Experiments
Screening of Chitosanase
— AKTA pure™ chromatography system
— LB liquid medium
— Kanamycin (50 mg/mL)
— IPTG (Isopropyl β-D-1-thiogalactopyranoside) (1 mol/L)
— Ultrasonic Cell Crusher
— 0.22-µm Micron Pore Filter Membrane
— Tecan Infinite M200 microplate reader
— Elution Solution A (20 mM Tris-HCl, 0.5 M NaCl, pH 8)
— Elution Solution B (20 mM Tris-HCl, 0.5 M NaCl, 0.08 M imidazole, pH 8)
— HisTrap HP His tag protein purification columns
— Centrifuge
1.Bacteria culture is inoculated into LB liquid mediums at 2% (v/v)
and kept at 37°C, 200 rpm and last 12-16 h.
2.Bacteria culture from step 1 is inoculated into LB liquid mediums
at 2% (v/v) and kept at 37°C, 200 rpm until the OD600 of bacteria
culture reaches the OD600 specified in the table below.
Table 1 OD600 for Inducing Chitosanase Expression
3.Corresponding inducer is added into the bacteria culture from step 2, which is then incubated under appropriate conditions for the specified duration.
Table 2 Conditions for Inducing Chitosanase Expression
4.Bacteria culture from step 3 is centrifuged at 6500 rpm at
4°C for 10 min to collect the cell and resuspended with 25-30 mL Elution Solution A.
5.Repeat step 4 once.
6.The liquid from step 5 is treated with ultrasonication to lyse the cell.
7.The lysis is centrifuged at 9000 rpm at 4°C for 10 min to remove
the cell debris and then the supernatant is filtered through a 0.22-µm Micron Pore Filter Membrane.
8.The target protein is purified from the supernatant using a
HisTrap column with AKTA pure™ chromatography system.
9.The eluate containing the target proteins is dialyzed
with Elution Solution A overnight to remove imidazole for subsequent uses.
— Chitosanase Solution
— 0.22-µm Micron Pore Filter Membrane
— LB liquid medium
— Tecan Infinite M200 microplate reader
— UV-Sterilized Chitosan powder
1. Add 2 mL chitosanase solution to a centrifuge tube pre-loaded with 0.01 g of chitosan powder, and keep it at 37°C, 200 rpm and last 18 h.
2. Hydrolysate from step 1 is centrifuged at 12000 rpm for 3 min to collect the supernatant and performs membrane filtration. Seal with parafilm and store at -20°C.
3. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm and last 12-16 h.
4. Bacteria culture from step 3 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm until the OD600 of bacteria culture reached 0.8.
5. Bacterial culture from step 4 is mixed with the supernatant from step 2 at a 1:1 (v/v) ratio and incubated at 37°C, 200 rpm. Reaction solution is collected at 1.5 h and 8 h.
6. The OD600 of the reaction solution is measured immediately by Tecan Infinite M200 microplate reader.
Biochemical Characterization of Chitosanase
— Chitosan powder
— 30% hydrogen peroxide(H2O2)
— 2.0% (w/w) acetic acid (HAc)
— Sterile water
— Catalase (CAT)
— Water bath
— Centrifuge
1. Mix the reagents according to the table 3 to prepare a 2% chitosan solution.
Table 3 Composition of Chitosan Solution
2. Incubate the mixed solution from step 1 at 65°C for 15 min.
3. Add catalase in small aliquots multiple times, and incubate at 40°C for 30 min until no further bubble formation is observed.
Note: Swirl the flask while adding catalase.
— DNS chromogen reagent (MY73006)
— Tecan Infinite M200 microplate reader
— Glucose
— Test sample solution
— Sterile water
— Water bath
— Centrifuge
1. Glucose solutions are prepared at concentrations of 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL, 1 mg/mL in a 15 mL centrifuge tube.
2. Separately take equal volumes of the glucose solution from step 1, sterile water (blank control), and the test sample solution, and mix each with an equal volume of DNS chromogen reagent.
3. Incubate the mixed solution from step 2 at 100°C for 5 min, then immediately transfer it to an ice-water bath for rapid cooling.
