http://2011.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=100&target=Hadidi&year=&month=2011.igem.org - User contributions [en]2024-03-28T11:14:12ZFrom 2011.igem.orgMediaWiki 1.16.0http://2011.igem.org/Team:Washington/Team/MembersTeam:Washington/Team/Members2011-09-23T23:07:01Z<p>Hadidi: /* Who we are */</p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
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<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------><br />
<br />
== '''Who we are''' ==<br />
<br />
<gallery caption="Undergraduate Team Members" widths="180px" heights="120px" perrow="4"><br />
Image:Washington Casey Ager Profile1.jpg|<center>Casey Ager <br/> Biochemistry</center><br />
Image:Washington Photo-0088A.png|<center>Juhye An <br/> Biochemistry </center><br />
Image: Profile_Michael_Brasino.jpg|<center>Michael Brasino <br/> Materials Science and Engineering</center><br />
Image:Washington_.jpg|<center>Marika Cheng <br/> Microbiology</center><br />
Image:Washington_choe_chris.png|<center>Chris Choe <br/> Major</center><br />
Image:Washington_.jpg|<center>Justin De Leon <br/> Microbiology</center><br />
File:Princess Peachy.JPG|<center>Sydney Gordon <br/> Biochemistry, Music</center><br />
Image:CIMG0021.jpg|<center>Daniel Hadidi <br/> Neurobiology</center><br />
Image:Washington_Matthew_Harger.JPG|<center>Matthew Harger <br/> Cellular and Molecular Biology</center><br />
Image:Washington_Elaine.jpg|<center>Elaine Lai <br/> Microbiology, Chemistry</center><br />
Image:Washington_2011_FirstWeekInLab.png|<center>Benjamin Mo <br/> Bioengineering</center><br />
Image:Washington 2011 CIMG0015-2.jpg|<center>Austin Moon <br/> Cellular and Molecular Biology, Microbiology</center><br />
Image:Washington_.jpg|<center>Rashmi Ravichandran <br/> Microbiology</center><br />
Image:Washington_.jpg|<center>Seth Sagulo <br/> Biochemistry</center><br />
File:Washington_Liz.png|<center>Liz Stanley <br/> Microbiology, Chemistry</center><br />
Image:Washington_.jpg|<center>Angus Toland <br/> Microbiology</center><br />
Image:Washington_.jpg|<center>Sarah Wolf <br/> Biochemistry</center><br />
Image:Washington_igem11whowearepicture.jpg|<center>SauShun (Alicia) Wong <br/> Materials Science and Engineering</center><br />
Image:Washington_Cindy.jpg|<center>Cindy Wu <br/> Cellular and Molecular Biology</center><br />
Image:Washington_Sean_Wu.jpg|<center>Sean Wu <br/> Computer Science and Engineering</center><br />
Image:Washington_2011_BATMAN.jpg|<center>Lei Zheng <br/> Biochemistry</center><br />
Image:Washington_david_zong.jpg|<center>David Zong <br/> Bioengineering</center><br />
<br />
</gallery><br />
<br />
<br />
<gallery caption="Advisors" widths="180px" heights="120px" perrow="4"><br />
Image:Washington_.jpg|<center>Aaron Chevalier <br/> Bioengineering </center><br />
Image:Regbert.jpg|<center>Rob Egbert <br/> Electrical Engineering </center><br />
Image:Washington_.jpg|<center>Chris Eiben <br/> Biochemistry </center><br />
Image:Washington_.jpg|<center>Jeremy Mills <br/> Biochemistry </center><br />
Image:Siegel_UW_2011.jpg|<center>Justin Siegel <br/> Biomolecular Structure and Design</center><br />
Image:Washington2011_Matthew-d-smith.jpg|<center>Matt Smith <br/> Molecular and Cellular Biology</center><br />
File:Washington_Ingrid_Pic_1.jpg|<center>Ingrid Swanson Pultz<br/> Microbiology </center><br />
</gallery><br />
<br />
<br />
<gallery caption="Faculty" widths="180px" heights="120px" perrow="5"><br />
image:Washington_David_Baker.jpg|<center>David Baker <br/> Biochemistry</center><br />
Image:Washington_Klavins.jpg|<center>Eric Klavins <br/> Electrical Engineering</center><br />
</gallery><br />
<br />
<br />
<gallery caption="Collaborators and Support" widths="140px" heights="100px" perrow="5"><br />
<br />
Image:Washington_OSLI.png|<center><b>Oil Sand Leadership Initiative</b> <br/> Funding to support Travel and Registration Costs</center><br />
<br />
Image:Washington_UniversitySeal.gif|<center><b>UW Biochemistry</b> <br/> Lab space</center><br />
<br />
Image:Washington_Anaspec.gif|<center><b>Anaspec</b> <br/> Peptide Discounts</center><br />
<br />
Image:Washington_ARPA-E_Logo.png|<center><b>Advanced Research Projects Agency - Energy </b><br/> Registration Support</center><br />
<br />
Image:Washington2011_Hhmi_362_72.jpg|<center><b>Howard Hughes Medical Institute</b> <br/> Lab materials and supplies</center><br />
<br />
<br />
</gallery><br />
<br />
== '''Who did what''' ==<br />
The teams were assembled during our winter quarter. During this term we went around to classes and had weekly meetings to introduce students to synthetic biology through a series of guest lectures. During the spring, students participated in brainstorming projects related to the sponsoring Faculty advisors labs (Baker and Klavins) and came up with the project they wanted to carry out. Students also planned and partcipated in community outreach events where we taught our community about synthetic biology. Over the summer, students completed all of the experimental work, but worked closely with both graduate students and faculty advisors to plan the most pertinent experiments and make the most of our limited time.<br />
<br />
=== Diesel Production ===<br />
After producing promising results, in future directions <br />
<br />
Casey Ager, Austin Moon, and Seth Sagulo worked on Enzyme Localization via Direct Fusion and Zinc Finger Fusion methods.<br />
<br />
Juhye An, Elaine Lai, and Benjamin Mo worked on Decarbonylase Redesign<br />
<br />
Marika Cheng and Justin De Leon worked on Alternative Chassis<br />
<br />
Chris Choe and David Zong worked on Alternate Aldehyde Production<br />
<br />
Matthew Harger worked on Branched Alkanes Production<br />
<br />
Matthew Harger and Lei Zheng worked on System Optimization<br />
<br />
=== Gluten Destruction ===<br />
Sydney Gordon, Daniel Hadidi, Liz Stanley, Angus Toland, Sarah Wolf, and Sean Wu designed, built, and tested Kumamolisin-As and over 100 mutants to combat gluten intolerance by increasing the activity on the PQLP antigenic peptide.<br />
<br />
=== iGEM Toolkits ===<br />
Michael Brasino, Rashmi Ravichandran, and Alicia Wong<br />
<br />
=== Community Outreach ===<br />
Cindy Wu headed and organized all iGEM 2011 Community Outreach events.</div>Hadidihttp://2011.igem.org/Team:Washington/Team/MembersTeam:Washington/Team/Members2011-09-23T23:06:27Z<p>Hadidi: /* Who we are */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------><br />
<br />
== '''Who we are''' ==<br />
<br />
<gallery caption="Undergraduate Team Members" widths="180px" heights="120px" perrow="4"><br />
Image:Washington Casey Ager Profile1.jpg|<center>Casey Ager <br/> Biochemistry</center><br />
Image:Washington Photo-0088A.png|<center>Juhye An <br/> Biochemistry </center><br />
Image: Profile_Michael_Brasino.jpg|<center>Michael Brasino <br/> Materials Science and Engineering</center><br />
Image:Washington_.jpg|<center>Marika Cheng <br/> Microbiology</center><br />
Image:Washington_choe_chris.png|<center>Chris Choe <br/> Major</center><br />
Image:Washington_.jpg|<center>Justin De Leon <br/> Microbiology</center><br />
File:Princess Peachy.JPG|<center>Sydney Gordon <br/> Biochemistry, Music</center><br />
Image:CIMG0021.jpg|<center>Daniel Hadidi <br/> Neurobiology</center><br />
Image:Washington_Matthew_Harger.JPG|<center>Matthew Harger <br/> Cellular and Molecular Biology</center><br />
Image:Washington_Elaine.jpg|<center>Elaine Lai <br/> Microbiology, Chemistry</center><br />
Image:Washington_2011_FirstWeekInLab.png|<center>Benjamin Mo <br/> Bioengineering</center><br />
Image:Washington 2011 CIMG0015-2.jpg|<center>Austin Moon <br/> Cellular and Molecular Biology, Microbiology</center><br />
Image:Washington_.jpg|<center>Rashmi Ravichandran <br/> Microbiology</center><br />
Image:Washington_.jpg|<center>Seth Sagulo <br/> Biochemistry</center><br />
File:Washington_Liz.png|<center>Liz Stanley <br/> Microbiology, Chemistry</center><br />
Image:Washington_.jpg|<center>Angus Toland <br/> Microbiology</center><br />
Image:Washington_.jpg|<center>Sarah Wolf <br/> Biochemistry</center><br />
Image:Washington_igem11whowearepicture.jpg|<center>SauShun (Alicia) Wong <br/> Materials Science and Engineering</center><br />
Image:Washington_Cindy.jpg|<center>Cindy Wu <br/> Cellular and Molecular Biology</center><br />
Image:Washington_Sean_Wu.jpg|<center>Sean Wu <br/> Computer Science and Engineering</center><br />
Image:Washington_2011_BATMAN.jpg|<center>Lei Zheng <br/> Biochemistry</center><br />
Image:Washington_david_zong.jpg|<center>David Zong <br/> Bioengineering</center><br />
<br />
</gallery><br />
<br />
<br />
<gallery caption="Advisors" widths="180px" heights="120px" perrow="4"><br />
Image:Washington_.jpg|<center>Aaron Chevalier <br/> Bioengineering </center><br />
Image:Regbert.jpg|<center>Rob Egbert <br/> Electrical Engineering </center><br />
Image:Washington_.jpg|<center>Chris Eiben <br/> Biochemistry </center><br />
Image:Washington_.jpg|<center>Jeremy Mills <br/> Biochemistry </center><br />
Image:Siegel_UW_2011.jpg|<center>Justin Siegel <br/> Biomolecular Structure and Design</center><br />
Image:Washington2011_Matthew-d-smith.jpg|<center>Matt Smith <br/> Molecular and Cellular Biology</center><br />
File:Washington_Ingrid_Pic_1.jpg|<center>Ingrid Swanson Pultz<br/> Microbiology </center><br />
</gallery><br />
<br />
<br />
<gallery caption="Faculty" widths="180px" heights="120px" perrow="5"><br />
image:Washington_David_Baker.jpg|<center>David Baker <br/> Biochemistry</center><br />
Image:Washington_Klavins.jpg|<center>Eric Klavins <br/> Electrical Engineering</center><br />
</gallery><br />
<br />
<br />
<gallery caption="Collaborators and Support" widths="140px" heights="100px" perrow="5"><br />
<br />
Image:Washington_OSLI.png|<center><b>Oil Sand Leadership Initiative</b> <br/> Funding to support Travel and Registration Costs</center><br />
<br />
Image:Washington_UniversitySeal.gif|<center><b>Biochemistry</b> <br/> Lab space</center><br />
<br />
Image:Washington_Anaspec.gif|<center><b>Anaspec</b> <br/> Peptide Discounts</center><br />
<br />
Image:Washington_ARPA-E_Logo.png|<center><b>Advanced Research Projects Agency - Energy </b><br/> Registration Support</center><br />
<br />
Image:Washington2011_Hhmi_362_72.jpg|<center><b>Howard Hughes Medical Institute</b> <br/> Lab materials and supplies</center><br />
<br />
<br />
</gallery><br />
<br />
== '''Who did what''' ==<br />
The teams were assembled during our winter quarter. During this term we went around to classes and had weekly meetings to introduce students to synthetic biology through a series of guest lectures. During the spring, students participated in brainstorming projects related to the sponsoring Faculty advisors labs (Baker and Klavins) and came up with the project they wanted to carry out. Students also planned and partcipated in community outreach events where we taught our community about synthetic biology. Over the summer, students completed all of the experimental work, but worked closely with both graduate students and faculty advisors to plan the most pertinent experiments and make the most of our limited time.<br />
<br />
=== Diesel Production ===<br />
After producing promising results, in future directions <br />
<br />
Casey Ager, Austin Moon, and Seth Sagulo worked on Enzyme Localization via Direct Fusion and Zinc Finger Fusion methods.<br />
<br />
Juhye An, Elaine Lai, and Benjamin Mo worked on Decarbonylase Redesign<br />
<br />
Marika Cheng and Justin De Leon worked on Alternative Chassis<br />
<br />
Chris Choe and David Zong worked on Alternate Aldehyde Production<br />
<br />
Matthew Harger worked on Branched Alkanes Production<br />
<br />
Matthew Harger and Lei Zheng worked on System Optimization<br />
<br />
=== Gluten Destruction ===<br />
Sydney Gordon, Daniel Hadidi, Liz Stanley, Angus Toland, Sarah Wolf, and Sean Wu designed, built, and tested Kumamolisin-As and over 100 mutants to combat gluten intolerance by increasing the activity on the PQLP antigenic peptide.<br />
<br />
=== iGEM Toolkits ===<br />
Michael Brasino, Rashmi Ravichandran, and Alicia Wong<br />
<br />
=== Community Outreach ===<br />
Cindy Wu headed and organized all iGEM 2011 Community Outreach events.</div>Hadidihttp://2011.igem.org/Team:Washington/Team/MembersTeam:Washington/Team/Members2011-09-23T03:15:22Z<p>Hadidi: /* Who we are */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------><br />
<br />
== '''Who we are''' ==<br />
<br />
<gallery caption="Undergraduate Team Members" widths="180px" heights="120px" perrow="4"><br />
Image:Washington Casey Ager Profile1.jpg|<center>Casey Ager <br/> Major</center><br />
Image:Washington Photo-0088A.png|<center>Juhye An <br/> Major</center><br />
Image: Profile_Michael_Brasino.jpg|<center>Michael Brasino <br/> Major</center><br />
Image:Washington_.jpg|<center>Marika Cheng <br/> Major</center><br />
Image:Washington_choe_chris.png|<center>Chris Choe <br/> Major</center><br />
Image:Washington_.jpg|<center>Justin De Leon <br/> Major</center><br />
File:Princess Peachy.JPG|<center>Sydney Gordon <br/> Major</center><br />
Image:CIMG0021.jpg|<center>Daniel Hadidi <br/> Neurobiology</center><br />
Image:Washington_Matthew_Harger.JPG|<center>Matthew Harger <br/> Major</center><br />
Image:Washington_Elaine.jpg|<center>Elaine Lai <br/> Major</center><br />
Image:Washington_2011_BATMAN.jpg|<center>Benjamin Mo <br/> Major</center><br />
Image:Washington 2011 CIMG0015-2.jpg|<center>Austin Moon <br/> Major</center><br />
Image:Washington_.jpg|<center>Rashmi Ravichandran <br/> Major</center><br />
Image:Washington_.jpg|<center>Seth Sagulo <br/> Major</center><br />
File:Washington_Liz.png|<center>Liz Stanley <br/> Major</center><br />
Image:Washington_.jpg|<center>Angus Toland <br/> Major</center><br />
Image:Washington_.jpg|<center>Sarah Wolf <br/> Major</center><br />
Image:Washington_.jpg|<center>Alicia Wong <br/> Major</center><br />
Image:Washington_Cindy.jpg|<center>Cindy Wu <br/> Major</center><br />
Image:Washington_Sean_Wu.jpg|<center>Sean Wu <br/> Major</center><br />
Image:Washington_2011_BATMAN.jpg|<center>Lei Zheng <br/> Major</center><br />
Image:Washington_david_zong.jpg|<center>David Zong <br/> Major</center><br />
<br />
</gallery><br />
<br />
<br />
<gallery caption="Advisors" widths="180px" heights="120px" perrow="4"><br />
Image:Washington_.jpg|<center>Aaron Chevalier <br/> Bioengineering </center><br />
Image:Washington_.jpg|<center>Rob Egbert <br/> Electrical Engineering </center><br />
Image:Washington_.jpg|<center>Chris Eiben <br/> Biochemistry </center><br />
Image:Washington_.jpg|<center>Jeremy Mills <br/> Biochemistry </center><br />
Image:Siegel_UW_2011.jpg|<center>Justin Siegel <br/> Biomolecular Structure and Design</center><br />
Image:Washington2011_Matthew-d-smith.jpg|<center>Matt Smith <br/> Molecular and Cellular Biology</center><br />
Image:Washington_.jpg|<center>Ingrid Swanson <br/> Microbiology </center><br />
</gallery><br />
<br />
<br />
<gallery caption="Faculty" widths="180px" heights="120px" perrow="5"><br />
image:Washington_David_Baker.jpg|<center>David Baker <br/> Biochemistry</center><br />
Image:Washington_Klavins.jpg|<center>Eric Klavins <br/> Electrical Engineering</center><br />
</gallery><br />
<br />
<br />
<gallery caption="Collaborators and Support" widths="140px" heights="100px" perrow="5"><br />
<br />
Image:Washington_OSLI.png|<center><b>Oil Sand Leadership Initiative</b> <br/> Funding to support Travel and Registration Costs</center><br />
<br />
Image:Washington_UniversitySeal.gif|<center><b>Biochemistry</b> <br/> Lab space</center><br />
<br />
Image:Washington_Anaspec.gif|<center>Anaspec <br/> <b>Peptide Discounts</b></center><br />
<br />
Image:Washington_ARPA-E_Logo.png|<center><b>Advanced Research Projects Agency - Energy </b><br/> Registration Support</center><br />
<br />
Image:Washington2011_Hhmi_362_72.jpg|<center><b>Howard Hughes Medical Institute</b> <br/> Lab materials and supplies</center><br />
<br />
<br />
</gallery><br />
<br />
== '''Who did what''' ==<br />
The teams were assembled during our winter quarter. During this term we went around to classes and had weekly meetings to introduce students to synthetic biology through a series of guest lectures. During the spring, students participated in brainstorming projects related to the sponsoring Faculty advisors labs (Baker and Klavins) and came up with the project they wanted to carry out. Students also planned and partcipated in community outreach events where we taught our community about synthetic biology. Over the summer, students completed all of the experimental work, but worked closely with both graduate students and faculty advisors to plan the most pertinent experiments and make the most of our limited time.<br />
<br />
=== Alkanes Production ===<br />
After producing promising results, in future directions <br />
<br />
Casey Ager, Austin Moon, and Seth Sagulo worked on Enzyme Localization via Direct Fusion and Zinc Finger Fusion methods.<br />
<br />
Juhye An, Elaine Lai, and Benjamin Mo worked on Decarbonylase Redesign<br />
<br />
Marika Cheng and Justin De Leon worked on Alternative Chassis<br />
<br />
Chris Choe and David Zong worked on Alternate Aldehyde Production<br />
<br />
Matthew Harger worked on Branched Alkanes Production<br />
<br />
Matthew Harger and Lei Zheng worked on System Optimization<br />
<br />
=== Gluten Degradation ===<br />
Sydney Gordon, Daniel Hadidi, Liz Stanley, Angus Toland, Sarah Wolf, and Sean Wu designed, built, and tested Kumamolisin-As and over 100 mutants to combat gluten intolerance by increasing the activity on the PQLP antigenic peptide.<br />
<br />
=== iGEM Toolkits ===<br />
Michael Brasino, Rashmi Ravichandran, and Alicia Wong<br />
<br />
=== Community Outreach ===<br />
Cindy Wu headed and organized all iGEM 2011 Community Outreach events.</div>Hadidihttp://2011.igem.org/Team:Washington/Alkanes/BackgroundTeam:Washington/Alkanes/Background2011-09-23T02:41:04Z<p>Hadidi: /* The Ideal Fuel is Diesel */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Background'''</big></big></big></big></center><br><br><br />
<br />
='''Petroleum, an Unfortunate Necessity'''=<br />
[[Image:Washington2011_PetroUsage.png|border|450px|right|thumb|Petroleum may be a necessity for modern civilization, but current extraction methods are unsustainable and non-cyclical in the long run.]]<br />
<br />
