http://2011.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=250&target=Mdsmith&year=&month=2011.igem.org - User contributions [en]2024-03-28T20:26:39ZFrom 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>Mdsmithhttp://2011.igem.org/Team:Washington/Team/SponsorsTeam:Washington/Team/Sponsors2011-09-29T00:50:55Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
===Thank you to our sponsors!===<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>Mdsmithhttp://2011.igem.org/Team:Washington/Team/MembersTeam:Washington/Team/Members2011-09-29T00:49:45Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------><br />
<br />
<center><big><big><big><big>'''Who we are'''</big></big></big></big></center><br><br />
<center><br />
<gallery caption="Undergraduate Team Members" widths="180px" heights="120px" perrow="4"><br />
Image:Washington Casey Ager Profile1.jpg|<center>Casey Ager <br/> Biochemistry</center><br />
Image:Washington Photo-0088A.png|<center>Juhye An <br/> Biochemistry </center><br />
Image: Profile_Michael_Brasino.jpg|<center>Michael Brasino <br/> Materials Science and Engineering</center><br />
Image:Washington MC profilepic1.jpg|<center>Marika Cheng <br/> Microbiology</center><br />
Image:Washington_choe_chris.png|<center>Chris Choe <br/> Undecided</center><br />
Image:Washington DeLeon 2011.jpg|<center>Justin De Leon <br/> Microbiology</center><br />
File:Princess Peachy.JPG|<center>Sydney Gordon <br/> Biochemistry, Music</center><br />
Image:CIMG0021.jpg|<center>Daniel Hadidi <br/> Neurobiology</center><br />
Image:Washington_Matthew_Harger.JPG|<center>Matthew Harger <br/> Cellular and Molecular Biology</center><br />
Image:Washington_Elaine.jpg|<center>Elaine Lai <br/> Microbiology, Chemistry</center><br />
Image:Washington_2011_Lab_Ben.png|<center>Benjamin Mo <br/> Bioengineering</center><br />
Image:Washington 2011 CIMG0015-A.jpg|<center>Austin Moon <br/> Molecular Biology</center><br />
Image:Washington_iGEM2011_Rashmi.jpg|<center>Rashmi Ravichandran <br/> Microbiology</center><br />
Image:rsz_cats.png|<center>Seth Sagulo <br/> Biochemistry</center><br />
File:Washington_Liz.png|<center>Liz Stanley <br/> Microbiology, Chemistry</center><br />
Image:Washington_Angus_Pic.jpg|<center>Angus Toland <br/> Microbiology</center><br />
Image:Washington_Sarah.jpg|<center>Sarah Wolf <br/> Biochemistry</center><br />
Image:Washington_igem11whowearepicture.jpg|<center>SauShun (Alicia) Wong <br/> Materials Science and Engineering</center><br />
Image:Washington_Cindy.jpg|<center>Cindy Wu <br/> Cellular and Molecular Biology</center><br />
Image:Washington_Sean_Wu.jpg|<center>Sean Wu <br/> Computer Science and Engineering</center><br />
Image:Washington_lei_zheg.png|<center>Lei Zheng <br/> Biochemistry</center><br />
Image:Washington_david_zong.jpg|<center>David Zong <br/> Bioengineering</center><br />
<br />
</gallery><br />
<br />
<br />
<gallery caption="Advisors" widths="180px" heights="120px" perrow="4"><br />
File:Aaron_bike_UW.png|<center>Aaron Chevalier <br/> Bioengineering </center><br />
Image:Regbert.jpg|<center>Rob Egbert <br/> Electrical Engineering </center><br />
Image:CEiben.jpg|<center>Chris Eiben <br/> Biochemistry </center><br />
Image:IMG_0129.jpg|<center>Jeremy Mills <br/> Biochemistry </center><br />
Image:Siegel_UW_2011.jpg|<center>Justin Siegel <br/> Biomolecular Structure and Design</center><br />
Image:Washington2011_Matthew-d-smith.jpg|<center>Matt Smith <br/> Molecular and Cellular Biology</center><br />
File:Washington_Ingrid_Pic_1.jpg|<center>Ingrid Swanson Pultz<br/> Microbiology </center><br />
</gallery><br />
<br />
<br />
<gallery caption="Faculty" widths="180px" heights="120px" perrow="5"><br />
image:Washington_David_Baker.jpg|<center>David Baker <br/> Biochemistry</center><br />
Image:Washington_Klavins.jpg|<center>Eric Klavins <br/> Electrical Engineering</center><br />
</gallery><br />
<br />
<br />
<gallery caption="Collaborators and Support" widths="140px" heights="100px" perrow="5"><br />
<br />
Image:Washington_OSLI.png|<center><b>Oil Sand Leadership Initiative</b> <br/> Funding to support Travel and Registration Costs</center><br />
<br />
Image:Washington_UniversitySeal.gif|<center><b>UW Biochemistry</b> <br/> Lab space</center><br />
<br />
Image:Washington_Anaspec.gif|<center><b>Anaspec</b> <br/> Peptide Discounts</center><br />
<br />
Image:Washington_ARPA-E_Logo.png|<center><b>Advanced Research Projects Agency - Energy </b><br/> Registration Support</center><br />
<br />
Image:Washington2011_Hhmi_362_72.jpg|<center><b>Howard Hughes Medical Institute</b> <br/> Lab materials and supplies</center><br />
<br />
<br />
</gallery><br />
</center><br />
<br><br />
== '''Who did what''' ==<br />
The teams were assembled during our winter quarter. During this term we went around to classes and had weekly meetings to introduce students to synthetic biology through a series of guest lectures. During the spring, students participated in brainstorming projects related to the sponsoring Faculty advisors labs (Baker and Klavins) and came up with the project they wanted to carry out. Students also planned and partcipated in community outreach events where we taught the local public about synthetic biology. Over the summer, students completed all of the experimental work, but worked closely with both graduate students and faculty advisors to plan the most pertinent experiments and make the most of our limited time. '''The work documented here in this wiki and on our presentation is entirely the work of iGEM students - it does not represent the project of any host lab, advisor, or instructor participating in iGEM.'''<br />
<br />
=== Diesel Production ===<br />
After producing promising results, in future directions:<br />
<br />
Casey Ager, Austin Moon, and Seth Sagulo worked on Enzyme Localization via Direct Fusion and Zinc Finger Fusion methods.<br />
<br />
Juhye An, Elaine Lai, and Benjamin Mo worked on Decarbonylase Redesign<br />
<br />
Marika Cheng and Justin De Leon worked on Alternative Chassis<br />
<br />
Chris Choe and David Zong worked on Alternate Aldehyde Production<br />
<br />
Matthew Harger worked on Branched Alkanes Production<br />
<br />
Matthew Harger and Lei Zheng worked on System Optimization<br />
<br />
=== Gluten Destruction ===<br />
Sydney Gordon, Daniel Hadidi, Liz Stanley, Angus Toland, Sarah Wolf, and Sean Wu designed, built, and tested Kumamolisin-As and over 100 mutants to combat gluten intolerance by increasing the activity on the PQLP antigenic peptide.<br />
<br />
=== iGEM Toolkits ===<br />
Michael Brasino, Rashmi Ravichandran, and Alicia Wong worked on extracting and BioBricking the essential genes for magnetosomes, made the fusion proteins, and did the experimental measurements. They also expanded on work that was started by UW iGEM students in 2010, by constructing more Gibson-friendly plasmid backbones and characterizing the Gibson-assembly efficiencies of pSB and pGA plasmids.<br />
<br />
=== Community Outreach ===<br />
Cindy Wu headed and organized all iGEM 2011 Community Outreach events.</div>Mdsmithhttp://2011.igem.org/Team:WashingtonTeam:Washington2011-09-29T00:44:02Z<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 />
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<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://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 />
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[[File:Washington2011_Hhmi_362_72.jpg|link=http://www.hhmi.org/]]</div>Mdsmithhttp://2011.igem.org/Team:WashingtonTeam:Washington2011-09-29T00:41:00Z<p>Mdsmith: </p>
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=<center>'''Make It or Break It: <br/> Diesel Production and Gluten Destruction, the Synthetic Biology Way'''</center>=<br />
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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 />
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[[Image:Washington_Fire.jpg|left|320px|borderless]]<br />
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[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 />
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[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 />
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[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 />
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[[Image:UW Diesel Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
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[[Image:UW Toolkits Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]<br />
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[[Image:UW Gluten Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
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[[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://http://www.washington.edu|University of Washington]]<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/Team:Washington/SafetyTeam:Washington/Safety2011-09-23T01:55:57Z<p>Mdsmith: </p>
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<center><big><big><big><big>Safety</big></big></big></big></center><br><br><br />
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Please use this page to answer the safety questions posed on the [[Safety | safety page]].<br />
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'''1. Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
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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 />
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'''2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?<br />
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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 />
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'''3. Is there a local biosafety group, committee, or review board at your institution?<br />
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The University of Washington has an Environmental Health and Safety(EHS) committee that deals with biosafety and other safety and health issues. The EHS committee has no concerns about our projects.<br />
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'''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 />
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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:Washington/Outreach/iGEM_CollaborationsTeam:Washington/Outreach/iGEM Collaborations2011-09-23T01:55:19Z<p>Mdsmith: </p>
