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

 
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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>
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<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>
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<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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     <p class="article-p">The Relationship between Cytochrome CYP3A4 in Vivo and the Toxicity of Aflatoxin B1 in Feedstuffs<br>ZHENG Lili1, ZHU Yujing1, SHAO Caimei2, ZHANG Yong1</p>
 
     <p class="article-p">The Relationship between Cytochrome CYP3A4 in Vivo and the Toxicity of Aflatoxin B1 in Feedstuffs<br>ZHENG Lili1, ZHU Yujing1, SHAO Caimei2, ZHANG Yong1</p>
 
   </article>
 
   </article>
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<article class="article">
 
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              <h3 class="article-title">Result</h3>
 
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<ol class="article-ol">
 
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<li>
 
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<span>Whether Aflatoxin can enter e.coli or not</span>
 
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<ol class="article-ol" type="A">
 
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<li><span>Method</span>
 
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<h4 class="article-h4">Detection of the amount of toxins in the e.coli.</h4>
 
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<ol class="article-ol" type="I">
 
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  <li>Add 100&#956;l of DH5&#945; and 900&#956;l of LB broth into the tube and incubate for 1hr.</li>
 
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  <li>Centrifuge at 4000rpm for 3min and clicard 800&#956;l of the supernatant</li>
 
−
  <li>
 
−
    <p class="article-p">Plate each 100&#956;l of the bacteria onto the dishes and spread.</p>
 
−
    <p class="article-p">Incubate the plates at 37&#8451; overnight</p>
 
−
    </li>
 
−
  <li>
 
−
    <p class="article-p">Prepare each concentration of the toxin.</p>
 
−
    <p class="article-p">Statutory standards *100 / *10 / *1 / *0.1 / *0.01</p>
 
−
  </li>
 
−
</ol>
 
−
<h4 class="article-h4">Next day</h4>
 
−
<ol class="article-ol" type="I">
 
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  <li>
 
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    <p class="article-p">Prepare 16 microcentrifuge tubes.(5 kinds of concentration *3 timings+control)</p>
 
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    <p class="article-p">Add 500&#956;l of DH5&#945; to each tube.</p>
 
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    <p class="article-p">Centrifuge all tubes at 4000rpm for 3min.</p>
 
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    <p class="article-p">Remove the supernatent.</p>
 
−
  </li>
 
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  <li>
 
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    <p class="article-p">Add 1000&#956;l of the toxic solution each time.</p>
 
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    <p class="article-p">Follow the concentration and 3 timings(0.5hr / 1hr / 1.5hr).</p>
 
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  </li>
 
−
  <li>
 
−
    <ol class="article-ol" type="i">
 
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      <li>Add 0.5cc of ddH<sub>2</sub>O and mix with the bacterias</li>
 
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      <li>Centrifuge at 13000rpm for 30 sec</li>
 
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      <li>Remove the water</li>
 
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      <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>
 
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    <p class="article-p">Centrifuge at 13000rpm for 30sec.</p>
 
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    <p class="article-p">Remove 700&#956;l of the supernatant</p>
 
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  </li>
 
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  <li>
 
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    <p class="article-p">Kill the bacteria:</p>
 
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      <ol class="article-ol" type="i">
 
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        <li>Put all the tubes in the Liquid nitrogen</li>
 
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        <li>When they freeze,heat them at 100&#8451;</li>
 
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        <li>Repeat step1~step2 for 3 times</li>
 
−
      </ol>
 
−
  </li>
 
−
</ol>
 
  
−
</li>
 
−
</ol>
 
−
</li>
 
−
<li>
 
−
<span>Whether e.coli is alive in the poisons, condition or not</span>
 
−
<ol class="article-ol" type="A">
 
−
<li><span>Method</span>
 
−
<div class="section note">
 
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  <h2 class="note-title">DH5&#945;-Pretest</h2>
 
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  <div class="note-content">
 
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    <h3 class="note-subtitle">Procedure</h3>
 
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  <p class="note-caption">Because we must test E.coli’s Survival in the environment there is Aflatoxin by counting the colonies,First we test how much concentration is the best.</p>
 
−
    <ol class="note-ordered-list" type="I">
 
−
      <li>
 
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        <p class="note-caption">culture</p>
 
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        <p class="note-caption">STEP1:take 1&#956;L DH5&#945; to spread the plate(no Antibiotic)</p>
 
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        <p class="note-caption">STEP2:put in 37 degree Celsius 12~16hr</p>
 
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      </li>
 
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      <li>
 
−
        <p class="note-caption">liquid culture</p>
 
−
      </li>
 
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      <li>
 
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        <p class="note-caption">STEP1:put 80&#956;L into 2ml LB broth </p>
 
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        <p class="note-caption">STEP2:recovering</p>
 
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        <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>
 
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        <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>
 
