1. Prepare the culture medium

1) LB liquid medium

① Weigh 2.5 grams of LB broth using a balance, pour it into an Erlenmeyer flask, and add approximately 80 milliliters of deionized water.

② Swirl the Erlenmeyer flask to dissolve the LB broth, then add deionized water to adjust the volume to 100 milliliters.

③ Seal the flask mouth with parafilm.

④ Sterilize using an autoclave (conditions: 121°C, 20 minutes).

2) LB solid medium

① Weigh 2.5 grams of LB broth using a balance, pour it into an Erlenmeyer flask, and add approximately 80 milliliters of deionized water.

② Swirl the Erlenmeyer flask to dissolve the LB broth, then add deionized water to adjust the volume to 100 milliliters.

③ Weigh another 1.5 grams of agar powder and add it to the Erlenmeyer flask.

④ Seal the flask mouth with parafilm.

⑤ Sterilize using an autoclave (conditions: 121°C, 20 minutes).

⑥ Disinfect the Erlenmeyer flask and petri dishes with 75% ethanol, then place them in a laminar flow hood and expose to UV irradiation for 15-20 minutes.

⑦ Wait for the LB solid medium to cool to 50-60°C, then add 100 microliters of ampicillin (Amp, 100 mg/ml) and gently swirl to mix, avoiding generating bubbles.

⑧ Open the disposable petri dishes and quickly pour approximately 20 milliliters of the medium into each dish.

⑨ Leave the petri dish lids slightly open to allow the medium to solidify for 30 minutes.

⑩ After solidification, collect the petri dishes, seal them with parafilm, label them properly, and store in a 4°C refrigerator.

3) MRS Liquid Medium

① Use a balance to weigh 10.44 grams of LB Broth, pour it into an Erlenmeyer flask, and add approximately 180 milliliters of deionized water.

② Swirl the Erlenmeyer flask to dissolve the LB Broth, then add deionized water to bring the volume up to 200 milliliters (to make the final volume 200 mL).

③ Seal the mouth of the flask tightly with parafilm.

④ Sterilize using an autoclave (sterilization conditions: 115°C, 30 minutes).

2. Plasmid Extraction

① Take 5 ml of overnight-cultured bacterial broth, centrifuge at 8000 × g for 2 minutes to collect the bacterial cells, and discard the supernatant.

② Add 250 µl of Buffer P1 to the pellet and thoroughly resuspend the bacterial cells.

③ Add 250 µl of Buffer P2, immediately invert the centrifuge tube gently 10 times to mix. Incubate at room temperature for 2-4 minutes.

④ Add 350 µl of Buffer P3, immediately invert the centrifuge tube gently 10 times.

⑤ Centrifuge at 12000 × g for 10 minutes. Transfer the supernatant to an adsorption column, centrifuge at 8000 × g for 30 seconds, and discard the liquid in the collection tube.

⑥ Add 500 µl of Wash Solution, centrifuge at 9000 × g for 30 seconds, and discard the liquid in the collection tube.

⑦ Repeat step 6.

⑧ Centrifuge the empty adsorption column at 9000 × g for 1 minute.

⑨ Place the adsorption column into a clean 1.5 ml centrifuge tube, then put the centrifuge tube in a 55℃ water bath (for approximately 10 minutes) to allow residual ethanol to evaporate completely.

⑩ Add 30 µl of Elution Buffer to the center of the adsorption column membrane, incubate at room temperature for 1 minute, then centrifuge for 1 minute. Store the DNA solution in the tube.

3. PCR

① Add ddH2O, forward primer, reverse primer, template, and 2x Super Pfx Master Mix to the PCR tube in sequence according to the PCR reaction system.

PCR Reaction System:

② Place the PCR tube in a mini centrifuge for centrifugation.

③ Set up the PCR instrument according to the PCR reaction program and place the PCR tube in it.

PCR Reaction Program:

④ After the PCR reaction is completed, perform agarose gel electrophoresis on the samples.

4. Agarose Gel Electrophoresis

① Prepare the gel mold: place the gel tray in the gel casting plate, and position the comb in the casting plate to form sample wells.

② Weigh 0.75 g of agarose and add it to an Erlenmeyer flask.

