Difference between revisions of "Team:HSNU-TAIPEI/projectBP"

 
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<li class="list-item"><a href="https://2015.igem.org/Team:HSNU-TAIPEI/Description">Overview</a></li>
 
<li class="list-item"><a href="https://2015.igem.org/Team:HSNU-TAIPEI/Description">Overview</a></li>
 
<li class="list-item"><a href="https://2015.igem.org/Team:HSNU-TAIPEI/projectBP">Benzo[A]Pyrene</a></li>
 
<li class="list-item"><a href="https://2015.igem.org/Team:HSNU-TAIPEI/projectBP">Benzo[A]Pyrene</a></li>
−
<li class="list-item"><a href="https://2015.igem.org/Team:HSNU-TAIPEI/projectcopper">Copper</a></li>
 
 
<li class="list-item"><a href="https://2015.igem.org/Team:HSNU-TAIPEI/projectcadmium">Cadmium</a></li>
 
<li class="list-item"><a href="https://2015.igem.org/Team:HSNU-TAIPEI/projectcadmium">Cadmium</a></li>
−
<li class="list-item"><a href="https://2015.igem.org/Team:HSNU-TAIPEI/projectmercury">Mercury</a></li>
 
 
<li class="list-item"><a href="https://2015.igem.org/Team:HSNU-TAIPEI/projectlead">Lead</a></li>
 
<li class="list-item"><a href="https://2015.igem.org/Team:HSNU-TAIPEI/projectlead">Lead</a></li>
 
<li class="list-item"><a href="https://2015.igem.org/Team:HSNU-TAIPEI/projectafla">Aflatoxin</a></li>
 
<li class="list-item"><a href="https://2015.igem.org/Team:HSNU-TAIPEI/projectafla">Aflatoxin</a></li>
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<main>
 
<main>
 
<div class="mc-container">
 
<div class="mc-container">
−
 
+
      <h1>Project<span><em>Benzo[A]Pyrene</em></span></h1>
−
<h1>Project<span><em>Benzo[A]Pyrene</em></span></h1>
+
 
<div class="section">
 
<div class="section">
 
   <article class="article">
 
   <article class="article">
Line 102: Line 105:
 
       <li>
 
       <li>
 
         <span>Why do we detect benzo[a]pyrene?</span>
 
         <span>Why do we detect benzo[a]pyrene?</span>
−
         <p class="article-p">Because when food,such as fried food ,which is heated above 300 degrees celcius,food will release benzo[a]pyrene which recycled oil usually contain[1].</p>
+
         <p class="article-p">Because when food, such as fried food , is heated above 300 degrees celcius, benzo[a]pyrene is released. That is why recycled oil usually contains benzo[a]pyrene [1].</p>
 
       </li>
 
       </li>
 
       <li>
 
       <li>
 
         <span>The harm of benzo[a]pyrene</span>
 
         <span>The harm of benzo[a]pyrene</span>
−
         <p class="article-p">Pathway:from skin,breathing in,eating Carcinogenic,environmental pollution, Skin irritation,eye irritation[1].</p>
+
         <p class="article-p">Pathway: from skin, breathing in, and eating. It is carcinogenic and causes environmental pollution. It causes skin irritation and eye irritation [1].</p>
 
       </li>
 
       </li>
 
       <li>
 
       <li>
 
         <span>Taiwanese regulation</span>
 
         <span>Taiwanese regulation</span>
 +
        <p class="article-p table-note">&#9660;Table1:The regulation of Benzo[a]pyrene in Taiwan.</p>
 
         <div class="article-img"><img src="https://static.igem.org/mediawiki/2015/9/94/2015hsnu-benzo-a-pyrene_5.png"></div>
 
         <div class="article-img"><img src="https://static.igem.org/mediawiki/2015/9/94/2015hsnu-benzo-a-pyrene_5.png"></div>
 
       </li>
 
       </li>
 
       <li>
 
       <li>
 
         <span>National regulation</span>
 
         <span>National regulation</span>
−
        <ul class="article-ul">
+
      </li>
−
           <li>Europe:The same to Taiwanese regulation[2]</li>
+
      <ul class="article-ul">
 +
           <li>Europe:same to Taiwanese regulation[2]</li>
 