Modern society is completely dependent on petroleum based fuels. Automobiles are slowly transitioning towards electric power. However, for the foreseeable future, batteries will not be able to hold the energy needed for applications that require long range (e.g. jet planes, maritime shipping, and long range trucking) or high horsepower (e.g. agriculture, construction, industry). Without the use of petroleum, society as we know it would crumble. Petroleum is not a viable long term fuel due because it a non-renewable resource. When petroleum based fuels are combusted, CO<sub>2</sub> is released into the atmosphere. Using current technology, it is impossible to turn this carbon dioxide back into fuel, meaning that the amount of petroleum based fuel is a finite commodity. In addition, this excess carbon dioxide is a potent greenhouse gas that contributes to global warming.<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
='''Todays biofuels are ''renewable'', but do not work as "drop-in" replacements'''=<br />
<br />
<br />
[[Image:Washington2011_BiofuelsAreRenewable.png|right|450px|frameless|thumb|Since carbon in biofuels can come from cellular respiration, biofuels may be carbon neutral. However, current biofuels have clear and problematic limits concerning energy production.]]<br />
<br />
Many different attempts have been made to produce a renewable, biologically derived fuel that would alleviate both the limited supply and emissions issues presented by petroleum based fuels. These efforts include alcohols (ethanol, butanol and other, higher alcohols), and biodiesel . Like petroleum based fuels, biofuels consist of combustible molecules that emit carbon dioxide. However, unlike petroleum based fuels, biofuels are renewable. CO<sub>2</sub> can be converted into more biofuel by feeding biofuel producing microbes (bacteria, yeast) photosynthetically derived plant biomass. Since the amount of CO<sub>2</sub> produced by burning a biofuel cannot exceed the CO<sub>2</sub> incorporated into plant biomass, a biofuel can be used indefinitely without any net carbon emissions.<br />
However, current biofuels consist of drastically different compounds from those found in petroleum. Petroleum consists of mostly long-chain length alkanes consisting of long hydrocarbon chains. Current biofuels contain either alcohols or long chain esters (biodiesel). Both of these molecules contain oxygen, which dramatically changes chemical properties. Both alcohols and biodiesel are more corrosive than unreactive alkanes. Alcohols are highly corrosive, both in pipelines ( [[#References | [1]]] ), and in engines not designed for the use of alcohols, even at concentrations as low as 20%( [[#References | [2]]] ). The corrosive property of alcohols in pipelines means that ethanol (the main alcohol in widespread use) is transported in vehicles (mostly by train)(cite), as opposed to by cheaper and less energy intensive pipelines( [[#References | [1]]] ). Transport of alcohols by pipeline would require retrofitting the entire fuel distribution infrastructure. The Esters in biodiesel are not directly as corrosive as alcohols, but can be biodegraded by anaerobic bacteria, producing hydrogen sulfide and other acids( [[#References | [3]]] ). Biodiesel has a higher freezing point than diesel, causing engine fuel filer clogging at low temperatures( [[#References | [4]]] ). Ethanol suffers from a much lower energy density than diesel(21.27-23.56 MJ/L vs 32.36-34.66 MJ/L)( [[#References | [5]]] ), resulting in lower gas mileage. The table below shows selected chemical properties of diesel, JP-8 (jet fuel), as well as the common biofuels ethanol, butanol, and biodiesel( data from [[#References | [5]]] and [[#References | [6]]]). <br />
{| {{table}}<br />
| align="center" style="background:#f0f0f0;"|'''Property'''<br />
| align="center" style="background:#f0f0f0;"|'''Diesel'''<br />
| align="center" style="background:#f0f0f0;"|'''Ethanol'''<br />
| align="center" style="background:#f0f0f0;"|'''Biodiesel'''<br />
|-<br />
| Specific gravity @ 15.5°C||0.85||0.794||0.88<br />
|-<br />
| Density @ 15.5°C(g/L)||848.25||792.05||878.09<br />
|-<br />
| Energy Density(MJ/L)||32.36-34.66 ||21.27-23.56 ||33.32-35.66<br />
|-<br />
| Cetane number||40-55||0-54||48-65<br />
|-<br />
| Freezing point(°C)||-40 - -1||-114||-3 -19<br />
|-<br />
| Viscosity( @ 20°C)(mm<sup>2</sup>/s)||2.8-5.0||1.5||6.4-6.6<br />
|-<br />
| Flash point(°C)||60-80||12.8||100-170<br />
|-<br />
| <br />
|} <br />
<br />
<br />
----<br />
<br />
<br />
='''The Ideal Fuel is Microbially-Produced Diesel'''=<br />
<br />
[[Image:Washington2011_PetroBiofuel.png|frameless|left|400px]]<br />
:::::::::::::::::<p>The ideal fuel would be compatible with modern engines and infrastructure, and also be able to be produced in a renewable manner. No current biofuel has the same identical enough to that of diesel to be able to fully integrate with current engines and infrastructure. No known alternative fuel is able to match the chemical properties of diesel. Currently, the only way to renewably produce a fuel with the chemical properties and compatibility of diesel would be to make a biofuel with a composition identical to that of diesel. This would require a biological pathway that is able to produce alkanes, the main class of compounds in diesel. Alkanes are simple chains of carbon and hydrogen. The majority of the alkanes found in diesel have a carbon chain of 10 to 20 carbons long. Alkanes make up approximately 62% of jet diesel (a fairly representative diesel fuel)([[#References | [7]]]). This 62% includes 34% straight chain alkanes that contain only one linear chain, and 28% branched chain alkanes that contain 1 or more carbon branches. The remaining 38% consists mostly of cyclic and aromatic hydrocarbons. If long (10+) chain length alkanes could be biologically produced, it would allow for the production of a fuel that is both renewable and fully compatible with current engines and infrastructure.</p><br />
<br />
<br />
----<br />
<br />
='''The Solution: a Microbial Alkane Production Pathway'''=<br />
[[Image:Washington2011_AlkaneAndBackCycle.png|right|400px|frameless|thumb|The use of AAR and ADC converts the fatty acid intermediate Acl-ACP into an aldehyde, then an alkane--biofuel. Acyl-ACP is naturally produced by all organisms, which increases host choice considerably]]<br />
A recent study([[#References | [8]]]),has shown the production of long chain length alkanes in ''E. coli'' using two genes found in many cyanobacteria species. The first gene codes for Acyl-ACP Reductase (AAR) which reduces long chain length acyl-ACPs into the corresponding fatty aldehydes. Acyl-ACPs are essential intermediates in fatty acid biosynthesis in every known organism, meaning that this system can work in a wide range of organisms. This long chain fatty acid acts as a substrate for Aldehyde Decarbonylase (ADC), the enzyme that removes the carbonyl group (C=O) from the fatty aldehyde, yielding an alkane one carbon shorter than the original Acyl-ACP and a molecule of formate. Since the vast majority of the fatty acyl-ACPs produced by ''E. coli'' have an even chain length, this system produces detectable amounts of only odd chain length alkanes. This study reported production of the C13, C15, and C17 alkanes, as well as the C17 alkene (unsaturated hydrocarbon). This chain length range fall well within the range of those found in diesel, so this system is theoretically able to make the alkane portion of a fuel compatible with current engines and infrastructure.<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
<br />
='''The PetroBrick: A modular and open platform for the biological production of diesel fuel'''=<br />
<br />
[[Image:Washington_2011_PetroBrick.png|200px|frameless|border|left|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
The goal of our "'''''Make It: Diesel Production'''''" portion of this summer's iGEM project is to turn convert this recently discovered set of enzymes (AAR and ADC) for microbial alkane production into an open and modular platform for iGEM teams to develop into a robust replacement for petrochemical fuels. Our alkane production system is specifically designed to be easily improved upon, and we have started work on improving this open system, both by increasing alkane yields and by changing the product produced. In addition, we have started to move this system into an alternative chassis, yeast.<br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
<br />
----<br />
<br />
<br />
==References:==<br />
1. http://ourenergypolicy.org/docs/2/biofuels-taskforce.pdf<br />
<br />
2. http://www.environment.gov.au/atmosphere/fuelquality/publications/2000hours-vehicle-fleet/pubs/2000-hours-vehicles.pdf<br />
<br />
3. Anaerobic Metabolism of Biodiesel and Its Impact on Metal Corrosion<br />
Deniz F. Aktas, Jason S. Lee, Brenda J. Little, Richard I. Ray, Irene A. Davidova, Christopher N. Lyles, Joseph M. Suflita Energy & Fuels 2010 24 (5), 2924-2928(http://pubs.acs.org/doi/full/10.1021/ef100084j)<br />
<br />
4. http://www.mda.state.mn.us/news/publications/renewable/biodiesel/biodieselcoldissues.pdf<br />
<br />
5. http://www.afdc.energy.gov/afdc/pdfs/fueltable.pdf<br />
<br />
6. The viscosities of three biodiesel fuels at temperatures up to 300°C<br />
R.E. Tate, K.C. Watts, C.A.W. Allen K.I. Wilkie Fuel 2006 85, 1010-1015 (https://netfiles.uiuc.edu/mccrady/shared/Biodiesel/The%20viscosities%20of%20three%20biodiesel%20fuels%20at%20temperatures%20up%20to%20300%208C.pdf)<br />
<br />
7. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
8. Microbial Biosynthesis of Alkanes Andreas Schirmer, Mathew A. Rude, Xuezhi Li, Emanuela Popova and Stephen B. del Cardayre Science 30 July 2010: Vol. 329 no. 5991 pp. 559-562 http://www.sciencemag.org/content/329/5991/559</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/FutureTeam:Washington/Celiacs/Future2011-09-23T02:37:51Z<p>Hadidi: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Gluten Destruction: Future Directions'''</big></big></big></big></center><br><br><br />
<br />
====Kinetic Characterization====<br />
<br />
We intend to develop a mass spectroscopy assay to measure the kcat (the rate of turnover per molecule of enzyme) and kM (the substrate binding constant) values for our best mutant.<br />
<br />
====Specificity Studies====<br />
<br />
====Crystal Structure====<br />
<br />
Protein structures can be obtained by crystallizing the protein, then bombarding it with X-rays and analyzing the diffraction pattern. Several crystal structures for wild-type Kumamolisin-As have already been published. To obtain further structural information, we hope to eventually obtain a crystal structure of our best mutated enzyme (G319S, D358G,D368H, N291D) bound to the PQLP model substrate. This will involve mutating one of the residues in the catalytic triad, so that the substrate will remain bound without being cleaved. The structural information that we may be able to glean from such a structure will allow us to better characterize, and perhaps further improve our mutant.<br />
<br />
====Biophysical Characterization==== <br />
<br />
Once the ideal mutations are isolated, we intend to test the best mutants at gastric pH and in the presence of other gastric enzymes for a short period of time, mimicking the environment after enzyme ingestion and prior to enzyme uptake by the small intestine. We suspect that the thermostable properties of Kumamolisin-As will render our mutant enzyme reasonably resistant to degradation by gastric enzymes such as pepsin. However, if this proves not to be the case, we intend to reengineer the mutant for enhanced resistance to pepsin and other such gastric enzymes. Once ensured that the mutated Kumamolisin-As remains active under stomach conditions, this ideal mutated enzyme will be ready for ''in vivo'' experimentation.<br />
<br />
[[File:Washington_Kumamolisin_VS_ScPEP_NEW.png|250px|thumb|right|Comparison of Theoretical Pills of ScPEP and Kumamolisin (G319S, D358G,D368H, N291D)]]<br />
<br />
====Therapeutic Promise====<br />
<br />
After successful completion of in vivo experimentation and clinical trials, treatment in capsule-form can be released for commercial use. Due to the much higher activity of our Kumamolisin mutant over SC-PEP, the amount of enzyme necessary for treatment can be greatly reduced.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/FutureTeam:Washington/Celiacs/Future2011-09-23T02:37:06Z<p>Hadidi: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Gluten Destruction: Future Directions'''</big></big></big></big></center><br><br><br />
<br />
====Kinetic Characterization====<br />
<br />
We intend to develop a mass spectroscopy assay to measure the kcat (the rate of turnover per molecule of enzyme) and kM (the substrate binding constant) values for our best mutant.<br />
<br />
====Specificity Studies====<br />
<br />
====Crystal Structure====<br />
<br />
Protein structures can be obtained by crystallizing the protein, then bombarding it with X-rays and analyzing the diffraction pattern. Several crystal structures for wild-type Kumamolisin-As have already been published. To obtain further structural information, we hope to eventually obtain a crystal structure of our best mutated enzyme (G319S, D358G,D368H, N291D) bound to the PQLP model substrate. This will involve mutating one of the residues in the catalytic triad, so that the substrate will remain bound without being cleaved. The structural information that we may be able to glean from such a structure will allow us to better characterize, and perhaps further improve our mutant.<br />
<br />
====Biophysical Characterization==== <br />
<br />
Once the ideal mutations are isolated, we intend to test the best mutants at gastric pH and in the presence of other gastric enzymes for a short period of time, mimicking the environment after enzyme ingestion and prior to enzyme uptake by the small intestine. We suspect that the thermostable properties of Kumamolisin-As will render our mutant enzyme reasonably resistant to degradation by gastric enzymes such as pepsin. However, if this proves not to be the case, we intend to reengineer the mutant for enhanced resistance to pepsin and other such gastric enzymes. Once ensured that the mutated Kumamolisin-As remains active under stomach conditions, this ideal mutated enzyme will be ready for ''in vivo'' experimentation.<br />
<br />
[[File:Washington_Kumamolisin_VS_ScPEP_NEW.png|250px|thumb|right|Comparison of Theoretical Pills of ScPEP and Kumamolisin (G319S, D358G,D368H, N291D)]]<br />
<br />
<br />
<br />
====Therapeutic Promise====<br />
<br />
After successful completion of in vivo experimentation and clinical trials, treatment in capsule-form can be released for commercial use. Due to the much higher activity of our Kumamolisin mutant over SC-PEP, the amount of enzyme necessary for treatment can be greatly reduced.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/FutureTeam:Washington/Celiacs/Future2011-09-23T02:36:20Z<p>Hadidi: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Gluten Destruction: Future Directions'''</big></big></big></big></center><br><br><br />
<br />
====Kinetic Characterization====<br />
<br />
We intend to develop a mass spectroscopy assay to measure the kcat (the rate of turnover per molecule of enzyme) and kM (the substrate binding constant) values for our best mutant.<br />
<br />
====Specificity Studies====<br />
<br />
====Crystal Structure====<br />
<br />
Protein structures can be obtained by crystallizing the protein, then bombarding it with X-rays and analyzing the diffraction pattern. Several crystal structures for wild-type Kumamolisin-As have already been published. To obtain further structural information, we hope to eventually obtain a crystal structure of our best mutated enzyme (G319S, D358G,D368H, N291D) bound to the PQLP model substrate. This will involve mutating one of the residues in the catalytic triad, so that the substrate will remain bound without being cleaved. The structural information that we may be able to glean from such a structure will allow us to better characterize, and perhaps further improve our mutant.<br />
<br />
====Biophysical Characterization==== <br />
<br />
Once the ideal mutations are isolated, we intend to test the best mutants at gastric pH and in the presence of other gastric enzymes for a short period of time, mimicking the environment after enzyme ingestion and prior to enzyme uptake by the small intestine. We suspect that the thermostable properties of Kumamolisin-As will render our mutant enzyme reasonably resistant to degradation by gastric enzymes such as pepsin. However, if this proves not to be the case, we intend to reengineer the mutant for enhanced resistance to pepsin and other such gastric enzymes. Once ensured that the mutated Kumamolisin-As remains active under stomach conditions, this ideal mutated enzyme will be ready for ''in vivo'' experimentation.<br />
<br />
[[File:Washington_Kumamolisin_VS_ScPEP_NEW.png|250px|thumb|right|Comparison of Theoretical Pills of ScPEP and Kumamolisin (G319S, D358G,D368H, N291D)]]<br />
<br />
<br />
<br />
<br />
<br />
====Therapeutic Promise====<br />
<br />
After successful completion of in vivo experimentation and clinical trials, treatment in capsule-form can be released for commercial use. Due to the much higher activity of our Kumamolisin mutant over SC-PEP, the amount of enzyme necessary for treatment can be greatly reduced.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/FutureTeam:Washington/Celiacs/Future2011-09-23T02:35:26Z<p>Hadidi: /* Therapeutic Promise */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>Gluten Destruction: Future Directions</big></big></big></big></center><br><br><br />
<br />
====Kinetic Characterization====<br />
<br />
We intend to develop a mass spectroscopy assay to measure the kcat (the rate of turnover per molecule of enzyme) and kM (the substrate binding constant) values for our best mutant.<br />
<br />
====Specificity Studies====<br />
<br />
====Crystal Structure====<br />
<br />
Protein structures can be obtained by crystallizing the protein, then bombarding it with X-rays and analyzing the diffraction pattern. Several crystal structures for wild-type Kumamolisin-As have already been published. To obtain further structural information, we hope to eventually obtain a crystal structure of our best mutated enzyme (G319S, D358G,D368H, N291D) bound to the PQLP model substrate. This will involve mutating one of the residues in the catalytic triad, so that the substrate will remain bound without being cleaved. The structural information that we may be able to glean from such a structure will allow us to better characterize, and perhaps further improve our mutant.<br />
