideas raise safety issues in terms of researcher safety, public safety, or environmental safety?''====<br />
None beyond the usual safety requirements as provided for in a level-2 laboratory (Canadian standard).<br />
<br />
====''Is there a local biosafety group, committee, or review board at our institution?''====<br />
Yes - The Research Training Centre at The Hospital for Sick Children.<br />
<br />
====''What does our local biosafety group think about your project?''====<br />
The Research Institute has approached us to profile our work in media promotions. We take this as a sign that they approve.<br />
<br />
====''Do any of the new BioBrick parts that we made this year raise any safety issues?''====<br />
No. Our key part is already native to bacteria. Our manipulations lay the groundwork for new technologies that may influence the cost of producing some medicines. This is generally seen as a positive.</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/SafetyTeam:TorontoMaRSDiscovery/Safety2009-10-21T19:55:22Z<p>Gcromar: /* Is there a local biosafety group, committee, or review board at our institution? */</p>
<hr />
<div>[[image:To_igem_wiki_banner.jpg|965px]]<br />
{| style="color:white;background-color:#99CCFF;" height:100px cellpadding="2" cellspacing="0" border="0" width="100%" align="center" class="menu"<br />
!align="center"|[[Team:TorontoMaRSDiscovery|Home]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Team|The Team]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Project|The Project]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Parts|BioBricks]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Modeling|Modeling]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Safety|Safety]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Notebook|Notebook]]<br />
|}<br />
<br><br />
=iGEM Safety=<br />
Members of our lab team were required to take biological and chemical safety training courses offered through the Department of Occupational Health and Safety at the Hospital for Sick Children in Toronto. Each safety seminar was divided into 2 parts, each part being 2 hours in length followed by a short exam. In addition, due to our proximity to patients and hospital staff each member of the team was required to present proof of immunizations and a recent TB test as well as attending a hospital orientation and sensitivity training. We were closely supervised by our instructors during further lab-specific training for the first 4-6 weeks of our summer work period. As we were involved in setting up a new lab this year, we created and actively maintained a binder of Material Safety Data Sheets (MSDS) as required by WHMIS (Workplace Hazardous Material Information System).<br />
<br />
''Do any of our project ideas raise safety issues in terms of researcher safety, public safety, or environmental safety?''<br />
None beyond the usual safety requirements as provided for in a level-2 laboratory (Canadian standard).<br />
<br />
====''Is there a local biosafety group, committee, or review board at our institution?''====<br />
Yes - The Research Training Centre at The Hospital for Sick Children.<br />
<br />
====What does our local biosafety group think about your project?====<br />
The Research Institute has approached us to profile our work in media promotions. We take this as a sign that they approve.<br />
<br />
====Do any of the new BioBrick parts that we made this year raise any safety issues?====<br />
No. Our key part is already native to bacteria. Our manipulations lay the groundwork for new technologies that may influence the cost of producing some medicines. This is generally seen as a positive.</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/SafetyTeam:TorontoMaRSDiscovery/Safety2009-10-21T19:54:58Z<p>Gcromar: /* Do any of our project ideas raise safety issues in terms of researcher safety, public safety, or environmental safety? */</p>
<hr />
<div>[[image:To_igem_wiki_banner.jpg|965px]]<br />
{| style="color:white;background-color:#99CCFF;" height:100px cellpadding="2" cellspacing="0" border="0" width="100%" align="center" class="menu"<br />
!align="center"|[[Team:TorontoMaRSDiscovery|Home]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Team|The Team]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Project|The Project]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Parts|BioBricks]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Modeling|Modeling]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Safety|Safety]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Notebook|Notebook]]<br />
|}<br />
<br><br />
=iGEM Safety=<br />