           <li>American:The MCL has been set at 0.2 ppb[3]</li>
 
           <li>American:The MCL has been set at 0.2 ppb[3]</li>
−
        </ul>
+
      </ul>
−
      </li>
+
 
     </ol>
 
     </ol>
 
   </article>
 
   </article>
Line 125: Line 129:
 
     <p class="article-p">So far,we don’t find the protein which can combine with the Benzo[a]pyrene,so first we must degradate it by Laccase. And its product is Bap-1,6-quinone which can combine with the protein,in order that we can detect Benzo[a]pyrene.[4] We put the gene fragment which can produce the Laccase in the E.coli. Let it produce itself.</p>
 
     <p class="article-p">So far,we don’t find the protein which can combine with the Benzo[a]pyrene,so first we must degradate it by Laccase. And its product is Bap-1,6-quinone which can combine with the protein,in order that we can detect Benzo[a]pyrene.[4] We put the gene fragment which can produce the Laccase in the E.coli. Let it produce itself.</p>
 
     <div class="article-img"><img src="https://static.igem.org/mediawiki/2015/e/ed/2015hsnu-benzo-a-pyrene_2.png"></div>
 
     <div class="article-img"><img src="https://static.igem.org/mediawiki/2015/e/ed/2015hsnu-benzo-a-pyrene_2.png"></div>
 +
    <p class="article-p">&#9650;Fig.1-1:The circuit of detecting Benzo[a]pyrene.</p>   
 
     <p class="article-p">QsrR is a protein which can combine with Bap-1,6-quinone.[5]As Laccase,we put the gene fragment in the E.coli. Let it produce itself.</p>
 
     <p class="article-p">QsrR is a protein which can combine with Bap-1,6-quinone.[5]As Laccase,we put the gene fragment in the E.coli. Let it produce itself.</p>
 
     <div class="article-img"><img src="https://static.igem.org/mediawiki/2015/7/7d/2015hsnu-benzo-a-pyrene_3.png"></div>
 
     <div class="article-img"><img src="https://static.igem.org/mediawiki/2015/7/7d/2015hsnu-benzo-a-pyrene_3.png"></div>
 +
    <p class="article-p">&#9650;Fig.1-2:The circuit of detecting Benzo[a]pyrene.</p>       
 
     <p class="article-p">We design a gene part make QsrR repress sfRFP’s production. Usually,QsrR is on the Binding Site,and red fluorescent protein will not be produced. When QsrR combine with Bap-1,6-quinone, QsrR will be activated and go away. And the transcription will carry on,then it will produce red fluorescent protein.</p>
 
     <p class="article-p">We design a gene part make QsrR repress sfRFP’s production. Usually,QsrR is on the Binding Site,and red fluorescent protein will not be produced. When QsrR combine with Bap-1,6-quinone, QsrR will be activated and go away. And the transcription will carry on,then it will produce red fluorescent protein.</p>
 
     <div class="article-img"><img src="https://static.igem.org/mediawiki/2015/8/8e/2015hsnu-benzo-a-pyrene_4.png"></div>
 
     <div class="article-img"><img src="https://static.igem.org/mediawiki/2015/8/8e/2015hsnu-benzo-a-pyrene_4.png"></div>
 +
    <p class="article-p">&#9650;Fig.1-3:The circuit of detecting Benzo[a]pyrene.</p>   
 