<br />
====Biophysical Characterization==== <br />
<br />
Once the ideal mutations are isolated, we intend to test the best mutants at gastric pH and in the presence of other gastric enzymes for a short period of time, mimicking the environment after enzyme ingestion and prior to enzyme uptake by the small intestine. We suspect that the thermostable properties of Kumamolisin-As will render our mutant enzyme reasonably resistant to degradation by gastric enzymes such as pepsin. However, if this proves not to be the case, we intend to reengineer the mutant for enhanced resistance to pepsin and other such gastric enzymes. Once ensured that the mutated Kumamolisin-As remains active under stomach conditions, this ideal mutated enzyme will be ready for ''in vivo'' experimentation.<br />
<br />
[[File:Washington_Kumamolisin_VS_ScPEP_NEW.png|250px|thumb|right|Comparison of Theoretical Pills of ScPEP and Kumamolisin (G319S, D358G,D368H, N291D)]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
====Therapeutic Promise====<br />
<br />
After successful completion of in vivo experimentation and clinical trials, treatment in capsule-form can be released for commercial use. Due to the much higher activity of our Kumamolisin mutant over SC-PEP, the amount of enzyme necessary for treatment can be greatly reduced.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/FutureTeam:Washington/Celiacs/Future2011-09-23T02:32:32Z<p>Hadidi: /* Therapeutic Promise */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>Gluten Destruction: Future Directions</big></big></big></big></center><br><br><br />
<br />
====Kinetic Characterization====<br />
<br />
We intend to develop a mass spectroscopy assay to measure the kcat (the rate of turnover per molecule of enzyme) and kM (the substrate binding constant) values for our best mutant.<br />
<br />
====Specificity Studies====<br />
<br />
====Crystal Structure====<br />
<br />
Protein structures can be obtained by crystallizing the protein, then bombarding it with X-rays and analyzing the diffraction pattern. Several crystal structures for wild-type Kumamolisin-As have already been published. To obtain further structural information, we hope to eventually obtain a crystal structure of our best mutated enzyme (G319S, D358G,D368H, N291D) bound to the PQLP model substrate. This will involve mutating one of the residues in the catalytic triad, so that the substrate will remain bound without being cleaved. The structural information that we may be able to glean from such a structure will allow us to better characterize, and perhaps further improve our mutant.<br />
<br />
====Biophysical Characterization==== <br />
<br />
Once the ideal mutations are isolated, we intend to test the best mutants at gastric pH and in the presence of other gastric enzymes for a short period of time, mimicking the environment after enzyme ingestion and prior to enzyme uptake by the small intestine. We suspect that the thermostable properties of Kumamolisin-As will render our mutant enzyme reasonably resistant to degradation by gastric enzymes such as pepsin. However, if this proves not to be the case, we intend to reengineer the mutant for enhanced resistance to pepsin and other such gastric enzymes. Once ensured that the mutated Kumamolisin-As remains active under stomach conditions, this ideal mutated enzyme will be ready for ''in vivo'' experimentation.<br />
<br />
====Therapeutic Promise====<br />
<br />
After successful completion of in vivo experimentation and clinical trials, treatment in capsule-form can be released for commercial use. Due to the much higher activity of our Kumamolisin mutant over SC-PEP, the amount of enzyme necessary for treatment can be greatly reduced.<br />
<br />
[[File:Washington_Kumamolisin_VS_ScPEP_NEW.png|250px|thumb|right|Comparison of Theoretical Pills of ScPEP and Kumamolisin (G319S, D358G,D368H, N291D)]]</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/FutureTeam:Washington/Celiacs/Future2011-09-23T02:28:43Z<p>Hadidi: /* Therapeutic Promise */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>Gluten Destruction: Future Directions</big></big></big></big></center><br><br><br />
<br />
====Kinetic Characterization====<br />
<br />
We intend to develop a mass spectroscopy assay to measure the kcat (the rate of turnover per molecule of enzyme) and kM (the substrate binding constant) values for our best mutant.<br />
<br />
====Specificity Studies====<br />
<br />
====Crystal Structure====<br />
<br />
Protein structures can be obtained by crystallizing the protein, then bombarding it with X-rays and analyzing the diffraction pattern. Several crystal structures for wild-type Kumamolisin-As have already been published. To obtain further structural information, we hope to eventually obtain a crystal structure of our best mutated enzyme (G319S, D358G,D368H, N291D) bound to the PQLP model substrate. This will involve mutating one of the residues in the catalytic triad, so that the substrate will remain bound without being cleaved. The structural information that we may be able to glean from such a structure will allow us to better characterize, and perhaps further improve our mutant.<br />
<br />
====Biophysical Characterization==== <br />
<br />
Once the ideal mutations are isolated, we intend to test the best mutants at gastric pH and in the presence of other gastric enzymes for a short period of time, mimicking the environment after enzyme ingestion and prior to enzyme uptake by the small intestine. We suspect that the thermostable properties of Kumamolisin-As will render our mutant enzyme reasonably resistant to degradation by gastric enzymes such as pepsin. However, if this proves not to be the case, we intend to reengineer the mutant for enhanced resistance to pepsin and other such gastric enzymes. Once ensured that the mutated Kumamolisin-As remains active under stomach conditions, this ideal mutated enzyme will be ready for ''in vivo'' experimentation.<br />
<br />
====Therapeutic Promise====<br />
<br />
After successful completion of in vivo experimentation and clinical trials, <br />
treatment in capsule-form can be released for commercial use. <br />
Due to the much higher activity of our Kumamolisin mutant over SC-PEP, <br />
drug volume necessary for treatment can be greatly reduced.<br />
<br />
[[File:Washington_Kumamolisin_VS_ScPEP_NEW.png|250px|thumb|right|Comparison of Theoretical Pills of ScPEP and Kumamolisin (G319S, D358G,D368H, N291D)]]</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/FutureTeam:Washington/Celiacs/Future2011-09-23T02:21:41Z<p>Hadidi: /* Therapeutic Promise */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>Gluten Destruction: Future Directions</big></big></big></big></center><br><br><br />
<br />
====Kinetic Characterization====<br />
<br />
We intend to develop a mass spectroscopy assay to measure the kcat (the rate of turnover per molecule of enzyme) and kM (the substrate binding constant) values for our best mutant.<br />
<br />
====Specificity Studies====<br />
<br />
====Crystal Structure====<br />
<br />
Protein structures can be obtained by crystallizing the protein, then bombarding it with X-rays and analyzing the diffraction pattern. Several crystal structures for wild-type Kumamolisin-As have already been published. To obtain further structural information, we hope to eventually obtain a crystal structure of our best mutated enzyme (G319S, D358G,D368H, N291D) bound to the PQLP model substrate. This will involve mutating one of the residues in the catalytic triad, so that the substrate will remain bound without being cleaved. The structural information that we may be able to glean from such a structure will allow us to better characterize, and perhaps further improve our mutant.<br />
<br />
====Biophysical Characterization==== <br />
<br />
Once the ideal mutations are isolated, we intend to test the best mutants at gastric pH and in the presence of other gastric enzymes for a short period of time, mimicking the environment after enzyme ingestion and prior to enzyme uptake by the small intestine. We suspect that the thermostable properties of Kumamolisin-As will render our mutant enzyme reasonably resistant to degradation by gastric enzymes such as pepsin. However, if this proves not to be the case, we intend to reengineer the mutant for enhanced resistance to pepsin and other such gastric enzymes. Once ensured that the mutated Kumamolisin-As remains active under stomach conditions, this ideal mutated enzyme will be ready for ''in vivo'' experimentation.<br />
<br />
====Therapeutic Promise====<br />
<br />
After successful completion of in vivo experimentation and clinical trials, treatment in capsule-form can be released for commercial use. Due to the much higher activity of our Kumamolisin mutant over SC-PEP, drug volume necessary for treatment can be greatly reduced.<br />
<br />
[[File:Washington_Kumamolisin_VS_ScPEP_NEW.png|250px|thumb|right|Comparison of Theoretical Pills of ScPEP and Kumamolisin (G319S, D358G,D368H, N291D)]]</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/FutureTeam:Washington/Celiacs/Future2011-09-23T02:20:27Z<p>Hadidi: /* Therapeutic Promise */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>Gluten Destruction: Future Directions</big></big></big></big></center><br><br><br />
<br />
====Kinetic Characterization====<br />
<br />
We intend to develop a mass spectroscopy assay to measure the kcat (the rate of turnover per molecule of enzyme) and kM (the substrate binding constant) values for our best mutant.<br />
<br />
====Specificity Studies====<br />
<br />
====Crystal Structure====<br />
<br />
Protein structures can be obtained by crystallizing the protein, then bombarding it with X-rays and analyzing the diffraction pattern. Several crystal structures for wild-type Kumamolisin-As have already been published. To obtain further structural information, we hope to eventually obtain a crystal structure of our best mutated enzyme (G319S, D358G,D368H, N291D) bound to the PQLP model substrate. This will involve mutating one of the residues in the catalytic triad, so that the substrate will remain bound without being cleaved. The structural information that we may be able to glean from such a structure will allow us to better characterize, and perhaps further improve our mutant.<br />
<br />
====Biophysical Characterization==== <br />
<br />
Once the ideal mutations are isolated, we intend to test the best mutants at gastric pH and in the presence of other gastric enzymes for a short period of time, mimicking the environment after enzyme ingestion and prior to enzyme uptake by the small intestine. We suspect that the thermostable properties of Kumamolisin-As will render our mutant enzyme reasonably resistant to degradation by gastric enzymes such as pepsin. However, if this proves not to be the case, we intend to reengineer the mutant for enhanced resistance to pepsin and other such gastric enzymes. Once ensured that the mutated Kumamolisin-As remains active under stomach conditions, this ideal mutated enzyme will be ready for ''in vivo'' experimentation.<br />
<br />
====Therapeutic Promise====<br />
<br />
After successful completion of in vivo experimentation and clinical trials, treatment in capsule-form can be released for commercial use. Due to the much higher activity of our Kumamolisin mutant over SC-PEP, drug volume necessary for treatment can be greatly reduced.<br />
<br />
[[File:Washington_Kumamolisin_VS_ScPEP_NEW.png|250px|thumb|left|Comparison of Theoretical Pills of ScPEP and Kumamolisin (G319S, D358G,D368H, N291D)]]</div>Hadidihttp://2011.igem.org/Team:Washington/Magnetosomes/PartsTeam:Washington/Magnetosomes/Parts2011-09-23T02:09:11Z<p>Hadidi: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>iGEM Toolkits: Parts Submitted</big></big></big></big></center><br><br><br />
<br />
iGem Toolkits: submitted a total twenty-two parts to the registry: <br />
*10 magnetosome gene-groups, <br />
*2 mam gene-sfGFP fusions, <br />
*3 parts of the essential magnetosome gene- assembly, and <br />
*5 pGA vectors.<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
! Gene <br />
! AMB Number<br />
! Cluster Membership<br />
! Member of 28 genes list? (specific*/related**)<br />
! Function Summary (Vesicle chain formation, and/or biomineralization)<br />
! Gene Function<br />
|-<br />
| mamH<br />
| amb0961<br />
| mamAB<br />
| Related<br />
|<br />
|<br />
|-<br />
| mamI<br />
| amb0962<br />
| mamAB<br />
| Specific<br />
| Vesicle, (Chain Formation?)<br />
| >berkeley 2010: Loss causes no membrane formation, is localized onto chains<br />
|-<br />
| mamE<br />
| amb0963<br />
| mamAB; mam Islet<br />
| Related<br />
|<br />
| >Membrane-bound serine protease required for magnetite formation; might control the localization of other magnetosome proteins<br />
|-<br />
| mamJ<br />
| amb0964<br />
| mamAB; mam Islet<br />
| Specific<br />
| Chain Formation<br />
| >Proper magnetosome chain organization/assembly<br />
|-<br />
| mamK<br />
| amb0965<br />
| mamAB; mam Islet<br />
| Related<br />
| Chain Formation<br />
| >required for proper magnetosome chain organization; *bacterial actin-like cytoskeleton protein required for proper alignment of the magnetosomes in a chain, shown to localize the mamI<br />
|- <br />
| mamL<br />
| amb0966<br />
| mamAB; mam Islet<br />
| Specific<br />
| Vesicle, biomineralization<br />
| >berkely 2010: Crucial to mangneosome membrane creation, shown to be spread across the cell membrane and sometimes forms lines<br />
|-<br />
| mamM<br />
| amb0967<br />
| mamAB<br />
| Related<br />
|<br />
| >biomineralization, involved in iron transport, magnetite nucleation, or establishement of the proper chemical enviornment for magnetite synthesis in the magnetosome<br />
|-<br />
| mamN<br />
| amb0968<br />
| mamAB<br />
| Related<br />
|<br />
| >biomineralization, involved in iron transport, magnetite nucleation, or establishement of the proper chemical enviornment for magnetite synthesis in the magnetosome<br />
|-<br />
| mamO<br />
| amb0969<br />
| mamAB<br />
| Related<br />
|<br />
| >biomineralization, involved in iron transport, magnetite nucleation, or establishement of the proper chemical enviornment for magnetite synthesis in the magnetosome<br />
|- <br />
| mamP<br />
| amb0970<br />
| mamAB<br />
| Related<br />
| Biomineralization<br />
| >berkeley 2010: loss causes weak magnetic response, with large but fewer crystals<br />
|-<br />
| mamA<br />
| amb0971<br />
| mamAB<br />
| Related<br />
|<br />
| >Required for magnetosome activation; activation of vessicles<br />
|-<br />
| mamQ<br />
| amb0972<br />
| mamAB; mam Islet<br />
| Related<br />
|<br />
| >ORF; formation/maintenance of magnetosome membranes<br />
|-<br />
| mamR<br />
| amb0973<br />
| mamAB<br />
| Specific<br />
| Chain formation, Biomineralization<br />
| >ORF; plays a role in controlling both particle number and size of magnetite cyrstals<br />
|-<br />
| mamB<br />
| amb0974<br />
| mamAB<br />
| Related<br />
| Vesicle, Biomineralization<br />
| >indirect role in magnetosome membrane invagination and biomineralization; magnetosome compartment formation<br />
|-<br />
| mamS<br />
| amb0975<br />
| mamAB<br />
| Specific<br />
| <br />
|<br />
|-<br />
| mamT<br />
| amb0976<br />
| mamAB<br />
| Specific<br />
| Biomineralization<br />
| >magnetite crystal growth; participates in different steps during magnetite synthesis<br />
|-<br />
| mamU<br />
| amb0977<br />
| mamAB<br />
| Related<br />
| <br />
| <br />
|-<br />
| mamV<br />
| amb0978<br />
| mamAB<br />
| N/A<br />
|<br />
|<br />
|-<br />
|}</div>Hadidihttp://2011.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2011-09-23T02:03:00Z<p>Hadidi: /* Break It: Gluten Destruction Protocols */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>Protocols</big></big></big></big></center><br><br><br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/gel_electrophoresis General Agarose Gel Electrophoresis]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/PCR General PCR Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Digestion General Digestion Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Ligation General Ligation Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Transformation General Transformation Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Colony Colony PCR Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Competent Competent Cell Prep Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Kunkel Kunkel Mutagensis]<br />
<br />
[http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of how Kunkel Mutagensis works]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/expression_purification Standard 1L Expression Purification]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/gene_assembly Gene Assembly With Oligos]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/sequencing Sequencing]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/CompDesign Computational Protein Design]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Glycerol_Stocks Glycerol Stocks]<br />
<br />
<br />
=Make It: Diesel Production Protocols=<br />
<br />
[https://2011.igem.org/Team:Washington/alkanebiosynthesis Alkane Biosynthesis media and extraction]<br />
<br />
[https://2011.igem.org/Team:Washington/alkanebiosynthesis_cloning Alkane Biosynthesis cloning]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/redesign_cell_lysate_assay Cell Lysate Assay by Decarbonylase Redesign Team]<br />
<br />
<br />
=Break It: Gluten Destruction Protocols=<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Cell_Lysate_Assay Whole Cell Lysate Assay]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/50mL_Scale Small Scale (50mL) Protein Expression and Purification]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_Assay Enzyme Assay]<br />
<br />
