Team:NCTU Formosa/Protocol

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Wet Lab Protocol
NCTU_Formosa Protocol
Experiment:
- Preparation for Potato dextrose agar (PDA) plates

Purpose:

     To prepare solid medium to civilize the fungi

Purpose:

     To prepare solid medium to civilize the fungi

Drugs and equipment:

  • PDA powder
  • ddH2O
  • Petri dishes
  • Serum bottle
  • Microwave oven
  • Autoclave
  • Alcohol burner

Process:

  1. The ratio of PDA powder and ddH2O is 39g : 1000mL. To prevent the liquid spill out from Serum bottles, we usually have only 300mL of the liquid in a 500mL Serum bottle.
  2. Loosen the bottle cap for a bit, and put it into the autoclave.
  3. Screw the bottle cap tight to cool down. If the liquid becomes solid, heat it up with a microwave oven.
  4. Bring the bottle into the Hood, disinfect bottleneck with alcohol burner. Pour out the liquid to plates, each plate can hold 15~20mL of the PDA liquid. Shake the plate smoothly and make the liquid surface flat.
  5. Keep the plates in normal temperature or in 4 °C refrigerator
Experiment:
- Dual culture technique

Purpose:

     To test the antifungal activity of peptides comparing to negative control.

Purpose:

     To test the antifungal activity of peptides comparing to negative control.

Drugs and equipment:

  • Drugs (ex: NaN3, peptides…)
  • Hole puncher (tips are recommended)
  • PDA (Potato dextrose agar) plate
  • Fungi plates
  • Parafilm
  • Alcohol burner
  • Tweezers

Process:

  1. Culture some plates of fungi.
  2. Remove all the drugs and tools inside the Hood, disinfect tools with an alcohol burner. You may take a PDA plate for cooling tools down.
  3. Pick up fungi on the outer cycle (peripheral part) of a plate (the latest part of fungi plate) with tip. Dig into the previous PDA plate and pick up a piece of agar with fungi, remove it onto a new PDA plate, put the piece in the center of the plate.
  4. Disinfect tools with an alcohol burner. Seal up the plate with Parafilm, and label on the name of fungi, date and so on.
  5. Remove all the drugs and tools inside the Hood, disinfect tools with an alcohol burner. You may take a PDA plate for cooling tools down.
  6. Dig holes on the plate with tweezers, the position of the holes should be 0.5cm far away along the radius from the position of latest mycelium as the picture shows.
  7. Introduce the testing drug into the hole (different concentration of peptides, HEPES for our experiments)
  8. Disinfect tools with an alcohol burner. Seal up the plate with Parafilm.
  9. Observe how the mycelium grow
Experiment:
- Spore germination percentage

Purpose:

     To test the concentration of spore suspension liquid and calculate germination rate.

Purpose:

     To test the concentration of spore suspension liquid and calculate germination rate.

Drugs and equipment:

  • 75% Alcohol
  • 2% glucose solution
  • ddH2O
  • HEPES buffer
  • Peptides
  • Alcohol burner
  • Hemocytometer
  • Fungi plates
  • Glass Cell Spreaders
  • 10μl pipet
  • Gauze
  • Centrifuge
  • Beaker*2
  • Petri dishes
  • Lens cleaning paper
  • Microscope
  • Slide glass with 2 Cavities
  • Counter

Process:

