http://2011.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=20&target=Mdsmith&year=&month=2011.igem.org - User contributions [en]2024-03-28T11:45:08ZFrom 2011.igem.orgMediaWiki 1.16.0http://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-10-21T22:12:19Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1<br />
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors<br />
* A table compiling individual gene functions from our literature search<br />
* A table of cloning efficiency <br />
<br> <br />
-----<br><br />
<br />
== '''Superfolder GFP-magnetosome gene protein fusions'''==<br />
<br />
The two genes we characterized as fusions with superfolder GFP are <i>mamK</i> and <i>mamI</i>. They each perform core functions of magnetosome formation. MamK is a bacterial actin-like cytoskeleton protein required for proper alignment of the magnetosomes in a chain. MamI is a membrane-localized protein required for magnetosome vesicle formation that has also been shown to localize on the MamK filament. For more information, see the <i>mamAB</i> description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page]. Using our two genes of interest, we created C-terminal sfGFP fusions so we could track the localization of each gene separately within ''E.coli.'' <br />
<br />
===sfGFP-MamK: Scaffold formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
The results we obtained with our sfGFP fusions inside ''E. coli'' were comparable to those done through other studies in the host organism ''Magnetospirillum magneticum''. Within AMB-1, MamK is a filament which runs along the long axis of the bacteria. In the our images of sfGFP-MamK, scaffold-like structures can be clearly seen running through the length of most of the cells, in some cases looping back within a single cell for "figure-8" shaped filaments. In other cases, the filaments seem to prevent the cells from dividing properly, resulting in long chains of ''E. coli''. In our experimental result, there was an over- expression of mamK which connected the ''E.coli'' cells together. <br />
<br />
Here is a video showing the filaments connecting the growing E coli:<br />
<br />
<html><center><iframe width="420" height="315" src="http://www.youtube.com/embed/BLLyUHrrcV4" frameborder="0" allowfullscreen></iframe></center></html><br />
<br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br><br />
<br />
===sfGFP-MamI: Membrane localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. The membrane localization is easily seen by the fluorescence profile analysis seen on the panel on the right. The graph shows that the fluorescence levels peak near the cell membrane and decrease to a minimum in the middle of the cytoplasm.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
After verifying that the construction of the sfGFP-MamK scaffold worked as expected, we proceeded to create a full assembly of the <i>mamAB</i> operon by building three super-assemblies: ''mamHIEJKL'', ''mamMNOPA'', and ''mamQRBSTUV''. The PCR products of these intermediate assemblies are shown below. The ''mamHIEJKL'' and ''mamQRBSTUV'' have been partially sequence-confirmed, and we are currently working on designing primers to fill in the gap sequences. Despite these gaps, when cells with the ''mamHIEJKL'' construct were imaged, they appeared to be forming chains.<br/><br />
<center>[[File:Washington_iGEM2011_magentosome_HIEJKL3k3.png|500px|middle]]:[[File:Washington_iGEM2011_magentosome_MNOPA.png|100px|middle]][[File:Washington_iGEM2011_magentosome_QRBSTUV.png|100px|middle]]<br />
</center><br />
<br/><br/><br />
<br />
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==<br />
<br />
Before piecing together the 16 kb genome of the mamAB gene cluster within the magnetosome island (MAI), we extracted out the genes in the following groups: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}.<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].<br />
<br />
=='''Table of computationally predicted cloning efficiencies ''' == <br />
The following results and analyses are preliminary. <br />
<br />
The following is a list of computationally predicted cloning efficiencies for each gene we cloned, from the publicly available RBS tool. <br />
<br />
{| class="wikitable"<br />
|-<br />
! mam gene<br />
! AMB-1<br />
! ''E.Coli''<br />
|-<br />
| mamH<br />
| 10821.63<br />
| 10821.63<br />
|-<br />
| mamI<br />
| 88.93<br />
| 88.93<br />
|-<br />
| mamE<br />
| 1.59<br />
| 1.62<br />
|-<br />
| mamJ<br />
| 105.9<br />
| 105.9<br />
|- <br />
| mamK<br />
| 1056.92<br />
| 1056.92<br />
|-<br />
| mamL<br />
| 584.21<br />
| 584.21<br />
|-<br />
| mamM<br />
| 930.1<br />
| 930.1<br />
|-<br />
| mamN<br />
| 413.73<br />
| 230.48<br />
|- <br />
| mamO<br />
| 138.73<br />
| 138.73<br />
|-<br />
| mamP<br />
| 198.85<br />
| 198.85<br />
|-<br />
| mamA<br />
| 14.29<br />
| 14.29<br />
|-<br />
| mamQ<br />
| 22.41<br />
| 12.49<br />
|-<br />
| mamR<br />
| XXX<br />
| XXX<br />
|- <br />
| mamB<br />
| 16.56<br />
| 16.56<br />
|- <br />
| mamS<br />
| 32.86<br />
| 25.09<br />
|- <br />
| mamT<br />
| 81.16<br />
| 81.16<br />
|- <br />
| mamU<br />
| 33.58<br />
| 33.58<br />
|- <br />
| mamV<br />
| 1.61<br />
| 1.61<br />
<br />
|}.</div>Mdsmithhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-10-21T21:24:14Z<p>Mdsmith: Adding MamK video</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1<br />
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors<br />
* A table compiling individual gene functions from our literature search<br />
* A table of cloning efficiency <br />
<br> <br />
-----<br><br />
<br />
== '''Superfolder GFP-magnetosome gene protein fusions'''==<br />
<br />
The two genes we characterized as fusions with superfolder GFP are <i>mamK</i> and <i>mamI</i>. They each perform core functions of magnetosome formation. MamK is a bacterial actin-like cytoskeleton protein required for proper alignment of the magnetosomes in a chain. MamI is a membrane-localized protein required for magnetosome vesicle formation that has also been shown to localize on the MamK filament. For more information, see the <i>mamAB</i> description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page]. Using our two genes of interest, we created C-terminal sfGFP fusions so we could track the localization of each gene separately within ''E.coli.'' <br />
<br />
===sfGFP-MamK: Scaffold formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
The results we obtained with our sfGFP fusions inside ''E. coli'' were comparable to those done through other studies in the host organism ''Magnetospirillum magneticum''. Within AMB-1, MamK is a filament which runs along the long axis of the bacteria. In the our images of sfGFP-MamK, scaffold-like structures can be clearly seen running through the length of most of the cells, in some cases looping back within a single cell for "figure-8" shaped filaments. In other cases, the filaments seem to prevent the cells from dividing properly, resulting in long chains of ''E. coli''. In our experimental result, there was an over- expression of mamK which connected the ''E.coli'' cells together. <br />
<br />
Here is a video showing the filaments connecting the growing E coli:<br />
<br />
<html><center><iframe width="420" height="315" src="http://www.youtube.com/embed/BLLyUHrrcV4" frameborder="0" allowfullscreen></iframe></center></html><br />
<br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br><br />
<br />
===sfGFP-MamI: Membrane localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. The membrane localization is easily seen by the fluorescence profile analysis seen on the panel on the right. The graph shows that the fluorescence levels peak near the cell membrane and decrease to a minimum in the middle of the cytoplasm.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
After verifying that the construction of the sfGFP-MamK scaffold worked as expected, we proceeded to create a full assembly of the <i>mamAB</i> operon by building three super-assemblies: ''mamHIEJKL'', ''mamMNOPA'', and ''mamQRBSTUV''. The PCR products of these intermediate assemblies are shown below. The ''mamHIEJKL'' and ''mamQRBSTUV'' have been partially sequence-confirmed, and we are currently working on designing primers to fill in the gap sequences. Despite these gaps, when cells with the ''mamHIEJKL'' construct were imaged, they appeared to be forming chains.<br/><br />
<center>[[File:Washington_iGEM2011_magentosome_HIEJKL3k3.png|500px|middle]]:[[File:Washington_iGEM2011_magentosome_MNOPA.png|100px|middle]][[File:Washington_iGEM2011_magentosome_QRBSTUV.png|100px|middle]]<br />
</center><br />
<br/><br/><br />
<br />
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==<br />
<br />
Before piecing together the 16 kb genome of the mamAB gene cluster within the magnetosome island (MAI), we extracted out the genes in the following groups: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}.<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].<br />
<br />
=='''A table of cloning efficiency ''' ==<br />
{| class="wikitable"<br />
|-<br />
! mam gene<br />
! AMB-1<br />
! ''E.Coli''<br />
|-<br />
| mamH<br />
| 10821.63<br />
| 10821.63<br />
|-<br />
| mamI<br />
| 88.93<br />
| 88.93<br />
|-<br />
| mamE<br />
| 1.59<br />
| 1.62<br />
|-<br />
| mamJ<br />
| 105.9<br />
| 105.9<br />
|- <br />
| mamK<br />
| 1056.92<br />
| 1056.92<br />
|-<br />
| mamL<br />
| 584.21<br />
| 584.21<br />
|-<br />
| mamM<br />
| 930.1<br />
| 930.1<br />
|-<br />
| mamN<br />
| 413.73<br />
| 230.48<br />
|- <br />
| mamO<br />
| 138.73<br />
| 138.73<br />
|-<br />
| mamP<br />
| 198.85<br />
| 198.85<br />