=Make It: iGem Toolkits=<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Cyto. Cytometry Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Elect. Electroporation (Transformation)]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Gib_Rxn Gibson Cloning/Assembly]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Gib_Purif. Gibson Purification]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/High_PCR High-Yield PCR (Full-Gene Assembly)]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Plas_DNA. Isolation of Plasmid DNA (miniprep)]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Induc_studies. Induction Studies of Proteins Fusions (mam-sfGFP)]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/PBS. PBS Stock Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Overnights. Preparation of Overnight Cultures]<br />
<br />
<br />
=Wiki Design=<br />
[https://2011.igem.org/Team:Washington/Protocols/Wiki_Design Wiki Design Tools (Wiki Markup, WikiDust, etc.)]</div>Hadidihttp://2011.igem.org/Team:Washington/SafetyTeam:Washington/Safety2011-09-23T02:00:40Z<p>Hadidi: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
<center><big><big><big><big>Safety</big></big></big></big></center><br><br><br />
<br />
Please use this page to answer the safety questions posed on the [[Safety | safety page]].<br />
<br />
<br />
'''1. Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
'''<br />
All projects are being conducted in lab-safe strains of ''E. coli''. All researchers have been trained in applicable lab safety to insure that no bacteria are inadvertently released into the environment. The researchers have also been trained in proper handling of chemicals, which is required due to the work with alkanes which requires use of chemicals not normally used in a molecular biology lab. In both the Celiac's disease and alkane production projects, the actual organisms being engineered are intended to be maintained in lab conditions ( cultures, bioreactors, etc.). The active ingredient in our Celiac's disease treatment would be used as a purified protein, like many current protein therapeutics, and is consistant with current FDA guidelines. Extraction of alkanes from our alkane producing ''E. coli'' would not result in any live bacterial carryover, and even if bacteria were to be present in the extraction, they would not be able to survive in the high alkane environment of gasoline. <br />
<br />
<br />
'''2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?<br />
'''<br />
None of the parts we made this year raise any particular safety issues that we can foresee. All of our major parts are found in non-pathogenic bacterial species( cyanobacteria for our alkane production, ''Alicyclobacillus sendaiensis'' for our gluten destruction project). None of our new parts would provide any foreseeable selective advantage, and these engineered bacteria would not be better able to compete with native bacteria. Thus, these parts would not increase bacterial survival in the case of accidental release.<br />
<br />
<br />
'''3. Is there a local biosafety group, committee, or review board at your institution?<br />
'''<br />
The University of Washington has an Environmental Health and Safety(EHS) committee that deals with biosafety and other safety and health issues. All procedures and materials used are standard, the EHS has no specific concerns. You can visit the EHS at http://www.ehs.washington.edu<br />
<br />
<br />
'''4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
'''<br />
One biosafety measure that would be helpful for many teams would be a standardized bacterial strain with knockout(s) that would require that media be supplemented with a relatively cheap chemical for bacterial growth to occur. This would greatly reduce any risks of accidental release, and virtually eliminate the chances of bacterial growth outside of controled lab environments. The main difficulty with this approach would be finding a knockout that would not have an impact on growth in controlled laboratory media. This would have an added effect of increasing comparability between projects, as tests could be done in standardized strains.</div>Hadidihttp://2011.igem.org/Team:Washington/SafetyTeam:Washington/Safety2011-09-23T01:57:32Z<p>Hadidi: /* Safety */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
<center><big><big><big><big>Safety</big></big></big></big></center><br><br><br />
<br />
Please use this page to answer the safety questions posed on the [[Safety | safety page]].<br />
<br />
<br />
'''1. Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
'''<br />
All projects are being conducted in lab-safe strains of ''E. coli''. All researchers have been trained in applicable lab safety to insure that no bacteria are inadvertently released into the environment. The researchers have also been trained in proper handling of chemicals, which is required due to the work with alkanes which requires use of chemicals not normally used in a molecular biology lab. In both the Celiac's disease and alkane production projects, the actual organisms being engineered are intended to be maintained in lab conditions ( cultures, bioreactors, etc.). The active ingredient in our Celiac's disease treatment would be used as a purified protein, like many current protein therapeutics, and is consistant with current FDA guidelines. Extraction of alkanes from our alkane producing ''E. coli'' would not result in any live bacterial carryover, and even if bacteria were to be present in the extraction, they would not be able to survive in the high alkane environment of gasoline. <br />
<br />
<br />
'''2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?<br />
'''<br />
None of the parts we made this year raise any particular safety issues that we can foresee. All of our major parts are found in non-pathogenic bacterial species( cyanobacteria for our alkane production, ''Alicyclobacillus sendaiensis'' for our gluten destruction project). None of our new parts would provide any foreseeable selective advantage, and these engineered bacteria would not be better able to compete with native bacteria. Thus, these parts would not increase bacterial survival in the case of accidental release.<br />
<br />
<br />
'''3. Is there a local biosafety group, committee, or review board at your institution?<br />
'''<br />
The University of Washington has an Environmental Health and Safety(EHS) committee that deals with biosafety and other safety and health issues. All procedures and materials used are standard, and upon review, the EHS has no concerns. You can visit the EHS at http://www.ehs.washington.edu<br />
<br />
<br />
'''4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
'''<br />
One biosafety measure that would be helpful for many teams would be a standardized bacterial strain with knockout(s) that would require that media be supplemented with a relatively cheap chemical for bacterial growth to occur. This would greatly reduce any risks of accidental release, and virtually eliminate the chances of bacterial growth outside of controled lab environments. The main difficulty with this approach would be finding a knockout that would not have an impact on growth in controlled laboratory media. This would have an added effect of increasing comparability between projects, as tests could be done in standardized strains.</div>Hadidihttp://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_AssayTeam:Washington/Protocols/Purified Enzyme Assay2011-09-23T01:11:41Z<p>Hadidi: /* Whole Cell Lysate Assay */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
=Purified Enzyme Assay=<br />
<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
var mainimg = document.getElementById("mainimg");<br />
var default_src = mainimg.src;<br />
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mainimg.src = "/wiki/images/e/ed/Main_graphic2_release.png";<br />
}, restore_mainimg);<br />
});<br />
</script><br />
</html><br />
<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------><br />
<br />
<br />
*'''Assay'''<br />
**Add 90uL of 5uM substrate (in NaAc pH 4.0 buffer) to each well of a black fluorescent microtiter assay plate<br />
**Start reaction by adding 0.0125mg/mL enzyme (try to avoid bubbles and pippette quickly, but accurately)<br />
***Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet!<br />
**Monitor the reaction with the SpectraMax<br />
<br />
<br />
<!---------------------------------------PAGE CONTENT GOES ABOVE THIS----------------------------------------><br />
<div style="text-align:center"><br />
<br />
<br />
'''&larr; [[Team:Washington/Protocols|Back to Protocols]]'''<br />
&nbsp; &nbsp; &nbsp;<br />
</div></div>Hadidihttp://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_AssayTeam:Washington/Protocols/Purified Enzyme Assay2011-09-23T01:11:00Z<p>Hadidi: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
=Whole Cell Lysate Assay=<br />
<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
var mainimg = document.getElementById("mainimg");<br />
var default_src = mainimg.src;<br />
var restore_mainimg = function() {<br />
mainimg.src = default_src;<br />
};<br />
$(document.getElementById("area_target")).hover(function() {<br />
mainimg.src = "/wiki/images/1/1d/Main_graphic2_target.png";<br />
}, restore_mainimg);<br />
$(document.getElementById("area_secretion")).hover(function() {<br />
mainimg.src = "/wiki/images/2/23/Main_graphic2_secretion.png";<br />
}, restore_mainimg);<br />
$(document.getElementById("area_display")).hover(function() {<br />
mainimg.src = "/wiki/images/a/aa/Main_graphic2_display.png";<br />
}, restore_mainimg);<br />
$(document.getElementById("area_release")).hover(function() {<br />
mainimg.src = "/wiki/images/e/ed/Main_graphic2_release.png";<br />
}, restore_mainimg);<br />
});<br />
</script><br />
</html><br />
<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------><br />
<br />
<br />
*'''Assay'''<br />
**Add 90uL of 5uM substrate (in NaAc pH 4.0 buffer) to each well of a black fluorescent microtiter assay plate<br />
**Start reaction by adding 0.0125mg/mL enzyme (try to avoid bubbles and pippette quickly, but accurately)<br />
***Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet!<br />
**Monitor the reaction with the SpectraMax<br />
<br />
<br />
<!---------------------------------------PAGE CONTENT GOES ABOVE THIS----------------------------------------><br />
<div style="text-align:center"><br />
<br />
<br />
'''&larr; [[Team:Washington/Protocols|Back to Protocols]]'''<br />
&nbsp; &nbsp; &nbsp;<br />
</div></div>Hadidihttp://2011.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2011-09-23T01:05:42Z<p>Hadidi: /* Break It: Gluten Destruction Protocols */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=General Protocols=<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/gel_electrophoresis General Agarose Gel Electrophoresis]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/PCR General PCR Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Digestion General Digestion Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Ligation General Ligation Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Transformation General Transformation Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Colony Colony PCR Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Competent Competent Cell Prep Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Kunkel Kunkel Mutagensis]<br />
<br />
[http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of how Kunkel Mutagensis works]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/expression_purification Standard 1L Expression Purification]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/gene_assembly Gene Assembly With Oligos]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/sequencing Sequencing]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/CompDesign Computational Protein Design]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Glycerol_Stocks Glycerol Stocks]<br />
<br />
<br />
=Make It: Diesel Production Protocols=<br />
<br />
[https://2011.igem.org/Team:Washington/alkanebiosynthesis Alkane Biosynthesis media and extraction]<br />
<br />
[https://2011.igem.org/Team:Washington/alkanebiosynthesis_cloning Alkane Biosynthesis cloning]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/redesign_cell_lysate_assay Cell Lysate Assay by Decarbonylase Redesign Team]<br />
<br />
<br />
=Break It: Gluten Destruction Protocols=<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Cell_Lysate_Assay Whole Cell Lysate Assay]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/50mL_Scale Small Scale (50mL) Protein Expression and Purification]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_Assay Purified Enzyme Assay]<br />
<br />
=Make It: iGem Toolkits=<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Cyto. Cytometry Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Elect. Electroporation (Transformation)]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Gib_Rxn Gibson Cloning/Assembly]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Gib_Purif. Gibson Purification]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/High_PCR High-Yield PCR (Full-Gene Assembly)]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Plas_DNA. Isolation of Plasmid DNA (miniprep)]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Induc_studies. Induction Studies of Proteins Fusions (mam-sfGFP)]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/PBS. PBS Stock Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Overnights. Preparation of Overnight Cultures]<br />
<br />
<br />
=Wiki Design=<br />
[https://2011.igem.org/Team:Washington/Protocols/Wiki_Design Wiki Design Tools (Wiki Markup, WikiDust, etc.)]</div>Hadidihttp://2011.igem.org/Team:Washington/Protocols/Cell_Lysate_AssayTeam:Washington/Protocols/Cell Lysate Assay2011-09-23T01:04:55Z<p>Hadidi: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
=Whole Cell Lysate Assay=<br />
<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
var mainimg = document.getElementById("mainimg");<br />
var default_src = mainimg.src;<br />
var restore_mainimg = function() {<br />
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};<br />
$(document.getElementById("area_target")).hover(function() {<br />
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$(document.getElementById("area_secretion")).hover(function() {<br />
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$(document.getElementById("area_display")).hover(function() {<br />
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}, restore_mainimg);<br />
});<br />
</script><br />
</html><br />
<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------><br />
Spin down cells from expression cultures at 4000rpm for 20min, pour off supernatant and perform either Triton Lysis or Sonication Lysis.<br />
*'''Triton Lysis'''<br />
**Triton Lysis Buffer (10mL, enough for 2 plates)<br />
***9mL of 1x PBS<br />
***10mg of Lsyozyme (5-20 is fine)<br />
***5mg of DNase (2-10 is fine)<br />
***1mL of 10% Triton X100<br />
**Add 50uL of Triton lysis buffer to each well<br />
**Shake on High Speed Plate Shaker (Set to 1500rpm, in J562) for 20-60 minutes<br />
**Add 250uL of 100mM NaOAc pH 4.0 to each well<br />
**Spin 40 minutes 4000rpm<br />
<br />
<br />
*'''Sonication Lysis'''<br />
**Add 300uL of 100mM NHAc pH 4.0 to each well<br />
***No lysozyme or DNase needed as sonication will break up the cell wall AND the genomic DNA<br />
**Use Plate Sonicator (Ask Chris)<br />
**Spin 40 minutes 4000rpm<br />
<br />
<br />
*'''2.Assay'''<br />
**Add 90uL of 5microM substrate (in NaAc pH 4.0 buffer) to each well of a black fluorescent microtiter assay plate<br />
**Start reaction by adding 10uL Supernatent (try to avoid bubbles and pippette quickly, but accurately)<br />
***Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet!<br />
**Monitor the reaction with the SpectraMax<br />
<br />
<br />
<!---------------------------------------PAGE CONTENT GOES ABOVE THIS----------------------------------------><br />
<div style="text-align:center"><br />
<br />
<br />
'''&larr; [[Team:Washington/Protocols|Back to Protocols]]'''<br />
&nbsp; &nbsp; &nbsp;<br />
</div></div>Hadidihttp://2011.igem.org/Team:Washington/Protocols/Cell_Lysate_AssayTeam:Washington/Protocols/Cell Lysate Assay2011-09-23T00:47:39Z<p>Hadidi: /* Whole Cell Lysate Assay */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
=Whole Cell Lysate Assay=<br />
<br />
<html><br />
<script type="text/javascript"><br />
$(function() {<br />
var mainimg = document.getElementById("mainimg");<br />
var default_src = mainimg.src;<br />
var restore_mainimg = function() {<br />
mainimg.src = default_src;<br />
};<br />
$(document.getElementById("area_target")).hover(function() {<br />
mainimg.src = "/wiki/images/1/1d/Main_graphic2_target.png";<br />
}, restore_mainimg);<br />
$(document.getElementById("area_secretion")).hover(function() {<br />
mainimg.src = "/wiki/images/2/23/Main_graphic2_secretion.png";<br />
}, restore_mainimg);<br />
$(document.getElementById("area_display")).hover(function() {<br />
mainimg.src = "/wiki/images/a/aa/Main_graphic2_display.png";<br />
}, restore_mainimg);<br />
$(document.getElementById("area_release")).hover(function() {<br />
mainimg.src = "/wiki/images/e/ed/Main_graphic2_release.png";<br />
}, restore_mainimg);<br />
});<br />
</script><br />
</html><br />
<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------><br />
Spin down cells from expression cultures at 4000rpm for 20min, pour off supernatant and perform either Triton Lysis or Sonication Lysis.<br />
*'''Triton Lysis'''<br />
**Triton Lysis Buffer (10mL, enough for 2 plates)<br />
***9mL of 1x PBS<br />
***10mg of Lsyozyme (5-20 is fine)<br />
***5mg of DNase (2-10 is fine)<br />
***1mL of 10% Triton X100<br />
**Add 50uL of Triton lysis buffer to each well<br />
**Shake on High Speed Plate Shaker (Set to 1500rpm, in J562) for 20-60 minutes<br />
**Add 250uL of 100mM NaOAc pH 4.0 to each well<br />
**Spin 40 minutes 4000rpm<br />
<br />
<br />
*'''Sonication Lysis'''<br />
**Add 300uL of 100mM NHAc pH 4.0 to each well<br />
***No lysozyme or DNase needed as sonication will break up the cell wall AND the genomic DNA<br />
**Use Plate Sonicator (Ask Chris)<br />
**Spin 40 minutes 4000rpm<br />
<br />
<br />
*'''2.Assay'''<br />
**Add 90uL of 5microM substrate (in NaAc pH 4.0 buffer) to each well of a black fluorescent microtiter assay plate<br />
**Start reaction by adding 10uL Supernatent (try to avoid bubbles and pippette quickly, but accurately)<br />
***Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet!<br />
**Monitor the reaction with the SpectraMax<br />
***Basic Settings = ???<br />
<br />
