1.1 Preparation of LB Medium

Purpose: To create a functional and nutritional liquid environment for bacteria to grow. Liquid media are used for various purposes, such as the propagation of microorganisms or other tests.

Materials:

Instruments and Equipment	Sterilized Duran bottles, with caps
	Autoclave
	Electronic Balance
	Weighing Paper
	Lab Refrigerator
	Parafilm
Chemicals and Reagents	Tryptone
	Yeast Extract
	Sodium Chloride
	Agar
	Double-distilled H2O

1L of LB medium was prepared in-house with the following components:

Component	Mass
Tryptone	10 g
Yeast Extract	5 g
Sodium Chloride	10 g
Double-distilled H2O	1 L
Agar (to prepare a solid medium)	Add 1.5 g per 100 ml of ddH₂O

Procedure:

For the Liquid media:

1) Dissolve all components in distilled H₂O.

2) Autoclave at 121°C for 20 minutes.

3) Store at 4°C (pre-use equilibration to room temperature).

For the solid media:

1) Dissolve all components in distilled H₂O.

2) Autoclave at 121°C for 20 minutes.

3) Cool to 50°C, add antibiotic.

4) Pour 15–20 mL per plate in sterile petri dishes.

5) Coagulate at room temperature, wrap in parafilm, and store inverted at 4°C.

1.2 Preparation of Antibiotics

Purpose: To establish a selective growth environment to allow only target cells to survive and proliferate, while eliminating non-target cells that lack specific antibiotic resistance genes, thereby enabling the accurate selection of positive clones required for the experiment.

Materials:

Instruments and Equipment	Sterilized Duran bottles, with caps
	Sterile Centrifuge Tubes
	0.22μm Filter
	Disposable Syringe
	Electronic Balance
	Weighing Paper
	Lab Refrigerator
Chemicals and Reagents	Antibiotic Powder
	Double-distilled H2O

Recipes:

Antibiotic	Concentration	Working Concentration	Preparation
Kanamycin	100mg/mL	50 µg/mL	Dissolve 100mg in 1mL sterile H₂O
Ampicillin	50mg/mL	100 µg/mL	Dissolve 50mg in 1mL sterile H₂O

Procedure:

1) Dissolve the component in distilled H₂O.

2) Filter sterilize using a 0.22 µm PVDF syringe filter.

3) Aliquot and store at -20°C.

2.1 PCR amplification

Purpose: To amplify target DNA for gel extraction/cloning.

Materials:

Instruments and Equipment	Centrifuge tube
	Centrifuge
	PCR thermal cycler
	Lab refrigerator
Chemicals and Reagents	2×PrimeMixStar
	Primer
	Template DNA
	ddH2O

Reaction Mix (50 µL Total):

Component	Volume (μL)
2×PrimeMixStar	25
Forward Primer	1
Reverse Primer	1
Template DNA	1
ddH2O	22
Total	50

Cycling Conditions (30 cycles):

Step	Temperature (°C)	Time	Number of cycles
Initial Denaturation	95	3 minutes	1
Denaturation	95	30 seconds	30
Annealing	56-60	30 seconds
Extension	72	1 minute
Final Extension	72	5 minutes	1
Hold	4	∞

Note: Annealing temperature is primer-specific.

Procedure:

Mix all components in labeled PCR tubes, followed by brief centrifugation. Load samples into a pre-programmed thermal cycler running through the PCR amplification reaction procedure above.

2.2 Agarose gel Electrophoresis

Purpose: To verify the DNA bands’ lengths and thus prepare for the next step of gel extraction to extract the target bands.

Materials:

Instruments and Equipment	Casting tray
	Electrophoresis chamber
	Microwave
	UV Transilluminator / Gel Imager
Chemicals and Reagents	Agarose
	1× TAE Buffer
	ddH2O
	Nucleic acid gel stain
	DNA ladder
	DNA sample
	Loading Buffer

Procedure:

1) Dissolve 1 g agarose in 100 mL 1× TAE buffer. Heat the material in a microwave at short intervals until the agarose dissolves completely.

