•Bacterial Polysaccharide Extraction Kit (Bolson Bioscience)
• Anhydrous Ethanol
• 80% Ethanol
• Polysaccharide Assay Kit (Grace Biosciences)
• Concentrated Sulfuric Acid
1. Cultivate the engineered bacteria in LB liquid medium to an OD600 of 0.6, then add 0.5mM IPTG to induce gene overexpression for 3 hours, and finally measure and record the OD600.
2. Dilute the aforementioned treated bacterial cultures to an OD600 of 0.6 with LB medium.
3. Take 2ml of bacterial culture in a microcentrifuge tube and centrifuge at 8000×g for 3 minutes, discard the supernatant, and collect bacterial cells.
4. Add 500μl of Reagent A to resuspend the cells, centrifuge at 8000×g for 3 minutes, and discard the supernatant.
5. Collect the pellet and repeat step 4.
6. Collect the pellet, add 500μl of Reagent B to resuspend the cell pellet, and incubate in a water bath at 80°C for 6 hours.
7. Centrifuge at 10000×g for 10 minutes.
8. Transfer the supernatant to another clean centrifuge tube to obtain the extracellular polysaccharide sample for testing.
1. Take 0.3mL of extracellular polysaccharide sample into a new EP tube, add 1.2mL of ethanol, mix well, and place at 4°C for 1 hour. After centrifugation at 8000rpm for 5 minutes, discard the supernatant and retain the pellet.
2. Add 1mL of 80% ethanol to the pellet from the previous step, mix briefly, centrifuge at 8000rpm for 5 minutes, discard the supernatant, and retain the pellet.
3. Add 2mL of distilled water to the pellet from the previous step and heat in a boiling water bath (95-100°C) until the pellet is completely dissolved (approximately 5 minutes) to obtain the bacterial polysaccharide sample for testing.
4. For each sample, follow the instructions in the table:
| Reagents (μL) | Test Tube |
|---|---|
| Sample for Testing | 100 |
| Reagent One | 50 |
| Concentrated Sulfuric Acid | 250 |
5. Mix the solution well, seal tightly with a membrane, place in a 95°C water bath for 20 minutes, cool to room temperature, transfer 200μL to a 96-well plate, and read the absorbance at 488nm.
1. Add 2ml of liquid culture medium to each well of a 6-well polystyrene microplate, inoculate the bacterial culture diluted to an OD of 0.05 in the total system, add 0.5mM IPTG, and incubate statically at 37°C for 6 hours.
2. Aspirate the culture medium, add 2ml of sterile PBS buffer to each well to wash the plate wells once (washing must be gentle, as the biofilm is at the bottom of the wells).
3. Add 2ml of 10% methanol to each well to fix for 15 minutes, then aspirate the methanol and air dry.
4. Add 2ml of 0.1% crystal violet solution to each well and stain at room temperature for 5 minutes (the concentration of crystal violet solution can vary depending on the thickness of the biofilm, generally controlled within an OD of 0.2-1 for accuracy).
5. Aspirate the crystal violet staining solution from the culture wells and rinse off excess dye with water.
6. Invert the culture plate on filter paper to remove residual water and dry in a 37°C oven or at room temperature.
7. After complete drying, add 2ml of 95% ethanol solution to each well and let it act for 30 minutes at 37°C to dissolve the crystal violet.
8. Measure the OD of the solution in the culture wells under 590nm (570nm/595nm) conditions using a microplate reader.
9. Use uninoculated culture medium as a negative control, with all other operations consistent with the experimental group.
10. Perform the experiment with three replicates for each bacterial strain in each trial, and take the average of three trials for the experimental values.
1. First, spray the laminar flow hood with alcohol, and then spray the relevant experimental equipment with alcohol before placing them into the laminar flow hood. Sterilize with UV light for 20 minutes.