<br />
<!---------------------------------------PAGE CONTENT GOES ABOVE THIS----------------------------------------><br />
<div style="text-align:center"><br />
<br />
<br />
'''&larr; [[Team:Washington/Protocols|Back to Protocols]]'''<br />
&nbsp; &nbsp; &nbsp;<br />
</div></div>Hadidihttp://2011.igem.org/Team:Washington/Protocols/Cell_Lysate_AssayTeam:Washington/Protocols/Cell Lysate Assay2011-09-23T00:45:32Z<p>Hadidi: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
==Whole Cell Lysate Assay== <br />
Spin down cells from expression cultures at 4000rpm for 20min, pour off supernatant and perform either Triton Lysis or Sonication Lysis.<br />
*'''Triton Lysis'''<br />
**Triton Lysis Buffer (10mL, enough for 2 plates)<br />
***9mL of 1x PBS<br />
***10mg of Lsyozyme (5-20 is fine)<br />
***5mg of DNase (2-10 is fine)<br />
***1mL of 10% Triton X100<br />
**Add 50uL of Triton lysis buffer to each well<br />
**Shake on High Speed Plate Shaker (Set to 1500rpm, in J562) for 20-60 minutes<br />
**Add 250uL of 100mM NaOAc pH 4.0 to each well<br />
**Spin 40 minutes 4000rpm<br />
<br />
<br />
*'''Sonication Lysis'''<br />
**Add 300uL of 100mM NHAc pH 4.0 to each well<br />
***No lysozyme or DNase needed as sonication will break up the cell wall AND the genomic DNA<br />
**Use Plate Sonicator (Ask Chris)<br />
**Spin 40 minutes 4000rpm<br />
<br />
<br />
*'''2.Assay'''<br />
**Add 90uL of 5microM substrate (in NaAc pH 4.0 buffer) to each well of a black fluorescent microtiter assay plate<br />
**Start reaction by adding 10uL Supernatent (try to avoid bubbles and pippette quickly, but accurately)<br />
***Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet!<br />
**Monitor the reaction with the SpectraMax<br />
***Basic Settings = ???</div>Hadidihttp://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_AssayTeam:Washington/Protocols/Purified Enzyme Assay2011-09-23T00:35:17Z<p>Hadidi: Created page with "{{Template:Team:Washington/Templates/Top}}"</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-16T04:48:02Z<p>Hadidi: /* Testing Kumamolysin against SC-PEP */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Testing Kumamolysin against SC-PEP=<br />
<br />
After finding Kumamolysin to be the ideal enzyme for our purposes, we used the assay described below to test it against SC-PEP to determine comparative activity levels, resulting in evidence that wild-type Kumamolysin is already over 7 fold better than SC-PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolysin has a much higher activity level than SC-PEP, in addition to being amenable to engineering and effective at gastric pH.]]<br />
<br />
=Testing mutants for activity on breaking down PQLP=<br />
<br />
==Using a whole cell lysate assay to screen a large number of mutants for good activity==<br />
<br />
In order to determine whether our proposed mutations to the wild-type Kumamolisin improved the ability of the enzyme to break down PQLP, we tested each mutant with a whole cell lysate fluorescence assay. Cells harboring the expressed mutants were lysed at pH 4, mimicking the gastric environment. The released enzymes, after being roughly separated from cell material, were added to a fluorescent PQLP that had been conjugated to a quencher. Thus, no fluorescence was achieved until the peptide had been cleaved and the fluorophore had been released from the quencher. This allowed a relative assessment of rate of enzyme activity by measuring increase in fluorescence of the system.<br />
<br />
As one might expect, our first screen of mutants showed some mutants with a decrease in activity from the wild-type, some showed no change, and some actually showed great increase in activity. One single point mutant showed close to a 1000% increase in activity from wild-type Kumamolisin!<br />
<br />
<br />
[[File:Washington Mutant Screen Percent IncDec.jpg|center|800px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate.]]<br />
<br />
==Purifying and characterizing promising mutants for accurate rate comparison==<br />
<br />
Once we had identified mutants that showed a promising increase in activity from the wild-type kumamolisin, we purified and characterized activity in concentration controlled fluorescence assays, identical to the fluorescence system used for the whole cell lysate assay. Our best mutant demonstrated an 11-fold increase in activity from the native enzyme.<br />
<br />
<br />
[[File:Washington Best Mutants.png|center|500px|thumb|We narrowed this down to a few of our best mutants.]]<br />
<br />
==Creating combinatorial mutants by combining successful mutations==<br />
<br />
In order to achieve even more rate improvement from the native, we repeated our mutagenesis, this time taking successful mutations and adding them all to make combinatorial variants. By combining two of our top groups of mutations from the first round, we achieved an over 100-fold increase in activity on breaking down PQLP from the wild-type enzyme. This variant enzyme is ultimately 784 times better at breaking down PQLP than SC PEP, the enzyme currently in clinical trials for treating gluten intolerance!<br />
<br />
<br />
[[File:Washington BestCombMutant.png|center|500px|thumb|Our final engineered enzyme showed activity over 100 fold higher than wild type Kumamolisin, and ~700 fold higher than SC-PEP.]]</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-16T04:45:48Z<p>Hadidi: /* Testing Kumamolysin against SC-PEP */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Testing Kumamolysin against SC-PEP=<br />
<br />
After finding Kumamolysin to be the ideal enzyme for our purposes, we used the assay described below to test it against SC-PEP to determine comparative activity levels, resulting in evidence that wild-type Kumamolysin is already over 7 fold better than SC-PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolysin has a much higher activity level than SC-PEP, in addition to be amenable to engineering and effective at gastric pH.]]<br />
<br />
=Testing mutants for activity on breaking down PQLP=<br />
<br />
==Using a whole cell lysate assay to screen a large number of mutants for good activity==<br />
<br />
In order to determine whether our proposed mutations to the wild-type Kumamolisin improved the ability of the enzyme to break down PQLP, we tested each mutant with a whole cell lysate fluorescence assay. Cells harboring the expressed mutants were lysed at pH 4, mimicking the gastric environment. The released enzymes, after being roughly separated from cell material, were added to a fluorescent PQLP that had been conjugated to a quencher. Thus, no fluorescence was achieved until the peptide had been cleaved and the fluorophore had been released from the quencher. This allowed a relative assessment of rate of enzyme activity by measuring increase in fluorescence of the system.<br />
<br />
As one might expect, our first screen of mutants showed some mutants with a decrease in activity from the wild-type, some showed no change, and some actually showed great increase in activity. One single point mutant showed close to a 1000% increase in activity from wild-type Kumamolisin!<br />
<br />
<br />
[[File:Washington Mutant Screen Percent IncDec.jpg|center|800px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate.]]<br />
<br />
==Purifying and characterizing promising mutants for accurate rate comparison==<br />
<br />
Once we had identified mutants that showed a promising increase in activity from the wild-type kumamolisin, we purified and characterized activity in concentration controlled fluorescence assays, identical to the fluorescence system used for the whole cell lysate assay. Our best mutant demonstrated an 11-fold increase in activity from the native enzyme.<br />
<br />
<br />
[[File:Washington Best Mutants.png|center|500px|thumb|We narrowed this down to a few of our best mutants.]]<br />
<br />
==Creating combinatorial mutants by combining successful mutations==<br />
<br />
In order to achieve even more rate improvement from the native, we repeated our mutagenesis, this time taking successful mutations and adding them all to make combinatorial variants. By combining two of our top groups of mutations from the first round, we achieved an over 100-fold increase in activity on breaking down PQLP from the wild-type enzyme. This variant enzyme is ultimately 784 times better at breaking down PQLP than SC PEP, the enzyme currently in clinical trials for treating gluten intolerance!<br />
<br />
<br />
[[File:Washington BestCombMutant.png|center|500px|thumb|Our final engineered enzyme showed activity over 100 fold higher than wild type Kumamolisin, and ~700 fold higher than SC-PEP.]]</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-16T04:42:44Z<p>Hadidi: /* Finding the ideal enzyme */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Testing Kumamolysin against SC-PEP=<br />
<br />
After finding Kumamolysin to be the ideal enzyme for our purposes, we used the assay described below to test it against SC-PEP to determine comparative activity levels, resulting in evidence that wild-type Kumamolysin is already over 7 fold better than SC-PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolysin has a much higher activity level than SC-PEP, is engineerable, and works well at gastric pH.]]<br />
<br />
=Testing mutants for activity on breaking down PQLP=<br />
<br />
==Using a whole cell lysate assay to screen a large number of mutants for good activity==<br />
<br />
In order to determine whether our proposed mutations to the wild-type Kumamolisin improved the ability of the enzyme to break down PQLP, we tested each mutant with a whole cell lysate fluorescence assay. Cells harboring the expressed mutants were lysed at pH 4, mimicking the gastric environment. The released enzymes, after being roughly separated from cell material, were added to a fluorescent PQLP that had been conjugated to a quencher. Thus, no fluorescence was achieved until the peptide had been cleaved and the fluorophore had been released from the quencher. This allowed a relative assessment of rate of enzyme activity by measuring increase in fluorescence of the system.<br />
<br />
As one might expect, our first screen of mutants showed some mutants with a decrease in activity from the wild-type, some showed no change, and some actually showed great increase in activity. One single point mutant showed close to a 1000% increase in activity from wild-type Kumamolisin!<br />
<br />
<br />
[[File:Washington Mutant Screen Percent IncDec.jpg|center|800px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate.]]<br />
<br />
==Purifying and characterizing promising mutants for accurate rate comparison==<br />
<br />
Once we had identified mutants that showed a promising increase in activity from the wild-type kumamolisin, we purified and characterized activity in concentration controlled fluorescence assays, identical to the fluorescence system used for the whole cell lysate assay. Our best mutant demonstrated an 11-fold increase in activity from the native enzyme.<br />
<br />
<br />
[[File:Washington Best Mutants.png|center|500px|thumb|We narrowed this down to a few of our best mutants.]]<br />
<br />
==Creating combinatorial mutants by combining successful mutations==<br />
<br />
In order to achieve even more rate improvement from the native, we repeated our mutagenesis, this time taking successful mutations and adding them all to make combinatorial variants. By combining two of our top groups of mutations from the first round, we achieved an over 100-fold increase in activity on breaking down PQLP from the wild-type enzyme. This variant enzyme is ultimately 784 times better at breaking down PQLP than SC PEP, the enzyme currently in clinical trials for treating gluten intolerance!<br />
<br />
<br />
[[File:Washington BestCombMutant.png|center|500px|thumb|Our final engineered enzyme showed activity over 100 fold higher than wild type Kumamolisin, and ~700 fold higher than SC-PEP.]]</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-16T04:31:40Z<p>Hadidi: /* Finding the ideal enzyme */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Finding the ideal enzyme=<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolysin has a much higher activity level than SC-PEP, is engineerable, and works well at gastric pH.]]<br />
<br />
=Testing mutants for activity on breaking down PQLP=<br />
<br />
==Using a whole cell lysate assay to screen a large number of mutants for good activity==<br />
<br />
In order to determine whether our proposed mutations to the wild-type Kumamolisin improved the ability of the enzyme to break down PQLP, we tested each mutant with a whole cell lysate fluorescence assay. Cells harboring the expressed mutants were lysed at pH 4, mimicking the gastric environment. The released enzymes, after being roughly separated from cell material, were added to a fluorescent PQLP that had been conjugated to a quencher. Thus, no fluorescence was achieved until the peptide had been cleaved and the fluorophore had been released from the quencher. This allowed a relative assessment of rate of enzyme activity by measuring increase in fluorescence of the system.<br />
<br />
As one might expect, our first screen of mutants showed some mutants with a decrease in activity from the wild-type, some showed no change, and some actually showed great increase in activity. One single point mutant showed close to a 1000% increase in activity from wild-type Kumamolisin!<br />
<br />
<br />
[[File:Washington Mutant Screen Percent IncDec.jpg|center|800px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate.]]<br />
<br />
==Purifying and characterizing promising mutants for accurate rate comparison==<br />
<br />
Once we had identified mutants that showed a promising increase in activity from the wild-type kumamolisin, we purified and characterized activity in concentration controlled fluorescence assays, identical to the fluorescence system used for the whole cell lysate assay. Our best mutant demonstrated an 11-fold increase in activity from the native enzyme.<br />
<br />
<br />
[[File:Washington Best Mutants.png|center|500px|thumb|We narrowed this down to a few of our best mutants.]]<br />
<br />
==Creating combinatorial mutants by combining successful mutations==<br />
<br />
In order to achieve even more rate improvement from the native, we repeated our mutagenesis, this time taking successful mutations and adding them all to make combinatorial variants. By combining two of our top groups of mutations from the first round, we achieved an over 100-fold increase in activity on breaking down PQLP from the wild-type enzyme. This variant enzyme is ultimately 784 times better at breaking down PQLP than SC PEP, the enzyme currently in clinical trials for treating gluten intolerance!<br />
<br />
<br />
[[File:Washington BestCombMutant.png|center|500px|thumb|Our final engineered enzyme showed activity over 100 fold higher than wild type Kumamolisin, and ~700 fold higher than SC-PEP.]]</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-16T04:30:53Z<p>Hadidi: /* Creating combinatorial mutants by combining successful mutations */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Finding the ideal enzyme=<br />
<br />
[[File:Washington InitialKumavSC.png|left|500px|thumb|Initial screenings revealed that Kumamolysin has a much higher activity level than SC-PEP, is engineerable, and works well at gastric pH.]]<br />
<br />
=Testing mutants for activity on breaking down PQLP=<br />
<br />
==Using a whole cell lysate assay to screen a large number of mutants for good activity==<br />
<br />
In order to determine whether our proposed mutations to the wild-type Kumamolisin improved the ability of the enzyme to break down PQLP, we tested each mutant with a whole cell lysate fluorescence assay. Cells harboring the expressed mutants were lysed at pH 4, mimicking the gastric environment. The released enzymes, after being roughly separated from cell material, were added to a fluorescent PQLP that had been conjugated to a quencher. Thus, no fluorescence was achieved until the peptide had been cleaved and the fluorophore had been released from the quencher. This allowed a relative assessment of rate of enzyme activity by measuring increase in fluorescence of the system.<br />
<br />
As one might expect, our first screen of mutants showed some mutants with a decrease in activity from the wild-type, some showed no change, and some actually showed great increase in activity. One single point mutant showed close to a 1000% increase in activity from wild-type Kumamolisin!<br />
<br />
<br />
[[File:Washington Mutant Screen Percent IncDec.jpg|center|800px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate.]]<br />
<br />
==Purifying and characterizing promising mutants for accurate rate comparison==<br />
<br />
Once we had identified mutants that showed a promising increase in activity from the wild-type kumamolisin, we purified and characterized activity in concentration controlled fluorescence assays, identical to the fluorescence system used for the whole cell lysate assay. Our best mutant demonstrated an 11-fold increase in activity from the native enzyme.<br />
<br />
<br />
[[File:Washington Best Mutants.png|center|500px|thumb|We narrowed this down to a few of our best mutants.]]<br />
<br />
==Creating combinatorial mutants by combining successful mutations==<br />
<br />
In order to achieve even more rate improvement from the native, we repeated our mutagenesis, this time taking successful mutations and adding them all to make combinatorial variants. By combining two of our top groups of mutations from the first round, we achieved an over 100-fold increase in activity on breaking down PQLP from the wild-type enzyme. This variant enzyme is ultimately 784 times better at breaking down PQLP than SC PEP, the enzyme currently in clinical trials for treating gluten intolerance!<br />
<br />
<br />
[[File:Washington BestCombMutant.png|center|500px|thumb|Our final engineered enzyme showed activity over 100 fold higher than wild type Kumamolisin, and ~700 fold higher than SC-PEP.]]</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-16T04:30:25Z<p>Hadidi: /* Purifying and characterizing promising mutants for accurate rate comparison */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Finding the ideal enzyme=<br />
