. Experiments .

● LB liquid medium
● Tecan Infinite M200 microplate reader

1. Revive the transformed bacteria by incubating them at 37 °C with shaking at 200 r.p.m. for 12 hours.
2. Inoculate the revived bacteria at 2% concentration into 10 mL of LB medium and shake at 37 °C for around 2 hours until the OD₆₀₀ values reaches 0.6-0.8.
3. Incubate the above bacteria separately at 4 °C, 15 °C, and 37 °C, each with shaking at 200 r.p.m. .
4. Use Tecan Infinite M200 microplate reader to measure the green fluorescence intensity and OD₆₀₀ values at 2 hours, 4 hours, and 6 hours.
Note: Excitation wavelength: 488 nm, Emission wavelength: 530 nm.
5. Calculate the ratio of green fluorescence intensity to OD₆₀₀ data.

● LB liquid medium
● Tecan Infinite M200 microplate reader
● TwistAmp® Liquid Basic Kit
● Chloramphenicol (34 mg/mL)

1. Revive the transformed bacteria by incubating them at 37 °C with shaking at 200 r.p.m. for 12 hours.
2. Inoculate the revived bacteria at 2% concentration into 10 mL of LB medium and shake at 37 °C with shaking at 200 r.p.m. for approximately 2 hours until the OD₆₀₀ values reaches 0.6-0.8.
3. Add 100 µL of 20% L-arabinose to the above bacterial culture and induce at 37 °C with shaking at 200 r.p.m. for 10 hours.
4. Add 10 µL of Chloramphenicol and 100 µL of the above bacterial culture to 10 mL of non-solidified LB agar medium, mix well and pour onto a Petri dish.
Note: Set up 3 parallel groups for both experimental and control groups.
5. After the agar medium has solidified, place 5 Oxford cups on the plate. 100 µL of 100, 75, 50, and 25 mol/L H₂O₂ were added to four cups around the perimeter, and 100 µL of ddH₂O was added to the center cup.
6. Incubate the plate overnight at 37 °C in an incubator.
7. Measure the diameter of the bacterial inhibition zones using a ruler.

● LB liquid medium
● Tecan Infinite M200 microplate reader
● Chloramphenicol (34 mg/mL)

1. Revive the transformed bacteria by incubating them at 37 °C with shaking at 200 r.p.m. for 12 hours.
2. Inoculate the revived bacteria at 2% concentration into 10 mL of LB liquid medium and
shake at 37 °C, 200 r.p.m. for approximately 2 hours until the OD₆₀₀ values reaches 0.6-0.8. 3. Add 100 μL of 20% L-arabinose to the above bacterial culture and induce at 37 °C with shaking at 200 r.p.m. for 10 hours.
4. Transfer the induced bacterial culture to -4 °C standing for 8 hours and measure the OD₆₀₀ values.
5. Transfer the culture to 37 °C with shaking at 200 r.p.m. and incubate for 1 hour, then measure the OD₆₀₀ values.
6. Generate bar graphs of the delta OD₆₀₀ values for the experimental and control groups.

● Differential scanning calorimeter
● 25 μL aluminum pan

Prepare 50 μM TmAFP, SfIBP, and BSA solution in advance.

1. Place the sample in the aluminum pan. Then, seal it and place it in the sample chamber. Place an empty aluminum pan in the reference chamber.
2. Adjust the temperature to 0 °C at a rate of 2 °C/min after that decreae it to -20 °C and hold for 5 minutes. Then, increase the temperature at a rate of 2 °C/min until the sample is fully melted. Determine the melting point (T_m) and freezing point (T_f) based on the heat capacity curve.
3. Adjust the temperature again to -20 °C at a rate of 2 °C/min and hold for 5 minutes. Then, increase the temperature at a rate of 2 °C/min until it reach 0.5 ℃ below Tm. Maintain this temperature for 5 minutes, and record it as T_h. Then the temperature is decreased to -20 ℃ again.
4. Calculate TH as TH = T_h - T_o

● 150 mM NaCl
● 10 mM Tris-HCl
● 10 μg/mL BSA
● 30% CaCl₂ solution
● Capillary tubes
● Polarizing filter

1. Prepare 0.5, 5, 10, 50, 100, 200, 500, and 1000 nM TmAFP and SfIBP solutions in advance.
2. Prepare the buffer containing 150 mM NaCl, 10 mM Tris-HCl, and 10 μg/mL BSA.

1. Use capillary tubes to draw samples and fill them completely.
2. Arrange capillary tubes containing the same protein side by side between two glass slides, secure both ends with metal clips and quickly place them in a -80 °C freezer for 1 hour.
3. Remove the glass slides and rapidly transfer them to a 30% CaCl₂ solution in a -20 °C freezer. Take pictures of ice crystals under a microscope.
4. Place the glass slides in the -20 °C freezer and keep them for 16 hours.
5. Remove the glass slides and quickly take pictures of the ice crystals under a microscope.
6. Compare the two ice crystal images, and the lowest concentration at which the ice crystals remain unchanged is the Ice Recrystallization Inhibition (IRI).

