iGEM 2025 — Team shanghai-hs

{% extends "layout.html" %} {% block title %}Notebook{% endblock %} {% block
lead %}This serves as a chronological record of your team's progress throughout
the season. It documents your daily activities, experiments, discussions, and
decisions.{% endblock %} {% block page\_content %} {% from
"/components/article-content.html" import articleContent %} {% call
articleContent("Notebook", [{"content": "Overview","anchor": "#title\_1"},
{"content": "Experiment Goals","anchor": "#title\_2"}]) %}

# 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 | 1. Prepare Petri dishes for *E. coli DH5α* and    *Pichia Pastoris* (using YPD and LB media). 2. Prepare zeocin solution. 3. Perform PCR amplification of T-Gal and GOD. 4. Prepare agarose gel. |
| Content | 1. **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. 2. **Prepare zeocin solution** 3. **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 4. **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 | ![PCR amplification of T-Gal and GOD](https://static.igem.wiki/teams/5923/images/note1.webp) **Fig 1. PCR amplification of T-Gal and GOD.** |

|  |  |
| --- | --- |
| Date | 8.12-14 |
| Participants | All members |
| Goal | 1. Extract plasmids 2. Perform double digestion 3. Conduct gel electrophoresis 4. Recycle gel 5. Carry out homologous recombination 6. Perform heat-shock transformation |

|  |  |
| --- | --- |
| Content | 1. **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. 2. **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. 3. **Gel Electrophoresis**: Use a    horizontal electrophoresis tank to subject the gel    to electrophoresis at 180 V for 15 min. 4. **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. 5. **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. 6. **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 | ![Gel electrophoresis of pPICZαA after double enzyme digestion](https://static.igem.wiki/teams/5923/images/note2.webp) **Fig 2. Gel electrophoresis of pPICZαA after double enzyme digestion**  ![E. coli after overnight culture of T-Gal](https://static.igem.wiki/teams/5923/images/note3.webp) **Fig 3. *E. coli* after overnight culture of T-Gal**  ![E. coli after overnight culture of GOD](https://static.igem.wiki/teams/5923/images/note4.webp) **Fig 4. *E. coli* after overnight culture of GOD** |

|  |  |
| --- | --- |
| Date | 8.14-15 |
| Participants | All members |
| Goal | 1. Quantify colonies on LB-zeocin plates. 2. Conduct genome sequencing. 3. Transform into *Pichia pastoris* GS115. |
| Content | 1. **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. 2. **Genome Sequencing**: Recombinant    plasmids were sequenced by Beijing Tsingke Biotech    Co., Ltd. 3. **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 | ![Gel electrophoresis of pPICZαA–T–Gal and pPICZαA–GOD after the PCR of E. coli DH5α colony](https://static.igem.wiki/teams/5923/images/note5.webp) **Fig 5. Gel electrophoresis of pPICZαA–T–Gal and pPICZαA–GOD after the PCR of *E. coli* DH5α colony**  ![Sequencing of pPICZαA–T–Gal](https://static.igem.wiki/teams/5923/images/note6.webp) **Fig 6. Sequencing of pPICZαA–T–Gal**  ![Sequencing of pPICZαA–GOD](https://static.igem.wiki/teams/5923/images/note7.webp) **Fig 7. Sequencing of pPICZαA–GOD**  ![Gel electrophoresis of pPICZαA–T–Gal after linearization](https://static.igem.wiki/teams/5923/images/note9.webp) **Fig 8. Gel electrophoresis of pPICZαA–T–Gal after linearization**  ![Gel electrophoresis of pPICZαA–GOD after linearization](https://static.igem.wiki/teams/5923/images/note8.webp) **Fig 9. Gel electrophoresis of pPICZαA–GOD after linearization** |

|  |  |
| --- | --- |
| Date | 8.16-17 |
| Participants | All members |
| Goal | 1. Extract recombinant plasmids and test their    concentration. 2. Perform qualitative and quantitative analysis of    colonies on YPD-Zeo agar plates. 3. Inoculate selected colonies into YPD liquid medium    and culture with shaking to prepare yeast cultures. 4. Scale up the cultivation of positive colonies and    induce protein expression. |
| Content | 1. **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 2. **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. 3. **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. 4. **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 | ![YPD plate was coated with Pichia Pastoris carrying the T–Gal plasmid](https://static.igem.wiki/teams/5923/images/notebook10.webp) **Fig 10. YPD plate was coated with *Pichia Pastoris* carrying the T–Gal plasmid**  ![YPD plate was coated with Pichia Pastoris carrying the GOD plasmid](https://static.igem.wiki/teams/5923/images/note11.webp) **Fig 11. YPD plate was coated with *Pichia Pastoris* carrying the GOD plasmid**  ![Gel electrophoresis of T–Gal–pPICZαA and GOD–pPICZαA after the PCR of Pichia Pastoris colony](https://static.igem.wiki/teams/5923/images/note12.webp) **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 | 1. Harvest intracellular functional proteins (T-Gal and    GOD) from GS115 cells. 2. Assess protein expression and quantify concentration    by SDS-PAGE. 3. Decolorize the protein gel. |
| Content | 1. **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. 2. **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. 3. **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 | ![Electrophoresis gel of stained T-Gal](https://static.igem.wiki/teams/5923/images/note13.webp) **Fig 13. Electrophoresis gel of stained T-Gal**  ![Electrophoresis gel of stained GOD](https://static.igem.wiki/teams/5923/images/note14.webp) **Fig 14. Electrophoresis gel of stained GOD** |

|  |  |
| --- | --- |
| Date | 8.20-8.21 |
| Participants | All members |
| Goal | 1. Test the hydrolysis activity of β-T-galactosidase    (T-Gal). |
| Content | 1. **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 | ![T-Gal Hydrolysis Activity Test](https://static.igem.wiki/teams/5923/images/notebook-fig15-tif.webp) **Fig 15. T-Gal Hydrolysis Activity Test** |

|  |  |
| --- | --- |
| Date | 8.22-8.24 |
| Participants | All members |
| Goal | 1. Test GOD activity. 2. Perform TLC test for β-T-galactosidase (T-Gal)    activity. |
| Content | 1. **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. 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 | ![GOD Abundance Activity Test Result](https://static.igem.wiki/teams/5923/images/notebook-fig16-tif.webp) **Fig 16. GOD Abundance Activity Test Result**  ![TLC Test for T-Gal Activity](https://static.igem.wiki/teams/5923/images/notebook-fig17.webp) **Fig 17. TLC Test for T-Gal Activity** |

|  |  |
| --- | --- |
| Date | 8.25-8.29 |
| Participants | All members |
| Goal | 1. Repeat the TLC experiment: shorten the heating time and improve drying to reduce tailing and    confirm T-Gal activity. 2. TLC Analysis of Dual-Enzyme Synergy. 3. Quantitative analysis of dual-enzyme synergistic products by HPLC. |
| Content | 1. **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. 2. **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. 3. **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 | ![TLC result of hydrolysis activity of T-Gal with different T-Gal concentrations](https://static.igem.wiki/teams/5923/images/notebook-fig18.webp) **Fig 18. TLC result of hydrolysis activity of T-Gal with different T-Gal concentrations**  ![Results of TLC for dual-enzyme synergy](https://static.igem.wiki/teams/5923/images/notebook-fig20.webp) **Fig 20. Results of HPLC for dual–enzyme synergy**  **A) at 25 ° C, B) at 37°C, C) at 42 ° C** |

{% endcall %} {% endblock %}

{% extends "layout.html" %} {% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your
project by going through at least one iteration of the engineering design
cycle.{% endblock %} {% block page\_content %} {% from
"/components/article-content.html" import articleContent %} {% call
articleContent("Engineering", [
{"content": "Overview", "anchor": "#title\_1"},
{"content": "Part 1: pPICZαA-β-T-Gal", "anchor": "#title\_2", "children": [
{"content": "Objective", "anchor": "#level2\_1"},
{"content": "Design", "anchor": "#level2\_2"},
{"content": "Build", "anchor": "#level2\_3"},
{"content": "Test", "anchor": "#level2\_4"},
{"content": "Learn", "anchor": "#level2\_5"}
]},
{"content": "Part 2: pPICZαA-GOD", "anchor": "#title\_3", "children": [
{"content": "Objective", "anchor": "#level2\_6"},
{"content": "Design", "anchor": "#level2\_7"},
{"content": "Build", "anchor": "#level2\_8"},
{"content": "Test", "anchor": "#level2\_9"},
{"content": "Learn", "anchor": "#level2\_10"}
]},
{"content": "Next Steps", "anchor": "#title\_4"}
]) %}