<br />
[[File:Washington InitialKumavSC.png|left|500px|thumb|Initial screenings revealed that Kumamolysin has a much higher activity level than SC-PEP, is engineerable, and works well at gastric pH.]]<br />
<br />
=Testing mutants for activity on breaking down PQLP=<br />
<br />
==Using a whole cell lysate assay to screen a large number of mutants for good activity==<br />
<br />
In order to determine whether our proposed mutations to the wild-type Kumamolisin improved the ability of the enzyme to break down PQLP, we tested each mutant with a whole cell lysate fluorescence assay. Cells harboring the expressed mutants were lysed at pH 4, mimicking the gastric environment. The released enzymes, after being roughly separated from cell material, were added to a fluorescent PQLP that had been conjugated to a quencher. Thus, no fluorescence was achieved until the peptide had been cleaved and the fluorophore had been released from the quencher. This allowed a relative assessment of rate of enzyme activity by measuring increase in fluorescence of the system.<br />
<br />
As one might expect, our first screen of mutants showed some mutants with a decrease in activity from the wild-type, some showed no change, and some actually showed great increase in activity. One single point mutant showed close to a 1000% increase in activity from wild-type Kumamolisin!<br />
<br />
<br />
[[File:Washington Mutant Screen Percent IncDec.jpg|center|800px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate.]]<br />
<br />
==Purifying and characterizing promising mutants for accurate rate comparison==<br />
<br />
Once we had identified mutants that showed a promising increase in activity from the wild-type kumamolisin, we purified and characterized activity in concentration controlled fluorescence assays, identical to the fluorescence system used for the whole cell lysate assay. Our best mutant demonstrated an 11-fold increase in activity from the native enzyme.<br />
<br />
<br />
[[File:Washington Best Mutants.png|center|500px|thumb|We narrowed this down to a few of our best mutants.]]<br />
<br />
==Creating combinatorial mutants by combining successful mutations==<br />
<br />
In order to achieve even more rate improvement from the native, we repeated our mutagenesis, this time taking successful mutations and adding them all to make combinatorial variants. By combining two of our top groups of mutations from the first round, we achieved an over 100-fold increase in activity on breaking down PQLP from the wild-type enzyme. This variant enzyme is ultimately 784 times better at breaking down PQLP than SC PEP, the enzyme currently in clinical trials for treating gluten intolerance!<br />
<br />
<br />
[[File:Washington BestCombMutant.png|left|500px|thumb|Our final engineered enzyme showed activity over 100 fold higher than wild type Kumamolisin, and ~700 fold higher than SC-PEP.]]</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-16T04:27:14Z<p>Hadidi: /* Testing enzymes for activity on breaking down PQLP */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Finding the ideal enzyme=<br />
<br />
[[File:Washington InitialKumavSC.png|left|500px|thumb|Initial screenings revealed that Kumamolysin has a much higher activity level than SC-PEP, is engineerable, and works well at gastric pH.]]<br />
<br />
=Testing mutants for activity on breaking down PQLP=<br />
<br />
==Using a whole cell lysate assay to screen a large number of mutants for good activity==<br />
<br />
In order to determine whether our proposed mutations to the wild-type Kumamolisin improved the ability of the enzyme to break down PQLP, we tested each mutant with a whole cell lysate fluorescence assay. Cells harboring the expressed mutants were lysed at pH 4, mimicking the gastric environment. The released enzymes, after being roughly separated from cell material, were added to a fluorescent PQLP that had been conjugated to a quencher. Thus, no fluorescence was achieved until the peptide had been cleaved and the fluorophore had been released from the quencher. This allowed a relative assessment of rate of enzyme activity by measuring increase in fluorescence of the system.<br />
<br />
As one might expect, our first screen of mutants showed some mutants with a decrease in activity from the wild-type, some showed no change, and some actually showed great increase in activity. One single point mutant showed close to a 1000% increase in activity from wild-type Kumamolisin!<br />
<br />
<br />
[[File:Washington Mutant Screen Percent IncDec.jpg|center|800px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate.]]<br />
<br />
==Purifying and characterizing promising mutants for accurate rate comparison==<br />
<br />
Once we had identified mutants that showed a promising increase in activity from the wild-type kumamolisin, we purified and characterized activity in concentration controlled fluorescence assays, identical to the fluorescence system used for the whole cell lysate assay. Our best mutant demonstrated an 11-fold increase in activity from the native enzyme.<br />
<br />
<br />
[[File:Washington Best Mutants.png|center|500px|thumb|Our final engineered enzyme showed activity over 100 fold higher than wild type Kumamolisin, and ~700 fold higher than SC-PEP.]]<br />
<br />
==Creating combinatorial mutants by combining successful mutations==<br />
<br />
In order to achieve even more rate improvement from the native, we repeated our mutagenesis, this time taking successful mutations and adding them all to make combinatorial variants. By combining two of our top groups of mutations from the first round, we achieved an over 100-fold increase in activity on breaking down PQLP from the wild-type enzyme. This variant enzyme is ultimately 784 times better at breaking down PQLP than SC PEP, the enzyme currently in clinical trials for treating gluten intolerance!<br />
<br />
<br />
[[File:Washington BestCombMutant.png|left|500px|thumb|Our final engineered enzyme showed activity over 100 fold higher than wild type Kumamolisin, and ~700 fold higher than SC-PEP.]]</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-16T04:15:04Z<p>Hadidi: /* Kunkel Mutagenesis */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Redesigning Kumamolisin to Have Higher Activity at Low pH=<br />
[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
==Using Foldit to Design Mutations==<br />
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. <br />
<br />
Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.<br />
<br />
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.<br />
<br />
==Mutagenizing Kumamolisin==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]<br />
<br />
===Producing ssDNA===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. <br />
<br />
We designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We:<br />
*isolated the single stranded DNA (ssDNA) of the sense strand of our gene, <br />
*harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form, and finally, <br />
*harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.<br />
<br />
<br />
<br />
===Kunkel Mutagenesis===<br />
<br />
We annealed and extended our mutagenic oligos to allow for specific binding to our template. This vector was transformed into E. coli that degraded Uracil-containing DNA and replaced them with sections complementary to the opposite strand that contain thymine.<br />
Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.<br />
<br />
==Using a Whole Cell Lysate Assay to Test Activity of Mutants==<br />
Repeated growth, incubation, and induction of cells, followed by lysation, allowed us to test the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.<br />
[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]<br />
<br />
==Testing Purified Mutants to Accurately Assess Activity==<br />
===Purification===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level, as high activity without purification could simply be the result of high protein concentration. Growth, induction, and lysation of single colonies allowed the enzymes to be released from the cells, followed by collection of the purified proteins.<br />
<br />
===Assay===<br />
Concentrations were taken of the purified proteins, and diluted to the same concentration, to produce an assay resulting in accurate data representing which mutants had higher activity than kumamolisin and by how much their activity was greater.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-16T04:13:51Z<p>Hadidi: /* Kunkel Mutagenesis */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Redesigning Kumamolisin to Have Higher Activity at Low pH=<br />
[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
==Using Foldit to Design Mutations==<br />
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. <br />
<br />
Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.<br />
<br />
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.<br />
<br />
==Mutagenizing Kumamolisin==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]<br />
<br />
===Producing ssDNA===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. <br />
<br />
We designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We:<br />
*isolated the single stranded DNA (ssDNA) of the sense strand of our gene, <br />
*harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form, and finally, <br />
*harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.<br />
<br />
<br />
<br />
===Kunkel Mutagenesis===<br />
<br />
We annealed and extended our mutagenic oligos to allow for specific binding to our template. This vector was transformed into E. coli that degraded Uracil-containing DNA and replaced them with sections complementary to the opposite strand that contain thymine. <br />
Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.<br />
<br />
==Using a Whole Cell Lysate Assay to Test Activity of Mutants==<br />
Repeated growth, incubation, and induction of cells, followed by lysation, allowed us to test the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.<br />
[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]<br />
<br />
==Testing Purified Mutants to Accurately Assess Activity==<br />
===Purification===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level, as high activity without purification could simply be the result of high protein concentration. Growth, induction, and lysation of single colonies allowed the enzymes to be released from the cells, followed by collection of the purified proteins.<br />
<br />
===Assay===<br />
Concentrations were taken of the purified proteins, and diluted to the same concentration, to produce an assay resulting in accurate data representing which mutants had higher activity than kumamolisin and by how much their activity was greater.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-16T04:00:25Z<p>Hadidi: /* */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Redesigning Kumamolisin to Have Higher Activity at Low pH=<br />
[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
==Using Foldit to Design Mutations==<br />
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. <br />
<br />
Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.<br />
<br />
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.<br />
<br />
==Mutagenizing Kumamolisin==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]<br />
<br />
===Producing ssDNA===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. <br />
<br />
We designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We:<br />
*isolated the single stranded DNA (ssDNA) of the sense strand of our gene, <br />
*harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form, and finally, <br />
*harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.<br />
<br />
<br />
<br />
===Kunkel Mutagenesis===<br />
<br />
We annealed and extended our mutagenic oligos to allow for specific binding to our template. This vector was transformed into E. coli that degraded Uracil-containing DNA and replaced them with sections complementary to the opposite strand that contain thymine. Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.<br />
<br />
==Using a Whole Cell Lysate Assay to Test Activity of Mutants==<br />
Repeated growth, incubation, and induction of cells, followed by lysation, allowed us to test the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.<br />
[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]<br />
<br />
==Testing Purified Mutants to Accurately Assess Activity==<br />
===Purification===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level, as high activity without purification could simply be the result of high protein concentration. Growth, induction, and lysation of single colonies allowed the enzymes to be released from the cells, followed by collection of the purified proteins.<br />
<br />
===Assay===<br />
Concentrations were taken of the purified proteins, and diluted to the same concentration, to produce an assay resulting in accurate data representing which mutants had higher activity than kumamolisin and by how much their activity was greater.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-16T03:59:01Z<p>Hadidi: /* Using a Whole Cell Lysate Assay to Test Activity of Mutants */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Redesigning Kumamolisin to Have Higher Activity at Low pH=<br />
[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
==Using Foldit to Design Mutations==<br />
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. <br />
<br />
Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.<br />
<br />
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.<br />
<br />
==Mutagenizing Kumamolisin==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]<br />
<br />
===Producing ssDNA===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. <br />
<br />
We designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We:<br />
*isolated the single stranded DNA (ssDNA) of the sense strand of our gene, <br />
*harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form, and finally, <br />
*harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.<br />
<br />
<br />
<br />
===Kunkel Mutagenesis===<br />
<br />
We annealed and extended our mutagenic oligos to allow for specific binding to our template. This vector was transformed into E. coli that degraded Uracil-containing DNA and replaced them with sections complementary to the opposite strand that contain thymine. Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.<br />
<br />
== ==<br />
<br />
==Using a Whole Cell Lysate Assay to Test Activity of Mutants==<br />
Repeated growth, incubation, and induction of cells, followed by lysation, allowed us to test the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.<br />
[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]<br />
<br />
==Testing Purified Mutants to Accurately Assess Activity==<br />
===Purification===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level, as high activity without purification could simply be the result of high protein concentration. Growth, induction, and lysation of single colonies allowed the enzymes to be released from the cells, followed by collection of the purified proteins.<br />
<br />
===Assay===<br />
Concentrations were taken of the purified proteins, and diluted to the same concentration, to produce an assay resulting in accurate data representing which mutants had higher activity than kumamolisin and by how much their activity was greater.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-16T03:57:49Z<p>Hadidi: /* Kunkel Mutagenesis */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Redesigning Kumamolisin to Have Higher Activity at Low pH=<br />
[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
==Using Foldit to Design Mutations==<br />
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. <br />
<br />
Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.<br />
<br />
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.<br />
<br />
==Mutagenizing Kumamolisin==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]<br />
<br />
===Producing ssDNA===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. <br />
<br />
We designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We:<br />
*isolated the single stranded DNA (ssDNA) of the sense strand of our gene, <br />
*harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form, and finally, <br />
*harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.<br />
<br />
<br />
<br />
===Kunkel Mutagenesis===<br />
<br />
We annealed and extended our mutagenic oligos to allow for specific binding to our template. This vector was transformed into E. coli that degraded Uracil-containing DNA and replaced them with sections complementary to the opposite strand that contain thymine. Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.<br />
<br />
==Using a Whole Cell Lysate Assay to Test Activity of Mutants==<br />
Repeated growth, incubation, and induction of cells, followed by lysation, allowed us to test the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.<br />
[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]<br />
<br />
==Testing Purified Mutants to Accurately Assess Activity==<br />
===Purification===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level, as high activity without purification could simply be the result of high protein concentration. Growth, induction, and lysation of single colonies allowed the enzymes to be released from the cells, followed by collection of the purified proteins.<br />
<br />
===Assay===<br />
Concentrations were taken of the purified proteins, and diluted to the same concentration, to produce an assay resulting in accurate data representing which mutants had higher activity than kumamolisin and by how much their activity was greater.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-16T03:53:34Z<p>Hadidi: /* Using Foldit to Design Mutations */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Redesigning Kumamolisin to Have Higher Activity at Low pH=<br />
[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
==Using Foldit to Design Mutations==<br />
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. <br />
<br />
Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.<br />
<br />
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.<br />
<br />
==Mutagenizing Kumamolisin==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]<br />
