http://2011.igem.org/wiki/index.php?title=Special:Contributions/LStanley&feed=atom&limit=50&target=LStanley&year=&month=2011.igem.org - User contributions [en]2024-03-29T05:03:03ZFrom 2011.igem.orgMediaWiki 1.16.0http://2011.igem.org/Team:Washington/Team/AmericasTeam:Washington/Team/Americas2011-10-23T03:57:45Z<p>LStanley: </p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
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<center><span style="font-size: 28pt">'''Americas Regional Jamboree'''</span></center><br><br><br />
<br />
We had a lot of fun at the Americas Regional Jamboree! It was an action packed weekend filled with great talks, posters and people, and overall lots of thought-provoking science. It was amazing to see the diversity of projects presented, as well as the immense amount of work that was completed by all teams over the course of the summer. Many thanks to iGEM and IBE for hosting the jamboree, as well as to all of the judges and coordinators who put in so many volunteer hours. We are honored to have been chosen as the Americas Regional Grand Champion, and to have our enzyme, that we engineered to break down gluten, named Best New Part, Engineered. We look forward to the opportunity to meet again and experience more of the fantastic iGEM community.<br />
<br />
<br />
<br />
<center><div style="width: 650px">[[File:Washington Americas Results.jpg|center|650px|]]<br />
<span style="font-size: 10pt">After the Americas Regional Jamboree we were excited to tell the rest of our teammates about our experience, as only five of us were able to travel to Indianapolis. Thanks to Joe Oh at The Daily for this team picture.</span></div></center><br />
<br/></div>LStanleyhttp://2011.igem.org/Team:Washington/Team/AmericasTeam:Washington/Team/Americas2011-10-23T03:56:08Z<p>LStanley: </p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
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<center><span style="font-size: 28pt">'''Americas Regional Jamboree'''</span></center><br><br><br />
<br />
We had a lot of fun at the Americas Regional Jamboree! It was an action packed weekend filled with great talks, posters and people, and overall lots of thought-provoking science. It was amazing to see the diversity of projects presented, as well as the immense amount of work that was completed by all teams over the course of the summer. Many thanks to iGEM and IBE for hosting the jamboree, as well as to all of the judges and coordinators who put in so many volunteer hours. We are honored to have been chosen as the Americas Regional Grand Champion, and to have our enzyme, engineered to break down gluten, named Best New Part, Engineered. We look forward to the opportunity to meet again and experience more of the fantastic iGEM community.<br />
<br />
<br />
<br />
<center><div style="width: 650px">[[File:Washington Americas Results.jpg|center|650px|]]<br />
<span style="font-size: 10pt">After the Americas Regional Jamboree we were excited to tell the rest of our teammates about our experience, as only five of us were able to travel to Indianapolis. Thanks to Joe Oh at The Daily for this team picture.</span></div></center><br />
<br/></div>LStanleyhttp://2011.igem.org/Team:Washington/Team/AmericasTeam:Washington/Team/Americas2011-10-23T03:30:07Z<p>LStanley: </p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
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<center><span style="font-size: 28pt">'''Americas Regional Jamboree'''</span></center><br><br><br />
<br />
We had a lot of fun at the Americas Regional Jamboree! It was an action packed weekend filled with great talks, posters and people, and overall lots of thought-provoking science. It was amazing to see the diversity of projects presented, as well as the immense amount of work that was completed by all teams over the course of the summer. Many thanks to iGEM and IBE for hosting the jamboree, as well as to all of the judges and coordinators who put in so many volunteer hours. We are honored to have been chosen as the Americas Regional Grand Champion, and look forward to the opportunity to meet again and experience more of the fantastic iGEM community.<br />
<br />
<br />
<br />
<center><div style="width: 650px">[[File:Washington Americas Results.jpg|center|650px|]]<br />
<span style="font-size: 10pt">After the Americas Regional Jamboree we were excited to tell the rest of our teammates about our experience, as only five of us were able to travel to Indianapolis. Thanks to Joe Oh at The Daily for this team picture.</span></div></center><br />
<br/></div>LStanleyhttp://2011.igem.org/Team:Washington/Team/AmericasTeam:Washington/Team/Americas2011-10-23T03:29:39Z<p>LStanley: </p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
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<br />
<center><span style="font-size: 28pt">'''Americas Regional Jamboree'''</span></center><br><br><br />
<br />
We had a lot of fun at the Americas Regional Jamboree! It was an action packed weekend filled with great talks, posters and people, and overall lots of thought-provoking science. It was amazing to see the diversity of projects presented, as well as the immense amount of work that was completed by all teams over the course of the summer. Many thanks to iGEM and IBE for hosting the jamboree, as well as to all of the judges and coordinators who put in so many volunteer hours. We are honored to have been chosen as the Americas Regional Grand Champion, and look forward to the opportunity to meet again and experience more of the fantastic iGEM community.<br />
<br />
<br />
<br />
<center><div style="width: 650px">[[File:Washington Americas Results.jpg|center|650px|]]<br />
<span style-"font-size: 8pt"><br/></span><br />
<span style="font-size: 10pt">After the Americas Regional Jamboree we were excited to tell the rest of our teammates about our experience, as only five of us were able to travel to Indianapolis. Thanks to Joe Oh at The Daily for this team picture.</span></div></center></div>LStanleyhttp://2011.igem.org/Team:Washington/Team/AmericasTeam:Washington/Team/Americas2011-10-23T03:28:22Z<p>LStanley: </p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
<center><span style="font-size: 28pt">'''Americas Regional Jamboree'''</span></center><br><br><br />
<br />
We had a lot of fun at the Americas Regional Jamboree! It was an action packed weekend filled with great talks, posters and people, and overall lots of thought-provoking science. It was amazing to see the diversity of projects presented, as well as the immense amount of work that was completed by all teams over the course of the summer. Many thanks to iGEM and IBE for hosting the jamboree, as well as to all of the judges and coordinators who put in so many volunteer hours. We are honored to have been chosen as the Americas Regional Grand Champion, and look forward to the opportunity to meet again and experience more of the fantastic iGEM community.<br />
<br />
<br />
<br />
<center><div style="width: 650px">[[File:Washington Americas Results.jpg|center|650px|]]<br />
<br/><br />
<span style="font-size: 10pt">After the Americas Regional Jamboree we were excited to tell the rest of our teammates about our experience, as only five of us were able to travel to Indianapolis. Thanks to Joe Oh at The Daily for this team picture.</span></div></center></div>LStanleyhttp://2011.igem.org/Team:Washington/Team/AmericasTeam:Washington/Team/Americas2011-10-23T03:27:46Z<p>LStanley: </p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
<center><span style="font-size: 28pt">'''Americas Regional Jamboree'''</span></center><br><br><br />
<br />
We had a lot of fun at the Americas Regional Jamboree! It was an action packed weekend filled with great talks, posters and people, and overall lots of thought-provoking science. It was amazing to see the diversity of projects presented, as well as the immense amount of work that was completed by all teams over the course of the summer. Many thanks to iGEM and IBE for hosting the jamboree, as well as to all of the judges and coordinators who put in so many volunteer hours. We are honored to have been chosen as the Americas Regional Grand Champion, and look forward to the opportunity to meet again and experience more of the fantastic iGEM community.<br />
<br />
<br />
<br />
<center><div style="width: 650px">[[File:Washington Americas Results.jpg|center|650px|]]<br />
<br />
<span style="font-size: 10pt">After the Americas Regional Jamboree we were excited to tell the rest of our teammates about our experience, as only five of us were able to travel to Indianapolis. Thanks to Joe Oh at The Daily for this team picture.</span></div></center></div>LStanleyhttp://2011.igem.org/Team:Washington/Team/AmericasTeam:Washington/Team/Americas2011-10-23T03:26:13Z<p>LStanley: </p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
<center><span style="font-size: 28pt">'''Americas Regional Jamboree'''</span></center><br><br><br />
<br />
We had a lot of fun at the Americas Regional Jamboree! It was an action packed weekend filled with great talks, posters and people, and overall lots of thought-provoking science. It was amazing to see the diversity of projects presented, as well as the immense amount of work that was completed by all teams over the course of the summer. Many thanks to iGEM and IBE for hosting the jamboree, as well as to all of the judges and coordinators who put in so many volunteer hours. We are honored to have been chosen as the Americas Regional Grand Champion, and look forward to the opportunity to meet again and experience more of the fantastic iGEM community.<br />
<br />
<br />
<br />
<center><div style="width: 650px">[[File:Washington Americas Results.jpg|center|650px|]]<br />
<span style="font-size: 10pt">After the Americas Regional Jamboree we were excited to tell the rest of our teammates about our experience, as only five of us were able to travel to Indianapolis. Thanks to Joe Oh at The Daily for this team picture.</span></div></center></div>LStanleyhttp://2011.igem.org/Team:Washington/Team/AmericasTeam:Washington/Team/Americas2011-10-23T03:25:37Z<p>LStanley: </p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
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<br />
<center><span style="font-size: 28pt">'''Americas Regional Jamboree'''</span></center><br><br><br />
<br />
<center>We had a lot of fun at the Americas Regional Jamboree! It was an action packed weekend filled with great talks, posters and people, and overall lots of thought-provoking science. It was amazing to see the diversity of projects presented, as well as the immense amount of work that was completed by all teams over the course of the summer. Many thanks to iGEM and IBE for hosting the jamboree, as well as to all of the judges and coordinators who put in so many volunteer hours. We are honored to have been chosen as the Americas Regional Grand Champion, and look forward to the opportunity to meet again and experience more of the fantastic iGEM community.</center><br />
<br />
<br />
<br />
<center><div style="width: 650px">[[File:Washington Americas Results.jpg|center|650px|]]<br />
<span style="font-size: 10pt">After the Americas Regional Jamboree we were excited to tell the rest of our teammates about our experience, as only five of us were able to travel to Indianapolis. Thanks to Joe Oh at The Daily for this team picture.</span></div></center></div>LStanleyhttp://2011.igem.org/Team:Washington/Team/AmericasTeam:Washington/Team/Americas2011-10-23T03:25:03Z<p>LStanley: </p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
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<br />
<center><span style="font-size: 28pt">'''Americas Regional Jamboree'''</span></center><br><br><br />
<br />
We had a lot of fun at the Americas Regional Jamboree! It was an action packed weekend filled with great talks, posters and people, and overall lots of thought-provoking science. It was amazing to see the diversity of projects presented, as well as the immense amount of work that was completed by all teams over the course of the summer. Many thanks to iGEM and IBE for hosting the jamboree, as well as to all of the judges and coordinators who put in so many volunteer hours. We are honored to have been chosen as the Americas Regional Grand Champion, and look forward to the opportunity to meet again and experience more of the fantastic iGEM community.<br />
<br />
<br />
<br />
<center><div style="width: 650px">[[File:Washington Americas Results.jpg|center|650px|]]<br />
<span style="font-size: 11pt">After the Americas Regional Jamboree we were excited to tell the rest of our teammates about our experience, as only five of us were able to travel to Indianapolis. Thanks to Joe Oh at The Daily for this team picture.</span></div></center></div>LStanleyhttp://2011.igem.org/File:Washington_Americas_Results.jpgFile:Washington Americas Results.jpg2011-10-23T02:20:21Z<p>LStanley: </p>
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<div></div>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-29T02:03:27Z<p>LStanley: /* Testing Kumamolisin-As against SC-PEP */</p>
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<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 identifying Kumamolisin as a good candidate for activity at low pH, we tested its activity on breaking down PQLP at pH 4 against the activity of SC PEP, the enzyme currently in clinical trials for breaking down gluten. Kumamolisin had never been tested for its ability to breakdown gluten, and so we began novel experimentation into the enzyme's activity on our gluten model. From tests using the fluorescent PQLP system described in our methods section, Kumamolisin showed about 7 times better activity on breaking down PQLP at pH 4 when compared to the activity of SC PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolisin 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 screened 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 over 10-fold increase in activity from wild-type Kumamolisin!<br />
<br />
[[File:Washington Vertical Initial Screen.png|center|700px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate. *"deg" in the data labels indicates use of a degenerate primer. Data for these points is representative of a group of variants, each with different substitutions at one residue. This accounts for the <100 data points on this graph, despite testing >100 novel mutants in total.]]<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, 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|Concentration controlled rate data relative to native Kumamolisin for three of our most active mutants.]]<br />
<br />
<br />
----<br />
<br />
<br />
='''Combining Mutations for the Construction of a Gluten Hydrolase'''=<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 together to make combinatorial variants.<br />
<br />
=='''Successful mutations were combined to construct a second library for screening'''==<br />
<br />
After designing a collection of combinatorial mutants, drawing from successful mutations discovered in the first round, we again performed a rough screen to identify promising combinations of mutations. From the initial screen on our combinatorial mutants, it appeared that we had achieved around 50 times better activity than native Kumamolisin on breaking down PQLP.<br />
