Experiment

Medium Preparation

LB Liquid Medium

1. Weigh 2.5 g of LB broth using a balance, place it into an Erlenmeyer flask, and add approximately 80 ml of deionized water.

2. Swirl the flask to dissolve the LB broth, then add deionized water to make up the volume to 100 ml.

3. Seal the flask with parafilm.

4. Sterilize using an autoclave (conditions: 121℃, 20 min).

LB Solid Medium

1. Weigh 2.5 g of LB broth using a balance, place it into an Erlenmeyer flask, and add approximately 80 ml of deionized water.
2. Swirl the flask to dissolve the LB broth, then add deionized water to make up the volume to 100 ml.
3. Weigh 1.5 g of agar powder and add it to the flask.
4. Seal the flask with parafilm.
5. Sterilize using an autoclave (conditions: 121℃, 20 min).
6. Disinfect the flask and petri dishes with 75% ethanol and place them in a biosafety cabinet. Expose them to UV light for 15-20 min.
7. After the LB solid medium has cooled to 50-60℃, add 100 µl of Amp (100 mg/ml) and gently swirl to mix, avoiding bubbles.
8. Quickly pour about 20 ml of the medium into each petri dish.
9. Leave the petri dishes uncovered to solidify for 30 minutes.
10. After solidification, seal the dishes with parafilm, label them, and store them in a 4℃ refrigerator.

Plasmid Extraction

1. Take 5 ml of overnight culture, centrifuge at 8000 xg for 2 min, and discard the supernatant to collect the cells.
2. Add 250 µl of Buffer P1 to the pellet and resuspend the cells thoroughly.
3. Add 250 µl of Buffer P2, gently invert the tube 10 times to mix, and incubate at room temperature for 2-4 minutes.
4. Add 350 µl of Buffer P3, gently invert the tube 10 times.
5. Centrifuge at 12000 xg for 10 min. Transfer the supernatant to the adsorption column and centrifuge at 8000 xg for 30 s. Discard the flow-through.
6. Add 500 µl of Wash Solution, centrifuge at 9000 xg for 30 s, and discard the flow-through.
7. Repeat step ⑥.
8. Centrifuge the empty adsorption column at 9000 xg for 1 minute.
9. Place the adsorption column in a clean 1.5 ml centrifuge tube and incubate in a 55℃ water bath for about 10 minutes to evaporate the ethanol completely.
10. Add 30 µl of Elution Buffer to the center of the membrane in the adsorption column, incubate at room temperature for 1 minute, and centrifuge for 1 minute. Store the DNA solution in the tube.

PCR

1. According to the PCR reaction system, sequentially add ddH2O, forward primer, reverse primer, template, and 2x Super Pfx Master Mix into a PCR tube.

PCR Reaction System

Component	Volume (µl)
2x Super Pfx Master Mix	10
Forward Primer (10 µM)	1
Reverse Primer (10 µM)	1
Template	1
ddH2O	7

2. Centrifuge in a mini centrifuge.
3. Set up the PCR instrument according to the PCR reaction program and place the PCR tube inside.

PCR Reaction Program

Pre-denaturation	Amplification				Final Extension
	Denaturation	Annealing	Extension	Cycle Count
98℃, 30 s	98℃, 30 s	55℃, 10 s	72℃, 30 s	35	72℃, 5 min

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

Agarose Gel Electrophoresis

1. Prepare the gel mold by placing the gel tray in the casting tray and inserting the comb 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 and add 49 ml of deionized water to dilute it to 1X TAE Buffer.
4. Pour the diluted solution into the Erlenmeyer flask.
5. Heat the flask in a microwave oven until the agarose is fully dissolved.
6. Cool to around 50℃ and add 3 µl of 4S Green Plus Nucleic Acid Stain, gently swirl to mix, avoiding bubbles.
7. Pour the solution into the casting tray and allow it to cool and solidify at room temperature.
8. Carefully remove the comb vertically and place the gel in the electrophoresis chamber with the wells facing the negative electrode.
9. Using a pipette, load 7 µl of PCR product and 1 µl of plasmid DNA into the wells.
10. Turn on the power and run the electrophoresis at 160V.
11. After electrophoresis, remove the gel and visualize the results using a gel imaging system.