<br />
===Producing ssDNA===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. <br />
<br />
We designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We:<br />
*isolated the single stranded DNA (ssDNA) of the sense strand of our gene, <br />
*harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form, and finally, <br />
*harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.<br />
<br />
===Kunkel Mutagenesis===<br />
<br />
We annealed and extended our mutagenic oligos to allow for specific binding to our template. This vector was transformed into E. coli that degraded Uracil-containing DNA and replaced them with sections complementary to the opposite strand that contain thymine. Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.<br />
<br />
==Using a Whole Cell Lysate Assay to Test Activity of Mutants==<br />
Repeated growth, incubation, and induction of cells, followed by lysation, allowed us to test the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.<br />
[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]<br />
<br />
==Testing Purified Mutants to Accurately Assess Activity==<br />
===Purification===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level, as high activity without purification could simply be the result of high protein concentration. Growth, induction, and lysation of single colonies allowed the enzymes to be released from the cells, followed by collection of the purified proteins.<br />
<br />
===Assay===<br />
Concentrations were taken of the purified proteins, and diluted to the same concentration, to produce an assay resulting in accurate data representing which mutants had higher activity than kumamolisin and by how much their activity was greater.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-16T03:48:43Z<p>Hadidi: /* Using Foldit to Design Mutations */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Redesigning Kumamolisin to Have Higher Activity at Low pH=<br />
[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
==Using Foldit to Design Mutations==<br />
<div class="center" style="width:auto; margin-left:auto; margin-right:auto;">In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure.</div> <br />
<br />
<div class="center" style="width:auto; margin-left:auto; margin-right:auto;">Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.</div><br />
<br />
<div class="center" style="width:auto; margin-left:auto; margin-right:auto;">Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.</div><br />
<br />
==Mutagenizing Kumamolisin==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]<br />
<br />
===Producing ssDNA===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. <br />
<br />
We designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We:<br />
*isolated the single stranded DNA (ssDNA) of the sense strand of our gene, <br />
*harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form, and finally, <br />
*harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.<br />
<br />
===Kunkel Mutagenesis===<br />
<br />
We annealed and extended our mutagenic oligos to allow for specific binding to our template. This vector was transformed into E. coli that degraded Uracil-containing DNA and replaced them with sections complementary to the opposite strand that contain thymine. Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.<br />
<br />
==Using a Whole Cell Lysate Assay to Test Activity of Mutants==<br />
Repeated growth, incubation, and induction of cells, followed by lysation, allowed us to test the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.<br />
[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]<br />
<br />
==Testing Purified Mutants to Accurately Assess Activity==<br />
===Purification===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level, as high activity without purification could simply be the result of high protein concentration. Growth, induction, and lysation of single colonies allowed the enzymes to be released from the cells, followed by collection of the purified proteins.<br />
<br />
===Assay===<br />
Concentrations were taken of the purified proteins, and diluted to the same concentration, to produce an assay resulting in accurate data representing which mutants had higher activity than kumamolisin and by how much their activity was greater.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-16T03:20:29Z<p>Hadidi: /* Using Foldit to Design Mutations */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Redesigning Kumamolisin to Have Higher Activity at Low pH=<br />
[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
==Using Foldit to Design Mutations==<br />
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. <br />
<br />
Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.<br />
<br />
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.<br />
<br />
==Mutagenizing Kumamolisin==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]<br />
<br />
===Producing ssDNA===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. <br />
<br />
We designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We:<br />
*isolated the single stranded DNA (ssDNA) of the sense strand of our gene, <br />
*harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form, and finally, <br />
*harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.<br />
<br />
===Kunkel Mutagenesis===<br />
<br />
We annealed and extended our mutagenic oligos to allow for specific binding to our template. This vector was transformed into E. coli that degraded Uracil-containing DNA and replaced them with sections complementary to the opposite strand that contain thymine. Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.<br />
<br />
==Using a Whole Cell Lysate Assay to Test Activity of Mutants==<br />
Repeated growth, incubation, and induction of cells, followed by lysation, allowed us to test the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.<br />
[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]<br />
<br />
==Testing Purified Mutants to Accurately Assess Activity==<br />
===Purification===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level, as high activity without purification could simply be the result of high protein concentration. Growth, induction, and lysation of single colonies allowed the enzymes to be released from the cells, followed by collection of the purified proteins.<br />
<br />
===Assay===<br />
Concentrations were taken of the purified proteins, and diluted to the same concentration, to produce an assay resulting in accurate data representing which mutants had higher activity than kumamolisin and by how much their activity was greater.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-16T03:15:16Z<p>Hadidi: /* Producing ssDNA */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Redesigning Kumamolisin to Have Higher Activity at Low pH=<br />
[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
==Using Foldit to Design Mutations==<br />
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.<br />
<br />
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.<br />
<br />
<br />
<br />
==Mutagenizing Kumamolisin==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]<br />
<br />
===Producing ssDNA===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. <br />
<br />
We designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We:<br />
*isolated the single stranded DNA (ssDNA) of the sense strand of our gene, <br />
*harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form, and finally, <br />
*harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.<br />
<br />
===Kunkel Mutagenesis===<br />
<br />
We annealed and extended our mutagenic oligos to allow for specific binding to our template. This vector was transformed into E. coli that degraded Uracil-containing DNA and replaced them with sections complementary to the opposite strand that contain thymine. Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.<br />
<br />
==Using a Whole Cell Lysate Assay to Test Activity of Mutants==<br />
Repeated growth, incubation, and induction of cells, followed by lysation, allowed us to test the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.<br />
[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]<br />
<br />
==Testing Purified Mutants to Accurately Assess Activity==<br />
===Purification===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level, as high activity without purification could simply be the result of high protein concentration. Growth, induction, and lysation of single colonies allowed the enzymes to be released from the cells, followed by collection of the purified proteins.<br />
<br />
===Assay===<br />
Concentrations were taken of the purified proteins, and diluted to the same concentration, to produce an assay resulting in accurate data representing which mutants had higher activity than kumamolisin and by how much their activity was greater.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-16T03:10:50Z<p>Hadidi: /* Producing ssDNA */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Redesigning Kumamolisin to Have Higher Activity at Low pH=<br />
[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
==Using Foldit to Design Mutations==<br />
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.<br />
<br />
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.<br />
<br />
<br />
<br />
==Mutagenizing Kumamolisin==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]<br />
<br />
===Producing ssDNA===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. <br />
<br />
We designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We isolated the single stranded DNA (ssDNA) of the sense strand of our gene. We then harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form. Finally, we harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.<br />
<br />
===Kunkel Mutagenesis===<br />
<br />
We annealed and extended our mutagenic oligos to allow for specific binding to our template. This vector was transformed into E. coli that degraded Uracil-containing DNA and replaced them with sections complementary to the opposite strand that contain thymine. Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.<br />
<br />
==Using a Whole Cell Lysate Assay to Test Activity of Mutants==<br />
Repeated growth, incubation, and induction of cells, followed by lysation, allowed us to test the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.<br />
[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]<br />
<br />
==Testing Purified Mutants to Accurately Assess Activity==<br />
===Purification===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level, as high activity without purification could simply be the result of high protein concentration. Growth, induction, and lysation of single colonies allowed the enzymes to be released from the cells, followed by collection of the purified proteins.<br />
<br />
===Assay===<br />
Concentrations were taken of the purified proteins, and diluted to the same concentration, to produce an assay resulting in accurate data representing which mutants had higher activity than kumamolisin and by how much their activity was greater.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-16T03:06:10Z<p>Hadidi: /* Producing ssDNA */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Redesigning Kumamolisin to Have Higher Activity at Low pH=<br />
[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
==Using Foldit to Design Mutations==<br />
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.<br />
<br />
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.<br />
<br />
<br />
<br />
==Mutagenizing Kumamolisin==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]<br />
<br />
===Producing ssDNA===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. We designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We isolated the single stranded DNA (ssDNA) of the sense strand of our gene.<br />
We then harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form. Finally, we harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.<br />
<br />
===Kunkel Mutagenesis===<br />
<br />
We annealed and extended our mutagenic oligos to allow for specific binding to our template. This vector was transformed into E. coli that degraded Uracil-containing DNA and replaced them with sections complementary to the opposite strand that contain thymine. Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.<br />
<br />
==Using a Whole Cell Lysate Assay to Test Activity of Mutants==<br />
Repeated growth, incubation, and induction of cells, followed by lysation, allowed us to test the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.<br />
[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]<br />
<br />
==Testing Purified Mutants to Accurately Assess Activity==<br />
===Purification===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level, as high activity without purification could simply be the result of high protein concentration. Growth, induction, and lysation of single colonies allowed the enzymes to be released from the cells, followed by collection of the purified proteins.<br />
<br />
===Assay===<br />
Concentrations were taken of the purified proteins, and diluted to the same concentration, to produce an assay resulting in accurate data representing which mutants had higher activity than kumamolisin and by how much their activity was greater.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-16T03:00:24Z<p>Hadidi: /* Assay */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Redesigning Kumamolisin to Have Higher Activity at Low pH=<br />
[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
==Using Foldit to Design Mutations==<br />
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.<br />
<br />
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.<br />
<br />
<br />
<br />
==Mutagenizing Kumamolisin==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]<br />
<br />
===Producing ssDNA===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. To do this, we designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We isolated the single stranded DNA (ssDNA) of the sense strand of our gene by growing the vector harboring our gene in a uracil-N-glycosidase (UNG) and deoxyuracil triphosphate pyrophosphatase (DUT) deficient E. coli- strain. These cells thus did not have the ability to maintain thymine in their DNA, and so all T’s become replaced by U’s in the DNA of these cells.<br />
We then harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form. Finally, we harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.<br />
<br />
<br />
===Kunkel Mutagenesis===<br />
<br />
We annealed and extended our mutagenic oligos to allow for specific binding to our template. This vector was transformed into E. coli that degraded Uracil-containing DNA and replaced them with sections complementary to the opposite strand that contain thymine. Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.<br />
<br />
==Using a Whole Cell Lysate Assay to Test Activity of Mutants==<br />
Repeated growth, incubation, and induction of cells, followed by lysation, allowed us to test the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.<br />
[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]<br />
<br />
==Testing Purified Mutants to Accurately Assess Activity==<br />
===Purification===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level, as high activity without purification could simply be the result of high protein concentration. Growth, induction, and lysation of single colonies allowed the enzymes to be released from the cells, followed by collection of the purified proteins.<br />
<br />
===Assay===<br />
Concentrations were taken of the purified proteins, and diluted to the same concentration, to produce an assay resulting in accurate data representing which mutants had higher activity than kumamolisin and by how much their activity was greater.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-16T02:58:59Z<p>Hadidi: /* Purification */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Redesigning Kumamolisin to Have Higher Activity at Low pH=<br />
[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
==Using Foldit to Design Mutations==<br />
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.<br />
<br />
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.<br />
<br />
<br />
<br />
==Mutagenizing Kumamolisin==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]<br />
<br />
===Producing ssDNA===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. To do this, we designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We isolated the single stranded DNA (ssDNA) of the sense strand of our gene by growing the vector harboring our gene in a uracil-N-glycosidase (UNG) and deoxyuracil triphosphate pyrophosphatase (DUT) deficient E. coli- strain. These cells thus did not have the ability to maintain thymine in their DNA, and so all T’s become replaced by U’s in the DNA of these cells.<br />
We then harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form. Finally, we harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.<br />
<br />
<br />
===Kunkel Mutagenesis===<br />
<br />
We annealed and extended our mutagenic oligos to allow for specific binding to our template. This vector was transformed into E. coli that degraded Uracil-containing DNA and replaced them with sections complementary to the opposite strand that contain thymine. Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.<br />
<br />
==Using a Whole Cell Lysate Assay to Test Activity of Mutants==<br />
Repeated growth, incubation, and induction of cells, followed by lysation, allowed us to test the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.<br />
[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]<br />
<br />
==Testing Purified Mutants to Accurately Assess Activity==<br />
===Purification===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level, as high activity without purification could simply be the result of high protein concentration. Growth, induction, and lysation of single colonies allowed the enzymes to be released from the cells, followed by collection of the purified proteins.<br />
<br />
===Assay===<br />
