Introduction

1. Why do we detect cadmium?

   Vegetable oil can be extracted in various ways. But if the plants’ resources are exposed to polluted soil, the vegetable oil may contain toxins. Once polluted or unstandardized oil is mixed with these polluted plants, those plants will contain pollutants. Moreover, we’ve noticed that the problem of cadmium-polluted planet is a common situation all over the world, especially in China. Recycled tainted oil also contains cadmium. Because of this reason, our group wants to detect cadmium.

2. The harm of cadmium

   Cadmium is an environmental hazard. Human exposures to environmental cadmium are primarily the result of fossil fuel combustion, phosphate fertilizers, natural sources, iron and steel production, cement production and so on. There have been a few instances of general population toxicity as the result of long-term exposure to cadmium in contaminated food and water, and ongoing studies have found that its estrogen mimicry may induce breast cancer. Some members of the agricultural communities who consumed cadmium-contaminated rice developed itai-itai disease and renal abnormalities, including proteinuria and glucosuria.

3. Taiwanese regulation
   • 3-1: The limit of cadmium in vegetable or mushroom
     Vegetable: 0.05~0.2ppm ; mushroom: 2ppm
   • 3-2: The limit of cadmium in rice
     Rice: 0.4 ppm
   • 3-3: The limit of cadmium in oil
     There is no law to standard.
4. National regulations

   Due to the adverse effects on environment and human health, the supply and use of cadmium is restricted in Europe under the REACH Regulation. The EFSA Panel on Contaminants in the Food Chain has set 2.5 μg/kg body weight as a tolerable weekly intake for humans. In comparison, the Joint FAO/WHO Expert Committee on Food Additives has established 7 μg/kg bw as a provisional tolerable weekly intake level.p>

   America:

   1. Restrict the release of cadmium to lack, river, arable land, rubbish tip.
   2. The limit of cadmium in water :10ppm
   3. The limit of cadmium in food coloring :15ppm

Circuit Design

Zintp K896008 is a promoter designed by the NYMU in 2013.It is highly specific to the cadmium. When cadmium attaches to Zintp k896008, the circuit will begins. When the fluorescence protein E1010/E0040 receives the message from zintp k896008, it will result in purple and red fluorescence. Finally, the terminator, B0015 completes the circuit.

Result

1. Whether Cadmium 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 Cd2+ 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 Cadmium 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 Cadmium 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 Cadmium(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] Metal contamination of soils and crops affected by the Chenzhou lead/zinc mine spill (Hunan, China).(Liu H, Probst A, Liao B.)
• [2] High levels of heavy metals in rice (Oryza sativa L.) from a typical E-waste recycling area in southeast China and its potential risk to human health (Jianjie Fu, Qunfang Zhou, Jiemin Liu, Wei LiuThanh Wang,Qinghua Zhang,Guibin Jiang)
• [3]Rancidity of Used Cooking Oil and Heavy Metal Analy ses on Selected Street-Vended Foods (Annabelle A. Callano( University of the Immaculate Conception)
• [4] Cadmium. (2015,august 02) In Wikipedia, the free encyclopedia. Retrieved August 14, 2015, from http://en.wikipedia.org/wiki/cadmium