<br />
[[File:Washington Comb Fold Change.png|center|500px|thumb|From initial whole cell lysate screens on combinatorial mutants, it appears that about 50-fold improvement over native Kumamolisin activity on PQLP has been achieved.]]<br />
<br />
=='''One of the combinatorial mutants resulted in over a 100-fold increase in activity'''==<br />
<br />
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 />
[[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>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-29T02:03:14Z<p>LStanley: /* Testing Kumamolisin-As against SC-PEP */</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 identifying Kumamolisin as a good candidate for activity at low pH, we tested its activity on breaking down PQLP at pH 4 against the activity of SC PEP, the enzyme currently in clinical trials for breaking down gluten. Kumamolisin had never been tested for its ability to breakdown gluten, and so we began novel experimentation into the enzyme's activity on our gluten model. From tests using the fluorescent PQLP system described in our methods section, Kumamolisin showed about 7 times better activity on breaking down PQLP at pH 4 when compared to the activity of SC PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolisin 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 screened 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 over 10-fold increase in activity from wild-type Kumamolisin!<br />
<br />
[[File:Washington Vertical Initial Screen.png|center|700px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate. *"deg" in the data labels indicates use of a degenerate primer. Data for these points is representative of a group of variants, each with different substitutions at one residue. This accounts for the <100 data points on this graph, despite testing >100 novel mutants in total.]]<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, 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|Concentration controlled rate data relative to native Kumamolisin for three of our most active mutants.]]<br />
<br />
<br />
----<br />
<br />
<br />
='''Combining Mutations for the Construction of a Gluten Hydrolase'''=<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 together to make combinatorial variants.<br />
<br />
=='''Successful mutations were combined to construct a second library for screening'''==<br />
<br />
After designing a collection of combinatorial mutants, drawing from successful mutations discovered in the first round, we again performed a rough screen to identify promising combinations of mutations. From the initial screen on our combinatorial mutants, it appeared that we had achieved around 50 times better activity than native Kumamolisin on breaking down PQLP.<br />
<br />
[[File:Washington Comb Fold Change.png|center|500px|thumb|From initial whole cell lysate screens on combinatorial mutants, it appears that about 50-fold improvement over native Kumamolisin activity on PQLP has been achieved.]]<br />
<br />
=='''One of the combinatorial mutants resulted in over a 100-fold increase in activity'''==<br />
<br />
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 />
[[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>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-29T02:01:41Z<p>LStanley: /* Redesigning Kumamolisin to Have Higher Activity at Low pH */</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>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-29T02:00:41Z<p>LStanley: /* Redesigning Kumamolisin to Have Higher Activity at Low pH */</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|500px|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>LStanleyhttp://2011.igem.org/Team:Washington/Alkanes/MethodsTeam:Washington/Alkanes/Methods2011-09-29T01:56:54Z<p>LStanley: /* Introducing the PetroBrick */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Methods'''</big></big></big></big></center><br><br><br />
<br />
='''Introducing the PetroBrick'''=<br />
<br />
<p>In order to produce alkanes, we need both [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Acyl-ACP Reductase] (AAR) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 Aldehyde Decarbonylase] (ADC) to work together in the cell. To achieve this goal, we used standard cloning methods combining both to construct the [http://partsregistry.org/Part:BBa_K590025 BBa_K590025] Biobrick which contains both AAR and ADC under a high constitutive promoter, each with its own Elowitz standard RBS. This construct successfully synthesized our target product; thus, we have created a new modular alkane-producing platform:</p><br />
[[File:Washington PetroBrick Construct.png|730px|center|frameless|border|bottom|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
<br />
<br><br />
<br><br />
<br />
----<br />
<br />
='''Alkane Production & Extraction'''=<br />
[[File:Washington_Alkane_extraction.png|right|400px|thumb|Diagram showing the process of extraction.]]<p>After we had the complete gene assembly in our hands, the next step was to transform it into cells and start them growing for alkane production. We let them grow in 37 degree shaker for 48-72 hours, in sealed glass tubes. After the cells have gone through the alkane production process, the next step is to extract the alkanes out of the cell broth. We add acyl acetate directly into the glass test tube for cell growth. Then we vortex until to everything is well mixed, to make sure all of the alkanes go directly into the ethyl acetate solvent. Next, we spin down the mixture by using a centrifuge at full speed to form three layers (cell pellet, media, and ethyl acetate supernatant). We use only the ethyl acetate layer to send for GCMS analysis. For a more extensive coverage of our media, growth, and extraction techniques, refer to our [https://2011.igem.org/Team:Washington/alkanebiosynthesis Protocols page]. </p><br />
<br />
<br />
----<br />
<br />
='''Alkane Detection'''=<br />
==Gas Chromatography and Mass Spectrometry==<br />
<br />
We utilized a Gas Chromatograph / Mass Spectrometer (GCMS) to analyze alkane production concentrations. The GCMS is considered a "specific" test, because it identifies compounds specifically, not just to a category of compounds. It works by separating the individual components of a sample through a capillary column based mainly on its boiling point, similar to fractional distillation. The separated compounds generally elute from the column at different retention times, and are passed to the mass spectrometer. <br />
<br />
Inside the mass spectrometer each compound is then broken down into its individual molecular components through electron beam ionization. These ions differ in mass-to-charge (m/z) ratios, creating a unique ion de-composition profile for each compound that can be used to identify it through comparison to known chemical standards. Because compounds occasionally have similar elution times or mass spec fingerprints, the combination of analyses results in reducing the chances for overlap.<br />
<br />
<br />
<center><gallery widths="400px" heights="300px" perrow="2"><br />
Image:Washington2011_Chrom.png|'''Gas chromatography:''' A method used to separate molecules from the media extraction based on boiling point. In this image the temperature is increasing over time, and molecules with a higher boiling point are being eluted and detected by a mass spectrometer. The ion abundance is concentration dependent and can be converted if using a standard curve<br />
<br />
Image:Washington2011 Spectra.png|'''Mass Spectrometry:''' Molecules exiting gas chromatography enter an electron impact mass spectrometer. The molecules are ionized and fragmented. The resulting spectra is compared to a database of molecules in order to predict its chemical identity. On the top (red) is an experimental spectra of our biologically produced C15 alkane, on the bottom (blue) is the NIST standard spectra for a C15 alkane. The fragmentation pattern and parent ion (blue arrow, bottom right) match perfectly.<br />
<br />
</gallery></center></div>LStanleyhttp://2011.igem.org/Team:Washington/Alkanes/MethodsTeam:Washington/Alkanes/Methods2011-09-29T01:56:36Z<p>LStanley: /* Introducing the PetroBrick */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Methods'''</big></big></big></big></center><br><br><br />
<br />
='''Introducing the PetroBrick'''=<br />
<br />
<p>In order to produce alkanes, we need both [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Acyl-ACP Reductase] (AAR) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 Aldehyde Decarbonylase] (ADC) to work together in the cell. To achieve this goal, we used standard cloning methods combining both to construct the [http://partsregistry.org/Part:BBa_K590025 BBa_K590025] Biobrick which contains both AAR and ADC under a high constitutive promoter, each with its own Elowitz standard RBS. This construct successfully synthesized our target product; thus, we have created a new modular alkane-producing platform:</p><br />
<br />
<br />
[[File:Washington PetroBrick Construct.png|730px|center|frameless|border|bottom|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
<br />
<br><br />
<br><br />
<br />
----<br />
<br />
='''Alkane Production & Extraction'''=<br />
[[File:Washington_Alkane_extraction.png|right|400px|thumb|Diagram showing the process of extraction.]]<p>After we had the complete gene assembly in our hands, the next step was to transform it into cells and start them growing for alkane production. We let them grow in 37 degree shaker for 48-72 hours, in sealed glass tubes. After the cells have gone through the alkane production process, the next step is to extract the alkanes out of the cell broth. We add acyl acetate directly into the glass test tube for cell growth. Then we vortex until to everything is well mixed, to make sure all of the alkanes go directly into the ethyl acetate solvent. Next, we spin down the mixture by using a centrifuge at full speed to form three layers (cell pellet, media, and ethyl acetate supernatant). We use only the ethyl acetate layer to send for GCMS analysis. For a more extensive coverage of our media, growth, and extraction techniques, refer to our [https://2011.igem.org/Team:Washington/alkanebiosynthesis Protocols page]. </p><br />
<br />
<br />
----<br />
<br />
='''Alkane Detection'''=<br />
==Gas Chromatography and Mass Spectrometry==<br />
<br />
We utilized a Gas Chromatograph / Mass Spectrometer (GCMS) to analyze alkane production concentrations. The GCMS is considered a "specific" test, because it identifies compounds specifically, not just to a category of compounds. It works by separating the individual components of a sample through a capillary column based mainly on its boiling point, similar to fractional distillation. The separated compounds generally elute from the column at different retention times, and are passed to the mass spectrometer. <br />
<br />
Inside the mass spectrometer each compound is then broken down into its individual molecular components through electron beam ionization. These ions differ in mass-to-charge (m/z) ratios, creating a unique ion de-composition profile for each compound that can be used to identify it through comparison to known chemical standards. Because compounds occasionally have similar elution times or mass spec fingerprints, the combination of analyses results in reducing the chances for overlap.<br />
<br />
<br />
<center><gallery widths="400px" heights="300px" perrow="2"><br />
Image:Washington2011_Chrom.png|'''Gas chromatography:''' A method used to separate molecules from the media extraction based on boiling point. In this image the temperature is increasing over time, and molecules with a higher boiling point are being eluted and detected by a mass spectrometer. The ion abundance is concentration dependent and can be converted if using a standard curve<br />
<br />
Image:Washington2011 Spectra.png|'''Mass Spectrometry:''' Molecules exiting gas chromatography enter an electron impact mass spectrometer. The molecules are ionized and fragmented. The resulting spectra is compared to a database of molecules in order to predict its chemical identity. On the top (red) is an experimental spectra of our biologically produced C15 alkane, on the bottom (blue) is the NIST standard spectra for a C15 alkane. The fragmentation pattern and parent ion (blue arrow, bottom right) match perfectly.<br />
<br />
</gallery></center></div>LStanleyhttp://2011.igem.org/File:Washington_PetroBrick_Construct.pngFile:Washington PetroBrick Construct.png2011-09-29T01:55:29Z<p>LStanley: uploaded a new version of &quot;File:Washington PetroBrick Construct.png&quot;</p>
<hr />
<div></div>LStanleyhttp://2011.igem.org/Team:Washington/Alkanes/MethodsTeam:Washington/Alkanes/Methods2011-09-29T01:52:07Z<p>LStanley: /* Introducing the PetroBrick */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Methods'''</big></big></big></big></center><br><br><br />
<br />
='''Introducing the PetroBrick'''=<br />
<br />
<p>In order to produce alkanes, we need both [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Acyl-ACP Reductase] (AAR) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 Aldehyde Decarbonylase] (ADC) to work together in the cell. To achieve this goal, we used standard cloning methods combining both to construct the [http://partsregistry.org/Part:BBa_K590025 BBa_K590025] Biobrick which contains both AAR and ADC under a high constitutive promoter, each with its own Elowitz standard RBS. This construct successfully synthesized our target product; thus, we have created a new modular alkane-producing platform:</p><br />
<br />
<br><br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;'''the <font size="5" weight=bold>PetroBrick.'''</font><br />
<br />
<br />
[[File:Washington PetroBrick Construct.png|730px|center|frameless|border|bottom|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
<br />
<br><br />
<br><br />
<br />
----<br />
<br />
='''Alkane Production & Extraction'''=<br />
[[File:Washington_Alkane_extraction.png|right|400px|thumb|Diagram showing the process of extraction.]]<p>After we had the complete gene assembly in our hands, the next step was to transform it into cells and start them growing for alkane production. We let them grow in 37 degree shaker for 48-72 hours, in sealed glass tubes. After the cells have gone through the alkane production process, the next step is to extract the alkanes out of the cell broth. We add acyl acetate directly into the glass test tube for cell growth. Then we vortex until to everything is well mixed, to make sure all of the alkanes go directly into the ethyl acetate solvent. Next, we spin down the mixture by using a centrifuge at full speed to form three layers (cell pellet, media, and ethyl acetate supernatant). We use only the ethyl acetate layer to send for GCMS analysis. For a more extensive coverage of our media, growth, and extraction techniques, refer to our [https://2011.igem.org/Team:Washington/alkanebiosynthesis Protocols page]. </p><br />
<br />
<br />
----<br />
<br />
='''Alkane Detection'''=<br />
==Gas Chromatography and Mass Spectrometry==<br />
<br />