|-<br />
| mamA<br />
| 14.29<br />
| 14.29<br />
|-<br />
| mamQ<br />
| 22.41<br />
| 12.49<br />
|-<br />
| mamR<br />
| XXX<br />
| XXX<br />
|- <br />
| mamB<br />
| 16.56<br />
| 16.56<br />
|- <br />
| mamS<br />
| 32.86<br />
| 25.09<br />
|- <br />
| mamT<br />
| 81.16<br />
| 81.16<br />
|- <br />
| mamU<br />
| 33.58<br />
| 33.58<br />
|- <br />
| mamV<br />
| 1.61<br />
| 1.61<br />
<br />
|}.</div>Mdsmithhttp://2011.igem.org/Team:Washington/googlefa08e57614f677f4.htmlTeam:Washington/googlefa08e57614f677f4.html2011-10-16T23:02:17Z<p>Mdsmith: Making google verification page</p>
<hr />
<div><html>google-site-verification: googlefa08e57614f677f4.html</html></div>Mdsmithhttp://2011.igem.org/Team:WashingtonTeam:Washington2011-10-16T22:19:20Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<html><meta name="google-site-verification" content="fg3_xZB6BF10NZTT7oSIbF6AmRx0o-b-VZdgok0O3Ok" /></html><br />
=<center>'''Make It or Break It: <br/> Diesel Production and Gluten Destruction, the Synthetic Biology Way'''</center>=<br />
<br />
<center>Synthetic biology holds great promise regarding the production of important compounds, and the degradation of harmful ones. This summer, we harnessed the power of synthetic biology to meet the world’s needs for fuel and medicine.</center><br />
<br/><br />
[[Image:Washington_Fire.jpg|left|320px|borderless|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[Image:Washington_Bottle.jpg|right|200px|borderless|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
<br />
[https://2011.igem.org/Team:Washington/Alkanes/Background '''Make It: Diesel Production'''] We constructed a strain of ''Escherichia coli'' that produces a variety of alkanes, the main constituents of diesel fuel, by introducing a pair of genes recently shown convert fatty acid synthesis intermediates into alkanes. <br />
<br />
[https://2011.igem.org/Team:Washington/Celiacs/Background '''Break It: Gluten Destruction'''] We identified a protease with gluten-degradation potential, and then reengineered it to have greatly increased gluten-degrading activity, allowing for the breakdown of gluten in the digestive track when taken in pill form. <br />
<br />
[https://2011.igem.org/Team:Washington/Magnetosomes/Background '''iGEM Toolkits'''] To enable next-generation cloning of standard biological parts, we built BioBrick vectors optimized for Gibson assembly and used them to create the Magnetosome Toolkit: a set of 18 genes from an essential operon in magnetotactic bacteria which we are characterizing to create magnetic ''E. coli''.<br />
<br />
<br />
[[File:Washington_Spacer.jpg|1px]]<br />
[[Image:UW Diesel Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Toolkits Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Gluten Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
<br />
<br />
<br/><br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_OSLI.png|frameless|border|link=http://www.osli.ca|Oil Sands Leadership Intiative]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_UniversitySeal.gif|frameless|border|110px|link=http://www.washington.edu|University of Washington]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_Anaspec.gif|frameless|border|120px|link=http://www.anaspec.com|Anaspec]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_ARPA-E_Logo.png|frameless|border|link=http://arpa-e.energy.gov/ProgramsProjects/Electrofuels.aspx|Advanced Research Projects Agency - Energy]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington2011_Hhmi_362_72.jpg|link=http://www.hhmi.org/|Howard Hughes Medical Institute]]</div>Mdsmithhttp://2011.igem.org/Team:WashingtonTeam:Washington2011-10-16T22:19:03Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<html><meta name="google-site-verification" content="fg3_xZB6BF10NZTT7oSIbF6AmRx0o-b-VZdgok0O3Ok" /><html/><br />
=<center>'''Make It or Break It: <br/> Diesel Production and Gluten Destruction, the Synthetic Biology Way'''</center>=<br />
<br />
<center>Synthetic biology holds great promise regarding the production of important compounds, and the degradation of harmful ones. This summer, we harnessed the power of synthetic biology to meet the world’s needs for fuel and medicine.</center><br />
<br/><br />
[[Image:Washington_Fire.jpg|left|320px|borderless|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[Image:Washington_Bottle.jpg|right|200px|borderless|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
<br />
[https://2011.igem.org/Team:Washington/Alkanes/Background '''Make It: Diesel Production'''] We constructed a strain of ''Escherichia coli'' that produces a variety of alkanes, the main constituents of diesel fuel, by introducing a pair of genes recently shown convert fatty acid synthesis intermediates into alkanes. <br />
<br />
[https://2011.igem.org/Team:Washington/Celiacs/Background '''Break It: Gluten Destruction'''] We identified a protease with gluten-degradation potential, and then reengineered it to have greatly increased gluten-degrading activity, allowing for the breakdown of gluten in the digestive track when taken in pill form. <br />
<br />
[https://2011.igem.org/Team:Washington/Magnetosomes/Background '''iGEM Toolkits'''] To enable next-generation cloning of standard biological parts, we built BioBrick vectors optimized for Gibson assembly and used them to create the Magnetosome Toolkit: a set of 18 genes from an essential operon in magnetotactic bacteria which we are characterizing to create magnetic ''E. coli''.<br />
<br />
<br />
[[File:Washington_Spacer.jpg|1px]]<br />
[[Image:UW Diesel Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Toolkits Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Gluten Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
<br />
<br />
<br/><br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_OSLI.png|frameless|border|link=http://www.osli.ca|Oil Sands Leadership Intiative]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_UniversitySeal.gif|frameless|border|110px|link=http://www.washington.edu|University of Washington]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_Anaspec.gif|frameless|border|120px|link=http://www.anaspec.com|Anaspec]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_ARPA-E_Logo.png|frameless|border|link=http://arpa-e.energy.gov/ProgramsProjects/Electrofuels.aspx|Advanced Research Projects Agency - Energy]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington2011_Hhmi_362_72.jpg|link=http://www.hhmi.org/|Howard Hughes Medical Institute]]</div>Mdsmithhttp://2011.igem.org/Team:WashingtonTeam:Washington2011-10-12T00:39:28Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=<center>'''Make It or Break It: <br/> Diesel Production and Gluten Destruction, the Synthetic Biology Way'''</center>=<br />
<br />
<center>Synthetic biology holds great promise regarding the production of important compounds, and the degradation of harmful ones. This summer, we harnessed the power of synthetic biology to meet the world’s needs for fuel and medicine.</center><br />
<br/><br />
[[Image:Washington_Fire.jpg|left|320px|borderless|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[Image:Washington_Bottle.jpg|right|200px|borderless|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
<br />
[https://2011.igem.org/Team:Washington/Alkanes/Background '''Make It: Diesel Production'''] We constructed a strain of ''Escherichia coli'' that produces a variety of alkanes, the main constituents of diesel fuel, by introducing a pair of genes recently shown convert fatty acid synthesis intermediates into alkanes. <br />
<br />
[https://2011.igem.org/Team:Washington/Celiacs/Background '''Break It: Gluten Destruction'''] We identified a protease with gluten-degradation potential, and then reengineered it to have greatly increased gluten-degrading activity, allowing for the breakdown of gluten in the digestive track when taken in pill form. <br />
<br />
[https://2011.igem.org/Team:Washington/Magnetosomes/Background '''iGEM Toolkits'''] To enable next-generation cloning of standard biological parts, we built BioBrick vectors optimized for Gibson assembly and used them to create the Magnetosome Toolkit: a set of 18 genes from an essential operon in magnetotactic bacteria which we are characterizing to create magnetic ''E. coli''.<br />
<br />
<br />
[[File:Washington_Spacer.jpg|1px]]<br />
[[Image:UW Diesel Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Toolkits Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Gluten Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
<br />
<br />
<br/><br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_OSLI.png|frameless|border|link=http://www.osli.ca|Oil Sands Leadership Intiative]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_UniversitySeal.gif|frameless|border|110px|link=http://www.washington.edu|University of Washington]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_Anaspec.gif|frameless|border|120px|link=http://www.anaspec.com|Anaspec]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_ARPA-E_Logo.png|frameless|border|link=http://arpa-e.energy.gov/ProgramsProjects/Electrofuels.aspx|Advanced Research Projects Agency - Energy]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington2011_Hhmi_362_72.jpg|link=http://www.hhmi.org/]]<br />
<br />
<html><br />
</html></div>Mdsmithhttp://2011.igem.org/Template:Team:Washington/Templates/TopTemplate:Team:Washington/Templates/Top2011-10-12T00:39:12Z<p>Mdsmith: Adding google analytics</p>
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<div id="ddnav" align="center"><br />
<ul><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington">Home</a><br />
<div><br />