2. Add bacterial culture with an OD600 of 0.6-0.8 to 0.5mM IPTG induction medium and cultivate for 3 hours.
3. Control group: Inoculate 10% of the culture into 5ml of LB liquid medium, mix well; Experimental group: Inoculate 10% of the culture into 5ml of LB liquid medium containing 0.5mM IPTG, mix well.
4. At regular intervals (every 2 hours), take a small amount of bacterial culture, mix it evenly with a vortex mixer, and measure the OD600 value of the culture (take the average of three parallel experimental data) to plot the growth curve graph.
• Sodium Heparin
• 0.45μm Sterile Microporous Membrane
• Sterile Syringe
• Anhydrous Ethanol
• 80% Ethanol
• Glycoprotein Assay Kit (Grace Biosciences)
• Concentrated Sulfuric Acid
1. Cultivate the modified strain, the wild-type strain, and the empty vector to an OD600 of 0.6. Add 20mg/ml sodium heparin solution (filtered and sterilized) to each bacterial culture, and add 0.5mM IPTG inducer to the modified strain, continue to culture for 3 hours, and finally measure and record the OD600.
2. Take 3ml of each bacterial culture and filter through a microporous membrane to remove bacteria.
3. Transfer 0.3mL of the filtrate into a new EP tube, use LB liquid medium as the blank group, add 1.2mL of ethanol to each, mix well, and place at 4°C for 1 hour. After centrifugation at 8000rpm for 5 minutes, discard the supernatant and retain the pellet.
4. Add 1mL of 80% ethanol to the pellet from the previous step, mix briefly, centrifuge at 8000rpm for 5 minutes, discard the supernatant, and retain the pellet.
5. Add 2mL of distilled water to the pellet from the previous step, heat in a boiling water bath (95-100°C) until the pellet is completely dissolved to obtain the supernatant sodium heparin solution for testing.
6. Perform three parallel measurements, and follow the instructions of the microplate method glycoprotein assay kit (phenol-sulfuric acid method):
| Reagents (μL) | Test Tube |
|---|---|
| Sample for Testing | 100 |
| Reagent One | 50 |
| Concentrated Sulfuric Acid | 250 |
7.Mix the solution well, seal tightly with a membrane, place in a 95°C water bath for 20 minutes, cool to room
temperature, transfer 200μL to a 96-well plate, and read the absorbance at 488nm.
Heparin to Add Design a gradient of sodium heparin addition at concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6mg/ml. After cultivation, take 3ml of bacterial culture, filter through a microporous membrane to remove bacteria, and perform heparin content determination; adjust the gradient addition to 0, 0.4, 0.5, 0.6, 0.7, and 0.8mg/ml, with other operations remaining the same.
1. Glucose Standard Solution: Weigh 2mg of glucose standard into a new EP tube, add 2mL of distilled water to dissolve and mix to make a 1mg/mL glucose solution. Dilute the mother liquor with distilled water to make six concentration gradients of standards: 0, 0.02, 0.04, 0.06, 0.08, and 0.1mg/mL, and create a standard curve.
2. Glycoprotein Content Calculation Formula:
(mg/mL liquid) = [(ΔA-0.0065) ÷ 86.454] ÷ (V2×V1÷V) × D = 0.2313 × (ΔA-0.0065) ÷ V2 × D
V---Total volume of sample extraction liquid, 2mL;
V1---Volume of sample solution for testing, 0.1mL;
V2---Volume of liquid sampling, 0.3mL.
1. Prepare semi-solid agar medium (0.5% agar), sterilize, and pour into plates in a laminar flow hood. Use a pipette tip to pick a single colony of the target bacterial strain from the plate, and inoculate it into the semi-solid medium by stabbing. Incubate at 37°C.
2. Observe under transmitted light; if the growth is confined to the stab line with a clear edge, it indicates that the bacteria are non-motile. If the growth spreads from the stab line in all directions with a cloudy edge, it indicates that the bacteria are motile. The extent of the spread from the stab line can be observed to assess bacterial motility, indirectly indicating changes in bacterial flagella.