We utilized a Gas Chromatograph / Mass Spectrometer (GCMS) to analyze alkane production concentrations. The GCMS is considered a "specific" test, because it identifies compounds specifically, not just to a category of compounds. It works by separating the individual components of a sample through a capillary column based mainly on its boiling point, similar to fractional distillation. The separated compounds generally elute from the column at different retention times, and are passed to the mass spectrometer. <br />
<br />
Inside the mass spectrometer each compound is then broken down into its individual molecular components through electron beam ionization. These ions differ in mass-to-charge (m/z) ratios, creating a unique ion de-composition profile for each compound that can be used to identify it through comparison to known chemical standards. Because compounds occasionally have similar elution times or mass spec fingerprints, the combination of analyses results in reducing the chances for overlap.<br />
<br />
<br />
<center><gallery widths="400px" heights="300px" perrow="2"><br />
Image:Washington2011_Chrom.png|'''Gas chromatography:''' A method used to separate molecules from the media extraction based on boiling point. In this image the temperature is increasing over time, and molecules with a higher boiling point are being eluted and detected by a mass spectrometer. The ion abundance is concentration dependent and can be converted if using a standard curve<br />
<br />
Image:Washington2011 Spectra.png|'''Mass Spectrometry:''' Molecules exiting gas chromatography enter an electron impact mass spectrometer. The molecules are ionized and fragmented. The resulting spectra is compared to a database of molecules in order to predict its chemical identity. On the top (red) is an experimental spectra of our biologically produced C15 alkane, on the bottom (blue) is the NIST standard spectra for a C15 alkane. The fragmentation pattern and parent ion (blue arrow, bottom right) match perfectly.<br />
<br />
</gallery></center></div>LStanleyhttp://2011.igem.org/Team:Washington/Alkanes/MethodsTeam:Washington/Alkanes/Methods2011-09-29T01:51:00Z<p>LStanley: /* Introducing the PetroBrick */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Methods'''</big></big></big></big></center><br><br><br />
<br />
='''Introducing the PetroBrick'''=<br />
<br />
<p>In order to produce alkanes, we need both [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Acyl-ACP Reductase] (AAR) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 Aldehyde Decarbonylase] (ADC) to work together in the cell. To achieve this goal, we used standard cloning methods combining both to construct the [http://partsregistry.org/Part:BBa_K590025 BBa_K590025] Biobrick which contains both AAR and ADC under a high constitutive promoter, each with its own Elowitz standard RBS. This construct successfully synthesized our target product; thus, we have created a new modular alkane-producing platform:</p><br />
<br />
<br><br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;'''the <font size="5" weight=bold>PetroBrick.'''</font><br />
<br />
<br><br />
<br />
[[File:Washington PetroBrick Construct.png|730px|center|frameless|border|bottom|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
<br />
<br><br />
<br><br />
<br />
----<br />
<br />
='''Alkane Production & Extraction'''=<br />
[[File:Washington_Alkane_extraction.png|right|400px|thumb|Diagram showing the process of extraction.]]<p>After we had the complete gene assembly in our hands, the next step was to transform it into cells and start them growing for alkane production. We let them grow in 37 degree shaker for 48-72 hours, in sealed glass tubes. After the cells have gone through the alkane production process, the next step is to extract the alkanes out of the cell broth. We add acyl acetate directly into the glass test tube for cell growth. Then we vortex until to everything is well mixed, to make sure all of the alkanes go directly into the ethyl acetate solvent. Next, we spin down the mixture by using a centrifuge at full speed to form three layers (cell pellet, media, and ethyl acetate supernatant). We use only the ethyl acetate layer to send for GCMS analysis. For a more extensive coverage of our media, growth, and extraction techniques, refer to our [https://2011.igem.org/Team:Washington/alkanebiosynthesis Protocols page]. </p><br />
<br />
<br />
----<br />
<br />
='''Alkane Detection'''=<br />
==Gas Chromatography and Mass Spectrometry==<br />
<br />
We utilized a Gas Chromatograph / Mass Spectrometer (GCMS) to analyze alkane production concentrations. The GCMS is considered a "specific" test, because it identifies compounds specifically, not just to a category of compounds. It works by separating the individual components of a sample through a capillary column based mainly on its boiling point, similar to fractional distillation. The separated compounds generally elute from the column at different retention times, and are passed to the mass spectrometer. <br />
<br />
Inside the mass spectrometer each compound is then broken down into its individual molecular components through electron beam ionization. These ions differ in mass-to-charge (m/z) ratios, creating a unique ion de-composition profile for each compound that can be used to identify it through comparison to known chemical standards. Because compounds occasionally have similar elution times or mass spec fingerprints, the combination of analyses results in reducing the chances for overlap.<br />
<br />
<br />
<center><gallery widths="400px" heights="300px" perrow="2"><br />
Image:Washington2011_Chrom.png|'''Gas chromatography:''' A method used to separate molecules from the media extraction based on boiling point. In this image the temperature is increasing over time, and molecules with a higher boiling point are being eluted and detected by a mass spectrometer. The ion abundance is concentration dependent and can be converted if using a standard curve<br />
<br />
Image:Washington2011 Spectra.png|'''Mass Spectrometry:''' Molecules exiting gas chromatography enter an electron impact mass spectrometer. The molecules are ionized and fragmented. The resulting spectra is compared to a database of molecules in order to predict its chemical identity. On the top (red) is an experimental spectra of our biologically produced C15 alkane, on the bottom (blue) is the NIST standard spectra for a C15 alkane. The fragmentation pattern and parent ion (blue arrow, bottom right) match perfectly.<br />
<br />
</gallery></center></div>LStanleyhttp://2011.igem.org/Team:Washington/Alkanes/MethodsTeam:Washington/Alkanes/Methods2011-09-29T01:49:01Z<p>LStanley: /* Introducing the PetroBrick */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Methods'''</big></big></big></big></center><br><br><br />
<br />
='''Introducing the PetroBrick'''=<br />
[[File:Washington PetroBrick.png|220px|frameless|border="2"|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025|left]]<br />
<br />
<br />
:::::::::<p>In order to produce alkanes, we need both [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Acyl-ACP Reductase] (AAR) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 Aldehyde Decarbonylase] (ADC) to work together in the cell. To achieve this goal, we used standard cloning methods combining both to construct the [http://partsregistry.org/Part:BBa_K590025 BBa_K590025] Biobrick which contains both AAR and ADC under a high constitutive promoter, each with its own Elowitz standard RBS. This construct successfully synthesized our target product; thus, we have created a new modular alkane-producing platform:</p><br />
<br />
<br><br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;'''the <font size="5" weight=bold>PetroBrick.'''</font><br />
<br />
<br><br />
<br />
[[Image:Washington2011_PetroBrick_Construct.png|730px|frameless|border|bottom|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
[[File:Washington PetroBrick Construct.png|730px|frameless|border|bottom|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
<br />
<br><br />
<br><br />
<br />
----<br />
<br />
='''Alkane Production & Extraction'''=<br />
[[File:Washington_Alkane_extraction.png|right|400px|thumb|Diagram showing the process of extraction.]]<p>After we had the complete gene assembly in our hands, the next step was to transform it into cells and start them growing for alkane production. We let them grow in 37 degree shaker for 48-72 hours, in sealed glass tubes. After the cells have gone through the alkane production process, the next step is to extract the alkanes out of the cell broth. We add acyl acetate directly into the glass test tube for cell growth. Then we vortex until to everything is well mixed, to make sure all of the alkanes go directly into the ethyl acetate solvent. Next, we spin down the mixture by using a centrifuge at full speed to form three layers (cell pellet, media, and ethyl acetate supernatant). We use only the ethyl acetate layer to send for GCMS analysis. For a more extensive coverage of our media, growth, and extraction techniques, refer to our [https://2011.igem.org/Team:Washington/alkanebiosynthesis Protocols page]. </p><br />
<br />
<br />
----<br />
<br />
='''Alkane Detection'''=<br />
==Gas Chromatography and Mass Spectrometry==<br />
<br />
We utilized a Gas Chromatograph / Mass Spectrometer (GCMS) to analyze alkane production concentrations. The GCMS is considered a "specific" test, because it identifies compounds specifically, not just to a category of compounds. It works by separating the individual components of a sample through a capillary column based mainly on its boiling point, similar to fractional distillation. The separated compounds generally elute from the column at different retention times, and are passed to the mass spectrometer. <br />
<br />
Inside the mass spectrometer each compound is then broken down into its individual molecular components through electron beam ionization. These ions differ in mass-to-charge (m/z) ratios, creating a unique ion de-composition profile for each compound that can be used to identify it through comparison to known chemical standards. Because compounds occasionally have similar elution times or mass spec fingerprints, the combination of analyses results in reducing the chances for overlap.<br />
<br />
<br />
<center><gallery widths="400px" heights="300px" perrow="2"><br />
Image:Washington2011_Chrom.png|'''Gas chromatography:''' A method used to separate molecules from the media extraction based on boiling point. In this image the temperature is increasing over time, and molecules with a higher boiling point are being eluted and detected by a mass spectrometer. The ion abundance is concentration dependent and can be converted if using a standard curve<br />
<br />
Image:Washington2011 Spectra.png|'''Mass Spectrometry:''' Molecules exiting gas chromatography enter an electron impact mass spectrometer. The molecules are ionized and fragmented. The resulting spectra is compared to a database of molecules in order to predict its chemical identity. On the top (red) is an experimental spectra of our biologically produced C15 alkane, on the bottom (blue) is the NIST standard spectra for a C15 alkane. The fragmentation pattern and parent ion (blue arrow, bottom right) match perfectly.<br />
<br />
</gallery></center></div>LStanleyhttp://2011.igem.org/File:Washington_PetroBrick_Construct.pngFile:Washington PetroBrick Construct.png2011-09-29T01:45:56Z<p>LStanley: </p>
<hr />
<div></div>LStanleyhttp://2011.igem.org/Team:WashingtonTeam:Washington2011-09-29T01:28:30Z<p>LStanley: </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 />
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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 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 />
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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 />
[[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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[[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>LStanleyhttp://2011.igem.org/Team:Washington/Alkanes/ResultsTeam:Washington/Alkanes/Results2011-09-29T00:30:13Z<p>LStanley: /* GCMS confirms The PetroBrick enables diesel production from sugar using E. coli */</p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
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<br />
<center><big><big><big><big>'''Diesel Production: Results Summary'''</big></big></big></big></center><br><br><br />
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='''GCMS confirms The PetroBrick enables diesel production from sugar using ''E. coli'''''=<br />
<br />
We preformed GC-MS analysis on extracts from cell cultures expressing both [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Acyl-ACP Reductase] (AAR) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 Aldehyde Decarbonylase] (ADC) (this composite part is designated [http://partsregistry.org/Part:BBa_K590025 The PetroBrick]). 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 />
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[[File:Washington 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 />
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'''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 />
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'''The PetroBrick Works! C13, C15, and C17 alkanes are produced when both AAR and ADC are expressed.''' <br />
[[File:Washington2011 Spectra.png|right|550px|thumb|Comparasion of the NIST reference C15 alkane spectrum( in blue) to the C15 peak spectrum taken from a PetroBrick culture (in red). Note the strong m/z peak at 212, corresponding to the parent ion of the C15 alkane. For spectra of the C13 alkane and C17 alkene, refer to our [https://2011.igem.org/Team:Washington/Alkanes/spectra Spectra Page.]]]<br />
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 C14 alcohol and the C17 alkene. The fact that the C17 alkene peak overlaps with the C14 alcohol peak makes exact 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 earlier section of the peak has an MS spectra consistent with the C16 alcohol, and the later 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(for these spectra, as well as the C17 alkene spectrum, refer to our [https://2011.igem.org/Team:Washington/Alkanes/spectra Spectra Page.]). 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. Using the PetroBrick, we can turn simple sugars into diesel, a fuel fully compatible with modern infrastructure.<br />
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='''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 yield. To determine how much alkane was being produced by the Petrobrick, we grew up 3 MG1655 cell cultures transformed with the PetroBrick in M9 production media ([https://2011.igem.org/Team:Washington/alkanebiosynthesis link]), and analyzed using GC-MS.<br />
<br />
[[File:Washington_alkanestandardcurve.png|right|450px|thumb|Standard curves for converting peak area to an absolute amount. Note that these curves is almost perfectly linear. In addition, the curve generated from each alkane is nearly identical, allowing us to use 1, average curve for all 3 different alkanes.]]<br />
[[File:Washinton 2011 Pre-Optimization Quant.png|left|450px|thumb|Diagram showing yields of the C13 and C15 alkanes. Note: The C17 alkene is not included due to inability to quantify.]]<br />