2) Add 7 µL nucleic acid gel stain.

3) Pour into mold, insert comb. Solidify for 30 minutes at room temperature.

4) Mix 5 μl of PCR product with 1μl of loading buffer, followed by loading into the wells alongside the DNA marker.

5) Perform electrophoresis at a constant voltage of 100V for 45 minutes in 1×TAE buffer.

6) After electrophoresis, place the gel in a UV gel imaging system for observation.

2.3 Bacterial Culture and Transformation

Purpose: To obtain a sufficient quantity of engineered strains with stable activity for subsequent experiments.

Materials:

Instruments and Equipment	Laminar flow hood
	Shaking incubator
	Shaking the culture tube
Chemicals and Reagents	LB liquid medium
	antibiotic(kan+)
	glycerol stock(pRSFDuet）

Procedure:

1) Add 5 µL antibiotic stock (e.g., Kanamycin for pET28a) to 5 mL sterile LB.

2) Inoculate with 5 µL glycerol stock (e.g., E. coli BL21 with plasmid).

3) Incubate at 37°C, 220 rpm for 16–18 hours.

2.5 Extraction of Plasmids

Experimental Workflow Overview:

Goal: To obtain high-purity plasmid DNA from host cells, including empty vectors or recombinant vectors.

Materials:

Instruments and Equipment	Centrifuge
	Centrifugate tube
	FastPure DNA Mini Column
	Collection Tubes
Chemicals and Reagents	Buffer P1
	Buffer P2
	Buffer P3
	Buffer PW1
	Buffer PW2
	ddH2O

Procedure:

1. Transfer 2 mL of the overnight culture into a 1.5 mL microcentrifuge tube. Centrifuge at 12000 rpm for 1 min, and drop the solution.
2. Add 250 μL Buffer P1 containing RNase A, and mix thoroughly by vortexing.
3. Add 250 μL Buffer P2, and gently invert the tube up and down 10 times to fully lyse the bacterial cells.
4. Add 350 μL Buffer P3, immediately invert gently up and down 10 times to mix thoroughly, then centrifuge at 12,000 rpm for 2 minutes.
5. Place the FastPure DNA Mini Columns spin column into the Collection Tubes, carefully pipette the supernatant into the adsorption columns, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid, and put the adsorption column back into the collection tube.
6. Add 500 μL of Buffer PW1, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid.
7. Add 600 μL of Buffer PW2, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid.
8. Repeat step 7.
9. Put the spin column back into the collection tube, and centrifuge at 12,000 rpm for 1 minute.
10. Place the spin column into a new clean 1.5 mL centrifuge tube. Add 40 μL of ddH₂O to the middle part of the adsorption membrane, let it stand at room temperature for 1 minute, then centrifuge at 12,000 rpm for 1 minute. Measure the concentration via Nanodrop and store the plasmid DNA solution at 4 ℃.

2.6 Restriction Enzyme Double Digestion

Purpose: To double-digest the plasmid vector to linearize it, providing a backbone for recombination and preventing self-ligation.

Materials:

Instruments and Equipment	Centrifuge tube
	Centrifuge
	PCR thermal cycler
	Lab refrigerator
	Water bath pot
Chemicals and Reagents	plasmid
	restriction enzyme
	10×cutsmart Buffer
	ddH2O

Reaction Mix (50 µL):

Component	Volume
plasmid	500 ng
Restriction Enzyme A	1 µL
Restriction Enzyme B	1 µL
10× Buffer	5 µL
ddH2O	To 50 µL

Procedure:

1. Add all the components to PCR tubes, followed by brief centrifugation at 12,000 rpm for 10 seconds.

Enzyme Pairs:

pETDuet-1 + AGE insert: NcoI/BamHI

pET28a + S insert: NcoI/HindIII

2. Incubate the reaction system in a 37 ℃ water bath for 30 minutes to ensure complete digestion of the vector plasmid into linearized fragments. Store at 4 ℃.