4. Dilute the reaction solution from step 3 with sterile water at a 1:2 (v/v) ratio and then measure OD540 of the diluted solution by Tecan Infinite M200 microplate reader.
5. Plot a standard curve with glucose standard concentration as the x-axis and the corresponding OD540 values as the y-axis, and calculate the reducing sugar content in the test sample solution based on this curve.
— 2% Chitosan solution
— Chitosanase solution
— 2.0% (w/w) acetic acid (HAc)
— 11% Sodium hydroxide solution
— DNS chromogen reagent (MY73006)
— Tecan Infinite M200 microplate reader
— Water bath
— Centrifuge
1. Mix the 2% chitosan solution with the chitosanase solution at a 1:1 (v/v) ratio, and add amounts of acetic acid or sodium hydroxide solution to adjust the pH to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0.
2. Incubate the mixed solution from step 1 at 45°C for 30 min.
3. Hydrolysate from step 2 is subjected to reducing sugar content determination.
4. The enzyme activity is calculated according to the following formula, and a curve of enzyme activity versus pH is plotted to determine the optimal pH for the enzyme reaction.
with C the reducing sugar concentration calculated from the standard curve (mg/mL), Vt the total volume of the enzymatic reaction system (mL),
n the dilution factor of the reaction solution after adding DNS reagent (if undiluted, n = 1), Ce the concentration (mg/mL) of enzyme solution, M the molecular weight of glucose (180.16 g/mol), t the enzymatic reaction time (min), and Ve the volume (mL) of enzyme solution added.
— 2% Chitosan solution
— Chitosanase solution
— 2.0% (w/w) acetic acid (HAc)
— 1% Ammonia water
— 0.05 M acetate buffer (pH 3.0-6.0)
— 0.05 M phosphate buffer (pH 6.0-8.0)
— 0.05 M Tris-HCl buffer (pH 9.0)
— DNS chromogen reagent (MY73006)
— Tecan Infinite M200 microplate reader
— Water bath
— Centrifuge
1. Adjust the pH of the chitosanase solution to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 using 0.05 M acetate buffer (pH 3.0-6.0), 0.05 M phosphate buffer (pH 6.0-8.0), and 0.05 M Tris-HCl buffer (pH 9.0).
2. Pre-incubate the chitosanase solution from step 1 at 0°C for 2 h.
3. Mix the pre-incubated chitosanase solution from step 2 with the 2% chitosan solution at a 1:1 (v/v) ratio, adjust the temperature and pH to the optimal values.
4. Incubate the mixed solution from step 3 at 45°C for 30 min.
5. Hydrolysate from step 4 is subjected to reducing sugar content determination and then the pH stability curve of the enzyme is plotted.
— 2% Chitosan solution
— Chitosanase solution
— 2.0% (w/w) acetic acid (HAc)
— 1% Ammonia water
— DNS chromogen reagent (MY73006)
— Tecan Infinite M200 microplate reader
— Water bath
— Centrifuge
1. Mix the 2% chitosan solution with the chitosanase solution at a 1:1 (v/v) ratio, and adjust the pH to approximately 5.0.
2. Incubate the mixed solution from step 1 at 25°C, 30°C, 35°C, 40°C, and 45°C for 30 min.
3. Hydrolysate from step 2 is subjected to reducing sugar content determination and then the temperature-activity curve of the enzyme is plotted.
— 2% Chitosan solution
— Chitosanase solution
— 2.0% (w/w) acetic acid (HAc)
— 1% Ammonia water
— 0.05 M acetate buffer (pH 3.0-6.0)
— 0.05 M phosphate buffer (pH 6.0-8.0)
— 0.05 M Tris-HCl buffer (pH 9.0)
— DNS chromogen reagent (MY73006)
— Tecan Infinite M200 microplate reader
— Water bath
— Centrifuge
1. Adjust the pH of the chitosanase solution to 7.5 using 0.05 M phosphate buffer (pH 6.0-8.0).
2. Pre-incubate the chitosanase solution (pH 7.5) from step 1 at 30°C, 35°C, 40°C, 45°C, and 50°C for 2 h.
3. Mix the pre-incubated chitosanase solution from step 2 with the 2% chitosan solution at a 1:1 (v/v) ratio, and adjust the temperature to the optimal values.