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<center><big><big><big><big>Community Outreach: iGEM Collaborations</big></big></big></big></center><br><br><br />
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=Primer Design Tool with LMU-Munich=<br />
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We worked with [https://2011.igem.org/Team:LMU-Munich LMU-Munich 2011] iGEM team this summer on the testing and development of their PrimerDesign tool. To learn more about it check out their [https://2011.igem.org/Team:LMU-Munich/Primer_Design/Design_Primers website].<br />
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==Software Testing==<br />
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We helped LMU-Munich test their primer design software by sending our primer data and comparing it with the output of their automated designer. To do this, we compared the sequences of the primers used to amplify out parts of the LuxBrick with the primers that their software designed and concluded that their software works as expected. Unfortunately, we did not have enough time to order the primers that the software designed to test it ourselves. Overall, we found the tool easy to use and potentially very useful for future primer design.<br />
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[[File:Washington_collab_test.png|left|550px|thumb|An example of the output from the tool based off of input from our luxC biobrick, multiple primers with various melting temperatures are given]]<br />
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==Implemented Bug Fixes & Features==<br />
#Added cyanobacteria to available genomes<br />
#Switch coding sequences in the primer designer to allow for the user to incude a stop codon in a coding sequence. Before, software insisted that coding sequences not include the stop codon<br />
#Made primer designer default to only check for biobrick restriction enzymes. Before, Primer Designer defaulted to checking for all restriction sites, making the user scroll to the bottom of the page for results<br />
#added support for FASTA files with headers<br />
#added desriptions for each column of the Primer Designer output.<br />
[[File:Washington_OldPrimerDesignerdefault.png|left|550px|thumb|Old Default Primer Designer Output]]</div>Mdsmithhttp://2011.igem.org/Team:Washington/OutreachTeam:Washington/Outreach2011-09-23T01:54:52Z<p>Mdsmith: </p>
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<center><big><big><big><big>Community Outreach: Local</big></big></big></big></center><br><br><br />
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During the off season we took had the opportunity to share our project with our community at both the university level and the K-12 level. The [http://exp.washington.edu/urp/symp/index.html UW Undergraduate Research Symposium], [http://www.engr.washington.edu/alumcomm/openhouse.html UW Engineering Discovery Days], and [http://schoolbennett.org/ysw.aspx Young Scientist Week] took place during the spring.<br />
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=How we introduced people to the awesomeness of Synthetic Biology=<br />
For each of the following events our goal was to help teach young students about the basics of synthetic biology. To do this we brought two primary tools. First, we had an interactive cloning project, in which students were able to "digest" and "ligate" different colored pieces of ribbon together. After making their synthetic ribbon construct, we had them clone the "vector" into the "balloon" chasis of their choice. This is generally illustrated below.<br />
[[Image:Cloning diagram.png|550px|left|We simulated cloning with balloons and ribbons, making the daunting topic of cloning more familiar for kids.]]<br />
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[[File:Cloning Diagram Finish.png|400px|left|]]<br />
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In addition we brought laptops running the interactive protein folding and design computer game Foldit. Using this tool we were able to introduce the students to how proteins functions and ways that we can begin to redesign them. For more information on Foldit please visit the [http://fold.it/portal/ website]<br />
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[[Image:Washington_2011_Uw_foldit_lofo.jpeg|center]]<br />
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==Bellevue School District Bennett Elementary School Young Scientist Week==<br />
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In April, participants from the 2010 and 2011 iGEM teams joined a multitude of presentations to expand the knowledge of K-5 students, their parents, and elementary school teachers in the general sciences. Presentations included robotics, atmospheric science, bioengineering, and crime scene investigation among other demonstrations along with posters about the experiments the elementary students conducted themselves. <br />
For the students, we presented our interactive activity from UW Engineering Discovery Days to teach students about synthetic biology and protein engineering by filling a balloon (representing a cell) with a ribbon (representing a plasmind) as well as allowed them to play with [http://fold.it FoldIt], the David Baker Group's protein folding game. We also displayed our work from 2010 on the Type 6 Secretion System and CapD, a potential anthrax therapeutic, so students could be exposed to recent scientific research.<br />
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==UW Engineering Discovery Days==<br />
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Sponsored by the University of Washington College of Engineering, the Engineering Discovery Days is an opportunity for undergraduates to introduce K-12 students to the vast world of engineering. The Engineering Discovery Days functions like an open house with booths spread about campus and is split into two days: day one for elementary and middle schoolers, day two for high schoolers. We partnered with the department of Bioengineering to teach children how synthetic biology works through an activity where the kids fill a balloon (representing a cell) with a ribbon (representing a plasmid). On day two, we introduced high schoolers to the basics of synthetic biology through a poster session with last year's project. Through this event we were able to engage with our local K-12 community and teach them about iGEM.<br />
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[[File:Washington_Urs_poster_session.png|left|300px|A group of students from this years and last year's team at the Undergraduate Research Symposium]]<br />
==UW Undergraduate Research Symposium==<br />
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During the spring, this years team worked with last year's team and participated in the annual University of Washington Undergraduate Research Symposium. This was the first time the iGEM team had participated in this event. The university sponsored event had over 700 student participants and 3500 attendees. The 700 students come from all disciplines, not just the sciences and the attendees include faculty, mentors and our peers. During the symposium, we were able to tell our community about the basics of iGEM and synthetic biology as well as walk them through last year's award winning work. We found this event to be important in rooting the University of Washington iGEM team in our school's undergraduate research community.</div>Mdsmithhttp://2011.igem.org/Template:Team:Washington/Templates/TopTemplate:Team:Washington/Templates/Top2011-09-23T01:54:25Z<p>Mdsmith: </p>
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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 />
<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 />
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<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/Magnet_Toolkit">Magnetosomes Toolkit</a><br />
<br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Future"> Future Directions </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Reference"> References </a> <a href="https://2011.igem.org/Team:Washington/Magnetosomes/Parts"> Parts Submitted </a><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:Washington/Outreach/iGEM_CollaborationsTeam:Washington/Outreach/iGEM Collaborations2011-09-23T01:52:49Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
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<center><big><big><big><big>iGEM Collaborations</big></big></big></big></center><br><br><br />
<br />
=Primer Design Tool with LMU-Munich=<br />
<br />
We worked with [https://2011.igem.org/Team:LMU-Munich LMU-Munich 2011] iGEM team this summer on the testing and development of their PrimerDesign tool. To learn more about it check out their [https://2011.igem.org/Team:LMU-Munich/Primer_Design/Design_Primers website].<br />
<br />
==Software Testing==<br />
<br />
We helped LMU-Munich test their primer design software by sending our primer data and comparing it with the output of their automated designer. To do this, we compared the sequences of the primers used to amplify out parts of the LuxBrick with the primers that their software designed and concluded that their software works as expected. Unfortunately, we did not have enough time to order the primers that the software designed to test it ourselves. Overall, we found the tool easy to use and potentially very useful for future primer design.<br />
<br />
[[File:Washington_collab_test.png|left|550px|thumb|An example of the output from the tool based off of input from our luxC biobrick, multiple primers with various melting temperatures are given]]<br />
<br />
==Implemented Bug Fixes & Features==<br />
#Added cyanobacteria to available genomes<br />
#Switch coding sequences in the primer designer to allow for the user to incude a stop codon in a coding sequence. Before, software insisted that coding sequences not include the stop codon<br />
#Made primer designer default to only check for biobrick restriction enzymes. Before, Primer Designer defaulted to checking for all restriction sites, making the user scroll to the bottom of the page for results<br />
#added support for FASTA files with headers<br />
#added desriptions for each column of the Primer Designer output.<br />
