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].
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].
Taiwanese regulation
▼Table1:The regulation of Benzo[a]pyrene in Taiwan.
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
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.
culture
STEP1:take 1μL DH5α to spread the plate(no Antibiotic)
STEP2:put in 37 degree Celsius 12~16hr
liquid culture
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.▲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)