   </article>
 
   </article>
−
<article class="article">
+
<article class="article">
−
              <h3 class="article-title">Result</h3>
+
    <h3 class="article-title">Result</h3>
−
<ol class="article-ol">
+
<p>Whether e.coli is alive in the poisons, condition or not</p>
−
<li>
+
    <ol type="A">
−
<span>Whether benzo[a]pyrene can enter e.coli or not</span>
+
      <li>Method</li>
−
<ol class="article-ol" type="A">
+
      <h3>DH5&#945;-Pretest</h3>
−
<li><span>Method</span>
+
      <h4>Procedure</h4>
−
<h4 class="article-h4">Detection of the amount of toxins in the e.coli.</h4>
+
      <p>Because we must test E.coli’s Survival in the environment there is benzo[a]pryene by counting the colonies,First we test how much concentration is the best.</p>
−
<ol class="article-ol">
+
      <ol>
−
  <li>Add 100&#956;l of DH5&#945; and 900&#956;l of LB broth into the tube and incubate for 1hr.</li>
+
        <li>
−
  <li>Centrifuge at 4000rpm for 3min and clicard 800&#956;l of the supernatant</li>
+
          <p>culture</p>
−
  <li>
+
          <p>STEP1:take 1&#956;L DH5&#945; to spread the plate(no Antibiotic)</p>
−
    <p class="article-p">Plate each 100&#956;l of the bacteria onto the dishes and spread.</p>
+
          <p>STEP2:put in 37 degree Celsius 12~16hr</p>
−
    <p class="article-p">Incubate the plates at 37&#8451; overnight</p>
+
        </li>
−
    </li>
+
        <li>
−
  <li>
+
          <p>liquid culture</p>
−
    <p class="article-p">Prepare each concentration of the toxin.</p>
+
        </li>
−
    <p class="article-p">Statutory standards *100 / *10 / *1 / *0.1 / *0.01</p>
+
        <li>
−
  </li>
+
          <p>STEP1:put 80&#956;L into 2ml LB broth </p>
−
</ol>
+
          <p>STEP2:recovering</p>
−
<h4 class="article-h4">Next day</h4>
+
          <p>STEP3: After 2hr,dilute it to 10<sup>-4</sup>,10<sup>-5</sup>,10<sup>-6</sup>,10<sup>-7</sup>,and then go to spread the plate (no Antibiotic)</p>
−
<ol class="article-ol">
+
          <p>STEP4: After 4hr dilute it to 10<sup>-4</sup>, 10<sup>-5</sup> ,10<sup>-6</sup> ,10<sup>-7</sup> ,and then go to spread the plate (no Antibiotic), 6hr and 8hr Similarly</p>
−
  <li>
+
          <p>STEP5:Take 200&#956;L out from the tube and spread the plate(AMP+)</p>
−
    <p class="article-p">Prepare 16 microcentrifuge tubes.(5 kinds of concentration *3 timings+control)</p>
+
          <p>STEP6: put in  37 degree Celsius 12~16hr</p>
−
    <p class="article-p">Add 500&#956;l of DH5&#945; to each tube.</p>
+
         </li>
−
    <p class="article-p">Centrifuge all tubes at 4000rpm for 3min.</p>
+
−
    <p class="article-p">Remove the supernatent.</p>
+
−
  </li>
+
−
  <li>
+
−
    <p class="article-p">Add 1000&#956;l of the toxic solution each time.</p>
+
−
    <p class="article-p">Follow the concentration and 3 timings(0.5hr / 1hr / 1.5hr).</p>
+
−
  </li>
+
−
  <li>
+
−
    <ol class="article-ol">
+
−
      <li>Add 0.5cc of ddH<sub>2</sub>O and mix with the bacterias</li>
+
−
      <li>Centrifuge at 13000rpm for 30 sec</li>
+
−
      <li>Remove the water</li>
+
−
      <li>Repeat step1~step3 for three times</li>
+
−
    </ol>
+
−
  </li>
+
−
  <li>
+
−
    <p class="article-p">Add 1cc of ddH<sub>2</sub>O and mix with the bacterias</p>
+
−
    <p class="article-p">Centrifuge at 13000rpm for 30sec.</p>
+
−
    <p class="article-p">Remove 700&#956;l of the supernatant</p>
+
−
  </li>
+
−
  <li>
+
−
    <p class="article-p">Kill the bacteria:</p>
+
−
      <ol class="article-ol">
+
−
         <li>Put all the tubes in the Liquid nitrogen</li>
+
−
        <li>When they freeze,heat them at 100&#8451;</li>
+
−
        <li>Repeat step1~step2 for 3 times</li>
+
 
