Difference between revisions of "Team:San Andres/Software"

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   <div class="gwd-div-7xzo gwd-a-1thu">
−
     <h1>Parts&nbsp;</h1>
+
     <h1>Enzymes</h1>
−
     <big>Throughout the project we started to learn the fundamental
+
     <big>During our investigation we sought the perfect enzyme to
−
principles of synthetic biology to get to work on our plasmid with
+
degrade the gluten, and we found:<br>
−
which we want to see gluten degradation via the enzyme Kumamax. For
+
−
this we going to insert in an e. coli the parts (Biobricks) needed to
+
−
make our future bacteria can degrade gluten. The parts are:<br>
+
 
</big>
 
</big>
 
     <ul>
 
     <ul>
−
       <li><big><span style="font-family: 'Arial','sans-serif';"><strong style="font-weight: bold;">Promoter
+
       <li><big>Prolyl Endopeptidase: <span style="" lang="EN-US">The prolyl
−
    <a href="http://parts.igem.org/Part:BBa_J23119">(BBa_J23119</a></strong><a href="http://parts.igem.org/Part:BBa_J23119"><span style="font-weight: bold;">)</span></a>:
+
endopeptidase (PEP), is a class of serine-protease able to break
−
    </span>Constitutive
+
peptide bonds
−
promoter (which works permanently) that is give in the relative
+
following a PROLINE residue terminal carboxyl group. Were studying it
−
fluorescence of these plasmids in the TG1 strain grown in LB medium.</big>
+
its use
 +
as a therapeutic agent against celiac disease (CD), characterized by
 +
atrophy of
 +
the intestinal villi and inflammation. These reactions are attributed
 +
to
 +
peptides rich in PROLINE that are generated during the digestion of
 +
gluten of
 +
some cereals. This enzyme could be used as a nutritional supplement for
 +
individuals who suffer from celiac disease or during the processing of
 +
starches
 +
produced from cereals containing gluten. While it was a candidate for a
 +
possible treatment failed to meet expectations, because its activity is
 +
to a
 +
high pH, which is not suitable for an average digestive. Another reason
 +
was
 +
that degrades the gluten slowly, which would have resulted in a longer
 +
and
 +
inefficient treatment.</span><big>&nbsp;</big></big>
 
       </li>
 
       </li>
−
       <li><big><strong style="font-weight: bold;">RBS</strong><strong style="font-weight: bold;">
+
       <br>
−
    <a href="http://parts.igem.org/Part:BBa_K1084103">(BBa_K1084103)</a></strong>:
+
−
Synthetic RBS with uplifting sequence</big>.</li>
+
−
      <li><big><strong>Vector:<span style="font-weight: normal;"> <a href="http://parts.igem.org/Part:pSB1C3">pSB1C3</a></span></strong></big>
+
−
      </li>
+
−
      <li><big><strong><span style="font-family: 'Arial','sans-serif';">Coding Region:
+
−
KumaMax <a href="http://parts.igem.org/Part:BBa_K590087">(BBa_K590087)</a>:</span></strong>
+
−
It
+
−
degrades gluten,
+
−
celiac disease leading cause. Enzyme generated by rational mutation for
+
−
the active site of it. It was created by the team IGEM <a href="https://2011.igem.org/Team:Washington/Celiacs/Background">Washington
+
−
2011.</a></big>
+
−
      </li>
+
−
      <li><big><strong><span style="font-family: 'Arial','sans-serif';">Reporter: RFP
+
−
    <a href="http://parts.igem.org/Part:BBa_J04450">(BBa_J04450)</a>:</span></strong><span style="font-family: Arial,sans-serif;"> Red f</span>luorescence
+
−
protein.</big>
+
−
      </li>
+
−
      <li><big><span style="font-family: Arial,sans-serif;"><strong>Terminator
+
−
    <a href="http://parts.igem.org/Part:BBa_B0015">(BBa_B0015)</a>:</strong>
+
−
    </span>Dual terminator consisting of
+
−
the B0010 and B0012 parties. It serves to give greater efficiency in
+
−
transcription</big>.</li>
+
 
     </ul>
 
     </ul>
 
     <div style="text-align: center;">
 
     <div style="text-align: center;">
−
       <img alt="File:Parts.jpg" src="https://static.igem.org/mediawiki/2015/e/e8/Parts.jpg" height="59" width="288">
+
       <img alt="File:Prolil.jpg" src="https://static.igem.org/mediawiki/2015/c/c0/Prolil.jpg" height="376" width="565">
 
       <br>
 
       <br>
−
      <img alt="File:Circuito.jpg" src="https://static.igem.org/mediawiki/2015/thumb/2/2a/Circuito.jpg/800px-Circuito.jpg" height="309" width="800">
 
