Medium
LB medium was used for E.coil culture, Blood plate was used for Porphyromonas gingivalis culture, BHI medium was used for Streptococcus mutans and Streptococcus salivarius K12 culture, and GM17 medium was used for lactobacillus MG1363 culture.
LB medium
Per liter of medium contains the following components.
Tryptone 10g
Yeast extract 5g
NaCl 10g
pH 7.2
GM17 medium
Per liter of medium contains the following components.
glucose 5g
M17 medium 42.25g
Note: M17 medium is the commonly used growth medium for L. lactis. This medium is commercially available without
carbon source
pH 7.2
BHI medium
Peptone 10g
Calf brain powder 5g
Mrinated beef heart powder 5g
D-glucose 2g
NaCl 5g
Disodium hydrogen phosphate 2.5g
Agar (solid medium) 15g
pH 7.4
Blood plate
Beef extract 1000ml
Peptone 10g
NaCl 5g
Agar (solid medium) 15g
Degerminated sheep blood 60ml
Preparation method: The first four ingredients were mixed, heated to melt, adjusted pH to 7.4. After sterilization (121 ℃ for 15min), cool to 50 ℃, and sterile degerminated sheep blood was added. The blood plate was made after the medium was fully shaken.
Molecular cloning experiment
Plasmid Extraction
Performed according to the Solarbio's Plasmid Extraction Mini Kit.
1. 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).
2. 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.)
3. 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.
4. 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.
5. 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.
6. 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.
7. 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 of nuclease activity or high carbohydrate content to remove trace nuclease activity.
8. 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.
9. Repeat step 8.
10. 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.
11. 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.
Agarose-gel electrophoresis
1.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.
2.Pour the gel and let it curdle.
3.Add 1ul 6X DNA loading buffer to sample(5ul).
4.Place the gel into the tank and add 1X TAE buffer so that the gel is fully covered.
5.Pipet sample and marker into the pockets.
6.Run the gel at 120 V for 60 min.
7.If necessary, cut out bands .
(4)Gel Extraction
Performed according to Magen’s Hipure Gel Pure DNA Micro Kit.
PCR
PCR reaction as follow:
| Component | 50μL Reaction |
|---|---|
| Q5 High-Fidelity 2X Master Mix | 25μL |
| Forward Primer | 2μL |
| Reverse Primer | 2μL |
| Template DNA | 1μL |
| Nuclease-Free Water | 20μL |
The PCR machine should be set to run the following steps:
| STEP | TEMP | TIME |
|---|---|---|
| Initial Denaturation | 98°C | 30 seconds |
| 25–35 Cycles |
98°C *50–72°C 72°C |
5 seconds 10 seconds 15 seconds/kb |
| Final Extension | 72°C | 2 minutes |
| Hold | 4–10°C |
PCR product purification
Performed according to TIANGEN’s quick Midi Purification Kit
Enzyme digestion
1.Set up the system as shown. Carry out the process on ice
2.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 |
Preparation of E.coil competent cells
1. Inoculate 10 mL of LB liquid medium with a single colony of E.coil host strain, grow at 37 °C, 200 r / min ,overnight (16 ~ 18 h).
2. 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.
3. Transfer the bacteria under sterile conditions to a sterile, ice-cold 50 mL centrifuge tube.
4. Spin down cells for 10 min at 4000 r/min, 4℃.
5. 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.
6. 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.
7. Spin down cells for 10 min at 4000 r/min, 4℃.
8. Discard the supernatant and pour it on the filter paper for 1 min to allow the last remaining liquid to flow out.
9. Resuspend the cells by gently shaking with 4 mL of ice-cold 100 mM CaCl 2 solution.
10. 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.
Ligation
1.Set up a system as required, all operations on ice
2. 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)
4.42˚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)
5. Ice bath for 5min (Cell shrinks and DNA is dragged through the cell membrane)
6. Pipette 200µL of full nutrient culture (in clean bench), place in a shaker at 37˚C for 1h
7. Spread the entire sample on the plate, observe after 16h~20h
| 10μl system： |
|---|
|
Vector DNA 3-30 fmol Insert DNA 9-90 fmol T4 DNA ligase 0.5 μl 5X T4 DNA Ligase Buffer 2 μl ddH20 up to 10 μl |
Electrotransformation
1. 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.
2. 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,25 μF,400 Ω
- 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.
Preparation of high purity inclusion body samples
1. 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.
2. 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.
3. 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.
Minimum inhibitory concentration (MIC) of antimicrobial peptides
The minimum inhibitory concentration (MIC) test determines antimicrobial activity of antimicrobial agents against a specific bacteria. Broth microdilution method is one of MIC determination methods, and the MIC of antimicrobial peptides were performed using this method.
1. Strains were inoculated into corresponding broth (BHI broth was used for Streptococcus mutans and Streptococcus salivarius K12 culture, and GM17 broth was used for lactobacillus MG1363 culture), followed by incubation with shaking at 37°C 180 rpm for 4-5 h until OD600 is about 0.6.
