Team:SZPT-CHINA/Project/Protocols


S Z P T - C H I N A

Protocol
PROTOCOL
BASIC OPERATION
formula
LB medium
Formulated per liter of medium should be added in 950 ml of deionized water:
  • Tryptone 10g
  • Yeast extract 5g
  • NaCl 10g
GM17 medium
  • 0.5% glucose
M17 medium(M17 medium is the commonly used growth medium for L. lactis. This medium is commercially available without carbon source)
Performed according to the Solarbio's Plasmid Extraction Mini Kit.
  • Place the spin column CP3 in a clean collection tube and add 500μl of Buffer BL to CP3. Centrifuge at 12,000 rpm(~ 13,400 x g) for 1 minute in a benchtop microcentrifuge. After discarding the waste in the collection tube, turn the rotary column CP3 back into the collection tube. (Use the rotating column on the day).
  • Centrifuge for 1 minute at 12,000 rpm (~ 13,400 x g) in a conventional benchtop microcentrifuge at room temperature (15-25℃), 1-5 ml of bacterial cells were harvested in a microcentrifuge tube, and then all traces The supernatant opens the centrifuge tube by reversing until all the media has drained (for large volumes of bacterial cells, collect a tube through several centrifugation steps.)
  • Use 250μl of Buffer P1 (to ensure that the RNase A has been added) to resuspend the bacteria. The bacteria should be completely resuspended by up or down rotation or blowing until there is no cell mass. Note: After the suspension is suspended again, the cell mass is not visible, otherwise incomplete dissolution will reduce yield and purity.
  • Add 250 μl of buffer solution P2 and mix gently with 6-8 inverting tubes. NOTE: Mix gently by pouring the tube. Do not whirl, as this will lead to cutting genomic DNA. If necessary, continue to turn the tube until the solution becomes viscous and slightly removed. Do not let the cracking reaction be carried out for more than 5 minutes. If the lysate is still unclear, reduce the bacterial precipitation.
  • Add 350μl of buffer P3 and mix gently with the tube upside down 6-8 times immediately. The solution should be cloudy. Centrifuge at 12,000 rpm for 10 minutes (~ 13,400 x g) in a benchtop centrifuge. Note: To avoid localized precipitation, the solution is thoroughly mixed immediately after adding buffer P3. If there is still white precipitate in the supernatant, please centrifuge again.
  • Transfer the supernatant from step 5 to spin column CP3 (place CP3 in the collection tube by decantation or pipetting). Centrifuge at 12,000 rpm (~ 13,400 x g) for 30-60 seconds. Dispose of the circulation and set the rotary column CP3 back to the header.
  • Optional, practically we have almost never used) Rotate column CP3 by washing 500μl of buffer PD and centrifuging at 12,000 rpm (~ 13,400 × g) for 30-60 seconds. Release the circulation and return the rotary column CP3 to the collection tube. It is recommended to use endA + strains such as JM series, HB101 and its derivatives or any wild-type strain with high levels f nuclease activity or high carbohydrate content to remove trace nuclease activity.
  • The rotary column CP3 was rinsed with 600μl of buffer PW (ensuring that ethanol was added (96% -100%) and centrifuged at 12,000 rpm (~ 13,400 × g) for 30-60 seconds. Release the Spin Column CP3 and return it to the collection.
  • Repeat step 8.
  • Centrifuge at 12,000 rpm (~ 13,400 x g) for another 2 minutes to remove residual wash buffer PW. Note that residua ethanol from buffer PW may inhibit subsequent enzymatic reactions. We recommend opening the CP3 lid and keeping it at room temperature for a period of time to remove residual ethanol.
  • Place the rotary column CP3 in a clean 1.5 ml microcentrifuge tube. To elute the DNA, 50-100 μl of buffer EB was added to the center of the rotary column CP3 for 2 minutes and centrifuged at 12,000 rpm (~ 13,400 × g) for 2 minutes. Note: If the volume of the elution buffer is less than 50ul, the recovery efficiency may be affected. The pH of the elution buffer has a certain effect on the eluate; buffer EB or distilled water (pH 7.0-8.5) suggests elution of plasmid DNA. For long-term preservation of DNA, it is recommended to elute in buffer EB and store at -20 ℃ because the DNA stored in water undergo acid hydrolysis. Step 11 is repeated to improve the efficiency of the recovery of the plasmid.