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='''Optimized Production'''=<br />
By optimizing the method of growth, media, vectors, and cell line we were able to successfully increase alkane yield over 80-fold. Note that this improvement doesn't include C17 alkene production, as we were unable to quantify the C17 alkene peak due to co-elution with the C14 alcohol. The parameters 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|Our optimized growth conditions resulted in an 80 fold increase in total alkane yield.]]</div>LStanleyhttp://2011.igem.org/Team:Washington/Alkanes/MethodsTeam:Washington/Alkanes/Methods2011-09-29T00:24:49Z<p>LStanley: /* Introducing the PetroBrick */</p>
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__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Methods'''</big></big></big></big></center><br><br><br />
<br />
='''Introducing the PetroBrick'''=<br />
[[File:Washington PetroBrick.png|220px|frameless|border="2"|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025|left]]<br />
<br />
<br />
:::::::::<p>In order to produce alkanes, we need both [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Acyl-ACP Reductase] (AAR) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 Aldehyde Decarbonylase] (ADC) to work together in the cell. In order to achieve this goal, we used standard cloning methods combining both to construct the [http://partsregistry.org/Part:BBa_K590025 BBa_K590025] Biobrick that contained both AAR and ADC under a high constitutive promoter, each with its own Elowitz standard RBS. This construct successfully synthesized our target product, and thus we have created a new modular alkane-producing platform:</p><br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;'''the <font size="5" weight=bold>PetroBrick.'''</font><br />
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[[Image:Washington2011_PetroBrick_Construct.png|730px|frameless|border|bottom|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
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<br />
='''Alkane Production & Extraction'''=<br />
[[File:Washington_Alkane_extraction.png|right|400px|thumb|Diagram showing the process of extraction.]]<p>After we had the complete gene assembly in our hands, the next step was to transform it into cells and start them growing for alkane production. We let them grow in 37 degree shaker for 48-72 hours, in sealed glass tubes. After the cells have gone through the alkane production process, the next step is to extract the alkanes out of the cell broth. We add acyl acetate directly into the glass test tube for cell growth. Then we vortex until to everything is well mixed, to make sure all of the alkanes go directly into the ethyl acetate solvent. Next, we spin down the mixture by using a centrifuge at full speed to form three layers (cell pellet, media, and ethyl acetate supernatant). We use only the ethyl acetate layer to send for GCMS analysis. For a more extensive coverage of our media, growth, and extraction techniques, refer to our [https://2011.igem.org/Team:Washington/alkanebiosynthesis Protocol page]. </p><br />
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<br />
='''Alkane Detection'''=<br />
==Gas Chromatography and Mass Spectrometry==<br />
<br />
We utilized a Gas Chromatograph / Mass Spectrometer (GCMS) to analyze alkane production concentrations. The GCMS is considered a "specific" test, because it identifies compounds specifically, not just to a category of compounds. It works by separating the individual components of a sample through a capillary column based mainly on its boiling point, similar to fractional distillation. The separated compounds generally elute from the column at different retention times, and are passed to the mass spectrometer. <br />
<br />
Inside the mass spectrometer each compound is then broken down into its individual molecular components through electron beam ionization. These ions differ in mass-to-charge (m/z) ratios, creating a unique ion de-composition profile for each compound that can be used to identify it through comparison to known chemical standards. Because compounds occasionally have similar elution times or mass spec fingerprints, the combination of analyses results in reducing the chances for overlap.<br />
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<center><gallery widths="400px" heights="300px" perrow="2"><br />
Image:Washington2011_Chrom.png|'''Gas chromatography:''' A method used to separate molecules from the media extraction based on boiling point. In this image the temperature is increasing over time, and molecules with a higher boiling point are being eluted and detected by a mass spectrometer. The ion abundance is concentration dependent and can be converted if using a standard curve<br />
<br />
Image:Washington2011 Spectra.png|'''Mass Spectrometry:''' Molecules exiting gas chromatography enter an electron impact mass spectrometer. The molecules are ionized and fragmented. The resulting spectra is compared to a database of molecules in order to predict its chemical identity. On the top (red) is an experimental spectra of our biologically produced C15 alkane, on the bottom (blue) is the NIST standard spectra for a C15 alkane. The fragmentation pattern and parent ion (blue arrow, bottom right) match perfectly.<br />
<br />
</gallery></center></div>LStanleyhttp://2011.igem.org/File:Washington2011_AlkaneAndBackCycle.pngFile:Washington2011 AlkaneAndBackCycle.png2011-09-29T00:23:33Z<p>LStanley: uploaded a new version of &quot;File:Washington2011 AlkaneAndBackCycle.png&quot;</p>
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<div></div>LStanleyhttp://2011.igem.org/Team:Washington/Alkanes/BackgroundTeam:Washington/Alkanes/Background2011-09-29T00:16:02Z<p>LStanley: /* The PetroBrick: A modular and open platform for the biological production of diesel fuel */</p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
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<center><big><big><big><big>'''Diesel Production: Background'''</big></big></big></big></center><br><br><br />
<br />
='''Petroleum, an Unfortunate Necessity'''=<br />
[[Image:Washington2011_PetroUsage.png|border|450px|right|thumb|Petroleum may be a necessity for modern civilization, but current extraction methods are unsustainable and non-cyclical in the long run.]]<br />
<br />
Modern society is completely dependent on petroleum based fuels. Automobiles are slowly transitioning towards electric power. However, for the foreseeable future, batteries will not be able to hold the energy needed for applications that require long range (e.g. jet planes, maritime shipping, and long range trucking) or high horsepower (e.g. agriculture, construction, industry). Without the use of petroleum, society as we know it would crumble. Petroleum is not a viable long term fuel due because it a non-renewable resource. When petroleum based fuels are combusted, CO<sub>2</sub> is released into the atmosphere. Using current technology, it is impossible to turn this carbon dioxide back into fuel, meaning that the amount of petroleum based fuel is a finite commodity. In addition, this excess carbon dioxide is a potent greenhouse gas that contributes to global warming.<br />
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<br />
='''Todays biofuels are ''renewable'', but do not work as "drop-in" replacements'''=<br />
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<br />
[[Image:Washington2011_BiofuelsAreRenewable.png|right|450px|frameless|thumb|Since carbon in biofuels can be derived from photosynthesis, biofuels are renewable. However, current biofuels have clear and problematic limits concerning energy production.]]<br />
<br />
Many different attempts have been made to produce a renewable, biologically derived fuel that would alleviate both the limited supply and emissions issues presented by petroleum based fuels. These efforts include alcohols (ethanol, butanol and other, higher alcohols), and biodiesel . Like petroleum based fuels, biofuels consist of combustible molecules that emit carbon dioxide. However, unlike petroleum based fuels, biofuels are renewable. CO<sub>2</sub> can be converted into more biofuel by feeding biofuel producing microbes (bacteria, yeast) photosynthetically derived plant biomass. Since the amount of CO<sub>2</sub> produced by burning a biofuel cannot exceed the CO<sub>2</sub> incorporated into plant biomass, a biofuel can be used indefinitely without any net carbon emissions.<br />
However, current biofuels consist of drastically different compounds from those found in petroleum. Petroleum consists of mostly long-chain length alkanes consisting of long hydrocarbon chains. Current biofuels contain either alcohols or long chain esters (biodiesel). Both of these molecules contain oxygen, which dramatically changes chemical properties. Both alcohols and biodiesel are more corrosive than unreactive alkanes. Alcohols are highly corrosive, both in pipelines ( [[#References | [1]]] ), and in engines not designed for the use of alcohols, even at concentrations as low as 20%( [[#References | [2]]] ). The corrosive property of alcohols in pipelines means that ethanol (the main alcohol in widespread use) is transported in vehicles (mostly by train), as opposed to by cheaper and less energy intensive pipelines( [[#References | [1]]] ). Transport of alcohols by pipeline would require retrofitting the entire fuel distribution infrastructure. The Fatty Acid Methyl Esters(FAMEs) in biodiesel are not directly as corrosive as alcohols, but can be biodegraded by anaerobic bacteria, producing hydrogen sulfide and other acids( [[#References | [3]]] ). Biodiesel has a higher freezing point than diesel, causing engine fuel filer clogging at low temperatures( [[#References | [4]]] ). Ethanol suffers from a much lower energy density than diesel(21.27-23.56 MJ/L vs 32.36-34.66 MJ/L)( [[#References | [5]]] ), resulting in lower gas mileage. The table below shows selected chemical properties of diesel, as well as the common biofuels ethanol, butanol, and biodiesel( data from [[#References | [5,6,9-12]]]). <br />
{| {{table}}<br />
| align="center" style="background:#f0f0f0;"|'''Property'''<br />
| align="center" style="background:#f0f0f0;"|'''Diesel'''<br />
| align="center" style="background:#f0f0f0;"|'''Ethanol'''<br />
| align="center" style="background:#f0f0f0;"|'''Biodiesel'''<br />
| align="center" style="background:#f0f0f0;"|'''n-Butanol'''<br />
|-<br />
| Specific gravity @ 15.5°C||0.85||0.794||0.88||.81(@20°C)<br />
|-<br />
| Density @ 15.5°C(g/L)||848.25||792.05||878.09||808.8(@20°C)<br />
|-<br />
| Energy Density(MJ/L)||32.36-34.66 ||21.27-23.56 ||33.32-35.66||27<br />
|-<br />
| Cetane number||40-55||0-54||48-65||17<br />
|-<br />
| Freezing point(°C)||-40 - -1||-114||-3 -19||-89.3<br />
|-<br />
| Viscosity( @ 20°C)(mm<sup>2</sup>/s)||2.8-5.0||1.5||6.4-6.6<br />
|-<br />
| Flash point(°C)||60-80||12.8||100-170||35<br />
|-<br />
| <br />
|} <br />
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='''The Ideal Fuel is Microbially-Produced Diesel'''=<br />
<br />
[[Image:Washington2011_PetroBiofuel.png|frameless|left|400px]]<br />
:::::::::::::::::<p>The ideal fuel would be compatible with modern engines and infrastructure, and also be able to be produced in a renewable manner. No current biofuel has the same identical enough to that of diesel to be able to fully integrate with current engines and infrastructure. No known alternative fuel is able to match the chemical properties of diesel. Currently, the only way to renewably produce a fuel with the chemical properties and compatibility of diesel would be to make a biofuel with a composition identical to that of diesel. This would require a biological pathway that is able to produce alkanes, the main class of compounds in diesel. Alkanes are simple chains of carbon and hydrogen. The majority of the alkanes found in diesel have a carbon chain of 10 to 20 carbons long. Alkanes make up approximately 62% of jet diesel (a fairly representative diesel fuel)([[#References | [7]]]). This 62% includes 34% straight chain alkanes that contain only one linear chain, and 28% branched chain alkanes that contain 1 or more carbon branches. The remaining 38% consists mostly of cyclic and aromatic hydrocarbons. If long (10+) chain length alkanes could be biologically produced, it would allow for the production of a fuel that is both renewable and fully compatible with current engines and infrastructure.</p><br />
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='''The Solution: a Microbial Alkane Production Pathway'''=<br />
[[Image:Washington2011_AlkaneAndBackCycle.png|right|400px|frameless|thumb|The use of AAR and ADC converts the fatty acid intermediate Acl-ACP into an aldehyde, then an alkane--biofuel. Acyl-ACP is naturally produced by all organisms, which increases host choice considerably]]<br />
A recent study([[#References | [8]]]),has shown the production of long chain length alkanes in ''E. coli'' using two genes found in many cyanobacteria species. The first gene codes for [http://partsregistry.org/Part:BBa_K590032 Acyl-ACP Reductase] (AAR) which reduces long chain length acyl-ACPs into the corresponding fatty aldehydes. Acyl-ACPs are essential intermediates in fatty acid biosynthesis in every known organism, meaning that this system can work in a wide range of organisms. This long chain fatty acid acts as a substrate for [http://partsregistry.org/Part:BBa_K590031 Aldehyde Decarbonylase] (ADC), the enzyme that removes the carbonyl group (C=O) from the fatty aldehyde, yielding an alkane one carbon shorter than the original Acyl-ACP and a molecule of formate. Since the vast majority of the fatty acyl-ACPs produced by ''E. coli'' have an even chain length, this system produces detectable amounts of only odd chain length alkanes. This study reported production of the C13, C15, and C17 alkanes, as well as the C17 alkene (unsaturated hydrocarbon). This chain length range falls well within the range of those found in diesel, so this system is theoretically able to make the alkane portion of a fuel compatible with current engines and infrastructure.<br />
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='''The PetroBrick: A modular and open platform for the biological production of diesel fuel'''=<br />
<br />
[[File:Washington PetroBrick.png|200px|frameless|border|left|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
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The goal of our "'''''Make It: Diesel Production'''''" portion of this summer's iGEM project is to turn convert this recently discovered set of enzymes ([http://partsregistry.org/Part:BBa_K590032 AAR] and [http://partsregistry.org/Part:BBa_K590031 ADC]) for microbial alkane production into an open and modular platform for iGEM teams to develop into a robust replacement for petrochemical fuels. Our alkane production system is specifically designed to be easily improved upon, and we have started work on improving this open system, both by increasing alkane yields and by changing the product produced. In addition, we have started to move this system into an alternative chassis, yeast.<br />
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<br />