2. Have antimicrobial peptides and plates ready before the cultures reach the desired growth phase.
3. The bacterial concentration was adjusted with a final concentration of approximately 5.0 ×105 CFU/ml.
4. MIC plates were prepared with 96-well microdilution plates. Antimicrobial peptides were dissolved, two fold dilutions of the antimicrobial peptides in broth were made, and each well contains 100 μL antimicrobial peptides. The concentration gradient of antimicrobial peptide was 0.5 to 256 μg/ml.
5. 100 µl of diluted bacterial was dispensed into the wells, which contained 100 µl of antimicrobial peptides solution.
6. The inoculated broth microdilution plates were incubated for 24 h at 37 °C in an incubator.
7. The plate was scanned with spectra Max M2 multi-functional plate reader at 490 nm. The MIC value of antimicrobial peptide was calculated according to the minimum mass concentration less than 50% of the control (Compared with the positive control, the concentration of antimicrobial peptide in the tube which inhibited 80% bacterial growth was the MIC).
NOTE: Put different antimicrobial peptides on different rows of the same plate, but try to avoid putting bacteria together, to prevent cross-contamination. Antimicrobial peptides were tested included Apep10, [D4K]B2RP, TP-I, Polyphenusin I, JCpep7, LL37, Oncocin, pv-26, Histatin-p113 .
Reference:
[1] CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. 11th ed. CLSI standard M07. Wayne, PA: Clinical and Laboratory Standards Institute; 2018.
[2] CLSI. Performance standards for antimicrobial susceptibility testing. 27th ed. CLSI supplement 100. Wayne, PA: Clinical and Laboratory Standards Institute; 2017.
Fluorescence detection
The fluorescence of E.coil B21 induced by CSP was detected using a method designed by our team.
1. Engineering strain E.coil BL21 were inoculated into LB broth, followed by incubation with shaking at 37°C 180 rpm for 12 h until OD600 is about 0.8.
2. The CSP solution was diluted from 50 mg / ml to 4 mg/ml.
3. Pipette 50 µl of LB broth into the wells of a sterile 96-well microtitre plates.
4. 100 μl of cultured E.coil BL21 and 50 μl of diluted CSP were successively added into the well containing LB broth. Water instead of CSP was used as negative control. The well with only LB broth were used as blank.
5. 96 well plate was placed in the spectra Max M2 multi-functional plate reader and cultured at 37 ℃. The fluorescence value was determined every 15 min (excitation wavelength: 515nm, emission wavelength: 570nm).
Note: In our improved scheme, galactosidase gene was used instead of fluorescent gene GFP. The detection method is basically the same as that description above, but the visible light wavelength scanning can be used to determine the result. What's more convenient is that the scheme can also be used to observe the results with naked eyes.
Protein purification
The fusion proteins GST-TP1M and GST-[D4K]B2RP were purified according to GST Fusion Protein Purification Kit, and minor modifications have been made.
Preparation of solution
Dithiothreitol DTT (1 mol/L)
Glutathione elution buffer
10 mmol/L Reduced glutathione
50 mmol/L Tris-Cl (pH 8.0)
Sodium phosphate buffer solution (PBS)
Triton X-100(0.2 V/V)
DNase (5 mg/ml)
Lysozyme
RNase (5 mg/ml)
Preparation of Cell Extract
1. Harvest cell by centrifugation at 3,000 g at 4°C for 10 min, remove and discard the supernatant.
2. Resuspend the cell pellet in 3 ml ice-cold 1×PBS buffer per 50 ml culture and centrifuge at 3,000 g at 4°C for 10 min. Remove and discard the supernatant.
3. Freeze the cell pellet at -80°C for 1 hour (This is also a convenient point to stop and one can continue the procedure later).
4. Thaw cell pellet on ice and resuspend cells in 3 ml of ice-cold 1×PBS buffer per 50 ml culture. If desired, add appropriate additives, such as non-ionic detergents (NP-40) or protease inhibitors (PMSF).
5. Disrupt cells by brief pulses of sonication on ice until the sample is no longer viscous.
6. Centrifuge at 12,000 g at 4°C for 10 min and carefully transfer the supernatant (soluble fraction) to a clean and pre-chilled tube and resuspend pellet (insoluble fraction) with 3 ml of ice-cold 1×PBS buffer per 50 ml culture.
7. Aliquot 10 µl samples from both soluble and insoluble fractions for SDS-PAGE analysis [ by adding equal volume of 2X SDS Sample Buffer (125 mM Tris-HCl, pH 6.8, 4% w/v SDS, 20% glycerol, 100 mM DTT,0.02% w/v bromophenol blue), boiling for 5 min and running SDS-PAGE to determine the amount and solubility of the GST-fusion protein].
Purification of Recombinant GST-Fusion Protein
1. Completely resuspend the Glutathione Resin by gently shaking the vial.
2. Transfer an appropriate amount of slurry to a disposable column (included in Kit L00207 and L00208). Usually 1 ml settled resin (2 ml 50% slurry) can bind 35-45mg GST-fusion protein (26 kDa).
3. Wash the Glutathione Resin with 10×bed volumes of cold (4°C) 1×PBS.
4. Apply clear solution (sonicate, etc) containing GST-fusion protein in cold 1×PBS to the equilibrated column with the flow rate at 10-15 cm/h.