2.7 Gel Extraction and Purification

Purpose: To isolate and purify linearized vector DNA fragments from agarose gel, while removing impurities such as uncut circular vectors, other restriction-generated fragments, proteins, and agarose.

Materials:

Instruments and Equipment	Centrifuge tube
	Centrifuge
	Scalpel
	Lab Refrigerator
	Water bath pot
Chemicals and Reagents	Buffer GDP
	Buffer GW
	ddH2O

Procedure:

1. After completing agarose gel electrophoresis, precisely excise the gel slice containing linearized vector DNA under UV illumination. Transfer the excised gel slice to a 1.5 mL centrifuge tube.
2. Add an equal volume of Buffer GDP. Incubate at 65°C in a metal bath until the gel becomes completely transparent.
3. Transfer the solution to the spin column and centrifuge at 12000 rpm for 60 seconds.
4. Discard the filtrate. Place the spin column in the collection tube. Add 300 μL Buffer GDP to the spin column, and let it stand for 1 minute. Centrifuge at 12000 rpm for 60 seconds.
5. Discard the filtrate. Place the spin column in the collection tube. Add 700 μL Buffer GW to the spin column. Centrifuge at 12000 rpm for 60 seconds.
6. Repeat Step 5.
7. Discard the filtrate. Place the spin column in the collection tube. Centrifuge at 12000 rpm for 2 minutes.
8. Place the spin column into a new clean 1.5 mL centrifuge tube. Add 20 μL of ddH₂O to the middle part of the adsorption membrane, let it stand for 2 minutes. Centrifuge at 12,000 rpm for 1 minute. Store the DNA solution at -20℃.

2.8 Homologous Recombination

Purpose： To construct a recombinant vector by ligating the target gene to the linearized vector via homologous recombination.

Materials:

Instruments and Equipment	PCR thermal cycler
	Centrifuge
	Incubator
	Water bath pot
Chemicals and Reagents	5×CE enzyme
	target gene
	Linearized vector
	ddH2O

Reaction Mix (10 µL):

Component	Volume	Example
Linearized vector	0.02 x bp (ng)	6.3 kb → 126 ng
target gene	0.04x bp (ng)	500bp → 20 ng
5×CE enzyme	2 μL
ddH2O	To 10 µL

Procedure:

1. Prepare the system:
2. Place the system in a 37°C water bath for 30 minutes, then cool to 4℃ immediately

2.9 Qualitative Transformation Check

Purpose: To quickly assess whether the transformation of target plasmids (e.g., AGE-pETDuet, S-pET28a) into bacterial strains (E. coli DH5α or BL21) was successful by observing colony growth on selective plates.

Procedure:

1) Following transformation, plate the cell suspension onto LB agar plates containing the appropriate antibiotic（Kanamycin for pET28a derivatives, Ampicillin for pETDuet-1 derivatives).

2) Incubate plates overnight at 37°C.

3) Observe and document colony formation. The presence of distinct colonies indicates a successful transformation, while no growth suggests failure.

Note: Always include a negative control (untransformed cells) to confirm the antibiotic's effectiveness.

3.1 Preparation for the gel of SDS-PAGE

Purpose: To separate proteins for subsequent qualitative, quantitative, or identification analysis of the proteins.

