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| + | <title>wiki</title> |
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| <div class="gwd-div-7xzo gwd-a-1thu"> | | <div class="gwd-div-7xzo gwd-a-1thu"> |
− | <h1>Enzymes</h1> | + | <h1>Results </h1> |
− | <big>During our investigation we sought the perfect enzyme to | + | <big>The DNA sequences of the proteins KumaMax and RFP are |
− | degrade the gluten, and we found:<br>
| + | isolated to begin building our plasmid.<br> |
| + | <br> |
| </big> | | </big> |
− | <ul>
| |
− | <li><big>Prolyl Endopeptidase: <span style="" lang="EN-US">The prolyl
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− | endopeptidase (PEP), is a class of serine-protease able to break
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− | peptide bonds
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− | following a PROLINE residue terminal carboxyl group. Were studying it
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− | its use
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− | as a therapeutic agent against celiac disease (CD), characterized by
| |
− | atrophy of
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− | the intestinal villi and inflammation. These reactions are attributed
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− | to
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− | peptides rich in PROLINE that are generated during the digestion of
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− | gluten of
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− | some cereals. This enzyme could be used as a nutritional supplement for
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− | individuals who suffer from celiac disease or during the processing of
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− | starches
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− | produced from cereals containing gluten. While it was a candidate for a
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− | possible treatment failed to meet expectations, because its activity is
| |
− | to a
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− | high pH, which is not suitable for an average digestive. Another reason
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− | was
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− | that degrades the gluten slowly, which would have resulted in a longer
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− | and
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− | inefficient treatment.</span><big> </big></big>
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− | </li>
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− | <br>
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− | </ul>
| |
| <div style="text-align: center;"> | | <div style="text-align: center;"> |
− | <img alt="File:Prolil.jpg" src="https://static.igem.org/mediawiki/2015/c/c0/Prolil.jpg" height="376" width="565"> | + | <img alt="File:Biobricks.jpg" src="https://static.igem.org/mediawiki/2015/c/c5/Biobricks.jpg" height="249" width="412"> |
− | <br> | + | <img style="width: 395px; height: 250px;" alt="File:Electroforesis.png" src="https://static.igem.org/mediawiki/2015/b/bc/Electroforesis.png"> |
| + | </div> |
| + | <div style="text-align: center;"> |
| <br> | | <br> |
| <div style="text-align: left;"> | | <div style="text-align: left;"> |
− | <ul> | + | <br> |
− | <li><big>Kumamolisin As: It is the first known example of
| + | <br> |
− | a collagenase derived from the family of the sedolisin. This operates
| + | |
− | at high temperatures and low pH levels. Its characteristics, together
| + | |
− | with those predicted are measured by comparison between a collagenase
| + | |
− | and a peptidase from serine, which are related to the enzyme
| + | |
− | preference, to thus Digest collagen as gluten.</big>
| + | |
− | </li>
| + | |
− | </ul> | + | |
| <div style="text-align: center;"> | | <div style="text-align: center;"> |
− | <img alt="File:2-2-2 2.jpg" src="https://static.igem.org/mediawiki/2015/b/be/2-2-2_2.jpg" height="243" width="470">
| |
| <br> | | <br> |
− | <ul style="text-align: left;">
| |
− | <li><big>KumaMax (G319S, D358G, D368H, N281D): It is a
| |
− | mutation of the Kumamolisin As, which is designed to digest way more
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− | efficient gluten, because that can work at pH levels much more lower
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− | than the original enzyme (a pH of 4.0) which is excellent for the
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− | average digestive system. <span class="hps">It was
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− | created by the team IGEM <a href="https://2011.igem.org/Team:Washington/Celiacs/Background">Washington
| |
− | 2011</a></span>. Other advantages
| |
− | are:</big>
| |
− | </li>
| |
− | </ul>
| |
− | <ol style="text-align: left;">
| |
− | <li><big>It is resistant to high temperatures and acidity
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− | of the stomach.</big>
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− | </li>
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− | <li><big>It is heat stable, in others words, it is
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− | resistant to all changes in their physical and chemical structure.</big>
| |
− | </li>
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− | <li><big>It is easily repairable and creable.</big>
| |
− | </li>
| |
− | </ol>
| |
− | <div style="text-align: center;">
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− | <img alt="File:175px-Washington Bottle.jpg" src="https://static.igem.org/mediawiki/2015/d/d0/175px-Washington_Bottle.jpg" height="263" width="175">
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− | <img alt="File:250px-Washington Kumamolisin VS SC-PEP.png" src="https://static.igem.org/mediawiki/2015/1/1b/250px-Washington_Kumamolisin_VS_SC-PEP.png" height="213" width="250">
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− | <img alt="File:250px-Washington Kuma Bonded triad.png" src="https://static.igem.org/mediawiki/2015/6/62/250px-Washington_Kuma_Bonded_triad.png" height="193" width="250">