[[File:Washington_OldPrimerDesignerdefault.png|left|550px|thumb|Old Default Primer Designer Output]]</div>Mdsmithhttp://2011.igem.org/Team:Washington/OutreachTeam:Washington/Outreach2011-09-23T01:52:12Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>Community Outreach</big></big></big></big></center><br><br><br />
<br />
During the off season we took had the opportunity to share our project with our community at both the university level and the K-12 level. The [http://exp.washington.edu/urp/symp/index.html UW Undergraduate Research Symposium], [http://www.engr.washington.edu/alumcomm/openhouse.html UW Engineering Discovery Days], and [http://schoolbennett.org/ysw.aspx Young Scientist Week] took place during the spring.<br />
<br />
----<br />
<br />
=How we introduced people to the awesomeness of Synthetic Biology=<br />
For each of the following events our goal was to help teach young students about the basics of synthetic biology. To do this we brought two primary tools. First, we had an interactive cloning project, in which students were able to "digest" and "ligate" different colored pieces of ribbon together. After making their synthetic ribbon construct, we had them clone the "vector" into the "balloon" chasis of their choice. This is generally illustrated below.<br />
[[Image:Cloning diagram.png|550px|left|We simulated cloning with balloons and ribbons, making the daunting topic of cloning more familiar for kids.]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[File:Cloning Diagram Finish.png|400px|left|]]<br />
<br />
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<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
In addition we brought laptops running the interactive protein folding and design computer game Foldit. Using this tool we were able to introduce the students to how proteins functions and ways that we can begin to redesign them. For more information on Foldit please visit the [http://fold.it/portal/ website]<br />
<br />
[[Image:Washington_2011_Uw_foldit_lofo.jpeg|center]]<br />
<br />
<br />
----<br />
<br />
<br />
==Bellevue School District Bennett Elementary School Young Scientist Week==<br />
<br />
In April, participants from the 2010 and 2011 iGEM teams joined a multitude of presentations to expand the knowledge of K-5 students, their parents, and elementary school teachers in the general sciences. Presentations included robotics, atmospheric science, bioengineering, and crime scene investigation among other demonstrations along with posters about the experiments the elementary students conducted themselves. <br />
For the students, we presented our interactive activity from UW Engineering Discovery Days to teach students about synthetic biology and protein engineering by filling a balloon (representing a cell) with a ribbon (representing a plasmind) as well as allowed them to play with [http://fold.it FoldIt], the David Baker Group's protein folding game. We also displayed our work from 2010 on the Type 6 Secretion System and CapD, a potential anthrax therapeutic, so students could be exposed to recent scientific research.<br />
<br />
<br />
==UW Engineering Discovery Days==<br />
<br />
Sponsored by the University of Washington College of Engineering, the Engineering Discovery Days is an opportunity for undergraduates to introduce K-12 students to the vast world of engineering. The Engineering Discovery Days functions like an open house with booths spread about campus and is split into two days: day one for elementary and middle schoolers, day two for high schoolers. We partnered with the department of Bioengineering to teach children how synthetic biology works through an activity where the kids fill a balloon (representing a cell) with a ribbon (representing a plasmid). On day two, we introduced high schoolers to the basics of synthetic biology through a poster session with last year's project. Through this event we were able to engage with our local K-12 community and teach them about iGEM.<br />
<br />
<br />
<br />
[[File:Washington_Urs_poster_session.png|left|300px|A group of students from this years and last year's team at the Undergraduate Research Symposium]]<br />
==UW Undergraduate Research Symposium==<br />
<br />
During the spring, this years team worked with last year's team and participated in the annual University of Washington Undergraduate Research Symposium. This was the first time the iGEM team had participated in this event. The university sponsored event had over 700 student participants and 3500 attendees. The 700 students come from all disciplines, not just the sciences and the attendees include faculty, mentors and our peers. During the symposium, we were able to tell our community about the basics of iGEM and synthetic biology as well as walk them through last year's award winning work. We found this event to be important in rooting the University of Washington iGEM team in our school's undergraduate research community.</div>Mdsmithhttp://2011.igem.org/Team:Washington/PartsTeam:Washington/Parts2011-09-23T01:51:45Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>Data Page</big></big></big></big></center><br><br><br />
<br />
<gallery caption="An Overview of the 2011 UW iGEM Teams Summer Projects" widths="435px" heights="300px" perrow="2"><br />
Image:Diesel Production for Wiki 2.png|<center>'''Make It: Diesel Production'''<br>Insert brief explanation of figure above here</center><br />
Image:Gluten Destruction for Wiki.png|<center>'''Break It: Gluten Destruction'''<br>Insert brief explanation of figure above here</center><br />
Image:Magnetosome Toolkit for Wiki.png|<center>'''The Magnetosome ToolKit'''<br>Insert brief explanation of figure above here</center><br />
Image:Gibson Toolkit for Wiki.png|<center>'''The GibsonBricks ToolKit'''<br>Insert brief explanation of figure above here</center><br />
</gallery><br />
<br />
<br />
<br />
<br />
='''Data Summary'''=<br />
<br />
==Data for Favorite New Parts==<br />
Fill Me In.. here is an example:<br />
# [http://partsregistry.org/Part:BBa_X0X0X Main Page] - '''BAR responsive promoter, BBa_X0X0X''': promoter can be induced to express a marker gene (GFP) when exposed to skunk smell (Butanethiol, C<sub>4</sub>H<sub>9</sub>SH), but not when exposed to pleasant scents<br />
# [http://partsregistry.org/Part:BBa_XXX00 Main Page] - '''Insulated Vector, BBa_XXX00''': un-induced leaky expression is blocked by transcriptional terminators<br />
# [http://partsregistry.org/Part:BBa_000XX Main Page] - '''BAR Bad Odor Receptor, BBa_000XX''': yellow fluorescent protein-tagged BAR (BBa_X00XX) shows that BAR is produced in E. coli<br />
<br />
<br />
==Data for Existing Parts==<br />
Fill Me In<br />
# [http://partsregistry.org/Part:BBa_J45119:Experience Experience] - '''Wintergreen odor enzyme generator, BBa_J45119''' (MIT, iGEM 2006): 98 out of 100 volunteer subjects standing up to 5 feet away from the bacterial cultures could distinguish wintergreen-producing bacteria from negative controls. <br />
# [http://partsregistry.org/Part:BBa_J61110:Experience Experience] - '''RBS, BBa_J61110''' (Arkin Lab, 2007): Of the 5 RBS Parts we tested, this RBS works best for expressing yellow fluorescent protein-tagged BAR<br />
<br />
<br />
==Improved Parts==<br />
Fill Me In<br />
# [http://partsregistry.org/Part:BBa_XXXXX Main Page] - '''Air Freshilizor, BBa_XXXXX''': Our mathematical model predicts that the threshold of activation is 10 parts per billion, the concentration of Butanethiol that humans can typically smell<br />
<br />
<br />
<br />
='''All Submitted Parts'''=<br />
<br />
<groupparts>iGEM011 Washington</groupparts></div>Mdsmithhttp://2011.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2011-09-23T01:51:32Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>Protocols</big></big></big></big></center><br><br><br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/gel_electrophoresis General Agarose Gel Electrophoresis]<br />
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[https://2011.igem.org/Team:Washington/Protocols/PCR General PCR Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Digestion General Digestion Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Ligation General Ligation Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Transformation General Transformation Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Colony Colony PCR Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Competent Competent Cell Prep Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Kunkel Kunkel Mutagensis]<br />
<br />
[http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of how Kunkel Mutagensis works]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/expression_purification Standard 1L Expression Purification]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/gene_assembly Gene Assembly With Oligos]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/sequencing Sequencing]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/CompDesign Computational Protein Design]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Glycerol_Stocks Glycerol Stocks]<br />
<br />
<br />
=Make It: Diesel Production Protocols=<br />
<br />
[https://2011.igem.org/Team:Washington/alkanebiosynthesis Alkane Biosynthesis media and extraction]<br />
<br />
[https://2011.igem.org/Team:Washington/alkanebiosynthesis_cloning Alkane Biosynthesis cloning]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/redesign_cell_lysate_assay Cell Lysate Assay by Decarbonylase Redesign Team]<br />
<br />
<br />
=Break It: Gluten Destruction Protocols=<br />
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[https://2011.igem.org/Team:Washington/Protocols/Cell_Lysate_Assay Whole Cell Lysate Assay]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/50mL_Scale Small Scale (50mL) Protein Expression and Purification]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_Assay Purified Enzyme Assay]<br />
<br />
=Make It: iGem Toolkits=<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Cyto. Cytometry Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Elect. Electroporation (Transformation)]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Gib_Rxn Gibson Cloning/Assembly]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Gib_Purif. Gibson Purification]<br />