  • Prepare a 1 % agarose-gel: Dissolve the agarose in 1X TAE by boiling and add gelred (5 µl per 50 ml agarose solution). For short DNA fragments, gels with higher agarose percentage should be used.
  • Pour the gel and let it curdle.
  • Add 1ul 6X DNA loading buffer to sample(5ul).
  • Place the gel into the tank and add 1X TAE buffer so that the gel is fully covered.
  • Pipet sample and marker into the pockets.
  • Run the gel at 120 V for 60 min.
  • If necessary, cut out bands.
Performed according to Magen’s Hipure Gel Pure DNA Micro Kit.
  • Set up a PCR system
  • Use microcentrifuge to annihilate bubbles in PCR tube
  • Blend with Vortex
  • Place in PCR machine and set the program as needed
PCR reaction as follow:
F(10uM) F(10uM)
R(10uM) 0.8ul
DNA(1~20ng/20μL) 0.2ul
KOD plus 0.4ul
10xKOD plus buffer 2ul
dNTPs 2ul
MgSO4 0.8ul
DMSO 1.6ul
ddH2O up to 20μl
PCR machine should be set to run the following steps:
step Temperature(℃) Time
Initial denaturation 94 3 minutes
30 cycles

94 (denaturation)

55-60 (annealing)

68 (extension)

30 seconds

30 seconds

1 minute per kb
Performed according to TIANGEN’s quick Midi Purification Kit.
  • set up the system as shown. Carry out the process on ice
  • Blend with vortex then place in a metal bath at 37˚C for 1h
20μl system：
DNA（1000ng）
Enzyme 1 0.6μl
Enzyme 2 0.6μl
10X Buffer 2μl
ddH20 up to 20μl
  • Inoculate 10 mL of LB liquid medium with a single colony of E. coli host strain, grow at 37 °C, 200 r / min ,overnight (16 ~ 18 h).
  • Inoculate 100 mL of LB liquid medium with 1 mL of the bacterial solution, cultured at 37 ° C for 2 to 3 hours. Grow the culture until OD600 is 0.5-0.6, the conical flask is ice bathed for 10 minutes.
  • Transfer the bacteria under sterile conditions to a sterile, ice-cold 50 mL centrifuge tube.
  • Spin down cells for 10 min at 4000 r/min, 4℃.
  • Pour out the culture solution, and place the centrifuge tube on the filter paper for 1 min to allow the last remaining culture solution to be drained.
  • Resuspend the cells with about 20 mL (10 mL per tube) with ice-cold 100 mM CaCl2 solution (light operation when resuspending), and ice bath for 30 min.
  • Spin down cells for 10 min at 4000 r/min, 4℃.
  • Discard the supernatant and pour it on the filter paper for 1 min to allow the last remaining liquid to flow out.
  • Resuspend the cells by gently shaking with 4 mL of ice-cold 100 mM CaCl2 solution.
  • Adding 30% volume of glycerol preservation solution, dispensing the competent cells in an Eppendorf tube, each 100 μL, and storing at -80 ° C for use.
  • Set up a system as required, all operations on ice
  • Mix ligation system with 100µL of competent and ice bath for at least 30min (thorough contact of DNA and cell, enabling DNA to get inside cell walls)
  • 42˚C heat shock for 90s (competent cell swell under high temperature, cell membrane and cell wall fully contact, enabling DNA to flow inside the cell)
  • Ice bath for 5min (Cell shrinks and DNA is dragged through the cell membrane)
  • Pipette 200µL of full nutrient culture (in clean bench), place in a shaker at 37˚C for 1h
  • Spread the entire sample on the plate, observe after 16h~20h
10μl system：
Vector DNA 3-30fmol
Insert DNA 9-90fmol
T4 DNA ligase 0.5μl
5X T4 DNA Ligase Buffer 2μl
ddH20 up to 10μl
Preparation of the cells:
Day 1: Inoculate 5 ml of G/L-SGM17 medium with L. lactis glycerol stock from -80 ℃ and grow at 30 ℃,without aeration, overnight.
