Team:XMU-China/Experiments
● LB liquid medium with 0.1mg/mL chloramphenicol
● Pbs buffer
● Nail polish
● Slide glass and cover glass
● centrifuge
● Leica TCS SP8 X microscope (Leica Microsystems)
● SmartGain: 800 V, 1% Laser Intensity, resolution: 2048 bp, excitation wavelength: 488 nm.
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
Prepare aqueous solution below.
2.0 mg/mL Glyphosate
2 mM NADPH·Na4 pH=6.8, 50 mM Tris-HCl buffer
Prepare these reaction systems according to the table below. (Unit: μL)
| Tris-HCl buffer | NADPH·Na4 | Glyphosate | Purified GRHPR enzyme | Purified GOX enzyme | |
|---|---|---|---|---|---|
| A | 80 | 30 | 30 | 30 | 30 |
| B | 110 | 30 | 30 | 30 | 0 |
Prepare aqueous solution below.
80mM Glyoxylic acid
2mM NADPH·Na4
pH=7.5, 50mM Tris-HCl buffer
Prepare these reaction systems according to the table below. (Unit: μL)
| Tris-HCl buffer | NADPH·Na4 | Glyoxylic acid | Purified GRHPR enzyme | |
|---|---|---|---|---|
| A | 50 | 50 | 50 | 50 |
| B | 100 | 50 | 50 | 0 |
| Shaking Duration | 5 sec |
|---|---|
| Mode | Linear |
| Amplitude | 1 mm |
| Frequency | 886.9 rpm |
| Number of Cycles | 45 |
| Time interval | 10s |
| Wavelength | 340 nm |
| Temperature | 30℃ |
| Number of flash | 25 |
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)
| Tris-HCl buffer | NADPH·Na4 | Glyoxylic acid | E.Coli Anchored with GOX | Purified GRHPR enzyme | |
|---|---|---|---|---|---|
| A(Blank) | 50 | 50 | 50 | 0 | 50 |
| B (INPNC-GOX) | 50 | 50 | 50 | 50 | 0 |
| C (GOX-AIDA) | 50 | 50 | 50 | 50 | 0 |
| D (GOX-Brka) | 50 | 50 | 50 | 50 | 0 |
2. Surface display properties of GRHPR
●Prepare aqueous solution below.
●80mM Glyoxylic acid
●2mM NADPH·Na4 ●pH=7.5, 50mM Tris-HCl buffer
● Prepare these reaction systems according to the table below. (Unit: μL)
| Tris-HCl buffer | NADPH·Na4 | Glyoxylic acid | E.Coli Anchored with GOX | E.Coli (J23100-RBS-pSB1C3) | |
|---|---|---|---|---|---|
| A(Blank) | 50 | 50 | 50 | 0 | 50 |
| B (INPNC-GRHPR) | 50 | 50 | 50 | 50 | 0 |
| C (GRHPR-AIDA) | 50 | 50 | 50 | 50 | 0 |
| D (GRHPR-Brka) | 50 | 50 | 50 | 50 | 0 |
Program setting of Microplate Reader：
| Shaking Duration | 5 sec |
|---|---|
| Mode | Linear |
| Amplitude | 1 mm |
| Frequency | 886.9 rpm |
| Number of Cycles | 45 |
| Time interval | 10s |
| Wavelength | 340 nm |
| Temperature | 30℃ |
| Number of flash | 25 |
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
| Duration | 5 sec |
|---|---|
| Mode | Linear |
| Amplitude | 1 mm |
| Frequency | 886.9 rpm |
| Excitation wavelength | 420 nm |
|---|---|
| Emission wavelength | 528 nm |
| Mode | Top |
| Z-Position | Manual(20000 μm) |
| Number of flash | 25 |
| Gain | Manual / Optimal / Calculated from well |
● 1×PBS buffer
● E.coli BL21 (DE3) culture (J23100-B0034-phnE1E2_pSB1C3 and J23100-B0034_pSB1C3 plasmid, OD600=2.0)
● Glyphosate (Solarbio®)
● Isopropanol (Shanghai Institute of Chemical Reagents)
● Copper sulfate pentahydrate (Aldrich®)
● Double distilled water (ddH2O)
● Agilent Technologies 1200 Series (Degasser, Quat Pump, ALS, TCC)
● Agilent Technologies 1260 Inifinity (VWD)
● Column (Chirex 3126 (D)-penicillamin Column, 250×4.60 mm)
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) :
| Components | Concentration |
|---|---|
| HEPES | 100 mM |
| (NH4)2SO4 | 100 mM |
| 4,4′-dithiodipyridine (DTDP) | 200 μM |
| Ni2+ (NiCl2) | 100 μM |
| AcCoA | 0.1 mM |
| aminomethylphosphonic acid (AMPA) | 2 mM |
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:
| Shaking Duration | 5 sec |
|---|---|
| Mode | Linear |
| Amplitude | 1 mm |
| Frequency | 886.9 rpm |
| Number of Cycles | 45 |
| Time interval | 10s |
| Wavelength | 340 nm |
| Temperature | 30℃ |
| Number of flash | 25 |
●Culture dish
●LB solid medium
●LB liquid medium
●Chloramphenicol solution (50 mg/mL)
●phosphate buffer saline (PBS)
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).
