First Week

July 7

1. Medium Preparation

Liquid Medium

1. Prepare and clean conical flasks thoroughly.

2. Weigh 1.25 g of LB broth using a balance, pour it into a conical flask, and add approximately 40 ml of deionized water.

3. Shake the conical flask to dissolve the LB broth completely, then add deionized water to bring the total volume to 50 ml (volume make-up).

4. Seal the mouth of the conical flask with parafilm.

5. Sterilization

   • Preliminary preparation: Ensure there is a sufficient amount of water in the autoclave.

   • Sterilize at 121°C for 20 minutes.

Solid Medium

1. Prepare and clean conical flasks thoroughly.

2. Weigh 5 g of LB broth using a balance, pour it into a conical flask, and add approximately 180 ml of deionized water.

3. Shake the conical flask to dissolve the LB broth completely, then add deionized water to bring the total volume to 200 ml (volume make-up).

4. Weigh 4 g of agar powder using a balance and add it to the conical flask.

5. Seal the mouth of the conical flask with parafilm.

6. Sterilization

   • Preliminary preparation: Ensure there is a sufficient amount of water in the autoclave.

   • Sterilize at 121°C for 20 minutes.

7. Plate Pouring

   1. Disinfect the conical flask and Petri dishes with alcohol, place them in a clean bench, and expose to ultraviolet (UV) light for 15-20 minutes.

   2. After the LB solid medium cools down to 50-60°C, add 100 μl of Amp (100 mg/ml), and gently shake to mix well, avoiding bubble formation.

   3. Open the disposable Petri dishes and quickly pour approximately 20 ml of the medium into each Petri dish.

   4. Leave the lids of the Petri dishes slightly open to allow the medium to solidify for 30 minutes.

   5. After solidification, collect the Petri dishes, seal them with parafilm, and store them in a refrigerator at 4°C.

2. Strain Expansion Culture

1. Disinfect the conical flask and Petri dishes with alcohol, place them in a clean bench, and expose to UV light for 15-20 minutes.

2. Pipette 20 mL of LB medium and add 20 μL of ampicillin antibiotic.

3. Add 400 μl of two types of seed cultures (each containing the pET-22b(+) plasmid).

4. Place the flask in a shaker and incubate for 16-20 hours (37°C, 220 rpm).

July 8

1. Plasmid Extraction

Extraction of the pET-22b(+) plasmid

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

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. Let it stand 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 place the centrifuge tube in a 55°C water bath for approximately 10 minutes to allow complete evaporation of alcohol.

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

2. PCR

Amplify AgaE and Inulinase gene fragments separately using high-fidelity enzymes via PCR.

PCR Reaction Program

3. Agarose Gel Electrophoresis - Gel Extraction

Perform electrophoresis on the PCR products followed by gel extraction.

Electrophoresis

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

2. Weigh 0.75 g of agarose and add it to a conical flask.

3. Pour 1 ml of 50X TAE Buffer and 49 ml of deionized water into a graduated cylinder for dilution.

4. Pour the diluted solution into the conical flask.

5. Place the conical flask in a microwave oven and heat until the agarose is completely dissolved.

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

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

8. Carefully pull out the comb vertically upwards, and place the gel into the electrophoresis tank.

9. Use a pipette to aspirate the aforementioned PCR products and load them into the sample wells.

10. Turn on the power supply and perform electrophoresis at 180 V.

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

12. Capture images of the gel.

Gel Extraction

1. Cut out the target bands from the gel separately.

2. Transfer each gel piece into a 2 ml EP tube.

3. Add Buffer B2 to each gel sample (just enough to submerge the gel block).

4. Place the tube in a 50°C water bath until the gel is completely dissolved (approximately 5 minutes).

5. Transfer the gel-dissolved solution onto 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 place the centrifuge tube in a 50°C water bath for approximately 10 minutes to allow complete evaporation of alcohol.

10. Add 30 μl of ddH₂O to the center of the membrane in the adsorption column, let it stand at room temperature for 1 minute, then centrifuge for 1 minute. Store the DNA solution in the tube.

July 9

4. Double Enzyme Digestion

Perform double enzyme digestion on the gel-extracted products and plasmids separately, followed by gel extraction.