Day 2: Inoculate 50 ml of G/L-SGM17 with pre-culture in a dilution of 1:100 and grow at 30 ℃, withoutaeration, overnight.
Day 3:
  • Add 50 ml full-grown culture to 400 ml of G/L-SGM17 medium.
  • Grow the culture until OD600 is 0.2-0.3 (ca. 3 h).
  • Spin down cells for 20 min at 6000 x g, 4 ℃.
  • Wash cells with 400 ml of 0.5 M sucrose, 10% glycerol (4 ℃) and spin down at 6000 x g (centrifugation speed may need to be increased during successive washing steps).
  • Resuspend the cells in 200 ml of 0.5 M sucrose, 10% glycerol, 50 mM EDTA (4 ℃), keep the suspension on ice for 15 min and spin down.
  • Wash cells with 100 ml of 0.5 M sucrose, 10% glycerol (4 ℃) and spin down (6000 x g) - Resuspend the cells in 4 ml of 0.5 M sucrose, 10% glycerol (4 ℃):
    • Use 40 μl per electropopration (keep on ice).
    • Or store the cells in small portions at -80 ℃, let them defreeze on ice before use.
Electroporation:
  • Place 40 μl cells in a pre-chilled electroporation cuvette with 1 μl DNA (100-500 ng vector DNA reconstituted in TE-, Tris-buffer, or distilled water; for transforming cells with ligation product use 500-1000 ng DNA) and keep the cuvette on ice.
  • Use Biorad Genepulser with following adjustments: 2000 V , 25μF , 400Ω
  • Pulse (normal reading is 4.5-5 msec).
  • Add 1 ml of G/L-M17+ 500µl of 2 M MgCl2 +100µl of 1 M CaCl2.
  • Keep the cuvette for 5 min on ice and incubate 2 h at 30 ℃.
  • Plate 10 µl, 100 µl, 900 µl on M17agar with glucose or lactose and antibiotics (depends on plasmid).
  • Incubate 1-2 days at 30 °C.
Induced fermentation and bacterial cell lysis
  • The fermentation broth is induced according to optimized culture conditions and induction conditions. Pre-weigh the centrifuge tube, collect the cells at 4℃ and 6000 rpm for 10 min, try to pour off the liquid, and weigh the calculated wet weight of the cells.
  • The collected wet bacteria are frozen and thawed at -20℃ for three times, and the corresponding amount is added in a ratio of 5 ml of BugBuster Master Mix protein extracting reagent per gram of cell paste, and the cells are resuspended at room temperature. The resuspended cell suspension was incubated at 25℃ for 30 min with shaking, and the cells were fully lysed.
  • centrifugation at 4℃ and 12,000 rpm for 20 min, separating the insoluble cell extract and the supernatant containing a soluble protein mixture.
  • Precipitation was stored at -20℃as a material for purification of inclusion bodies. Transfer the supernatant soluble protein solution to another new centrifuge tube for affinity chromatography on the column.
Preparation of high purity inclusion body samples
  • The insoluble precipitate obtained after cell lysis was resuspended in the inclusion body washing solution, and the washed inclusion body precipitate was collected at 4℃ and 12,000 rpm for 20 minutes. Repeat washing twice, each time the inclusion body washing liquid is 1/10 of the culture volume.
  • Calculate the wet weight of the inclusion body, and use 1 ml of inclusion body lysis buffer per 10-20 mg of protein precipitation, add the corresponding volume of inclusion body lysis buffer to resuspend the protein precipitate, incubate for 15 min at room temperature, and completely dissolve the inclusion body protein precipitate.
  • Centrifuge at 4℃ and 12,000 rpm for 20 min, discard the precipitate, and use the supernatant for affinity chromatography.