# **Overview**

Lactose intolerance is a prevalent
condition characterized by the inability to digest or absorb dairy products, with symptoms including
diarrhea, nausea, and abdominal pain (Mayo Clinic). This digestive impairment stems from the body’s
failure to break down lactose, a disaccharide found in dairy. Lactase, the enzyme responsible for
hydrolyzing lactose into galactose and glucose, is deficient in individuals with lactose intolerance,
leading to the onset of symptoms. Current commercial solutions primarily involve incorporating
galactosidases into dairy products. Specifically, β-T-galactosidase—an enzyme that catalyzes the
breakdown
of lactose into galactose and glucose—has been used to mitigate lactose intolerance. However, this
approach faces significant limitations in widespread adoption: the glucose generated by
β-T-galactosidase
activity makes these modified dairy products unsuitable for consumers with strict glucose intake
restrictions, such as individuals with diabetes. To address this gap, we developed a synergistic
dual-enzyme system comprising β-T-galactosidase and glucose oxidase. The former hydrolyzes lactose,
while
the latter oxidizes the resulting glucose to eliminate its presence. To implement this system, we
utilized the β-T-Gal gene and the GOD gene for the synthesis of β-T-galactosidase and glucose oxidase,
respectively. **The success of our engineering efforts was validated
through Design-Build-Test-Learn (DBTL) cycles, encompassing plasmid engineering, protein
expression and purification, and dairy product (milk) optimization.**

# **Part 1 pPICZαA-β-T-Gal**

## **Objective:**

Engineer the yeast *Pichia
pastoris* to express and purify recombinant β-T-galactosidase (β-T-Gal), and validate the
lactose-hydrolyzing activity of the purified enzyme.

## **Design:**

**Construction of the
pPICZαA-β-T-Gal Plasmid**

The pPICZαA-β-T-Gal plasmid consists
of two key components: the pPICZαA vector and the β-T-Gal gene. The pPICZαA vector is a commonly used
vector for yeast expression. The β-T-Gal gene encodes β-T-galactosidase, which facilitates the
hydrolysis
of lactose into galactose and glucose. Homologous recombination was employed to construct the
pPICZαA-β-T-Gal plasmid.

The plasmid consists of the β-T-Gal
gene, AOX1 promoter and terminator, and two endonuclease binding sites for EcoRI and SalI. The AOX1
promoter is activated by the inducer methanol, resulting in transcriptional induction. The two
endonuclease binding sites for ECORI and SalI enable homologous recombination of β-T-Gal and the vector.
Another part is the α-factor secretion signal, which helps mature and process the synthesized
protein.

![](https://static.igem.wiki/teams/5923/images/engineering-1.webp)

**Fig 1. Plasmid map of
pPICZαA-β-T-Gal**

## **Build:**

The recombinant expression plasmid
pPICZαA-β-T-Galactosidase was successfully constructed for heterologous expression in Pichia pastoris.
The gene encoding the synthetic β-T-Galactosidase was amplified via PCR, generating a distinct fragment
of approximately 2209 bp. As shown in Fig. 2, agarose gel electrophoresis analysis of the PCR product
revealed a single, clear band that aligned with the expected molecular size, confirming the specificity
and success of the amplification. The correct fragment was subsequently ligated into the pPICZαA vector
for downstream applications.

![](https://static.igem.wiki/teams/5923/images/engineering-2.webp)

**Fig 2. Electrophoresis result of
β-T-Gal**

Following
transformation into *E. coli* DH5α, positive clones were selected from the zeocin-resistant
colonies (Fig. 3A) and subjected to colony PCR for initial verification. The analysis revealed a
specific amplification product of approximately 2209 bp, which corresponds to the expected size of the
β-T-Gal gene (Fig. 3B). Subsequent DNA sequencing of the plasmid from a positive clone confirmed the
precise insertion of the target sequence into the pPICZαA vector without mutations, thereby validating
the successful construction of the pPICZαA-β-T-Gal plasmid (Fig. 3C).

![](https://static.igem.wiki/teams/5923/images/engineering-3.webp)

**Fig 3. pPICZαA-β-T-Gal Recombinant Plasmid
Identification Map**

## **Test:**

Before yeast
transformation, the recombinant plasmids were linearized via restriction digestion. Fig. 4 presents the
electrophoresis result of the linearized DNA, with bands matching the expected size (~5693 bp for
pPICZαA-β-T-Gal).

![](https://static.igem.wiki/teams/5923/images/engineering-4.webp)

**Fig 4. Electrophoresis Result of
Linearized** **pPICZαA-β-T-Gal**

The linearized plasmid was
transformed into Pichia pastoris cells pre-cultured in YPD medium. Positive transformants were selected
on YPD plates containing zeocin, as shown in Fig. 5A, indicating successful integration of the
expression cassette into the yeast genome. To further verify this, genomic DNA was extracted from the
transformants and analyzed by PCR. As demonstrated in Fig. 5B, a DNA fragment of approximately 2209 bp,
consistent with the expected size of the β-T-Gal gene, was amplified, confirming the stable
incorporation of the target expression cassette into the yeast chromosome.

![](https://static.igem.wiki/teams/5923/images/engineering-5.webp)

**Fig 5. Validation of yeast
transformation results**

The expression and
purification of β-T-Galactosidase were assessed by SDS-PAGE. As depicted in Fig. 6, the purified protein
preparation exhibited a single, distinct band migrating at a position consistent with the theoretical
molecular weight of ~80 kDa when compared to the protein ladder, confirming the integrity and
homogeneity of the recombinant product.

![](https://static.igem.wiki/teams/5923/images/engineering-6.webp)

**Fig 6.**  **β-T-Gal Protein Expression
Map**

To evaluate the
catalytic activity of the purified β-T-galactosidase (β-T-Gal), this study utilized its specific
hydrolysis of o-nitrophenyl-β-D-galactoside (ONPG) to produce the yellow product o-nitrophenol (ONP).
Enzyme activity was quantified by measuring the characteristic absorbance of ONP at 420 nm. The
experimental method is briefly described as follows: Enzyme solutions of β-T-Gal at different
concentrations were reacted with ONPG substrate at 40°C and pH 7.0 for a specific duration. The reaction
was terminated by adding sodium carbonate solution, followed by immediate absorbance measurement using a
microplate reader. As shown in **Table 1**, three parallel experiments were conducted at each
concentration to ensure data reliability.

**Table 1. Absorbance measurement at
420 nm**

|  |  |  |  |
| --- | --- | --- | --- |
| **mg/ml** | **A420** | | |
| **0** | 0.032 | 0.034 | 0.031 |
| **1** | 0.071 | 0.078 | 0.077 |
| **2** | 0.084 | 0.089 | 0.091 |
| **5** | 0.126 | 0.133 | 0.132 |
| **10** | 0.232 | 0.239 | 0.239 |
| **15** | 0.305 | 0.289 | 0.295 |
| **20** | 0.296 | 0.299 | 0.298 |

Results demonstrated
that the purified β-T-Gal exhibited significant catalytic activity. As shown in Fig. 7, the absorbance
value of the reaction system increased significantly with rising enzyme concentration, exhibiting a good
linear relationship between the two. This linear trend clearly confirmed that ONP production is
proportional to enzyme concentration, indicating the successful purification of β-T-Gal with highly
efficient and concentration-dependent catalytic activity.

![](https://static.igem.wiki/teams/5923/images/engineering-7.webp)

**Fig 7. Results of
β-T-Gal Hydrolysis Activity Test**

Thin-layer
chromatography (TLC) was used to assess β-T-Gal activity under two experimental conditions: (1) fixed
β-T-Gal concentration with gradient lactose concentrations; (2) fixed lactose concentration with
gradient time. Fig 8 shows the results of the lactose-gradient experiment: the migration distance of
samples increased with increasing lactose concentration. This indicates that when the substrate is
insufficient and β-T-Gal is not saturated, the lactose concentration acts as the limiting factor for the
hydrolysis reaction, and the amount of product increases with the increase in substrate. As the reaction
time extends from 0 min to 60 min, there is little difference in the intensity of the "lactose substrate
spot" on the TLC plate, and no obvious distinction in the resolution of spots between adjacent time
points. This may be because β-T-Gal has completed the hydrolysis of most lactose within 15 min; the
change in the amount of substrate is minimal in the subsequent time, resulting in insignificant
differences between spots at adjacent time points and making it impossible to clearly distinguish the
degree of hydrolysis. The time gradient needs to be adjusted in subsequent experiments to enable such a
distinction.