1. PCR Products (AgaE or Inulinase)

2. Plasmid DNA (pET-22b)

Flick the tube wall to mix well. Heat in a 37°C water bath for 1 hour.

3. Electrophoresis

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

2. Weigh 0.75 g of agarose and add it to a conical flask.

3. Pour 1 ml of 50X TAE Buffer and 49 ml of deionized water into a graduated cylinder for dilution.

4. Pour the diluted solution into the conical flask.

5. Place the conical flask in a microwave oven and heat until the agarose is completely dissolved.

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

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

8. Carefully pull out the comb vertically upwards, and place the gel into the electrophoresis tank.

9. Use a pipette to aspirate the aforementioned PCR products and plasmid DNA, and load them into the sample wells.

10. Turn on the power supply and perform electrophoresis at 180 V.

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

12. Capture images of the gel.

4. Gel Extraction

1. Cut out the target fragments (AgaE or Inulinase) and pET-22b from the gel.

2. Add 500 μl of Buffer B2 to each of them separately.

3. Place the tubes in a 50°C water bath until the gel is completely dissolved (approximately 5 minutes).

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

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

6. Repeat Step 5.

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

8. Place the adsorption column into a clean 1.5 ml centrifuge tube, then place the centrifuge tube in a 50°C water bath for approximately 10 minutes to allow complete evaporation of alcohol.

9. Add 30 μl of ddH₂O to the center of the membrane in the adsorption column, let it stand at room temperature for 1 minute, then centrifuge for 1 minute. Store the DNA solution in the tube.

July 10

5. Ligation and Transformation

Ligate the digested target fragments with the plasmid.

Transformation (Competent E. coli TOP10)

1. Take the competent cells out of the -80°C refrigerator and thaw them on ice.

2. Divide the competent cells into two equal groups, with approximately 50 μl of competent cells in each group.

3. Add 5 μl of the ligation product to each group respectively and pipette gently to mix well.

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 incubate for 1 hour (37°C, 220 rpm).

8. Centrifuge at 4000 rpm for 2 minutes, discard the excess supernatant, and leave approximately 200 μl of the supernatant remaining.

9. Resuspend the bacterial cells.

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

11. Invert the Petri dish and place it in a 37°C constant-temperature incubator for 12-16 hours of cultivation.

July 11

6. Colony Verification

Picking Single Colonies

1. Turn on the clean bench in advance, and place LB medium, pipette tips, and 10 μl/1000 μl pipettes inside.

2. Spray gloves with alcohol, then reach into the clean bench and light an alcohol lamp.

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

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

5. Use a sterilized pipette tip to pick a single colony from the solid medium.

6. Place the 2 ml EP tube in a shaker and incubate for 2-4 hours until the sample becomes turbid (37°C, 220 rpm).

PCR Verification

1. Prepare the conventional PCR reaction system by adding ddH₂O, forward primer, reverse primer, and Tap-Plus PCR Forest Mix into the PCR tube respectively.

2. Turn on the clean bench, place the bacterial solution inside, and add it to the PCR tube.

3. Place the PCR tube in a palm centrifuge for centrifugation.

4. Set the PCR instrument according to the conventional PCR reaction program and place the PCR tube inside.

5. Place the remaining bacterial solution in a shaker for continued cultivation.

PCR Reaction System

PCR Reaction Program

Electrophoresis

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

2. Weigh 0.6 g of regular agarose G-10 and add it to a conical flask.

3. Pour 1.2 ml of 50X TAE Buffer into a graduated cylinder and make up the volume to 60 ml.

4. Pour the diluted solution into the conical flask.

5. Place the conical flask in a microwave oven and heat until the agarose is completely dissolved.

6. After cooling to approximately 50°C, add 3.5 μl of 4S Green Plus Nucleic Acid Stain, and gently shake to mix well, avoiding bubble formation.