Once we had pure protein, we determined the concentrations. We then diluted the proteins to the same concentration. We also used purified wild-type kumamolisin and ScPEP and ran the assay at pH 4. Using the resulting data, we were able to determine which mutants had higher activity than kumamolisin and by how much their activity was greater.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-16T02:57:40Z<p>Hadidi: /* Kunkel Mutagenesis */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Redesigning Kumamolisin to Have Higher Activity at Low pH=<br />
[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
==Using Foldit to Design Mutations==<br />
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.<br />
<br />
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.<br />
<br />
<br />
<br />
==Mutagenizing Kumamolisin==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]<br />
<br />
===Producing ssDNA===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. To do this, we designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We isolated the single stranded DNA (ssDNA) of the sense strand of our gene by growing the vector harboring our gene in a uracil-N-glycosidase (UNG) and deoxyuracil triphosphate pyrophosphatase (DUT) deficient E. coli- strain. These cells thus did not have the ability to maintain thymine in their DNA, and so all T’s become replaced by U’s in the DNA of these cells.<br />
We then harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form. Finally, we harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.<br />
<br />
<br />
===Kunkel Mutagenesis===<br />
<br />
We annealed and extended our mutagenic oligos to allow for specific binding to our template. This vector was transformed into E. coli that degraded Uracil-containing DNA and replaced them with sections complementary to the opposite strand that contain thymine. Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.<br />
<br />
==Using a Whole Cell Lysate Assay to Test Activity of Mutants==<br />
Repeated growth, incubation, and induction of cells, followed by lysation, allowed us to test the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.<br />
[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]<br />
<br />
==Testing Purified Mutants to Accurately Assess Activity==<br />
===Purification===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level, as high activity without purification could simply be the result of high protein concentration. Growth, induction, and lysation of single colonies allowed the enzymes to be released from the cells. The proteins were then collected on a column, washed with buffer, and eluted off the column. Finally, we dialyzed the protein in a pH 4 buffer.<br />
===Assay===<br />
Once we had pure protein, we determined the concentrations. We then diluted the proteins to the same concentration. We also used purified wild-type kumamolisin and ScPEP and ran the assay at pH 4. Using the resulting data, we were able to determine which mutants had higher activity than kumamolisin and by how much their activity was greater.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-16T02:48:30Z<p>Hadidi: /* Testing Purified Mutants to Accurately Assess Activity */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Redesigning Kumamolisin to Have Higher Activity at Low pH=<br />
[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
==Using Foldit to Design Mutations==<br />
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.<br />
<br />
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.<br />
<br />
<br />
<br />
==Mutagenizing Kumamolisin==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]<br />
<br />
===Producing ssDNA===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. To do this, we designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We isolated the single stranded DNA (ssDNA) of the sense strand of our gene by growing the vector harboring our gene in a uracil-N-glycosidase (UNG) and deoxyuracil triphosphate pyrophosphatase (DUT) deficient E. coli- strain. These cells thus did not have the ability to maintain thymine in their DNA, and so all T’s become replaced by U’s in the DNA of these cells.<br />
We then harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form. Finally, we harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.<br />
<br />
<br />
===Kunkel Mutagenesis===<br />
<br />
Using our isolated ssDNA as a template, we annealed our mutagenic oligos to allow for specific binding of our oligos to our template. The oligo harboring the desired point mutations was then extended, using the wild-type gene and associated vector as a template, by DNA polymerase. This hybrid vector, with one strand of the native Kumamolisin gene and the other a variant containing point mutations, was transformed into UNG and DUT wild-type E. coli, which have the ability to degrade sections of DNA that contain uracil, and replace them with sections complementary to the opposite strand that contain thymine. Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.<br />
<br />
==Using a Whole Cell Lysate Assay to Test Activity of Mutants==<br />
Repeated growth, incubation, and induction of cells, followed by lysation, allowed us to test the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.<br />
[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]<br />
<br />
==Testing Purified Mutants to Accurately Assess Activity==<br />
===Purification===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level, as high activity without purification could simply be the result of high protein concentration. Growth, induction, and lysation of single colonies allowed the enzymes to be released from the cells. The proteins were then collected on a column, washed with buffer, and eluted off the column. Finally, we dialyzed the protein in a pH 4 buffer.<br />
===Assay===<br />
Once we had pure protein, we determined the concentrations. We then diluted the proteins to the same concentration. We also used purified wild-type kumamolisin and ScPEP and ran the assay at pH 4. Using the resulting data, we were able to determine which mutants had higher activity than kumamolisin and by how much their activity was greater.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-16T02:48:01Z<p>Hadidi: /* Using a Whole Cell Lysate Assay to Test Activity of Mutants */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Redesigning Kumamolisin to Have Higher Activity at Low pH=<br />
[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
==Using Foldit to Design Mutations==<br />
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.<br />
<br />
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.<br />
<br />
<br />
<br />
==Mutagenizing Kumamolisin==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]<br />
<br />
===Producing ssDNA===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. To do this, we designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We isolated the single stranded DNA (ssDNA) of the sense strand of our gene by growing the vector harboring our gene in a uracil-N-glycosidase (UNG) and deoxyuracil triphosphate pyrophosphatase (DUT) deficient E. coli- strain. These cells thus did not have the ability to maintain thymine in their DNA, and so all T’s become replaced by U’s in the DNA of these cells.<br />
We then harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form. Finally, we harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.<br />
<br />
<br />
===Kunkel Mutagenesis===<br />
<br />
Using our isolated ssDNA as a template, we annealed our mutagenic oligos to allow for specific binding of our oligos to our template. The oligo harboring the desired point mutations was then extended, using the wild-type gene and associated vector as a template, by DNA polymerase. This hybrid vector, with one strand of the native Kumamolisin gene and the other a variant containing point mutations, was transformed into UNG and DUT wild-type E. coli, which have the ability to degrade sections of DNA that contain uracil, and replace them with sections complementary to the opposite strand that contain thymine. Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.<br />
<br />
==Using a Whole Cell Lysate Assay to Test Activity of Mutants==<br />
Repeated growth, incubation, and induction of cells, followed by lysation, allowed us to test the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.<br />
[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]<br />
<br />
==Testing Purified Mutants to Accurately Assess Activity==<br />
===Purification===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level. For instance, if the whole cell lysate assay showed one mutant to have ten times the activity level as wild-type kumamolisin we cannot assume that there has been an increase in activity because there could simply be ten times the amount of protein with the same level of activity. To purify our mutants, we first grew them in TB and kanamycin with a single colony of the mutant. This inoculation was grown over 24 hours at 37 degrees celcius and then expanded to 50 mililiters (TB+kanamycin). We then allowed the culture to grow to a specific optical density of cells before inducing the culture using IPTG. We then allowed the cells to grow again to express kumamolisin. We then lysed the cells using a lysis buffer and centrifugation. This allowed the enzymes to be released from the cells. The proteins were then collected on a column, washed with buffer, and eluted off the column. Finally, wwe dialyzed the protein in a sodium acetate buffer (pH 4).<br />
===Assay===<br />
Once we had pure protein, we determined the concentration of each using a NanoDrop machine. With the concentration of each, we then diluted the proteins to the same concentration. For the assay, we also used purified wild-type kumamolisin and ScPEP. The assay was again run for 30 minutes at pH 4. Using the data from the purified assays, we were able to deermine which mutants had higher activity than kumamolisin and by how much their activity was greater.</div>Hadidihttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-16T02:47:37Z<p>Hadidi: /* Mutagenizing Kumamolisin */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=Redesigning Kumamolisin to Have Higher Activity at Low pH=<br />
[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
==Using Foldit to Design Mutations==<br />
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.<br />
<br />
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.<br />
<br />
<br />
<br />
==Mutagenizing Kumamolisin==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]<br />
<br />
===Producing ssDNA===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. To do this, we designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We isolated the single stranded DNA (ssDNA) of the sense strand of our gene by growing the vector harboring our gene in a uracil-N-glycosidase (UNG) and deoxyuracil triphosphate pyrophosphatase (DUT) deficient E. coli- strain. These cells thus did not have the ability to maintain thymine in their DNA, and so all T’s become replaced by U’s in the DNA of these cells.<br />
We then harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form. Finally, we harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.<br />
<br />
<br />
===Kunkel Mutagenesis===<br />
<br />
Using our isolated ssDNA as a template, we annealed our mutagenic oligos to allow for specific binding of our oligos to our template. The oligo harboring the desired point mutations was then extended, using the wild-type gene and associated vector as a template, by DNA polymerase. This hybrid vector, with one strand of the native Kumamolisin gene and the other a variant containing point mutations, was transformed into UNG and DUT wild-type E. coli, which have the ability to degrade sections of DNA that contain uracil, and replace them with sections complementary to the opposite strand that contain thymine. Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.<br />
<br />
==Using a Whole Cell Lysate Assay to Test Activity of Mutants==<br />
After the cells had been allowed to grow overnight, colonies were picked and used to inoculate a 96 well plate containing LB and kanamycin. This step allowed us to grow a representative amount of cells containing each mutation. After growing overnight at 37 degrees celcius cells from each well were transferred to another 96 well plate containing TB and kanamycin. These plates were incubated at 37 degrees celcius and later induced using IPTG. After induction, we incubated the plates at 18 degrees celcius overnight. We then lysed the cells and tested the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation over a period of 30 minutes. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.<br />
[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]<br />
<br />
==Testing Purified Mutants to Accurately Assess Activity==<br />
===Purification===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level. For instance, if the whole cell lysate assay showed one mutant to have ten times the activity level as wild-type kumamolisin we cannot assume that there has been an increase in activity because there could simply be ten times the amount of protein with the same level of activity. To purify our mutants, we first grew them in TB and kanamycin with a single colony of the mutant. This inoculation was grown over 24 hours at 37 degrees celcius and then expanded to 50 mililiters (TB+kanamycin). We then allowed the culture to grow to a specific optical density of cells before inducing the culture using IPTG. We then allowed the cells to grow again to express kumamolisin. We then lysed the cells using a lysis buffer and centrifugation. This allowed the enzymes to be released from the cells. The proteins were then collected on a column, washed with buffer, and eluted off the column. Finally, wwe dialyzed the protein in a sodium acetate buffer (pH 4).<br />
===Assay===<br />
Once we had pure protein, we determined the concentration of each using a NanoDrop machine. With the concentration of each, we then diluted the proteins to the same concentration. For the assay, we also used purified wild-type kumamolisin and ScPEP. The assay was again run for 30 minutes at pH 4. Using the data from the purified assays, we were able to deermine which mutants had higher activity than kumamolisin and by how much their activity was greater.</div>Hadidihttp://2011.igem.org/File:CIMG0021.jpgFile:CIMG0021.jpg2011-09-16T02:26:08Z<p>Hadidi: </p>
<hr />
<div></div>Hadidihttp://2011.igem.org/Team:Washington/Team/MembersTeam:Washington/Team/Members2011-09-16T02:24:14Z<p>Hadidi: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------><br />
<br />
== '''Who we are''' ==<br />
<br />
<gallery caption="Undergraduate Team Members" widths="180px" heights="120px" perrow="4"><br />
Image:Washington Casey Ager Profile1.jpg|<center>Casey Ager</center><br />
Image:Washington_.jpg|<center>Ju Hye An</center><br />
Image: Profile_Michael_Brasino.jpg|<center>Michael Brasino</center><br />
Image:Washington_.jpg|<center>Marika Cheng</center><br />
Image:Washington_.jpg|<center>Chris Choe</center><br />
Image:Washington_.jpg|<center>Justin De Leon</center><br />
Image:Washington_.jpg|<center>Sydney Gordon</center><br />
Image:CIMG0021.jpg|<center>Daniel Hadidi</center><br />
Image:Washington_Matthew_Harger.JPG|<center>Matthew Harger</center><br />
Image:Washington_.jpg|<center>Elaine Lai</center><br />
Image:Washington_.jpg|<center>Benjamin Mo</center><br />
Image:Washington 2011 CIMG0015-2.jpg|<center>Austin Moon</center><br />
Image:Washington_.jpg|<center>Rashmi Ravichandran</center><br />
Image:Washington_.jpg|<center>Seth Sagulo</center><br />
File:Washington_Liz.png|<center>Liz Stanley</center><br />
Image:Washington_.jpg|<center>Angus Toland</center><br />
Image:Washington_.jpg|<center>Sarah Wolf</center><br />
Image:Washington_.jpg|<center>Alicia Wong</center><br />
Image:Washington_.jpg|<center>Cindy Wu</center><br />
Image:Washington_Sean_Wu.jpg|<center>Sean Wu</center><br />
Image:Washington_.jpg|<center>Lei Zheng</center><br />
Image:Washington_david_zong.jpg|<center>David Zong</center><br />
<br />
</gallery><br />
<br />
<br />
<gallery caption="Advisors" widths="180px" heights="120px" perrow="4"><br />
Image:Washington_.jpg|<center>Aaron Chevalier <br/> Bioengineering </center><br />
Image:Washington_.jpg|<center>Rob Egbert <br/> Electrical Engineering </center><br />
Image:Washington_.jpg|<center>Chris Eiben <br/> Biochemistry </center><br />
Image:Washington_.jpg|<center>Jeremy Mills <br/> Biochemistry </center><br />
Image:Siegel_UW_2011.jpg|<center>Justin Siegel <br/> Biomolecular Structure and Design</center><br />
Image:Washington2011_Matthew-d-smith.jpg|<center>Matt Smith <br/> Molecular and Cellular Biology</center><br />
Image:Washington_.jpg|<center>Ingrid Swanson <br/> Microbiology </center><br />
</gallery><br />
<br />
<br />
<gallery caption="Faculty" widths="180px" heights="120px" perrow="4"><br />
image:Washington_David_Baker.jpg|<center>David Baker <br/> Biochemistry</center><br />
Image:Washington_Klavins.jpg|<center>Eric Klavins <br/> Electrical Engineering</center><br />
</gallery><br />
<br />
<br />
<gallery caption="Collaborators and Support" widths="180px" heights="120px" perrow="4"><br />
Image:Washington_Biochemistry.jpg|<center>Biochemistry <br/> Lab space</center><br />
</gallery><br />
<br />
== '''Who did what''' ==<br />
The teams were assembled during our winter quarter. During this term we went around to classes and had weekly meetings to introduce students to synthetic biology through a series of guest lectures. During the spring, students participated in brainstorming projects related to the sponsoring Faculty advisors labs (Baker and Klavins) and came up with the project they wanted to carry out. Over the summer, students completed all of the experimental work, but worked closely with both graduate students and faculty advisors to plan the most pertinent experiments and make the most of our limited time.<br />
<br />
=== Alkanes Production ===<br />
<br />
=== iGem Toolkits ===<br />
<br />
=== Gluten Degradation ===</div>Hadidihttp://2011.igem.org/File:Washington_.jpgFile:Washington .jpg2011-09-16T01:59:39Z<p>Hadidi: uploaded a new version of &quot;File:Washington .jpg&quot;</p>
<hr />
<div></div>Hadidihttp://2011.igem.org/File:Washington_.jpgFile:Washington .jpg2011-09-16T01:40:47Z<p>Hadidi: uploaded a new version of &quot;File:Washington .jpg&quot;</p>
<hr />
<div></div>Hadidi