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[https://2011.igem.org/Team:Washington/Protocols/High_PCR High-Yield PCR (Full-Gene Assembly)]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Plas_DNA. Isolation of Plasmid DNA (miniprep)]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Induc_studies. Induction Studies of Proteins Fusions (mam-sfGFP)]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/PBS. PBS Stock Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Overnights. Preparation of Overnight Cultures]<br />
<br />
<br />
=Wiki Design=<br />
[https://2011.igem.org/Team:Washington/Protocols/Wiki_Design Wiki Design Tools (Wiki Markup, WikiDust, etc.)]</div>Mdsmithhttp://2011.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2011-09-23T01:51:08Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>UW iGEM 2011: Protocols</big></big></big></big></center><br><br><br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/gel_electrophoresis General Agarose Gel Electrophoresis]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/PCR General PCR Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Digestion General Digestion Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Ligation General Ligation Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Transformation General Transformation Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Colony Colony PCR Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Competent Competent Cell Prep Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Kunkel Kunkel Mutagensis]<br />
<br />
[http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of how Kunkel Mutagensis works]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/expression_purification Standard 1L Expression Purification]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/gene_assembly Gene Assembly With Oligos]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/sequencing Sequencing]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/CompDesign Computational Protein Design]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Glycerol_Stocks Glycerol Stocks]<br />
<br />
<br />
=Make It: Diesel Production Protocols=<br />
<br />
[https://2011.igem.org/Team:Washington/alkanebiosynthesis Alkane Biosynthesis media and extraction]<br />
<br />
[https://2011.igem.org/Team:Washington/alkanebiosynthesis_cloning Alkane Biosynthesis cloning]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/redesign_cell_lysate_assay Cell Lysate Assay by Decarbonylase Redesign Team]<br />
<br />
<br />
=Break It: Gluten Destruction Protocols=<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Cell_Lysate_Assay Whole Cell Lysate Assay]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/50mL_Scale Small Scale (50mL) Protein Expression and Purification]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_Assay Purified Enzyme Assay]<br />
<br />
=Make It: iGem Toolkits=<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Cyto. Cytometry Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Elect. Electroporation (Transformation)]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Gib_Rxn Gibson Cloning/Assembly]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Gib_Purif. Gibson Purification]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/High_PCR High-Yield PCR (Full-Gene Assembly)]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Plas_DNA. Isolation of Plasmid DNA (miniprep)]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Induc_studies. Induction Studies of Proteins Fusions (mam-sfGFP)]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/PBS. PBS Stock Protocol]<br />
<br />
[https://2011.igem.org/Team:Washington/Protocols/Overnights. Preparation of Overnight Cultures]<br />
<br />
<br />
=Wiki Design=<br />
[https://2011.igem.org/Team:Washington/Protocols/Wiki_Design Wiki Design Tools (Wiki Markup, WikiDust, etc.)]</div>Mdsmithhttp://2011.igem.org/Team:Washington/PartsTeam:Washington/Parts2011-09-23T01:48:52Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>UW iGEM 2011: Data Page</big></big></big></big></center><br><br><br />
<br />
<gallery caption="An Overview of the 2011 UW iGEM Teams Summer Projects" widths="435px" heights="300px" perrow="2"><br />
Image:Diesel Production for Wiki 2.png|<center>'''Make It: Diesel Production'''<br>Insert brief explanation of figure above here</center><br />
Image:Gluten Destruction for Wiki.png|<center>'''Break It: Gluten Destruction'''<br>Insert brief explanation of figure above here</center><br />
Image:Magnetosome Toolkit for Wiki.png|<center>'''The Magnetosome ToolKit'''<br>Insert brief explanation of figure above here</center><br />
Image:Gibson Toolkit for Wiki.png|<center>'''The GibsonBricks ToolKit'''<br>Insert brief explanation of figure above here</center><br />
</gallery><br />
<br />
<br />
<br />
<br />
='''Data Summary'''=<br />
<br />
==Data for Favorite New Parts==<br />
Fill Me In.. here is an example:<br />
# [http://partsregistry.org/Part:BBa_X0X0X Main Page] - '''BAR responsive promoter, BBa_X0X0X''': promoter can be induced to express a marker gene (GFP) when exposed to skunk smell (Butanethiol, C<sub>4</sub>H<sub>9</sub>SH), but not when exposed to pleasant scents<br />
# [http://partsregistry.org/Part:BBa_XXX00 Main Page] - '''Insulated Vector, BBa_XXX00''': un-induced leaky expression is blocked by transcriptional terminators<br />
# [http://partsregistry.org/Part:BBa_000XX Main Page] - '''BAR Bad Odor Receptor, BBa_000XX''': yellow fluorescent protein-tagged BAR (BBa_X00XX) shows that BAR is produced in E. coli<br />
<br />
<br />
==Data for Existing Parts==<br />
Fill Me In<br />
# [http://partsregistry.org/Part:BBa_J45119:Experience Experience] - '''Wintergreen odor enzyme generator, BBa_J45119''' (MIT, iGEM 2006): 98 out of 100 volunteer subjects standing up to 5 feet away from the bacterial cultures could distinguish wintergreen-producing bacteria from negative controls. <br />
# [http://partsregistry.org/Part:BBa_J61110:Experience Experience] - '''RBS, BBa_J61110''' (Arkin Lab, 2007): Of the 5 RBS Parts we tested, this RBS works best for expressing yellow fluorescent protein-tagged BAR<br />
<br />
<br />
==Improved Parts==<br />
Fill Me In<br />
# [http://partsregistry.org/Part:BBa_XXXXX Main Page] - '''Air Freshilizor, BBa_XXXXX''': Our mathematical model predicts that the threshold of activation is 10 parts per billion, the concentration of Butanethiol that humans can typically smell<br />
<br />
<br />
<br />
='''All Submitted Parts'''=<br />
<br />
<groupparts>iGEM011 Washington</groupparts></div>Mdsmithhttp://2011.igem.org/Team:Washington/Magnetosomes/PartsTeam:Washington/Magnetosomes/Parts2011-09-23T01:48:16Z<p>Mdsmith: </p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
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<center><big><big><big><big>iGEM Toolkits: Parts Submitted</big></big></big></big></center><br><br><br />
<br />
iGem Toolkits: submitted a total twenty-two parts to the registry: 10 magnetosome gene-groups, 2 mam gene-sfGFP fusions, 3 parts of the essential magnetosome gene- assembly, and 5 pGA vectors.</div>Mdsmithhttp://2011.igem.org/Team:Washington/Magnetosomes/ReferenceTeam:Washington/Magnetosomes/Reference2011-09-23T01:47:48Z<p>Mdsmith: </p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
<center><big><big><big><big>iGEM Toolkits: References</big></big></big></big></center><br><br><br />
<br />
=References:=<br />
<br />
<br />
# Matsunaga, T., Okamura, Y., Fukuda, Y., Wahyudi, A.T., Murase, Y., Takeyama, H. (2005). Complete genome sequence of the facultative anaerobic Magnetotactic bacterium Magnetospirillum sp. strain AMB-1. ''DNA research''; 12: 157-166. Doi:10.1093/dnares/dsi002. <br />
# Murat, D., Quinlan, A., Vali, H., Komeili, A. (2010). Comprehensive genetic dissection of the magnetosome gene island reveals the step-wise assembly of a prokaryotic organelle. ''PNAS''; 107 (12): 5593-5598. Doi:10.1073/pnas.0914439107.<br />
# Murat, D., Quinlan, A., Vali, H., Komeili, A. (2010). Supporting Information. ''PNAS''; 107 (12): 5593-5598. Doi:10.1073/pnas.0914439107.<br />
# Quinlan, A., Murat, D., Vali, H., Komeili, A. (2011).The HtrA/DegP family protease MamE is a bifunctional protein with roles in magnetosome protein localization and magnetite biomineralization. ''Molecular Microbiology''; 80 (4): 855-1131. Doi:10.1111/j.1365-2958.2011.07631.x.<br />
# Richter, M., Kube, M., Bazylinski, D.A., Lombardot, T.,Glockner, F.O., Reinhardt, R., Shuler, D. (2007). Comparative genome analysis of four Magnetotactic bacteria reveals a complex set of group-specific genes implicated in magnetosome biomineralization and function. ''Journal of Bacteriology''; 189(13): 4899-4910. Doi:10.1128/JB.00119-07.<br />
# Rioux, J.B., Philippe, N., Pereia, S., Pignol, D., Wu, L.F., Ginet, N. (2010). A second actin-like mamK protein in Magnetospirillum magneticum AMB-1 encoded outside the genomic magnetosome island. ''PLoS ONE''; 5(2): e9151. Doi:10.1371/journal.pone.0009151.</div>Mdsmithhttp://2011.igem.org/Team:Washington/Magnetosomes/ReferenceTeam:Washington/Magnetosomes/Reference2011-09-23T01:47:38Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<center><big><big><big><big>iGEM Toolkits: References</big></big></big></big></center><br><br><br />
<br />
=References:=<br />
<br />
<br />
# Matsunaga, T., Okamura, Y., Fukuda, Y., Wahyudi, A.T., Murase, Y., Takeyama, H. (2005). Complete genome sequence of the facultative anaerobic Magnetotactic bacterium Magnetospirillum sp. strain AMB-1. ''DNA research''; 12: 157-166. Doi:10.1093/dnares/dsi002. <br />
# Murat, D., Quinlan, A., Vali, H., Komeili, A. (2010). Comprehensive genetic dissection of the magnetosome gene island reveals the step-wise assembly of a prokaryotic organelle. ''PNAS''; 107 (12): 5593-5598. Doi:10.1073/pnas.0914439107.<br />
# Murat, D., Quinlan, A., Vali, H., Komeili, A. (2010). Supporting Information. ''PNAS''; 107 (12): 5593-5598. Doi:10.1073/pnas.0914439107.<br />
# Quinlan, A., Murat, D., Vali, H., Komeili, A. (2011).The HtrA/DegP family protease MamE is a bifunctional protein with roles in magnetosome protein localization and magnetite biomineralization. ''Molecular Microbiology''; 80 (4): 855-1131. Doi:10.1111/j.1365-2958.2011.07631.x.<br />
# Richter, M., Kube, M., Bazylinski, D.A., Lombardot, T.,Glockner, F.O., Reinhardt, R., Shuler, D. (2007). Comparative genome analysis of four Magnetotactic bacteria reveals a complex set of group-specific genes implicated in magnetosome biomineralization and function. ''Journal of Bacteriology''; 189(13): 4899-4910. Doi:10.1128/JB.00119-07.<br />
# Rioux, J.B., Philippe, N., Pereia, S., Pignol, D., Wu, L.F., Ginet, N. (2010). A second actin-like mamK protein in Magnetospirillum magneticum AMB-1 encoded outside the genomic magnetosome island. ''PLoS ONE''; 5(2): e9151. Doi:10.1371/journal.pone.0009151.</div>Mdsmithhttp://2011.igem.org/Team:Washington/Magnetosomes/FutureTeam:Washington/Magnetosomes/Future2011-09-23T01:47:07Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>iGEM Toolkits: Future Directions</big></big></big></big></center><br><br><br />
<br />
