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

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               <h3 class="article-title">Result</h3>
 
               <h3 class="article-title">Result</h3>
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<span>Whether benzo[a]pyrene can enter e.coli or not</span>
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<li><span>Method</span></li>
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<h4 class="article-h4">Detection of the amount of toxins in the e.coli.</h4>
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  <li>(1)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>(2)Centrifuge at 4000rpm for 3min and clicard 800&#956;l of the supernatant</li>
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    <li>(3)Plate each 100&#956;l of the bacteria onto the dishes and spread.</li>
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    <li>(4)Incubate the plates at 37&#8451; overnight</li>
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    <li>(5)Prepare each concentration of the toxin.</li>
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    <li>(6)Statutory standards *100 / *10 / *1 / *0.1 / *0.01</li>
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<h4 class="article-h4">Next day</h4>
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    <p class="article-p">(1)Prepare 16 microcentrifuge tubes.(5 kinds of concentration *3 timings+control)</p>
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    <p class="article-p">(2)Add 500&#956;l of DH5&#945; to each tube.</p>
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    <p class="article-p">(3)Centrifuge all tubes at 4000rpm for 3min.</p>
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    <p class="article-p">(4)Remove the supernatent.</p>
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    <p class="article-p">(5)Add 1000&#956;l of the toxic solution each time.</p>
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    <p class="article-p">(6)Follow the concentration and 3 timings(0.5hr / 1hr / 1.5hr).</p>
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      <p class="article-p">(7)Add 0.5cc of ddH<sub>2</sub>O and mix with the bacterias</p>
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      <p class="article-p">(8)Centrifuge at 13000rpm for 30 sec</p>
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      <p class="article-p">(9)Remove the water</p>
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      <p class="article-p">(10)Repeat step1~step3 for three times</p>
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    <p class="article-p">(11)Add 1cc of ddH<sub>2</sub>O and mix with the bacterias</p>
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    <p class="article-p">(12)Centrifuge at 13000rpm for 30sec.</p>
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    <p class="article-p">(13)Remove 700&#956;l of the supernatant</p>
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    <p class="article-p">Kill the bacteria:</p>
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      <p class="article-p">(1)Put all the tubes in the Liquid nitrogen</p>
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      <p class="article-p">(2)When they freeze,heat them at 100&#8451;</p>
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      <p class="article-p">(3)Repeat step1~step2 for 3 times</p>
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<span>Whether e.coli is alive in the poisons, condition or not</span>
 
<span>Whether e.coli is alive in the poisons, condition or not</span>
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         <p class="note-caption">STEP6: put in  37 degree Celsius 12~16hr</p>
 
         <p class="note-caption">STEP6: put in  37 degree Celsius 12~16hr</p>
 
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Revision as of 06:29, 15 October 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:

Take 80μL into 2ml LB broth × 6 tubes and then culture 1 hr.

After 1hr,add 20μL benzo[a]pryene into three tubes(conc. Is 2000ppb(A thousand times the standard value))

And add 20μL DMSO into the other tubes.Then,culture for 3hr.

After 3hr,dilute the broth to 10-6

And take 200μL to spread the plate.

B:

Take 80μL into 2ml LB broth in a tube And then culture 1 hr.

After 1hr, put them into 6 tubes equally.

Dilute the broth to 5×10-4

Add 0.4μL benzo[a]pryene(2×10-4) in three tubes.

Add 0.4μL DMSO in the other three tubes.

Go to 37 degree Celsius shaking for 10min.

Take 200μL to spread the plate.

▼Table2: E. coli on the agar plate.

  • Results

    ▼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.

    2hr plate from left to right is 10-4,10-5,10-6,10-7

    ▲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