![](https://static.igem.wiki/teams/5923/images/engineering-8.webp)

**Fig 8. TLC
Analysis of β-T-Gal Activity**

At a fixed enzyme
concentration, increasing lactose concentration not only significantly elevated the yield of the primary
hydrolysis product glucose. Notably, in the high-concentration lactose system (5%),
galacto-oligosaccharides (GOS) were also observed to form alongside glucose. Moreover, GOS production
increased with rising lactose concentration, indicating that high substrate concentration promotes
β-T-Gal-catalyzed glycosylation reactions, thereby synthesizing GOS.

To investigate the
efficiency of lactose hydrolysis under the synergistic action of β-T-Gal and GOD, we comparatively
analyzed the hydrolysis effects in both pure lactose solution and fresh milk. Thin Layer Chromatography
(TLC) results demonstrated that, under the synergistic action of the two enzymes, lactose was hydrolyzed
into glucose and galactooligosaccharides (GOS). Moreover, the hydrolysis effect in fresh milk was
identical to that in the pure lactose solution (Fig. 9). This confirms that the synergistic effect of
β-T-Gal and GOD is feasible in fresh milk.

![](https://static.igem.wiki/teams/5923/images/engineering-9.webp)

**Fig 9. TLC Results
of Dual-Enzyme (β-T-Gal + GOD) Synergistic Activity**

To quantitatively
compare the decomposition efficiency of lactose in milk under the synergistic action of dual enzymes
(β-T-Gal and GOD), we employed High-Performance Liquid Chromatography (HPLC) to dynamically monitor the
decrease in lactose concentration and the increase in glucose concentration in milk during the reaction.
The experimental setup was as follows: β-T-Gal and GOD were mixed at a 1:1 ratio and added to the milk
system; the reaction was initiated under three temperature conditions (25°C, 37°C, and 42°C), and
samples were taken at 10-minute, 20-minute, and 60-minute intervals after the start of the reaction. The
concentrations of lactose and glucose at each time point were determined by HPLC (Table 2 and Table 3),
and the relevant results are shown in Fig. 10.

**Table 2.****Lactose Concentration Detected by
HPLC**

|  |  |  |  |
| --- | --- | --- | --- |
| **Time (min)** | **25℃** | **37℃** | **42℃** |
| **0** | 3.5 | 2.7 | 2.4 |
| **10** | 2.6 | 2.1 | 2.5 |
| **20** | 2.5 | 1.7 | 1.4 |
| **60** | 2.1 | 2.1 | 1.2 |

**Table 3.****Glucose Concentration Detected by
HPLC**

|  |  |  |  |
| --- | --- | --- | --- |
| **Time (min)** | **25℃** | **37℃** | **42℃** |
| **0** | 0 | 0 | 0 |
| **10** | 0 | 2 | 3.5 |
| **20** | 0 | 3.4 | 6.1 |
| **60** | 3.6 | 5.6 | 6.7 |

In terms of the effect
of temperature on reaction efficiency, both the lactose decomposition rate and glucose production rate
at 42°C were significantly higher than those at 25°C and 37°C. **This indicates that 42°C is the
optimal reaction temperature for the dual-enzyme synergistic system to catalyze lactose
decomposition in milk.** A further analysis of the reaction kinetic characteristics at the optimal
temperature (42°C) revealed that the first 20 minutes of the reaction were the stage with the fastest
glucose production rate. This phenomenon suggests that during the initial stage of dual-enzyme synergy
(the first 20 minutes), the binding efficiency between the enzymes and the substrate (lactose) is the
highest, and the catalytic activity is at its peak, allowing lactose to be rapidly decomposed into
glucose. However, as the reaction proceeds, the glucose production rate gradually slows down, which may
be attributed to the decrease in substrate (lactose) concentration, the feedback inhibition caused by
the accumulation of the product (glucose), or a slight decline in enzyme activity over the reaction
time.

![](https://static.igem.wiki/teams/5923/images/contribution-14-1.webp)

**Fig 10.****Quantitative Analysis of
Dual-Enzyme Synergistic Products by HPLC**

In summary, this study
determined that the optimal reaction conditions for dual-enzyme synergistic lactose decomposition are
42°C, identifying the initial 20 minutes as the critical phase for high-efficiency conversion. During
this period, enzyme-substrate binding is highly efficient, catalytic activity reaches its peak, and
rapid lactose degradation is achievable. Therefore, controlling the reaction at 42°C for 20 minutes
represents an optimized strategy balancing efficiency and energy consumption.

## **Learn:**

Refining the
expression of β-T-Gal in *Pichia pastoris* revealed that procedural rigor, specifically
temperature control and aseptic technique, is fundamental to experimental success. Building on this
foundation, the expression construct itself will be optimized through two targeted strategies: codon
optimization to align with host tRNA pools, and rational engineering of the α-factor secretion signal to
enhance the efficiency of protein translocation and secretion, with the ultimate goal of maximizing
functional enzyme yield.

# **Part 2 pPICZαA-GOD**

## **Objective:**

Engineer *Pichia pastoris* to express
and purify glucose oxidase (GOD), and validate its glucose-reducing activity.

## **Design:**

**Construction of the pPICZαA-GOD
Plasmid**

To achieve efficient expression of
glucose oxidase in Pichia pastoris, this study constructed the recombinant expression plasmid
pPICZαA-GOD based on the established pPICZαA vector system. This plasmid adopts a modular design
philosophy, continuing the construction strategy consistent with our group's previous work: the vector
backbone is a high-efficiency expression vector for Pichia pastoris, and the target gene GOD encodes
glucose oxidase, which catalyzes the oxidation of glucose to produce hydrogen peroxide and D-gluconic
acid-δ-lactone. This reaction is the key step in glucose clearance within the dual-enzyme system.

The
core functional elements of the plasmid were systematically engineered to include: - A
methanol-inducible expression unit comprising the AOX1 promoter and terminator - EcoRⅠ and SalⅠ
restriction sites for homologous recombination - An α-factor signal peptide directing protein secretion
- A 6×His tag for convenient purification. The synergistic interaction of these elements ensures
efficient secretory expression and convenient purification of the recombinant GOD protein in Pichia
pastoris GS115.

The
ultimate goal of this design is to achieve co-expression of GOD and β-T-Gal via this plasmid,
establishing a dual-enzyme system capable of continuously catalyzing lactose degradation and glucose
clearance. This provides critical technological support for developing low-lactose dairy
products.

![](https://static.igem.wiki/teams/5923/images/engineering-11.webp)

**Fig 11. Plasmid map of
pPICZαA-GOD**

## **Build:**

The recombinant expression plasmid
pPICZαA-Glucose Oxidase (pPICZαA-GOD) was constructed using homologous recombination. The gene encoding
glucose oxidase (GOD) was amplified by polymerase chain reaction (PCR), resulting in a DNA fragment of
approximately 1284 bp, which corresponds to the expected length of the full-length coding sequence. As
shown in Fig. 12, agarose gel electrophoresis analysis of the PCR product displayed a single, clear band
of the correct size, confirming the successful and specific amplification of the target gene prior to
its insertion into the linearized pPICZαA vector.

![](https://static.igem.wiki/teams/5923/images/engineering-12.webp)

**Fig 12. Electrophoresis result of
GOD**

The recombinant plasmid was
transformed into *E. coli* DH5α competent cells, and transformants were selected on
zeocin-containing plates, as shown by the single colonies in Fig. 13A. Multiple colonies were initially
screened by colony PCR to identify those carrying an insert of the expected size. A positive clone
identified by PCR was subsequently subjected to DNA sequencing. The sequencing results confirmed that
the glucose oxidase (GOD) gene was accurately inserted into the pPICZαA vector without mutations,
thereby verifying the successful construction of the pPICZαA-Glucose Oxidase plasmid.