<a href="https://2011.igem.org/Main_Page"> iGEM Homepage </a><br />
<a href="https://2011.igem.org/Team:Washington">UW 2011</a><br />
<a href="https://2010.igem.org/Team:Washington"> UW 2010 </a><br />
<a href="https://2009.igem.org/Team:Washington"> UW 2009 </a><br />
<a href="https://2008.igem.org/Team:University_of_Washington"> UW 2008 </a><br />
<a href="http://synbio.washington.edu/"> UW SynBio </a><br />
</div><br />
<br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Team/Members">About Us</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Team/Members">The team</a><br />
<a href="https://2011.igem.org/Team:Washington/Team/Sponsors"> Sponsors </a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Background">Diesel Production</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Background">Background</a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Methods"> Methods </a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Results"> Results Summary </a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Future"> Future Directions </a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Parts"> Parts Submitted </a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Background">Gluten Destruction</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Background">Background</a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Methods"> Methods </a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Results"> Results Summary</a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Future"> Future Directions </a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Parts"> Parts Submitted </a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Background">iGEM Toolkits</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Background"> Background </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors">Gibson Assembly Toolkit </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults">Gibson Toolkit Results</a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit">Magnetosome Toolkit</a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Results">Magnetosome Results</a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Parts"> Parts Submitted </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Future"> Future Directions </a><br />
<br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Parts">Data Page</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Parts">Data Page</a><br />
<a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2011&group=Washington"> Parts Registry </a><br />
</div><br />
<br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Protocols">Protocols</a><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Outreach">Outreach</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Outreach">Local Outreach</a><br />
<br />
<a href="https://2011.igem.org/Team:Washington/Outreach/iGEM_Collaborations">iGEM Collaborations</a></div><br />
<br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Safety">Safety</a><br />
</li><br />
</ul><br />
</div><br />
</div><br />
</html></div>Mdsmithhttp://2011.igem.org/Team:WashingtonTeam:Washington2011-10-12T00:23:26Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=<center>'''Make It or Break It: <br/> Diesel Production and Gluten Destruction, the Synthetic Biology Way'''</center>=<br />
<br />
<center>Synthetic biology holds great promise regarding the production of important compounds, and the degradation of harmful ones. This summer, we harnessed the power of synthetic biology to meet the world’s needs for fuel and medicine.</center><br />
<br/><br />
[[Image:Washington_Fire.jpg|left|320px|borderless|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[Image:Washington_Bottle.jpg|right|200px|borderless|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
<br />
[https://2011.igem.org/Team:Washington/Alkanes/Background '''Make It: Diesel Production'''] We constructed a strain of ''Escherichia coli'' that produces a variety of alkanes, the main constituents of diesel fuel, by introducing a pair of genes recently shown convert fatty acid synthesis intermediates into alkanes. <br />
<br />
[https://2011.igem.org/Team:Washington/Celiacs/Background '''Break It: Gluten Destruction'''] We identified a protease with gluten-degradation potential, and then reengineered it to have greatly increased gluten-degrading activity, allowing for the breakdown of gluten in the digestive track when taken in pill form. <br />
<br />
[https://2011.igem.org/Team:Washington/Magnetosomes/Background '''iGEM Toolkits'''] To enable next-generation cloning of standard biological parts, we built BioBrick vectors optimized for Gibson assembly and used them to create the Magnetosome Toolkit: a set of 18 genes from an essential operon in magnetotactic bacteria which we are characterizing to create magnetic ''E. coli''.<br />
<br />
<br />
[[File:Washington_Spacer.jpg|1px]]<br />
[[Image:UW Diesel Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Toolkits Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Gluten Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
<br />
<br />
<br/><br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_OSLI.png|frameless|border|link=http://www.osli.ca|Oil Sands Leadership Intiative]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_UniversitySeal.gif|frameless|border|110px|link=http://www.washington.edu|University of Washington]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_Anaspec.gif|frameless|border|120px|link=http://www.anaspec.com|Anaspec]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_ARPA-E_Logo.png|frameless|border|link=http://arpa-e.energy.gov/ProgramsProjects/Electrofuels.aspx|Advanced Research Projects Agency - Energy]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington2011_Hhmi_362_72.jpg|link=http://www.hhmi.org/]]<br />
<br />
<html><br />
<script type="text/javascript"><br />
<br />
var _gaq = _gaq || [];<br />
_gaq.push(['_setAccount', 'UA-26265208-1']);<br />
_gaq.push(['_trackPageview']);<br />
<br />
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</script></html></div>Mdsmithhttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-29T02:13:46Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Gluten Destruction: Methods'''</big></big></big></big></center><br><br><br />
<br />
='''Redesigning Kumamolisin to Have Higher Activity at Low pH'''=<br />
<br />
<br />
[[File:Washington Foldit.png|550px|thumb|right|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 />
<br />
----<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|left|Overview of how Kunkel Mutagenesis works]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
=='''Kunkel Mutagenesis'''==<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. <br />
<br />
To incorporate these mutations, we first isolated single stranded DNA (ssDNA) of our vector harboring the wild-type Kumamolisin gene. To do this we infected cells with bacteriophage M13, which packages its own ssDNA genome identified by length, and so in tandem packaged our vector in single stranded form. We then harvested the phage from the lysed culture of E. coli, and extracted our single stranded vector DNA.<br />
<br />
Next, we annealed and extended our mutagenic oligos to incorporate the specified mutations into the newly synthesized antisense strand. This hybrid vector was transformed into E. coli that degraded the original uracil-containing DNA and replaced it with sections complementary to the mutagenized strand.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
='''Using a Whole Cell Lysate Assay to Test Activity of Mutants'''=<br />
To test our designs, we developed a whole cell lysate assay that allowed us to perform a rough screen of a large number of mutants. In this assay, we expressed our mutant enzymes in <i>E. coli</i>, lysed the cells and separated the enzymes from large cell particulate. We then performed the assay at pH 4, mimicking the gastric environment. We added our model PQLP peptide, conjugated to both a fluorophore and a quencher so that no fluorescence would be achieved until after the peptide had been enzymatically cleaved. We then measured the fluorescence of each reaction at 30 second intervals, and were thereby able to estimate relative activity on breaking down PQLP by increase in fluorescence of the system.<br />
<br />
[[File:Washington Whole Cell Lysate Assay.jpg|center|General Overview of the Whole Cell Lysate]]<br />
<br />
<br />
----<br />
<br />
<br />
='''Testing Purified Mutants to Accurately Assess Activity'''=<br />
<br />
[[File:Washington First Raw Data.png|right|500px|thumb|We measured fluorescence of each reaction at 30 second intervals to see the rate at which each mutant cleaved PQLP.]]<br />
<br />
=='''Purification'''==<br />
From our whole cell lysate screen of each design, we identified mutants that showed the most increase in activity from the wild-type Kumamolisin. We then proceeded to purify these most promising variants and test them against the wild-type and against SC PEP using the same fluorescence metric designed for the whole cell lysate assay. The key difference between the whole cell assay and the purified protein assay is that in the latter we were able to control the concentration of enzyme in each well, adjusting for the possibility of varying expression levels and thus enzyme concentrations in the whole cell lysate assay.<br />
<br />
Purification was performed via Nickel-affinity chromatography, and resulting protein concentrations were measured using ultraviolet-visible spectrophotometry.<br />
<br />
=='''Assay'''==<br />