Members of our lab team were required to take biological and chemical safety training courses offered through the Department of Occupational Health and Safety at the Hospital for Sick Children in Toronto. Each safety seminar was divided into 2 parts, each part being 2 hours in length followed by a short exam. In addition, due to our proximity to patients and hospital staff each member of the team was required to present proof of immunizations and a recent TB test as well as attending a hospital orientation and sensitivity training. We were closely supervised by our instructors during further lab-specific training for the first 4-6 weeks of our summer work period. As we were involved in setting up a new lab this year, we created and actively maintained a binder of Material Safety Data Sheets (MSDS) as required by WHMIS (Workplace Hazardous Material Information System).<br />
<br />
''Do any of our project ideas raise safety issues in terms of researcher safety, public safety, or environmental safety?''<br />
None beyond the usual safety requirements as provided for in a level-2 laboratory (Canadian standard).<br />
<br />
====Is there a local biosafety group, committee, or review board at our institution?====<br />
Yes - The Research Training Centre at The Hospital for Sick Children.<br />
<br />
====What does our local biosafety group think about your project?====<br />
The Research Institute has approached us to profile our work in media promotions. We take this as a sign that they approve.<br />
<br />
====Do any of the new BioBrick parts that we made this year raise any safety issues?====<br />
No. Our key part is already native to bacteria. Our manipulations lay the groundwork for new technologies that may influence the cost of producing some medicines. This is generally seen as a positive.</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/SafetyTeam:TorontoMaRSDiscovery/Safety2009-10-21T19:54:32Z<p>Gcromar: /* Do any of our project ideas raise safety issues in terms of researcher safety, public safety, or environmental safety? */</p>
<hr />
<div>[[image:To_igem_wiki_banner.jpg|965px]]<br />
{| style="color:white;background-color:#99CCFF;" height:100px cellpadding="2" cellspacing="0" border="0" width="100%" align="center" class="menu"<br />
!align="center"|[[Team:TorontoMaRSDiscovery|Home]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Team|The Team]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Project|The Project]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Parts|BioBricks]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Modeling|Modeling]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Safety|Safety]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Notebook|Notebook]]<br />
|}<br />
<br><br />
=iGEM Safety=<br />
Members of our lab team were required to take biological and chemical safety training courses offered through the Department of Occupational Health and Safety at the Hospital for Sick Children in Toronto. Each safety seminar was divided into 2 parts, each part being 2 hours in length followed by a short exam. In addition, due to our proximity to patients and hospital staff each member of the team was required to present proof of immunizations and a recent TB test as well as attending a hospital orientation and sensitivity training. We were closely supervised by our instructors during further lab-specific training for the first 4-6 weeks of our summer work period. As we were involved in setting up a new lab this year, we created and actively maintained a binder of Material Safety Data Sheets (MSDS) as required by WHMIS (Workplace Hazardous Material Information System).<br />
<br />
====Do any of our project ideas raise safety issues in terms of researcher safety, public safety, or environmental safety?====<br />
None beyond the usual safety requirements as provided for in a level-2 laboratory (Canadian standard).<br />
<br />
====Is there a local biosafety group, committee, or review board at our institution?====<br />
Yes - The Research Training Centre at The Hospital for Sick Children.<br />
<br />
====What does our local biosafety group think about your project?====<br />
The Research Institute has approached us to profile our work in media promotions. We take this as a sign that they approve.<br />
<br />
====Do any of the new BioBrick parts that we made this year raise any safety issues?====<br />
No. Our key part is already native to bacteria. Our manipulations lay the groundwork for new technologies that may influence the cost of producing some medicines. This is generally seen as a positive.</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/SafetyTeam:TorontoMaRSDiscovery/Safety2009-10-21T19:53:21Z<p>Gcromar: /* iGEM Safety */</p>
<hr />
<div>[[image:To_igem_wiki_banner.jpg|965px]]<br />
{| style="color:white;background-color:#99CCFF;" height:100px cellpadding="2" cellspacing="0" border="0" width="100%" align="center" class="menu"<br />