![](https://static.igem.wiki/teams/5923/images/engineering-13.webp)

**Fig 13. pPICZαA-GOD Recombinant Plasmid
Identification Map**

## **Test:**

Before transforming into yeast, the
recombinant plasmids were linearized by restriction digestion. Fig 14 shows the electrophoresis result
of the linearized DNA, with bands of the expected size (~4778 bp for pPICZαA-GOD).

![](https://static.igem.wiki/teams/5923/images/engineering-14.webp)

**Fig 14. Electrophoresis result of pPICZαA-GOD after
DNA linearization**

The linearized pPICZαA-GOD plasmid
was transformed into Pichia pastoris host cells pre-cultured in YPD medium. Successful transformants
were selected on YPD plates supplemented with zeocin, with colony growth evident in Fig. 15A, indicating
positive integration of the expression cassette into the yeast genome. To molecularly confirm the
integration, genomic DNA was extracted from selected transformants and analyzed by PCR using
gene-specific primers. As shown in Fig. 15B, an amplification product of approximately 1294 bp,
corresponding to the expected size of the GOD gene fragment, was detected, verifying the precise and
stable incorporation of the GOD expression cassette into the yeast chromosome.

![](https://static.igem.wiki/teams/5923/images/engineering-15.webp)

**Fig 15. Validation of yeast transformation
results**

The transformed yeast strains were
cultured under inducing conditions to express the recombinant GOD protein. Subsequently, the secreted
protein was purified from the culture supernatant using C-terminal His-tag technology. Analysis of the
purification process by SDS-PAGE revealed a predominant band at approximately 48 kDa, which aligns with
the expected molecular weight of the glycosylated GOD protein. The electrophoretic profile demonstrates
successful purification, with the target protein band being the major component in the elution fraction,
indicating a high purity level suitable for subsequent functional characterization.

![](https://static.igem.wiki/teams/5923/images/engineering-16.webp)

**Fig 16. Result of the GOD Protein
purification**

The activity of
purified glucose oxidase (GOD) was determined using a glucose oxidase (GOD)-peroxidase (POD) coupled
reaction system. The principle is as follows: GOD catalyzes the oxidation of glucose to produce H₂O₂.
Subsequently, POD catalyzes the decomposition of H₂O₂ in the presence of oxygen. The resulting reactive
oxygen species oxidize colorless o-anisidine to form a brownish-yellow product. The absorbance of this
product at a specific wavelength is positively correlated with GOD activity. A series of GOD dilutions
was measured for reaction absorbance using a Multiskan™ FC microplate reader.

**Table 4.
Absorbance measurement at 500 nm**

|  |  |  |  |
| --- | --- | --- | --- |
| **mg/ml** | **A500** | | |
| **0** | 0.076 | 0.074 | 0.075 |
| **0.5** | 0.178 | 0.179 | 0.178 |
| **1** | 0.184 | 0.183 | 0.183 |
| **2.5** | 0.469 | 0.467 | 0.465 |
| **5** | 0.725 | 0.723 | 0.721 |
| **10** | 1.251 | 1.249 | 1.247 |
| **20** | 1.508 | 1.503 | 1.5 |

Results demonstrated
that although the absorbance curve was not strictly linear, its overall trend of significant increase
with rising enzyme concentration clearly confirmed that the purified GOD retained potent catalytic
activity.

![](https://static.igem.wiki/teams/5923/images/engineering-17.webp)

**Fig 17. Results of
the GOD Hydrolysis Activity Test**

To investigate the
efficiency of lactose hydrolysis under the synergistic action of β-T-Gal and GOD, we comparatively
analyzed the hydrolysis effects in both pure lactose solution and fresh milk. Thin Layer Chromatography
(TLC) results demonstrated that, under the synergistic action of the two enzymes, lactose was hydrolyzed
into glucose and galactooligosaccharides (GOS). Moreover, the hydrolysis effect in fresh milk was
identical to that in the pure lactose solution. This confirms that the synergistic effect of β-T-Gal and
GOD is feasible in fresh milk.

![](https://static.igem.wiki/teams/5923/images/engineering-18.webp)

**Fig 18. TLC
Results of Dual-Enzyme (β-T-Gal + GOD) Synergistic Activity**

To quantitatively
compare the decomposition efficiency of lactose in milk under the synergistic action of dual enzymes
(β-T-Gal and GOD), we employed High-Performance Liquid Chromatography (HPLC) to dynamically monitor the
decrease in lactose concentration and the increase in glucose concentration in milk during the reaction.
The experimental setup was as follows: β-T-Gal and GOD were mixed at a 1:1 ratio and added to the milk
system; the reaction was initiated under three temperature conditions (25°C, 37°C, and 42°C), and
samples were taken at 10-minute, 20-minute, and 60-minute intervals after the start of the reaction. The
concentrations of lactose and glucose at each time point were determined by HPLC, and the relevant
results are shown in Fig. 19.

**Table 5.****Lactose Concentration Detected by
HPLC**

|  |  |  |  |
| --- | --- | --- | --- |
| **Time (min)** | **25℃** | **37℃** | **42℃** |
| **0** | 3.5 | 2.7 | 2.4 |
| **10** | 2.6 | 2.1 | 2.5 |
| **20** | 2.5 | 1.7 | 1.4 |
| **60** | 2.1 | 2.1 | 1.2 |

**Table 6.****Glucose Concentration Detected by
HPLC**

|  |  |  |  |
| --- | --- | --- | --- |
| **Time (min)** | **25℃** | **37℃** | **42℃** |
| **0** | 0 | 0 | 0 |
| **10** | 0 | 2 | 3.5 |
| **20** | 0 | 3.4 | 6.1 |
| **60** | 3.6 | 5.6 | 6.7 |

In terms of the effect
of temperature on reaction efficiency, both the lactose decomposition rate and glucose production rate
at 42°C were significantly higher than those at 25°C and 37°C. **This indicates that 42°C is the
optimal reaction temperature for the dual-enzyme synergistic system to catalyze lactose
decomposition in milk.** A further analysis of the reaction kinetic characteristics at the optimal
temperature (42°C) revealed that the first 20 minutes of the reaction were the stage with the fastest
glucose production rate. This phenomenon suggests that during the initial stage of dual-enzyme synergy
(the first 20 minutes), the binding efficiency between the enzymes and the substrate (lactose) is the
highest, and the catalytic activity is at its peak, allowing lactose to be rapidly decomposed into
glucose. However, as the reaction proceeds, the glucose production rate gradually slows down, which may
be attributed to the decrease in substrate (lactose) concentration, the feedback inhibition caused by
the accumulation of the product (glucose), or a slight decline in enzyme activity over the reaction
time.

![](https://static.igem.wiki/teams/5923/images/contribution-14-1.webp)

**Fig 19.****Quantitative Analysis of
Dual-Enzyme Synergistic Products by HPLC**

In summary, this study
determined that the optimal reaction conditions for dual-enzyme synergistic lactose decomposition are
42°C, identifying the initial 20 minutes as the critical phase for high-efficiency conversion. During
this period, enzyme-substrate binding is highly efficient, catalytic activity reaches its peak, and
rapid lactose degradation is achievable. Therefore, controlling the reaction at 42°C for 20 minutes
represents an optimized strategy balancing efficiency and energy consumption.

## **Learn:**

In the construction and expression
practice of the pPICZαA-GOD plasmid, we have summarized the following key insights: Adopting
transformation and purification protocols consistent with the pPICZαA-β-T-Gal system significantly
enhances the standardization of experimental procedures and the reproducibility of results. Using the
identical pPICZαA vector backbone to express different enzyme proteins provides systematic comparability
for evaluating the expression levels and secretion efficiency of the two recombinant proteins. Future
optimization will focus on analyzing expression differences between β-T-Gal and GOD under identical
induction conditions to balance the yield ratios of components within the dual-enzyme system.
Furthermore, systematic optimization of key parameters such as methanol concentration and induction
duration will be pursued to further enhance the enzymatic yield of recombinant
GOD.

# **Next Steps:**

Although this study yielded
encouraging results, several limitations exist that point to directions for future research. Currently,
TLC analysis can only provide qualitative and semi-quantitative analysis of the products. Subsequent
studies could achieve precise quantification of the target prebiotics by spotting GOS, glucose, and
glucuronic acid standards as internal references. Furthermore, this study tested only the synergistic
effect of a 1:1 mixture of β-T-Gal and GOD. Optimizing enzyme ratios (e.g., 2:1, 1:2) may further
enhance overall system efficiency and reduce costs. Simultaneously, refining the temperature gradient
(e.g., 35–50°C) would aid in more precisely identifying the optimal temperature point. Finally,
assessing the stability and shelf life of the developed dairy product, followed by clinical trials to
validate its efficacy in alleviating lactose intolerance symptoms, represents a critical step in
translating laboratory findings into practical applications.

