Team:Hong Kong JSS/Experiments


Hong Kong JSS

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Experiments

Generating The Standard Curve

Aim
A standard curve is a type of graph used as a quantitative research technique. It is used for quantifying the copper level of our samples in different experiments. According to Beer’s Law, A=Ebc, under ideal conditions, a substance’s concentration and its absorbance are directly proportional. Making use of the commercially available API copper test kit, absorbance of multiple samples with known copper concentration is measured and graphed, which then allows the concentration of the unknown samples to be determined by interpolation on the graph.

Materials and Equipments
  • 500ml volumetric flask
  • 10ml pipette
  • 200ul and 1000ul micropipette
  • Electronic balance
    		•
  • Colorimeter and cuvettes
  • API copper testing kit
  • Anhydrous copper (II) sulphate
  • Distilled water
    		•
Procedure
Preparation of copper standard solutions:
  1. 500ml of 1000mg/L copper solution was prepared by adding 1.255g anhydrous copper solution to a 500ml volumetric flask.
  2. Distilled water was added to the flask until it reached the graduation marking.
  3. The flask was gently inverted several times until all copper (II) sulphate dissolved.
  4. A serial dilution was done to prepare standard copper solution of 10, 8, 6, and 2 mg/L

Measuring the absorbance of standard copper solutions:
  1. 2.5ml of copper standard copper solution was added to a cuvette.
  2. 175ul of API copper testing solution was added to the cuvette.
  3. The solution was mixed by pipetting up and down for 10 seconds.
  4. Wait 20 seconds for the color to develop.
  5. The absorbance (440nm) of the solution was measured by a colorimeter.

Determine the copper level of a sample:
  1. Absorbance several copper standard solutions were measured.
  2. The graph was plotted by absorbance against concentration.
  3. The absorbance of the sample was measured.
  4. Copper concentration of the samples was determined by interpolation the absorbance measured on the graph.

An example of stand curve used in our experiments.



Quantifying Copper Concentration in Water Samples

Aim
Water samples collected from various locations in Hong Kong were tested for the copper level. We aimed to investigate the severity of copper pollution in different types of waters, coming from freshwater habitats, aquarium/aquaculture, seawater and tap water.

Water samples collected in Hong Kong.

Materials and Equipments
  • 200ul and 1000ul micropipette
  • Colorimeter and cuvettes
  • Water samples
  • API copper testing kit
    		•
Procedure
  1. 2.5ml of water samples was added to a cuvette.
  2. 175ul of API copper testing solution was added to the cuvette.
  3. The solution was mixed by pipetting up and down for 10 seconds.
  4. Wait 20 seconds for the color to develop.
  5. The absorbance (440nm) of the solution was measured by a colorimeter.
  6. The copper concentration of the samples was determined by interpolating the absorbance measured on the standard curve



Bacterial Copper Adsorption Test

Aim
In this experiment, we aimed to test the copper adsorption ability of our genetically modified bacteria. The bacteria will be mixed with copper solution and the concentration of the mixture will be measured at different time intervals.

Materials and Equipments
  • 200ul and 1000ul micropipette
  • Colorimeter
  • Electronic balance
  • Conical flasks
  • API copper testing kit
  • SOB medium
  • Ampicillin / Kanamycin
  • IPTG (optional)
  • 100mg/L copper solution
  • Distilled water
Procedure
Preparation of bacterial culture:
  1. 7.02 g SOB powder was dissolved in 250 ml distilled water to prepare the SOB medium.
  2. 40 ml SOB medium was added to 250 ml conical flask.
  3. Each of the conical flask with SOB medium was sterilized by autoclave.
  4. Prepare 1000X ampicillin (100mg/ml) by dissolving 1g ampicillin in 10 ml distilled water. Caution: Wear mask, safety goggles and gloves when handling Ampicillin powder as it is corrosive and irritating.
  5. 40 uL of 1000X ampicillin was added into each flask with 40 ml SOB medium.
  6. A single colonie of respective cell culture was inoculated into conical flask with SOB medium.
  7. The flasks were incubated in a shaking incubator at 37℃ for 18 hours.
Remarks:
  1. According to the selection marker of the plasmid or bacteria strain, Kanamycin or other antibiotics may be used instead of ampicillin.
  2. In some experiments, the bacteria needed to be induced by IPTG. To prepare IPTG stock (1000X), 2.38 g of IPTG was dissolved in 10 mL of distilled water. The solution was filter sterilized with a 0.22μm syringe filter.

Part 1 of the experiment: Grow up of cell culture and incubation with copper solution.