Gel Extraction

1. Select the correct target band and cut the gel, measuring the gel weight (0.912 g).
2. Divide the gel into two equal parts, each weighing 0.456 g.
3. Add 1.3 ml of Buffer B2 to each gel piece (three times the weight of the gel) and incubate in a 50℃ water bath until fully dissolved (about 5 minutes).
4. Transfer the dissolved gel solution onto an adsorption column and centrifuge at 8000 xg for 30 s, discarding the flow-through.
5. Add 500 µl of Wash Solution, centrifuge at 9000 xg for 30 s, and discard the flow-through.
6. Repeat step ⑤.
7. Centrifuge the empty adsorption column at 9000 xg for 1 minute.
8. Place the adsorption column in a clean 1.5 ml centrifuge tube and incubate in a 50℃ water bath for about 10 minutes to completely evaporate the ethanol.
9. Add 30 µl of ddH₂O to the center of the membrane in the adsorption column, incubate at room temperature for 1 minute, and centrifuge for 1 minute. Store the DNA solution in the tube.

Double Digestion

1. Prepare the double digestion reaction system according to the following systems:
   1. PCR Product (30 µl) (AgaA (70.2 ng/µl)):

Reagent	Volume (µl)
BamHI	1
EcoRI	1
Substrate DNA (AgaA template)	7
10X FuniCut tm Colour Buffer	3
ddH2O	18

2. Plasmid DNA (20 µl) (pET-32a(+) (98.5 ng/µl)):

Reagent	Volume (µl)
BamHI	1
EcoRI	1
Plasmid	10
10X FuniCut tm Colour Buffer	2
ddH2O	6

2. Incubate in a 37℃ water bath for 1 hour.
3. Perform agarose gel electrophoresis on the digestion products.
4. Select the correct target band and perform gel extraction.

Ligation and Transformation

Ligation

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

Component	Volume (µl)
Linearized Vector	6.5
Insert DNA Fragment	7
10X T4 Ligase Buffer	2
T4 DNA Ligase	1
ddH2O	3.5

2. Place the ligation mixture in a PCR instrument at 22℃ for 3 hours.
3. Heat inactivate the ligation mixture at 65℃ for 10 minutes.

Transformation

1. Take the competent cells from a -80℃ freezer and thaw them on ice.
2. Divide the competent cells into two groups, with approximately 50 µl of competent cells in each group.
3. Add 5 µl of the ligation product and mix by gently pipetting.
4. Incubate on ice for 30 minutes.
5. Heat shock at 42℃ for 90 s, then place on ice for 5 minutes.
6. Add 1 ml of LB medium (without antibiotics).
7. Incubate in a shaker at 37℃, 220 rpm for 1 hour.
8. Centrifuge at 4000 rpm for 2 minutes, discard the excess supernatant, leaving about 200 µl.
9. Resuspend the bacterial cells.
10. Plate 200 µl of the bacterial suspension onto LB solid medium using the spread plate method.
11. Invert the plates and incubate at 37℃ in an incubator for 12-16 hours.

Colony Verification

1. Turn on the biosafety cabinet and place the necessary experimental consumables inside. Sterilize with UV light for 20 minutes.
2. Pipette 1 ml of LB liquid medium into a 2 ml centrifuge tube.
3. Pipette 1 µl of ampicillin into the 2 ml centrifuge tube.
4. Pick a single colony from the solid medium using a sterilized pipette tip.
5. Place the 2 ml centrifuge tube into a shaker and culture for 3 hours (37℃, 220 rpm).
6. According to the conventional PCR reaction system, sequentially add ddH2O, forward primer, reverse primer, Tap-Plus PCR Forest Mix, into a PCR tube.