==Gibson Assembly Toolkit==<br />
<br />
1. Remove XhoI site from pGA3K3 vector<br />
<br />
2. Remove homologous regions from pGA4A5, pGA4C5 vectors<br />
<br />
We will keep working on adding more components into the toolkits at UW and hope that iGEM community will be able to use toolkits to join in on the fun!<br />
<br />
-----<br />
<br/><br />
;Gibson Assembly Toolkit<br />
We have developed and submitted several vectors that are Gibson Cloning friendly(see the "parts submitted" page). More of such vectors should be developed and added to the toolkit in the future.<br />
<br />
;Magnetosome Toolkit<br />
We still have a long way to achieve our goal of making magnetic ''E.coli'' but this project is certainly worth investigating. We have a ton of project ideas for the future teams...<br />
<br/><br />
*Express the rest of the gene in the MAI region in E.coli<br />
*Co-express the genes and study their interaction<br />
*Build the scaffold structure in E.coli<br />
*Express the full assembly in E.coli<br />
*Develop assay for the magnet formation<br />
*Determine the optimal cell growth condition......<br />
<br />
<br />
In addition, the ability to produce and control uniform, nano-sized magnetic particles is attractive in areas such as medical imaging and nano-electronics where scientists and engineers are actively seeking innovative solutions for breakthrough in size and accuracy. Thus, if we are able to produce magnets in an organism that are thoroughly understood in a controlled manner, and be able to extract the magnets from them, this indeed is going to be very useful for a lot of areas.</div>Mdsmithhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ToolkitTeam:Washington/Magnetosomes/Magnet Toolkit2011-09-23T01:46:32Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>iGEM Toolkits: Magnetosome Toolkit</big></big></big></big></center><br><br><br />
<br />
===What are magnetosomes? Where do they come from?===<br />
<br />
<br />
<br />
[[File:Magnetosome_chain.png|thumb|Fig. 1: A Chain of Magnetosomes within ''Magnetospirillum magneticum'' AMB-1]]<br />
<br />
Magnetotactic Bacteria are prokaryotic organisms which possess the unique ability to align themselves along a magnetic field. This form of taxis is made possible by the formation of a magnetosome formation. Magnetosomes are small invaginations of the bacterial cell membrane that contain magnetite particles<br />
<br />
These particles range in size between 20 and several hundred nanometers and are aligned in one or several chains along the long axis of the bacteria. These particles act together to form a magnetic dipole across the bacteria, allowing it to perceive the earth’s magnetic field. Magnetotactic bacteria are microaerophilic; therefore, the magnetosome is currently thought to help aid the organism in its search for the perfect oxygen level from a three dimensional space (in all directions) to a one dimensional space along a single path.<br />
<br />
<br />
===A Closer look at Magnetosome Formation ===<br />
<br />
The formation of the magnetosome organelle is a highly regulated, step-wise process requiring a cascade of essential genes. The process is generally hypothesized as four stages: i) membrane invagination, ii) acquiring minerals for magnetite formation, iii) iron-oxidation and reduction, iv) magnetite nucleation and morphology regulation. Earlier gene products must be present for later gene products to be formed as shown in the diagram below: [http://www.pnas.org/content/107/12/5593.full.pdf+html]:<br />
[[File:F6.medium.png|center|350px|thumb|Fig. 2: Diagram of stepwise magnetosome construction within AMB-1.]]<br />
<br />
===What did the UW iGEM team do with Magnetotactic Bacteria?===<br />
<br />
It is thought that many of the essential genes associated with magnetosome formation are located within a well-conserved region known as the magnetosome island (MAI). The MAI consists of 14 gene clusters labeled R1-R14 (see diagram below).Our team focused on the genes of the mamAB gene cluster (R5) as they were previously shown to be essential for magnetosome membrane biogenesis in AMB-1 (diagram show below).[http://www.pnas.org/content/107/12/5593/F1.expansion.html].<br />
<br />
The goal of our project was to extract all the essential genes from (R5) required for magnetosome formation and express them in E.coli. This was done in order to understand more about magnetosome formation and the magnet synthesis mechanism because many of the genes' functions are still unknown in the host species. Using the information we have gained, we have organized a '''Magnetosome Toolkit''' containing most of the essential genes for proper magnetosome formation. Ultimately, we would like to continue expanding the magnetosome toolkit to have enough parts to show complete magnetosome formation within E.coli.<br />
[[File:MamAB.png|center|500px|thumb|Fig. 3: The mamAB operon (R5) located in the magnetosome island (MAI).]]<br />
<br />
===About the Magnetosome Toolkit:===<br />
<br />
Using standard synthetic biology protocols and the vectors we created in our Gibson Assembly Toolkit, our team was able to create a '''"Magnetosome Toolkit"''' consisting of the most basic parts required for magnetosome formation. Providing this toolkit will help allow future iGem teams to manipulate and further understand magnetosome formation to eventually synthesize magnets in various types of bacteria. <br />
<br />
<br />
<br />
=== Toolkit construction and mamAB assembly in E.coli ===<br />
<br />
== Individual Magnetosome (mam) genes==<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 />
{| 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 />
[[File:Washington Methode image.jpg|right|500px]]<br />
<br />
Using standard protocols and our high-copy pGA vectors, these genes were extracted from the host genome and characterized to confirm their accuracy. <br />
<br />
As previously noted, magnetosome formation within the host-organism, ''Magnetospirillium magneticum'', strain AMB-1, is a highly regulated step-wise process. As shown in Fig. 2, some genes encode for an invagination in the inner membrane, other genes which help align the magnetosomes into their characteristics chains, and others which regulate the biomineralization of magnetic particles. Our team chose to focus on genes specifically related to magnetosome scaffolding/alignment since they are the essential foundation for magnetosome development. In addition, the creation of a scaffold to which other genes localize is highly applicable to systems in synthetic biology. (for more information, please see our Future Directions page)<br />
<br />
Our genes of interest were mamK and mamI as they have functions related to localization of the magnetosome. Specifically, mamK is a bacterial actin-like cytoskeleton protein required for proper alignment of the magnetosomes in a chain. mamK is also shown to localize the mamI, which is loss inhibits membrane formation. <br />
(for other gene functions, see the table below):<br />
<br />
{| class="wikitable"<br />
|-<br />
! Gene <br />
! AMB Number<br />
! Cluster Membership<br />
! Member of 28 genes list? (specific*/related**)<br />
! Function Summary (Vesicle chain formation, and/or biomineralization)<br />
! Gene Function<br />
|-<br />
| mamH<br />
| amb0961<br />
| mamAB<br />
| Related<br />
|<br />
|<br />
|-<br />
| mamI<br />
| amb0962<br />
| mamAB<br />
| Specific<br />
| Vesicle, (Chain Formation?)<br />
| >berkeley 2010: Loss causes no membrane formation, is localized onto chains<br />
|-<br />
| mamE<br />
| amb0963<br />
| mamAB; mam Islet<br />
| Related<br />
|<br />
| >Membrane-bound serine protease required for magnetite formation; might control the localization of other magnetosome proteins<br />
|-<br />
| mamJ<br />
| amb0964<br />
| mamAB; mam Islet<br />
| Specific<br />
| Chain Formation<br />
| >Proper magnetosome chain organization/assembly<br />
|-<br />
| mamK<br />
| amb0965<br />
| mamAB; mam Islet<br />
| Related<br />
| Chain Formation<br />
| >required for proper magnetosome chain organization; *bacterial actin-like cytoskeleton protein required for proper alignment of the magnetosomes in a chain, shown to localize the mamI<br />
|- <br />
| mamL<br />
| amb0966<br />
| mamAB; mam Islet<br />
| Specific<br />
| Vesicle, biomineralization<br />
| >berkely 2010: Crucial to mangneosome membrane creation, shown to be spread across the cell membrane and sometimes forms lines<br />
|-<br />
| mamM<br />
| amb0967<br />
| mamAB<br />
| Related<br />
|<br />
| >biomineralization, involved in iron transport, magnetite nucleation, or establishement of the proper chemical enviornment for magnetite synthesis in the magnetosome<br />
|-<br />
| mamN<br />
| amb0968<br />
| mamAB<br />
| Related<br />
|<br />
| >biomineralization, involved in iron transport, magnetite nucleation, or establishement of the proper chemical enviornment for magnetite synthesis in the magnetosome<br />
|-<br />
| mamO<br />
| amb0969<br />
| mamAB<br />
| Related<br />
|<br />
| >biomineralization, involved in iron transport, magnetite nucleation, or establishement of the proper chemical enviornment for magnetite synthesis in the magnetosome<br />
|- <br />
| mamP<br />
| amb0970<br />
| mamAB<br />
| Related<br />
| Biomineralization<br />
| >berkeley 2010: loss causes weak magnetic response, with large but fewer crystals<br />
|-<br />
| mamA<br />
| amb0971<br />
| mamAB<br />
| Related<br />
|<br />
| >Required for magnetosome activation; activation of vessicles<br />
|-<br />
| mamQ<br />
| amb0972<br />
| mamAB; mam Islet<br />
| Related<br />
|<br />
| >ORF; formation/maintenance of magnetosome membranes<br />
|-<br />
| mamR<br />
| amb0973<br />
| mamAB<br />
| Specific<br />
| Chain formation, Biomineralization<br />
| >ORF; plays a role in controlling both particle number and size of magnetite cyrstals<br />
|-<br />
| mamB<br />
| amb0974<br />
| mamAB<br />
| Related<br />
| Vesicle, Biomineralization<br />