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

8. Carefully pull out the comb vertically upwards, and place the gel into the electrophoresis tank.

9. Use a pipette to aspirate 8 μl of the bacterial solution and load it into the sample well.

10. Turn on the power supply and perform electrophoresis at 180 V.

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

12. Capture images of the gel.

Expansion Culture of E. coli (E. coli TOP10 AgaE_pET22b/E. coli TOP10 Inulinase_pET22b)

1. Select the bacteria verified to be correct as the seed culture.

2. Inoculate into 10 ml of fresh LB liquid medium (Amp 100 μg/ml) at a 2% inoculation volume.

3. Incubate in a shaker for 12-16 hours (37°C, 220 rpm).

Second Week

July 14

7. Bacterial Shaking

Cultivate 15 mL of E. coli TOP10 AgaE_pET22b and 15 mL of E. coli TOP10 Inulinase_pET22b separately (37°C, 220 rpm).

July 15

8. Plasmid Extraction

Extract the constructed AgaE_pET22b and Inulinase_pET22b plasmids respectively according to the previously described method. Take a portion of the plasmids for sequencing to prevent mutations during the construction process.

July 16

9. Transformation

Transform the plasmids into competent cells (Rosetta)

1. Take the competent cells out of the -80°C refrigerator and thaw them on ice.

2. Divide the competent cells into four equal groups, with approximately 25 μl of competent cells in each group.

3. Add 1.5 μl of plasmids to each group respectively (two groups for AgaE_pET22b plasmid and two groups for Inulinase_pET22b plasmid), and pipette gently to mix well.

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 incubate for 1 hour (37°C, 220 rpm).

8. Pre-place four solid media in the incubator for 20 minutes of preheating and turn on the clean bench in advance.

9. Centrifuge at 4000 rpm for 2 minutes, discard the excess supernatant, and leave approximately 200 μl of the supernatant remaining.

10. Resuspend the bacterial cells.

11. Evenly spread 200 μl of the bacterial solution onto the LB solid medium using the spreading method.

12. Invert the Petri dish and place it in a 37°C constant-temperature incubator for 12-16 hours of cultivation.

July 17

10. Colony Verification

Pick single colonies and perform PCR verification.

July 18

11. Bacterial Shaking

Perform expansion culture of the verified correct E. coli Rosetta AgaE and E. coli Rosetta Inulinase separately (5 mL each).

Third Week

July 21

Activate the verified correct E. coli Rosetta AgaE and E. coli Rosetta Inulinase from the previous week separately (1 mL each).

July 22

12. Optimization of Induced Expression

Optimize the expression of E. coli Rosetta AgaE and E. coli Rosetta Inulinase under different IPTG concentrations and temperatures.

Expansion Culture of Strains (Rosetta)

1. Pipette several groups of 1000 μl LB medium respectively.

2. Add 1 μl of ampicillin antibiotic to each group in sequence.

3. Add 20 μl of the activated seed culture (E. coli Rosetta AgaE or 15 mL of E. coli Rosetta Inulinase) to each group respectively.

4. Place the samples in a shaker and incubate for 2-3 hours (37°C, 220 rpm).

5. Add IPTG inducers of different concentrations (0.05, 0.1, 0.5, 1 mM) to each group respectively.

6. Place the groups in shakers at different temperatures for 16-20 hours of cultivation (220 rpm, 20°C/37°C).

July 23

13. SDS-PAGE

Verify using SDS-PAGE.

Bacterial Harvesting

1. Take the bacterial cultures out of the shaker.

2. Place them in a centrifuge (8000 × g, 5 minutes, 4°C).

3. Discard the supernatant, add 500 μl of PB buffer to each sample, and resuspend the bacterial cells (AgaE: pH 7.0; Inulinase: pH 5.5).

4. Place in a centrifuge (8000 × g, 5 minutes, 4°C) and discard the supernatant.

5. Repeat Steps 3 and 4.

6. Add 150 μl of buffer to each sample, resuspend, and store in a refrigerator at 4°C.

Protein Gel Preparation

1. Clean the glass plates.

2. Assemble the gel-making mold.

3. Add water to test for leaks, then pour out the water.

4. Prepare 4 ml of 10% lower gel for each sample according to the table below:

1. Add 3 ml of the SDS-PAGE lower gel and add ultrapure water for liquid sealing.

2. After the lower gel solidifies, pour out the excess ultrapure water.

3. Prepare 2 ml of 5% upper gel for each sample according to the table below:

1. Add 1 ml of the SDS-PAGE upper gel.

2. Gently insert the comb.

3. Prepare 1 L of protein electrophoresis buffer according to the table below:

1. Add 10 μl of loading buffer and 20 μl of protein sample to a 1.5 ml EP tube, and inactivate in a metal bath at 98°C for 10 minutes.