Conventional PCR Reaction System:

Component	Volume (µl)
Tap-Plus PCR Forest Mix (2x)	10
Forward Primer (10 µM)	1
Reverse Primer (10 µM)	1
Bacterial Suspension	1
ddH2O	7

7. Place the bacterial suspension into the PCR tube inside the biosafety cabinet.
8. Centrifuge in a mini centrifuge.
9. Set up the PCR instrument according to the conventional PCR reaction program and place the PCR tube inside.
10. Place the remaining bacterial suspension back into the shaker for continued culture.

1. Conventional PCR Reaction Program:

Pre-denaturation	Amplification				Final Extension
	Denaturation	Annealing	Extension	Cycle Count
94℃, 10 min	94℃, 30 s	55℃, 30 s	72℃, 70 s	30	72℃, 5 min

Induced Expression of E.coli Rosetta AgaA_pET-32a(+)

Strain Expansion (Rosetta)

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 AgaA_pET-32a(+).
3. Place E. coli Rosetta AgaA_pET-32a(+) into a shaker and culture for 12 hours (37℃, 220 rpm).
4. Take 50 ml of LB liquid medium and add 50 µl of Amp (100 mg/ml).
5. Add 1 ml of the seed culture of E. coli Rosetta AgaA_pET-32a(+).
6. Place in a shaker (37℃, 220 rpm) and culture until OD600 reaches approximately 0.5.
7. Add 25 µl of 1 M IPTG inducer (final concentration: 0.5 mM).
8. Place in a shaker and culture for 16-20 hours (16℃, 220 rpm).

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

Cell Harvesting

1. Remove the culture from the shaker.
2. Transfer to a 50 ml centrifuge tube and centrifuge (5000 xg, 10 min, 4℃).
3. Discard the supernatant and resuspend the cells in 30 ml of Tris-HCl (20 mM, pH 7.0).
4. Centrifuge again (5000 xg, 10 min, 4℃) and discard the supernatant.
5. Repeat steps ③ and ④.
6. Resuspend in 7 ml of Tris-HCl and store at 4℃.

Cell Lysis

1. Take 7 ml of the cell suspension from the 4℃ refrigerator and place it in an ice-water mixture.
2. Turn on the ultrasonic cell disruptor, set the lysis conditions (1 s on, 2 s off, power 195W), and sonicate for 8 minutes until the cells are completely lysed.
3. Centrifuge in a refrigerated centrifuge (10000 xg, 30 min).
4. The supernatant is the soluble protein, which is temporarily stored at 4℃, while the pellet is the inclusion body.
5. Add buffer and wash the inclusion body twice according to the cell harvesting method.
6. Resuspend the inclusion body in 3 ml of buffer.

SDS-PAGE

Gel Preparation

1. Clean the glass plates and assemble the gel casting mold. Test for leaks by adding water, then pour out the water.
2. Prepare a 10% separating gel according to the following table:

Component	Volume (ml)
Distilled Water	1.63
30% Acr-Bis (29:1)	1.33
Gel Buffer A	1
10% APS	0.04
TEMED	0.0024

3. Pour 3 ml of the SDS-PAGE separating gel, then add ultrapure water for sealing.
4. After the gel solidifies, pour off the ultrapure water on top.
5. Prepare a 5% stacking gel according to the following table:

Component	Volume (ml)
Distilled Water	0.67
30% Acr-Bis (29:1)	0.33
Gel Buffer B	1
10% APS	0.02
TEMED	0.002

6. Add the prepared stacking gel to the gel casting mold and gently insert the comb.

SDS-PAGE Electrophoresis

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

Component	Weight (g)
Glycine	18.8
Tris	3
SDS	1

2. Add 10 µl of protein loading buffer and 20 µl of protein sample to a 1.5 ml centrifuge tube, incubate in a metal bath at 98℃ for 10 minutes, then centrifuge at 8000 xg for 5 minutes.
3. Align the prepared gel with the slots and clamp it into the vertical electrophoresis apparatus. Fill the inner chamber with 1X protein electrophoresis buffer until completely full, and check for leaks.
4. Loading: Carefully remove the comb, and load 5 µl of marker and 10 µl of protein sample into the wells.
5. Electrophoresis: Run the electrophoresis at 130V, and when the samples reach the separating gel, increase the voltage to 150V.
6. After electrophoresis, carefully disassemble the gel and place it in a container. Add staining solution and place it on a shaker (30 rpm) until the gel is fully stained. The staining solution is prepared as follows:

