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

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<p class="article-p">&#9650;Fig1-2:Circuit design of detecting Copper ion.</p>
 
<p class="article-p">&#9650;Fig1-2:Circuit design of detecting Copper ion.</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 Mercury 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>
 
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  <li>
 
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    <p class="article-p">Plate each 100&#956;l of the bacteria onto the dishes and spread.</p>
 
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    <p class="article-p">Incubate the plates at 37&#8451; overnight</p>
 
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  </li>
 
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  <li>
 
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    <p class="article-p">Prepare each concentration of the toxin.</p>
 
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    <p class="article-p">Statutory standards *100 / *10 / *1 / *0.1 / *0.01</p>
 
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  </li>
 
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</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>
 
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  </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>
 
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    <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>
 
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    </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>
 
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    <p class="article-p">Remove 700&#956;l of the supernatant</p>
 
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  </li>
 
−
  <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>
 
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      </ol>
 
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  </li>
 
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</ol>
 
  
−
</li>
 
−
<li><span>Result</span></li>
 
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  <img src="https://static.igem.org/mediawiki/2015/d/dc/2015_hsnu_hg_1111111.jpg" width="70%">
 
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<p class="article-p">&#9650;Fig3:The absorption by using gold nanoparticles in different Hg<sup>+</sup> concentration.</p>
 
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    <p class="article-p">With this figure, we can know that the higher Hg<sup>2+</sup> concentration, the lower fluorescence intensity it showed.</p>
 
−
 
−
 
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</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>
 
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<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 Mercury by counting the colonies,First we test how much concentration is the best.</p>
 
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    <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>
 
−
      <li>
 
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        <p class="note-caption">liquid culture</p>
 
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      </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>
 
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        <p class="note-caption">STEP6: put in  37 degree Celsius 12~16hr</p>
 
−
      </li>
 
−
    </ol>
 
−
 
−
  </div>
 
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</div>
 
−
 
−
<div class="section note">
 
−
  <h2 class="note-title">Survival</h2>
 
−
  <div class="note-content">
 
−
    <h3 class="note-subtitle">Procedure</h3>
 
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      <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>
 
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      <p class="note-caption">We divided two categories  A and B.</p>
 
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    <h3 class="note-subtitle">A:</h3>
 
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      <p class="note-caption">Take 80&#956;L into 2ml LB broth &#215; 6 tubes and then culture 1 hr.</p>
 
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      <p class="note-caption">After 1hr,add 20&#956;L Mercury into three tubes(conc. Is 2000ppb(A thousand times the standard value))</p>
 
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      <p class="note-caption">And add 20&#956;L DMSO into the other tubes.Then,culture for 3hr.</p>
 
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      <p class="note-caption">After 3hr,dilute the broth to 10<sup>-6</sup></p>
 
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      <p class="note-caption">And take 200&#956;L to spread the plate.</p>
 
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    <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>
 
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      <p class="note-caption">Dilute the broth to 5&#215;10<sup>-4</sup></p>
 
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      <p class="note-caption">Add 0.4&#956;L Mercury(2&#215;10<sup>-4</sup>) in three tubes.</p>
 
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      <p class="note-caption">Add 0.4&#956;L DMSO in the other three tubes.</p>
 
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      <p class="note-caption">Go to 37 degree Celsius shaking for 10min.</p>
 
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      <p class="note-caption">Take 200&#956;L to spread the plate.</p>
 
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<p class="article-p">&#9660;Table1: E. coli on the agar plate.</p>
 
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    <img src="https://static.igem.org/mediawiki/2015/b/b0/HSNU-TAIPEI-reasult-mercury.jpg" width="70%">
 
−
 
−
  </div>
 
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</div>
 
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</li>
 
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</ol>
 
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</li>
 
−
<ol>
 
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            </article>
 
 
   <article class="article">
 
   <article class="article">
 
     <h3 class="article-title">Reference</h3>
 
     <h3 class="article-title">Reference</h3>

Revision as of 06:37, 15 October 2015

ProjectMercury

Introduction

  1. Why do we detect mercury?

    Normally, there is no excess heavy metal in edible oil. Ingested mercury will hoard in the organs. Some recycled oil is made from animal internal organs. After mercury enters our body, it cannot be metabolized effectively and this leads to serious diseases. Due to the reasons above, we decided to detect this heavy metal.[1]

  2. The harm of mercury

    Methyl mercury is a type of neurotoxin. It represses cell division and restricts motility of electronic ions. It can also interfere with growing brain structure. Besides, methyl mercury can result in cardiovascular diseases like myocardial infarction, ischemic heart disease, hypertension and irregular pulse.[2]

  3. Taiwanese regulations
    • Edible Fat and Oil Sanitary Standards Article 2: The maximum allowance for Heavy Metal and Erucic acid: Mercury 0.05 ppm
    • Edible Rice and Heavy Metal Restriction Standard Article 2: permissible percentage limits for edible rice and heavy metal: less than 0.05ppm

Circuit Design

▲Fig1-1:Circuit design of detecting Mercury ion.

MerB can turn Methyl mercury into Hg2+. MerR can combine with Hg2+ and change the structure of DNA. And DNA can be transcripted easier. PmerT is controlled by MerR. PmerT cannot be transcripted because MerR changes the shape of PmerT. In short, the whole reaction will continue if MerR combines with Hg2+.[3][4]

▲Fig1-2:Circuit design of detecting Copper ion.

Reference

  • [1]J. Agric. Food Chem., 1975, “Metabolism of mercury, administered as methylmercuric chloride or mercuric chloride, by lactating ruminants”.23 (4), pp 803–808,DOI: 10.1021/jf60200a013 Publication Date: July 1975
  • [2]Knowledge、Attitude、Practice and risk assessment of mercury exposure through the consumption of fish from the mercury containminated area邱宇昕,JULY/2007
  • [3]Brown, N. L., J. V. Stoyanov, et al. (2003). "The MerR family of transcriptional regulators." FEMS Microbiol Rev 27(2-3): 145-163.
  • [4]Park, S. J., J. Wireman, et al. (1992). "Genetic analysis of the Tn21 mer operator-promoter." J Bacteriol 174(7): 2160-2171.

Project