●1×PBS solution
●Buffer A
●Buffer B
●GE AKTA Prime Plus FPLC System
A. Buffer preparation
1. Buffer A
| Na3PO4·12H2O | 7.6g |
|---|---|
| NaCl | 29.2g |
| Imidazole | 1.36g |
| H2O | 1L |
2.Buffer B
| Na3PO4·12H2O | 7.6g |
|---|---|
| NaCl | 29.2g |
| Imidazole | 34g |
| H2O | 1L |
3.30 % ethanol
4.ddH2O Be filtered through 0.22-μm filter membrane and ultrasonic defoam for 30 min.
B. Sampling
1.40 mL bacterial solution was taken from each group in a 50-mL centrifuge tube, and centrifuged at 6000 rpm for 10 min.
2.Discard the supernatant, then add 40 mL PBS buffer solution to each tube. Solution is resuspended by shock, and centrifuge it at 6000 rpm for 10 min.
3.Add 40 mL Buffer A to shake and resuspended, and place on the ice.
4.Ultrasonic crush.
5.After crushing, centrifuge at 10000 rpm for 20 min to collect supernatant and filter.
C. AKTA purification
1.Clean the pipes
Flush the pipe with water at a flow rate of 50 mL/min , and set the pressure Limit as 1.0 MPa.
2.Packing column
a)Set the flow rate at 0.5ml /min and pressure Limit at 1.0mpa.
b)Load the Histrap TM purified column into the pipeline.
3.Balance column
a)Replace the mobile phase of pipeline A with Buffer A and that of pipeline B with Buffer B.
b)Wash 25 mL of pipeline B, and then 50 mL of pipeline A to the baseline horizontally.
4.Load sample
Put the pipeline A into the sample and set the flow rate at 3 mL/min. Pay attention not to let the air enter the pipeline.
5.Wash the miscellaneous
Put pipeline A back into Buffer A and set the flow rate at 5.0 mL/min until the baseline become horizontal.
6.Elute
a) Set Gradient, Length: 100 mL, Target: 100 mL.
b) Set flow rate at 5 mL/min.
c) Set Fraction size at 5 mL/min.
d) Collect protein fluid where the peak is located, then desalt protein fluid with Tris-HCl ( pH7.5)
7.Rebalance the column
8.Dismantle the column
a)Pipeline A and B were rinsed and placed in water. Set the flow rate at 5 mL/min. 25 mL was rinsed with water and 25 mL was rinsed with ethanol.
b)Set the flow rate at 0.5 mL /min and dismantle the HistrapTM column
Set the flow rate at 50 mL/min and rinse 75 mL
●LB liquid medium with 0.1mg/mL chloramphenicol
●Membrane and Cytosol Protein Extraction Kit from BestBio company.
●Water bath
1. 200μL bacteria solution and 10μL chloramphenicol were inoculated into 10mL LB liquid mediums at 37 °C, 200 rpm and last 12-16h.
1. centrifuge all bacteria solution at 6,500rpm for 15 minutes at room temperature. Decant or aspirate medium and discard.
2. Add 20mL pbs buffer, pipet up and down to mix thoroughly. And centrifuge at 6,500rpm for 15 minutes at 4 °C. Decant or aspirate medium and discard.
3. Blot the supernatant.
4. add 1mL Membrane protein extract A, 10μL Membrane protein extract B and 4μL Protease inhibitor, and pipet up and down to mix thoroughly. Then transform the solution into new 1.5mL tubes.
5. place the tubes in refrigerator at 4 °C and resuspend every 10 minutes lasting about 2~2.5h.
6. centrifuge the solution at 12,000xg for 5 minutes at 4 °C. and inoculate supernatant into new 1.5 mL tubes.
7. place the tubes at 37 °C water bath for 30 minutes. Decant or aspirate supernatant and discard.
8. add 100μL Membrane Protein Solubilizer and leave overnight at 4°C.
●Solarbio® SDS-PAGE Gel preparation kit
●Bio-Rad® Mini-PROTEAN System
●Double distilled water (ddH2O)
●Glycine (produced by Solarbio®)
●Tris Powder (produced by Solarbio®)
●SDS (produced by Solarbio®)
●Coomassie Blue R-250
●Isopropanol
●Glacial acetic acid
●Absolute ethyl alcohol
●Protein samples
●Prestained protein marker (produced by TransGen®)
●6 × Protein loaddng buffer (produced by TransGen®)
●50-mL centrifuge tube
●Electrophoresis power supply
●Microwave oven
1. Wash the Bio-Rad® Mini-PROTEAN System with ddH2O.
2. Prepare samples for electrophoresis.
A.Hand casting Polyacrylamide Gels
1.Wash the glass plate and dry with a mirror paper, assemble the glass cassette sandwich.