       </ol>
 
       </ol>
−
  </li>
+
      <h3>Survival</h3>
−
</ol>
+
      <h4>Procedure</h4>
−
 
+
      <p>First we culture DH5α with LB only plate for 15hr. Then,pick one colony in the LB broth,and liquid culture for 15hr. We divided two categories A and B.</p>
−
</li>
+
      <h5>A:</h5>
−
<li><span>Result</span></li>
+
      <ol>
−
</ol>
+
        <li>Take 80&#956;L into 2ml LB broth &#215; 6 tubes and then culture 1 hr.</li>
−
</li>
+
        <li>After 1hr,add 20&#956;L benzo[a]pryene into three tubes(conc. Is 2000ppb(A thousand times the standard value))</li>
−
<li>
+
        <li>And add 20&#956;L DMSO into the other tubes.Then,culture for 3hr.</li>
−
<span>Whether e.coli is alive in the poisons, condition or not</span>
+
        <li>After 3hr,dilute the broth to 10<sup>-6</li>
−
<ol class="article-ol" type="A">
+
        <li>And take 200&#956;L to spread the plate.</li>
−
<li><span>Method</span>
+
      </ol>
−
<div class="section note">
+
      <h5>B:</h5>
−
  <h2 class="note-title">DH5&#945;-Pretest</h2>
+
      <ol>
−
  <div class="note-content">
+
        <li>Take 80&#956;L into 2ml LB broth  in a tube And then culture 1 hr.</li>
−
    <h3 class="note-subtitle">Procedure</h3>
+
        <li>After 1hr, put them into 6 tubes equally.</li>
−
  <p class="note-caption">Because we must test E.coli’s Survival in the environment there is benzo[a]pryene by counting the colonies,First we test how much concentration is the best.</p>
+
        <li>Dilute the broth to 5&#215;10<sup>-4</sup></li>
−
    <ol class="note-ordered-list">
+
        <li>Add 0.4&#956;L benzo[a]pryene(2&#215;10<sup>-4</sup>) in three tubes.</li>
−
      <li>
+
        <li>Add 0.4&#956;L DMSO in the other three tubes.</li>
−
         <p class="note-caption">culture</p>
+
         <li>Go to 37 degree Celsius shaking for 10min.</li>
−
         <p class="note-caption">STEP1:take 1&#956;L DH5&#945; to spread the plate(no Antibiotic)</p>
+
         <li>Take 200&#956;L to spread the plate.</li>
−
         <p class="note-caption">STEP2:put in 37 degree Celsius 12~16hr</p>
+
      </ol>
−
       </li>
+
      <figure>
−
       <li>
+
        <figcaption>&#9660;Table2: E. coli on the agar plate.</figcaption>
−
         <p class="note-caption">liquid culture</p>
+
         <img src="https://static.igem.org/mediawiki/2015/e/ee/HSNU-TAIPEI-BZP-904-1.jpg" width="70%">
−
       </li>
+
      </figure>
−
       <li>
+
       <li>Results</li>
−
         <p class="note-caption">(8/19)</p>
+
       <figure>
−
         <p class="note-caption">STEP1:put 80&#956;L into 2ml LB broth </p>
+
         <figcaption>&#9660;Table3: E. coli on the agar plate.</figcaption>
−
        <p class="note-caption">STEP2:recovering</p>
+
        <img src="https://static.igem.org/mediawiki/2015/2/2c/HSNU-TAIPEI-BZP-820-4.jpg">
−
         <p class="note-caption">STEP3: After 2hr,dilute it to 10<sup>-4</sup>,10<sup>-5</sup>,10<sup>-6</sup>,10<sup>-7</sup>,and then go to spread the plate (no Antibiotic)</p>
+
      </figure>
−
        <p class="note-caption">STEP4: After 4hr dilute it to 10<sup>-4</sup>, 10<sup>-5</sup> ,10<sup>-6</sup> ,10<sup>-7</sup> ,and then go to spread the plate (no Antibiotic), 6hr and 8hr Similarly</p>
+
      <p>
−
        <p class="note-caption">STEP5:Take 200&#956;L out from the tube and spread the plate(AMP+)</p>
+
        The number of the colonies in the AMP+ plate is zero.<br>
−
         <p class="note-caption">STEP6: put in 37 degree Celsius 12~16hr</p>
+
        According to the result, 2hr 10-5 and 4hr 10-6 is the best.
−
       </li>
+
       </p>
 +
       <figure>
 +
         <figcaption>&#9660;Table4:Line Chart of Table 3.</figcaption>
 +
        <img src="https://static.igem.org/mediawiki/2015/b/bc/HSNU-TAIPEI-BZP-result.jpg">
 +
      </figure>
 +
      <figure>
 +
         <img src="https://static.igem.org/mediawiki/2015/6/6b/HSNU-TAIPEI-BZP-820-1.jpg" width="50%">
 +
        <figcaption>&#9650;Fig.2:2hr agar only plate from left to rights are10<sup>-4</sup>, 10<sup>-5</sup>, 10<sup>-6</sup>, 10<sup>-7</sup>.</figcaption>
 +
      </figure>
 +
      <figure>
 +
         <img src="https://static.igem.org/mediawiki/2015/e/ef/HSNU-TAIPEI-BZP-820-2.jpg" width="50%">
 +
        <figcaption>&#9650;Fig3: 4hr agar only plate from left to rights are10<sup>-4</sup>, 10<sup>-5</sup>, 10<sup>-6</sup>, 10<sup>-7</sup>.</p>
 +
          <p class="note-caption">4hr plate from left to right is 10<sup>-4</sup>,10<sup>-5</sup>,10<sup>-6</sup>,10<sup>-7</sup></figcaption>
 +
      </figure>
 +
      <figure>
 +
        <img src="https://static.igem.org/mediawiki/2015/f/f7/HSNU-TAIPEI-BZP-820-3.jpg" width="50%">
 +
        <figcaption>&#9650;Fig4:Ampicillin Plate.</figcaption>
 +
      </figure>
 +
      <p>AMP+ Plate</p>
 +
      <p>According to the result, Beno[a]pryene does not affect E.coli’s survival.</p>
 +
      <p>But Category B is failed because its number of colony is too much.</p>
 +
      <figure>
 +
        <img src="https://static.igem.org/mediawiki/2015/d/d3/HSNU-TAIPEI-BZP-904-2.jpg" width="50%">
 +
        <figcaption>&#9650;Fig5: Benzo[a]pyrene Category A</figcaption>
 +
      </figure>
 +
      <p>Benzo[a]pryene Category A</p>
 +
      <figure>
 +
         <img src="https://static.igem.org/mediawiki/2015/6/66/HSNU-TAIPEI-BZP-904-3.jpg" width="50%">
 +
        <figcaption>&#9650;Fig6:Control Category A</figcaption>
 +
      </figure>
 +
      <p>Control Category A</p>
 +
      <figure>
 +
        <img src="https://static.igem.org/mediawiki/2015/6/69/HSNU-TAIPEI-BZP-904-4.jpg" width="50%">
 +
        <figcaption></figcaption>
 +
      </figure>
 +
      <p>&#9650;Fig7: Benzo[a]pyrene Category B</p>
 +
      <p>Benzo[a]pryene CategoryB</p>
 +
       <figure>
 +
        <img src="https://static.igem.org/mediawiki/2015/c/ce/HSNU-TAIPEI-BZP-904-5.jpg" width="50%">
 +
        <figcaption>&#9650;Fig8: Control Category B</figcaption>
 +
      </figure>
 +
      <p>Control  CategoryB</p>
 