4. Incubate the mixed solution from step 3 at 45°C for 30 min.
5. Hydrolysate from step 4 is subjected to reducing sugar content determination and then the thermal stability curve of the enzyme is plotted.
Thin Layer Chromatography (TLC)
— Test sample
— Developing solvent (Isopropanol: sterile water: 1% Ammonia Water = 60: 30: 4, v/v/v)
— TLC silica gel plate
— Microcapillary tube
— 0.5% Ninhydrin
1. Using a microcapillary tube, spot test sample onto the TLC plate, positioning the spots approximately 1.0 cm above the bottom edge along the starting line.
2. Add an appropriate amount of developing solvent to the TLC chamber until the liquid depth reaches approximately 0.5 cm. Carefully place the spotted TLC plate into the chamber, ensuring the starting line is positioned above the solvent level. Remove the TLC plate when the solvent front has migrated to approximately 1.0 cm from the top edge, and immediately mark the solvent front position with a pencil.
3. The TLC plate from step 2 is air-dried, sprayed with 0.5% ninhydrin, and then heated to visualize the separated chitooligosaccharides.
4. Compare the spot positions between the hydrolysate and the standard, and analyze the ability of each mutant to produce low-degree polymerization COSs; simultaneously, observe the color and intensity of each spot to preliminarily quantify the COSs of different polymerization degrees in the hydrolysate.
High Performance Liquid Chromatography (HPLC)
— Test sample
— Shodex Asahipak NH2P-50 4E Column (4.6 mm × 250 mm,5 μm)
— Mobile phase (acetonitrile : 1% ammonia solution (v/v) = 7:3)
— Methanol (HPLC Grade)
— 0.22-µm Micron Pore Filter Membrane
— Chitooligosaccharides (DP 3-7)
— Essentia Prep LC-16P (Shimadzu, Japan)
— Glucosamine
— Chitobiose solution
1. Chitooligosaccharides solutions are prepared at concentrations of 5 mg/mL, 10 mg/mL, 15 mg/mL, 25 mg/mL, and 50 mg/mL.
2. Analytical-grade glucosamine and chitobiose solutions are prepared at a concentration of 5 mg/mL.
3. The test sample, chitooligosaccharides, glucosamine and chitobiose solution from step 1 and step 2 are filtered through a 0.22-µm Micron Pore Filter Membrane into a sample vial for HPLC analysis.
4. The HPLC parameters are set as follows:
Table 4 HPLC Parameters
5. The standard curve is generated by recording the retention times and peak areas for the chitooligosaccharides standards with various polymerization degrees. Subsequently, the retention times corresponding to chitooligosaccharides with different polymerization degrees are identified, and a linear standard curve is constructed for each polymerization degree based on the relationship between concentration and peak area.
6. The filtered test sample from step 3 is analyzed to determine the peak area and retention time.
7. Data analysis involves qualitative analysis of the polymerization degree of the main products by comparing retention times with standard references, and quantitative analysis is performed using integrated peak areas to calculate the yields of chitooligosaccharides with different polymerization degrees in the degradation products.
Characterization of the Secretion System
— Chloramphenicol (34 mg/mL)
— LB liquid medium
— Tecan Infinite M200 microplate reader
— Centrifuge
1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm and last 12-16 h.
2. Bacteria culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm until the OD600 of bacteria culture reached 0.6.
3. Bacteria culture from step 2 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm.
4. 1 mL of the bacterial cultures is collected per hour until 10 h.
5. The fluorescence intensity of the bacteria culture is measured immediately by Tecan Infinite M200 microplate reader.
6. The secretion efficiency is calculated by dividing the fluorescence intensity of the supernatant by the fluorescence intensity of the bacteria culture.
Characterization for the Combination of Secretion System and Chitosanase
— Tecan Infinite M200 microplate reader
— Centrifuge
— Kanamycin (50 mg/mL)
— LB liquid medium
— sterile water
— IPTG (Isopropyl β-D-1-thiogalactopyranoside) (1 mol/L)
— Anhydrous ethanol
1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm and last 12-16 h.
2. Bacteria culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm until the OD600 of bacteria culture reached at 0.6.