{% endcall %} {% endblock %}

{% extends "layout.html" %} {% block title %}Experiments{% endblock %} {% block
lead %}Describe the research, experiments, and protocols you used in your
project. It is designed to provide sufficient information for other teams to
replicate our work.{% endblock %} {% block page\_content %} {% from
"/components/article-content.html" import articleContent %} {% call
articleContent("Experiments", [
{
"content": "Media Preparation","anchor": "#title\_1",
"children": [
{
"content": "YPD Liquid Medium","anchor": "#title\_1\_1"
},
{
"content": "YPD Solid Medium","anchor": "#title\_1\_2"
},
{
"content": "LB Solid Medium","anchor": "#title\_1\_3"
},
{
"content": "LB Liquid Medium","anchor": "#title\_1\_4"
}
]
},
{
"content": "Plasmid Construction","anchor": "#title\_2",
"children": [
{
"content": "PCR Amplification of Target Genes (T-Gal/GOD)","anchor": "#title\_2\_1"
},
{
"content": "Agarose Gel Electrophoresis and DNA Recovery","anchor": "#title\_2\_2"
},
{
"content": "Plasmid Extraction of Empty Vector","anchor": "#title\_2\_3"
},
{
"content": "Double Digestion of Empty Vector","anchor": "#title\_2\_4"
},
{
"content": "Homologous Recombination","anchor": "#title\_2\_5"
}
]
},
{
"content": "Transformation and Screening","anchor": "#title\_3",
"children": [
{
"content": "Transformation into E. coli DH5α","anchor": "#title\_3\_1"
},
{
"content": "Colony PCR Verification","anchor": "#title\_3\_2"
},
{
"content": "Recombinant Plasmid Extraction and Sequencing","anchor": "#title\_3\_3"
},
{
"content": "Transformation into Pichia pastoris GS115","anchor": "#title\_3\_4"
}
]
},
{
"content": "Protein Expression and Purification","anchor": "#title\_4",
"children": [
{
"content": "Expansion Culture of Positive Clones","anchor": "#title\_4\_1"
},
{
"content": "Methanol-Induced Expression","anchor": "#title\_4\_2"
},
{
"content": "Protein Extraction","anchor": "#title\_4\_3"
},
{
"content": "Protein Purification and SDS-PAGE Verification","anchor": "#title\_4\_4"
}
]
},
{
"content": "Functional Assays","anchor": "#title\_5",
"children": [
{
"content": "β-T-galactosidase Hydrolytic Activity Assay","anchor": "#title\_5\_1"
},
{
"content": "β-T-galactosidase Transglycosylation Activity Assay","anchor": "#title\_5\_2"
},
{
"content": "Dual-Enzyme Catalysis Analysis","anchor": "#title\_5\_3"
},
{
"content": "Glucose Oxidase Activity Assay","anchor": "#title\_5\_4"
}
]
},
]) %}

## **Outline**

### 1. Media Preparation

* 1.1 YPD Liquid Medium
* 1.2 YPD Solid Medium
* 1.3 LB Solid Medium
* 1.4 LB Liquid Medium

### 2. Plasmid Construction

* 2.1 PCR Amplification of Target Genes (T-Gal/GOD)
* 2.2 Agarose Gel Electrophoresis and DNA Recovery
* 2.3 Plasmid Extraction of Empty Vector
* 2.4 Double Digestion of Empty Vector
* 2.5 Homologous Recombination

### 3. Transformation and Screening

* 3.1 Transformation into E. coli DH5α
* 3.2 Colony PCR Verification
* 3.3 Recombinant Plasmid Extraction and Sequencing
* 3.4 Transformation into Pichia pastoris GS115

### 4. Protein Expression and Purification

* 4.1 Expansion Culture of Positive Clones
* 4.2 Methanol-Induced Expression
* 4.3 Protein Extraction
* 4.4 Protein Purification and SDS-PAGE Verification

### 5. Functional Assays

* 5.1 β-T-galactosidase Hydrolytic Activity Assay
* 5.2 β-T-galactosidase Transglycosylation Activity Assay
* 5.3 Dual-Enzyme Catalysis Analysis
* 5.4 Glucose Oxidase Activity Assay

# 1. Media Preparation

**Reagents:** Peptone, Yeast Extract, Glucose, diluted water, Agar, Zeocin (antibiotics)

**Instruments:** Erlenmeyer flask, Pipette, Autoclave, Laminar flow hood, Graduated cylinder,
Disposable gloves.

## **1.1 YPD liquid medium**

**Table 1. YPD liquid medium**

| Ingredients | Concentration | Amount |
| --- | --- | --- |
| Peptone/Tryptone | 2% w/v | 20 g |
| Yeast Extract | 1% w/v | 10 g |
| Glucose | 2% w/v | 20 g |
| Distilled Water | — | 800 mL+200 mL |

**Steps:**

1. Dissolve 20 g peptone/tryptone and 10 g yeast extract into about 800 mL of distilled water;
2. Put 20 g of glucose into 200ml of distilled water;
3. Sterilize the glucose solution and the yeast-peptone solution respectively (121°C, 20 min); (Send
   them to the autoclave room before 2:00 p.m., including student safety warnings)
4. Cool to room temperature after sterilization and mix the solutions together. Add antibiotics under
   laminar flow hood for standby use (Zeocin, final concentration: 100 µg/mL)

## **1.2 YPD solid medium**

**Table 2. YPD solid medium**

| Ingredients | Concentration | Amount |
| --- | --- | --- |
| Peptone/Tryptone | 2% w/v | 2 g |
| Yeast Extract | 1% w/v | 1 g |
| Glucose | 2% w/v | 2 g |
| Agar | 2% | 2 g |
| Distilled water | — | 80 mL+20 mL |

**Steps:**

1. Dissolve 2 g peptone/tryptone, 1 g yeast extract, and 2 g Agar into about 80 mL of distilled water;
2. Put 2 g of glucose into 20ml of distilled water;
3. Sterilize the glucose solution and the yeast-peptone solution respectively (121°C, 20 min), before
   2:00 p.m.;

## **1.3 LB solid medium**

**Table 3. LB solid medium**

| Ingredients | Concentration | Amounts |
| --- | --- | --- |
| Tryptone | 1% w/v | 1 g |
| Yeast Extract | 0.5% w/v | 0.5 g |
| NaCl | 1% w/v | 1 g |
| Agar | 1.5% | 1.5 g |
| Distilled water | — | 100 mL |

**Steps:**

1. Put all of the powder (All the ingredients except distilled water) and dissolve it in about 80 mL of
   distilled water, and bring to the volume of 100 mL;

## **1.4 LB liquid medium**

**Table 4. LB liquid medium**

| Ingredients | Concentration | Amount |
| --- | --- | --- |
| Tryptone | 1% w/v | 1 g |
| Yeast Extract | 0.5% w/v | 0.5 g |
| NaCl | 1% w/v | 1 g |
| Distilled water | — | 100 mL |

**Steps:**

1. Dissolve all the powder (Ingredients other than water) into about 80 mL of distilled water, and
   bring to the volume of 100 mL;

# 2. Plasmid Construction

**Reagents:** Plasmid extraction kit (ex.: Qiagen Plasmid Mini Kit), EcoRI, SalI enzyme,
10×Buffer, dd H2O, Agarose, 1×TAE Buffer, Nucleic acid strain Gel Red, Buffer B2, Elution
Buffer, Exnase II, 5x CE Buffer, ddH₂O

**Consumables:** Consumables in the plasmid extraction kit, Agarose gel related consumables,
Electrophoresis related consumables, Centrifuge tube, Gel extraction kit, PCR tubes, Centrifuge tubes

**Instruments:** Centrifuge, Microwave (To prepare the agarose gel), Electrophoresis powering
supply, blue light transilluminator, Water bath, PCR machine.

