Team:HSNU-TAIPEI/projectBP

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

  1. Whether benzo[a]pyrene can enter e.coli or not
    1. Method

      2. Detection of the amount of toxins in the e.coli.

      • (1)Add 100μl of DH5α and 900μl of LB broth into the tube and incubate for 1hr.
      • (2)Centrifuge at 4000rpm for 3min and clicard 800μl of the supernatant
      • (3)Plate each 100μl of the bacteria onto the dishes and spread.
      • (4)Incubate the plates at 37℃ overnight
      • (5)Prepare each concentration of the toxin.
      • (6)Statutory standards *100 / *10 / *1 / *0.1 / *0.01

      Next day

      • (1)Prepare 16 microcentrifuge tubes.(5 kinds of concentration *3 timings+control)

        (2)Add 500μl of DH5α to each tube.

        (3)Centrifuge all tubes at 4000rpm for 3min.

        (4)Remove the supernatent.

        (5)Add 1000μl of the toxic solution each time.

        (6)Follow the concentration and 3 timings(0.5hr / 1hr / 1.5hr).

        (7)Add 0.5cc of ddH2O and mix with the bacterias

        (8)Centrifuge at 13000rpm for 30 sec

        (9)Remove the water

        (10)Repeat step1~step3 for three times

        (11)Add 1cc of ddH2O and mix with the bacterias

        (12)Centrifuge at 13000rpm for 30sec.

        (13)Remove 700μl of the supernatant

      • Kill the bacteria:

        (1)Put all the tubes in the Liquid nitrogen

        (2)When they freeze,heat them at 100℃

        (3)Repeat step1~step2 for 3 times

  2. 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.

    2. 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)

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