| >indirect role in magnetosome membrane invagination and biomineralization; magnetosome compartment formation<br />
|-<br />
| mamS<br />
| amb0975<br />
| mamAB<br />
| Specific<br />
| <br />
|<br />
|-<br />
| mamT<br />
| amb0976<br />
| mamAB<br />
| Specific<br />
| Biomineralization<br />
| >magnetite crystal growth; participates in different steps during magnetite synthesis<br />
|-<br />
| mamU<br />
| amb0977<br />
| mamAB<br />
| Related<br />
| <br />
| <br />
|-<br />
| mamV<br />
| amb0978<br />
| mamAB<br />
| N/A<br />
|<br />
|<br />
|-<br />
|}<br />
<br />
== Magnetosome gene-protein Fusions==<br />
<br />
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 />
<br />
[[File:Igem2011_mamK_and_I.png|700px|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''. In the image of mamK, a filament is seen running through the length of many bacteria. For mamI, the gene product is seen to fluoresce around the cell membrane of the bacteria but mostly concentrated at the ends. Similarly, the graph shows that as the arrow cross the cell membrane, the fluorescent peaks are at a maximum, and through the center of the cell, the level of fluorescence decreases.<br />
<br />
==Construction of the R5 region of the Magnetosome Island in ''E.coli''==<br />
<br />
After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJLK, mamMNOPA, and mamQRBSTUV. The first, and the third part of the assembly are shown below. They have been sequence confirmed...<br />
<br />
------>Pictures of mamHIEJLK and QRBSTUV<-------</div>Mdsmithhttp://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectorsTeam:Washington/Magnetosomes/GibsonVectors2011-09-23T01:44:26Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>iGEM Toolkits: Gibson Assembly Toolkit</big></big></big></big></center><br><br><br />
<br />
===About Gibson Cloning/Assembly===<br />
Gibson Cloning/Assembly is a cloning method that allows multiple inserts in one time isothermal reaction for assembling overlapping DNA fragments. During the cloning process, this does not only saves a lot of time and efforts, but also allows more complexity when building the circuit. <br/> [[File:Igem2011_GibsonReacion.png|center|400px]] <br />
<br />
===What happened last year?===<br />
The Gibson Cloning method is definitely not a new terminology to the iGEM community. This cloning method was published in Nature protocols 2009 ( Please see Enzymatic assembly of DNA molecules up to several hundred kilobases, Gibson et al. (2009).) and it has gained a lot of attention from the iGEM community since then. <br />
<br />
In 2010, the Cambridge iGEM team proposed the proposed a BioBrick standard for Gibson Assembly in RFC 57 which enables simultaneous assembly of multiple fragments with no scar sequences. We found that this Biobrick Standard was incapable of giving high yields even in two-fragments assembly. The primary problem is the self-complementarity of the two NotI sequences embedded in the BioBrick prefix and suffix which prevents the insert from being incorporated into the vector. <br/><br />
[[File:Igem2011 biobrick NotI.png|400px|center]]<br />
*E=ECoRI, N=NotI, X=XohI, S=SpeI, P=PstI<br />
<br />
Therefore, to combat the problem, a “gibson reaction compatible” prefix and suffix were designed based on BglBrick standard to increase the cloning efficiency. <br/><br />
[[File:Igem2011_gibsonbrick.png|400px|center]]<br />
<br />
*E=ECoRI,S1=Spacer 1, Bg=Bgl S2=Spacer2, P=PstI<br />
<br />
=== What about this year? ===<br />
<br />
Seeing that this is such a great method to do cloning...we continued with the investigation and made the '''Gibson Assembly Toolkit'''! <br />
<br />
We call our new vectors plamid Gisbon Assembly (pGA) vectors. And we were able to show that the cloning efficiency of pGA vector is better than the pSB vector. <br />
<br />
(Stay tuned for our results)<br />
<br />
<br />
We submitted 5 pGA vectors of different copy numbers and antibiotic resistances to the Registry. (All of them have pLac GFP) <br />
<br />
pGA1A3: high copy plasmid backbone with Amp resistance. <br />
pGA1C3: high copy plasmid backbone, with Chloramphenicol resistance. <br />
pGA3K3, pGA 4C5 and pGA 4A5 are low copy plasmid backbone, which are good for more gene fragment inserts.<br />
<br />
pGA4C5_pLacGFP: a low copy plasmid backbone which has Chloramphenicol resistance<br />
<br />
<br />
Talk about how we made them?<br />
<br />
a diagram<br />
a table showing the antibiotics, copy number<br />
<br />
=== Next level of iGEM project: more complex circuit.===<br />
And we decided to look at magnetosome<br />
<br />
====References====<br />
<br />
1.</div>Mdsmithhttp://2011.igem.org/Team:Washington/Magnetosomes/BackgroundTeam:Washington/Magnetosomes/Background2011-09-23T01:18:42Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>iGEM Toolkits: Background</big></big></big></big></center><br><br><br />
<br />
As with the expansion of the iGEM competition, many iGEM teams have started to investigate the possibility of working with large-scale genomes. Large-scale gene manipulation often requires the use of tools which allow multiple gene inserts as to bring the cloning project from single gene level to a multiple gene level. However, the current BioBrick standard vectors available through iGEM are not designed for multiple-insert cloning. Therefore, the UW iGEM team decided to research methods to improve cloning efficiency and as a result, two "toolkits" were submitted to the registry. <br />
<br />
-----<br />
<br />
<br />
=== Gibson Assembly Toolkit ===<br />
As a continuation of the [https://2010.igem.org/Team:Washington/Tools_Used/Next-Gen_Cloning 2010 UW IGEM project], this year we developed and submitted several plasmid backbones that are Gibson cloning method friendly-- aka pGA vectors. It is called the [https://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors Gibson Assembly Toolkit] <br />
<br />
<br />
<br />
[[File:Igem2011 GibsonToolkit.png|left|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors]] <br />
;'''What's in the Gibson Assembly Toolkit?'''<br/><br />
*Five plasmid backbones<br />
* 2 High copy extraction vectors: pGA1A3, pGA1C3<br />
* 3 low copy assembly vectors: pGA3K3, pGA4A5, pGA4C5<br />
<br />
<br><br />
<br />
<br/><br />
<br/><br />
<br/> <br />
<br/><br />
<br/><br />
<br/><br />
<br/><br />
<br/><br />
<br/><br />
=== Magnetosome Toolkit ===<br />
In addition, we were also ambitious about assembling a large gene-construct of over 16 kb. Therefore, utilizing our pGA vectors and Gibson cloning methods, the [https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit Magnetosome Toolkit] was developed with the goal to build magnetic ''E.Coli''; a novel characteristic expressed solely by magnetotactic bacteria, such as ''Magnetospirillum magneticum'' strain AMB-1.<br />
<br />
<br />
<br />
[[File:Igem2011 MagnetToolkit.png|left|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit]]<br />
'''What’s in the Magnetosome Toolkit?'''<br />
<br />
*A set of the 18 essential genes for the various steps of magnetosome formation. <br />
*Our favorite genes in pGA vectors<br />
*A table compiling individual gene functions from our literature search</div>Mdsmithhttp://2011.igem.org/Team:Washington/Magnetosomes/BackgroundTeam:Washington/Magnetosomes/Background2011-09-23T01:18:07Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>iGEM Toolkits</big></big></big></big></center><br><br><br />
<br />
As with the expansion of the iGEM competition, many iGEM teams have started to investigate the possibility of working with large-scale genomes. Large-scale gene manipulation often requires the use of tools which allow multiple gene inserts as to bring the cloning project from single gene level to a multiple gene level. However, the current BioBrick standard vectors available through iGEM are not designed for multiple-insert cloning. Therefore, the UW iGEM team decided to research methods to improve cloning efficiency and as a result, two "toolkits" were submitted to the registry. <br />
<br />
-----<br />
<br />
<br />
=== Gibson Assembly Toolkit ===<br />
As a continuation of the [https://2010.igem.org/Team:Washington/Tools_Used/Next-Gen_Cloning 2010 UW IGEM project], this year we developed and submitted several plasmid backbones that are Gibson cloning method friendly-- aka pGA vectors. It is called the [https://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors Gibson Assembly Toolkit] <br />
<br />
<br />
<br />
[[File:Igem2011 GibsonToolkit.png|left|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors]] <br />
;'''What's in the Gibson Assembly Toolkit?'''<br/><br />
*Five plasmid backbones<br />
* 2 High copy extraction vectors: pGA1A3, pGA1C3<br />
* 3 low copy assembly vectors: pGA3K3, pGA4A5, pGA4C5<br />
<br />
<br><br />
<br />
<br/><br />
<br/><br />
<br/> <br />
<br/><br />
<br/><br />
<br/><br />
<br/><br />
<br/><br />
<br/><br />
=== Magnetosome Toolkit ===<br />
In addition, we were also ambitious about assembling a large gene-construct of over 16 kb. Therefore, utilizing our pGA vectors and Gibson cloning methods, the [https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit Magnetosome Toolkit] was developed with the goal to build magnetic ''E.Coli''; a novel characteristic expressed solely by magnetotactic bacteria, such as ''Magnetospirillum magneticum'' strain AMB-1.<br />
<br />
<br />
<br />
[[File:Igem2011 MagnetToolkit.png|left|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit]]<br />
'''What’s in the Magnetosome Toolkit?'''<br />
<br />
*A set of the 18 essential genes for the various steps of magnetosome formation. <br />
*Our favorite genes in pGA vectors<br />
*A table compiling individual gene functions from our literature search</div>Mdsmithhttp://2011.igem.org/Team:Washington/Celiacs/PartsTeam:Washington/Celiacs/Parts2011-09-23T01:17:12Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>Gluten Destruction: Parts Submitted</big></big></big></big></center><br><br><br />
<br />
Gluten Destruction submitted four parts to the registry: Wild-type Kumamolisin-As and three of our promising mutants. A short description for each part is provided below.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590021 BBa_K590021: '''Kumamolisin-As''']<br />
<br />
An enzyme from the sedolisin family native to ''Alicyclobacillus sendaiensis'' with known collagenase activity at low pH and elevated temperatures.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590022 BBa_K590022: '''Kumamolisin-As_G319S, D358G, D368H''']<br />
