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

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:

1. Take 80μL into 2ml LB broth × 6 tubes and then culture 1 hr.
2. After 1hr,add 20μL benzo[a]pryene into three tubes(conc. Is 2000ppb(A thousand times the standard value))
3. And add 20μL DMSO into the other tubes.Then,culture for 3hr.
4. After 3hr,dilute the broth to 10^-6
5. And take 200μL to spread the plate.

B:

1. Take 80μL into 2ml LB broth in a tube And then culture 1 hr.
2. After 1hr, put them into 6 tubes equally.
3. Dilute the broth to 5×10^-4
4. Add 0.4μL benzo[a]pryene(2×10^-4) in three tubes.
5. Add 0.4μL DMSO in the other three tubes.
6. Go to 37 degree Celsius shaking for 10min.
7. Take 200μL to spread the plate.



2. Results



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.









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.



Benzo[a]pryene Category A



Control Category A



▲Fig7: Benzo[a]pyrene Category B

Benzo[a]pryene CategoryB



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)