  1. Choose fungi plates that have produced spores.
  2. (It is recommended to experiment in a laminar flow (hood)). Add ddH2O to the plate until water covers the surface of the plate.
  3. Disinfect the glass cell spreaders with an alcohol burner, after cooling down, scrape the plate softly so the spore would be in the water.
  4. Remove the water in the plate to a beaker (You may filter out impurity with gauze). Now you got a spore suspension liquid with unknown concentration.
  5. Clean the Hemocytometer with 75%alcohol and wipe it clean with lens cleaning paper, so as not to make any scratch on it. Put the coverslip on the Hemocytometer, and inject 10mL spore suspension liquid from the tiny chamber beside. The spore suspension should cover all the square of the Hemocytometer.
  6. Put the Hemocytometer under a microscope, and observe the spore.
  7. Count the amount of the spore in the square, and calculate the concentration. Add water if it’s concentration is too high; centrifuge the liquid if the concentration is too low. Finally, you got a spore suspension liquid with concentration you know. We would like to prepare spore suspension liquid with the concentration of 1.5*10^5 spores/mL for our experiments.
  8. Mixed 5μl spore suspension liquid with 5μl 2% glucose solution and 5μl peptide together into a PCR tube (It is recommended for pipetting 50 times to ensure that there is a mix of uniform).
  9. Draw 15μl of the solution from PCR tube to the double concave slide, and put the slide into a Petri dish. Add some ddH2O around the slide, in order to create an environment of 100% relative humidity so that the liquid would note evaporate easily.
  10. After incubating under 20℃ for 6 hours, we observe and classify the spores into four grades according to the length of the germination tube, calculate the percentage of each germination grade of each sample.
Experiment:
- Separation of infected plant tissue

Purpose:

     To separate infected plant tissue and cultivate pathogen fungi

Purpose:

     To separate infected plant tissue and cultivate pathogen fungi

Drugs and equipment:

  • 75% Alcohol
  • 0.5%~1% Sodium hypochlorite (NaClO)
  • ddH2O
  • PDA (Potato dextrose agar) plate
  • Parafilm
  • Alcohol burner
  • Plants
  • Knifes
  • Tweezers

Process:

  1. Soak the plant tissue to sterilization: All parts of the plant should soak in alcohol for 1 minute. For leaves, skim over NaClO for a few times; for roots, soak in NaClO for 1 minute; for mature stem parts, soak in NaClO for 20~30 seconds; for tender stems, skim over NaClO for a few times. All of these parts should be washed with ddH2O.
  2. Remove all the drugs and tools inside the Hood, disinfect knives and tweezers with an alcohol burner. You may take a PDA plate for cool down.
  3. Cut the infected plant tissue for an area of 5mm2, remove them all to new PDA plates. The side with hypha should put downward, and be towed on the plate. The amount of tissue which is put on one single plate depends on the range of cutting area.
  4. Seal up the plate with Parafilm, and label on the name of fungi, date and so on.
  5. Put the plate in the best environment to observe if the fungi or other species of microorganism grow.
Experiment:
E. coli DH5α growth rate in the arsenic solution

Purpose:

     Measure the E. coli DH5α growth rate arsenic solution.

Purpose:

     Measure the E. coli DH5α growth rate arsenic solution.

Drugs and equipment:

  • DH5α pc DNA (ctrl) on LB agar plates
  • SOC broth
  • Amp (1000 X)
  • 15 mL tube, 50 mL tube
  • 96 well plate

Process:


    Rpm:
  1. Culture 3 tubes of 2mL E. coli overnight by 15mL centrifuge tubes. (2mL SOC, 2uL AMP)
  2. Put them to the same tube at the next day, then add 6mL of SOC broth (water bath 37°C for 5 min. before use) and 12uL AMP for a 2-fold dilution.
  3. Take 5mL SOC broth for to a 15mL centrifuge tube for blank.
  4. Aliquot the bacterial broth into six 15mL centrifuge tubes (2mL for each), then culture for 2 hours.
  5. Measure OD 595
  6. Dilute to OD 0.312. More than 12mL

    Rpm80:
  1. 100, 50, 30, 10, 1ppm arsenic solutions, use ddH2O in control group
  2. Dilute the E. coli broth to 50 fold by SOC broth (0.6mL E. coli broth+0.3mL Arsenic solutuin+30uL AMP 29.1mL SOC broth)
  3. Measure OD at 0min. measure every 30 min. Blank is needed every time.
Experiment:
- Growth curve of E. coli with fMT plasmid in arsenic solution of different concentration

Purpose:

     Measure the time curve of E. coli chelating arsenic of 20 ppm.

