Notebook

Overview

This document records all experimental data of the 2025 Lactose Killer iGEM team from August 10 to August 29.

Experiment Goals

Construct target plasmids (pPICZαA-T-Gal and pPICZαA-GOD).
Transform the target genes into E. coli DH5α.
Linearize the target plasmids and transform them into Pichia Pastoris (GS115).
Use methanol to induce the expression of β-T-galactosidase and glucose oxidase.
Purify the target proteins by utilizing the high-affinity coordination between Ni²⁺ and histidine.
Employ the Multiskan™ FC Microplate Photometer (Thermo Fisher) to test the hydrolysis activity of both β-T-galactosidase (T-Gal) and glucose oxidase (GOD).
Corroborate substrate conversion and product formation via thin-layer chromatography (TLC)
Confirm the identity and purity of products through high-performance liquid chromatography (HPLC).

Date	8.10-8.11
Participants	All members
Goal	Prepare Petri dishes for E. coli DH5α and Pichia Pastoris (using YPD and LB media). Prepare zeocin solution. Perform PCR amplification of T-Gal and GOD. Prepare agarose gel.
Content	*Prepare Petri dishes for E. coli* DH5α and Pichia Pastoris (using YPD and LB media) A. Prepare LB solid and liquid media I. LB Solid medium: Dissolve 1 g tryptone, 0.5 g yeast extract, 1 g sodium chloride, and 1.5 g agar in a glass flask, then add deionized (ddH₂O) water to reach a final volume of 100 mL. II. LB Liquid medium: Dissolve 1 g tryptone, 0.5 g yeast extract, and 1 g sodium chloride in a glass flask, then add deionized water to make a final volume of 100 mL. B. Prepare YPD solid and liquid media I. YPD solid medium: Dissolve 2 g tryptone, 1 g yeast extract, 2 g glucose, and 2 g agar in a glass flask, then add deionized water to a final volume of 100 mL. II. YPD liquid medium: Dissolve 2 g tryptone, 1 g yeast extract, and 2 g glucose in a glass flask, then add deionized water to a final volume of 100 mL. C. High-temperature and high-pressure sterilization: Sterilize all media by autoclaving at 121 °C and 103.4 kPa for 30 minutes. Prepare zeocin solution Perform PCR amplification of T-Gal and GOD A. PCR amplification system of T-Gal and GOD: Transfer 25 μL of PCR master mix, 1 μL of template DNA, and 1 μL each of the upstream and downstream primers into a PCR tube. Add ddH₂O to bring the final volume to 50 μL. B. PCR amplification procedure: I. Initial denaturation: 98 °C for 5 min. II. Denaturation: 98 °C for 30 s. III. Annealing: 56 °C for 30 s. IV. Extension: 72 °C for 2 min 20 s. V. Repeat steps II–IV for 30 cycles Prepare agarose gel**: Transfer 200 mL of TAE buffer and 2 g of agarose to a glass flask. Heat the mixture in a microwave oven at high power for 2 minutes, swirling gently to ensure complete dissolution. Allow the solution to cool to 50–60 °C, then add 20 μL of Gel Red and mix thoroughly.
Result	Fig 1. PCR amplification of T-Gal and GOD.

Date	8.12-14
Participants	All members
Goal	Extract plasmids Perform double digestion Conduct gel electrophoresis Recycle gel Carry out homologous recombination Perform heat-shock transformation