    • The inclusion body solution was shaken and mixed with the filler at 4℃for 1 hour. Operate according to the soluble protein purification method. The corresponding components were collected for SDS-PAGE electrophoresis analysis. The protein was purified using binding buffer, wash buffer, and elution buffer to collect a higher purity protein solution.
Use the Amicon"Ultra-2 Centrifugal Filter Devices kit
  • Insert the device into the filtrate collection tube, making sure that the device is fully seated in the tube.
  • Add to 2 mL of sample to the device and cover with concentrate collection tube, Push the tube firmly onto the device.
  • Place filter device into the centrifuge rotor with one membrane panel facing the center of the rotor. Maker sure the device is seated on the bottom of the rotor well and that the rim of the concentrate collection tube is completely inside the well.
  • Spin for approximately 10-60 minutes depending on the NMWL of the device used.
  • Remove the assembled device from the centrifuge and separate the filter device from the filtrate collection tube.
  • To recover the concentrated solute, invert the filter device and concentrate collection tube. Place in centrifuge and counterbalance with a similar device. Spin for 2 minutes at 1,000 g to transfer the concentrated sample from the device to the tube.
30% acrylamide solution:
29 g acrylamide, 1 g methylene acrylamide, dissolved to a volume of 100 mL at 37℃, and stored in a brown bottle at 4℃;
Separation gel buffer (1.5 mol/L Tris-HCl):
18.2 g Tris, concentrated hydrochloric acid to pH 8.8, distilled water to a volume of 100mL;
concentrated gel buffer (0.5 mol / L Tris-HCl):
6.0 g Tris, concentrated hydrochloric acid to pH 6.8., distilled water to a volume of 100mL;
10 × SDS-PAGE running buffer:
30.2g Tris base, 188g Gly, 10g SDS to a volume of 1L;
  • Centrifugal bacterial collection of cells. Centrifuge at 4℃ and 12,000 rpm for 1 minute.
  • Add PBS to resuspend the cells.
  • Take 30 μ l suspension and add 10 μ L 4 X protein sample buffer.
  • Heated at 100 ℃ for 5 minutes.
  • Electrophoresed on SDS-PAGE.80 V for 20 minutes, then 120 V until the band go to the bottom.
  • Add appropriate commassie solution to the SDS-PAGE.
  • Shake at the room temperature for 30 minutes.
  • Take 30 μ l suspension and add 10 μ L 4 X protein sample buffer.
  • Remove the dye, then wash the gel with Bleaching liquid.
Coomassie brilliant blue staining solution
Dissolve 2.5g Coomassie bright blue r-250 in 450ml 50% methanol solution, add 100mL glacial acetic acid, and adjust the volume to 1000mL with distilled water.
Bleaching liquid
Weigh 80mL glacial acetic acid, 250mL 95% ethanol, and adjust the volume to 1000mL with distilled water.
1、SDS-PAGE performed according to protocol talked before
2、Membrane transfer:
  • Prepare sufficient transfer buffer to fill the transfer tank, plus an additional 200 mL to equilibrate the gel and membrane, and wet the filter paper.
  • Remove the gel from its cassette; trim away any stacking gel and wells.
  • Immerse the gel in transfer buffer for 10 to 30 minutes.
  • Soak filter papers in transfer buffer for at least 30 seconds.
  • Prepare the membrane:
    • let the PVDF membrane in methanol for 15 seconds. The membrane should uniformly change from opaque to semi-transparent.
    • Carefully place the membrane in Milli-Q® water and soak for 2 minutes.
    • Carefully place the membrane in transfer buffer and let equilibrate for at least 5 minutes.
3、Transfer Stack Assembly:
  • Open the cassette holder.
  • Place a foam (fiber) pad on one side of the cassette.
  • Place one sheet of filter paper on top of the pad.
  • Place the gel on top of the filter paper.
  • Place the membrane on top of the gel.
  • Place a second sheet of filter paper on top of the stack.
  • Place second foam pad on top of the filter paper.