Materials:

Instruments and Equipment	Electronic Balance
	pH Meter
	Fume Hood
	Reagent Bottles
	Gel Casting Plates
	Gel Combs
	Sealing Gaskets
	Pipette
Chemicals and Reagents	Acrylamide
	N, N'-Methylenebisacrylamide
	1.5M Tris-HCl Buffer
	0.5M Tris-HCl Buffer
	10% Sodium Dodecyl Sulfate Solution
	10% Ammonium Persulfate (APS)
	N, N, N', N'-Tetramethylethylenediamine (TEMED)
	ddH2O

Procedure:

1. Mix 4 mL lower gel (2x) and 4 mL lower gel buffer (2x).
2. Add 80 μL gel accelerator and quickly add the liquid into the glass slit.
3. Use CH3-COH-CH3 to seal the lower gel.
4. Wait until the gel is completely set (6-10 minutes).
5. Mix 1 mL upper gel (2x) and 1 mL upper gel buffer (2x).
6. Add 20 μL gel accelerator and add the liquid into the glass slit until it is full.
7. Insert the sample comb and wait for 15 minutes.
8. When the upper gel is completely concrete, plug out the comb.

3.2 Transformation into E. coli Origami 2(DE3) Expression Strain

Purpose: To introduce the target plasmid into the expression strain for protein production.

Materials:

Instruments and Equipment	Centrifuge
	Incubator
	Water bath pot
	Ice-making machine
Chemicals and Reagents	pRSFDuet-PDI-Thaumatian A/B/C/D plasmid
	E. coli Origami 2(DE3) competent cells
	LB liquid medium (K⁺)

Procedure:

1. Thaw competent cells on ice for 10 minutes.
2. Add 1-5 µL plasmid DNA to 50 µL cells, mix gently, then incubate on ice for 30 min.
3. Heat-shock at 42°C for 45 seconds, then immediately put on ice for 2 minutes.
4. Add 500 µL LB medium (no antibiotic), incubate at 37°C for 1 hour.
5. Plate 100 µL of the transformed cells on LB agar plates (antibiotic), and incubate at 37℃ for 12-16 hours.

3.3 Ultrasonic Disruption

Purpose: To break the cell apart by sonicating, and to remove cell debris and insoluble components by centrifugation, obtaining a crude protein supernatant containing the target protein as the starting material for subsequent affinity purification.

Materials:

Instruments and Equipment	Centrifuge
	Ultrasonic Cell Disruptor
	Ice-making machine
Chemicals and Reagents	Bacterial pellet
	Lysis buffer

Procedure:

1. Place the bacterial solution on ice for 5 minutes to cool it down.
2. Setting parameters: 20kHz, amplitude 40%, pulse 5 seconds on/5 seconds off, total time 3-5 minutes.
3. Keep an ice bath during the process, and avoid the temperature exceeding 4℃.
4. A 10 μL sample was examined under a microscope (to check if the cells were lysed).
5. Centrifuge at 5000 rpm, 4 °C for 15 min, and collect the supernatant.

3.4 Protein Purification

Purpose: To selectively bind and purify His-tagged target proteins from the crude protein supernatant using Ni-affinity chromatography, while including a control group to evaluate the efficiency and specificity of the purification process.

Materials:

Instruments and Equipment	Gravity column
	Centrifuge tube
Chemicals and Reagents	Ni-NTA resin
	Wash buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 40 mM imidazole)
	Elution buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 250 mM imidazole)

Procedure:

1) Transfer the crude protein supernatants into two micro-centrifuge tubes for each sample. Load 4 mL of crude protein supernatant from one tube into an affinity chromatography column.

2) Add 1 mL of Ni solution to the affinity chromatography column, and collect the flow-through for later analysis.

3) Wash to remove nonspecific proteins by adding 1 mL of 25 mM imidazole solution, repeating the wash step five times.

4) Elute the target protein by adding 250 μL of 500 mM imidazole solution, repeating the elution step twice.

3.5 SDS-PAGE

Purpose: To confirm protein size and purity.

Materials:

Instruments and Equipment	Vertical electrophoresis system
	Microcentrifuge
	Water bath
	Vortex mixer
Chemicals and Reagents	12.5% SDS-PAGE gel
	Running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS)
	Protein samples
	Protein ladder
	Coomassie Blue stain
	Destaining solution

Procedure:

1. Mix 10 µL protein sample with 2× Laemmli buffer in clean centrifuge tubes, Vortex for 10 seconds. Boil for 5 minutes.
2. Load the ladder and samples into the lanes. Run at 120 V for 90 min.
3. Stain with Coomassie Blue stain for 30 minutes. Incubate the gel in destaining solution overnight (12-16 hours).