Content

Extract plasmids
- A. Aliquot 1 mL of overnight bacterial culture into each of five 2 mL microcentrifuge tubes (5 mL total), then centrifuge at 10,000 rpm for 1 min and discard the supernatant.
- B. Add 250 µL of Buffer P1 (from the FastPure EndoFree Plasmid Mini Kit, Vazyme, Cat. No. DC201-01) containing RNase A to each tube and resuspend the pellet by vigorous pipetting.
- C. Add 250 µL of Buffer P2 to each tube and gently invert 8–10 times until the suspension clears, indicating complete bacterial lysis.
- D. Immediately add 350 µL of Buffer P3 to each tube and invert gently 8–10 times to neutralize Buffer P2. Centrifuge at 12,000 rpm for 10 minutes.
- E. Insert each FastPure DNA Mini Column into a fresh 2 mL Collection Tube. Transfer the clarified supernatant onto the column and centrifuge at 12,000 rpm for 30–60 s. Discard the flow-through and place the column back into the same Collection Tube.
- F. Add 500 µL of Buffer PW1 to each column and centrifuge at 12,000 rpm for 30–60 s. Discard the flow-through and place the column back in the same Collection Tube.
- G. Add 600 µL of Buffer PW2 (supplied with absolute ethanol) to each column and centrifuge at 12,000 rpm for 30–60 s. Discard the flow-through and return the column to the same Collection Tube.
- H. Repeat step G.
- I. Return the column to the collection tube and centrifuge at 12,000 rpm for 1 minute to dry the membrane and remove any residual wash buffer.
- J. Transfer the column to a clean 1.5 mL microcentrifuge tube. Add 100 μL of Elution Buffer (or sterile ddH₂O) directly onto the membrane. Let stand at room temperature (25 °C) for 2 min, then centrifuge at 12,000 rpm for 1 min to elute the DNA.
- K. Remove the column.
Double Digestion: Add 10 μL of plasmid (pPICZαA) into a PCR tube. Then add 5 μL of 10× buffer. Next, add 1 μL of EcoRI and 1 μL of SalI. Finally, add ddH₂O to bring the solution to a final volume of 50 μL. Incubate in a thermal cycler at 37 °C for 30 minutes.
Gel Electrophoresis: Use a horizontal electrophoresis tank to subject the gel to electrophoresis at 180 V for 15 min.
Gel Recycle (Follow the FastPure Gel DNA Extraction Mini Kit (Vazyme, DC301-01) operation instructions exactly.)
- A. After electrophoresis, quickly excise the target DNA band under UV transillumination. Blot the slice dry with absorbent paper, then weigh it (tare the tube first).
- B. Add an equal volume of Buffer GDP to the gel slice. Incubate at 50–55 °C for 7–10 min, inverting the tube twice to ensure complete dissolution of the gel matrix.
- C. Briefly centrifuge to collect any droplets. Insert a FastPure DNA Mini Columns-G adsorption column into a 2 mL collection tube. Load the dissolved gel mixture onto the column and centrifuge at 12,000 rpm (≈ 13,800 × g) for 30–60 s.
- D. Discard the flow-through. Re-insert the column into the same collection tube. Add 300 µL of Buffer GDP to the column, let stand for 1 min, then centrifuge at 12,000 rpm for 30–60 s.
- E. Discard the flow-through. Re-insert the column into the same collection tube. Add 700 µL of Buffer GW (prepared with absolute ethanol) and centrifuge at 12,000 rpm for 30–60 s.
- F. Repeat step E once more.
- G. Discard the flow-through. Return the column to the collection tube and centrifuge at 12,000 rpm for 2 min to dry the membrane.
- H. Transfer the column to a new sterile 1.5 mL microcentrifuge tube. Add 20–30 µL of Elution Buffer directly onto the center of the membrane. Let stand for 2 min at room temperature, then centrifuge at 12,000 rpm for 1 min. Discard the column; the eluted DNA is ready for use.
Homologous Recombination: Add 2 μL of target gene (T-Gal and GOD), 5 μL of pPICZαA, 1 μL of Exnase II, and 2 μL of 5×CE buffer into a PCR tube. Then incubate in a thermal cycler at 37 °C for 30 minutes.
Heat-Shock Transformation
- A. Place the tube containing E. coli DH5α (Sangon Biotech) competent cells on ice, then add 5 μL of ligation product (pPICZαA-T-Gal and pPICZαA-GOD).
- B. Transfer the tube to a 42 °C water bath for 90 seconds, then place it back on ice.
- C. Spread the suspension onto LB agar plates and incubate at 37 °C for 18 h.