<br />
A mutated Kumamolisin-As enzyme aimed to combat gluten intolerance by increased activity with the PQLP peptide, an antigenic epitope in gliadin. This mutant has point mutations at residues 319, 358, and 368 from Glycine to Serine, Aspartate to Glycine, and Aspartate to Histidine, respectively.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590023 BBa_K590023: '''Kumamolisin-As_N291D''']<br />
<br />
A mutated Kumamolisin-As enzyme aimed to combat gluten intolerance by increased activity with the PQLP peptide, an antigenic epitope in gliadin. This mutant has a point mutation at residue 291 from Asparagine to Aspartate.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590024 BBa_K590024: '''Kumamolisin-As_S354N, D358G, D368H''']<br />
<br />
A mutated Kumamolisin-As enzyme aimed to combat gluten intolerance by increased activity with the PQLP peptide, an antigenic epitope in gliadin. This mutant has point mutations at residue 354, 358, and 368 from Serine to Asparagine, Aspartate to Glycine, and Aspartate to Histidine, respectively.</div>Mdsmithhttp://2011.igem.org/Team:Washington/Celiacs/FutureTeam:Washington/Celiacs/Future2011-09-23T01:16:31Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>Gluten Destruction: Future Directions</big></big></big></big></center><br><br><br />
<br />
====Kinetic Characterization====<br />
<br />
We intend to develop a mass spectroscopy assay to measure the kcat (the rate of turnover per molecule of enzyme) and kM (the substrate binding constant) values for our best mutant.<br />
<br />
====Crystal Structure====<br />
<br />
To obtain further structural information, we hope to eventually obtain a crystal structure of our best mutated enzyme bound to the PQLP substrate. This will involve mutating one of the residues in the catalytic triad, so that the substrate will remain bound without being cleaved. The structural information that we may be able to glean from such a structure will allow us to better characterize, and perhaps further improve our mutant.<br />
<br />
====Biophysical Characterization==== <br />
<br />
Once the ideal mutations are isolated, we intend to test the best mutants at gastric pH and in the presence of other gastric enzymes for a short period of time, mimicking the environment after enzyme ingestion and prior to enzyme uptake by the small intestine. We suspect that the thermostable properties of Kumamolisin-As will render our mutant enzyme reasonably resistant to degradation by gastric enzymes such as pepsin. However, if this proves not to be the case, we intend to reengineer the mutant for enhanced resistance to pepsin and other such gastric enzymes. Once ensured that the mutated Kumamolisin-As remains active under stomach conditions, this ideal mutated enzyme will be ready for ''in vivo'' experimentation.<br />
<br />
====Therapeutic Promise====</div>Mdsmithhttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-23T01:16:02Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>Gluten Destruction: Results Summary</big></big></big></big></center><br><br><br />
<br />
=Testing Kumamolisin-As against SC-PEP=<br />
<br />
After finding Kumamolisin-As to be the ideal enzyme for our purposes, we used the assay described below to test it against SC-PEP to determine comparative activity levels, resulting in evidence that wild-type Kumamolisin-As is already over 7 fold better than SC-PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolysin has a much higher activity level than SC-PEP, in addition to being amenable to engineering and effective at gastric pH.]]<br />
<br />
----<br />
<br />
=Testing mutants for activity on breaking down PQLP=<br />
<br />
==Using a whole cell lysate assay to screen a large number of mutants for good activity==<br />
<br />
In order to determine whether our proposed mutations to the wild-type Kumamolisin improved the ability of the enzyme to break down PQLP, we tested each mutant with a whole cell lysate fluorescence assay. Cells harboring the expressed mutants were lysed and the assay was performed at pH 4, mimicking the gastric environment. The released enzymes, after being roughly separated from cell material, were added to a fluorescent PQLP that had been conjugated to a quencher. Thus, no fluorescence was achieved until the peptide had been cleaved and the fluorophore had been released from the quencher. This allowed a relative assessment of rate of enzyme activity by measuring increase in fluorescence of the system.<br />
<br />
As one might expect, our first screen of mutants showed some mutants with a decrease in activity from the wild-type, some showed no change, and some actually showed great increase in activity. One single point mutant showed close to a 1000% increase in activity from wild-type Kumamolisin!<br />
<br />
<br />
[[File:Washington Mutant Screen Percent IncDec.jpg|center|800px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate.]]<br />
<br />
==Purifying and characterizing promising mutants for accurate rate comparison==<br />
<br />
Once we had identified mutants that showed a promising increase in activity from the wild-type kumamolisin, we purified and characterized activity in concentration controlled fluorescence assays, identical to the fluorescence system used for the whole cell lysate assay. Our best mutant demonstrated an 11-fold increase in activity from the native enzyme.<br />
<br />
<br />
[[File:Washington Best Mutants.png|center|500px|thumb|We narrowed this down to a few of our best mutants.]]<br />
<br />
<br />
----<br />
<br />
<br />
<br />
=Combining Mutants for the construction of a Gluten Hydrolase=<br />
==A second library of based on the first round of mutagensis was constructed and tested==<br />
<br />
==One of the combinatorial mutants resulted in over a 100-fold increase in activity==<br />
<br />
In order to achieve even more rate improvement from the native, we repeated our mutagenesis, this time taking successful mutations and adding them all to make combinatorial variants. By combining two of our top groups of mutations from the first round, we achieved an over 100-fold increase in activity on breaking down PQLP from the wild-type enzyme. This variant enzyme is ultimately 784 times better at breaking down PQLP than SC PEP, the enzyme currently in clinical trials for treating gluten intolerance!<br />
<br />
<br />
[[File:Washington BestCombMutant.png|center|500px|thumb|Our final engineered enzyme showed activity over 100 fold higher than wild type Kumamolisin, and ~700 fold higher than SC-PEP.]]</div>Mdsmithhttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-23T01:15:48Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>Gluten Destruction: Result Summary</big></big></big></big></center><br><br><br />
<br />
=Testing Kumamolisin-As against SC-PEP=<br />
<br />
After finding Kumamolisin-As to be the ideal enzyme for our purposes, we used the assay described below to test it against SC-PEP to determine comparative activity levels, resulting in evidence that wild-type Kumamolisin-As is already over 7 fold better than SC-PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolysin has a much higher activity level than SC-PEP, in addition to being amenable to engineering and effective at gastric pH.]]<br />
<br />
----<br />
<br />
=Testing mutants for activity on breaking down PQLP=<br />
<br />
==Using a whole cell lysate assay to screen a large number of mutants for good activity==<br />
<br />
In order to determine whether our proposed mutations to the wild-type Kumamolisin improved the ability of the enzyme to break down PQLP, we tested each mutant with a whole cell lysate fluorescence assay. Cells harboring the expressed mutants were lysed and the assay was performed at pH 4, mimicking the gastric environment. The released enzymes, after being roughly separated from cell material, were added to a fluorescent PQLP that had been conjugated to a quencher. Thus, no fluorescence was achieved until the peptide had been cleaved and the fluorophore had been released from the quencher. This allowed a relative assessment of rate of enzyme activity by measuring increase in fluorescence of the system.<br />
<br />
As one might expect, our first screen of mutants showed some mutants with a decrease in activity from the wild-type, some showed no change, and some actually showed great increase in activity. One single point mutant showed close to a 1000% increase in activity from wild-type Kumamolisin!<br />
<br />
<br />
[[File:Washington Mutant Screen Percent IncDec.jpg|center|800px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate.]]<br />
<br />
==Purifying and characterizing promising mutants for accurate rate comparison==<br />
<br />
Once we had identified mutants that showed a promising increase in activity from the wild-type kumamolisin, we purified and characterized activity in concentration controlled fluorescence assays, identical to the fluorescence system used for the whole cell lysate assay. Our best mutant demonstrated an 11-fold increase in activity from the native enzyme.<br />
<br />
<br />
[[File:Washington Best Mutants.png|center|500px|thumb|We narrowed this down to a few of our best mutants.]]<br />
<br />
<br />
----<br />
<br />
<br />
<br />
=Combining Mutants for the construction of a Gluten Hydrolase=<br />
==A second library of based on the first round of mutagensis was constructed and tested==<br />
<br />
==One of the combinatorial mutants resulted in over a 100-fold increase in activity==<br />
<br />
In order to achieve even more rate improvement from the native, we repeated our mutagenesis, this time taking successful mutations and adding them all to make combinatorial variants. By combining two of our top groups of mutations from the first round, we achieved an over 100-fold increase in activity on breaking down PQLP from the wild-type enzyme. This variant enzyme is ultimately 784 times better at breaking down PQLP than SC PEP, the enzyme currently in clinical trials for treating gluten intolerance!<br />
<br />
<br />
[[File:Washington BestCombMutant.png|center|500px|thumb|Our final engineered enzyme showed activity over 100 fold higher than wild type Kumamolisin, and ~700 fold higher than SC-PEP.]]</div>Mdsmithhttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-23T01:14:19Z<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 />