3. IPTG is added into the bacterial culture to a final concentration of 1 mM, and expression is induced at 16°C, 200 rpm for 36 hours.
4. 2 mL of the bacterial cultures is collected at 0 h, 5 h, 8 h, 12 h, 16 h and 20 h.
5. Bacteria culture from step 4 is centrifuged at 13000 rpm at 4°C for 10 min to collect the supernatant.
6. The collected supernatant from step 5 is mixed with 2 mL of anhydrous ethanol by gentle inversion, and stored at 4°C for 12 h.
7. Mixture from step 6 is centrifuged at 13000 rpm at 4°C for 10 min and then discard the supernatant.
8. 400 µL sterile water is added into the precipitate from step 7 to dissolve and obtain the extracellular supernatant, which is then subjected to SDS-PAGE.
9. The supernatant from the 18 h is taken for preservation experiment.
Preservation Efficacy Validation
It should be noted that the fruits and fish used in our preservation experiment have been approved by the Safety and Security Committee (Safety Form).
— Tryptone powder
— Yeast extract powder
— D-glucose
— Agar powder
— sterile water
— Fresh back meat of fish
— Tomato
— Sterile sampling bag
— Normal saline solution (0.9% sodium chloride)
— Chitooligosaccharides (MW < 1 kDa)
— Chitooligosaccharides (MW < 2 kDa)
— Chitooligosaccharides (MW < 3 kDa)
— Potassium sorbate
1. Prepare the PCA medium by mixing reagents according to the table 5, and sterilize it at 121°C for 15 min.
Table 5 Composition of PCA Medium
2. Pour the medium into plates while in liquid state, allow it to cool and solidify, and store at 4°C.
1. Fresh back meat of fish is cut into uniformly sized and equal-weight pieces, immersed in different solutions for 10 minutes, drained of excess liquid, packaged in sterile sampling bags, and stored at 4°C for refrigeration.
2. Samples are retrieved and weighed on day 0, day 1, day 2, day 3, and day 4.
3. In a biosafety cabinet, 10 mL of sterile physiological saline is added to the samples retrieved in step 2. After sealing the sampling bags, the fish meat is homogenized by grinding.
4. 10 µL of the homogenized fish sample is mixed thoroughly with 90 µL of sterile physiological saline.
5. 10 µL of the diluted homogenate is mixed with 90 µL of sterile physiological saline. This dilution process is repeated 5 times, resulting in 6 dilution levels.
6. 10 µL of each diluted homogenate is taken and spotted onto pre-prepared PCA solid plates. The plates are then incubated at 37°C for 48 hours.
7. Viable bacterial count is calculated. The colony forming units (CFU)/g is calculated as follows: CFU/g = colony count × dilution factor × 10 / sample weight (g) (a single dilution factor is 10, two dilutions correspond to 102, and so on).
Note: When the viable bacterial count reaches 108 CFU/g or higher, the food is considered to be in the initial stage of spoilage.
1. Fresh tomatoes are immersed in different solutions for 10 minutes, drained of excess liquid, packaged in sterile sampling bags (one tomato per bag), and stored at room temperature.
2. Samples are retrieved on day 0, day 1, day 2, day 3, and day 4, weighed, and the diameter measured to calculate surface area.
3. In a biosafety cabinet, 10 mL of sterile water is added to the samples retrieved in step 2 to collect the surface bacteria of the tomato.
4. 10 µL of the water containing surface bacteria is mixed thoroughly with 90 µL of sterile water.
5. 10 µL of the diluted solution is mixed with 90 µL of sterile water. This dilution process is repeated 5 times, resulting in 6 dilution levels.
6. 10 µL of each diluted solution is taken and spotted onto pre-prepared PCA solid plates. The plates are then incubated at 37°C for 48 hours.
7. After water collection in step 4, the tomato is mashed to prepare a homogenate.
8. 10 µL of the tomato homogenate is mixed thoroughly with 90 µL of sterile water.
9. 10 µL of the diluted homogenate is mixed with 90 µL of sterile water. This dilution process is repeated 5 times, resulting in 6 dilution levels.
10. 10 µL of each diluted homogenate is taken and spotted onto pre-prepared PCA solid plates. The plates are then incubated at 37°C for 48 hours.