3. For fast-growing bacteria, observe after 1 day of incubation, and if necessary, extend the observation to 2-3 days or 5-6 days.
1. Inoculate the predator and prey bacterial cultures into LB liquid medium at a defined volume and cultivate at a constant temperature until the OD600 reaches 0.6 (the concentrations of both cultures should be similar). Add 0.5mM IPTG and induce at 37°C with 220rpm for 3 hours.
2. Preliminary experiment: Mix the predator and prey cells at certain ratios (1:1, 1:100, 1:10000, 10000:1, 100:1) and cultivate for 3 hours. Perform serial dilutions (dilution factors of 1, 100, 10^4, and 20^4), then spread 20μl of the mixed culture on plates with varying antibiotic resistances, and include a blank control.
3. Based on the preliminary experiment, the best results were obtained when the predator and prey cells were mixed at a ratio of 1:100 or 100:1 and cultivated for 3 hours, then diluted to 20,000-fold.
| Media and Bacteria Volume Calculator: | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Strain | Final OD600 | Final Volume(ml) | Measured OD600 | Volume to Add(ml) | |||||
| Prey | 0.1 | 5 | Prey OD600 | 0.1*5/Prey OD600 | |||||
| Predator | 50 | 0.1 | Predator OD600 | 50*0.1/Predator OD600 | |||||
4. Then pour it onto the regular LB agar plates and allow it to cool and solidify.
5. Transfer the calculated volume of the predator strain to a centrifuge tube, centrifuge at 13000 RPM for 1 minute, discard the supernatant, and resuspend in 100μl of LB liquid medium.
6. Make holes in the solidified mixed-strain agar plate, add 100μl of the centrifuged and resuspended predator strain to one hole, and add 100μl of LB liquid medium to another hole as a blank sample.
7.Incubate at the appropriate temperature for 12-24 hours and record the size of the inhibitory zone around the predator strain.
1. Inoculate the predator and prey bacterial cultures into LB liquid medium at a defined volume and cultivate at a constant temperature until the OD600 reaches 0.6 (the concentrations of both cultures should be similar). Add 0.5mM IPTG and induce at 37°C with 220rpm for 3 hours.
2. Preliminary experiment: Mix the predator and prey cells at certain ratios (1:1, 1:100, 1:10000, 10000:1, 100:1) and cultivate for 3 hours. Perform serial dilutions (dilution factors of 1, 100, 10^4, and 20^4), then spread 20μl of the mixed culture on plates with varying antibiotic resistances, and include a blank control.
3. Based on the preliminary experiment, the best results were obtained when the predator and prey cells were mixed at a ratio of 1:100 or 100:1 and cultivated for 3 hours, then diluted to 20,000-fold.
4. Formal experiment: ① Inoculate the predator and prey bacterial cultures into LB liquid medium at a defined volume and cultivate at a constant temperature until the OD600 reaches 0.6 (the concentrations of both cultures should be similar). Add 0.5mM IPTG and induce at 37°C with 220rpm for 3 hours. ② Mix the predator and prey cells at a certain ratio (1:100 or 100:1) and cultivate for 3 hours, then dilute the culture to 20,000-fold. ③ Spread 20μl of the mixed culture on plates with varying antibiotic resistances, and include a blank control.
5. After overnight incubation in an incubator, calculate the CFU values for the predator and prey cell plates. [CFU = (number of colonies counted) / (volume of sample applied × dilution factor)]
1. Take 100μL of the killing bacteria into 5mL of SOC medium and incubate in a 37°C constant temperature shaker until the OD reaches 0.6.
2. After cultivation, add 5μL of IPTG inducer to the bacterial culture, then incubate in a 37°C constant temperature shaker for 3 hours.
3. Record the OD value after the 3-hour incubation, then continue to record the OD value every 2 hours for a period of 12 hours.