Result

Fig 2. Gel electrophoresis of pPICZαA after double enzyme digestion

Fig 3. E. coli after overnight culture of T-Gal

Fig 4. E. coli after overnight culture of GOD

Date	8.14-15
Participants	All members
Goal	Quantify colonies on LB-zeocin plates. Conduct genome sequencing. Transform into Pichia pastoris GS115.
Content	*Quantification of E. coli* DH5α from a culture plate** A. Pick a single colony of E. coli DH5α from the agar plate with a sterile tip. B. Suspend the colony in 10 µL of sterile ddH₂O to serve as template DNA. C. Prepare a 50 µL PCR reaction system: 1 µL of template, 1 µL of forward primer, 1 µL of reverse primer, 22 µL of ddH₂O and 25 µL of 2× PCR master mix. D. Program the thermal cycler: initial denaturation at 98 °C for 5 min, followed by 30 cycles of 98 °C for 30 s, 56 °C for 30 s, and 72 °C for 2 min 20 s. E. Load the PCR products into a gel lane and run horizontal electrophoresis at 180 V for 15 min. Genome Sequencing: Recombinant plasmids were sequenced by Beijing Tsingke Biotech Co., Ltd. *Transformation into Pichia pastoris* GS115 A. Linearization of recombinant plasmids and gel electrophoresis I. Prepare the digestion mixture in a 0.2 mL PCR tube: 2 µL of 10× restriction buffer 1 µL of linearized pPICZαA-T-Gal or pPICZαA-GOD plasmid (~ 1 µg) 1 µL of SacI enzyme (10 U) 16 µL of ddH₂O (total volume 20 µL) II. Incubate both reactions at 37 °C for 30 min in a thermal cycler. III. Load 5–10 µL of each digestion product into separate wells of a 1% agarose gel and perform electrophoresis at 180 V for 15 min. B. Gel Recycle I. Excise the linearized plasmid bands under UV transillumination; blot excess buffer with absorbent paper. II. Purify the DNA using the FastPure Gel DNA Extraction Mini Kit (Vazyme, Cat. No. DC301-01) according to the manufacturer's instructions. C. Plasmid Transformation I. Prepare GS115 competent cells by removing them from the -80 °C freezer and thawing to 0 °C on ice. II. Heat 20 µL of carrier DNA in a 95 °C water bath for 3 min, then place it back on ice at 0 °C. III. In a clean bench, mix 50 µL of GS115 competent cells with 5 μL of linearized plasmid (pPICZαA-T-Gal and pPICZαA-GOD), respectively. IV. Add 10 µL of carrier DNA and 1.4 mL of PD buffer to the mixture. V. Incubate the mixture in a 30 °C water bath for 20 min, flipping it 6–8 times during incubation. VI. Incubate the mixture in a 42 °C water bath for 20 min, flipping it 6–8 times during incubation. VII. Centrifuge the mixture at 5,000 rpm for 40 seconds. Discard the supernatant and add 14 mL of PN buffer. VIII.** Add 0.2 mL of PN buffer to resuspend the mixture, then spread the mixture onto YPD-zeocin Petri dishes. D. Prepare 2 bottles of 100 mL YPD liquid medium for yeast culturing. E. Prepare bacterial solutions of DH5α-pPICZαA-T-Gal and DH5α-pPICZαA-GOD. Incubate them overnight with shaking at 220 rpm at 30 °C.
Results	*Fig 5. Gel electrophoresis of pPICZαA–T–Gal and pPICZαA–GOD after the PCR of E. coli* DH5α colony Fig 6. Sequencing of pPICZαA–T–Gal Fig 7. Sequencing of pPICZαA–GOD Fig 8. Gel electrophoresis of pPICZαA–T–Gal after linearization Fig 9. Gel electrophoresis of pPICZαA–GOD after linearization**

Date	8.16-17
Participants	All members
Goal	Extract recombinant plasmids and test their concentration. Perform qualitative and quantitative analysis of colonies on YPD-Zeo agar plates. Inoculate selected colonies into YPD liquid medium and culture with shaking to prepare yeast cultures. Scale up the cultivation of positive colonies and induce protein expression.
Content	Recombinant Plasmid Extraction and Gel Electrophoresis A. Plasmid Extraction: Follow the operation instructions of the FastPure EndoFree Plasmid Mini Kit (Vazyme, DC201-01) exactly. B. Concentration Test: The extracted pPICZαA-T-Gal and pPICZαA-GOD plasmids were quantified using a NanoDrop spectrophotometer. The concentrations are as follows: pPICZαA-T-GAL: 26.2, 45.9, and 42.2 ng/μL pPICZαA-GOD: 10.55, 12.6, and 10.35 ng/μL Qualitative and Quantitative Analysis of Colonies on YPD-Zeo Agar Plates A. Pick colonies from YPD-Zeo agar plates and transfer them into microcentrifuge tubes containing 10 μL of ddH₂O. Add lysis buffer, then incubate the tubes in a boiling water bath at 100 °C for 10 min. B. Prepare a 50 µL PCR reaction system by combining 25 µL of 2× PCR master mix, 1 µL of forward primer (F), 1 µL of reverse primer (R), 1 µL of template DNA, and 22 µL of nuclease-free water. C. Load 5–10 µL of each digestion product into separate wells of a 1% agarose gel and perform electrophoresis at 180 V for 15 min. Inoculate Selected Colonies into YPD Liquid Medium and Culture with Shaking to Prepare Yeast Cultures A. Dispense 4 mL of YPD medium into each of two 15 mL culture tubes. B. Add 4 µL of Zeocin to each tube. C. Transfer the GOD and T-Gal cultures into separate tubes. D. Incubate the tubes at 30 °C with shaking at 220 rpm overnight. Scale Up Cultivation of Positive Colonies and Induce Protein Expression A. Add 100 µL of Zeocin to sterile YPD medium. B. Inoculate the medium with 1 mL of cell culture containing T-GAL or GOD. C. Incubate at 30 °C with orbital shaking at 220 rpm for 3 h. D. Add 100 µL of anhydrous ethanol to each flask. E. Continue incubation at 30 °C with shaking at 220 rpm overnight.
Results	*Fig 10. YPD plate was coated with Pichia Pastoris* carrying the T–Gal plasmid Fig 11. YPD plate was coated with Pichia Pastoris carrying the GOD plasmid Fig 12. Gel electrophoresis of T–Gal–pPICZαA and GOD–pPICZαA after the PCR of Pichia Pastoris colony**