  • Close the cassette holder.
4、 Method for Protein Transfer
  • Place the cassette holder in the transfer tank so that the gel side of the cassette holder is facing the cathode (–) and the membrane side is facing the anode (+).
  • Add adequate buffer into the tank to cover the cassette holder.
  • Insert the black cathode lead (–) into the cathode jack and the red anode lead (+) into the anode jack on the transfer unit.
  • Connect the anode lead and cathode lead to their corresponding power outputs.
  • Turn on the system for 1 to 2 hours at 6 to 8 V/cm inter-electrode distance. Follow the tank manufacturer’s guidelines, for optimization details.
  • After the transfer is complete, remove the cassette holder from the tank.
  • Using forceps, carefully disassemble the transfer stack.
5、Wash the PVDF membrane by TBST for 5 minutes
6、Blocking：put the membrane in the blocking liquid, shake at room temperature for 1h.
7、Primary antibody hybridization: prepare a primary antibody dilution solution in a suitable ratio with blocking solution; incubate the membrane with the antibody dilution at room temperature for 1 h, or incubate at 4 ° C overnight;
8、Wash PVDF membrane for 5 minutes
9、Repeat step 7 twice
10、Secondary antibody hybridization: prepare a secondary antibody dilution solution in a suitable ratio with blocking solution; and incubate the membrane and antibody dilution solution at room temperature for 1 hour;
11、Wash PVDF membrane for 8 minutes
12、Repeat step 10 twice
13、Development of PVDF membrane
IN VITRO ACTIVITY DETECTION OF ANTIHYPERTENSIVE PEPTIDE(HPLC)
Principle
ACE can catalyze the decomposition of HHL to produce hippuric acid, while the blood pressure lowering peptide reduces the production of hippuric acid by inhibiting ACE activity, and the absorption of hippuric acid at 228 nm is determined by reverse phase HPLC to calculate the inhibition rate of the polypeptide of interest.
Method
Add 1.667×10-6moL/(s ∙ kg) ACE 25μL to the sample tube, normal tube and blank tube, respectively, 0.1 mol/L boric acid-borax buffer solution with a pH value of 8.3, 160, 170, 170 μL
Add 10uL of blood pressure lowering peptide sample to the sample tube, and the other two tubes are not added and mixed.
The blank tube was sterilized for 5 min at 100 ° C, and the other two tubes were reacted at 37 ° C for 5 min.
Add 5 μL of HHL substrate to each tube, the total volume is 200 μL, and react at 37 ° C for 30 min.
100 ° C inactivated enzyme.
Mobile phase composition:
  • Phase A is 100% ACN plus 0.1% TFA
  • Phase B is 0.1% TFA with 5% ACN.
Chromatographic conditions: flow rate 0.8ml / min, column temperature 30 ° C, elution gradient 30% ~ 60% (A phase) 30min, hip uric acid retention time is 4.1min
VERIFICATION OF H+ REGULATION MECHANISM
Content
Inoculate the recombinant bacteria to LB medium with pH values of 3,4,5,6 in a dilution of 1:50 and grow at 30 ℃ for 30h.
Identified by SDS-PAGE electrophoresis.
SECURITY MECHANISM VERIFICATION
Content
Inoculate 50 ml of M17 medium with the recombinant bacteria and the empty bacteria respectively stock from -80 ℃ and grow at 30 ℃, overnight.
Centrifuge at 5000 r / min for 10 min to collect the cells, wash three times with 0.9% NaCl and adjust the OD600 to the same level.
Inoculate to the prepared M17 medium with glucose concentration of 0, 0.05, 0.1, 0.2% in a dilution of 1:50 and grow at 30 ℃ for 24h.
Centrifuge at 12000 r / min for 3 min to collect the cells, remove the supernatant, and wash the cells with 0.9% NaCl.
Dry at 105 ° C, weigh and calculate the dry weight of the bacteria.
We compared the dry weight of the empty bacteria with the recombinant bacteria and found the lowest concentration that MG1363 can grow healthily.