③ Take 1 ml of 50X TAE Buffer, add 49 ml of deionized water, and dilute it to 1× TAE Buffer.

④ Pour the diluted solution into the Erlenmeyer flask.

⑤ Heat the Erlenmeyer flask in a microwave oven until the agarose is completely dissolved.

⑥ Cool to approximately 50°C, then add 3 µl of 4S Green Plus Nucleic Acid Stain, and gently shake to mix, avoiding bubble formation.

⑦ Pour into the gel tray and cool to solidify at room temperature.

⑧ Carefully pull out the comb vertically upwards, place the gel into the electrophoresis tank with the sample wells facing the negative electrode.

⑨ Use a pipette to take 7 µl of PCR products and 1 µl of plasmid DNA respectively, and load them into the sample wells.

⑩ Turn on the power supply for electrophoresis (160V).

⑪ After electrophoresis, take out the gel and place it in a gel imager to observe the electrophoresis results.

5. Gel Extraction

① Select the correct target band and perform gel cutting.

② Transfer the gel blocks into 2 mL EP tubes respectively.

③ Add Buffer B2 to each gel sample (just enough to submerge the gel block), then place the tubes in a 50°C water bath until the gel blocks are completely dissolved (approximately 5 minutes).

④ Transfer the dissolved gel solution onto an adsorption column, centrifuge at 8000 Xg for 30 seconds, and discard the liquid in the collection tube.

⑤ Add 500 µL Wash Solution, centrifuge at 9000 Xg for 30 seconds, and discard the liquid in the collection tube.

⑥ Repeat Step ⑤.

⑦ Centrifuge the empty adsorption column at 9000 Xg for 1 minute.

⑧ Place the adsorption column into a clean 1.5 mL centrifuge tube, then put the centrifuge tube in a 50°C water bath (for approximately 10 minutes) to fully evaporate the ethanol.

⑨ Add 30 µL ddH₂O (double-distilled water) to the center of the adsorption column membrane, let it stand at room temperature for 1 minute, then centrifuge for 1 minute. Store the DNA solution in the tube.

6. Double Digestion

① Prepare the double digestion systems according to the following formulations respectively:

a) PCR product (30 µl) (AgaE/Inulinase)

b) Plasmid DNA (20 µl) (pET-22b(+))

② Incubate in a 37°C water bath for 1 hour.

③ Perform agarose gel electrophoresis on the digestion products.

④ Excise and recover the correct target band from the gel.

7. Ligation and Transformation

1) Ligation

① Add the recovered target DNA fragment and linearized vector according to the following system:

② Place the ligation system in a PCR instrument (22°C for 3 hours).

③ Inactivate the ligation system in a 65°C PCR instrument for 10 minutes.

2) Transformation

① Take competent cells from the -80°C refrigerator and thaw on ice.

② Divide the competent cells equally into two groups, with approximately 50 µl of competent cells per group.

③ Add 5 µl of the ligation product and mix gently by pipetting.

④ Incubate on ice for 30 minutes.

⑤ Perform heat shock in a 42°C water bath for 90 seconds, then place on ice for 5 minutes.

⑥ Add 1 ml of LB medium (without antibiotics).

⑦ Place in a shaker and culture for 1 hour (37°C, 220 rpm).

⑧ Centrifuge at 4000 rpm for 2 minutes, discard the excess supernatant, leaving approximately 200 µl.

⑨ Resuspend the bacterial cells.

⑩ Evenly spread 200 µl of the bacterial suspension onto LB solid medium using the spreading method.

⑪ Incubate upside down in a 37°C constant temperature incubator for 12-16 hours.

8. Colony Verification

① Turn on the laminar flow hood in advance, place the required experimental consumables inside, and turn on the UV lamp for disinfection for 20 minutes.

② Pipette 1 ml of LB liquid medium into a 2 ml EP tube.

③ Pipette 1 µl of ampicillin into the 2 ml EP tube.

④ Pick a single colony from the solid medium using a sterilized pipette tip.

⑤ Place the 2 ml EP tube in a shaker and culture for 3 hours (37°C, 220 rpm).