2. Align the prepared gel with the card slot, clamp it into the vertical electrophoresis apparatus, and pour 1X protein electrophoresis buffer into the inner tank until it is completely full. Check for leaks.

3. Sample loading: Pull out the comb vertically, and add 5 μl of marker and 10 μl of protein sample into the wells.

4. Electrophoresis: Perform electrophoresis at 130 V. When the sample reaches the lower gel, increase the voltage to 150 V.

5. Coomassie Brilliant Blue Staining: Prepare the staining solution.

1. After electrophoresis, take out the protein gel, place it in a box, pour the staining solution into the box, and place it on a decolorizing shaker for 30 minutes (30 rpm).

2. Prepare the decolorizing solution according to the table below:

1. Pour out the staining solution from the box and rinse with clean water.

2. Pour the decolorizing solution into the box and place it on the decolorizing shaker.

3. Replace the decolorizing solution every 30 minutes to 1 hour until the decolorization is complete.

July 24

Expansion Culture of Strains (Rosetta)

1. Take two bottles of 200 mL LB medium.

2. Add 200 μl of ampicillin antibiotic to each bottle respectively.

3. Add 4 mL of E. coli Rosetta AgaE seed culture and 4 mL of E. coli Rosetta Inulinase seed culture to the two bottles respectively.

4. Place the bottles in a shaker and incubate for 2 hours (37°C, 220 rpm) until the OD₆₀₀ reaches approximately 0.6.

5. Add IPTG inducer to each bottle respectively (0.025 mM for AgaE; 0.5 mM for Inulinase).

6. Place the bottles in a shaker and incubate for 16-20 hours (220 rpm, 20°C).

July 25

Bacterial Harvesting

1. Take the bacterial cultures out of the 20°C shaker and pour them into 50 ml centrifuge tubes respectively.

2. Place the tubes in a centrifuge (5000 × g, 10 minutes).

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. Place the tubes in a centrifuge (5000 × g, 10 minutes) and discard the supernatant.

5. Repeat Steps 3 and 4.

6. Add 25 ml of PB buffer to each tube, resuspend, and store in a refrigerator at 4°C.

7. Harvest the bacterial cells of the control group (without inducer added) using the same method.

Bacterial Lysis

1. Take 25 ml of each bacterial solution (Inulinase) from the 4°C refrigerator and place them in an ice-water mixture.

2. Turn on the ultrasonic homogenizer, set the lysis parameters (5 seconds of sonication, 2 seconds of pause, power 195 W), and sonicate for 10 minutes until the bacterial cells are completely lysed and the solution becomes clear.

3. Place the lysate in a low-temperature refrigerated centrifuge (10000 × g, 30 minutes).

4. Pour out the supernatant, which is the soluble protein, and temporarily store it in a refrigerator at 4°C.

5. The precipitate is the inclusion body.

6. Add buffer to wash the inclusion body twice, and centrifuge (10000 × g, 5 minutes) after each wash.

7. Adjust the volume to 25 ml.

Lyse AgaE using the same method described above.

Fourth Week

July 28

Protein Gel Preparation

1. Clean the glass plates.

2. Assemble the gel-making mold.

3. Add water to test for leaks, then pour out the water.

4. Prepare 4 ml of 12% and 10% lower gels respectively according to the tables below:

1. Add 3 ml of the SDS-PAGE lower gel and add ultrapure water for liquid sealing.

2. After the lower gel solidifies, pour out the excess ultrapure water.

3. Prepare two portions of 2 ml 5% upper gel according to the table below:

1. Add 1 ml of the SDS-PAGE upper gel to each mold.

2. Gently insert the comb.

3. Prepare 1 L of protein electrophoresis buffer according to the table below:

1. Add 10 μl of loading buffer and 20 μl of protein sample (various samples prepared on July 25) to a 1.5 ml EP tube, and inactivate in a metal bath at 98°C for 10 minutes.

2. Align the prepared gel with the card slot, clamp it into the vertical electrophoresis apparatus, and pour 1X protein electrophoresis buffer into the inner tank until it is completely full. Check for leaks.