     </ol>
 
     </ol>
−
 
+
   </article>
−
   </div>
+
−
</div>
+
−
 
+
−
<div class="section note">
+
−
  <h2 class="note-title">Survival</h2>
+
−
  <div class="note-content">
+
−
    <h3 class="note-subtitle">Procedure</h3>
+
−
      <p class="note-caption">First we culture DH5α with LB only plate for 15hr. Then,pick one colony in the LB broth,and liquid culture for 15hr.</p>
+
−
      <p class="note-caption">We divided two categories  A and B.</p>
+
−
    <h3 class="note-subtitle">A:</h3>
+
−
      <p class="note-caption">Take 80&#956;L into 2ml LB broth &#215; 6 tubes and then culture 1 hr.</p>
+
−
      <p class="note-caption">After 1hr,add 20&#956;L benzo[a]pryene into three tubes(conc. Is 2000ppb(A thousand times the standard value))</p>
+
−
      <p class="note-caption">And add 20&#956;L DMSO into the other tubes.Then,culture for 3hr.</p>
+
−
      <p class="note-caption">After 3hr,dilute the broth to 10<sup>-6</sup></p>
+
−
      <p class="note-caption">And take 200&#956;L to spread the plate.</p>
+
−
    <h3 class="note-subtitle">B:</h3>
+
−
      <p class="note-caption">Take 80&#956;L into 2ml LB broth  in a tube And then culture 1 hr.</p>
+
−
      <p class="note-caption">After 1hr, put them into 6 tubes equally.</p>
+
−
      <p class="note-caption">Dilute the broth to 5&#215;10<sup>-4</sup></p>
+
−
      <p class="note-caption">Add 0.4&#956;L benzo[a]pryene(2&#215;10<sup>-4</sup>) in three tubes.</p>
+
−
      <p class="note-caption">Add 0.4&#956;L DMSO in the other three tubes.</p>
+
−
      <p class="note-caption">Go to 37 degree Celsius shaking for 10min.</p>
+
−
      <p class="note-caption">Take 200&#956;L to spread the plate.</p>
+
−
    <img src="https://static.igem.org/mediawiki/2015/e/ee/HSNU-TAIPEI-BZP-904-1.jpg" width="50%">
+
−
 