Component	Volume
Acetic acid	5 ml
Methanol	22.5 ml
Coomassie Brilliant Blue R-250	0.05 g
Water	22.5 ml

7. Pour off the staining solution and wash the gel with water.
8. Pour the destaining solution into the container and place it on a shaker for destaining. The destaining solution is prepared as follows:

Component	Volume
Acetic acid	5 ml
Methanol	22.5 ml
Water	22.5 ml

9. Replace the destaining solution every 30 minutes to 1 hour until the gel is completely destained.

Protein Purification (Ni-NTA Affinity Chromatography)

1. Take 2 ml of the supernatant containing the soluble AgaA protein after sonication and add it to the nickel column.
2. Place the nickel column in an ice box and then on a shaker (50 rpm) for approximately 3 hours to allow sufficient binding of the target protein to the resin.
3. Sequentially elute the column with 7 ml of imidazole solutions at concentrations of 0 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, and 300 mM, collecting the first 2 ml of eluate in each case.
4. Take several 2 ml centrifuge tubes, add 20 µl of 2.5×SDS-PAGE loading buffer, and then add 30 µl of the eluates obtained from the previous step.
5. Incubate in a 96℃ metal bath for 10 minutes.
6. Perform SDS-PAGE electrophoresis. After electrophoresis, disassemble the gel, stain with the staining solution, and once staining is complete, destain using the destaining solution.

Enzyme Activity Verification

Standard Curve Preparation

Prepare fructose standard solutions with concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1 mg/mL, and pipette 200 µL of each solution into 2 ml centrifuge tubes. Add an equal volume of DNS solution to each tube, mix, and incubate the samples in a boiling water bath for 5 minutes. Cool the reaction in an ice bath to stop the reaction. Dilute each sample with 1 ml of deionized water and measure the absorbance at 540 nm using a microplate reader. The linear regression equation and correlation coefficient are obtained by plotting the fructose concentration on the x-axis and the OD value on the y-axis, thus creating a standard curve for reducing sugars.

Enzyme Activity Assay

1. Add 100 µl of agarose solution.
2. Add 100 µl of AgaA bacterial suspension.
3. Incubate in a 55℃ water bath for 30 minutes.
4. Add 200 µl of DNS reagent.
5. Incubate in a metal bath at 98℃ for 5 minutes.
6. Observe the color change to confirm the production of neoagaro-oligosaccharides, thereby confirming the activity of the agarase AgaA.

Product Analysis

1. Weigh 0.00322 g of NA2, 0.0063 g of NA4, and 0.00936 g of NA6 standards, place them in a 2 ml centrifuge tube, and add 1 ml of ultrapure water to dissolve completely, preparing a mixed standard solution with a concentration of 10 mM for each component.
2. Take 1 ml of 2% agarose solution and add 1 ml of AgaA enzyme (the control group uses inactivated enzyme).
3. Incubate in a 55℃ water bath for 3 hours.
4. Inactivate in a 95℃ water bath for 10 minutes.
5. Transfer 100 µl of the solution from the control and experimental groups into two separate 2 ml centrifuge tubes.
6. Centrifuge the standard solution, experimental, and control group samples at 12000 xg for 10 minutes.
7. Filter the samples using a 0.22 µm membrane filter.
8. Send the processed samples to the laboratory platform for HPLC analysis.

Antioxidant Activity Analysis

This experiment uses the ABTS assay kit to detect the antioxidant activity of the NAOS produced by AgaA hydrolysis. Upon oxidation, ABTS generates a stable blue-green cation radical ABTS+, which is soluble in aqueous or acidic ethanol solutions and has maximum absorbance at 734 nm. When the test substance is added to the ABTS+ solution, the antioxidant components in the substance react with ABTS+, resulting in a decrease in color intensity. The change in absorbance at 734 nm is measured to assess the antioxidant capacity of the substance.