⑥ Add ddH₂O, forward primer, reverse primer, and Tap-Plus PCR Forest Mix to the PCR tube respectively according to the conventional PCR reaction system:

Conventional PCR Reaction System

⑦ Turn on the laminar flow hood, place the bacterial suspension in it, and add it to the PCR tube.

⑧ Place in a mini centrifuge for centrifugation.

⑨ Set up the PCR instrument according to the conventional PCR reaction program and place the PCR tube in it.

⑩ Place the remaining bacterial suspension in a shaker for continued culture.

Conventional PCR Reaction Program

9. Inducible Expression of E. coli Rosetta AgaE_pET-22b(+) and E. coli Rosetta Inulinase_pET-22b(+)

1) Strain Expansion - E. coli Rosetta Inulinase_pET-22b(+)

① Take 1 ml of LB liquid medium and add 1 µl of Amp (100 mg/ml).

② Add 20 µl of E. coli Rosetta Inulinase_pET-22b(+) seed culture.

③ Place the bacterial culture in a shaker for 12 hours (37°C, 220 rpm).

④ Take 50 ml of LB liquid medium and add 50 µl of Amp (100 mg/ml).

⑤ Add 1 ml of E. coli Rosetta Inulinase_pET-22b(+) seed culture.

⑥ Incubate in a shaker (37°C, 220 rpm) until the OD600 reaches approximately 0.6.

⑦ Add 25 µl of 1 M IPTG inducer (final concentration: 0.5 mM) respectively.

⑧ Incubate in a shaker for 16-20 hours (20°C, 220 rpm).

(The control group is the bacterial culture without the inducer under the same culture conditions.)

2) Strain Expansion - E. coli Rosetta AgaE_pET-22b(+)

⑨ Take 1 ml of LB liquid medium and add 1 µl of Amp (100 mg/ml).

⑩ Add 20 µl of E. coli Rosetta AgaE_pET-22b(+) seed culture.

⑪ Place the bacterial culture in a shaker for 12 hours (37°C, 220 rpm).

⑫ Take 50 ml of LB liquid medium and add 50 µl of Amp (100 mg/ml).

⑬ Add 1 ml of E. coli Rosetta AgaE_pET-22b(+) seed culture.

⑭ Incubate in a shaker (37°C, 220 rpm) until the OD600 reaches approximately 0.6.

⑮ Add 1.25 µl of 1 M IPTG inducer (final concentration: 0.025 mM) respectively.

⑯ Incubate in a shaker for 16-20 hours (20°C, 220 rpm).

(The control group is the bacterial culture without the inducer under the same culture conditions.)

3) Bacterial Harvesting

① Remove the induced bacterial culture from the shaker.

② Transfer to a 50 mL centrifuge tube and centrifuge (5000 xg, 10 min, 4°C).

③ Discard the supernatant, add 30 ml of 20 mM PB buffer to each tube, and resuspend the bacterial cells (AgaE: pH 7.0; Inulinase: pH 5.5).

④ Centrifuge (5000 xg, 10 min, 4°C) and discard the supernatant.

⑤ Repeat steps ③ and ④.

⑥ Add 7 ml of buffer, resuspend the cells, and store in a 4°C refrigerator.

4) Bacterial Lysis

⑦ Take 7 ml of the bacterial culture from the 4°C refrigerator and place it in an ice-water bath.

⑧ Turn on the ultrasonic homogenizer, set the lysis parameters (1s on, 2s off, power 195W), and sonicate for 8 minutes until the bacterial cells are completely lysed and the solution becomes clear.

⑨ Place in a low-temperature refrigerated centrifuge (10,000 xg, 30 min).

⑩ Pour off the supernatant, which is the soluble protein, and temporarily store it in a 4°C refrigerator; the pellet is the inclusion bodies.

⑪ Add buffer and wash the inclusion bodies twice using the bacterial harvesting method.

⑫ Add 3 mL of buffer and resuspend.10. SDS-PAGE

10. SDS-PAGE

1) Preparation of Protein Gel

① Clean the glass plates, assemble the gel casting mold, add water to test for leaks, then pour out the water.

② Prepare 4 ml of 10% separating gel according to the following table:

③ Add 3 ml of SDS-PAGE separating gel, then add ultrapure water for liquid sealing.