Testing of the copper absorption ability of genetically engineered E. coli:
To take the measurement of the initial copper concentration,
  1. 4.5 ml of SOB medium was mixed with 0.5 ml of 100mg/L copper solution inside a test tube.
  2. 2.5 ml of the mixture was transferred to a cuvette.
  3. 175ul of API copper testing solution was added to the cuvette.
  4. The solution was mixed by pipetting up and down for 10 seconds.
  5. Wait 20 seconds for the color to develop.
  6. The absorbance (440nm) of the solution was measured by a colorimeter.
  7. Copper concentration of the samples was determined by interpolation the absorbance measured on the standard curve.

To measure the copper absorption ability of the bacteria,
  1. 4.5 ml of bacterial culture was added into 2 test tubes. (labeled as 2 hrs and 4 hrs)
  2. 0.5 ml of 100mg/L copper solution was added into each of the test tubes. The final copper concentration of each test tube was 10mg/L.
  3. All of the tubes were incubated in a shaking incubator at 37℃.
  4. At 2 and 4 hours, the mixture inside each test tube was transferred to two 1.5ml microcentrifuge tubes.
  5. The bacteria inside microcentrifuge tubes were spun down at 6000g for 3 minutes.
  6. 2.5 ml of supernatant from the two microcentrifuge tubes were extracted to a cuvette.
  7. 175ul of API copper testing solution was added to the cuvette.
  8. The solution was mixed by pipetting up and down for 10 seconds.
  9. Wait 20 seconds for the color to develop.
  10. The absorbance (440nm) of the solution was measured by a colorimeter.
  11. Copper concentration of the samples was determined by interpolation the absorbance measured on the standard curve.

Part 2 of the experiment: Measuring the copper concentration of the mixture.



Testing the Bacterial Copper Adsorption Device (B-CAD)

Aim
In this experiment, we aimed to test our B-CAD in a small scale but realistic situation. We obtain aquaponic water from a local aquaponic system and adjust the copper concentration (to 5, 10 or 15 mg/L) by adding copper (II) sulphate, this simulate copper polluted aquaponic water. We load our genetically modified bacteria to the B-CAD and circulate them through the “copper polluted” aquaponic water. The copper level of the system was measured every 2 hours for 2 days to evaluate if our B-CAD can lower copper level in water.

Materials and Equipments
  • 200ul and 1000ul micropipette
  • Colorimeter
  • Electronic balance
  • Conical flasks
  • Test tubes
  • API copper testing kit
    		•
  • SOB medium
  • Ampicillin / Kanamycin
  • IPTG (optional)
  • 100mg/L copper solution
  • Aquaponic water
  • Distilled water
    		•
Procedure
Preparation of bacterial culture:
  1. 7.02 g SOB powder was dissolved in 250 ml distilled water to prepare the SOB medium.
  2. 40 ml SOB medium was added to 250 ml conical flask.
  3. Each of the conical flask with SOB medium was sterilized by autoclave.
  4. Prepare 1000X ampicillin (100mg/ml) by dissolving 1g ampicillin in 10 ml distilled water. Caution: Wear mask, safety goggles and gloves when handling Ampicillin powder as it is corrosive and irritating.
  5. 40 uL of 1000X ampicillin was added into each flask with 40 ml SOB medium.
  6. A single colonie of respective cell culture was inoculated into conical flask with SOB medium.
  7. The flasks were incubated in a shaking incubator at 37℃ for 18 hours.
Remarks:
  1. According to the selection marker of the plasmid or bacteria strain, Kanamycin or other antibiotics may be used instead of ampicillin.
  2. In some experiments, the bacteria needed to be induced by IPTG. To prepare IPTG stock (1000X), 2.38 g of IPTG was dissolved in 10 mL of distilled water. The solution was filter sterilized with a 0.22μm syringe filter.

Testing of the B-CAD,
  1. 5 L of aquaponic water was added to a water tank.
  2. 1000mg/L copper solution was added to the tank until the final concentration of copper reach 10mg/L.
  3. 950 ml of aquaponic water was added to the reservoir of the B-CAD.
  4. 50 ml of bacterial culture was added to the reservoir of the B-CAD.
  5. The peristaltic pump and magnetic stirrer of the B-CAD was switched on.
  6. 3 ml of water sample was collected from the water tank and B-CAD every 12 hours for water testing.
  7. The 3 ml of water sample was separated into two 1.5ml microcentrifuge tubes.
  8. The bacteria inside microcentrifuge tubes were spun down at 6000g for 3 minutes.
  9. 2.5 ml of supernatant from the two microcentrifuge tubes were extracted to a cuvette.
  10. 175ul of API copper testing solution was added to the cuvette.
  11. The solution was mixed by pipetting up and down for 10 seconds.
  12. Wait 20 seconds for the color to develop.
  13. The absorbance (440nm) of the solution was measured by a colorimeter.
  14. Copper concentration of the samples was determined by interpolating the absorbance measured on the standard curve.