!align="center"|[[Team:TorontoMaRSDiscovery|Home]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Team|The Team]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Project|The Project]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Parts|BioBricks]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Modeling|Modeling]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Safety|Safety]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Notebook|Notebook]]<br />
|}<br />
<br><br />
=iGEM Safety=<br />
Members of our lab team were required to take biological and chemical safety training courses offered through the Department of Occupational Health and Safety at the Hospital for Sick Children in Toronto. Each safety seminar was divided into 2 parts, each part being 2 hours in length followed by a short exam. In addition, due to our proximity to patients and hospital staff each member of the team was required to present proof of immunizations and a recent TB test as well as attending a hospital orientation and sensitivity training. We were closely supervised by our instructors during further lab-specific training for the first 4-6 weeks of our summer work period. As we were involved in setting up a new lab this year, we created and actively maintained a binder of Material Safety Data Sheets (MSDS) as required by WHMIS (Workplace Hazardous Material Information System).<br />
<br />
====Do any of our project ideas raise safety issues in terms of researcher safety, public safety, or environmental safety?====<br />
None beyond the usual safety requirements as provided for in a (Canadian) level-2 laboratory.<br />
<br />
====Is there a local biosafety group, committee, or review board at our institution?====<br />
Yes - The Research Training Centre at The Hospital for Sick Children.<br />
<br />
====What does our local biosafety group think about your project?====<br />
The Research Institute has approached us to profile our work in media promotions. We take this as a sign that they approve.<br />
<br />
====Do any of the new BioBrick parts that we made this year raise any safety issues?====<br />
No. Our key part is already native to bacteria. Our manipulations lay the groundwork for new technologies that may influence the cost of producing some medicines. This is generally seen as a positive.</div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/PartsTeam:TorontoMaRSDiscovery/Parts2009-10-21T18:31:58Z<p>Gcromar: /* Parts in Progress */</p>
<hr />
<div>[[image:To_igem_wiki_banner.jpg|965px]]<br />
{| style="color:white;background-color:#99CCFF;" height:100px cellpadding="2" cellspacing="0" border="0" width="100%" align="center" class="menu"<br />
!align="center"|[[Team:TorontoMaRSDiscovery|Home]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Team|The Team]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Project|The Project]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Parts|BioBricks]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Modeling|Modeling]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Safety|Safety]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Notebook|Notebook]]<br />
|}<br />
<br><br />
<br />
=BioBricks=<br />
<br />
==Parts Submitted to the Registry==<br />
<br />
Encapsulin from T. ''maritima''<br />
<partinfo>BBa_K192000</partinfo><br />
<br />
==Parts in Progress==<br />
<br />
A fluorescent protein (CFP) onto which we added an LVA degradation tag and an N-terminal targeting sequence.<br />
<partinfo>BBa_K192001</partinfo></div>Gcromarhttp://2009.igem.org/Team:TorontoMaRSDiscovery/PartsTeam:TorontoMaRSDiscovery/Parts2009-10-21T18:28:14Z<p>Gcromar: /* Parts Submitted to the Registry */</p>
<hr />
<div>[[image:To_igem_wiki_banner.jpg|965px]]<br />
{| style="color:white;background-color:#99CCFF;" height:100px cellpadding="2" cellspacing="0" border="0" width="100%" align="center" class="menu"<br />
!align="center"|[[Team:TorontoMaRSDiscovery|Home]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Team|The Team]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Project|The Project]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Parts|BioBricks]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Modeling|Modeling]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Bioinformatics|Bioinformatics]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Safety|Safety]]<br />
!align="center"|[[Team:TorontoMaRSDiscovery/Notebook|Notebook]]<br />
|}<br />
<br><br />
<br />
=BioBricks=<br />
<br />
==Parts Submitted to the Registry==<br />
<br />
Encapsulin from T. ''maritima''<br />
<partinfo>BBa_K192000</partinfo><br />
<br />
==Parts in Progress==<br />
<partinfo>BBa_K192001</partinfo></div>Gcromar