④ After the lower gel solidifies, pour out the ultrapure water on top.

⑤ Prepare 2 ml of 5% stacking gel according to the following table:

⑥ Add an appropriate amount of the prepared stacking gel to the gel casting plate, and gently insert the comb.

2) SDS-PAGE Electrophoresis

① Prepare 1 L of protein electrophoresis buffer according to the following table:

② Add 10 µl of protein loading buffer and 20 µl of protein sample to a 1.5 ml EP tube, inactivate in a dry bath at 98°C for 10 minutes, and centrifuge at 8000 × g for 5 minutes.

③ Align the prepared gel with the 卡槽，clamp it into the vertical electrophoresis apparatus, and pour 1X protein electrophoresis buffer into the inner tank until it is completely full to check for leaks.

④ Loading: Pull out the comb vertically, add 5 µl of marker and 10 µl of protein sample into the wells.

⑤ Electrophoresis: Perform electrophoresis at 130V; when the sample runs into the separating gel, increase the voltage to 150V.

⑥ After electrophoresis, carefully remove the protein gel, place it in a box, pour in staining solution, and place on a shaker (30 rpm) until the protein gel is fully stained. The formula of the staining solution is as follows:

⑦ Pour out the staining solution in the box and rinse with clean water.

⑧ Pour in decolorizing solution and place on a decolorizing shaker. The formula of the decolorizing solution is as follows:

⑨ Change the decolorizing solution every 30 minutes to 1 hour until decolorization is complete.

11. Protein Purification (Ni-NTA Affinity Chromatography)

Inulinase

① Take 2 mL of the supernatant (soluble protein) of Inulinase after ultrasonic disruption and add it to the nickel column.

② Place the nickel column in an ice box, then place it on a shaker (50 rpm) for binding for about 3 hours to allow sufficient binding of the target protein to the filler.

③ Sequentially pass 7 mL of 0 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, and 300 mM imidazole solutions through the nickel column, and collect the first 2 mL of effluent respectively.

④ Take several 2 mL EP tubes, add 20 µL of 2.5×SDS-PAGE loading buffer, and add 30 µL of the effluent obtained in the above steps respectively.

⑤ Incubate in a 96°C dry bath for 10 minutes.

⑥ Perform SDS-PAGE electrophoresis; after electrophoresis, remove the gel, stain with staining solution, and after the staining effect is achieved, decolorize with decolorizing solution.

AgaE

① Take 2 mL of the supernatant (soluble protein) of AgaE after ultrasonic disruption and add it to the nickel column.

② Place the nickel column in an ice box, then place it on a shaker (50 rpm) for binding for about 3 hours to allow sufficient binding of the target protein to the filler.

③ Sequentially pass 7 mL of 0 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, and 300 mM imidazole solutions through the nickel column, and collect the first 2 mL of effluent respectively.

④ Take several 2 mL EP tubes, add 20 µL of 2.5×SDS-PAGE loading buffer, and add 30 µL of the effluent obtained in the above steps respectively.

⑤ Incubate in a 96°C dry bath for 10 minutes.

⑥ Perform SDS-PAGE electrophoresis; after electrophoresis, remove the gel, stain with staining solution, and after the staining effect is achieved, decolorize with decolorizing solution.

12. Enzyme Activity Verification:

1) Drawing the Standard Curve:

Prepare fructose standard solutions with different concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1 mg/mL. Take 200 μL of each into 2 mL centrifuge tubes, and add an equal volume of DNS solution to mix. Place the mixed samples in a boiling water bath for 5 minutes, then cool in an ice water bath to terminate the reaction. Add 1 mL of deionized water to dilute each sample, then use a microplate reader to measure the absorbance at 540 nm. Finally, obtain the linear regression equation and correlation coefficient with fructose concentration as the abscissa and OD value as the ordinate, and draw the reducing sugar standard curve.

2) Enzyme Activity Assay:

Verify the enzymatic activities of AgaE and Inulinase using the DNS method, respectively.

AgaE Enzyme Activity Verification

Prepare a 2% agarose solution.

Add 2 mL of the agarose solution, then add 3 mL of AgaE soluble protein (the control group uses inactivated protein samples).