## **2.1 PCR Amplification of Target Genes (T-Gal/GOD)**

### 1. Amplification of T-Gal sequence

**(1) T-Gal sequence** 783AA 2199 bp

MRPLIDFNSGWLFEGSETVRLPHNAVELPFSYFDERSYQRPFTYEKRFAADPSWEGKEVSIVFDGAMADCKVSLNGNETQSHRDGYTPFEVRLTGKLQPGENVLTVAIDGSENPEIPPFGGQIDYLTYAGIYRDVWLRVTSPIFVENAKIETPNVLSDHKSVRARIDVANPQNLPLSGTLLAILTDSQGNTIASTDASVDGASIEIAFDDLTGISLWDLDNPALYTLAITIDTTHGQDESAFRFGFRSVEFTHEGFRLNGKPLKLRGLNRHQSFPYSGYAMGRAAQERDAEILKHQLHLNIVRTSHYPQSHWFLDRCDELGLLVFEEIPGWQHIGGEAWKDESVENVRRMITRDWNHPSIVIWGVRINESQDDHDFYVRTNKLARELDTTRPTGGVRYITESELLEDVYTMNDFILGNEELGGNRPRTPLRPQQEVTGLDRVVPYMITEYGGHMYPTKVHDEEQRQAEHVRRHLEVMNAAYGDPQIAGCIGWCAFDYNTHKDFGSGDRIVHHGVMDMFREPKFAAYAYASQCEPDEEVILRPVTFWARGERNIGGVLPLIVLTNCDEVELRYGTNPPKRFRPDRDAFPHLPHAPVVMDRKDFTADELGRWGMSWESATITGYVGGKAVKTIDFVADPVPATLQVEADTPDLPGLGETVRVMVRALDQAGNKLPFFSDPVSIEVSGAAERLGPALVPLRAGATGFWVRATGQGPVTVTVINERLGRQSITLTAN

**(2) PCR components**

**Table 5. PCR components**

| Components | Volume(μL) |
| --- | --- |
| 2× Hieff PCR Master Mix | 25 |
| T-Gal-F | 1 |
| T-Gal-R | 1 |
| Template DNA (Bacterial culture) | 1 |
| dd H2O | 22 |
| Total Volume | 50 |

**(3) Program Setup**

* 95°C 3 min Initial denaturation
* 30 cycles: 95°C 30 s → 55°C 30 s → 72°C 1 min/kb (2 min 10 s)
* 72°C 5 min extension

### 2. Amplification of GOD sequence

**(1) Glucose oxidase sequence** 428 AA 1284 bp

MSEDIFDAIIVGAGLAGSVAALVLAREGAQVLVIERGNSAGAKNVTGGRMYAHSLERIIPGFAEQAPIERIITHEKLAFMTDKGAMTIDYCNGEEAASSQVSYSVLRSKFDAWLMEQAEEAGAQLITGIRVDNVVQREGKVVGVEADGDILEAKVVILADGVNSLLAEKLGMAKRVEASHVAVGVKELIELPKSVIEDRFQLQGNEGAACLFAGSPTDGLMGGGFLYTNETTLSLGLVCGLHHLKDAKKSVPQMLEDFKQHPAVAPLIAGGKLVEYAAHVVPEAGMNMQPELVGDGVLIAGDAAGMCMNLGFTIRGMDLAISAGEAAAKTVLSAMKRDDFSKQSLGEYRQHLDDGPMRDMRMYQKLPAFLDNPRMFTAYPEMAISIARDLFTVDGSAPVPMRKKILRHAKKVGFINLMKDGLKGVTVL

**(2) PCR Components**

**Table 6. PCR components**

| Components | Volume(μL) |
| --- | --- |
| 2× Hieff PCR Master Mix | 25 |
| GOD-F | 1 |
| GOD-R | 1 |
| Template DNA (Bacterial culture) | 1 |
| dd H2O | 22 |
| Total Volume | 50 |

**(3) Program Setup**

* 95°C 3 min Initial Denaturation
* 30 cycles: 95°C 30 s → 55°C 30 s → 72°C 1 min/kb (1 min 10s)
* 72°C 5 min

## **2.2 Agarose Gel Electrophoresis and DNA Recovery**

1. **Preparation of agarose gel:** weight and take 0.35 g of Agarose and put it into a
   Erlenmeyer flask. Add 35 mL of 1×TAE buffer into the flask and microwave it for about 3 min
   (until all the agarose melts). Take the flask out and shake it well, then take 5 μL of nucleic
   acid strain Gel Red and add it to the agarose gel. Install the gel plate and insert the gel
   comb. Pour the liquid gel onto the plate (For about 3-4 mm high, avoid generating bubbles), and
   wait until the gel solidify. When the gel completely turns into solid, take out the comb, and
   put the gel into the electrophoresis tank. Add 1×TAE Buffer until it completely immerse the gel;
2. **Sample loading:** put the PCR samples into those wells in the gel, and add 10 μL of
   DNA Marker at last in another well, (Be careful not to damage the wells around or pushing the
   pipette too deep);
3. **Electrophoresis:** close the electrophoresis chamber, the direction of the wells
   should be toward the negative electrode of the electric field. Switch the electrophoresis
   machine on (160 V, 20 min);
4. **Blue light observation:** shut the instrument down, take the gel out, and observe it
   under the blue light transilluminator. The substance with orange-red fluorescence is DNA.
5. **Cut the part of the gel that contains target DNA sequences off and weight it.** Add 3
   × (100 μL Buffer B2/100 mg gel cut). Put the sample at 50℃ in a water bath for 10 min, mix by
   inversion for 3 times during the water bath and let the gel completely dissolve;
6. **Recover Fragments (Following the kit protocol):** Transfer 700 µL of gel solution
   into a spin column and centrifuge it at 8000 ×g for 30 s. Remove the liquid from the collection
   tube; Add 500 µL of Wash Solution, centrifuge it at 9000 ×g for 30 s. Remove the liquid from the
   collection tube, and repeat it 2 times; Then, centrifuge the column without adding the wash
   solution at 9000 ×g for 2 min;. Put the column into a clean 1.5 mL centrifuge tube, and add
   15-40 µL of Elution Buffer on the center of the absorption membrane. Incubate the tube at room
   temperature for 1 min, and then centrifuge for 2 min. Lastly, use Nanodrop to measure the DNA
   concentration.

## **2.3 Plasmid Extraction of Empty Vector**

1. Open a plasmid extraction kit (ex.: Qiagen Plasmid Mini Kit).
2. Follow the instruction book in it and extract plasmid DNA.
3. Use Nanodrop to measure the plasmid DNA's concentration and purity.

## **2.4 Double Digestion of Empty Vector**

**Table 7. Double digestion components**

| Components | Volume/Mass(μL/μg) |
| --- | --- |
| Plasmid DNA | 1-2 |
| 10×Buffer | 5 |
| EcoRI | 1 |
| SalI | 1 |
| dd H₂O | 50 |

Let the components react under the temperature of 37°C for 30 min, and use the Agarose gel electrophoresis to
verify the effect of digestion. Then recover the fragments and verify their size.

## **2.5 Homologous Recombination**

**Table 8. Homologous Recombination components**

| Components | Volume(μL) |
| --- | --- |
| Gene (DNA) | 0.04×bp/concentration |
| Vector | 0.02×bp/concentration |
| Exnase II | 1 |
| 5x CE Buffer | 2 |
| ddH₂O | Add until the whole mixture is to 10 |

Put them under the temperature of 37°C to react for at least 30 min.

# 3. Transformation and Screening

**Reagents:** Plasmid extraction kit (Ex.: Qiagen Plasmid Mini Kit), SacI enzyme, 10×Buffer, dd
H2O, competent cells, YPD medium, Zeocin (antibiotics)

**Consumables:** Consumables in the plasmid extraction kit, Centrifuge tube

**Instruments:** Centrifuge, PCR machine, Shaking incubator, Incubator

## **3.1 Transformation into *E. coli* DH5α**

1. Take 100 μL of competent cells, separated it into 2 equal parts, and add 1-2 μL of the recombination
   product;
2. Put them in ice bath for 30 min;
3. Heat shock them under the temperature of 42°C for 45-60 s, and immediately put it back into the ice
   bath for 2 min;
4. Add 800 μL of LB medium (The medium does not contain antibiotics), thaw the samples under the
   temperature of 37°C for 1 h;
5. Coat them on a LB-medium plate that contains Zeocin, culture them at 37°C for 12-16 h.