● LB liquid medium
● Tecan Infinite M200 UV-Visible spectrophotometer

1. Resuscitate the transformed bacteria overnight at 37 °C with shaking at 200 r.p.m. .
2. In conditions of 37 °C and 200 r.p.m., inoculate the resuscitated bacteria (2%) into 10 mL LB liquid culture medium and shake for approximately 2 hours until the OD₆₀₀ values reaches 0.6-0.8.
3. Add L-arabinose to the above bacterial cultures to a final concentration of 0.2%. Set up three parallel control groups for the experimental and control groups, and incubate them at 37 °C with shaking at 200 r.p.m. for 12 hours.
4. Take 1.5 mL of the above bacterial cultures into 2.0-mL centrifuge tubes, centrifuge at room temperature at 12,000 r.p.m. for 2 minutes.
5. Measure the fluorescence intensity of the original bacterial culture and supernatant separately. Express the signal peptide secretion efficiency as the ratio of the fluorescence intensity of the supernatant to the bacterial culture.

● LB liquid medium
● Tecan Infinite M200 UV-Visible spectrophotometer
● Chloramphenicol (34 mg/mL)
● PBS buffer
● FITC labelled anti-his tag Antibody
● 5% skim milk
● 1.5-mL centrifuge tubes

1. Resuscitate the transformed bacteria overnight at 37 °C with shaking at 200 r.p.m..
2. In conditions of 37 °C and 200 r.p.m., inoculate the resuscitated bacteria (2%) into 10 mL LB liquid culture medium and shake for approximately 2 hours until the OD₆₀₀ values reaches 0.6-0.8.
3. Add L-arabinose to the above bacterial cultures to a final concentration of 0.2%. Keep shaking at 37 °C and 200 r.p.m. for an additional 6 hours.
4. Take six clean 1.5-mL centrifuge tubes and add 1 mL of bacterial culture to each tube. Centrifuge at 6500 r.p.m. for 3 minutes. Then, discard the supernatant to collect the pellet.
Note: Perform three parallel experiments for the experimental group and positive control.
5. Add 1 mL of 1×PBS buffer to the centrifuge tubes containing the pellets and mix well.
6. Centrifuge at 6500 r.p.m. for 3 minutes, discard the supernatant, and repeat this step twice.
7. Add 750 μL of 5% skim milk to each centrifuge tube and mix well. Then, incubate at 37 °C for 0.5 hours.
8. Centrifuge at 6500 r.p.m. for 3 minutes and discard the supernatant.
9. Add 600 μL of 1×PBS buffer and mix well. Then, add FITC labelled anti-his Antibody (1:3000). Incubate at 37 °C for 1 hour (or overnight at 4 °C).
10. Repeat step 8 once, add 1 mL 1×PBS solution into the centrifuge tube, mix well. Then, centrifuge at 6500 r.p.m. for 3min, discard the supernatant, and repeat twice.
11. Pipette 200 μL of the bacterial culture from each tube into a 96-well microplate. Use a microplate reader to measure the fluorescence intensity and OD₆₀₀.
Note: Excitation wavelength: 492 nm, Emission wavelength: 518 nm.
12. Calculate the fluorescence intensity/OD₆₀₀ for both the negative control group and the experimental group.
Note: Steps 8-11 should be performed in the dark, and subtract the value of PBS blank.

Expected Results:
The fluorescence intensity/OD₆₀₀ value in the experimental group should be higher than that in the negative control group, indicating successful expression of mv¹⁴⁰ and surface display of CBM on the bacterial surface.

● LB liquid medium
● Chloramphenicol (34 mg/mL)
● 96-well plate
● L-arabinose stock solution (0.2 g/mL)
● D-glucose stock solution (500 g/L)

1. Resuscitate the transformed bacteria overnight at 37 °C with shaking at 200 r.p.m. .
2. Inoculate the overnight revived bacterial culture into fresh LB liquid culture medium at 2% ratio.
3. In the experimental group, add L-arabinose stock solution to achieve a final concentration of 0.2%, while in the control group, add D-glucose stock solution to achieve a final concentration of 0.5%.
4. After culturing for 6 hours at 37 °C, 200 r.p.m., take 100 μL of the bacterial culture and centrifuge it at room temperature at 6,500 r.p.m. for 3 minutes. Discard the supernatant and resuspend the pellet in 100 μL of fresh LB liquid culture medium with the same antibiotic concentration. Repeat this process once.
5. Perform a gradient dilution using a 96-well plate. Take 20 μL of the resuspended bacterial culture and add it to 180 μL of fresh LB liquid culture medium with the same antibiotic concentration, mixing thoroughly. Repeat this dilution step six times to create a total of six dilution levels.
6. From each dilution, spot 5 μL of the diluted culture onto a solid agar plate prepared with the same antibiotic concentration. Allow the spots to air dry and then incubate the plates upside down in a 37 °C constant temperature incubator for 12 hours. After incubation, count the bacterial colonies.
7. Calculate the survival ratio.