[[File:Washington Foldit.png|400px|thumb|left|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
==Using Foldit to Design Mutations==<br />
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. <br />
<br />
Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.<br />
<br />
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.<br />
<br />
==Mutagenizing Kumamolisin==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|right|Overview of how Kunkel Mutagenesis works]]<br />
<br />
===Producing ssDNA===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. <br />
<br />
We designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. We:<br />
*isolated the single stranded DNA (ssDNA) of the sense strand of our gene, <br />
*harvested the ssDNA of the sense strand by infecting the cells with a bacteriophage that packages its own ssDNA genome, identified by length, and so in tandem also packaged our vector in single stranded form, and finally, <br />
*harvested the phage from the lysed culture of E. coli, and isolated our single stranded vector DNA.<br />
<br />
<br />
<br />
===Kunkel Mutagenesis===<br />
<br />
We annealed and extended our mutagenic oligos to allow for specific binding to our template. This vector was transformed into E. coli that degraded Uracil-containing DNA and replaced them with sections complementary to the opposite strand that contain thymine.<br />
Thus, the native Kumamolisin strand that still contained the U’s from the UNG-/DUT- strain was degraded, and the new cells incorporated our desired mutation when synthesizing new DNA from the variant strand.<br />
<br />
==Using a Whole Cell Lysate Assay to Test Activity of Mutants==<br />
Repeated growth, incubation, and induction of cells, followed by lysation, allowed us to test the supernatant for proteolytic activity towards PQLP in an assay which measured PQLP degradation. The assay was done at pH 4 in accordance with the assays done to test ScPEP according to the literature. The mutants were tested against wild-type kumamolisin and ScPEP, an enzyme currently used for the treatment of gluten intolerance via proteolysis. The assay we used was not highly accurate in terms of actual activity. However, what the assay allowed us to do was determine activity relative to our controls. This allowed us to determine which mutants were worth purifying to get more accurate activity data.<br />
[[File:Washington Assay.png|center|General Overview of the Whole Cell Lysate]]<br />
<br />
==Testing Purified Mutants to Accurately Assess Activity==<br />
===Purification===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level, as high activity without purification could simply be the result of high protein concentration. Growth, induction, and lysation of single colonies allowed the enzymes to be released from the cells, followed by collection of the purified proteins.<br />
<br />
===Assay===<br />
Concentrations were taken of the purified proteins, and diluted to the same concentration, to produce an assay resulting in accurate data representing which mutants had higher activity than kumamolisin and by how much their activity was greater.</div>Mdsmithhttp://2011.igem.org/Team:Washington/Alkanes/ResultsTeam:Washington/Alkanes/Results2011-09-23T01:12:46Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
<center><big><big><big><big>Diesel Production: Results Summary</big></big></big></big></center><br><br><br />
<br />
=GCMS confirms that The PetroBrick enables diesel production from sugar using ''E. coli''=<br />
<br />
We preformed GC-MS analysis on extracts from cell cultures expressing both Acyl-ACP reductase ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 AAR]) and Aldehyde Decarbonylase ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 ADC]). To act as controls and in order to show that the alkane production system is working as expected, we also analyzed cell cultures expressing either only AAR, or only ADC.<br />
<br />
<br />
[[Image:Washington_2011_PetroBrick.png|300px|frameless|border|left|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
[[Image:Washinton_AARADCGC-MS.png|center|600px|thumb|GCMS confirms PetroBrick diesel production from sugar using E. coli]]<br />
<br />
<br><br />
<br />
'''No significant hydrocarbons peaks in samples extracted from cells expressing only ADC.''' The blue GCMS chromatagram trace on the bottom show no significant peaks are found on the ADC only control within the time range that we expect alkane signal based upon GC-MS runs on chemical standards (8-10.5 minutes). This is expected, as ''E. coli'' does not normally produce any long chain length aldehyde substrates.<br />
<br />
'''C16 alcohols are observed when AAR is expressed on its own.''' The red GCMS chromatagram trace in the middle has a significant peak at 10.2 minutes corresponding to the C16 alcohol (as confirmed by comparison of the peak's MS spectra to a reference library) is observed when AAR is expressed in MG1655 E. coli. The production of C16 alcohols in cells expressing AAR (but not in cells expressing only ADC) is consistent with AAR reducing even chain length Acyl-ACPs into even chain length fatty aldehydes, which are further reduced by aldehyde dehydrogenases to the alcohol.<br />
<br />
'''The PetroBrick Works! C13, C15, and C17 alkanes are produced when both AAR and ADC are expressed.''' The green GCMS chromatagram trace in the middle has a significant new peaks corresponding to the C13 (8.2min) and C15 (9.2min) alkanes, as well as a peak at 10.2 minutes that contains both the C16 alcohol and the C17 alkene. The fact that the C17 alkene and C16 alcohol peak overlaps with the C16 alcohol peak makes quantification of C17 akene yields impossible, but we have sufficient evidence to determine that both molecules are present in the 10.2 minute peak( the later section of the peak has an MS spectra consistent with the C16 alcohol, and the earlier section is consistent with the C17 alkene). The C13 and C15 alkane peaks showed MS spectra highly consistent with C13 and C15 alkane reference spectra. In addition, both alkane molecules eluted off of the GC at the same time as C13 and C15 alkane standards, further increasing confidence that the 8.2 min and 9.2 min peaks do correspond to the C13 and C15 alkanes. Odd chain length alkane production is what is expected from our system, as ADC removes a carbonyl group from even chain length aldehydes produced by AAR, yielding an odd chain length alkane.The mass spectra for the peaks at 8.2 and 9.3 elute and have the same MS spectra as our standards, as well as the NIST compound library. The peak at 10.2 corresponded to a mixture of C17 alkene and C16 alcohol. Therefore quantifying the amount of C13 and C15 alkanes was readily achieved by constructing a standard curve. The coelution of the C17 with the significant alcohol peaks made it difficult to quantify the level of C17 alkene produced.<br />
<br />
<br />
----<br />
<br />
=Initial Quantization of Alkane Production=<br />
In order to be able to know how much alkane was being produced by our ''E. coli'', we spiked known amounts of alkane into cell cultures known to not produce alkanes. We then extracted using ethyl acetate, and analyzed extracts using GC-MS. Since peak area corresponds to the amount of each substance present, we used these GC plots to make a standard curve that allows us to convert peak area into an absolute amount. To determine how much alkane was being produced by our alkane production construct, we grew up 3 E. coli cultures expressing ADC and AAR in a standard M9 media ([https://2011.igem.org/Team:Washington/alkanebiosynthesis link]), and analyzed using GC-MS.<br />
<br />
<br />
[[File:Washinton 2011 Pre-Optimization Quant.png|center|550px|thumb|Diagram showing yields of the C13 and C15 alkanes, and C17 alkenes. Note: This C17 quantification is a rough approximation.]]<br />
<br />
<br />
----<br />
<br />
=Optimized Production=<br />
By optimizing the method of growth, media, vectors, and cell line we were able to succesfull increase yeilds of alkane production over 50-fold. In addition, the peak at C17 no longer had any trace of alcohol and all three peaks were clearly the desired alkenes, as determined by comparrsion to our product standards and the standard NIST reference spectrum library. The paramters we adjusted for optimization are discussed in detail at [https://2011.igem.org/Team:Washington/Alkanes/Future/Vector our systems optimization] page under future directions, as this is an on going process.<br />
<br />
[[File:Washinton 2011 Optimization Quant.png|center|550px|thumb|Diagram showing yields of the C13 and C15 alkanes, and C17 alkenes.]]</div>Mdsmithhttp://2011.igem.org/Team:Washington/Celiacs/BackgroundTeam:Washington/Celiacs/Background2011-09-23T01:11:51Z<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|200px|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: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: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/Alkanes/PartsTeam:Washington/Alkanes/Parts2011-09-23T01:10:57Z<p>Mdsmith: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>Diesel Production: Parts Submitted</big></big></big></big></center><br><br><br />
<br />
Below are interactive image maps of some of the parts we submitted created using Tinkercell along with the plugin Wikidust. <br />
<br />
==Composite==<br />
<br />
The Petrobrick: A modular and open platform for the biological production of diesel fuel<br />
<br />
<html><br />
<img src=https://static.igem.org/mediawiki/2011/3/3d/Washington_wikidust_petrobrick.png width="600" height="282" usemap="#washington_wikidust_petrobrick.png"/><br />
<map name="washington_wikidust_petrobrick.png"><br />
<area shape="rect" coords="435,59,601,224" title="Part:BBa_K590032" href="http://partsregistry.org/Part:Part:BBa_K590032"/> <br />
<area shape="rect" coords="187,59,353,224" title="Part:Part:BBa_K590031" href="http://partsregistry.org/Part:BBa_K590031"/> <br />
<area shape="rect" coords="105,100,187,183" title="Part:BBa_B0034" href="http://partsregistry.org/Part:BBa_B0034"/> <br />
<area shape="rect" coords="353,100,435,183" title="Part:BBa_B0034" href="http://partsregistry.org/Part:BBa_B0034"/> <br />
<area shape="rect" coords="2,68,105,171" title="Part:BBa_J23100" href="http://partsregistry.org/Part:BBa_J23100"/> <br />
</map><br />
</html><br />
<br />
==Coding==</div>Mdsmith