==References==<br />
1. http://ourenergypolicy.org/docs/2/biofuels-taskforce.pdf<br />
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2. http://www.environment.gov.au/atmosphere/fuelquality/publications/2000hours-vehicle-fleet/pubs/2000-hours-vehicles.pdf<br />
<br />
3. Anaerobic Metabolism of Biodiesel and Its Impact on Metal Corrosion<br />
Deniz F. Aktas, Jason S. Lee, Brenda J. Little, Richard I. Ray, Irene A. Davidova, Christopher N. Lyles, Joseph M. Suflita Energy & Fuels 2010 24 (5), 2924-2928(http://pubs.acs.org/doi/full/10.1021/ef100084j)<br />
<br />
4. http://www.mda.state.mn.us/news/publications/renewable/biodiesel/biodieselcoldissues.pdf<br />
<br />
5. http://www.afdc.energy.gov/afdc/pdfs/fueltable.pdf<br />
<br />
6. The viscosities of three biodiesel fuels at temperatures up to 300°C<br />
R.E. Tate, K.C. Watts, C.A.W. Allen K.I. Wilkie Fuel 2006 85, 1010-1015 (https://netfiles.uiuc.edu/mccrady/shared/Biodiesel/The%20viscosities%20of%20three%20biodiesel%20fuels%20at%20temperatures%20up%20to%20300%208C.pdf)<br />
<br />
7. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
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8. Microbial Biosynthesis of Alkanes Andreas Schirmer, Mathew A. Rude, Xuezhi Li, Emanuela Popova and Stephen B. del Cardayre Science 30 July 2010: Vol. 329 no. 5991 pp. 559-562 http://www.sciencemag.org/content/329/5991/559<br />
<br />
9.http://msdssearch.dow.com/PublishedLiteratureDOWCOM/dh_0119/0901b803801195a5.pdf?filepath=oxysolvents/pdfs/noreg/327-00014.pdf&fromPage=GetDoc<br />
<br />
10.Metabolic engineering of Escherichia coli for 1-butanol production Shota Atsumi, Anthony F. Cann, Michael R. Connor, Claire R. Shen, Kevin M. Smith, Mark P. Brynildsen, Katherine J.Y. Chou, Taizo Hanai1, James C. Liao Metabolic Engineering Volume 10, Issue 6, November 2008, Pages 305-311<br />
<br />
11. http://www.nrel.gov/vehiclesandfuels/pdfs/sr368051.pdf<br />
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12. http://www.arpltd.com/n-butano.htm</div>LStanleyhttp://2011.igem.org/Team:Washington/Alkanes/BackgroundTeam:Washington/Alkanes/Background2011-09-29T00:15:10Z<p>LStanley: /* The PetroBrick: A modular and open platform for the biological production of diesel fuel */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Background'''</big></big></big></big></center><br><br><br />
<br />
='''Petroleum, an Unfortunate Necessity'''=<br />
[[Image:Washington2011_PetroUsage.png|border|450px|right|thumb|Petroleum may be a necessity for modern civilization, but current extraction methods are unsustainable and non-cyclical in the long run.]]<br />
<br />
Modern society is completely dependent on petroleum based fuels. Automobiles are slowly transitioning towards electric power. However, for the foreseeable future, batteries will not be able to hold the energy needed for applications that require long range (e.g. jet planes, maritime shipping, and long range trucking) or high horsepower (e.g. agriculture, construction, industry). Without the use of petroleum, society as we know it would crumble. Petroleum is not a viable long term fuel due because it a non-renewable resource. When petroleum based fuels are combusted, CO<sub>2</sub> is released into the atmosphere. Using current technology, it is impossible to turn this carbon dioxide back into fuel, meaning that the amount of petroleum based fuel is a finite commodity. In addition, this excess carbon dioxide is a potent greenhouse gas that contributes to global warming.<br />
<br />
<br />
<br />
<br />
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----<br />
<br />
='''Todays biofuels are ''renewable'', but do not work as "drop-in" replacements'''=<br />
<br />
<br />
[[Image:Washington2011_BiofuelsAreRenewable.png|right|450px|frameless|thumb|Since carbon in biofuels can be derived from photosynthesis, biofuels are renewable. However, current biofuels have clear and problematic limits concerning energy production.]]<br />
<br />
Many different attempts have been made to produce a renewable, biologically derived fuel that would alleviate both the limited supply and emissions issues presented by petroleum based fuels. These efforts include alcohols (ethanol, butanol and other, higher alcohols), and biodiesel . Like petroleum based fuels, biofuels consist of combustible molecules that emit carbon dioxide. However, unlike petroleum based fuels, biofuels are renewable. CO<sub>2</sub> can be converted into more biofuel by feeding biofuel producing microbes (bacteria, yeast) photosynthetically derived plant biomass. Since the amount of CO<sub>2</sub> produced by burning a biofuel cannot exceed the CO<sub>2</sub> incorporated into plant biomass, a biofuel can be used indefinitely without any net carbon emissions.<br />
However, current biofuels consist of drastically different compounds from those found in petroleum. Petroleum consists of mostly long-chain length alkanes consisting of long hydrocarbon chains. Current biofuels contain either alcohols or long chain esters (biodiesel). Both of these molecules contain oxygen, which dramatically changes chemical properties. Both alcohols and biodiesel are more corrosive than unreactive alkanes. Alcohols are highly corrosive, both in pipelines ( [[#References | [1]]] ), and in engines not designed for the use of alcohols, even at concentrations as low as 20%( [[#References | [2]]] ). The corrosive property of alcohols in pipelines means that ethanol (the main alcohol in widespread use) is transported in vehicles (mostly by train), as opposed to by cheaper and less energy intensive pipelines( [[#References | [1]]] ). Transport of alcohols by pipeline would require retrofitting the entire fuel distribution infrastructure. The Fatty Acid Methyl Esters(FAMEs) in biodiesel are not directly as corrosive as alcohols, but can be biodegraded by anaerobic bacteria, producing hydrogen sulfide and other acids( [[#References | [3]]] ). Biodiesel has a higher freezing point than diesel, causing engine fuel filer clogging at low temperatures( [[#References | [4]]] ). Ethanol suffers from a much lower energy density than diesel(21.27-23.56 MJ/L vs 32.36-34.66 MJ/L)( [[#References | [5]]] ), resulting in lower gas mileage. The table below shows selected chemical properties of diesel, as well as the common biofuels ethanol, butanol, and biodiesel( data from [[#References | [5,6,9-12]]]). <br />
{| {{table}}<br />
| align="center" style="background:#f0f0f0;"|'''Property'''<br />
| align="center" style="background:#f0f0f0;"|'''Diesel'''<br />
| align="center" style="background:#f0f0f0;"|'''Ethanol'''<br />
| align="center" style="background:#f0f0f0;"|'''Biodiesel'''<br />
| align="center" style="background:#f0f0f0;"|'''n-Butanol'''<br />
|-<br />
| Specific gravity @ 15.5°C||0.85||0.794||0.88||.81(@20°C)<br />
|-<br />
| Density @ 15.5°C(g/L)||848.25||792.05||878.09||808.8(@20°C)<br />
|-<br />
| Energy Density(MJ/L)||32.36-34.66 ||21.27-23.56 ||33.32-35.66||27<br />
|-<br />
| Cetane number||40-55||0-54||48-65||17<br />
|-<br />
| Freezing point(°C)||-40 - -1||-114||-3 -19||-89.3<br />
|-<br />
| Viscosity( @ 20°C)(mm<sup>2</sup>/s)||2.8-5.0||1.5||6.4-6.6<br />
|-<br />
| Flash point(°C)||60-80||12.8||100-170||35<br />
|-<br />
| <br />
|} <br />
<br />
<br />
----<br />
<br />
='''The Ideal Fuel is Microbially-Produced Diesel'''=<br />
<br />
[[Image:Washington2011_PetroBiofuel.png|frameless|left|400px]]<br />
:::::::::::::::::<p>The ideal fuel would be compatible with modern engines and infrastructure, and also be able to be produced in a renewable manner. No current biofuel has the same identical enough to that of diesel to be able to fully integrate with current engines and infrastructure. No known alternative fuel is able to match the chemical properties of diesel. Currently, the only way to renewably produce a fuel with the chemical properties and compatibility of diesel would be to make a biofuel with a composition identical to that of diesel. This would require a biological pathway that is able to produce alkanes, the main class of compounds in diesel. Alkanes are simple chains of carbon and hydrogen. The majority of the alkanes found in diesel have a carbon chain of 10 to 20 carbons long. Alkanes make up approximately 62% of jet diesel (a fairly representative diesel fuel)([[#References | [7]]]). This 62% includes 34% straight chain alkanes that contain only one linear chain, and 28% branched chain alkanes that contain 1 or more carbon branches. The remaining 38% consists mostly of cyclic and aromatic hydrocarbons. If long (10+) chain length alkanes could be biologically produced, it would allow for the production of a fuel that is both renewable and fully compatible with current engines and infrastructure.</p><br />
<br />
<br />
----<br />
<br />
='''The Solution: a Microbial Alkane Production Pathway'''=<br />
[[Image:Washington2011_AlkaneAndBackCycle.png|right|400px|frameless|thumb|The use of AAR and ADC converts the fatty acid intermediate Acl-ACP into an aldehyde, then an alkane--biofuel. Acyl-ACP is naturally produced by all organisms, which increases host choice considerably]]<br />
A recent study([[#References | [8]]]),has shown the production of long chain length alkanes in ''E. coli'' using two genes found in many cyanobacteria species. The first gene codes for [http://partsregistry.org/Part:BBa_K590032 Acyl-ACP Reductase] (AAR) which reduces long chain length acyl-ACPs into the corresponding fatty aldehydes. Acyl-ACPs are essential intermediates in fatty acid biosynthesis in every known organism, meaning that this system can work in a wide range of organisms. This long chain fatty acid acts as a substrate for [http://partsregistry.org/Part:BBa_K590031 Aldehyde Decarbonylase] (ADC), the enzyme that removes the carbonyl group (C=O) from the fatty aldehyde, yielding an alkane one carbon shorter than the original Acyl-ACP and a molecule of formate. Since the vast majority of the fatty acyl-ACPs produced by ''E. coli'' have an even chain length, this system produces detectable amounts of only odd chain length alkanes. This study reported production of the C13, C15, and C17 alkanes, as well as the C17 alkene (unsaturated hydrocarbon). This chain length range falls well within the range of those found in diesel, so this system is theoretically able to make the alkane portion of a fuel compatible with current engines and infrastructure.<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
='''The PetroBrick: A modular and open platform for the biological production of diesel fuel'''=<br />
<br />
[[File:Washington PetroBrick.png|200px|frameless|border|left|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
The goal of our "'''''Make It: Diesel Production'''''" portion of this summer's iGEM project is to turn convert this recently discovered set of enzymes ([http://partsregistry.org/Part:BBa_K590032 AAR] and [http://partsregistry.org/Part:BBa_K590031 ADC]) for microbial alkane production into an open and modular platform for iGEM teams to develop into a robust replacement for petrochemical fuels. Our alkane production system is specifically designed to be easily improved upon, and we have started work on improving this open system, both by increasing alkane yields and by changing the product produced. In addition, we have started to move this system into an alternative chassis, yeast.<br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
<br />
----<br />
<br />
<br />
==References==<br />
1. http://ourenergypolicy.org/docs/2/biofuels-taskforce.pdf<br />
<br />
2. http://www.environment.gov.au/atmosphere/fuelquality/publications/2000hours-vehicle-fleet/pubs/2000-hours-vehicles.pdf<br />
<br />
3. Anaerobic Metabolism of Biodiesel and Its Impact on Metal Corrosion<br />
Deniz F. Aktas, Jason S. Lee, Brenda J. Little, Richard I. Ray, Irene A. Davidova, Christopher N. Lyles, Joseph M. Suflita Energy & Fuels 2010 24 (5), 2924-2928(http://pubs.acs.org/doi/full/10.1021/ef100084j)<br />
<br />
4. http://www.mda.state.mn.us/news/publications/renewable/biodiesel/biodieselcoldissues.pdf<br />
<br />
5. http://www.afdc.energy.gov/afdc/pdfs/fueltable.pdf<br />
<br />
6. The viscosities of three biodiesel fuels at temperatures up to 300°C<br />
R.E. Tate, K.C. Watts, C.A.W. Allen K.I. Wilkie Fuel 2006 85, 1010-1015 (https://netfiles.uiuc.edu/mccrady/shared/Biodiesel/The%20viscosities%20of%20three%20biodiesel%20fuels%20at%20temperatures%20up%20to%20300%208C.pdf)<br />
<br />
7. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
8. Microbial Biosynthesis of Alkanes Andreas Schirmer, Mathew A. Rude, Xuezhi Li, Emanuela Popova and Stephen B. del Cardayre Science 30 July 2010: Vol. 329 no. 5991 pp. 559-562 http://www.sciencemag.org/content/329/5991/559<br />
<br />
9.http://msdssearch.dow.com/PublishedLiteratureDOWCOM/dh_0119/0901b803801195a5.pdf?filepath=oxysolvents/pdfs/noreg/327-00014.pdf&fromPage=GetDoc<br />
<br />
10.Metabolic engineering of Escherichia coli for 1-butanol production Shota Atsumi, Anthony F. Cann, Michael R. Connor, Claire R. Shen, Kevin M. Smith, Mark P. Brynildsen, Katherine J.Y. Chou, Taizo Hanai1, James C. Liao Metabolic Engineering Volume 10, Issue 6, November 2008, Pages 305-311<br />
<br />
11. http://www.nrel.gov/vehiclesandfuels/pdfs/sr368051.pdf<br />
<br />
12. http://www.arpltd.com/n-butano.htm</div>LStanleyhttp://2011.igem.org/File:Washington_PetroBrick.pngFile:Washington PetroBrick.png2011-09-29T00:14:26Z<p>LStanley: </p>
<hr />
<div></div>LStanleyhttp://2011.igem.org/Team:Washington/Team/SponsorsTeam:Washington/Team/Sponsors2011-09-29T00:07:13Z<p>LStanley: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
===Thank you to our sponsors!===<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>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-26T19:22:33Z<p>LStanley: /* Combining Mutants for the Construction of a Gluten Hydrolase */</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 identifying Kumamolisin as a good candidate for activity at low pH, we tested its activity on breaking down PQLP at pH 4 against the activity of SC PEP, the enzyme currently in clinical trials for breaking down gluten. Kumamolisin had never been tested for its ability to breakdown gluten, and so we began novel experimentation into the enzyme's activity on our gluten model. From tests using the fluorescent PQLP system described in our methods section, Kumamolisin showed about 7 times better activity on breaking down PQLP at pH 4 when compared to the activity of SC PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolisin 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 />
<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 screened 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 over 10-fold increase in activity from wild-type Kumamolisin!<br />
<br />
[[File:Washington Vertical Initial Screen.png|center|700px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate. *"deg" in the data labels indicates use of a degenerate primer. Data for these points is representative of a group of variants, each with different substitutions at one residue. This accounts for the <100 data points on this graph, despite testing >100 novel mutants in total.]]<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, 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|Concentration controlled rate data relative to native Kumamolisin for three of our most active mutants.]]<br />
