Introduction

1. Why do we detect Copper?

   Copper is a compulsory ingredient in respiratory pigment, and have been identified in many other proteins and enzymes. In 1847, Harless found that copper has a great action in Molluscs, and in 1878 Frederig first separated copper from protein contamination in blood of octopus, and he named it as ceraloplasim. In 1928, Hart’s report said copper is a compulsory element.

   According to the study, since copper can effect the absorption of iron, lack of copper will cause neutropenia, osteoporosis and growth retardation, or damage human’s immune system.

2. The harm of Copper

   Too much copper harms human body. Inhale of high concentration of copper will result in irritation of the nose and throat. Ingesting high concentration of copper also lead to liver and kidney damage. Excess copper causes toxicity and the presenting symptoms include nausea, queasiness, vomit and dirrahea. Other severe symptoms like hematuria, jaundice, and urine oligonucleotide may appear too. Copper poisoning could lead to hemolysis, decrease of hemoglobin and increase of serum lactate dehydrogenase.

3. Taiwanese regulation
   • Drinking 1.0 ppm
   • Cooking oil 0.4 ppm
   • Egg 5 ppm
   • Drinks 5 ppm
4. National regulation
   • USEPD request that the concentration of copper in drinking water shouldn’t over 1.3 mg/L.
   • US Department of Agriculture suggests people over 8 years old should only take less than 900 mg per day.
   • US OSHA demands that in workplace, the concentration of copper in either air or dust should lower than 0.1 mg/m3.

Circuit Design

▲Fig1:Circuit design of detecting Copper ion.

Just like detecting cadmium, the same principle is applied on copper. First, we use the CopA promoter, which can detect copper and is more sensitive than CueO Promoter. When CopA promoter detects copper in oil, it activates the reversed translation.

Result

1. Whether Copper 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
   

   ▲Fig2:The absorption by using gold nanoparticles in different Cu²⁺ concentration.

   With this figure, we can know that the higher Cu2+ 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 Cppper 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:

      Take 80μL into 2ml LB broth × 6 tubes and then culture 1 hr.

      After 1hr,add 20μL Copper 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 Copper(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.

      ▼Table1: E. coli on the agar plate.

      

Reference

• [1]John H. Duffus: "Heavy Metals"- A Meaningless Term, Chemistry International, November 2001