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        <p class="note-caption">STEP5:Take 200&#956;L out from the tube and spread the plate(AMP+)</p>
 
−
        <p class="note-caption">STEP6: put in  37 degree Celsius 12~16hr</p>
 
−
      </li>
 
−
    </ol>
 
−
 
−
  </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 Aflatoxin 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 Aflatoxin(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><p class="article-p">&#9660;Table1: E. coli on the agar plate.</p>
 
−
    <img src="https://static.igem.org/mediawiki/2015/4/46/HSNU-TAIPEI-reasult-Aflatoxin.jpg" width="70%">
 
−
 
−
  </div>
 
−
</div>
 
−
</li>
 
−
</ol>
 
−
</li>
 
−
 
−
<ol>
 
−
            </article>
 
 
   <article class="article">
 
   <article class="article">
 
     <h3 class="article-title">Reference</h3>
 
     <h3 class="article-title">Reference</h3>

Latest revision as of 07:43, 26 November 2015

ProjectAflatoxin

Introduction

  1. Why do we detect Aflatoxin?

    Gutter oil contains aflatoxin which is very toxic and will accumulate in people’s liver and kidney.

    In Taiwan, there are many people who are the asymptomatic carriers of hepatitis B, therefore if they be poisoned oil again, it will be easier for them to get liver disease or resulting in kidney dialysis.

    In our experiment, we try to detect Afb1 because it is the most toxic of aflatoxin. Our experiment was to find out more convenient detection method, so that everyone can easily detect their own oil if it is safe to eat.

  2. Taiwanese regulations

    ▼Table1: The regulation of Aflatoxin in Taiwan..

  3. National regulations
    1. groundnuts, nuts, dried fruit and processed products of such products or food additives: B1 aflatoxin not exceed 2ug / kg (ppb), and B1, B2, G1, G2 sum not exceeding 4ug / kg.
    2. for the storage or not for direct human consumption's peanuts: B1 aflatoxin 8ug / kg, and B1, B2, G1, G2 sum shall not exceed 15ug / kg.
    3. for the storage or not for direct human consumption's nuts and dried fruits: B1 aflatoxin 5ug / kg, and B1, B2, G1, G2 sum shall not exceed 10ug / kg.
    4. Grains class for the direct human consumption: B1 aflatoxin 2ug / kg, or B1, B2, G1, G2 sum not exceeding 4ug / kg.
    5. Dairy: M1 aflatoxin not exceed 0.05 ug / kg.[4]

Circuit Design

▲Fig.1-1: Circuit design of detecting Lead ion.

When aflatoxin goes into the cell, CYP1A2 will oxidize the Aflatoxin B1, and then it will turn into Aflatoxin oxidation AFBO[5].

▲Fig.1-2: Circuit design of detecting Lead ion.

Single-strand DNA(ssDNA) can activate protein RecA. Ecoli has a sos response system, and there is a repressor LexA which inhibits the next reaction, so the fluorescent gene won’t express.But when the activated protein RecA comes and hydrolysis the represser LexA, the next reaction can work.[7]

The Relationship between Cytochrome CYP3A4 in Vivo and the Toxicity of Aflatoxin B1 in Feedstuffs
ZHENG Lili1, ZHU Yujing1, SHAO Caimei2, ZHANG Yong1

Reference

  • [1]Veronica S. Mary a, Ana Valdehita b, Jose M. Navas b, Hector R. Rubinstein a, Maria L. Fernandez-Cruz. Effects of aflatoxin B1, fumonisin B1 and their mixture on the aryl hydrocarbon receptor and cytochrome P450 1A induction
  • [2]Yan-Ping Du Hui-Lian Zhu. The possible mechanism of Aflatoxin B1 Hepatocarcinogenesis and preventive effects of phytochemicals
  • [3]Food hygiene standards - Food in aflatoxin limits (human) 82 1.4.4 Health Department Announcement fresh word No. 8189322
  • [4]EU animal and plant inspection and quarantine of food hygiene regulations cum Profile
  • [5]Veronica S. Mary a, Ana Valdehita b, Jose M. Navas b, Hector R. Rubinstein a,*Maria L. Fernandez-Cruz b. Effects of aflatoxin B1, fumonisin B1 and their mixture on the aryl hydrocarbon receptor and cytochrome P450 1A induction
  • [6]ZHENG Lili, ZHU Yujing, SHAO Caimei, ZHANG Yong The Relationship between Cytochrome CYP3A4 in Vivo and the Toxicity of Aflatoxin B1 in Feedstuffs
  • [7]Nejc Paulič2,†,Adrijana Leonardi1, Vesna Hodnik2,Gregor Anderluh2,3, Zdravko Podlesek2, Darja Žgur-Bertok2,Igor Križaj1,4,5 Matej Butala2. Structural insight into LexA–RecA* interaction Lidija Kovačič1

Project