<br />
<br />
----<br />
<br />
<br />
='''Combining Mutations for the Construction of a Gluten Hydrolase'''=<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 together to make combinatorial variants.<br />
<br />
=='''Successful mutations were combined to construct a second library for screening'''==<br />
<br />
After designing a collection of combinatorial mutants, drawing from successful mutations discovered in the first round, we again performed a rough screen to identify promising combinations of mutations. From the initial screen on our combinatorial mutants, it appeared that we had achieved around 50 times better activity than native Kumamolisin on breaking down PQLP.<br />
<br />
[[File:Washington Comb Fold Change.png|center|500px|thumb|From initial whole cell lysate screens on combinatorial mutants, it appears that about 50-fold improvement over native Kumamolisin activity on PQLP has been achieved.]]<br />
<br />
=='''One of the combinatorial mutants resulted in over a 100-fold increase in activity'''==<br />
<br />
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 />
[[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>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-26T19:21:12Z<p>LStanley: </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 identifying Kumamolisin as a good candidate for activity at low pH, we tested its activity on breaking down PQLP at pH 4 against the activity of SC PEP, the enzyme currently in clinical trials for breaking down gluten. Kumamolisin had never been tested for its ability to breakdown gluten, and so we began novel experimentation into the enzyme's activity on our gluten model. From tests using the fluorescent PQLP system described in our methods section, Kumamolisin showed about 7 times better activity on breaking down PQLP at pH 4 when compared to the activity of SC PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolisin 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 />
<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 screened 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 over 10-fold increase in activity from wild-type Kumamolisin!<br />
<br />
[[File:Washington Vertical Initial Screen.png|center|700px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate. *"deg" in the data labels indicates use of a degenerate primer. Data for these points is representative of a group of variants, each with different substitutions at one residue. This accounts for the <100 data points on this graph, despite testing >100 novel mutants in total.]]<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, 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|Concentration controlled rate data relative to native Kumamolisin for three of our most active mutants.]]<br />
<br />
<br />
----<br />
<br />
<br />
='''Combining Mutants for the Construction of a Gluten Hydrolase'''=<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 together to make combinatorial variants.<br />
<br />
=='''A second library based on the first round of mutagenesis was constructed and tested'''==<br />
<br />
After designing a collection of combinatorial mutants, drawing from successful mutations discovered in the first round, we again performed a rough screen to identify promising combinations of mutations. From the initial screen on our combinatorial mutants, it appeared that we had achieved around 50 times better activity than native Kumamolisin on breaking down PQLP.<br />
<br />
[[File:Washington Comb Fold Change.png|center|500px|thumb|From initial whole cell lysate screens on combinatorial mutants, it appears that about 50-fold improvement over native Kumamolisin activity on PQLP has been achieved.]]<br />
<br />
=='''One of the combinatorial mutants resulted in over a 100-fold increase in activity'''==<br />
<br />
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 />
[[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>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-26T19:20:02Z<p>LStanley: /* A second library based on the first round of mutagenesis was constructed and tested */</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 identifying Kumamolisin as a good candidate for activity at low pH, we tested its activity on breaking down PQLP at pH 4 against the activity of SC PEP, the enzyme currently in clinical trials for breaking down gluten. Kumamolisin had never been tested for its ability to breakdown gluten, and so we began novel experimentation into the enzyme's activity on our gluten model. From tests using the fluorescent PQLP system described in our methods section, Kumamolisin showed about 7 times better activity on breaking down PQLP at pH 4 when compared to the activity of SC PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolisin 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 screened 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 over 10-fold increase in activity from wild-type Kumamolisin!<br />
<br />
[[File:Washington Vertical Initial Screen.png|center|700px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate. *"deg" in the data labels indicates use of a degenerate primer. Data for these points is representative of a group of variants, each with different substitutions at one residue. This accounts for the <100 data points on this graph, despite testing >100 novel mutants in total.]]<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, 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|Concentration controlled rate data relative to native Kumamolisin for three of our most active mutants.]]<br />
<br />
<br />
----<br />
<br />
='''Combining Mutants for the Construction of a Gluten Hydrolase'''=<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 together to make combinatorial variants.<br />
<br />
=='''A second library based on the first round of mutagenesis was constructed and tested'''==<br />
<br />
After designing a collection of combinatorial mutants, drawing from successful mutations discovered in the first round, we again performed a rough screen to identify promising combinations of mutations. From the initial screen on our combinatorial mutants, it appeared that we had achieved around 50 times better activity than native Kumamolisin on breaking down PQLP.<br />
<br />
[[File:Washington Comb Fold Change.png|center|500px|thumb|From initial whole cell lysate screens on combinatorial mutants, it appears that about 50-fold improvement over native Kumamolisin activity on PQLP has been achieved.]]<br />
<br />
=='''One of the combinatorial mutants resulted in over a 100-fold increase in activity'''==<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.]]<br />
<br />
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!</div>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-26T19:19:17Z<p>LStanley: /* Combining Mutants for the Construction of a Gluten Hydrolase */</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 identifying Kumamolisin as a good candidate for activity at low pH, we tested its activity on breaking down PQLP at pH 4 against the activity of SC PEP, the enzyme currently in clinical trials for breaking down gluten. Kumamolisin had never been tested for its ability to breakdown gluten, and so we began novel experimentation into the enzyme's activity on our gluten model. From tests using the fluorescent PQLP system described in our methods section, Kumamolisin showed about 7 times better activity on breaking down PQLP at pH 4 when compared to the activity of SC PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolisin 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 screened 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 over 10-fold increase in activity from wild-type Kumamolisin!<br />
<br />
[[File:Washington Vertical Initial Screen.png|center|700px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate. *"deg" in the data labels indicates use of a degenerate primer. Data for these points is representative of a group of variants, each with different substitutions at one residue. This accounts for the <100 data points on this graph, despite testing >100 novel mutants in total.]]<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, 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|Concentration controlled rate data relative to native Kumamolisin for three of our most active mutants.]]<br />
<br />
<br />
----<br />
<br />
='''Combining Mutants for the Construction of a Gluten Hydrolase'''=<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 together to make combinatorial variants.<br />
<br />
=='''A second library based on the first round of mutagenesis was constructed and tested'''==<br />
<br />
After designing a collection of combinatorial mutants, drawing from successful mutations discovered in the first round, we again performed a rough screen to identify promising combinations of mutations. From the initial screen on our combinatorial mutants, it appeared that we had achieved around 50 times better activity than native Kumamolisin on breaking down PQLP.<br />
<br />
[[File:Washington Comb Fold Change.png|center|500px|thumb|From initial whole cell lysate screens on combinatorial mutants, it appears that about 50-fold improvement over native Kumamolisin activity on PQLP.]]<br />
<br />
<br />
=='''One of the combinatorial mutants resulted in over a 100-fold increase in activity'''==<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.]]<br />
<br />
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!</div>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-26T19:18:41Z<p>LStanley: /* Purifying and characterizing promising mutants for accurate rate comparison */</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 identifying Kumamolisin as a good candidate for activity at low pH, we tested its activity on breaking down PQLP at pH 4 against the activity of SC PEP, the enzyme currently in clinical trials for breaking down gluten. Kumamolisin had never been tested for its ability to breakdown gluten, and so we began novel experimentation into the enzyme's activity on our gluten model. From tests using the fluorescent PQLP system described in our methods section, Kumamolisin showed about 7 times better activity on breaking down PQLP at pH 4 when compared to the activity of SC PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolisin 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 screened 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 over 10-fold increase in activity from wild-type Kumamolisin!<br />
<br />
[[File:Washington Vertical Initial Screen.png|center|700px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate. *"deg" in the data labels indicates use of a degenerate primer. Data for these points is representative of a group of variants, each with different substitutions at one residue. This accounts for the <100 data points on this graph, despite testing >100 novel mutants in total.]]<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, 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|Concentration controlled rate data relative to native Kumamolisin for three of our most active mutants.]]<br />
<br />
<br />
----<br />
<br />
='''Combining Mutants for the Construction of a Gluten Hydrolase'''=<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 together to make combinatorial variants.<br />
<br />
=='''A second library based on the first round of mutagensis was constructed and tested'''==<br />
<br />
After designing a collection of combinatorial mutants, drawing from successful mutations discovered in the first round, we again performed a rough screen to identify promising combinations of mutations. From the initial screen on our combinatorial mutants, it appeared that we had achieved around 50 times better activity than native Kumamolisin on breaking down PQLP.<br />
<br />
[[File:Washington Comb Fold Change.png|center|500px|thumb|From initial whole cell lysate screens on combinatorial mutants, it appears that about 50-fold improvement over native Kumamolisin activity on PQLP.]]<br />
<br />
<br />
=='''One of the combinatorial mutants resulted in over a 100-fold increase in activity'''==<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.]]<br />
<br />
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!</div>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-26T19:16:31Z<p>LStanley: /* Using a whole cell lysate assay to screen a large number of mutants for good activity */</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 identifying Kumamolisin as a good candidate for activity at low pH, we tested its activity on breaking down PQLP at pH 4 against the activity of SC PEP, the enzyme currently in clinical trials for breaking down gluten. Kumamolisin had never been tested for its ability to breakdown gluten, and so we began novel experimentation into the enzyme's activity on our gluten model. From tests using the fluorescent PQLP system described in our methods section, Kumamolisin showed about 7 times better activity on breaking down PQLP at pH 4 when compared to the activity of SC PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolisin 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 screened 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 over 10-fold increase in activity from wild-type Kumamolisin!<br />
<br />
[[File:Washington Vertical Initial Screen.png|center|700px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate. *"deg" in the data labels indicates use of a degenerate primer. Data for these points is representative of a group of variants, each with different substitutions at one residue. This accounts for the <100 data points on this graph, despite testing >100 novel mutants in total.]]<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, 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 />
='''Combining Mutants for the Construction of a Gluten Hydrolase'''=<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 together to make combinatorial variants.<br />
<br />
=='''A second library based on the first round of mutagensis was constructed and tested'''==<br />
<br />
After designing a collection of combinatorial mutants, drawing from successful mutations discovered in the first round, we again performed a rough screen to identify promising combinations of mutations. From the initial screen on our combinatorial mutants, it appeared that we had achieved around 50 times better activity than native Kumamolisin on breaking down PQLP.<br />
<br />
[[File:Washington Comb Fold Change.png|center|500px|thumb|From initial whole cell lysate screens on combinatorial mutants, it appears that about 50-fold improvement over native Kumamolisin activity on PQLP.]]<br />
<br />
<br />
=='''One of the combinatorial mutants resulted in over a 100-fold increase in activity'''==<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.]]<br />
<br />