5. Add 1×PBS to wash the column just after all the protein solution get into the column, use 20×bed volumes of PBS for wash. Protease inhibitors such as PMSF are better added to wash solution to inhibit protease activity.
6. Elute the fusion protein with 10-15×bed volumes of freshly made 10 mM glutathione elution buffer (0.154 g of reduced glutathione dissolved in 50 ml of 50 mM Tris-HCl, pH 8.0.).
7. Monitor elution of the fusion protein using absorbance readings at 280 nm.
8. Aliquot 10-20 µl supernatant containing GST-fusion protein, flow-through, wash and the eluted protein, respectively, and analyze all the samples by running SDS-PAGE to confirm the presence of the target protein.
9. Pool eluted fractions containing target protein. Remove free glutathione by dialysis at 4°C against a buffer of choice or by using a G15 Sephadex desalt column.
SDS-PAGE
Reagents
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;
Gel preparation
1. Clean and completely dry the plates, combs, and any other experiment materials. Assemble the system according to manufacturer’s instruction.
2. Prepare the separating gel solution according to the following formula. Ammonium persulfate and TEMED are added when the gel is ready to be polymerized, stir gently, then immediately apply to the sandwich until the gel height is lower 1.5 cm than the short plate. Allowing the gel to polymerize 30~60min by covering gently with a layer of water (1cm).
3. Until a sharp optical discontinuity at the watet/gel interface is visible, pour off the water.
4. Prepare the stacking gel solution in a similar way to the separating gel . Insert the comb and allow the gel to polymerize completely.
| Reagents | 12% Seperation Gel (ml) | 5% Stacking Gel (ml) |
|---|---|---|
| Distilled water | 4.8 | 2.72 |
| 1.5 mol/L Tris-Cl buffer, pH 8.8 | 3.8 | |
| 0.5 mol/L Tris-Cl buffer, pH 6.8 | 1.25 | |
| 30% Acrylamide stock solution | 6.0 | 0.83 |
| 10% (W/V) SDS | 0.15 | 0.05 |
| 10% (W/V) APS | 0.15 | 0.05 |
| 10% (W/V) TEMED | 0.1 | 0.1 |
| Total | 15 | 5 |
Preparation of the sample and Electrophoresis
1.Centrifugal bacterial collection of cells. Centrifuge at 4℃ and 12,000 rpm for 1 minute
2.Add PBS to resuspend the cells
3.Take 30 μ l suspension and add 10 μ L 4 X protein sample buffer
4.Heated at 100 ℃ for 5 minutes
5.Electrophoresed on SDS-PAGE.80 V for 20 minutes, then 120 V until the band go to the bottom
Coomassie blue staining
1. Add appropriate commassie solution to the SDS-PAGE
2. Shake at the room temperature for 30 minutes
3. 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
Western Blot
1.SDS-PAGE performed according to protocol talked before
2.Membrane transfer:
(1)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.
(2)Remove the gel from its cassette; trim away any stacking gel and wells.
(3)Immerse the gel in transfer buffer for 10 to 30 minutes.
(4)Soak filter papers in transfer buffer for at least 30 seconds.
(5)Prepare the membrane:
a. let the PVDF membrane in methanol for 15 seconds. The membrane should uniformly change from opaque to semi-transparent.
b. Carefully place the membrane in Milli-Q® water and soak for 2 minutes.
c. Carefully place the membrane in transfer buffer and let equilibrate for at least 5 minutes.
3. Transfer Stack Assembly:
(1) Open the cassette holder.
(2) Place a foam (fiber) pad on one side of the cassette.
(3)Place one sheet of filter paper on top of the pad.
(4)Place the gel on top of the filter paper.
(5)Place the membrane on top of the gel.
(6)Place a second sheet of filter paper on top of the stack.
(7)Place second foam pad on top of the filter paper.
(8)Close the cassette holder.
4. Method for Protein Transfer
(1) 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 (+).
(2) Add adequate buffer into the tank to cover the cassette holder.
(3) Insert the black cathode lead (–) into the cathode jack and the red anode lead (+) into the anode jack on the transfer unit.
(4) Connect the anode lead and cathode lead to their corresponding power outputs.
(5) 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.
(6) After the transfer is complete, remove the cassette holder from the tank.
(7) 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
Antimicrobial activity test of antimicrobial peptides
Antimicrobial activity of expressed antimicrobial TP-I and [D4K]B2RP were assayed by the Oxford cup method.
1. Bacterial were inoculated into corresponding broth (BHI broth was used for Streptococcus mutans and Streptococcus salivarius K12 culture, and GM17 broth was used for lactobacillus MG1363 culture), followed by incubation with shaking at 37°C 180 rpm for 12 h.
2. BHI plate and GM17 plate were made, 0.1 ml bacterial solution was evenly coated on the plate.
3. Four oxford cups were put vertically on every plate. Every cup was poured into 100 μL antimicrobial peptides.
4. Bacterial was cultured in an incubator under 37 °C. The results were checked after 24 hours, measuring the diameter (DM) of pathogens and calculating antimicrobial rate.