+
−
  </div>
+
−
</div>
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    <img src="https://static.igem.org/mediawiki/2015/2/2c/HSNU-TAIPEI-BZP-820-4.jpg">
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    <p class="note-caption">The number of the colonies in the AMP+ plate is zero.</p>
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    <p class="note-caption">According to the result, 2hr 10<sup>-5</sup> and 4hr 10<sup>-6</sup> is the best. </p>
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    <img src="https://static.igem.org/mediawiki/2015/b/bc/HSNU-TAIPEI-BZP-result.jpg">
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    <img src="https://static.igem.org/mediawiki/2015/6/6b/HSNU-TAIPEI-BZP-820-1.jpg" width="50%">
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    <p class="note-caption">2hr plate from left to right is 10<sup>-4</sup>,10<sup>-5</sup>,10<sup>-6</sup>,10<sup>-7</sup></p>
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    <img src="https://static.igem.org/mediawiki/2015/e/ef/HSNU-TAIPEI-BZP-820-2.jpg" width="50%">
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    <p class="note-caption">4hr plate from left to right is 10<sup>-4</sup>,10<sup>-5</sup>,10<sup>-6</sup>,10<sup>-7</sup></p>
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    <p class="note-caption">AMP+ Plate</p>
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      <p class="note-caption">According to the result, Beno[a]pryene does not affect E.coli’s survival.</p>
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      <p class="note-caption">But Category B is failed because its number of colony is too much.</p>
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      <p class="note-caption">Benzo[a]pryene Category A</p>
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      <img src="https://static.igem.org/mediawiki/2015/6/66/HSNU-TAIPEI-BZP-904-3.jpg" width="50%">
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      <p class="note-caption">Control Category A</p>
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      <img src="https://static.igem.org/mediawiki/2015/6/69/HSNU-TAIPEI-BZP-904-4.jpg" width="50%">
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      <p class="note-caption">Benzo[a]pryene  CategoryB</p>
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      <img src="https://static.igem.org/mediawiki/2015/c/ce/HSNU-TAIPEI-BZP-904-5.jpg" width="50%">
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      <p class="note-caption">Control  CategoryB</p>
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     <h3 class="article-title">Reference</h3>
 
     <h3 class="article-title">Reference</h3>
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Latest revision as of 07:21, 26 November 2015

ProjectBenzo[A]Pyrene

Introduction

  1. Why do we detect benzo[a]pyrene?

    Because when food, such as fried food , is heated above 300 degrees celcius, benzo[a]pyrene is released. That is why recycled oil usually contains benzo[a]pyrene [1].

  2. The harm of benzo[a]pyrene

    Pathway: from skin, breathing in, and eating. It is carcinogenic and causes environmental pollution. It causes skin irritation and eye irritation [1].

  3. Taiwanese regulation

    ▼Table1:The regulation of Benzo[a]pyrene in Taiwan.

  4. National regulation
    • Europe:same to Taiwanese regulation[2]
    • American:The MCL has been set at 0.2 ppb[3]

Circuit Design

So far,we don’t find the protein which can combine with the Benzo[a]pyrene,so first we must degradate it by Laccase. And its product is Bap-1,6-quinone which can combine with the protein,in order that we can detect Benzo[a]pyrene.[4] We put the gene fragment which can produce the Laccase in the E.coli. Let it produce itself.

▲Fig.1-1:The circuit of detecting Benzo[a]pyrene.

QsrR is a protein which can combine with Bap-1,6-quinone.[5]As Laccase,we put the gene fragment in the E.coli. Let it produce itself.

▲Fig.1-2:The circuit of detecting Benzo[a]pyrene.