2.Prepare the resolving gel solutions without APS and TEMED, mix gently .
| Resolving Gel | Stacking Gel | |
|---|---|---|
| 12% | 5% | |
| Range | 12~60 kDa | — |
| ddH2O | 3.3mL | 3.42mL |
| 30% Acrylamide/Bis | 4 mL | 0.83 mL |
| 1 M Tris-HCl, pH 6.8 | — | 0.625 mL |
| 1.5 M Tris-HCl, pH 8.8 | 2.5 mL | — |
| 10% SDS | 100 μL | 50 μL |
| 10% APS | 100 μL | 75 μL |
| TEMED | 10 μL | 7.5 μL |
| Total Volume | 10 mL | 5 mL |
4.Using a pipet immediately overlay the monomer solution with ddH2O.
5.Allow the gel to polymerize 45-60 min, the gel is polymerized once you see a line from between the deionized water and the resolving gel. Pour off the overlay water.
6.Dry the area above the resolving gel with filter paper before pouring the stacking gel.
7.Prepare the stacking gel solutions without APS and TEMED, mix gently.
8.Add the APS and TEMED to stacking gel solution, pour the solution above the resolving gel using a pipet, pour until the liquid level reaches the upper edge.
9.Place the comb into the stacking gel and allow the gel to polymerize 30-45 min.
10.Remove the comb by pulling it straight up slowly and gently, rinse the wells completely with ddH2O.
* Polymerized gel can be storage at 4 °C no more than 1 week when soaking in ddH2O without removing the comb if the electrophoresis not be applied immediately after preparation of the gel.
B.Performing Electrophoresis 1.Prepare 2000 mL running buffer:
| Total Volume | 1000 mL | 2000 mL |
|---|---|---|
| Glycine | 18.8 g | 37.6 g |
| Tris Powder | 3.0 g | 6.0 g |
| SDS | 1.0 g | 2.0 g |
| ddH2O | 1000 mL | 2000 mL |
a.Remove the comb form the gels and assemble the electrophoresis cell
b.Fill the inner and outer buffer chambers with running buffer. Fill the upper buffer chamber of each core with 200 mL of running buffer; fill the lower buffer chamber to the indicator mark for 2 gels (550 mL) or 4 gels (800 mL) with running buffer
3.Prepare samples
a.Add 6 × Protein loading buffer into 5 times the volume of protein sample, mix gently
b.Heat samples with boiled water bath for 8 minutes
c.Load the appropriate volume of the sample on the gel (~30 μL)
4.Connect the electrophoresis cell to the power supply and perform electrophoresis according to the following conditions:
a.Stage 1: 100V, 80 mA for 30 min
b.Stage 2: 150V, 120 mA for 40 min
5.After electrophoresis is complete, turn the power supply off and disconnect the electrical leads. Pop open the gel cassettes and remove the gel by floating it off the plate into water
6.Stain and image the gel
C.Coomassie Blue Stain
1.Prepare Coomassie Blue dyeing liquid and bleaching liquid:
| Dyeing liquid | Bleaching liquid | |
|---|---|---|
| Coomassie Blue R-250 | 1.0 g | –– |
| Absolute ethyl alcohol | –– | 100 mL |
| Isopropanol | 250 mL | –– |
| Glacial acetic acid | 100 mL | 200 mL |
| ddH2O | 650 mL | 700 mL |
| Glacial acetic acid | 1000 mL | 1000 mL |
a.Wash gels three times in ddH2O
b.Remove all water from staining container and add Coomassie blue dyeing liquid over the gel
c.Heat with microwave oven in medium-high heat in 1 minute
d.Agitate in 40~60 rpm on a shaking table for 8 min
3.Bleaching the staining
a.Remove all water from staining container and add dyeing liquid back into container
b.Wash gels three times in ddH2O
c.Add bleaching liquid over the gel
d.Heat with microwave oven in medium-high heat in 1 minute
e.Agitate in 40~60 rpm on a shaking table for 20 min
f.Pour the bleaching liquid back to container with activated carbon filtration
g.Repeat step 3d-3f for 2 times
4.Wash the gel with ddH2O and image the gel
- Guide to Polyacrylamide Gel Electrophoresis
[1] Bio-Rad. A Guide to Polyacrylamide Gel Electrophoresis and Detection. [M]. Bio-Rad Laboratories, Inc., 2019.
https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf
[2] Green M R, Sambrook J. Molecular cloning. A Laboratory Manual 4th, 2012.
- Usage of 6 × Loading Buffer
[1] Manual of 6 × Protein loading buffer
http://www.transgen.com.cn/data/upload/pdf/DL101_2019-10-07.pdf
[2] Chen X, Lu X, Shu N, et al. Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system. Scientific Reports, 2017, 7:44304.
[3] Yang S, Meng J, Yang Y, et al. A HSP60-targeting peptide for cell apoptosis imaging. Oncogenesis, 2016, 5(2):e201.
[4] Cheng Y, Liu Y, Wang Y, et al. Chicken DNA virus sensor DDX41 activates IFN-β signaling pathway dependent on chSTING. Developmental & Comparative Immunology, 2017, 76:334-342.
Xiamen University, Fujian, China
No.422, Siming South Road, Fujian, P.R.China 361005