Concentration dependent assays were performed for each promising mutant. We measured the fluorescence of each reaction at 30 second intervals to see the rate at which fluorescence increased, thus obtaining a relative rate of cleavage of PQLP by increase in fluorescence of the system. Raw data appeared as shown right, and the slope of each line was calculated, giving us relative rate information that could be used in conjunction with rate information obtained in the same assay for native Kumamolisin to determine fold change in activity.</div>Mdsmithhttp://2011.igem.org/Team:Washington/Celiacs/BackgroundTeam:Washington/Celiacs/Background2011-09-29T02:11:54Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Gluten Destruction: Background'''</big></big></big></big></center><br><br><br />
<br />
='''What is Gluten Intolerance?'''=<br />
<br />
[[File:Washington CD Diagram.png|right|250px|thumb|Proline(P) and glutamine(Q) -rich peptide fragments of gluten provoke an immune response which causes painful inflammation in the digestive tract of gluten intolerant individuals.]]<br />
<br />
People who suffer from gluten intolerance have a adverse reaction to gluten proteins found in wheat, barley, and rye products. The glutens invoke an immune response in the digestive tract of genetically predisposed individuals resulting in inflammation of the gut, impeding the absorption of nutrients. Symptoms can appear in early childhood or later in life, and range widely in severity, from diarrhea, fatigue and weight loss to abdominal distension, anemia, and neurological symptoms. There are currently no effective therapies for this lifelong disease except the total elimination of glutens from the diet. Although celiac sprue remains largely underdiagnosed, its prevalence in the US and Europe is estimated at 0.5-1.0% of the population. With this in mind, we set out to design an enzyme therapeutic for gluten intolerance that could be taken in pill form.<br />
<br />
Proline (P)- and glutamine (Q)-rich components of gluten known as ‘gliadins’ appear to be responsible for the bulk of the immune response in most patients. Their high PQ content protects gliadin oligopeptides from degradation by gastrointenstinal endoproteases, but also presents a target for drug design. Any peptidase capable of cleaving at or near the P-Q bond while remaining active at the temperature and harsh pH of the stomach would have pharmacological potential as a therapy for celiac sprue.<br />
<br />
----<br />
<br />
='''There is currently a protein therapeutic in clinical trials, but a second generation is needed'''=<br />
<br />
[[File:Washington_SC-PEP.png|left|250px|thumb|SC-PEP, a prolyl endopeptidase from ''Sphingomonas capsulata'']]<br />
<br />
One candidate enzyme, currently in clinical trials, utilizes a prolyl endopeptidase (PEP) from ''Sphingomonas capsulata'' (SC) to hydrolyze gliadins. This enzyme was a logical drug candidate as it has a native specificity for the proline rich gliadin peptides. Unfortunately, the enzyme’s optimal activity is at pH 7, and engineering attempts to enhance its activity at relevant gastric pH levels has not yet been met with significant success. It is therefore pertinent to identify new candidate enzymes that have both activity on the gliadin PQ structural motif, as well as optimal activity at gastric pH levels during digestion around pH 4. <br />
<br />
<br />
Here, we describe an alternative approach to identifying and engineering an enzyme therapeutic for celiac sprue. Rather than focusing primarily on substrate specificity when choosing our candidate enzyme, we identified an enzyme already capable of catalyzing peptide hydrolysis at gastric pH levels, regardless of peptide substrate specificity. Upon identification of such an enzyme we used computational tools to reengineer its substrate specificity for enhanced activity on immunogenic gliadin peptides with the common PQ structural motif.<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[File:Washington_Kumamolisin-As.png|right|250px|thumb|Kumamolisin-As, a protease from the thermoacidophilic bacterium ''Alicyclobacillus sendaiensis'']]<br />
<br />
='''We have identified an enzyme that could potentially act as a therapeutic for gluten intolerance'''=<br />
<br />
When searching for a protease with optimal activity at gastric pH levels that could be produced in a recombinant host, we identified an enzyme known as Kumamolisin-As. Kumamolisin-As, isolated from the thermoacidophilic bacterium ''Alicyclobacillus sendaiensis'' strain NTAP-1, has been shown to be produced in a recombinant host, and exhibits significant enzymatic activity at pH 2.5 and above. Its maximal activity is at about pH 4.0. It is this robust activity under acidic, gastric conditions that makes Kumamolisin-As so promising for the development of a pill for gluten intolerance.<br />
<br />
='''A special set of catalytic residues enables high activity at gastric pH levels'''=<br />
<br />
One of the primary reasons that Kumamolisin-As is more active than SC-PEP at low pH is due its catalytic triad, the three amino acid residues most heavily involved in conducting the chemistry of cleaving peptides. SC-PEP is part of a large class of enzymes known as serine proteases, which make use of the catalytic triad Serine-Histidine-Aspartate. Acidic conditions can impede the histidine's ability to play its role in this triad, rendering most serine proteases inactive at low pH. Kumamolisin-As, on the other hand, is a member of the sedolisin family of serine-carboxyl peptidases, which utilize the key catalytic triad Serine-Glutamate-Aspartate to hydrolyze their substrates. It is the acidic Glutamate, as opposed to Histidine, in the triad that makes Kumamolisin-As optimal for catalyzing peptide cleavage at low pH. The native substrate for this enzyme in ''Alicyclobacillus sendaiensis'' is unknown. In addition, this enzyme has been shown to be thermostable, with an ideal temperature at 60&ordm;C, but still showing significant activity at 37&ordm;C. <br />
<br />
[[File:Washington_Kuma Bonded triad.png|left|250px|thumb|Kumamolisin-As' unusual catalytic triad Ser278-Glu78-Asp82 makes the enzyme well suited to a low pH environment.]]<br />
[[File:Washington_serine protease triad2.png|left|250px|thumb|The traditional Ser-His-Asp catalytic triad found in serine proteases like SC-PEP does not function optimally at low pH.]]<br />
<br />
='''Kumamolisin-As is already active for the dipeptide motif PR, we just need to change it to PQ'''=<br />
<br />
:::::::::::::::::::::<p>Specificity studies have been conducted on the native enzyme, which is most efficient at hydrolyzing peptides at the carboxyl side after the PR dipeptide motif (where the P1 and P2 subsites are arginine and proline, respectively, in standard proteolysis nomenclature). The enzyme shows little preference for what is on the amino S1 and S2 subsites. The combination of Kumamolisin-As having the desired enzymatic activity under gastric pH levels, and the P2 subsite already having the specificity for the proline from the desired PQ motif makes it a highly attractive candidate for further engineering. In addition, several crystal structures of this enzyme have been solved, allowing for the use of computational design techniques.</p><br />
<br />
----<br />
<br />
==References==<br />
<br />
1.Shan, Lu, et al. "Structural Basis for Gluten Intolerance in Celiac Sprue." Science 297.5590 (2002): 2275-2279.<br />
<br />
2.Mustalahti, Kirsi, et al. "The prevalence of celiac disease in Europe: Results of a centralized, international mass screening project." Annals of Medicine 42.8 (2010): 587-595.<br />
<br />
3.Ehren, Jennifer, et al. "Protein engineering of improved prolyl endopeptidases for celiac sprue therapy." Protein Engineering, Design & Selection 21.12 (2008): 699-707.<br />
<br />
4.Wlodawer, Alexander, et al. "Crystallographic and Biochemical Investigations of Kumamolisin-As, a Serine-Carboxyl Peptidase with Collagenase Activity." The Journal of Biological Chemistry 279.20 (2004): 21500-21510.</div>Mdsmithhttp://2011.igem.org/Team:Washington/Celiacs/BackgroundTeam:Washington/Celiacs/Background2011-09-29T02:11:20Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Gluten Destruction: Background'''</big></big></big></big></center><br><br><br />
<br />
='''What is Gluten Intolerance?'''=<br />
<br />
[[File:Washington CD Diagram.png|right|250px|thumb|Proline(P) and glutamine(Q) -rich peptide fragments of gluten provoke an immune response which causes painful inflammation in the digestive tract of gluten intolerant individuals.]]<br />
<br />
People who suffer from gluten intolerance have a adverse reaction to gluten proteins found in wheat, barley, and rye products. The glutens invoke an immune response in the digestive tract of genetically predisposed individuals resulting in inflammation of the gut, impeding the absorption of nutrients. Symptoms can appear in early childhood or later in life, and range widely in severity, from diarrhea, fatigue and weight loss to abdominal distension, anemia, and neurological symptoms. There are currently no effective therapies for this lifelong disease except the total elimination of glutens from the diet. Although celiac sprue remains largely underdiagnosed, its prevalence in the US and Europe is estimated at 0.5-1.0% of the population. With this in mind, we set out to design an enzyme therapeutic for gluten intolerance that could be taken in pill form.<br />
<br />
Proline (P)- and glutamine (Q)-rich components of gluten known as ‘gliadins’ appear to be responsible for the bulk of the immune response in most patients. Their high PQ content protects gliadin oligopeptides from degradation by gastrointenstinal endoproteases, but also presents a target for drug design. Any peptidase capable of cleaving at or near the P-Q bond while remaining active at the temperature and harsh pH of the stomach would have pharmacological potential as a therapy for celiac sprue.<br />
