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.

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.

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.


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

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.

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.

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

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.