3. Sample loading: Pull out the comb vertically, and add 5 μl of marker and 10 μl of protein sample into the wells.

4. Electrophoresis: Perform electrophoresis at 130 V. When the sample reaches the lower gel, increase the voltage to 150 V.

5. Coomassie Brilliant Blue Staining: Prepare the staining solution.

1. After electrophoresis, take out the protein gel, place it in a box, pour the staining solution into the box, and place it on a decolorizing shaker for 30 minutes (30 rpm).

2. Prepare the decolorizing solution according to the table below:

1. Pour out the staining solution from the box and rinse with clean water.

2. Pour the decolorizing solution into the box and place it on the decolorizing shaker.

3. Replace the decolorizing solution every 30 minutes to 1 hour until the decolorization is complete.

July 28

Protein Purification: Protein Purification Using Ni-NTA Affinity Chromatography

For AgaE

1. Prepare imidazole solutions of different concentrations (0 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM) using 3 M imidazole solution (pH 7.0).

2. Take 2 mL of the supernatant (soluble protein) of AgaE after ultrasonic lysis and add it to a nickel column. Place the nickel column in an ice box, then place the ice box on a shaker (50 rpm) for binding for approximately 3 hours to allow sufficient binding between the target protein and the filler.

3. Pass 7 mL of imidazole solutions (0 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM) through the nickel column in sequence, and collect the first 2 mL of effluent for each concentration in separate tubes.

4. Take several 2 mL EP tubes, add 20 μL of 2.5×SDS-PAGE loading buffer to each tube, and then add 30 μL of the effluent obtained in the above step to each corresponding tube.

5. Incubate the tubes in a metal bath at 96°C for 10 minutes.

6. Perform electrophoresis using a 10% protein gel. After electrophoresis, disassemble the gel, stain it with the staining solution, and after the staining effect is achieved, decolorize it with the decolorizing solution.

For Inulinase

1. Prepare imidazole solutions of different concentrations (0 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM) using 3 M imidazole solution (pH 5.5).

2. Take 2 mL of the supernatant (soluble protein) of Inulinase after ultrasonic lysis and add it to a nickel column. Place the nickel column in an ice box, then place the ice box on a shaker (50 rpm) for binding for approximately 3 hours to allow sufficient binding between the target protein and the filler.

3. Pass 7 mL of imidazole solutions (0 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM) through the nickel column in sequence, and collect the first 2 mL of effluent for each concentration in separate tubes.

4. Take several 2 mL EP tubes, add 20 μL of 2.5×SDS-PAGE loading buffer to each tube, and then add 30 μL of the effluent obtained in the above step to each corresponding tube.

5. Incubate the tubes in a metal bath at 96°C for 10 minutes.

6. Perform electrophoresis using a 12% protein gel. After electrophoresis, disassemble the gel, stain it with the staining solution, and after the staining effect is achieved, decolorize it with the decolorizing solution.

July 30

14. Enzyme Activity Verification

1) Standard Curve Drawing

Prepare fructose standard solutions of different concentrations (0, 0.2, 0.4, 0.6, 0.8, and 1 mg/mL). Pipette 200 μL of each standard solution into a 2 ml centrifuge tube, and add an equal volume of DNS solution to mix. Place the mixed samples in a boiling water bath for 5 minutes of reaction, then cool them in an ice water bath to terminate the reaction. Add 1 ml of deionized water to each sample for dilution, 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 Determination

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

AgaE Enzyme Activity Verification

1. Prepare a 2% agarose solution.

2. Add 2 ml of the agarose solution and 3 ml of AgaE soluble protein (the control group uses inactivated protein samples).

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

4. Pipette 200 μl of the reaction solution and add 200 μl of DNS reagent.

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

6. After cooling, add 1 ml of water.

7. Add the samples to a 96-well plate, with 0.2 ml added to each well.

8. Use a microplate reader to measure the OD₅₄₀ value.

Inulinase Enzyme Activity Verification

1. Prepare a 2% inulin solution.

2. Add 2 ml of the inulin solution and 3 ml of Inulinase soluble protein (the control group uses inactivated protein samples).