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− | <br>
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− | <br>
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− | <h1 style="border-bottom: 1px solid rgb(170, 170, 170); margin: 0px 0px 0.6em; background: transparent none repeat scroll 0% 50%; -moz-background-clip: initial; -moz-background-origin: initial; -moz-background-inline-policy: initial; color: rgb(0, 0, 0); font-weight: normal; padding-top: 0.5em; padding-bottom: 0.17em; font-size: 23.876px; font-family: sans-serif; font-style: normal; font-variant: normal; letter-spacing: normal; line-height: 19.05px; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 1; word-spacing: 0px;">Metodology </h1>
| |
− | <div style="text-align: left;"><big style="color: rgb(0, 0, 0); font-family: sans-serif; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.05px; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 1; word-spacing: 0px;">The
| |
− | methodology consists of:<br>
| |
− | </big>
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− | <p class="MsoNormal"><big><span style="" lang="EN-US">- For our
| |
− | Plasmid construction, we decided to take the method standard of
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− | Assembly
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− | biobrick, based on grafts and vectors, front and reverse.<o:p></o:p></span></big>
| |
− | </p>
| |
− | <p class="MsoNormal"><big><span style="" lang="EN-US">- Then we
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− | cut the two genes and making them again an insert front, which we took
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− | to the
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− | vector of the terminator making as well as the genes a front vector.<o:p></o:p></span></big>
| |
− | </p>
| |
− | <p class="MsoNormal"><big><span style="" lang="EN-US">- After
| |
− | making two front inserts, we proceed to start with two reverse inserts
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− | for
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− | where we cut the three genes (Kumamax, RFP, terminator) and we make
| |
− | them a
| |
− | reverse insert, and we took him to the inverse vector of the RBS.<o:p></o:p></span></big>
| |
− | </p>
| |
− | <p class="MsoNormal"><span style="" lang="EN-US"><big>-
| |
− | Finally
| |
− | we proceed to cut the four genes (RBS, Kumamax, RFP, terminator) as
| |
− | insert
| |
− | reverse, to take them to the inverse vector of the promoter, and we
| |
− | finished
| |
− | building our final plasmid, the "Kumamax Plux".</big><o:p></o:p></span>
| |
− | </p>
| |
− | <p class="MsoNormal"><span style="" lang="EN-US"><o:p></o:p></span>
| |
− | </p>
| |
− | </div>
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− | <br style="color: rgb(0, 0, 0); font-family: sans-serif; font-size: 12.7px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.05px; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 1; word-spacing: 0px;">
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− | <div style="color: rgb(0, 0, 0); font-family: sans-serif; font-size: 12.7px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.05px; text-indent: 0px; text-transform: none; white-space: normal; widows: 1; word-spacing: 0px; text-align: center;">
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− | <img class="shrinkToFit" alt="https://static.igem.org/mediawiki/2015/c/c8/Metodology_2.jpg" src="https://static.igem.org/mediawiki/2015/c/c8/Metodology_2.jpg" style="border: medium none ; vertical-align: middle;" height="657" width="755">
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− | <div style="text-align: left;"><big>At this time we
| |
− | are innovating ideas to add a circuit that will allow us in the future
| |
− | to obtain a method of detecting and quantifying the presence of gluten,
| |
− | which can also be checked by a commercial kit.</big>
| |
− | <br>
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− | <br>
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− | <div style="text-align: center;">
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− | <img alt="File:Kit gluten.jpg" src="https://static.igem.org/mediawiki/2015/0/09/Kit_gluten.jpg" style="border: medium none ; vertical-align: middle;" height="513" width="593">
| |
− | </div>
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− | </div>
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− | </div>
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− | <big style="color: rgb(0, 0, 0); font-family: sans-serif; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.05px; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 1; word-spacing: 0px;"><br>
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− | </big><span style="color: rgb(0, 0, 0); font-family: sans-serif; font-size: 12.7px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.05px; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 1; word-spacing: 0px; display: inline ! important; float: none; background-color: rgb(255, 255, 255);"></span>
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− | <br>
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− | <br>
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− | </div>
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− | <div style="color: rgb(0, 0, 0); font-family: sans-serif; font-size: 12.7px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 19.05px; text-indent: 0px; text-transform: none; white-space: normal; widows: 1; word-spacing: 0px; text-align: center;">
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− | <br>
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− | </div>
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− | </div>
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| </div> | | </div> |
| </div> | | </div> |
| + | </div> |
| + | <big><br> |
| + | </big> |
| + | <div style="text-align: center;"> |
| + | <br> |
| + | <br> |
| + | </div> |
| + | <div style="text-align: center;"> |
| + | <br> |
| + | <br> |
| </div> | | </div> |
| </div> | | </div> |
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