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

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               <p class="article-p">Just like detecting cadmium, the same principle is applied on copper. First, we use the CopA promoter, which can detect copper and is more sensitive than CueO Promoter. When CopA promoter detects copper in oil, it activates the reversed translation.</p>
 
               <p class="article-p">Just like detecting cadmium, the same principle is applied on copper. First, we use the CopA promoter, which can detect copper and is more sensitive than CueO Promoter. When CopA promoter detects copper in oil, it activates the reversed translation.</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 Copper 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>
 
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<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>
 
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  <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>
 
−
    </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>
 
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      </ol>
 
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  </li>
 
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</ol>
 
  
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</li>
 
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<li><span>Result</span></li>
 
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    <img src="https://static.igem.org/mediawiki/2015/8/8c/2015_hsnu_cu_1111111.jpg" width="70%">
 
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<p class="article-p">&#9650;Fig2:The absorption by using gold nanoparticles in different Cu<sup>2+</sup> concentration.</p>
 
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<p class="article-p">With this figure, we can know that the higher Cu<sup>2+</sup> concentration, the lower fluorescence intensity it showed</p>
 
−
 
−
 
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</ol>
 
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</li>
 
−
<li>
 
−
<span>Whether e.coli is alive in the poisons, condition or not</span>
 
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<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 Cppper 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">
 
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      <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>
 
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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>
 
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      </li>
 
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    </ol>
 
−
 
−
  </div>
 
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</div>
 
−
 
−
<div class="section note">
 
−
  <h2 class="note-title">Survival</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">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 Copper 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>
 
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      <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 Copper(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>
 
−
      <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/2015/4/47/HSNU-TAIPEI-reasult-copper.jpg" width="70%">
 
−
 
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  </div>
 
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</div>
 
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</li>
 
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</ol>
 
−
</li>
 
−
 
−
            </article>
 
 
             <article class="article">
 
             <article class="article">
 
<h3 class="article-title">Reference</h3>
 
<h3 class="article-title">Reference</h3>

Revision as of 06:36, 15 October 2015

ProjectCopper

Introduction

  1. Why do we detect Copper?

    Copper is a compulsory ingredient in respiratory pigment, and have been identified in many other proteins and enzymes. In 1847, Harless found that copper has a great action in Molluscs, and in 1878 Frederig first separated copper from protein contamination in blood of octopus, and he named it as ceraloplasim. In 1928, Hart’s report said copper is a compulsory element.

    According to the study, since copper can effect the absorption of iron, lack of copper will cause neutropenia, osteoporosis and growth retardation, or damage human’s immune system.

  2. The harm of Copper

    Too much copper harms human body. Inhale of high concentration of copper will result in irritation of the nose and throat. Ingesting high concentration of copper also lead to liver and kidney damage. Excess copper causes toxicity and the presenting symptoms include nausea, queasiness, vomit and dirrahea. Other severe symptoms like hematuria, jaundice, and urine oligonucleotide may appear too. Copper poisoning could lead to hemolysis, decrease of hemoglobin and increase of serum lactate dehydrogenase.

  3. Taiwanese regulation
    • Drinking 1.0 ppm
    • Cooking oil 0.4 ppm
    • Egg 5 ppm
    • Drinks 5 ppm
  4. National regulation
    • USEPD request that the concentration of copper in drinking water shouldn’t over 1.3 mg/L.
    • US Department of Agriculture suggests people over 8 years old should only take less than 900 mg per day.
    • US OSHA demands that in workplace, the concentration of copper in either air or dust should lower than 0.1 mg/m3.

Circuit Design

▲Fig1:Circuit design of detecting Copper ion.

Just like detecting cadmium, the same principle is applied on copper. First, we use the CopA promoter, which can detect copper and is more sensitive than CueO Promoter. When CopA promoter detects copper in oil, it activates the reversed translation.

Reference

  • [1]John H. Duffus: "Heavy Metals"- A Meaningless Term, Chemistry International, November 2001

Project