11. Viable bacterial count is calculated.
Surface bacterial count of tomato: The colony forming units (CFU)/cm2 is calculated as follows: CFU/cm² = colony count × dilution factor × 10 / sample surface area (cm2) (a single dilution factor is 10, two dilutions correspond to 102, and so on).
Total bacterial count of tomato: The colony forming units (CFU)/g is calculated as follows: CFU/g = colony count × dilution factor × 10 / sample weight (g).
Biosafety
— Chloramphenicol (34 mg/mL)
— LB liquid medium
— LB solid plates
— IPTG (Isopropyl β-D-1-thiogalactopyranoside) (1 mol/L)
1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm and last 12-16 h.
2. Bacteria culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm. Two groups are set: one group (IPTG) is added IPTG to reach the concentration of 1 mM and the other group (NO IPTG) is without IPTG.
Note: Each group should have three parallel cultures.
3. After culturing for 6 h, 20 μL of the bacteria culture is taken and mixed thoroughly with 180 μL of fresh LB medium containing the same antibiotic concentration.
4. 20 μL of the diluted culture is taken and mixed with another 180 μL of fresh LB medium with the same antibiotic concentration. Repeat this dilution process 7 times, resulting in 8 dilution levels.
5. 5 μL of each diluted culture is taken and spotted onto pre-prepared solid plates containing the same antibiotic concentration. Then the plates are incubated at 37°C for 12 h.
6. The survival ratio is calculated. First, the colony forming units (CFU)/mL is calculated: CFU/mL = colony count × dilution factor / 0.005 mL (a single dilution has a dilution factor of 10, two dilutions would be 102, and so on). The survival ratio (log10) is calculated as: log10[(CFU/mL of the IPTG group)/(CFU/mL of the NO IPTG group)].
— Chloramphenicol (34 mg/mL)
— LB liquid medium
— LB solid plates
— IPTG (Isopropyl β-D-1-thiogalactopyranoside) (1 mol/L)
1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm and last 12-16 h.
2. Bacteria culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm. Groups are set: groups (IPTG) are added IPTG to reach the concentration of 0.001 mM, 0.003 mM, 0,01 mM, 0.03 mM, 0.1 mM, 0.5 mM, 1 mM and the control group (NO IPTG) is without IPTG.
Note: Each group should have three parallel cultures.
3. Bacterial culture is collected at 0 h, 2 h, 4 h, 6 h and 8 h.
4. 20 μL of the bacteria culture is taken and mixed thoroughly with 180 μL of fresh LB medium containing the same antibiotic concentration.
5. 20 μL of the diluted culture is taken and mixed with another 180 μL of fresh LB medium with the same antibiotic concentration. Repeat this dilution process 7 times, resulting in 8 dilution levels.
6. 5 μL of each diluted culture is taken and spotted onto pre-prepared solid plates containing the same antibiotic concentration. Then the plates are incubated at 37°C for 12 h.
7. The survival ratio is calculated. First, the colony forming units (CFU)/mL is calculated: CFU/mL = colony count × dilution factor / 0.005 mL (a single dilution has a dilution factor of 10, two dilutions would be 102, and so on). The survival ratio (log10) is calculated as: log10[(CFU/mL of the IPTG group)/(CFU/mL of the NO IPTG group)].
— Chloramphenicol (34 mg/mL)
— LB liquid medium
— LB solid plates
— Cuminic acid stock solution (0.1 mol/L)
1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm and last 12-16 h.
2. Bacteria culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm. Two groups are set: one group (Cuma) is added Cuminic acid stock solution to reach the concentration of 0.1 mM and the other group (NO Cuma) is without Cuminic acid.
Note: Each group should have three parallel cultures.
3. After culturing for 4 h, 20 μL of the bacteria culture is taken and mixed thoroughly with 180 μL of fresh LB medium containing the same antibiotic concentration.
4. 20 μL of the diluted culture is taken and mixed with another 180 μL of fresh LB medium with the same antibiotic concentration. Repeat this dilution process 7 times, resulting in 8 dilution levels.