<br />
----<br />
<br />
='''There is currently a protein therapeutic in clinical trials, but a second generation is needed'''=<br />
<br />
[[File:Washington_SC-PEP.png|left|250px|thumb|SC-PEP, a prolyl endopeptidase from ''Sphingomonas capsulata'']]<br />
<br />
One candidate enzyme, currently in clinical trials, utilizes a prolyl endopeptidase (PEP) from ''Sphingomonas capsulata'' (SC) to hydrolyze gliadins. This enzyme was a logical drug candidate as it has a native specificity for the proline rich gliadin peptides. Unfortunately, the enzyme’s optimal activity is at pH 7, and engineering attempts to enhance its activity at relevant gastric pH levels has not yet been met with significant success. It is therefore pertinent to identify new candidate enzymes that have both activity on the gliadin PQ structural motif, as well as optimal activity at gastric pH levels during digestion around pH 4. <br />
<br />
<br />
Here, we describe an alternative approach to identifying and engineering an enzyme therapeutic for celiac sprue. Rather than focusing primarily on substrate specificity when choosing our candidate enzyme, we identified an enzyme already capable of catalyzing peptide hydrolysis at gastric pH levels, regardless of peptide substrate specificity. Upon identification of such an enzyme we used computational tools to reengineer its substrate specificity for enhanced activity on immunogenic gliadin peptides with the common PQ structural motif.<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[File:Washington_Kumamolisin-As.png|right|250px|thumb|Kumamolisin-As, a protease from the thermoacidophilic bacterium ''Alicyclobacillus sendaiensis'']]<br />
<br />
='''We have identified an enzyme that could potentially act as a therapeutic for gluten intolerance'''=<br />
<br />
When searching for a protease with optimal activity at gastric pH levels that could be produced in a recombinant host, we identified an enzyme known as Kumamolisin-As. Kumamolisin-As, isolated from the thermoacidophilic bacterium ''Alicyclobacillus sendaiensis'' strain NTAP-1, has been shown to be produced in a recombinant host, and exhibits significant enzymatic activity at pH 2.5 and above. Its maximal activity is at about pH 4.0. It is this robust activity under acidic, gastric conditions that makes Kumamolisin-As so promising for the development of a pill for gluten intolerance.<br />
<br />
='''A special set of catalytic residues enables high activity at gastric pH levels'''=<br />
<br />
One of the primary reasons that Kumamolisin-As is more active than SC-PEP at low pH is due its catalytic triad, the three amino acid residues most heavily involved in conducting the chemistry of cleaving peptides. SC-PEP is part of a large class of enzymes known as serine proteases, which make use of the catalytic triad Serine-Histidine-Aspartate. Acidic conditions can impede the histidine's ability to play its role in this triad, rendering most serine proteases inactive at low pH. Kumamolisin-As, on the other hand, is a member of the sedolisin family of serine-carboxyl peptidases, which utilize the key catalytic triad Serine-Glutamate-Aspartate to hydrolyze their substrates. It is the acidic Glutamate, as opposed to Histidine, in the triad that makes Kumamolisin-As optimal for catalyzing peptide cleavage at low pH. The native substrate for this enzyme in ''Alicyclobacillus sendaiensis'' is unknown. In addition, this enzyme has been shown to be thermostable, with an ideal temperature at 60&ordm;C, but still showing significant activity at 37&ordm;C. <br />
<br />
[[File:Washington_Kuma Bonded triad.png|left|250px|thumb|Kumamolisin-As' unusual catalytic triad Ser278-Glu78-Asp82 makes the enzyme well suited to a low pH environment.]]<br />
[[File:Washington_serine protease triad2.png|left|250px|thumb|The traditional Ser-His-Asp catalytic triad found in serine proteases like SC-PEP does not function optimally at low pH.]]<br />
<br />
='''Kumamolisin-As is already active for the dipeptide motif PR, we just need to change it to PQ'''=<br />
<br />
:::::::::::::::::::::<p>Specificity studies have been conducted on the native enzyme, which is most efficient at hydrolyzing peptides at the carboxyl side after the PR dipeptide motif (where the P1 and P2 subsites are arginine and proline, respectively, in standard proteolysis nomenclature). The enzyme shows little preference for what is on the amino S1 and S2 subsites. The combination of Kumamolisin-As having the desired enzymatic activity under gastric pH levels, and the P2 subsite already having the specificity for the proline from the desired PQ motif makes it a highly attractive candidate for further engineering. In addition, several crystal structures of this enzyme have been solved, allowing for the use of computational design techniques.</p><br />
<br />
----<br />
<br />
='''References'''=<br />
<br />
1.Shan, Lu, et al. "Structural Basis for Gluten Intolerance in Celiac Sprue." Science 297.5590 (2002): 2275-2279.<br />
<br />
2.Mustalahti, Kirsi, et al. "The prevalence of celiac disease in Europe: Results of a centralized, international mass screening project." Annals of Medicine 42.8 (2010): 587-595.<br />
<br />
3.Ehren, Jennifer, et al. "Protein engineering of improved prolyl endopeptidases for celiac sprue therapy." Protein Engineering, Design & Selection 21.12 (2008): 699-707.<br />
<br />
4.Wlodawer, Alexander, et al. "Crystallographic and Biochemical Investigations of Kumamolisin-As, a Serine-Carboxyl Peptidase with Collagenase Activity." The Journal of Biological Chemistry 279.20 (2004): 21500-21510.</div>Mdsmithhttp://2011.igem.org/Team:Washington/Celiacs/BackgroundTeam:Washington/Celiacs/Background2011-09-29T02:09:13Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Gluten Destruction: Background'''</big></big></big></big></center><br><br><br />
<br />
='''What is Gluten Intolerance?'''=<br />
<br />
[[File:Washington CD Diagram.png|right|250px|thumb|Proline(P) and glutamine(Q) -rich peptide fragments of gluten provoke an immune response which causes painful inflammation in the digestive tract of gluten intolerant individuals.]]<br />
<br />
People who suffer from gluten intolerance have a adverse reaction to gluten proteins found in wheat, barley, and rye products. The glutens invoke an immune response in the digestive tract of genetically predisposed individuals resulting in inflammation of the gut, impeding the absorption of nutrients. Symptoms can appear in early childhood or later in life, and range widely in severity, from diarrhea, fatigue and weight loss to abdominal distension, anemia, and neurological symptoms. There are currently no effective therapies for this lifelong disease except the total elimination of glutens from the diet. Although celiac sprue remains largely underdiagnosed, its prevalence in the US and Europe is estimated at 0.5-1.0% of the population. With this in mind, we set out to design an enzyme therapeutic for gluten intolerance that could be taken in pill form.<br />
<br />
Proline (P)- and glutamine (Q)-rich components of gluten known as ‘gliadins’ appear to be responsible for the bulk of the immune response in most patients. Their high PQ content protects gliadin oligopeptides from degradation by gastrointenstinal endoproteases, but also presents a target for drug design. Any peptidase capable of cleaving at or near the P-Q bond while remaining active at the temperature and harsh pH of the stomach would have pharmacological potential as a therapy for celiac sprue.<br />
<br />
----<br />
<br />
=There is currently a protein therapeutic in clinical trials, but a second generation is needed=<br />
<br />
[[File:Washington_SC-PEP.png|left|250px|thumb|SC-PEP, a prolyl endopeptidase from ''Sphingomonas capsulata'']]<br />
<br />
One candidate enzyme, currently in clinical trials, utilizes a prolyl endopeptidase (PEP) from ''Sphingomonas capsulata'' (SC) to hydrolyze gliadins. This enzyme was a logical drug candidate as it has a native specificity for the proline rich gliadin peptides. Unfortunately, the enzyme’s optimal activity is at pH 7, and engineering attempts to enhance its activity at relevant gastric pH levels has not yet been met with significant success. It is therefore pertinent to identify new candidate enzymes that have both activity on the gliadin PQ structural motif, as well as optimal activity at gastric pH levels during digestion around pH 4. <br />
<br />
<br />
Here, we describe an alternative approach to identifying and engineering an enzyme therapeutic for celiac sprue. Rather than focusing primarily on substrate specificity when choosing our candidate enzyme, we identified an enzyme already capable of catalyzing peptide hydrolysis at gastric pH levels, regardless of peptide substrate specificity. Upon identification of such an enzyme we used computational tools to reengineer its substrate specificity for enhanced activity on immunogenic gliadin peptides with the common PQ structural motif.<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[File:Washington_Kumamolisin-As.png|right|250px|thumb|Kumamolisin-As, a protease from the thermoacidophilic bacterium ''Alicyclobacillus sendaiensis'']]<br />
<br />
=We have identified an enzyme that could potentially act as a therapeutic for gluten intolerance=<br />
<br />
