Difference between revisions of "Team:XMU-China/Experiments"

1. 200μL bacteria solution and 10μL chloramphenicol were inoculated into 10mL LB liquid mediums at 37 °C, 200 rpm and last 12-16h.
2. Pellet 1mL bacteria solution from step 1 in a clean 1.5-mL microcentrifuge tube by centrifugation at 5,000rpm for 1 minute at room temperature. Decant or aspirate medium and discard.
3. Add 1mL pbs buffer, pipet up and down to mix thoroughly. And centrifuge at 5,000rpm for 1 minute at room temperature. Decant or aspirate medium and discard.
4. Repeat step 3 once.
5. Add 500μL pbs buffer, pipet up and down to mix thoroughly.
6. 5μL bacteria solution from step 5 was inoculated in the center of cover glass. And place the slide glass on the cover glass gently.
7. Turn over the slide glass gently and paste nail polish around the edges of cover glass.
8. Turn over the slide glass gently again until nail polish is solidified and last 30min.
9. Turn over the slide glass and make a marker.

1. Use Leica TCS SP8 X microscope at appropriate vision and take a picture. Related parameters: SmartGain: 800 V,
1% Laser Intensity
resolution: 2048 bp
excitation wavelength: 488 nm.

● LB liquid medium with 0.1 mg/mL chloramphenicol

1. 200 μL bacteria solution and 10μL chloramphenicol were inoculated into 10mL LB liquid mediums at 37 °C, 200 rpm and last 12-16h.

1. inoculate 200 μL bacteria solution into 96-well microtiter plates.
2. measure the value of OD₆₀₀.
3. adjust OD₆₀₀ of remaining bacterial fluid to 1 with pbs buffer.
4. centrifuge the bacterial fluid at 6,500 rpm for 5 min at 4 °C, decant or aspirate supernatant and discard and add equivalent pbs buffer and pipet up and down to mix thoroughly. And take a picture.
5. inoculate 200 μL bacteria solution into 96-well microtiter plates and measure fluorescence with excitation wavelength 475 nm and emission wavelength 545 nm.
6. use ultrasonic waves to lysis cells at 25 times.
7. centrifuge the bacterial fluid at 20,000 rpm for 20 min at 4 °C. and take a picture.
8. inoculate 200 μL supernatant into 96-well microtiter plates and measure fluorescence with excitation wavelength 475 nm and emission wavelength 545 nm.
9. Decant or aspirate supernatant and discard, add equivalent pbs buffer and pipet up and down to mix thoroughly.
10. inoculate 200 μL supernatant into 96-well microtiter plates and measure fluorescence with excitation wavelength 475 nm and emission wavelength 545 nm.

● TECAN^® Infinite M200 Pro Microplate Reader
● 96 well microtiter plates
● Glyphosate, NADPH·Na₄, Tris-HCl buffer
● Purified GOX enzyme
● Purified GRHPR enzyme

1. Start testing immediately after preparing the reaction system. Record the value of fluorescence in real time.
2. Analyze the slope.

● TECAN^® Infinite M200 Pro Microplate Reader
● 96 well microtiter plates
● Glyoxylic acid, NADPH·Na₄,

1. Surface display properties of GOX
●Prepare aqueous solution below.
●2.0 mg/mL Glyphosate
●2mM NADPH·Na₄ ●pH=6.8, 50mM Tris-HCl buffer
● Prepare these reaction systems according to the table below. (Unit: μL)

1. Start testing immediately after preparing the reaction system. Record the value of fluorescence in real time.
2. Analyze the slope.

●Purified iNAP Protein
●Macklin^® NADPH
●Corning^® Costar 96-Well Black flat-Bottom Plates
●TECAN^® Infinite M200 Pro ELIASA

A. Preparation of NADPH Solution
1.9 mg NAPDH powder was dissolved in 6 mL purified water and prepared into 2000 μM solution.
2.Dilute in turn and prepare solutions of concentration of 0.01, 0.05, 0.1, 0.25, 0.5, 1,2.5, 5,10,25,50,100,200 μM
B．Determination
1.100 μL of solutions of 0.01, 0.05, 0.1, 0.25, 0.5, 0.5, 5, 2.5, 50, 50, 100, 200 μM are added to a group of wells in the 96-well plate, and 100 μL of purified iNAP protein fluid are added to the group of Wells for detection. Put the 96-well plate into ELIASA.
2.Program setting of ELIASA：
a)Shaking

