Team:HSNU-TAIPEI/projectlead

ProjectLead

Introduction

  1. Why do we detect Lead?

    There are two possible reasons that lead were found in recycled oil. One is that the animal feed oil is made from animal’s internal organs, and those organs are to conduct metabolism in animal’s bodies, so they may contain little lead. The other is that the oil container is made of metals like lead, and it may dissolve lead into the oil. There’s lead in recycled oil.[1] Human body can hardly merabolize and degrade lead. To the human body, lead is a chronic and cumulative poison; it may easily lead to poisoning or even carcinogenic. Therefore, we decided to detect lead.

  2. The harm of Lead

    Lead can cause several unwanted effects, such as:

    • Disruption of the biosynthesis of haemoglobin and anaemia
    • A rise in blood pressure
    • Kidney damage
    • Miscarriages and subtle abortions
    • Disruption of nervous systems
    • Brain damage
    • Declined fertility of men through sperm damage
    • Diminished learning abilities of children
    • Behavioural disruptions of children, such as aggression, impulsive behavior and hyperactivity

    Lead can enter a foetus through the placenta of the mother. Because of this it can cause serious damage to the nervous system and the brains of unborn children.[2]

  3. Taiwanese regulation

    The maximum allowance of lead in edible oil is 0.1 ppm.[3]

Circuit Design

We mainly divide the whole experiment into 2 major parts.

▲Fig1-1:Circuit design of detecting Lead ion.

The former fragment can consistently produce PbRr.[4]

▲Fig1-2:Circuit design of detecting Lead ion.

If the oil contains lead ions, they can integrate with proteins PbRr to further activate proteins.

▲Fig1-3:Circuit design of detecting Lead ion.

As for the later fragment, the lead promoter can be controlled by activated protein PbRr to further trigger the GFP.

Result

  1. Whether lead 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).

        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

      3. We put all the result into fluorescent reader.

      By using gold nanoparticles(AU-NPs), we measured the lead absorption of e.coli. The higher fluorescence intensity it shows, the less lead enters e.coli.

      ▲Fig.2:The Pb absorption of E.coli in 1 ppm Pb2+ in different timings.

      With this figure, we can know that the longer e.coli put in 1 ppm Pb2+ , the more lead entered the e.coli.

      ▲Fig.3:The Pb absorption of E.coli in 0.1 ppm Pb2+ in different timings.

      With this figure, we can know that the longer e.coli put in 0.1 ppm Pb2+ , the more lead entered the e.coli.

      ▲Fig.4: The Pb absorption of E.coli in 0.01 ppm Pb2+ in different timings.

      With this figure, we can know that the longer e.coli put in 0.01 ppm Pb2+ , the more lead entered the e.coli.

      ▲Fig.5: The Pb absorption of E.coli in 0.001 ppm Pb2+ in different timings.

      With this figure, we can know that the longer e.coli put in 0.001 ppm Pb2+ , the more lead entered the e.coli.

      ▲Fig:6: The Pb absorption of E.coli in 0.0001 ppm Pb2+ in different timings.

      With this figure, we can know that the longer e.coli put in 0.0001 ppm Pb2+ , the more lead entered the e.coli

      ▲Fig.7: The Pb absorption of E.coli in 0.5hr in different Pb2+ concentration.

      With this figure, we can know that the higher Pb2+ concentration in 0.5 hr, the more lead entered the e.coli.

      ▲Fig.8: The Pb absorption of E.coli in 1hr in different Pb2+ concentration.

      With this figure, we can know that the higher Pb2+ concentration in 1 hr, the more lead entered the e.coli.

      ▲Fig.9: The Pb absorption of E.coli in 1.5hr in different Pb2 concentration.

      With this figure, we can know that the higher Pb2+ concentration in 1.5 hr, the more lead entered the e.coli.

  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 Lead 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 Lead 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 Lead(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] Rancidity of Used Cooking Oil and Heavy Metal Analyses on Selected Street-Vended Foods (Annabelle A. Callano, 2012)
  • [2] Health effects of lead
  • [3] Edible Fat and Oil Sanitary Standards Article 17
  • [4] An Exceptionally Selective Lead(ii)-Regulatory Protein from Ralstonia Metallidurans: Development of a Fluorescent Lead(ii) Probe (Peng Chen, Bill Greenberg, Safiyh Taghavi, Christine Romano, Daniel van der Lelie, and Chuan He)

Project