Introduction

1. Why do we detect benzo[a]pyrene?

   Because when food,such as fried food ,which is heated above 300 degrees celcius,food will release benzo[a]pyrene recycled oil usually contain benzo[a]pyrene[1].

2. The harm of benzo[a]pyrene

   Pathway:from skin,breathing in,eating Carcinogenic,environmental pollution, Skin irritation,eye irritation[1].

3. Taiwanese regulation
   
4. National regulation
   • Europe:The 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.

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.

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.

Result

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

      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.

         Incubate the plates at 37℃ overnight

      4. Prepare each concentration of the toxin.

         Statutory standards *100 / *10 / *1 / *0.1 / *0.01

      Next day

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

         Add 500μl of DH5α to each tube.

         Centrifuge all tubes at 4000rpm for 3min.

         Remove the supernatent.

      2. Add 1000μl of the toxic solution each time.

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

      3. 1. Add 0.5cc of ddH₂O and mix with the bacterias
         2. Centrifuge at 13000rpm for 30 sec
         3. Remove the water
         4. Repeat step1~step3 for three times

      4. Add 1cc of ddH₂O and mix with the bacterias

         Centrifuge at 13000rpm for 30sec.

         Remove 700μl of the supernatant

      5. 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. Result
   3. Discussion
2. Whether e.coli is alive in the poisons, condition or not
   1. Method
   2. Results
   3. Discussion
3. The relation between the concentration of benzo[a]pyrene and illumination of RFP
   1. Method
   2. Results
   3. Discussion

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)