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!</div>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-26T19:11:43Z<p>LStanley: /* Testing Purified Mutants to Accurately Assess Activity */</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|600px|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>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-26T19:11:16Z<p>LStanley: /* Testing Purified Mutants to Accurately Assess Activity */</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|600px|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|600px|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 />
<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 />
<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>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-26T19:10:16Z<p>LStanley: /* Testing Purified Mutants to Accurately Assess Activity */</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|600px|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 />
<br />
==='''Purification'''===<br />
<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 />
<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 />
==='''Assay'''===<br />
<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 below, 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>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-26T19:09:40Z<p>LStanley: /* Testing Purified Mutants to Accurately Assess Activity */</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|600px|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 />
<br />
==='''Purification'''===<br />
<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 />
<br />
[[File:Washington First Raw Data.png|left|500px|thumb|We measured fluorescence of each reaction at 30 second intervals to see the rate at which each mutant cleaved PQLP.]]<br />
<br />
==='''Assay'''===<br />
<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 below, 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>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-26T19:08:35Z<p>LStanley: /* Testing Purified Mutants to Accurately Assess Activity */</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|600px|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 />
<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 />
<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 below, 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.<br />
<br />
[[File:Washington First Raw Data.png|center|500px|thumb|We measured fluorescence of each reaction at 30 second intervals to see the rate at which each mutant cleaved PQLP.]]</div>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-26T19:08:19Z<p>LStanley: /* Redesigning Kumamolisin to Have Higher Activity at Low pH */</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|600px|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 />
<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 />
<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 below, 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.<br />
<br />
[[File:Washington First Raw Data.png|center|500px|thumb|We measured fluorescence of each reaction at 30 second intervals to see the rate at which each mutant cleaved PQLP.]]</div>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-26T19:07:34Z<p>LStanley: /* Testing Purified Mutants to Accurately Assess Activity */</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|600px|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 />
=='''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 />
=='''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 />
=='''Testing Purified Mutants to Accurately Assess Activity'''==<br />
<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 />
<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 below, 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.<br />
<br />
[[File:Washington First Raw Data.png|center|500px|thumb|We measured fluorescence of each reaction at 30 second intervals to see the rate at which each mutant cleaved PQLP.]]</div>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-26T19:07:17Z<p>LStanley: /* Testing Purified Mutants to Accurately Assess Activity */</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|600px|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 />
=='''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 />
=='''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 />
=='''Testing Purified Mutants to Accurately Assess Activity'''==<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 below, 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.<br />
<br />
[[File:Washington First Raw Data.png|center|500px|thumb|We measured fluorescence of each reaction at 30 second intervals to see the rate at which each mutant cleaved PQLP.]]</div>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-26T19:06:47Z<p>LStanley: /* Testing Purified Mutants to Accurately Assess Activity */</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|600px|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 />
=='''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 />
=='''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 />
=='''Testing Purified Mutants to Accurately Assess Activity'''==<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 below, 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.<br />
<br />
[[File:Washington First Raw Data.png|center|500px|thumb|We measured fluorescence of each reaction at 30 second intervals to see the rate at which each mutant cleaved PQLP.]]</div>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-26T19:02:55Z<p>LStanley: /* Testing Purified Mutants to Accurately Assess Activity */</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|600px|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 />
=='''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 />
=='''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 />
=='''Testing Purified Mutants to Accurately Assess Activity'''==<br />
<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 />
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 below, 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.<br />
<br />
[[File:Washington First Raw Data.png|center|500px|thumb|We measured fluorescence of each reaction at 30 second intervals to see the rate at which each mutant cleaved PQLP.]]</div>LStanleyhttp://2011.igem.org/File:Washington_Liz.pngFile:Washington Liz.png2011-09-26T18:34:04Z<p>LStanley: uploaded a new version of &quot;File:Washington Liz.png&quot;</p>
<hr />
<div></div>LStanleyhttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-25T20:25:41Z<p>LStanley: /* Testing Kumamolisin-As against SC-PEP */</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 identifying Kumamolisin as a good candidate for activity at low pH, we tested its activity on breaking down PQLP at pH 4 against the activity of SC PEP, the enzyme currently in clinical trials for breaking down gluten. Kumamolisin had never been tested for its ability to breakdown gluten, and so we began novel experimentation into the enzyme's activity on our gluten model. From tests using the fluorescent PQLP system described in our methods section, Kumamolisin showed about 7 times better activity on breaking down PQLP at pH 4 when compared to the activity of SC PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolisin 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 screened 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 over 10-fold increase in activity from wild-type Kumamolisin!<br />
<br />
[[File:Washington Vertical Initial Screen.png|center|700px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate.]]<br />
<br />
<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, 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 />
='''Combining Mutants for the Construction of a Gluten Hydrolase'''=<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 together to make combinatorial variants.<br />
<br />
=='''A second library based on the first round of mutagensis was constructed and tested'''==<br />
<br />
After designing a collection of combinatorial mutants, drawing from successful mutations discovered in the first round, we again performed a rough screen to identify promising combinations of mutations. From the initial screen on our combinatorial mutants, it appeared that we had achieved around 50 times better activity than native Kumamolisin on breaking down PQLP.<br />
<br />
[[File:Washington Comb Fold Change.png|center|500px|thumb|From initial whole cell lysate screens on combinatorial mutants, it appears that about 50-fold improvement over native Kumamolisin activity on PQLP.]]<br />
<br />
<br />
=='''One of the combinatorial mutants resulted in over a 100-fold increase in activity'''==<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.]]<br />
<br />
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!</div>LStanleyhttp://2011.igem.org/Team:Washington/Alkanes/MethodsTeam:Washington/Alkanes/Methods2011-09-23T05:18:14Z<p>LStanley: /* Gas Chromatography and Mass Spectroscopy */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Methods'''</big></big></big></big></center><br><br><br />
<br />
='''Introducing the PetroBrick'''=<br />
[[Image:Washington_2011_PetroBrick.png|220px|frameless|border="2"|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025|left]]<br />
<br />
<br />
:::::::::<p>In order to produce alkanes, we need both <partinfo>BBa_K590031</partinfo> acyl-ACP reductase ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 ADC]) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Part:BBa_K590031] aldehyde decarbonylase ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 AAR]) to work together in the cell. In order to achieve this goal, we used standard cloning methods combining both to construct the [http://partsregistry.org/Part:BBa_K590025 BBa_K590025] Biobrick that contained both AAR and ADC under a high constitutive promoter, each with its own Elowitz standard RBS. This construct successfully synthesized our target product, and thus we have created a new modular alkane-producing platform:</p><br />
<br />
<br><br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;'''the <font size="5" weight=bold>PetroBrick.'''</font><br />
<br />
<br><br />
<br />
[[Image:Washington2011_PetroBrick_Construct.png|730px|frameless|border|bottom|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
<br><br />
<br><br />
<br />
----<br />
<br />
='''Alkane Production & Extraction'''=<br />
[[File:Washington_Alkane_extraction.png|right|400px|thumb|Diagram showing the process of extraction.]]<p>After we had the complete gene assembly in our hands, the next step was to transform it into cells and start them growing for alkane production. We let them grow in 37 degree shaker for 48-72 hours, in sealed glass tubes. After the cells have gone through the alkane production process, the next step is to extract the alkanes out of the cell broth. We add acyl acetate directly into the glass test tube for cell growth. Then we vortex until to everything is well mixed, to make sure all of the alkanes go directly into the ethyl acetate solvent. Next, we spin down the mixture by using a centrifuge at full speed to form three layers (cell pellet, media, and ethyl acetate supernatant). We use only the ethyl acetate layer to send for GCMS analysis.</p><br />
<br />
<br />
----<br />
<br />
<br />
='''Alkane Detection'''=<br />
==Gas Chromatography and Mass Spectrometry==<br />
<br />
We utilized a Gas Chromatograph / Mass Spectrometer (GCMS) to analyze alkane production concentrations. The GCMS is considered a "specific" test, because it identifies compounds specifically, not just to a category of compounds. It works by separating the individual components of a sample through a capillary column based mainly on it's boiling point, similar to fractional distillation. The separated compounds generally elute from the column at different retention times, and are passed to the mass spectrometer. <br />
<br />
Inside the mass spectrometer each compound is then broken down into it's individual molecular components through electron stream ionization. These ions differ in mass-to-charge (m/z) ratios, creating a unique ion de-composition profile for each compound that can be used to identify it through comparison to known chemical standards. Because compounds occasionally have similar elution times or mass spec fingerprints, the combination of analyses results in reducing the chances for overlap.<br />
<br />
<br />
<center><gallery widths="400px" heights="300px" perrow="2"><br />
Image:Washington2011_Chrom.png|'''Gas chromatography:''' A method used to separate molecules from the media extraction based on boiling point. In this image the temperature is increasing over time, and molecules with a higher boiling point are being eluted and detected by a mass spectrometer. The ion abundance is concentration dependent and can be converted if using a standard curve<br />
<br />
Image:Washington2011 Spectra.png|'''Mass Spectrometry:''' Molecules exiting gas chromatography enter an electron impact mass spectrometer. The molecules are ionized and fragmented. The resulting spectra is compared to a database of molecules in order to predict its chemical identity. On the top (red) is an experimental spectra of our biologically produced C15 alkane, on the bottom (blue) is the NIST standard spectra for a C15 alkane. The fragmentation pattern and parent ion (blue arrow, bottom right) match perfectly.<br />
<br />
</gallery></center></div>LStanleyhttp://2011.igem.org/Team:Washington/Team/MembersTeam:Washington/Team/Members2011-09-23T05:15:43Z<p>LStanley: /* Gluten Degradation */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------><br />
<br />
== '''Who we are''' ==<br />
<br />
<gallery caption="Undergraduate Team Members" widths="180px" heights="120px" perrow="4"><br />
Image:Washington Casey Ager Profile1.jpg|<center>Casey Ager <br/> Biochemistry</center><br />
Image:Washington Photo-0088A.png|<center>Juhye An <br/> Biochemistry </center><br />
Image: Profile_Michael_Brasino.jpg|<center>Michael Brasino <br/> Materials Science and Engineering</center><br />
Image:Washington_.jpg|<center>Marika Cheng <br/> Microbiology</center><br />
Image:Washington_choe_chris.png|<center>Chris Choe <br/> Major</center><br />
Image:Washington_.jpg|<center>Justin De Leon <br/> Microbiology</center><br />
File:Princess Peachy.JPG|<center>Sydney Gordon <br/> Biochemistry, Music</center><br />
Image:CIMG0021.jpg|<center>Daniel Hadidi <br/> Neurobiology</center><br />
Image:Washington_Matthew_Harger.JPG|<center>Matthew Harger <br/> Cellular and Molecular Biology</center><br />
Image:Washington_Elaine.jpg|<center>Elaine Lai <br/> Microbiology, Chemistry</center><br />
Image:Washington_2011_BATMAN.jpg|<center>Benjamin Mo <br/> Bioengineering</center><br />
Image:Washington 2011 CIMG0015-2.jpg|<center>Austin Moon <br/> Cellular and Molecular Biology, Microbiology</center><br />
Image:Washington_.jpg|<center>Rashmi Ravichandran <br/> Microbiology</center><br />
Image:Washington_.jpg|<center>Seth Sagulo <br/> Biochemistry</center><br />
File:Washington_Liz.png|<center>Liz Stanley <br/> Microbiology, Chemistry</center><br />
Image:Washington_.jpg|<center>Angus Toland <br/> Microbiology</center><br />
Image:Washington_.jpg|<center>Sarah Wolf <br/> Biochemistry</center><br />
Image:Washington_.jpg|<center>SauShun (Alicia) Wong <br/> Materials Science and Engineering</center><br />
Image:Washington_Cindy.jpg|<center>Cindy Wu <br/> Cellular and Molecular Biology</center><br />