Date	8.18-19
Participants	All members
Goal	Harvest intracellular functional proteins (T-Gal and GOD) from GS115 cells. Assess protein expression and quantify concentration by SDS-PAGE. Decolorize the protein gel.
Content	Harvest Intracellular Functional Proteins (T-Gal and GOD) from GS115 Cells A. Centrifuge the overnight-cultured Pichia pastoris GS115 at 4 °C and 4,000 rpm for 20 min. B. Cell Lysis I. Resuspend the pellet in 10 mL of ice-cold PBS. II. Add lysozyme (final concentration 1 mg/mL) and shake gently at 4 °C for 1 h. III. Lyse the cells on ice using an ultrasonic cell disruptor (... amplitude, 3 s on/3 s off, total 15 min). IV. Clarify the lysate by centrifugation at 4 °C and 4,500 rpm for 30 min; the supernatant is the crude protein extract. C. Protein Purification (His-Ni Affinity Chromatography) I. Take the pre-packed His-Ni affinity chromatography column. II. Load the clarified supernatant onto the column; collect the flow-through. III. Use washing buffer containing 25 mM imidazole to remove impurities. IV. Repeat step III once. V. Use high-concentration imidazole for elution, then filter the collected eluate through an ultrafiltration tube and centrifuge at 4 °C and 3,000–5,000 rpm to obtain the target protein. Assess Protein Expression and Quantify Concentration by SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) A. Prepare SDS-PAGE Gel I. Assemble the gel-making mold properly. II. Add 2 mL of lower gel solution (2×) and 2 mL of lower gel buffer (2×) to the gel-casting cup and mix thoroughly. III. Add 40 μL of improved accelerator to the mixed solution from Step II. IV. Carefully pour the mixed lower gel solution (from Step III) into the gel-casting cassette. Immediately overlay the solution with a thin layer of water or isopropanol to ensure a flat, even surface; avoid creating any bubbles (indentations). V. Allow to stand undisturbed at room temperature (25 °C) for 6–10 min. VI. Slowly and carefully remove the overlay. Combine equal volumes of upper gel solution (2×) and colored upper gel buffer (2×) by adding 0.5 mL of each to a fresh casting cup and mix gently. VII. Add 10 μL of improved accelerator to the mixed solution from Step VI and swirl gently to mix. VIII. Carefully overlay the lower gel with the upper gel solution until the cassette is filled to the top of the short plate, then slowly insert the comb into the gel. IX. Let the cassette stand undisturbed for 10–15 min until the upper gel has fully polymerized, then gently remove the comb. B. Process Samples I. Transfer 24 µL each of the crude protein, flow-through, washing buffer, and eluate into four separate PCR tubes. II. Add 6 μL of loading buffer (Bromophenol blue). III. Place the samples in a constant-temperature water bath at 95 °C for 10 min to denature the proteins. C. SDS-PAGE: Place the gel into a vertical electrophoresis tank and perform electrophoresis at 180 V for 15 minutes. D. Staining: Place the protein gel in a box containing Coomassie Brilliant Blue staining solution and leave it on a shaker overnight. Protein Gel Decolorization A. Transfer the Coomassie Brilliant Blue solution from the kit containing the Coomassie Brilliant Blue-stained denaturing polyacrylamide gels of GOD and T-Gal into a reagent tube. B. Add double-distilled water (ddH₂O) to the kit until the water level covers the denaturing polyacrylamide gel, then gently agitate the kit. Repeat this step 5–7 times until excess Coomassie Brilliant Blue is eluted from the denaturing polyacrylamide gel. C. Place the eluted denaturing polyacrylamide gel in a developing apparatus for development to obtain the result.
Results	Fig 13. Electrophoresis gel of stained T-Gal Fig 14. Electrophoresis gel of stained GOD