1. Take 1 ml of 2% agarose solution and add 1 ml of AgaA enzyme (the control group uses inactivated enzyme).
2. Incubate in a 55℃ water bath for 3 hours.
3. Inactivate in a 95℃ water bath for 10 minutes.
4. Transfer 50 µl of the solution from the control and experimental groups into two separate 2 ml centrifuge tubes.
5. Add 950 µl of antioxidant reagent.
6. Allow the mixture to stand at room temperature for 20 minutes and observe the color change.

Contribution

Weigh 0.2 g of sodium alginate and agarose and 0.5 g of CaCl₂, respectively, into 15 mL centrifuge tubes, add an appropriate amount of 20 mM of Tris HCl (pH 7.0) solution in turn, and fully dissolve (agarose solution needs to be heated at high temperature to dissolve), then set the volume to 10 mL respectively. Sodium alginate solution and calcium chloride solution can be temporarily stored at room temperature, agarose solution needs to be kept in a 55℃ water bath to prevent early solidification.

1. Antioxidant Activity Experiment: Different types of hydrogels are prepared according to the following systems: Group 1: 200 µL agarose +300 µL Tris HCl buffer Group 2: 200 µL agarose +90 µL AgaA+210 µL Tris HCl buffer Group 3: 200 µL sodium alginate +200 µL NAOS+10 µL CaCl₂+90 µL EGF-R Group 4: 200 µL sodium alginate +90 µL EGF-R+10 µL CaCl₂+200 µL Tris HCl buffer Control Group: 500 µl Tris-HCl buffer
2. Allow the mixture to stand at room temperature for 30 minutes.
3. Take 1/10th of each group.
4. Add 950 µl of antioxidant assay reagent.
5. Allow the mixture to stand at room temperature for 20 minutes.
6. Measure the antioxidant value at 734 nm wavelength.

OPA Method for Measuring AgaA Degradation

a) Preparation of Standard Solution Accurately weigh 0.0131 g of L-leucine using an electronic balance and dissolve in a centrifuge tube to make up to 10 ml (10 mM). Sequentially dilute to 0, 0.2, 0.4, 0.6, 0.8, and 1 mM.

b) Preparation of OPA Solution Dissolve 80 mg of ortho-phthalaldehyde (OPA) in 2 ml of anhydrous ethanol in a dark place. Then, dissolve 1.9068 g of sodium tetraborate and 0.1 g of SDS (sodium dodecyl sulfate) in water, and add 88 mg of DTT (dithiothreitol). Transfer the solution to a brown volumetric flask and make up the volume to 100 ml with water.

c) Measurement of Standard Solution Absorbance The specific method for measuring absorbance using the OPA method is as follows: First, set the wavelength of the microplate reader to 340 nm. Sequentially pipette 5 µl of water, 20 µl of different concentrations of L-leucine, and 150 µl of OPA solution into a 96-well plate. Start the stopwatch and measure the absorbance after 2 minutes, then plot the standard curve.

d) Measurement Method

(1) Solution Preparation: Weigh 1 g of bovine serum albumin (BSA) and 1 g of skim milk powder, dissolve in 20 mM Tris-HCl buffer (pH 7.0, 3 mM Ca²⁺), and make up the volume to 100 ml. Prepare 1% BSA solution and 1% skim milk solution, then dilute to appropriate multiples as mother solutions, and further dilute to a concentration similar to the target protein concentration (0.05 mg/ml).

(2) Absorbance Measurement: Set up the reaction groups according to the following system, with three parallel samples for each group:

1. 400 µl BSA + 80 µl buffer
2. 400 µl BSA + 80 µl proteinase K
3. 400 µl Milk + 80 µl buffer
4. 400 µl Milk + 80 µl proteinase K
5. 400 µl AgaA + 80 µl buffer
6. 400 µl AgaA + 80 µl proteinase K

Incubate at room temperature and take 25 µl of the reaction solution at 0, 5, 10, 15, 20, 25, 30, 45, and 60 minutes. Add the samples to a 96-well plate, and then add 150 µl of OPA solution to each sample well. Place the plate in the microplate reader, start the stopwatch, and read the absorbance at 340 nm after 2 minutes.