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

4. Pipette 200 μl of the reaction solution and add 200 μl of DNS reagent.

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

6. After cooling, add 1 ml of water.

7. Add the samples to a 96-well plate, with 0.2 ml added to each well.

8. Use a microplate reader to measure the OD₅₄₀ value.

15. Probiotic Activity Analysis

Verify the prebiotic effects of AgaE and Inulinase.

Strain Activation and Seed Culture Preparation

Isolate single colonies from the Lactobacillus acidophilus preparation using the spreading method. Pick typical single colonies and inoculate them into liquid MRS medium, then culture anaerobically at 37°C for 24 hours to obtain the seed culture in the logarithmic growth phase.

Experimental Grouping and Cultivation

Take fresh MRS medium and perform treatments according to the following grouping settings (three replicates are set for each 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 culture at 37°C.

Absorbance Detection

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

16. Contribution Experiment

Strain Activation

1. Pipette 1000 μl of LB medium.

2. Add 1 μl of Amp antibiotic.

3. Add 20 μl of E. coli Rosetta AgaA-pET-32a seed culture.

4. Place the bacterial solution in a shaker and incubate for 12 hours (37°C, 220 rpm).

July 31

17. 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 standard substances respectively, place them in a 2 ml EP tube, add 1 ml of ultrapure water, and fully dissolve to prepare a mixed standard solution with a concentration of 10 mM each.

2. Centrifuge the standard solution, the aforementioned experimental groups, and the control group at 12000 × g for 10 minutes.

3. Filter all the above samples through a 0.22 μm filter membrane.

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

HPLC Detection of Inulinase Products

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

2. Centrifuge the standard solution, the aforementioned experimental groups, and the control group at 12000 × g for 10 minutes.

3. Filter all the above samples through a 0.22 μm filter membrane.

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

18. Contribution Experiment

Expansion Culture of Strains (Rosetta)

1. Take 100 ml of LB medium.

2. Add 100 μl of ampicillin antibiotic.

3. Add 2000 μl of E. coli Rosetta AgaA-pET-32a seed culture.

4. Place the bacterial solution in a shaker and incubate for 2 hours (37°C, 220 rpm).

5. Add IPTG inducer (0.5 mM) respectively.

6. Place in a shaker and incubate for 16-20 hours (220 rpm, 16°C).

August 1

Bacterial Harvesting

Take out the induced E. coli Rosetta AgaA-pET-32a bacterial solution, pour it into two test tubes (A and B), balance them, place them in a centrifuge, centrifuge at 5000 rpm for 10 minutes, discard the supernatant, and obtain the precipitate (bacterial cells).

Bacterial Washing

Dilute 1 M Tris-HCl solution to 20 mM, adjust the pH from 8 to 7 by adding concentrated hydrochloric acid. Add the buffer to the bacterial cells, resuspend, centrifuge again to separate the bacterial cells, and repeat the above steps twice.

Lyse the bacterial solution using an ultrasonic homogenizer. After lysis, separate the soluble protein from the inclusion bodies using a centrifuge.

Inclusion Body Washing

Place the separated inclusion bodies in a centrifuge, centrifuge at 5000 rpm for 5 minutes, take them out, remove the supernatant, add Tris-HCl solution (pH 7.0), resuspend, place in the centrifuge, discard the supernatant, and obtain the precipitate. Repeat the operation twice. Add Tris-HCl to the precipitate again and resuspend.

Fifth Week

August 4

19. Contribution Experiment

SDS-PAGE Verification

Verify the protein samples prepared last week using SDS-PAGE.

August 5

Effect of Different Metal Ions on AgaA Enzyme Activity

Prepare 5 mL of solutions of NaCl, KCl, CuSO₄, CaCl₂, MgCl₂, FeSO₄, and FeCl₃ with a concentration of 50 mM each, using 20 mM Tris-HCl buffer (pH 7.0) as the solvent.

Reaction System Setup

Add 50 μL of 2% agarose solution (substrate) to each of multiple groups of 2 mL EP tubes, then add 50 μL of each of the aforementioned metal ion solutions to the corresponding tubes; the control group adds 50 μL of Tris-HCl buffer (without metal ions). Add 400 μL of AgaA enzyme solution to each tube to initiate the reaction, and incubate at a constant temperature of 35°C for 30 minutes.