We design a gene part make QsrR repress sfRFP’s production. Usually,QsrR is on the Binding Site,and red fluorescent protein will not be produced. When QsrR combine with Bap-1,6-quinone, QsrR will be activated and go away. And the transcription will carry on,then it will produce red fluorescent protein.

▲Fig.1-3:The circuit of detecting Benzo[a]pyrene.

Result

Whether e.coli is alive in the poisons, condition or not

  1. Method

    2. DH5α-Pretest

    Procedure

    Because we must test E.coli’s Survival in the environment there is benzo[a]pryene by counting the colonies,First we test how much concentration is the best.

    1. culture

      STEP1:take 1μL DH5α to spread the plate(no Antibiotic)

      STEP2:put in 37 degree Celsius 12~16hr

    2. liquid culture

    3. STEP1:put 80μL into 2ml LB broth

      STEP2:recovering

      STEP3: After 2hr,dilute it to 10-4,10-5,10-6,10-7,and then go to spread the plate (no Antibiotic)

      STEP4: After 4hr dilute it to 10-4, 10-5 ,10-6 ,10-7 ,and then go to spread the plate (no Antibiotic), 6hr and 8hr Similarly

      STEP5:Take 200μL out from the tube and spread the plate(AMP+)

      STEP6: put in 37 degree Celsius 12~16hr

    Survival

    Procedure

    First we culture DH5α with LB only plate for 15hr. Then,pick one colony in the LB broth,and liquid culture for 15hr. We divided two categories A and B.

    A:
    1. Take 80μL into 2ml LB broth × 6 tubes and then culture 1 hr.
    2. After 1hr,add 20μL benzo[a]pryene into three tubes(conc. Is 2000ppb(A thousand times the standard value))
    3. And add 20μL DMSO into the other tubes.Then,culture for 3hr.
    4. After 3hr,dilute the broth to 10-6
    5. And take 200μL to spread the plate.
    B:
    1. Take 80μL into 2ml LB broth in a tube And then culture 1 hr.
    2. After 1hr, put them into 6 tubes equally.
    3. Dilute the broth to 5×10-4
    4. Add 0.4μL benzo[a]pryene(2×10-4) in three tubes.
    5. Add 0.4μL DMSO in the other three tubes.
    6. Go to 37 degree Celsius shaking for 10min.
    7. Take 200μL to spread the plate.
    ▼Table2: E. coli on the agar plate.
  2. Results
    3. ▼Table3: E. coli on the agar plate.

    The number of the colonies in the AMP+ plate is zero.
    According to the result, 2hr 10-5 and 4hr 10-6 is the best.

    ▼Table4:Line Chart of Table 3.
    ▲Fig.2:2hr agar only plate from left to rights are10-4, 10-5, 10-6, 10-7.
    ▲Fig3: 4hr agar only plate from left to rights are10-4, 10-5, 10-6, 10-7.

    4hr plate from left to right is 10-4,10-5,10-6,10-7

    ▲Fig4:Ampicillin Plate.

    AMP+ Plate

    According to the result, Beno[a]pryene does not affect E.coli’s survival.

    But Category B is failed because its number of colony is too much.

    ▲Fig5: Benzo[a]pyrene Category A

    Benzo[a]pryene Category A

    ▲Fig6:Control Category A

    Control Category A

    ▲Fig7: Benzo[a]pyrene Category B

    Benzo[a]pryene CategoryB

    ▲Fig8: Control Category B

    Control CategoryB

Reference

  • [1] Smoked foods Mechanism of benzo (a) pyrene and prevention methods
  • [2] Reduce operating guidelines in food polycyclic aromatic hydrocarbon content of the (draft)
  • [3] Basic Information about Benzo(a)pyrene in Drinking Water
  • [4] Hadibarata T, Kristanti RA. Identification of metabolites from benzo[a]pyrene oxidation by ligninolytic enzymes of Polyporus sp. S133. J Environ Manage. 2012 11/30;111(0):115-9.(2013 IGEM CUHK)
  • [5] Ji Q, Zhang L, Jones MB, Sun F, Deng X, Liang H, et al. Molecular mechanism of quinone signaling mediated through S-quinonization of a YodB family repressor QsrR. Proceedings of the National Academy of Sciences. 2013 March 26;110(13):5010-5. (2013 IGEM CUHK)

Project