Date	8.20-8.21
Participants	All members
Goal	Test the hydrolysis activity of β-T-galactosidase (T-Gal).
Content	β-T-galactosidase (T-Gal) Hydrolysis Activity Test A. Prepare 1 mg/mL ONPG Solution I. Weigh 20 mg of ONPG powder using an electronic balance, then add 20 mL of PBS buffer (20 mmol/L) into a 15 mL tube. II. Place the tube in a constant-temperature water bath at 50 °C for 10 min. B. Prepare Test Samples I. Add 3 mL of ONPG solution to each of five 15 mL centrifuge tubes. II. Transfer 0, 0.25, 0.50, 1.25, and 2.50 mL of T-Gal liquid enzyme solution into the five separate test tubes containing ONPG solution, resulting in final T-Gal concentrations of 0, 1, 2, 5, and 10 mg/mL, respectively. III. Incubate in a constant-temperature water bath at 45 °C for 15 min. IV. Add 2 mL of Na₂CO₃ solution to denature the enzyme. C. Activity Test I. Transfer the samples into 96-well microplates and 8-well break-apart strips (ELISA-grade, BBI brand, Sangon Biotech). II. Use the Multiskan™ FC Microplate Photometer to test the hydrolytic activity of T-Gal.
Results	Fig 15. T-Gal Hydrolysis Activity Test

Date	8.22-8.24
Participants	All members
Goal	Test GOD activity. Perform TLC test for β-T-galactosidase (T-Gal) activity.
Content	Test GOD Activity (using Glucose Oxidase Activity Assay Kit) A. Preheat the Multiskan™ FC Microplate Photometer for 30 minutes, adjust the wavelength to 500 nm, and zero the instrument with distilled water. B. Prepare Reagents 1 and 2 from the Glucose Oxidase (GOD) Test Kit / Spectrophotometric Method (mlBio ml076383). C. Prepare Boiled Samples: Take 200 μL of the sample and transfer it to a new EP tube. Boil it in a 95 °C water bath for 10 minutes, then cool it to room temperature and centrifuge at 8,000×g for 10 minutes at 4 °C. Collect the supernatant as the boiled sample for the control tube. D. Reaction System: Total volume of 200 µL. Control tubes (5 tubes with concentrations 0/1/2/5/10 mg/mL): 50 µL of sample, 75 µL of Reagent 1, and 75 µL of Reagent 2. Test tubes (5 tubes with concentrations 0/1/2/5/10 mg/mL): 50 µL of boiled sample, 75 µL of Reagent 1, and 75 µL of Reagent 2. TLC Test for β-T-galactosidase (T-Gal) Activity A. Prepare Reaction Mixtures I. 5% galactose: 0.5 g galactose + 10 mL PBS II. 15% galactose: 1.5 g galactose + 10 mL PBS III. 20% galactose: 2 g galactose + 10 mL PBS B. Add 8 mL of PBS to a beaker containing the corresponding mass of lactose, then place the mixture in a water bath at 40–50 °C with continuous stirring to ensure complete dissolution. Cool to room temperature and adjust the final volume to 10 mL by dilution. C. Reaction Conditions: Use galactose solutions with concentrations ranging from 5% to 20%. Mix 2 mL of galactose solution with 0.1 mL of liquid T-Gal, incubate at 40 °C for 15–20 minutes, then heat at 65 °C for 5 minutes to inactivate the enzyme. D. TLC Operation I. Prepare the developing solvent by mixing n-butanol, glacial acetic acid, ethanol, and water in a volume ratio of 2:1:1:1, then transfer it to the development chamber. Line the chamber walls with filter paper to pre-saturate the chamber, and let it stand for 15–30 minutes to establish a stable gas phase. II. Cut a TLC plate and draw a starting line approximately 1 cm from the bottom edge using a pencil. Centrifuge the sample at 10,000×g for 1 minute, then spot the supernatant. Apply the sample at distinct positions along the starting line using a capillary tube or a 2 µL micropipette to avoid tailing. III. Place the plate vertically in the development chamber, ensuring the solvent front remains below the spotting line. Cover the chamber and allow the solvent to migrate until it has traveled 5–8 cm. Immediately remove the plate once development is complete, and dry it quickly with a hair dryer or at room temperature. IV. Immerse the dried plate in staining solution (5% sulfuric acid in ethanol), then allow it to air-dry or accelerate drying with a hair dryer. Subsequently, heat the plate in an oven at 130 °C for 5 minutes.