## **3.2 Colony PCR Verification**

Pick a single colony from the plate and put it in a 5 mL LB liquid medium (4 groups, so we made 4 samples).
Confirm that the final concentration of Zeocin is 100 µg/mL, then put it in the shaking incubator at 30℃
and 220 rpm for 12-18 h

**Table 9. Colony PCR Verification components**

| Components | Volume(μL) |
| --- | --- |
| 2× Hieff PCR Master Mix | 25 |
| T-Gal-F/GOD-F | 1 |
| T-Gal-R/GOD-R | 1 |
| Template (Bacterial culture) | 1 |
| dd H2O | 22 |
| Total Volume | **50** |

**PCR Program:**

* Pre-degeneration: 95°C 3 min
* 30× cycle: 95°C 30 s → 55°C 30 s → 72°C 1 min/kb(6 min)
* Elongation: 72°C 5 min

## **3.3 Recombinant Plasmid Extraction and Sequencing**

1. Open a plasmid extraction kit (Ex.: Qiagen Plasmid Mini Kit).
2. Follow the instruction book in it and extract plasmid DNA.
3. Use Nanodrop to measure the plasmid DNA's concentration and purity.
4. Send to test.

Pick 500μL of sample and go test its sequence of pPICZαA-T-Gal/pPICZαA-GOD

## **3.4 Transformation into Pichia pastoris GS115**

### 1. Plasmid linearization

Use SacI to digest the recombinant plasmid, then recover the linear fragments.

**Table 10. Plasmid linearization components**

| Components | Volume(μL) |
| --- | --- |
| 10× Buffer | 2 |
| DNA (Plasmid DNA, less than 1 μg) | 1 |
| SacI enzyme | 0.5 - 1 |
| ddH2O | Add until the volume is 20 |
| Total volume | 20 |

React at 37℃ for 20 min

### 2. Transformation

Pick 100 μL of competent cells, and add 1-22 μL of linear plasmid. Ice bath for 30 min, then heat shock it at
42°C for 90s, and immediately put it back in the ice bath for 2min. Add 800 μL of YPD medium (Not
including antibiotics), and thaw the samples at 30°C for 1h.

### 3. YPD-zeocin Plate screening

Spread the sample on a YPD plate with Zeocin (100 µg/mL) and culture the samples at 30°C for 2-3 days.

### 4. Colony PCR test

**Table 11. Colony PCR components**

| Components | Volume(μL) |
| --- | --- |
| 2× Hieff PCR Master Mix | 25 |
| T-Gal-F/GOD-F | 1 |
| T-Gal-R/GOD-R | 1 |
| Template (Bacterial culture) | 1 |
| dd H2O | 22 |
| Total Volume | 50 |

# 4. Protein Expression and Purification

**Reagents:** YPD medium, Antibiotics (Zeocin), PBS buffer, Methanol, Glycerol, SDS-PAGE sample
buffer (5×), 0.25% Coomassie Brilliant Blue staining solution, Destaining solution (40% Ethanol, 10%
acetic acid, 50% distilled water)

**Consumables:** Centrifuge tubes, Plates, Ni beads, Consumables used to prepare the SDS-PAGE
gel (Mold, gel casting cassette, etc.)

**Instruments:** Shaking incubator, Incubator, Centrifuge, Ultrasonic cell disruptor, 4℃
refrigerator, SDS-PAGE electrophoresis powering supply

## **4.1 Expansion Culture of Positive Clones**

Pick positive colonies and transfer them into 100 mL of YPD medium, put them in a shaking incubator at 30°C,
220 rpm overnight.

## **4.2 Methanol-Induced Expression**

When OD600 reaches 1.0, add 1000× methanol, leave it at 30℃ overnight to let the methanol induce the
expression.

## **4.3 Protein Extraction**

### 1. Ultrasonic disruption of yeast for protein extraction

1. Centrifuge the sample at 4℃, 4000 rpm for 20 min, collect the cells, and abandon the medium.
2. Cell disruption by ultrasonic waves: take out the sample and add 10 mL PBS Buffer and resuspend
   cells thoroughly. Put the suspension into a small beaker, ultrasonic disrupt on ice
   (Recommended: 500W (30%), stop for 3 s every 3 s of disrupting, making a total time of 10-15
   min). Centrifuge the supernatant at 4℃, 15000 rpm for 10 min.

### 2. Preparation of SDS-PAGE gel

1. Assemble the gel casting mold, and prepare the lower layer first (Ex.: 0.75 / 1.00 / 1.50 mm thick
   gel);
2. Extract lower layer gel solution (2×) and lower running buffer (2×) 2.0 / 2.7 / 4 mL each, put them
   into a gel casting cassette to mix them;
3. Add 40 / 55 / 80 µL of modified coagulant into the cassette in step 2, mix them carefully, make sure
   that there are no bubbles formed.
4. Add the mixture into the mold until the liquid level is about 1.5 cm less than the highest amount.
   Then, put water or alcohol in the mold to keep the liquid level smooth. Make sure not to create
   bubbles;
5. Put the mold under room temperature (25ºC) for 6-10 min. The gel is well solidified when the bound
   between the overlay phase is clear;
6. Then, prepare the upper layer gel (Ex.: 0.75 / 1.00 / 1.5 mm thick mini gel);
7. Slowly pour the overlay phase out and extract equal amount of upper layer gel solution (2×) and the
   upper
   running buffer (2×, with colors) each for 0.5 / 0.75 / 1.0 mL. Put them in a new cassette and
   mix it;
8. Add 10 / 15 / 20 µL of modified coagulant to the cassette, mix it carefully so that there are no
   bubbles
   created;
9. Add the upper layer gel solution onto the lower layer gel until the liquid level reaches the maximum
   level.
   Slowly put the comb into the mold (with an angle), avoid creating any bubbles.
10. Let the gel stand for 10-15 min, and wait for the upper layer gel to solidify. Carefully take out
    the comb,
    and use pipette tip or syringe to extract some buffer to wash the wells.

## **4.4 Protein Purification and SDS-PAGE Verification**

### 1. Binding

Extract 200 μL of Ni beads and put it in the supernatant to bind for 1 - 2 h

### 2. Wash and elution

Use 25 mM of imidazole for washing and 300mM imidazole for elution, store the samples in PBS buffer +
glycerol at -20℃, separate them into several 1mL tubes for storing.

### 3. SDS-PAGE Electrophoresis

#### (1) SDS-PAGE Electrophoresis

Extract 40 μL of Protein sample and add it into the 10 μL protein loading buffer (5×), then water bath it at
100˚C for 5-10 min. Take it out and cool to room temperature, and centrifuge at 9000rpm for 1 min;

**Loading:** Extract 10-15 µL of samples, add the samples orderly;

**Electrophoresis:** After loading the samples, close the electrophoresis chamber, connect it to
the power source, and open the power source. Maintain the voltage at 200-120V before the sample enter
the gel for about 15-20 min; When the straining indicator reaches the separating gel, increase the
voltage to 200 V, and make sure that the voltage is stable. Stop the electrophoresis when the indicator
is about 1-2 cm higher than the bottom of the gel (About 1 h later).

#### (2) Staining

**Staining:** After the electrophoresis, shut the powering supply down, take out the mold, and
use a tool to separate the gel and the mold. Using the copper wire at the center of the indicator band
as a marker, put the gel into a large container to strain, and add 0.25% Coomassie brilliant blue
straining solution. Strain for 1-2 h, and it can be put overnight if needed (Optional: use a microwave
to speed up the process, heat 1-2 min)

#### (3) Destaining

**Destaining:** Abandon the straining solution, use distilled water to wash the gel several
times, and add destaining solution to destain. Change the destaining solution frequently until the
protein band is clear, and destaining process can be overnight. (Destaining solution formula: 40%
Ethanol, 10% acetic acid, 50% distilled water)

#### (4) Gel imaging

# 5. Functional Assays

**Reagents:** ONPG (2-Nitrophenyl β-D-galactopyranoside), 1 M Na2CO3, PBS buffer, Lactose, PBS
buffer, milk, Glucose, o-Dianisidine solution, Horseradish peroxidase solution (HRP solution),

**Consumables:** Centrifuge tubes, 96-well plate (optional, used for microplate reader
measurements), TLC plate (Kieselgel 60 F254), Mobile phase (N-butanol : ethanol : acetic acid : water =
2 : 1 : 1 : 1), Chromogenic agent (5% Sulfuric acid-ethanol)

**Instruments:** microplate reader (optional), Spectrophotometer, Water bath, Oven,
Spectrophotometer,

## **5.1 β-T-galactosidase Hydrolytic Activity Assay**

### 1. Preparation of ONPG solution (1 mg/mL)

Use 20 mM phosphate buffer (PBS, pH 7.0) to dissolve ONPG until the concentration is 1 mg/mL. Preheat at 50°C
for 10 min before use

### 2. Prepare 1 M Na₂CO₃ (100 mL)

* Na₂CO₃: 10.6 g
* Distilled water: Add until the solution reaches 100mL

### 3. Substrate: 1 mg/mL ONPG (2-Nitrophenyl β-D-galactopyranoside)

### 4. Reaction system

3 mL of ONPG solution (50˚C preheated), 0.1 mL of enzyme template(T-Gal), put it in 40°C water bath and let
it react for 15 min, add 2 mL 1M Na2CO3 solution to stop the reaction, and test the absorbency at 420nm.