Image:Washington_Sean_Wu.jpg|<center>Sean Wu <br/> Computer Science and Engineering</center><br />
Image:Washington_2011_BATMAN.jpg|<center>Lei Zheng <br/> Biochemistry</center><br />
Image:Washington_david_zong.jpg|<center>David Zong <br/> Bioengineering</center><br />
<br />
</gallery><br />
<br />
<br />
<gallery caption="Advisors" widths="180px" heights="120px" perrow="4"><br />
Image:Washington_.jpg|<center>Aaron Chevalier <br/> Bioengineering </center><br />
Image:Regbert.jpg|<center>Rob Egbert <br/> Electrical Engineering </center><br />
Image:Washington_.jpg|<center>Chris Eiben <br/> Biochemistry </center><br />
Image:Washington_.jpg|<center>Jeremy Mills <br/> Biochemistry </center><br />
Image:Siegel_UW_2011.jpg|<center>Justin Siegel <br/> Biomolecular Structure and Design</center><br />
Image:Washington2011_Matthew-d-smith.jpg|<center>Matt Smith <br/> Molecular and Cellular Biology</center><br />
Image:Washington_.jpg|<center>Ingrid Swanson Pultz<br/> Microbiology </center><br />
</gallery><br />
<br />
<br />
<gallery caption="Faculty" widths="180px" heights="120px" perrow="5"><br />
image:Washington_David_Baker.jpg|<center>David Baker <br/> Biochemistry</center><br />
Image:Washington_Klavins.jpg|<center>Eric Klavins <br/> Electrical Engineering</center><br />
</gallery><br />
<br />
<br />
<gallery caption="Collaborators and Support" widths="140px" heights="100px" perrow="5"><br />
<br />
Image:Washington_OSLI.png|<center><b>Oil Sand Leadership Initiative</b> <br/> Funding to support Travel and Registration Costs</center><br />
<br />
Image:Washington_UniversitySeal.gif|<center><b>Biochemistry</b> <br/> Lab space</center><br />
<br />
Image:Washington_Anaspec.gif|<center>Anaspec <br/> <b>Peptide Discounts</b></center><br />
<br />
Image:Washington_ARPA-E_Logo.png|<center><b>Advanced Research Projects Agency - Energy </b><br/> Registration Support</center><br />
<br />
Image:Washington2011_Hhmi_362_72.jpg|<center><b>Howard Hughes Medical Institute</b> <br/> Lab materials and supplies</center><br />
<br />
<br />
</gallery><br />
<br />
== '''Who did what''' ==<br />
The teams were assembled during our winter quarter. During this term we went around to classes and had weekly meetings to introduce students to synthetic biology through a series of guest lectures. During the spring, students participated in brainstorming projects related to the sponsoring Faculty advisors labs (Baker and Klavins) and came up with the project they wanted to carry out. Students also planned and partcipated in community outreach events where we taught our community about synthetic biology. Over the summer, students completed all of the experimental work, but worked closely with both graduate students and faculty advisors to plan the most pertinent experiments and make the most of our limited time.<br />
<br />
=== Diesel Production ===<br />
After producing promising results, in future directions <br />
<br />
Casey Ager, Austin Moon, and Seth Sagulo worked on Enzyme Localization via Direct Fusion and Zinc Finger Fusion methods.<br />
<br />
Juhye An, Elaine Lai, and Benjamin Mo worked on Decarbonylase Redesign<br />
<br />
Marika Cheng and Justin De Leon worked on Alternative Chassis<br />
<br />
Chris Choe and David Zong worked on Alternate Aldehyde Production<br />
<br />
Matthew Harger worked on Branched Alkanes Production<br />
<br />
Matthew Harger and Lei Zheng worked on System Optimization<br />
<br />
=== Gluten Destruction ===<br />
Sydney Gordon, Daniel Hadidi, Liz Stanley, Angus Toland, Sarah Wolf, and Sean Wu designed, built, and tested Kumamolisin-As and over 100 mutants to combat gluten intolerance by increasing the activity on the PQLP antigenic peptide.<br />
<br />
=== iGEM Toolkits ===<br />
Michael Brasino, Rashmi Ravichandran, and Alicia Wong<br />
<br />
=== Community Outreach ===<br />
Cindy Wu headed and organized all iGEM 2011 Community Outreach events.</div>LStanleyhttp://2011.igem.org/Team:Washington/Team/MembersTeam:Washington/Team/Members2011-09-23T05:15:33Z<p>LStanley: /* Alkanes Production */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------><br />
<br />
== '''Who we are''' ==<br />
<br />
<gallery caption="Undergraduate Team Members" widths="180px" heights="120px" perrow="4"><br />
Image:Washington Casey Ager Profile1.jpg|<center>Casey Ager <br/> Biochemistry</center><br />
Image:Washington Photo-0088A.png|<center>Juhye An <br/> Biochemistry </center><br />
Image: Profile_Michael_Brasino.jpg|<center>Michael Brasino <br/> Materials Science and Engineering</center><br />
Image:Washington_.jpg|<center>Marika Cheng <br/> Microbiology</center><br />
Image:Washington_choe_chris.png|<center>Chris Choe <br/> Major</center><br />
Image:Washington_.jpg|<center>Justin De Leon <br/> Microbiology</center><br />
File:Princess Peachy.JPG|<center>Sydney Gordon <br/> Biochemistry, Music</center><br />
Image:CIMG0021.jpg|<center>Daniel Hadidi <br/> Neurobiology</center><br />
Image:Washington_Matthew_Harger.JPG|<center>Matthew Harger <br/> Cellular and Molecular Biology</center><br />
Image:Washington_Elaine.jpg|<center>Elaine Lai <br/> Microbiology, Chemistry</center><br />
Image:Washington_2011_BATMAN.jpg|<center>Benjamin Mo <br/> Bioengineering</center><br />
Image:Washington 2011 CIMG0015-2.jpg|<center>Austin Moon <br/> Cellular and Molecular Biology, Microbiology</center><br />
Image:Washington_.jpg|<center>Rashmi Ravichandran <br/> Microbiology</center><br />
Image:Washington_.jpg|<center>Seth Sagulo <br/> Biochemistry</center><br />
File:Washington_Liz.png|<center>Liz Stanley <br/> Microbiology, Chemistry</center><br />
Image:Washington_.jpg|<center>Angus Toland <br/> Microbiology</center><br />
Image:Washington_.jpg|<center>Sarah Wolf <br/> Biochemistry</center><br />
Image:Washington_.jpg|<center>SauShun (Alicia) Wong <br/> Materials Science and Engineering</center><br />
Image:Washington_Cindy.jpg|<center>Cindy Wu <br/> Cellular and Molecular Biology</center><br />
Image:Washington_Sean_Wu.jpg|<center>Sean Wu <br/> Computer Science and Engineering</center><br />
Image:Washington_2011_BATMAN.jpg|<center>Lei Zheng <br/> Biochemistry</center><br />
Image:Washington_david_zong.jpg|<center>David Zong <br/> Bioengineering</center><br />
<br />
</gallery><br />
<br />
<br />
<gallery caption="Advisors" widths="180px" heights="120px" perrow="4"><br />
Image:Washington_.jpg|<center>Aaron Chevalier <br/> Bioengineering </center><br />
Image:Regbert.jpg|<center>Rob Egbert <br/> Electrical Engineering </center><br />
Image:Washington_.jpg|<center>Chris Eiben <br/> Biochemistry </center><br />
Image:Washington_.jpg|<center>Jeremy Mills <br/> Biochemistry </center><br />
Image:Siegel_UW_2011.jpg|<center>Justin Siegel <br/> Biomolecular Structure and Design</center><br />
Image:Washington2011_Matthew-d-smith.jpg|<center>Matt Smith <br/> Molecular and Cellular Biology</center><br />
Image:Washington_.jpg|<center>Ingrid Swanson Pultz<br/> Microbiology </center><br />
</gallery><br />
<br />
<br />
<gallery caption="Faculty" widths="180px" heights="120px" perrow="5"><br />
image:Washington_David_Baker.jpg|<center>David Baker <br/> Biochemistry</center><br />
Image:Washington_Klavins.jpg|<center>Eric Klavins <br/> Electrical Engineering</center><br />
</gallery><br />
<br />
<br />
<gallery caption="Collaborators and Support" widths="140px" heights="100px" perrow="5"><br />
<br />
Image:Washington_OSLI.png|<center><b>Oil Sand Leadership Initiative</b> <br/> Funding to support Travel and Registration Costs</center><br />
<br />
Image:Washington_UniversitySeal.gif|<center><b>Biochemistry</b> <br/> Lab space</center><br />
<br />
Image:Washington_Anaspec.gif|<center>Anaspec <br/> <b>Peptide Discounts</b></center><br />
<br />
Image:Washington_ARPA-E_Logo.png|<center><b>Advanced Research Projects Agency - Energy </b><br/> Registration Support</center><br />
<br />
Image:Washington2011_Hhmi_362_72.jpg|<center><b>Howard Hughes Medical Institute</b> <br/> Lab materials and supplies</center><br />
<br />
<br />
</gallery><br />
<br />
== '''Who did what''' ==<br />
The teams were assembled during our winter quarter. During this term we went around to classes and had weekly meetings to introduce students to synthetic biology through a series of guest lectures. During the spring, students participated in brainstorming projects related to the sponsoring Faculty advisors labs (Baker and Klavins) and came up with the project they wanted to carry out. Students also planned and partcipated in community outreach events where we taught our community about synthetic biology. Over the summer, students completed all of the experimental work, but worked closely with both graduate students and faculty advisors to plan the most pertinent experiments and make the most of our limited time.<br />
<br />
=== 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 Degradation ===<br />
Sydney Gordon, Daniel Hadidi, Liz Stanley, Angus Toland, Sarah Wolf, and Sean Wu designed, built, and tested Kumamolisin-As and over 100 mutants to combat gluten intolerance by increasing the activity on the PQLP antigenic peptide.<br />
<br />
=== iGEM Toolkits ===<br />
Michael Brasino, Rashmi Ravichandran, and Alicia Wong<br />
<br />
=== Community Outreach ===<br />
Cindy Wu headed and organized all iGEM 2011 Community Outreach events.</div>LStanleyhttp://2011.igem.org/Team:Washington/Team/MembersTeam:Washington/Team/Members2011-09-23T05:14:53Z<p>LStanley: /* Who we are */</p>
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__NOTOC__<br />
<br />
<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------><br />
<br />
== '''Who we are''' ==<br />
<br />
<gallery caption="Undergraduate Team Members" widths="180px" heights="120px" perrow="4"><br />
Image:Washington Casey Ager Profile1.jpg|<center>Casey Ager <br/> Biochemistry</center><br />
Image:Washington Photo-0088A.png|<center>Juhye An <br/> Biochemistry </center><br />
Image: Profile_Michael_Brasino.jpg|<center>Michael Brasino <br/> Materials Science and Engineering</center><br />
Image:Washington_.jpg|<center>Marika Cheng <br/> Microbiology</center><br />
Image:Washington_choe_chris.png|<center>Chris Choe <br/> Major</center><br />
Image:Washington_.jpg|<center>Justin De Leon <br/> Microbiology</center><br />
File:Princess Peachy.JPG|<center>Sydney Gordon <br/> Biochemistry, Music</center><br />
Image:CIMG0021.jpg|<center>Daniel Hadidi <br/> Neurobiology</center><br />
Image:Washington_Matthew_Harger.JPG|<center>Matthew Harger <br/> Cellular and Molecular Biology</center><br />
Image:Washington_Elaine.jpg|<center>Elaine Lai <br/> Microbiology, Chemistry</center><br />
Image:Washington_2011_BATMAN.jpg|<center>Benjamin Mo <br/> Bioengineering</center><br />
Image:Washington 2011 CIMG0015-2.jpg|<center>Austin Moon <br/> Cellular and Molecular Biology, Microbiology</center><br />
Image:Washington_.jpg|<center>Rashmi Ravichandran <br/> Microbiology</center><br />
Image:Washington_.jpg|<center>Seth Sagulo <br/> Biochemistry</center><br />
File:Washington_Liz.png|<center>Liz Stanley <br/> Microbiology, Chemistry</center><br />
Image:Washington_.jpg|<center>Angus Toland <br/> Microbiology</center><br />
Image:Washington_.jpg|<center>Sarah Wolf <br/> Biochemistry</center><br />
Image:Washington_.jpg|<center>SauShun (Alicia) Wong <br/> Materials Science and Engineering</center><br />
Image:Washington_Cindy.jpg|<center>Cindy Wu <br/> Cellular and Molecular Biology</center><br />
Image:Washington_Sean_Wu.jpg|<center>Sean Wu <br/> Computer Science and Engineering</center><br />
Image:Washington_2011_BATMAN.jpg|<center>Lei Zheng <br/> Biochemistry</center><br />
Image:Washington_david_zong.jpg|<center>David Zong <br/> Bioengineering</center><br />
<br />
</gallery><br />
<br />
<br />
<gallery caption="Advisors" widths="180px" heights="120px" perrow="4"><br />
Image:Washington_.jpg|<center>Aaron Chevalier <br/> Bioengineering </center><br />
Image:Regbert.jpg|<center>Rob Egbert <br/> Electrical Engineering </center><br />
Image:Washington_.jpg|<center>Chris Eiben <br/> Biochemistry </center><br />
Image:Washington_.jpg|<center>Jeremy Mills <br/> Biochemistry </center><br />
Image:Siegel_UW_2011.jpg|<center>Justin Siegel <br/> Biomolecular Structure and Design</center><br />
Image:Washington2011_Matthew-d-smith.jpg|<center>Matt Smith <br/> Molecular and Cellular Biology</center><br />
Image:Washington_.jpg|<center>Ingrid Swanson Pultz<br/> Microbiology </center><br />
</gallery><br />
<br />
<br />
<gallery caption="Faculty" widths="180px" heights="120px" perrow="5"><br />
image:Washington_David_Baker.jpg|<center>David Baker <br/> Biochemistry</center><br />
Image:Washington_Klavins.jpg|<center>Eric Klavins <br/> Electrical Engineering</center><br />
</gallery><br />
<br />
<br />
<gallery caption="Collaborators and Support" widths="140px" heights="100px" perrow="5"><br />
<br />
Image:Washington_OSLI.png|<center><b>Oil Sand Leadership Initiative</b> <br/> Funding to support Travel and Registration Costs</center><br />
<br />
Image:Washington_UniversitySeal.gif|<center><b>Biochemistry</b> <br/> Lab space</center><br />
<br />
Image:Washington_Anaspec.gif|<center>Anaspec <br/> <b>Peptide Discounts</b></center><br />
<br />
Image:Washington_ARPA-E_Logo.png|<center><b>Advanced Research Projects Agency - Energy </b><br/> Registration Support</center><br />
<br />
Image:Washington2011_Hhmi_362_72.jpg|<center><b>Howard Hughes Medical Institute</b> <br/> Lab materials and supplies</center><br />
<br />
<br />
</gallery><br />
<br />
== '''Who did what''' ==<br />
The teams were assembled during our winter quarter. During this term we went around to classes and had weekly meetings to introduce students to synthetic biology through a series of guest lectures. During the spring, students participated in brainstorming projects related to the sponsoring Faculty advisors labs (Baker and Klavins) and came up with the project they wanted to carry out. Students also planned and partcipated in community outreach events where we taught our community about synthetic biology. Over the summer, students completed all of the experimental work, but worked closely with both graduate students and faculty advisors to plan the most pertinent experiments and make the most of our limited time.<br />
<br />
=== Alkanes Production ===<br />
After producing promising results, in future directions <br />
<br />
Casey Ager, Austin Moon, and Seth Sagulo worked on Enzyme Localization via Direct Fusion and Zinc Finger Fusion methods.<br />
<br />
Juhye An, Elaine Lai, and Benjamin Mo worked on Decarbonylase Redesign<br />
<br />
Marika Cheng and Justin De Leon worked on Alternative Chassis<br />
<br />
Chris Choe and David Zong worked on Alternate Aldehyde Production<br />
<br />
Matthew Harger worked on Branched Alkanes Production<br />
<br />
Matthew Harger and Lei Zheng worked on System Optimization<br />
<br />
=== Gluten Degradation ===<br />
Sydney Gordon, Daniel Hadidi, Liz Stanley, Angus Toland, Sarah Wolf, and Sean Wu designed, built, and tested Kumamolisin-As and over 100 mutants to combat gluten intolerance by increasing the activity on the PQLP antigenic peptide.<br />
<br />
=== iGEM Toolkits ===<br />
Michael Brasino, Rashmi Ravichandran, and Alicia Wong<br />
<br />
=== Community Outreach ===<br />
Cindy Wu headed and organized all iGEM 2011 Community Outreach events.</div>LStanley