Introduction

1. Why do we detect mercury?

   Normally, there is no excess heavy metal in edible oil. Ingested mercury will hoard in the organs. Some recycled oil is made from animal internal organs. After mercury enters our body, it cannot be metabolized effectively and this leads to serious diseases. Due to the reasons above, we decided to detect this heavy metal.[1]

2. The harm of mercury

   Methyl mercury is a type of neurotoxin. It represses cell division and restricts motility of electronic ions. It can also interfere with growing brain structure. Besides, methyl mercury can result in cardiovascular diseases like myocardial infarction, ischemic heart disease, hypertension and irregular pulse.[2]

3. Taiwanese regulations
   • Edible Fat and Oil Sanitary Standards Article 2: The maximum allowance for Heavy Metal and Erucic acid: Mercury 0.05 ppm
   • Edible Rice and Heavy Metal Restriction Standard Article 2: permissible percentage limits for edible rice and heavy metal: less than 0.05ppm

Circuit Design

MerB can turn Methyl mercury into Hg2+. MerR can combine with Hg2+ and change the structure of DNA. And DNA can be transcripted easier. PmerT is controlled by MerR. PmerT cannot be transcripted because MerR changes the shape of PmerT. In short, the whole reaction will continue if MerR combines with Hg2+.[3][4]

Result

1. Whether Mercury 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
   

   With this figure, we can know that the higher Hg2+ concentration, the lower fluorescence intensity it showed.

2. 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 Mercury 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. (8/19)

         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 Mercury 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 Mercury(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.

      

Reference

• [1]J. Agric. Food Chem., 1975, “Metabolism of mercury, administered as methylmercuric chloride or mercuric chloride, by lactating ruminants”.23 (4), pp 803–808,DOI: 10.1021/jf60200a013 Publication Date: July 1975
• [2]Knowledge、Attitude、Practice and risk assessment of mercury exposure through the consumption of fish from the mercury containminated area邱宇昕,JULY/2007
• [3]Brown, N. L., J. V. Stoyanov, et al. (2003). "The MerR family of transcriptional regulators." FEMS Microbiol Rev 27(2-3): 145-163.
• [4]Park, S. J., J. Wireman, et al. (1992). "Genetic analysis of the Tn21 mer operator-promoter." J Bacteriol 174(7): 2160-2171.