Purpose:

     Measure the time curve of E. coli chelating arsenic of 20 ppm.

Drugs and equipment:

  • DH5α as plasmid on LB agar plates
  • E. coli (chelate) on LB agar plates
  • E. coli (GFP positive control) on LB agar plates
  • LB broth
  • CP (1000 X)
  • Arsenic stock (10^5ppm)
  • 15 mL tube, 50 mL tube
  • 96 well plate

Process:

  1. Culture 6 tubes of 2 mL E. coli(chelate) and 2 tubes of E. coli(GFP positive control) overnight by 15 mL centrifuge tubes (2 mL SOC, 2uL CP)
  2. Prepare another 8 tubes and mark them as the tubes cultured last night
  3. Perform a two-fold dilution for the tubes cultured last night and take out 2 mL bacteria liquid of each group to the corresponding tube prepared
  4. Add 2uL CP in each tube.
  5. Culture the tubes for another two hours
  6. Measure the OD of each tube (wavelenght 595)
  7. Dilute all tubes to OD 0.5 by LB. (More than )
  8. Prepare and mark 50 mL tubes as chart below. Culture the tubes.
  9. Take out the tubes from incubator.

    1. Each tube contains
    2. 29.1 mL LB
    3. 0.6 mL bacteria liquid OD=0.5
    4. 0.3 mL arsenic solution
    5. 30uL CP

    Step 10-18 should be performed after Xhr X=the time marked on the tube
  10. Centrifuge the 50 mL tubes by 6000 g/10 min
  11. Take out the supernatant and preserve in -80°C
  12. Add 10 mL PBS to each tube
  13. Centrifuge by 6000 g/10 min
  14. Abandon PBS supernatant and add in 15 mL ddH2O
  15. Separate the liquid into fifteen full eppendorfs. Centrifuge by 12000 g/5 min
  16. Abandon ddH2O supernatant and add in 10 mL ddH2O
  17. Centrifuge by 12000 g/5 min
  18. Combine the bacteria into one tube
Experiment:
- Function test of GFP biosensor in different concentration of Chinese medicine

Purpose:

     Test the fluorescence intensity of GFP biosensor in different concentration of Chinese medicine.

Purpose:

     Test the fluorescence intensity of GFP biosensor in different concentration of Chinese medicine.

Drugs and equipment:

  • DH5α pc DNA (ctrl) on agar plates
  • LB broth
  • Cp (1000 X)
  • 15 mL tube
  • 96 well plate
  • Pipette
  • As solution
  • Chinese medicine

Process:

  1. Culture three tubes of 2mL E. coli GFP biosensor with 2uL CP+ , and culture one tube of 2mL E. coli positive control with 2uL CP+ in the other tube.
  2. Put both tubes into 37°C incubator for 16hr
  3. Make a 2-fold dilution, separate the GFP biosensor into eight tubes and the positive control into two tubes. add in 2uL CP+ in each tube
  4. Culture the ten tubes for another 2 hours
  5. Measure the Obstacle Density of the GFP biosensor and the positive control
  6. Prepare a tube that contains 12mL bacteria liquid, which has a 0.500 Obstacle Density
    • Note: v=original bacteria amount(uL) , X=LB amount (uL)
    • 0.5=v*bacteria OD/v+X
      v+X=3000
  7. Prepare tubes as the chart below


  8. Add in 4.85mL 37°C LB, 0.1mL bacteria liquid, 0.05mL Chinese medicine and 5uL CP+
  9. Put these tubes into 37°C incubator
  10. Measure the Obstacle Density and Fluorescence of each tube after 4, 5, 6 hours.
Experiment:
- Growth rate test of LacZ α biosensor on different concentration of As5+

Purpose:

     Test the growth rate of LacZ α biosensor on different concentration of As5+.

Purpose:

     Test the growth rate of LacZ α biosensor on different concentration of As5+.