 
       <br>
 
       <br>
−
       <div style="text-align: left;"><big>This is a
+
       <div style="text-align: left;">
−
graphic model of as it has be our plasmid where we can visualize the
+
        <ul>
−
promoter, the RBS, the enzyme KumaMax, the RFP and the terminator,
+
          <li><big>Kumamolisin As: It is the first known example of
−
joined by means of prefixes and suffixes that indicate the locations of
+
a collagenase derived from the family of the sedolisin. This operates
−
court.</big>
+
at high temperatures and low pH levels. Its characteristics, together
−
        <br>
+
with those predicted are measured by comparison between a collagenase
−
         <br>
+
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:Plasmido.jpg" src="https://static.igem.org/mediawiki/2015/thumb/c/ce/Plasmido.jpg/664px-Plasmido.jpg" height="600" width="664">
+
           <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>
 +
          <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
 +
efficient gluten, because that can work at pH levels much more lower
 +
than the original enzyme (a pH of 4.0) which is excellent for the
 +
average digestive system. <span class="hps">It was
 +
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
 +
of the stomach.</big>
 +
            </li>
 +
            <li><big>It is heat stable, in others words, it is
 +
resistant to all changes in their physical and chemical structure.</big>
 +
            </li>
 +
            <li><big>It is easily repairable and creable.</big>
 +
            </li>
 +
          </ol>
 +
          <div style="text-align: center;">
 +
            <img alt="File:175px-Washington Bottle.jpg" src="https://static.igem.org/mediawiki/2015/d/d0/175px-Washington_Bottle.jpg" height="263" width="175">
 +
            <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">&nbsp;
 +
            <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">
 +
            <br>
 +
            <br>
 +
            <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&nbsp;</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>
 +
              <p class="MsoNormal"><big><span style="" lang="EN-US">- For our
 +
Plasmid construction, we decided to take the method standard of
 +
Assembly
 +
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
 +
cut the two genes and making them again an insert front, which we took
 +
to the
 +
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
 +
for
 +
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>
 +
            <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;">
 +
            <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;">
 +
              <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">
 +
              <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>
 +
                <br>
 +
                <div style="text-align: center;">
 +
                  <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>
 +
              </div>
 +
            </div>
 +
            <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>
 +
</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>
 +
            <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;">
 +
              <br>
 +
              <br>
 +
            </div>
 +
            <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;">
 +
              <br>
 +
            </div>
 +
          </div>
 
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−
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Revision as of 00:11, 14 August 2015

wiki exp wiki wiki wiki 2   File:Gluten-s-Job.jpeg

Enzymes

During our investigation we sought the perfect enzyme to degrade the gluten, and we found:
  • Prolyl Endopeptidase: The prolyl endopeptidase (PEP), is a class of serine-protease able to break peptide bonds following a PROLINE residue terminal carboxyl group. Were studying it its use as a therapeutic agent against celiac disease (CD), characterized by atrophy of the intestinal villi and inflammation. These reactions are attributed to peptides rich in PROLINE that are generated during the digestion of gluten of some cereals. This enzyme could be used as a nutritional supplement for individuals who suffer from celiac disease or during the processing of starches produced from cereals containing gluten. While it was a candidate for a possible treatment failed to meet expectations, because its activity is to a high pH, which is not suitable for an average digestive. Another reason was that degrades the gluten slowly, which would have resulted in a longer and inefficient treatment. 

File:Prolil.jpg

  • Kumamolisin As: It is the first known example of 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.
File:2-2-2 2.jpg
  • KumaMax (G319S, D358G, D368H, N281D): It is a mutation of the Kumamolisin As, which is designed to digest way more efficient gluten, because that can work at pH levels much more lower than the original enzyme (a pH of 4.0) which is excellent for the average digestive system. It was created by the team IGEM Washington 2011. Other advantages are:
  1. It is resistant to high temperatures and acidity of the stomach.
  2. It is heat stable, in others words, it is resistant to all changes in their physical and chemical structure.
  3. It is easily repairable and creable.
File:175px-Washington Bottle.jpg File:250px-Washington Kumamolisin VS SC-PEP.png  File:250px-Washington Kuma Bonded triad.png

Metodology 

The methodology consists of:

- For our Plasmid construction, we decided to take the method standard of Assembly biobrick, based on grafts and vectors, front and reverse.

- Then we cut the two genes and making them again an insert front, which we took to the vector of the terminator making as well as the genes a front vector.

- After making two front inserts, we proceed to start with two reverse inserts for 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.

- 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".


https://static.igem.org/mediawiki/2015/c/c8/Metodology_2.jpg
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.

File:Kit gluten.jpg