Results

Fig 16. GOD Abundance Activity Test Result

Fig 17. TLC Test for T-Gal Activity

Date	8.25-8.29
Participants	All members
Goal	Repeat the TLC experiment: shorten the heating time and improve drying to reduce tailing and confirm T-Gal activity. TLC Analysis of Dual-Enzyme Synergy. Quantitative analysis of dual-enzyme synergistic products by HPLC.
Content	TLC Assay for T-Gal Activity with Different Lactose Concentrations A. Prepare lactose solutions with concentrations of 2%, 5%, 10%, and 20%, respectively, and take 2 mL of each. B. Add 100 μL of T-Gal enzyme solution to each 2 mL portion of lactose solution. C. Incubate the reactions at 40 °C for 15 min, then transfer the tubes to a 65 °C water bath for 5 min to terminate the reaction. D. TLC Detection I. Gently draw a starting line 1 cm from the bottom edge of the TLC plate with a pencil. Use a micropipette to aspirate the sample solution for spotting (be careful to avoid excessive spotting volume, which may cause tailing). II. Prepare the developing solvent (n-butanol: glacial acetic acid: ethanol: water = 2:1:1:1), pour it into the developing tank, and place the spotted TLC plate into the tank. Immerse the lower end of the plate in the developing solvent (ensure the solvent front is below the spotting line). Once the developing solvent has migrated 5–8 cm upward, remove the TLC plate and dry it with a hair dryer. III. Uniformly stain the dried TLC plate with a 5% sulfuric acid–ethanol solution. After staining, air-dry the plate, then place it in an oven at 130 °C. Once clear bands appear, remove the plate immediately, observe, and record the band patterns. TLC Analysis of Dual-Enzyme Synergy A. Prepare lactose solutions with concentrations of 5% and take 2 mL solution. B. Add 100 μL of T-Gal enzyme solution and GOD enzyme solution to 2 mL lactose solution. C. Incubate the reactions at 40 °C for 15 min, then transfer the tubes to a 65 °C water bath for 5 min to terminate the reaction. D. Gently draw a starting line 1 cm from the bottom edge of the TLC plate with a pencil. Use a micropipette to aspirate the sample solution for spotting (be careful to avoid excessive spotting volume, which may cause tailing). E. Prepare the developing solvent (n-butanol: glacial acetic acid: ethanol: water = 2:1:1:1), pour it into the developing tank, and place the spotted TLC plate into the tank. Immerse the lower end of the plate in the developing solvent (ensure the solvent front is below the spotting line). Once the developing solvent has migrated 5–8 cm upward, remove the TLC plate and dry it with a hair dryer. F. Uniformly stain the dried TLC plate with a 5% sulfuric acid–ethanol solution. After staining, air-dry the plate, then place it in an oven at 130 °C. Once clear bands appear, remove the plate immediately, observe, and record the band patterns. Quantitative analysis of dual-enzyme synergistic products by HPLC A. Prepare 3 reaction systems. Each system contains 3 mL of milk, with a total volume of 10 mL, and has a T-Gal concentration of 1 mg/mL, a GOD concentration of 1 mg/mL, and a lactose concentration of 5% (w/v). B. Place the tubes with the reaction systems into water baths at different temperatures: 25 °C, 37 °C, and 42 °C. C. Every 10 minutes, transfer 1.5 mL of liquid from each reaction system tube to a new tube. Place the new tube in a 65 °C water bath for 5 minutes to denature the enzymes and terminate the reaction. After the water bath, centrifuge the tube and collect the supernatant. For each temperature, 6 sample tubes are obtained. D. Submit the collected samples for HPLC analysis, analyze the peak pattern results, and plot the lactose decomposition rate curve under the dual-enzyme synergy.
Results	Fig 18. TLC result of hydrolysis activity of T-Gal with different T-Gal concentrations Fig 20. Results of HPLC for dual–enzyme synergy A) at 25 ° C, B) at 37°C, C) at 42 ° C