Reaction Termination and Detection

Place all samples in a 95°C water bath and heat for 10 minutes to inactivate the enzyme activity. Pipette 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 add 1 mL of ddH₂O to each tube after cooling. Use a microplate reader to measure the absorbance at a wavelength of 540 nm. Evaluate the effect of different metal ions on AgaA enzyme activity by comparing the absorbance differences among the groups. Calculate the relative enzyme activity of each experimental group with the enzyme activity of the control group set as 100%.

August 6

Effect of Different Concentrations of Mg²⁺ on AgaA Enzyme Activity

To investigate the effect of different concentrations of Mg²⁺ on AgaA enzyme activity, the experiment was conducted as follows:

First, 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 50 μL of MgCl₂ solutions of different concentrations mentioned above to the corresponding tubes; the control group adds 50 μL of Tris-HCl buffer (without Mg²⁺). Add 400 μL of AgaA enzyme solution to each tube to initiate the reaction, and incubate at a constant temperature of 35°C for 30 minutes.

Reaction Termination and Detection

Place all samples in a 95°C water bath and heat for 10 minutes to inactivate the enzyme activity. Pipette 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 add 1 mL of ddH₂O to each tube after cooling. Use a microplate reader to measure the absorbance at a wavelength of 540 nm. Evaluate the effect of different concentrations of Mg²⁺ on AgaA enzyme activity by comparing the absorbance differences among the groups. Calculate the relative enzyme activity of each experimental group with the enzyme activity of the control group set as 100%.

August 7

20. Model Experiment

Expansion Culture of Strains (E. coli Rosetta AgaE-pET-22b)

1. Take 100 ml of LB medium.

2. Add 100 μl of ampicillin antibiotic.

3. Add 2000 μl of seed culture.

4. Place the bacterial solution in a shaker and incubate for 2 hours (37°C, 220 rpm).

5. Add IPTG inducer (0.025 mM) respectively.

6. Place in a shaker and incubate for 16-20 hours (220 rpm, 20°C).

August 8

Bacterial Harvesting

Take out the induced E. coli Rosetta AgaE-pET-22b bacterial solution, pour it into a 50 mL centrifuge tube, balance it, place it in a centrifuge, centrifuge at 5000 rpm for 10 minutes, discard the supernatant, and obtain the precipitate (bacterial cells).

Bacterial Washing

Add 20 mL of PB (pH 7.0) buffer to the bacterial cells, resuspend, centrifuge again to separate the bacterial cells, and repeat the above steps twice.

Lyse the bacterial solution using an ultrasonic homogenizer. After lysis, separate the soluble protein from the inclusion bodies using a centrifuge, and store them in a -20°C refrigerator.

Sixth Week

August 11

21. Model Experiment

Substrate Preparation

Prepare agarose substrate solutions with concentrations of 0.5%, 1%, 2%, 2.5%, 3%, and 4% respectively: weigh 0.05 g, 0.1 g, 0.2 g, 0.25 g, 0.3 g, and 0.4 g of agarose respectively, place them in 15 mL centrifuge tubes, add 10 mL of PB (pH 7.0) buffer to each tube, place the tubes in a boiling water bath for dissolution, shake while heating until the agarose is completely dissolved (i.e., the solution is colorless and transparent), and place the tubes in a 60°C oven for later use.

AgaE Enzyme Activity Determination

Fix the concentration of AgaE, pipette 100 μL of substrate solutions with different concentrations (0.5%-4%) into 2 mL EP tubes in sequence, add 800 μL of AgaE solution to each tube respectively, and add seven groups of 100 μL NaCl solutions with different concentrations (0-150 mM gradient) to the corresponding tubes according to the above system. Incubate at 35°C for 3 minutes. Inactivate the enzyme in a boiling water bath for 5 minutes.

August 12

Pipette 200 μL of the reaction solution, mix it with an equal volume of DNS reagent, heat in a boiling water bath for 5 minutes for color development, then cool in an ice bath. Add 1 mL of deionized water for dilution. Use a microplate reader to measure the absorbance at a wavelength of 540 nm. Calculate the reducing sugar production using the standard curve and process the data.

August 13 - August 15

Determine the enzymatic reaction kinetic parameters of AgaE under the action of 43.7 mM Na⁺ using the above method.