Incubate in a 35°C water bath for 30 minutes.

Take 200 µL of the reaction solution and add 200 µL of DNS reagent.

Place in a metal bath at 98°C for 5 minutes.

After cooling, add 1 mL of water.

Add the samples to a 96-well plate, use a microplate reader to measure the OD540 value, and substitute it into the standard curve to calculate the product yield.

Inulinase Enzyme Activity Verification

Prepare a 2% inulin solution.

Add 2 mL of the inulin solution, then add 3 mL of Inulinase soluble protein (the control group uses inactivated protein samples).

Incubate in a 55°C water bath for 30 minutes.

Take 200 µL of the reaction solution and add 200 µL of DNS reagent.

Place in a metal bath at 98°C for 5 minutes.

After cooling, add 1 mL of water.

Add the samples to a 96-well plate, 0.2 mL per well.

Use a microplate reader to measure the OD540 value, and substitute it into the standard curve to calculate the product yield.

13. Product Analysis

HPLC Detection of AgaE Products

① Weigh 0.00322 g of A2, 0.0063 g of A4, and 0.00936 g of A6 standards separately, place them in a 2 mL EP tube, add 1 mL of ultrapure water, and fully dissolve to prepare a mixed standard with a concentration of 10 mM for each component.

② Centrifuge the standards, as well as the above experimental and control groups, at 12,000 Xg for 10 minutes.

③ Filter the centrifuged supernatant through a 0.22 µm filter membrane.

④ Send the processed samples to the experimental platform for HPLC detection.

HPLC Detection of Inulinase Products

① Weigh 0.01 g each of GF2 and GF3 standards, place them in a 2 mL EP tube, add 1 mL of ultrapure water, and fully dissolve to prepare a mixed standard with a concentration of 10 mg/mL for each component.

② Centrifuge the standards, as well as the above experimental and control groups, at 12,000 Xg for 10 minutes.

③ Filter the centrifuged supernatant through a 0.22 µm filter membrane.

④ Send the processed samples to the experimental platform for HPLC detection.

14. Prebiotic Activity Analysis

Strain Activation and Seed Culture Preparation:

Isolate single colonies from Lactobacillus acidophilus preparations or commercial yogurt drinks using the spread plate method. Pick typical single colonies and inoculate them into liquid MRS medium, then culture anaerobically at 37°C for 24 hours to obtain seed cultures in the logarithmic growth phase.

Experimental Grouping and Culture:

Take fresh MRS medium and treat according to the following group settings (three replicates per group):

Control group: 6 mL MRS medium + 700 μL PBS buffer + 100 μL seed culture;

FOS group: 6 mL MRS medium + 600 μL PBS buffer + 100 μL FOS solution (5.41 mg/mL) + 100 μL seed culture;

AOS group: 6 mL MRS medium + 100 μL PBS buffer + 600 μL AOS solution (0.81 mg/mL) + 100 μL seed culture;

FOS+AOS group: 6 mL MRS medium + 100 μL FOS solution + 600 μL AOS solution + 100 μL seed culture.

Place all groups in anaerobic culture bags and incubate at 37°C.

Absorbance Detection:

After 24 hours and 48 hours of culture, respectively, take 200 μL of the culture solution into a 96-well plate, and measure the OD₆₀₀ value using a microplate reader. Use uninoculated MRS medium as a blank for zero adjustment.

15. Contribution

① Prepare 50 mM solutions of NaCl, KCl, CuSO₄, CaCl₂, MgCl₂, FeSO₄, and FeCl₃ respectively. The specific steps are as follows: accurately weigh the required mass of each solute using an electronic balance, dissolve in an appropriate amount of 20 mM Tris-HCl buffer (pH 7.0), stir thoroughly until completely dissolved, and finally bring the volume to 5 mL for later use.