Note:
1. CFU/mL = Number of colonies × Dilution factor / 0.005 mL
2. Survival Ratio (log₁₀) = log₁₀[(CFU/mL in the experimental group)/(CFU/mL in the control group)]

● Tecan Infinite M200 microplate reader
● LB liquid medium
● Kanamycin antibiotic (50 mg/mL)
● Chloramphenicol antibiotic (34 mg/mL)

1. Revive the transformed bacteria by incubating them at 37 °C with shaking at 200 r.p.m. for 12 hours.
2. Inoculate the revived bacteria at 2% concentration into 10 mL of LB medium and shake at 37 °C.
3. Every hour record the OD₆₀₀ values and fluorescence intensity until the curve tend to be stable.

● Recombinant E. coli BL21(DE3) strain
● LB liquid medium
● Ampicillin antibiotic (100 mg/mL)
● 500 g/L glucose solution
● 37 °C shaking incubator
● 0.1% sodium dodecyl sulfate (SDS)
● 0.2 M acetate buffer (pH=6)
● 2.5 g/L CTAB solution containing 2% NaOH
● Tecan Infinite M200 microplate reader
● 96-well plate

1. Prepare the HA standard solutions with concentrations of 0, 20, 40, 60, and 80 mg/L. Perform five replicates for each concentration.
2. Add 150 μL of HA standard solution and 350 μL of 0.2 M acetate buffer in a 2-mL centrifuge tube. Then mix them well.
3. Add 1 mL CTAB solution to the above mixture.
4. After incubation for five minutes, transfer 200 μL to a 96-well plate and measure the OD₄₀₀ values.
5. Plot a standard curve with absorbance values to HA concentrations.

1. Take 500 μL of the overnight culture and add it to a 1.5-mL centrifuge tube, add 500 μL of 0.1% SDS to each sample.
2. Incubate the mixture at 37 °C for 15 minutes.
3. Centrifuge at 4,600 r.p.m. for 10 minutes, and pipette out 150 μL of the supernatant into a new 2-mL centrifuge tube.
4. Add 350 μL of 0.2 M acetate buffer and mix well.
5. Add 1 mL of CTAB solution.
6. After five minutes of incubation, transfer 200 μL to a 96-well plate and measure the OD₄₀₀ values.
Note: Be careful to ensure that the final data falls within the standard curve range.

● Recombinant E. coli Nissle 1917 (EcNP) strain
● LB liquid medium
● Ampicillin (100 mg/mL)
● 500 g/L glucose solution
● 37 °C shaking incubator
● Ampicillin (100 mg/mL)
● Kanamycin (50 mg/mL)
● Chloramphenicol (34 mg/mL)

1. Inoculate the recombinant BL21(DE3) strains in 2% ratio into 10 mL of LB medium containing corresponding antibiotics and glucose (20 g/L) at 37 °C and 200 r.p.m. for 20 h.
Note: Monitor the concentration of glucose in case of product consumption.
2. Centrifugate the bacteria above to obtain the supernatant containing HA.
3. Inoculate the recombinant EcNP strains in 2% ratio into 10 mL of LB medium containing corresponding antibiotics and glucose (30 g/L) at 37 °C and 200 r.p.m..
4. Add IPTG at final concentrations of 0.5 mM and the supernatant containing HA to it when the OD₆₀₀ reaches 0.8.
5. Shake the cultures at 37 °C with shaking at 200 r.p.m. for several hours.
6. Observe the morphology of product and test the water-retention ability.

1. Take same amount of soil in bottle caps. Add the water (V₁), co-culture solution (V₂) and the mixture of them (V₃=V₁+V₂) into the soil.
Note: Make sure the water is evenly mixed with soil and volume will not exceed the rim.
2. Place the mixture in 37 ℃ environment for 0.5 hours.
3. Calculate Δweight carefully. Δweight _{experiment group} = the Δweight of soil with co-culture solution and pure water minus the Δweight of soil with co-culture solution.

●Fourier Transform Infrared Spectroscopy (FTIR)

1. Prepare the samples.
2. In adsorption mode, acquire 32 scans at a resolution of 4 cm^⁻¹ within the wave number range of 4000 to 400 cm^⁻¹ using a VERTEX 70 V spectrometer.

● Field Emission Scanning Electron Microscope (FESEM)

1. Prepare freeze-dried samples.
2. Apply a brief (~5 nm) iridium sputter coating for 30 seconds to all freeze-dried samples before imaging.
3. During imaging, employ an acceleration voltage of 5.0 kV.