5. 5 μL of each diluted culture is taken and spotted onto pre-prepared solid plates containing the same antibiotic concentration. Then the plates are incubated at 37°C for 12 h.
6. The survival ratio is calculated. First, the colony forming units (CFU)/mL is calculated: CFU/mL = colony count × dilution factor / 0.005 mL (a single dilution has a dilution factor of 10, two dilutions would be 102, and so on). The survival ratio (log10) is calculated as: log10[(CFU/mL of the Cuma group)/(CFU/mL of the NO Cuma group)].
— Chloramphenicol (34 mg/mL)
— LB liquid medium
— LB solid plates
— IPTG (Isopropyl β-D-1-thiogalactopyranoside) (1 mol/L)
— Cuminic acid stock solution (0.1 mol/L)
1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm and last 12-16 h.
2. Bacteria culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm. Four groups are set:
Table 6 Kill Switch Condition Setting
Note: Each group should have three parallel cultures.
3. After culturing for 4 h, 20 μL of the bacteria culture is taken and mixed thoroughly with 180 μL of fresh LB medium containing the same antibiotic concentration.
4. 20 μL of the diluted culture is taken and mixed with another 180 μL of fresh LB medium with the same antibiotic concentration. Repeat this dilution process 7 times, resulting in 8 dilution levels.
5. 5 μL of each diluted culture is taken and spotted onto pre-prepared solid plates containing the same antibiotic concentration. Then the plates are incubated at 37°C for 12 h.
6. The survival ratio is calculated. First, the colony forming units (CFU)/mL is calculated: CFU/mL = colony count × dilution factor / 0.005 mL (a single dilution has a dilution factor of 10, two dilutions would be 102, and so on). The survival ratio (log10) is calculated as: log10[(CFU/mL of the other groups)/(CFU/mL of the NO IPTG and NO Cuma group)].
Others
— LB liquid medium
— Tecan Infinite M200 microplate reader
— UV-Sterilized chitooligosaccharides (MW < 1 kDa)
— UV-Sterilized chitooligosaccharides (MW < 2 kDa)
— UV-Sterilized chitooligosaccharides (MW < 3 kDa)
— sterile water
— Kanamycin (50 mg/mL)
1. Chitooligosaccharides with different degrees of molecular weights (MW) is weighed and added into an appropriate amount of sterile water to prepare 50% (w/v) stock solution.
2. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm and last 12-16 h.
3. Bacteria culture from step 2 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm until the OD600 of bacteria culture reached 0.6.
4. Bacterial culture from step 3 is added chitooligosaccharides stock solution from step 1 to reach the concentration of 7%, 1.2%, 0.012%, 0% (w/v) and kept at 37°C, 200 rpm. Reaction solution is collected at different time points.
5. The OD600 of the reaction solution is measured immediately by Tecan Infinite M200 microplate reader.
— LB liquid medium
— LB solid plates
— UV-Sterilized chitooligosaccharides (MW < 1 kDa)
— UV-Sterilized chitooligosaccharides (MW < 2 kDa)
— UV-Sterilized chitooligosaccharides (MW < 3 kDa)
— Kanamycin (50 mg/mL)
— IPTG (Isopropyl β-D-1-thiogalactopyranoside) (1 mol/L)
1. After autoclaving and cooling to 50°C, the LB solid medium is supplemented with chitooligosaccharides standards (MW < 1 kDa, MW < 2 kDa and MW < 3 kDa) at 0%, 0.25%, 0.5%, 1%, 1.25%, 1.5% (w/v), Kanamycin (50 µg/mL) and IPTG (0.1 mM) prior to pouring plates.
2. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm and last 12-16 h.
3. Bacteria culture from step 2 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm until the OD600 of bacteria culture reached 0.6.
4. Bacteria culture from step 3 is diluted 104-fold and plated onto LB solid plates containing Kanamycin (50 µg/mL) and IPTG (0.1 mM). Then the plates are incubated at 37°C for 12 h.
5. Colonies from the plate in step 4 are picked and spotted onto the plates containing different concentrations of chitooligosaccharides from step 1, using the same colony for all plates. Then the plates are incubated at 37°C for 12 h.
6. The survival frequency (%) is calculated as: (the number of colonies formed)/(total number of colonies).