When searching for a protease with optimal activity at gastric pH levels that could be produced in a recombinant host, we identified an enzyme known as Kumamolisin-As. Kumamolisin-As, isolated from the thermoacidophilic bacterium ''Alicyclobacillus sendaiensis'' strain NTAP-1, has been shown to be produced in a recombinant host, and exhibits significant enzymatic activity at pH 2.5 and above. Its maximal activity is at about pH 4.0. It is this robust activity under acidic, gastric conditions that makes Kumamolisin-As so promising for the development of a pill for gluten intolerance.<br />
<br />
=A special set of catalytic residues enables high activity at gastric pH levels=<br />
<br />
One of the primary reasons that Kumamolisin-As is more active than SC-PEP at low pH is due its catalytic triad, the three amino acid residues most heavily involved in conducting the chemistry of cleaving peptides. SC-PEP is part of a large class of enzymes known as serine proteases, which make use of the catalytic triad Serine-Histidine-Aspartate. Acidic conditions can impede the histidine's ability to play its role in this triad, rendering most serine proteases inactive at low pH. Kumamolisin-As, on the other hand, is a member of the sedolisin family of serine-carboxyl peptidases, which utilize the key catalytic triad Serine-Glutamate-Aspartate to hydrolyze their substrates. It is the acidic Glutamate, as opposed to Histidine, in the triad that makes Kumamolisin-As optimal for catalyzing peptide cleavage at low pH. The native substrate for this enzyme in ''Alicyclobacillus sendaiensis'' is unknown. In addition, this enzyme has been shown to be thermostable, with an ideal temperature at 60&ordm;C, but still showing significant activity at 37&ordm;C. <br />
<br />
[[File:Washington_Kuma Bonded triad.png|left|250px|thumb|Kumamolisin-As' unusual catalytic triad Ser278-Glu78-Asp82 makes the enzyme well suited to a low pH environment.]]<br />
[[File:Washington_serine protease triad2.png|left|250px|thumb|The traditional Ser-His-Asp catalytic triad found in serine proteases like SC-PEP does not function optimally at low pH.]]<br />
<br />
=Kumamolisin-As is already active for the dipeptide motif PR, we just need to change it to PQ=<br />
<br />
:::::::::::::::::::::<p>Specificity studies have been conducted on the native enzyme, which is most efficient at hydrolyzing peptides at the carboxyl side after the PR dipeptide motif (where the P1 and P2 subsites are arginine and proline, respectively, in standard proteolysis nomenclature). The enzyme shows little preference for what is on the amino S1 and S2 subsites. The combination of Kumamolisin-As having the desired enzymatic activity under gastric pH levels, and the P2 subsite already having the specificity for the proline from the desired PQ motif makes it a highly attractive candidate for further engineering. In addition, several crystal structures of this enzyme have been solved, allowing for the use of computational design techniques.</p><br />
<br />
----<br />
<br />
=References=<br />
<br />
1.Shan, Lu, et al. "Structural Basis for Gluten Intolerance in Celiac Sprue." Science 297.5590 (2002): 2275-2279.<br />
<br />
2.Mustalahti, Kirsi, et al. "The prevalence of celiac disease in Europe: Results of a centralized, international mass screening project." Annals of Medicine 42.8 (2010): 587-595.<br />
<br />
3.Ehren, Jennifer, et al. "Protein engineering of improved prolyl endopeptidases for celiac sprue therapy." Protein Engineering, Design & Selection 21.12 (2008): 699-707.<br />
<br />
4.Wlodawer, Alexander, et al. "Crystallographic and Biochemical Investigations of Kumamolisin-As, a Serine-Carboxyl Peptidase with Collagenase Activity." The Journal of Biological Chemistry 279.20 (2004): 21500-21510.</div>Mdsmithhttp://2011.igem.org/Team:Washington/Celiacs/BackgroundTeam:Washington/Celiacs/Background2011-09-29T02:08:32Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Gluten Destruction: Background'''</big></big></big></big></center><br><br><br />
<br />
=What is Gluten Intolerance?=<br />
<br />
[[File:Washington CD Diagram.png|right|250px|thumb|Proline(P) and glutamine(Q) -rich peptide fragments of gluten provoke an immune response which causes painful inflammation in the digestive tract of gluten intolerant individuals.]]<br />
<br />
People who suffer from gluten intolerance have a adverse reaction to gluten proteins found in wheat, barley, and rye products. The glutens invoke an immune response in the digestive tract of genetically predisposed individuals resulting in inflammation of the gut, impeding the absorption of nutrients. Symptoms can appear in early childhood or later in life, and range widely in severity, from diarrhea, fatigue and weight loss to abdominal distension, anemia, and neurological symptoms. There are currently no effective therapies for this lifelong disease except the total elimination of glutens from the diet. Although celiac sprue remains largely underdiagnosed, its prevalence in the US and Europe is estimated at 0.5-1.0% of the population. With this in mind, we set out to design an enzyme therapeutic for gluten intolerance that could be taken in pill form.<br />
<br />
Proline (P)- and glutamine (Q)-rich components of gluten known as ‘gliadins’ appear to be responsible for the bulk of the immune response in most patients. Their high PQ content protects gliadin oligopeptides from degradation by gastrointenstinal endoproteases, but also presents a target for drug design. Any peptidase capable of cleaving at or near the P-Q bond while remaining active at the temperature and harsh pH of the stomach would have pharmacological potential as a therapy for celiac sprue.<br />
<br />
----<br />
<br />
=There is currently a protein therapeutic in clinical trials, but a second generation is needed=<br />
<br />
[[File:Washington_SC-PEP.png|left|250px|thumb|SC-PEP, a prolyl endopeptidase from ''Sphingomonas capsulata'']]<br />
<br />
One candidate enzyme, currently in clinical trials, utilizes a prolyl endopeptidase (PEP) from ''Sphingomonas capsulata'' (SC) to hydrolyze gliadins. This enzyme was a logical drug candidate as it has a native specificity for the proline rich gliadin peptides. Unfortunately, the enzyme’s optimal activity is at pH 7, and engineering attempts to enhance its activity at relevant gastric pH levels has not yet been met with significant success. It is therefore pertinent to identify new candidate enzymes that have both activity on the gliadin PQ structural motif, as well as optimal activity at gastric pH levels during digestion around pH 4. <br />
<br />
<br />
Here, we describe an alternative approach to identifying and engineering an enzyme therapeutic for celiac sprue. Rather than focusing primarily on substrate specificity when choosing our candidate enzyme, we identified an enzyme already capable of catalyzing peptide hydrolysis at gastric pH levels, regardless of peptide substrate specificity. Upon identification of such an enzyme we used computational tools to reengineer its substrate specificity for enhanced activity on immunogenic gliadin peptides with the common PQ structural motif.<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[File:Washington_Kumamolisin-As.png|right|250px|thumb|Kumamolisin-As, a protease from the thermoacidophilic bacterium ''Alicyclobacillus sendaiensis'']]<br />
<br />
=We have identified an enzyme that could potentially act as a therapeutic for gluten intolerance=<br />
<br />
When searching for a protease with optimal activity at gastric pH levels that could be produced in a recombinant host, we identified an enzyme known as Kumamolisin-As. Kumamolisin-As, isolated from the thermoacidophilic bacterium ''Alicyclobacillus sendaiensis'' strain NTAP-1, has been shown to be produced in a recombinant host, and exhibits significant enzymatic activity at pH 2.5 and above. Its maximal activity is at about pH 4.0. It is this robust activity under acidic, gastric conditions that makes Kumamolisin-As so promising for the development of a pill for gluten intolerance.<br />
<br />
=A special set of catalytic residues enables high activity at gastric pH levels=<br />
<br />
One of the primary reasons that Kumamolisin-As is more active than SC-PEP at low pH is due its catalytic triad, the three amino acid residues most heavily involved in conducting the chemistry of cleaving peptides. SC-PEP is part of a large class of enzymes known as serine proteases, which make use of the catalytic triad Serine-Histidine-Aspartate. Acidic conditions can impede the histidine's ability to play its role in this triad, rendering most serine proteases inactive at low pH. Kumamolisin-As, on the other hand, is a member of the sedolisin family of serine-carboxyl peptidases, which utilize the key catalytic triad Serine-Glutamate-Aspartate to hydrolyze their substrates. It is the acidic Glutamate, as opposed to Histidine, in the triad that makes Kumamolisin-As optimal for catalyzing peptide cleavage at low pH. The native substrate for this enzyme in ''Alicyclobacillus sendaiensis'' is unknown. In addition, this enzyme has been shown to be thermostable, with an ideal temperature at 60&ordm;C, but still showing significant activity at 37&ordm;C. <br />
<br />
[[File:Washington_Kuma Bonded triad.png|left|250px|thumb|Kumamolisin-As' unusual catalytic triad Ser278-Glu78-Asp82 makes the enzyme well suited to a low pH environment.]]<br />
[[File:Washington_serine protease triad2.png|left|250px|thumb|The traditional Ser-His-Asp catalytic triad found in serine proteases like SC-PEP does not function optimally at low pH.]]<br />
<br />
=Kumamolisin-As is already active for the dipeptide motif PR, we just need to change it to PQ=<br />
<br />
