Team:XMU-China/Experiments
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1. MD® Spectra max190 Molecular Device
2. 96 well microtiter plates
3. L-histidine, Tris-HCl buffer, MnCl2, reduced glutathione (GSH) and water
4. Purified HutH enzyme
Prepare aqueous solution below.
5 mmol•L-1 L-histidine, 100 mmol•L-1 Tris-HCl buffer pH 9.0, 0.1 mmol•L-1 MnCl2, 1.7mmol•L-1 reduced glutathione (GSH)
Prepare these reaction systems according to the table below. (Unit: μL)
| L-histidine | Tris-HCl buffer | MnCl2 | GSH | H2O | Purified HutH enzyme | BSA | |
|---|---|---|---|---|---|---|---|
| A | 80 | 16 | 0.16 | 8 | 55.84 | 40 | 0 |
| B | 32 | 16 | 0.16 | 8 | 103.84 | 40 | 0 |
| C | 16 | 16 | 0.16 | 8 | 119.84 | 40 | 0 |
| D | 8 | 16 | 0.16 | 8 | 127.84 | 40 | 0 |
| E | 3.2 | 16 | 0.16 | 8 | 132.84 | 40 | 0 |
| F | 16 | 16 | 0.16 | 8 | 119.84 | 0 | 40 |
1. Start testing immediately after preparing the reaction system. Record the value of fluorescence in real time.
2. Analyze the slope.
Erlenmeyer flask; Hemocytometer; Optical microscope; Incubator; light source; 0.22µm membrane; filter; Milli Q water; Sea salt; Penicillin-Streptomycin Solution
Get sea water ready: add commercial sea salt to Milli Q water to get 3 % seawater, which is filtered through a 0.22µm pore filter membrane to prevent contamination by algae, sterilized at 110℃ for 30 minutes, and cooled at room temperature for later usage. The other components of L1-Si medium need to be filtered through 0.22µm membrane filters, and the whole preparation process needs to be aseptically operated in a Flow Clean Bench.
Prepare L1-Si medium (Silicate-free L1 medium):
●Purified TnaA enzyme
●TECAN® Infinite M200 Pro Microplate Reader
●96-well microtiter plates
A. Measurement of standard curve
1. 5 mg indole powder was dissolved in toluene and prepared into 0.1 mg/mL solution.
2. Dilute in turn and prepare solutions of concentration of 1, 2, 3, 4, 5 µg/mL.
3. Add 0.4 mL distilled water, 3 mL mixed chromogenic solution (5% PDAB, 5% concentrated HCl) into 1 mL of each indole solution prepared in step 2, stand for 30 min.
4. Measure the samples at 570 nm using microplate reader.
5. Draw the OD570 - Concentration curve.
B. Determination of enzyme activity
1. Add 20 µL 200 µg/mL PLP solution, 10 µL 5 mM GSH solution, and 270 µL enzyme solution in a 15mL centrifuge tube in order.
2. Add 1.0 mL toluene to cover the mixed solution in step 1.
3. Stand for 5 min at 37℃.
4. Add 100 µL tryptophan solution of different concentration (0.25, 0.5, 1, 2.5, 5, 10, 20 mg/mL)
5. Incubate the solution at 37 °C with agitation at 200 r.p.m for 10 min.
6. Add 0.4 mL distilled water, 3 mL mixed chromogenic solution (5% PDAB, 5% concentrated HCl), stand for 30min.
7. Measure the samples at 570 nm using microplate reader.
●Dry bath
●Shaker
●Rotary evaporator
●Benchtop centrifuge
●Vortex mixer
●Microplate reader
●96-well microtiter plates
●0.22-µm filter
Inoculate 200 µL bacteria glycerol stock into 10 mL LB liquid medium with 0.1 mg/mL kanamycin, 37 ℃, 200 rpm overnight.
Determine standard curve of rhamnose: Prepare 100 µL rhamnose solution of different concentration (0.01, 0.05, 0.10, 0.25, 0.05, 0.1, 0.2, 0.5 mM), 100 µL of 1.6% orcinol solution and 800 µL of 60% sulphuric acid in distilled water was added in each sample. The mixture was incubated at 80 ℃, 1000 rpm for 30 min, then cooled to room temperature. Measure the absorbance of the samples at 420 nm using microplate reader.