A. Sampling
1．Take 20 mg of glyphosate in a 15-mL centrifuge tube, and 10 mL of ddH₂O is added.
2．Each group was taken 1 mL of glyphosate solution as blank.
3．Add 19 mL E.coli BL21 (DE3) culture (experimental group 100-RBS-phnE₁E₂_pSB1C3 and control group 100-RBS_pSB1C3) and 1 ml of glyphosate solution to the 50-ml centrifuge tube.
4．Add 2 mL mixture that mentioned above to each of the experimental group, and mark the number. 5. Take the first group of mixture that the glyphosate solution is just added as blank. The reaction is then carried out at 37 °C, 200 rpm.
6. Centrifugal the 15-mL centrifuge tube with the reaction mixture at 6500 rpm for 5min, take the upper layer, mark the name, number and time period, resuspend the bacterium with PBS buffer, and place the samples in a boiling water bath as soon as possible to quench the reaction.
Gradient : 0, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300 min
B. Instrument pretreatment
1. Preparation of mobile phase
a) Add 950ml of water and 50ml Isopropanol to the beaker, and add 0.500g CuSO4·5H₂O and transfer them to the beaker.
b) Filter by vacuum pump and ultrasonic defoam for 30 min.
2. Preparation of glucose and cellobiose standard working curve samples (0.01, 0.02, 0.05, 0.1, 0.2, 0.5 g/L)
a) Prepare 2.0 g/L glucose and cellobiose solution.
b) Dilute to 0.1, 0.2, 0.5, 1.0, 2.0 g/L with other volumetric flasks based on a 2.0g/L.
3. Instrument pre-cleaning
a) Loosen the pump valve and clean the outer passage to no air bubbles.
b) Tighten the pump valve. The flow rate was adjusted to 0.8 mL/min. After the baseline was substantially leveled, the flow rate was set to 0.5 mL/min and the pump was turned off.
c) Replace the mobile phase (pre-made i-PrOH-CuSO4-H₂O mobile phase) and start the pump.
d) Adjust the solution fill and open the pump valve. (counterclockwise)
f) VMD—Turn on
g) After the baseline is basically leveled, adjust the rectification speed to 0.2 mL/min and the upper pressure limit to 85 bar.
h) Install the column (Chirex 3126 (D)-penicillamin Column, 250×4.60 mm).
i) Open the column oven at 35 °C, adjust the flow rate to 0.8 mL/min, and clean the reference cell for 20 min until the baseline is stable.
C. Detection
1. Put in the wash bottle, sequence the parameters, set the path, name and operator, then fill in the sequence table.
2. Take a 1 mL of sample from the syringe and inject it into the sample tube through a 0.22-μm filter.
3. Place all samples in the sample tray according to the sequence table and feed them into the machine.
D. Post-processing
1. After running the last sample, manually integrate and save the relevant data file.
2. Rinse for 1 hour and adjust the flow rate to 0.2 mL/min.
3. Close the column oven and turn off the pump and unload the column after the column temperature drops.
4. VMD—Turn off.
5. The flow rate is adjusted to 0.2 mL/min.
6. Turn off the pump, software, liquid meter, and computer in sequence.

●Purified PhnO enzyme
●TECAN® Infinite M200 Pro Microplate Reader
●96-well microtiter plates
●Reaction system (1 mL) :

1. Add 180 μL reaction system into one well in 96-well microtiter plates, and then add 20 μL PhnO enzyme solution into the same well. Put the plate into Microplate Reader.
2.Program setting of Microplate Reader:

1. Add 200 μL glycerin bacteria into 10 mL LB liquid medium containing 50 mg/mL chloramphenicol. Incubate the culture in a shaker overnight at 37 °C, 200 rpm.
2. Add 1 mL of the culture above into 50 mL LB liquid medium containing 50 mg/mL chloramphenicol, and incubate it in a shaker of 37 °C and 200 rpm until OD₆₀₀ reaches 1.0.
3. Samples are serially diluted in PBS over a 6-log range and spotted 50μL bacteria solution onto LB solid plate, incubate it at 37°C for 12h.
4. Count the number of colonies, and then calculate the CFU. (CFU/mL=(numbers of colonies)×(dilution factor)/0.05mL)(1)
Reference. C. T. Chan, J. W. Lee, D. E. Cameron, C. J. Bashor, J. J. Collins, 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat Chem Biol 12, 82-86 (2016).