:::::::::::::::::::::<p>Specificity studies have been conducted on the native enzyme, which is most efficient at hydrolyzing peptides at the carboxyl side after the PR dipeptide motif (where the P1 and P2 subsites are arginine and proline, respectively, in standard proteolysis nomenclature). The enzyme shows little preference for what is on the amino S1 and S2 subsites. The combination of Kumamolisin-As having the desired enzymatic activity under gastric pH levels, and the P2 subsite already having the specificity for the proline from the desired PQ motif makes it a highly attractive candidate for further engineering. In addition, several crystal structures of this enzyme have been solved, allowing for the use of computational design techniques.</p><br />
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<br />
=References=<br />
<br />
1.Shan, Lu, et al. "Structural Basis for Gluten Intolerance in Celiac Sprue." Science 297.5590 (2002): 2275-2279.<br />
<br />
2.Mustalahti, Kirsi, et al. "The prevalence of celiac disease in Europe: Results of a centralized, international mass screening project." Annals of Medicine 42.8 (2010): 587-595.<br />
<br />
3.Ehren, Jennifer, et al. "Protein engineering of improved prolyl endopeptidases for celiac sprue therapy." Protein Engineering, Design & Selection 21.12 (2008): 699-707.<br />
<br />
4.Wlodawer, Alexander, et al. "Crystallographic and Biochemical Investigations of Kumamolisin-As, a Serine-Carboxyl Peptidase with Collagenase Activity." The Journal of Biological Chemistry 279.20 (2004): 21500-21510.</div>Mdsmithhttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-29T02:01:51Z<p>Mdsmith: </p>
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__NOTOC__<br />
<br />
<center><big><big><big><big>'''Gluten Destruction: Methods'''</big></big></big></big></center><br><br><br />
<br />
===='''Redesigning Kumamolisin to Have Higher Activity at Low pH'''====<br />
<br />
<br />
[[File:Washington Foldit.png|550px|thumb|right|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 />
<br />
----<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|left|Overview of how Kunkel Mutagenesis works]]<br />
<br />
<br />
<br />
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<br />
<br />
<br />
==='''Kunkel Mutagenesis'''===<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. <br />
<br />
To incorporate these mutations, we first isolated single stranded DNA (ssDNA) of our vector harboring the wild-type Kumamolisin gene. To do this we infected cells with bacteriophage M13, which packages its own ssDNA genome identified by length, and so in tandem packaged our vector in single stranded form. We then harvested the phage from the lysed culture of E. coli, and extracted our single stranded vector DNA.<br />
<br />
Next, we annealed and extended our mutagenic oligos to incorporate the specified mutations into the newly synthesized antisense strand. This hybrid vector was transformed into E. coli that degraded the original uracil-containing DNA and replaced it with sections complementary to the mutagenized strand.<br />
<br />
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<br />
<br />
<br />
<br />
<br />
<br />
=='''Using a Whole Cell Lysate Assay to Test Activity of Mutants'''==<br />
To test our designs, we developed a whole cell lysate assay that allowed us to perform a rough screen of a large number of mutants. In this assay, we expressed our mutant enzymes in <i>E. coli</i>, lysed the cells and separated the enzymes from large cell particulate. We then performed the assay at pH 4, mimicking the gastric environment. We added our model PQLP peptide, conjugated to both a fluorophore and a quencher so that no fluorescence would be achieved until after the peptide had been enzymatically cleaved. We then measured the fluorescence of each reaction at 30 second intervals, and were thereby able to estimate relative activity on breaking down PQLP by increase in fluorescence of the system.<br />
<br />
[[File:Washington Whole Cell Lysate Assay.jpg|center|General Overview of the Whole Cell Lysate]]<br />
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<br />
----<br />
<br />
<br />
=='''Testing Purified Mutants to Accurately Assess Activity'''==<br />
<br />
[[File:Washington First Raw Data.png|right|500px|thumb|We measured fluorescence of each reaction at 30 second intervals to see the rate at which each mutant cleaved PQLP.]]<br />
<br />
==='''Purification'''===<br />
From our whole cell lysate screen of each design, we identified mutants that showed the most increase in activity from the wild-type Kumamolisin. We then proceeded to purify these most promising variants and test them against the wild-type and against SC PEP using the same fluorescence metric designed for the whole cell lysate assay. The key difference between the whole cell assay and the purified protein assay is that in the latter we were able to control the concentration of enzyme in each well, adjusting for the possibility of varying expression levels and thus enzyme concentrations in the whole cell lysate assay.<br />
<br />
Purification was performed via Nickel-affinity chromatography, and resulting protein concentrations were measured using ultraviolet-visible spectrophotometry.<br />
<br />
==='''Assay'''===<br />
Concentration dependent assays were performed for each promising mutant. We measured the fluorescence of each reaction at 30 second intervals to see the rate at which fluorescence increased, thus obtaining a relative rate of cleavage of PQLP by increase in fluorescence of the system. Raw data appeared as shown right, and the slope of each line was calculated, giving us relative rate information that could be used in conjunction with rate information obtained in the same assay for native Kumamolisin to determine fold change in activity.</div>Mdsmithhttp://2011.igem.org/Team:WashingtonTeam:Washington2011-09-29T01:22:47Z<p>Mdsmith: </p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
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=<center>'''Make It or Break It: <br/> Diesel Production and Gluten Destruction, the Synthetic Biology Way'''</center>=<br />
<br />
Synthetic biology holds great promise regarding the production of important compounds, and the degradation of harmful ones. This summer, we harnessed the power of synthetic biology to meet the world’s needs for fuel and medicine. <br />
<br/><br />
[[Image:Washington_Fire.jpg|left|320px|borderless|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[Image:Washington_Bottle.jpg|right|200px|borderless|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
<br />
[https://2011.igem.org/Team:Washington/Alkanes/Background '''Make It: Diesel Production'''] We constructed a strain of ''Escherichia coli'' that produces a variety of alkanes, the main constituents of diesel fuel, by introducing a pair of genes recently shown convert fatty acid synthesis intermediates into alkanes. <br />
<br />
[https://2011.igem.org/Team:Washington/Celiacs/Background '''Break It: Gluten Destruction'''] We identified a protease with gluten-degradation potential, and then reengineered it to have greatly increased gluten-degrading activity, allowing for the breakdown of gluten in the digestive track when taken in pill form. <br />
<br />
[https://2011.igem.org/Team:Washington/Magnetosomes/Background '''iGEM Toolkits'''] To enable next-generation cloning of standard biological parts, we built BioBrick vectors optimized for Gibson assembly and used them to create the Magnetosome Toolkit: a set of 18 genes from an essential operon in magnetotactic bacteria which we are characterizing to create magnetic ''E. coli''.<br />
<br />
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[[File:Washington_Spacer.jpg|1px]]<br />
[[Image:UW Diesel Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
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[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_OSLI.png|frameless|border|link=http://www.osli.ca|Oil Sands Leadership Intiative]]<br />
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[[File:Washington_UniversitySeal.gif|frameless|border|110px|link=http://www.washington.edu|University of Washington]]<br />
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[[File:Washington_Anaspec.gif|frameless|border|120px|link=http://www.anaspec.com|Anaspec]]<br />
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[[File:Washington_ARPA-E_Logo.png|frameless|border|link=http://arpa-e.energy.gov/ProgramsProjects/Electrofuels.aspx|Advanced Research Projects Agency - Energy]]<br />
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[[File:Washington2011_Hhmi_362_72.jpg|link=http://www.hhmi.org/]]</div>Mdsmithhttp://2011.igem.org/Template:Team:Washington/Templates/TopTemplate:Team:Washington/Templates/Top2011-09-29T01:14:27Z<p>Mdsmith: </p>
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<ul><br />
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<a href="https://2011.igem.org/Team:Washington">Home</a><br />
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<a href="https://2011.igem.org/Main_Page"> iGEM Homepage </a><br />
<a href="https://2011.igem.org/Team:Washington">UW 2011</a><br />
<a href="https://2010.igem.org/Team:Washington"> UW 2010 </a><br />
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<li><br />
<a href="https://2011.igem.org/Team:Washington/Team/Members">About Us</a><br />