1. Inoculate 1 mL of the culture into 100 mL LB liquid medium with 0.1 mg/mL kanamycin, culture at 37 ℃, 200 rpm for 72 hours, take 200 µL of culture at 0 h, 3 h, 5 h, 7 h, 9 h, 12 h, 18 h, 24 h, 36 h, 48 h, centrifuge at 16000×g for 5 min, then filter the supernatant with 0.22-µm filter, finally store the samples at 4 ℃ for further treatment.
2. 100 µL culture was extracted three times with 500 µL of ethyl acetate. The sample was mixed by vortex mixer for 2 min, then centrifuge at 10000 rpm for 1 min. The upper phase was collected in rotary evaporator sample bottles.
3. The organic solvent was removed by evaporation, the remaining was dissolved in 100 µL distilled water, subsequently 100 µL of 1.6% orcinol solution and 800 µL of 60% sulphuric acid in distilled water was added.
4. The mixture was incubated at 80 ℃, 1000 rpm for 30 min, then cooled to room temperature.
5. Measure the absorbance of the samples at 420 nm using microplate reader.
500-mL conical flasks; LBv2 medium; LB medium; Agar; LB agar plates; 96-well microtiter plates;
IPTG-induced blrA-GFP light * 3
IPTG non-induced blrA-GFP light * 3
1.Add 100 mL LBv2 medium to each of the six 500-mL conical flasks to sterilize.
2. Add 300 mL LB medium and 3 g agar to both of the 500-mL conical flasks to sterilize.
3. Preculture 15 mL bacteria culture to OD600 > 0.6 in dark.
4. Take samples of the bacteria culture in dark to measure the OD600 value until it is between 0.8 and 1.0.
5. Add kanamycin (100 µg/mL) at a dilution of 1:1000 in conical flasks and mix. Replace the tinfoil wrapped around the bottleneck of the flask with sterile six-layer gauze.
6. Inoculate the bacteria culture into the conical flasks at a volume ratio of 2-3%
7. Take 200 µL of samples twice at the same time after manual mixing into two wells of 96-well microtiter plates to measure OD600 and GFP, and take average of the measurement results.
8. Incubate the culture at 37 ℃ with agitation at 200 r.p.m., then take samples every two hours to measure OD600 and GFP fluorescence intensity, and replace them to the shaker as soon as possible after sampling.
9. Samples are serially diluted in LBv2 over a 6-log range and spot 100µL bacteria solution onto LBv2 solid plate.
11. Repeat step 10 after two hours, add IPTG at a dilution of 1:1000 to the induced group, at the same time turn on the blue light.
10.Take samples every two hours after induction, measure OD600, GFP fluorescence intensity and repeat step 10 to measure the colony forming unit (CFU). Repeat those steps 3-4 times in total.
LBv2 medium; LB medium; Agar; LB agar plates; 96-well microtiter plates
Light induced J23106-EL222-pBLind-GFP*3;
Light non-induced blrA-GFP*3;
Light induced pBLind-EL222-pBLind-GFP*3;
Light non-induced pBLind-EL222-pBLind-GFP*3.
1. Preculture 15 mL bacteria culture to reach an OD600 value between 0.8 and 1.0 in dark.
2. Add kanamycin (100 µg/mL) at a dilution of 1:1000 in conical flasks and mix. Replace the tinfoil wrapped around the bottleneck of the flask with sterile six-layer gauze.
3. Inoculate the bacteria culture into the conical flasks at a volume ratio of 2-3%
4. Take 200 µL of samples twice at the same time after manual mixing into two wells of 96-well microtiter plates to measure OD600 and GFP, and take average of the measurement results.
5. Incubate the culture at 37 ℃ with agitation at 200 r.p.m, then take samples every two hours to measure OD600 and GFP fluorescence intensity, and replace them to the shaker as soon as possible after sampling.
6. When OD600 value exceeds 0.6, turn on the blue light for the light group after sampling.
7. Keep the blue light on, continuously measure the OD600 and GFP fluorescence intensity for 10-12 hours with a 2-hour interval.
LBv2 medium; LB medium; Agar; LBv2 agar plate, 96-well microtiter plates
J23106-EL222-pBLind-blrA-GFP
pBLind-EL222-pBLind-blrA-GFP
1. Preculture 15 mL bacteria culture to reach an OD600 value between 0.8 and 1.0 in dark.
2. Add 10 mL LBv2 liquid culture into the 50 mL centrifuge tube, add Kanamycin (100 µg/mL) at a dilution of 1:1000 and mix. Cover the tube but do not tighten it, wrap the tube with tin foil.
3, Incubate the bacteria culture into the conical flasks at a volume ratio of 2-3%.