4.1 Thin Layer Chromatography (TLC)

Purpose: To qualitatively detect the activities of AGE and NAL.

Materials:

Instruments and Equipment	Silica gel TLC plates
Chemicals and Reagents	Solvent system
	Sample solution
	Standards
	Staining reagent

Procedure:

1. Prepare the mobile phase (n-butanol: glacial acetic acid: ethanol: water = 211: 1) and saturate the chamber for 15–30 minutes.
2. Spot 2 μL of the supernatant on a silica plate (about 1 cm from the bottom), dry briefly with a hair dryer.
3. Develop the TLC plate until the solvent front reaches 5 – 8 cm, and dry the plate.
4. Dip the plate in 5% sulfuric acid ethanol, dry and heat it at 130°C for 5 minutes.

4.2 Enzyme-Linked Immunosorbent Assay (ELISA)

Purpose: To produce respective enzymes using the three different plasmids that have been constructed.

Materials:

Instruments and Equipment	Shaking incubator
	Pipettes
	Autoclave
	Microtubes
	Vortex
	ELISA plate
Chemicals and Reagents	Standards
	Sample Diluent
	Antibody
	Washing Buffer
	Substrate Solution
	Stop Solution

Procedure:

1. Add 50 μL of standard substances with different concentrations to each standard well.
2. For sample wells, first add 10 μL of the test sample, and then add 40 μL of sample diluent; add nothing to the blank wells.
3. Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each standard well and sample well. Seal the reaction wells with a plate sealer and incubate in a 37°C water bath or incubator for 60 minutes.
4. Discard the liquid, pat dry on absorbent paper. Fill each well with washing solution, let it stand for 1 minute, shake off the washing solution, and pat dry on absorbent paper. Repeat the plate washing 5 times (a plate washer can also be used).
5. Add 50 μL of substrate A and 50 μL of substrate B to each well, and incubate at 37°C in the dark for 15 minutes.
6. Add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 minutes.

4.3 High Performance Liquid Chromatography (HPLC)

Purpose: To produce respective enzymes using the three different plasmids that have been constructed.

Materials:

Instruments and Equipment	HPLC System
	Chromatography Workstation
	Pipette
	HPLC Column
	Injection Needles
Chemicals and Reagents	Mobile Phase Solvents (HPLC Grade)
	Standard Substance
	Sample Solvents

Procedure:

1) Preparation of Sialic Acid Standard Solution: Weigh 20 mg of sialic acid standard powder and dissolve it in the mobile phase. Dilute the solution to a final volume of 100 mL in a volumetric flask to obtain a standard stock solution with a concentration of 0.2 mg/mL. Perform gradient dilution of this stock solution to prepare a series of standard solutions with different concentrations. After filtration through a 0.22 μm filter membrane, transfer the standard solutions to sample vials for subsequent use.

2) Sample Preparation: Take the sample to be tested and centrifuge it at 15,000 rpm and 4 °C for 15 minutes. Collect the supernatant and filter it through a 0.22 μm cellulose acetate filter membrane. Transfer the filtered supernatant into HPLC vials for detection.

3) HPLC Instrument Startup and Equilibration: Set the chromatographic conditions (mobile phase: 5 mmol/L H₂SO₄; flow rate: 0.6 mL/min; RID detector temperature: 35°C; column temperature: 65°C). Pump the mobile phase to flush the chromatographic column for 30 minutes until the baseline on the workstation stabilizes (fluctuation ≤ 0.1 mAU).

4) Detection of Standards and Samples: Inject the series of standard solutions into the injector in ascending order of concentration. For each concentration, perform the detection in triplicate. Record the retention time and peak area of the standard in the chromatogram. Record the chromatogram of the sample.