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<a href="https://2011.igem.org/Team:Washington/Alkanes/Background">Diesel Production</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Background">Background</a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Methods"> Methods </a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Results"> Results Summary </a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Future"> Future Directions </a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Parts"> Parts Submitted </a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Background">Gluten Destruction</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Background">Background</a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Methods"> Methods </a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Results"> Results Summary</a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Future"> Future Directions </a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Parts"> Parts Submitted </a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Background">iGEM Toolkits</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Background"> Background </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors">Gibson Assembly Toolkit </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults">Gibson Toolkit Results</a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit">Magnetosome Toolkit</a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Results">Magnetosome Results</a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Parts"> Parts Submitted </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Future"> Future Directions </a><br />
<br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Parts">Data Page</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Parts">Data Page</a><br />
<a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2011&group=Washington"> Parts Registry </a><br />
</div><br />
<br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Protocols">Protocols</a><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Outreach">Community Outreach</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Outreach">Local Outreach</a><br />
<br />
<a href="https://2011.igem.org/Team:Washington/Outreach/iGEM_Collaborations">iGEM Collaborations</a></div><br />
<br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Safety">Safety</a><br />
</li><br />
</ul><br />
</div><br />
</div><br />
</html></div>Mdsmithhttp://2011.igem.org/Team:WashingtonTeam:Washington2011-09-29T01:02:14Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Make It or Break It: <br/><br/> Diesel Production and Gluten Destruction, <br/><br/>the Synthetic Biology Way'''</big></big></big></big></center><br><br />
<br />
Synthetic biology holds great promise regarding the production of important compounds, and the degradation of harmful ones. This summer, we harnessed the power of synthetic biology to meet the world’s needs for fuel and medicine. <br />
<br/><br />
[[Image:Washington_Fire.jpg|left|320px|borderless|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[Image:Washington_Bottle.jpg|right|200px|borderless|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
<br />
[https://2011.igem.org/Team:Washington/Alkanes/Background '''Make It: Diesel Production'''] We constructed a strain of ''Escherichia coli'' that produces a variety of alkanes, the main constituents of diesel fuel, by introducing a pair of genes recently shown convert fatty acid synthesis intermediates into alkanes. <br />
<br />
[https://2011.igem.org/Team:Washington/Celiacs/Background '''Break It: Gluten Destruction'''] We identified a protease with gluten-degradation potential, and then reengineered it to have greatly increased gluten-degrading activity, allowing for the breakdown of gluten in the digestive track when taken in pill form. <br />
<br />
[https://2011.igem.org/Team:Washington/Magnetosomes/Background '''iGEM Toolkits'''] To enable next-generation cloning of standard biological parts, we built BioBrick vectors optimized for Gibson assembly and used them to bring to the Parts Registry the Magnetosome Toolkit: a set of 18 genes from an essential operon in magnetotactic bacteria which we are characterizing to create magnetic ''E. coli''.<br />
<br />
<br />
[[File:Washington_Spacer.jpg|1px]]<br />
[[Image:UW Diesel Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Toolkits Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Gluten Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
<br />
<br />
<br/><br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_OSLI.png|frameless|border|link=http://www.osli.ca|Oil Sands Leadership Intiative]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_UniversitySeal.gif|frameless|border|110px|link=http://www.washington.edu|University of Washington]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_Anaspec.gif|frameless|border|120px|link=http://www.anaspec.com|Anaspec]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_ARPA-E_Logo.png|frameless|border|link=http://arpa-e.energy.gov/ProgramsProjects/Electrofuels.aspx|Advanced Research Projects Agency - Energy]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington2011_Hhmi_362_72.jpg|link=http://www.hhmi.org/]]</div>Mdsmithhttp://2011.igem.org/Team:Washington/SafetyTeam:Washington/Safety2011-09-29T01:00:26Z<p>Mdsmith: </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><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><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><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><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>Mdsmithhttp://2011.igem.org/Team:WashingtonTeam:Washington2011-09-29T00:52:59Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Make It or Break It: <br/><br/> Diesel Production and Gluten Destruction, <br/><br/>the Synthetic Biology Way'''</big></big></big></big></center><br><br />
<br />
Synthetic biology holds great promise regarding the production of important compounds, and the degradation of harmful ones. This summer, we harnessed the power of synthetic biology to meet the world’s needs for fuel and medicine. <br />
<br/><br />
[[Image:Washington_Fire.jpg|left|320px|borderless]]<br />
[[Image:Washington_Bottle.jpg|right|200px|borderless]]<br />
<br />
[https://2011.igem.org/Team:Washington/Alkanes/Background '''Make It: Diesel Production'''] We constructed a strain of ''Escherichia coli'' that produces a variety of alkanes, the main constituents of diesel fuel, by introducing a pair of genes recently shown convert fatty acid synthesis intermediates into alkanes. <br />
<br />
[https://2011.igem.org/Team:Washington/Celiacs/Background '''Break It: Gluten Destruction'''] We identified a protease with gluten-degradation potential, and then reengineered it to have greatly increased gluten-degrading activity, allowing for the breakdown of gluten in the digestive track when taken in pill form. <br />
<br />
[https://2011.igem.org/Team:Washington/Magnetosomes/Background '''iGEM Toolkits'''] To enable next-generation cloning of standard biological parts, we built BioBrick vectors optimized for Gibson assembly and used them to bring to the Parts Registry the Magnetosome Toolkit: a set of 18 genes from an essential operon in magnetotactic bacteria which we are characterizing to create magnetic ''E. coli''.<br />
<br />
<br />
[[File:Washington_Spacer.jpg|1px]]<br />
[[Image:UW Diesel Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Toolkits Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Gluten Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
<br />
<br />
<br/><br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_OSLI.png|frameless|border|link=http://www.osli.ca|Oil Sands Leadership Intiative]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_UniversitySeal.gif|frameless|border|110px|link=http://www.washington.edu|University of Washington]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_Anaspec.gif|frameless|border|120px|link=http://www.anaspec.com|Anaspec]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_ARPA-E_Logo.png|frameless|border|link=http://arpa-e.energy.gov/ProgramsProjects/Electrofuels.aspx|Advanced Research Projects Agency - Energy]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington2011_Hhmi_362_72.jpg|link=http://www.hhmi.org/]]</div>Mdsmithhttp://2011.igem.org/Team:Washington/Team/SponsorsTeam:Washington/Team/Sponsors2011-09-29T00:52:30Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Thank you to our sponsors!'''</big></big></big></big></center><br><br />
<br />
[[File:Washington_HHMI.jpg|frameless|border|link=http://www.hhmi.org|Howard Hughes Medical Institute]]<br />
[[File:Washington_OSLI.png|frameless|border|link=http://www.osli.ca|Oil Sands Leadership Intiative]]<br />
[[File:Washington_ARPA-E_Logo.png|frameless|border|link=http://arpa-e.energy.gov/ProgramsProjects/Electrofuels.aspx|Advanced Research Projects Agency - Energy]]<br />
[[File:Washington_UniversitySeal.gif|frameless|border|link=http://www.washington.edu|University of Washington]]<br />
[[File:Washington_Anaspec.gif|frameless|border|link=http://www.anaspec.com|Anaspec]]</div>Mdsmithhttp://2011.igem.org/Team:Washington/Team/SponsorsTeam:Washington/Team/Sponsors2011-09-29T00:51:24Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Thank you to our sponsors!'''</big></big></big></big></center><br><br />
<br />
[[File:Washington_HHMI.jpg|frameless|border|link=http://www.hhmi.org|Howard Hughes Medical Institute]]<br />
[[File:Washington_OSLI.png|frameless|border|link=http://www.osli.ca|Oil Sands Leadership Intiative]]<br />
[[File:Washington_ARPA-E_Logo.png|frameless|border|link=http://arpa-e.energy.gov/ProgramsProjects/Electrofuels.aspx|Advanced Research Projects Agency - Energy]]<br />
[[File:Washington_UniversitySeal.gif|frameless|border|link=http://http://www.washington.edu|University of Washington]]<br />
[[File:Washington_Anaspec.gif|frameless|border|link=http://www.anaspec.com|Anaspec]]</div>Mdsmith