② To investigate the effect of different metal ions on AgaE enzyme activity, take 50 µL of 2% agarose solution (substrate) into multiple groups of 2 mL EP tubes. Then sequentially add 50 µL of 50 mM NaCl, KCl, CuSO₄, CaCl₂, MgCl₂, FeSO₄, or FeCl₃ solution to each tube. Subsequently, add 400 µL of AgaE enzyme solution to initiate the reaction, and incubate at 35°C for 30 minutes; meanwhile, set a control group with 50 µL Tris-HCl buffer (without metal ions). After the reaction, heat the samples in a 95°C water bath for 10 minutes to inactivate the enzyme. Finally, use a microplate reader to measure the absorbance of each reaction product at 540 nm, and evaluate the effect of different metal ions on enzyme activity by comparing the absorbance. Calculate the relative enzyme activity of each experimental group with the control group's enzyme activity set as 100%.

③ To investigate the effect of different concentrations of Mg²⁺ on AgaA enzyme activity, the experimental operation is as follows:

Prepare a series of MgCl₂ solutions with concentrations (0, 0.5, 1, 5, 10, 20, 50, 100, 150 mM) using 20 mM Tris-HCl buffer (pH 7.0) as the solvent.

Reaction system setup: Add 50 μL of 2% agarose solution to each of multiple groups of 2 mL EP tubes, then add 50μL of the above MgCl₂solutions with different concentrations; the control group adds 50μL of Tris-HCl buffer (without Mg²⁺). Add 400 μL of AgaA enzyme solution to each tube to initiate the reaction, and incubate at 35°C for 30 minutes.

Reaction termination and detection: Heat all samples in a 95°C water bath for 10 minutes to inactivate the enzyme. Take 200 μL of the reaction solution into a new 2 mL EP tube, add 200 μL of DNS reagent, heat in a 95°C water bath for 5 minutes, and after cooling, add 1 mL of ddH₂O to each tube. Use a microplate reader to measure the absorbance at 540 nm, evaluate the effect of different concentrations of Mg²⁺ on AgaA enzyme activity by comparing the absorbance differences among groups, and calculate the relative enzyme activity of each experimental group with the control group's enzyme activity set as 100%.

16. Model

1. Solution Preparation

a) Substrate agarose solution: Accurately weigh 0.8 g of agarose using an electronic balance, place it in an Erlenmeyer flask, add 20 mM Tris-HCl buffer (pH 7.0) to bring the volume to 20 mL to prepare a 4% agarose solution. Heat in a microwave until completely dissolved to obtain a clear and transparent solution. Before the solution solidifies, sequentially dilute it to concentrations of 3%, 2.5%, 2%, 1%, and 0.5%, and keep each concentration of agarose solution in a 50°C water bath for later use.

b) Sodium chloride solution: Accurately weigh 1.1688 g of sodium chloride using an electronic balance, place it in a 50 mL centrifuge tube, add 20 mM Tris-HCl buffer (pH 7.0) to bring the volume to 20 mL (1000 mM), stir until completely dissolved, then use this as the stock solution to sequentially dilute to 100, 200, 300, 500, 700, 800 mM sodium chloride solutions. Use Tris-HCl buffer as the blank control for the 0 mM concentration.

2. Determination Method

a) Take 50 µL of agarose solutions (substrates) with concentrations of 4%, 3%, 2.5%, 2%, 1%, and 0.5% into multiple 2 mL EP tubes, add 50 µL of Tris-HCl buffer to each, then add 400 µL of AgaE enzyme solution to initiate the reaction, and react at 35°C for 1, 5, 10, 20, 25, 30, 40, 50, and 60 minutes respectively. After the reaction, heat the samples in a 95°C water bath for 10 minutes to terminate the enzyme activity. Finally, use a microplate reader to measure the absorbance of each reaction product at 540 nm.

b) Take 50 µL of agarose solutions with concentrations of 4%, 3%, 2.5%, 2%, 1%, and 0.5% as substrates, place them in 2 mL EP tubes, with 3 replicate tubes set for each substrate concentration to form an experimental group. Add 50 µL of sodium chloride solutions with concentrations of 0, 100, 200, 300, 500, 700, 800, or 1000 mM to each tube in each group, then add 400 µL of AgaE enzyme solution to initiate the reaction, and react at 35°C for 3 minutes. After the reaction, heat the samples in a 95°C water bath for 10 minutes to terminate the enzyme activity. Finally, use a microplate reader to measure the absorbance of each reaction product at 540 nm, which is used to analyze the effect of different sodium ion concentrations on the kinetic parameters of AgaE enzymatic reactions.