4. Inoculate the culture at 37 ℃ with agitation at 220 r.p.m, for 5 h until OD600=1.0-1.4.
5. The samples are diluted 107 times. Individually spot 100 µL of each sample onto four LBv2 agar plates, wrap two of them in tin foil, expose the other two to blue light and culture at 37℃ for 10-12 h.
6. Observe and compare the differences between the illuminated group and the non-illuminated group.
Molecular Devices SpectraMax® iD3 Multi-Mode Microplate Reader
Corning® Costar 96-Well Black flat-Bottom Plates
LB liquid medium with 0.1 mg/mL chloramphenicol
Prepare two groups of 50-mL conical flask, wrapped with tinfoil and newspaper to make it light-proof. 8-layer of sterilized gauze covers the bottleneck for shadowing. 10 mL of LB liquid medium with 0.1 mg/mL chloramphenicol was added to each flask (for strictly shadowed condition).
Prepare two groups of 50-mL conical flask, breathable sterile sealing film covers the bottleneck. 10 mL of LB liquid medium with 0.1 mg/mL chloramphenicol was added to each flask (for light-induced condition).
1. Inoculate 200 µL bacteria glycerol stock into 10 mL LB liquid medium with 0.1 mg/mL chloramphenicol, 37 ℃, 200 rpm overnight.
2. Inoculate 200 µL bacteria culture of experimental and control group into each group of flasks, shake the flasks and immediately take 200 µL out for detection.
3. When cultured at 37 ℃, 200 rpm for 20 hours, take 200 µL of culture at 1 h, 2 h, 3 h, 4 h, 5 h, 8 h, 11 h and 19 h for detecting OD600 and fluorescence intensity.
1. Parameter settings for measuring fluorescence intensity in microplate reader:
2. Relative fluorescent unit (RFU) = Fluorescence Intensity of Sample – Fluorescence Intensity of Blank (LB medium)
—96-well microtiter plates
—LAS buffer
—PBS buffer
—Shaking incubator of 37 °C
1. Prepare a clean 96-well microtiter plates. (Note: choose non-polymer plate, such as quartz plate)
2. Prepare LBv2 culture medium (3.7 g LB broth, 1.19 g NaCl, 0.031 g KCl and 0.47 g MgCl2·6H2O per 100ml).
3. Prepare PBS buffer (pH=7.4)
4. Prepare 0.05 mM LAS buffer and 0.25 mM LAS buffer (dissolved in PBS buffer, pH=7.4)
1. Cells are inoculated in fresh LBv2 medium at a dilution of 1:100 (overnight culture/fresh medium) and incubated overnight at 30 ℃ with agitation at 200 r.p.m.
2. Add ten 1 mm PP balls into each well in the first column of the plate. Add 200μL culture into the wells. For negative control, add 200 μL LBv2.
3. Incubate at 30 ℃ with agitation at 200 r.p.m. for 30 min.
4. Move the beads to the next column, add 500 μL PBS buffer, incubate with agitation at 200 r.p.m. for 1 min, discard the solution, repeat 5 times.
5. Move the beads to the next column, add 500 μL 0.05mM LAS buffer, incubate with agitation at 200 r.p.m. for 1 min.
6. Repeat step 5 once.
7. Move the beads to the next column, add 500 μL 0.05mM LAS buffer, incubate with agitation at 200 r.p.m. for 1 min.
8. Move the beads to the next column, add 200 μL LBv2 culture medium, incubate at 30 ℃ with agitation at 200 r.p.m. for 30 min.
8. Take 10 μL of culture from each well in step 7, move it to the next column, add 190 μL LBv2, measure the samples at 600 nm using microplate reader.
—96-well microtiter plates
—PBS buffer
1. Prepare LBv2 culture medium (3.7 g LB broth, 1.19 g NaCl, 0.031 g KCl and 0.47 g MgCl2·6H2O per 100ml).
2. Prepare PBS buffer (pH=7.4)
1. Cells are inoculated in fresh LBv2 medium at a dilution of 1:100 (overnight culture/fresh medium) and incubated overnight at 30 ℃ with agitation at 200 r.p.m.
2. Take 10 mL culture and cleave the cell, centrifuge at 5000 rpm for 20 min in 4 times to extract protein.
3. Condense the protein, drop 1 mL solution onto the 96-well microtiter plates placed horizontally, and stand for 10 min.
4. Wash 96-well microtiter plates with PBS buffer from the same direction for 1 min continuously.
5. Measure the fluorescence intensity under blue light.