Drugs and equipment:

  • DH5⍺ pc DNA(crtl) on agar plate
  • SOC broth
  • Cp (1000x)
  • As5+ solution 100,1000,3000,5000,10000 ppm
  • ddH2O
  • 15mL tube
  • 50mL tube
  • 96 well plate
  • pipet

Process:

  1. Take a 15mL tube. Culture 2mL E. coli LacZ ⍺ biosensor by SOC with 2µL Cp.
  2. Put the tube into 37°C incubator for 15 hr.
  3. Take 1mL of the bacteria liquid with 1mL SOC, and 2µL Cp into a new 15mL tube to perform a 2-fold solution.
  4. Put the tube into 37°C incubator for 2hr.
  5. Take 200µL of the solution to the 96 well plate.
  6. Measure OD595
  7. Dilute the bacteria liquid to OD=0.312/1.2mL
  8. Take 18 15mL tubes.(0ppm,1ppm,10ppm,30ppm,50ppm,100ppm. 3 tube for every concentration)
  9. Add 29.1mL SOC, 30µL Cp, 300µL As5+ solution, 600µL bacteria liquid to each tube.
  10. Measure OD595 every 30 minutes for 7.5 hours.
Experiment:
- LacZ ⍺ biosensor pretest

Purpose:

     Test if the LacZ ⍺ biosensor can produce blue deposit in different As5+ condition.

Purpose:

     Test if the LacZ ⍺ biosensor can produce blue deposit in different As5+ condition.

Drugs and equipment:

  • DH5⍺ pc DNA(crtl) on agar plate
  • LB broth
  • Cp (1000x)
  • X-gal
  • As5+ solution 1000ppm, 10000ppm
  • ddH2O
  • 15mL tube
  • pipet
  • PBS
  • 96 well plate

Process:

  1. Culture two tubes of E. coli LacZ ⍺ biosensor for four hours
  2. Prepare twenty one 15mL tubes, mark them as below

  3. Make each tube contain 1900uL LB, bacteria liquid, 40uL Xgal and 20uL metal concentration
  4. Culture the twenty one tubes for 16 hours
  5. Take 200µL of the solution to the 96 well plate.
  6. Measure OD595
  7. After culturing, centrifuge the bacteria liquid by12000G, 5min
  8. Remove the supernatant
Experiment:
- Specificity test of GFP biosensor in As5+, Cu2+, and Pb2+

Purpose:

     Test the specificity of GFP biosensor in different concentration of As5+, Cu2+, and Pb2+ solutions.

Purpose:

     Test the specificity of GFP biosensor in different concentration of As5+, Cu2+, and Pb2+ solutions.

Drugs and equipment:

  • DH5⍺ pc DNA(crtl) on agar plate
  • SOC broth
  • Cp (1000x)
  • 15 mL tube
  • 96 well plate
  • Pipette
  • As5+ solution
  • Cu2+ solution
  • Pb2+ solution

Process:

  1. Culture three tubes of 2mL E. coli GFP biosensor with 2uL CP+ , and culture one tube of 2mL E. coli positive control with 2uL CP+ in the other tube.
  2. Put both tubes into 37°C incubator for 16hr
  3. Make a 2-fold dilution, separate the GFP biosensor into eight tubes and the positive control into two tubes. add in 2uL CP+ in each tube
  4. Culture the ten tubes for another 2 hours
  5. Measure the Obstacle Density of the GFP biosensor and the positive control
  6. Prepare a tube that contains 12mL bacteria liquid, which has a 0.500 Obstacle Density
    • Note: v=original bacteria amount(uL) , X=SOC amount (uL)
    • 0.5=v*bacteria OD/v+X
      v+X=3000
  7. Prepare tubes as the chart below

  8. Add in 4.8mL 37°C SOC, 0.1mL bacteria liquid, 0.05 mL ion solution and 5uL CP+.
  9. Put these tubes into 37°C incubator
  10. Measure the Obstacle Density and Fluorescence of each tube after 4 hours.

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