**Unit definition:** 1 U = 1 µmol / 1 min.

## **5.2 β-T-galactosidase Transglycosylation Activity Assay**

### 1. Components

* 5% w/v = 0.5 g Lactose / 10 mL PBS solution (Heat it to help it dissolve)
* 10% w/v = 1g Lactose / 10 mL PBS solution (Heat it to help it dissolve)
* 15% w/v = 1.5 g Lactose / 10 mL PBS solution (High, needed to stirred and heated sufficiently)
* 20% w/v = 2 g Lactose / 10 mL PBS solution (High, needed to stirred and heated sufficiently)

### 2. Preparation

1. Weight the corresponding lactose powder into a beaker;
2. Add about 8 mL of PBS buffer;
3. Heat the samples in a water bath at 40-50˚C, and stir it when heating until the powder completely
   dissolve;
4. Wait for the sample to cool to room temperature and then adjust the final volume to 10 mL;

### 3. Experimental procedure

**Substrate:** 5%-20% lactose solution

**Reaction condition:** 2 mL of lactose solution + 0.1mL liquid enzyme (T-Gal), and put it into
40˚C water bath for 15-20 min → 65°C to inactivate for 5 min.

#### TLC Steps:

1. First, prepare the mobile phase (N-butanol : ethanol : acetic acid : water = 2 : 1 : 1 : 1), and
   pour it into the developing chamber (About 0.5-1 cm thick). Pre-satiated the filter paper on the
   chamber walls, and wait 15-30 min to let it form a stable gas phase.
2. Spotting: First, cut a piece of silica gel plate, and use a pencil to mark a starting line (about
   1cm from the bottom edge); After sampling, centrifuge the sample at 10,000 × g for 1 min and
   spot with the supernatant (If the concentration is high, dilute it); Use a small pipette or 2 µL
   applicator to add the samples on different locations on the line; Every spot is about 1-2 mm in
   diameter, and quickly dry the samples by using a hair dryer (To prevent streaking).
3. Development: Put the TLC plate into the developing chamber (And perpendicular to the chamber), then
   add mobile phase (Make sure that the liquid level is lower than the starting line); Close the
   chamber, and wait until the solvent front is about 5-8 cm higher than the starting line; When
   the development is over, use a hair dryer to dry the plate quickly or wait in room temperature
   until it is dry.
4. Visualization: Immerse the plate quickly in the straining solution (5% Sulfuric acid-ethanol); Use a
   hair dryer or let it dry naturally; Put the plate in an oven and heat it at 120˚C for 2 min; The
   Rf value of lactose is 0.12 – 0.18 (Optional);

## **5.3 Dual-Enzyme Catalysis Analysis**

**Table 12. Dual-Enzyme Catalysis components**

| Reagents | Components | Volume (μL) |
| --- | --- | --- |
| Substrate A | 5% Lactose solution | 1000 |
| Substrate B | Fresh milk (Filtered/centrifuged) | 1000 |
| T-Gal liquid enzyme | Maintain a specific activity unit | 100 |
| GOD liquid enzyme | Mix with T-Gal to a certain ratio 1:1 - 1:5 (10 µL – 50 µL) | 100-500 |
| PBS Buffer | Adjust the volume to 2mL (Change according to the actual volume) |  |

Use 5% lactose solution and fresh milk as reaction mixture, and add the 2 enzymes into the mixture. Then,
test the carbohydrate distribution using TLC test.

**Substrate:** 5% lactose solution/fresh milk.

**Enzyme ratio:** T-Gal : GOD = 1:1 to 1:3 (Used for gradient optimization).

**Reaction conditions:**

* Temperature: 25°C, 35°C, 45°C
* Time: 0, 20, 40, 60 min. Take a sample every 20 min.
* Water bath every sample for 5 min at 65˚C to stop the reaction; Centrifuge the samples at 5000 × g
  for 5 min;

#### TLC:

1. First, prepare the mobile phase (n-butanol : acetic acid : ethanol : water = 2:1:1:1), and pour it
   into a developing chamber (For about 0.5-1 cm thick). Pre-satiated the filter paper on the
   chamber walls, and wait 15-30 min to let it form a stable gas phase.
2. Spotting: First, cut a piece of silica gel plate, and use a pencil to mark a starting line (about
   1cm from the bottom edge); After sampling, centrifuge the sample at 10,000 × g for 1 min and
   spot with the supernatant (If the concentration is high, dilute it); Use a small pipette or 2 µL
   applicator to add the samples on different locations on the line; Every spot is about 1-2 mm in
   diameter, and quickly dry the samples by using a hair dryer (To prevent streaking).
3. Development: Put the TLC plate into the developing chamber (And perpendicular to the chamber), then
   add mobile phase (Make sure that the liquid level is lower than the starting line); Close the
   chamber, and wait until the solvent front is about 5-8 cm higher than the starting line; When
   the development is over, use a hair dryer to dry the plate quickly or wait in room temperature
   until it is dry.
4. Visualization: Immerse the plate quickly in the straining solution (5% Sulfuric acid-ethanol); Use a
   hair dryer or let it dry naturally; Put the plate in an oven and heat it at 120˚C for 2 min;

#### HPLC:

1. Sample Collection: At the preset time points of the dual-enzyme synergistic reaction, accurately
   aspirate 1.0 mL of the reaction solution as the test sample using a pipette.
2. Centrifugation for Impurity Removal: Transfer each aspirated sample to a clean centrifuge tube
   separately, and centrifuge at 15,000 rpm and 4°C for 15 minutes. Collect the supernatant.
3. Filtration for Clarification: Aspirate the centrifuged supernatant, pass it through a 0.22 μm
   cellulose acetate filter membrane, collect the clarified filtrate into a clean HPLC vial, cap
   and seal the vial, and set it aside for subsequent HPLC analysis.
4. HPLC System Equilibration: Connect the Prevail Carbohydrate ES chromatographic column for glucose
   detection system equilibration, pump the 65% (v/v) acetonitrile-water solution at a flow rate of
   1.0 mL/min. Set the column oven temperature to 35°C and the refractive index detector (RID)
   temperature to 35°C. Continue equilibrating the chromatographic column for at least 30 minutes
   until the detector baseline becomes stable. Replace with the Aminex HPX-87H ion-exchange column
   for lactose detection system equilibration. Pump the 5 mmol/L H₂SO₄ aqueous solution at a flow
   rate of 0.6 mL/min. Set the column oven temperature to 65°C and the detector temperature to
   35°C. Continue equilibrating the chromatographic column for at least 60 minutes until the
   baseline becomes stable.
5. Standard Curve Preparation: Inject the glucose and lactose standard working solutions into the HPLC
   system sequentially in the order of "from low concentration to high concentration", and record
   the chromatogram of each concentration of the standard. Draw the glucose and lactose standard
   curves respectively using the linear regression method, and calculate the regression equations.
6. Sample Detection: Inject each dual-enzyme reaction sample into the HPLC system separately. First,
   perform analysis under the glucose detection conditions, then switch to the lactose detection
   conditions for analysis. Record the chromatogram of each sample and the corresponding peak area.
7. Quantitative Calculation and Reaction Efficiency Analysis: Complete the quantitative calculation of
   glucose and lactose concentrations in the samples based on the standard curves, and analyze the
   efficiency of the dual-enzyme synergistic reaction through the time-concentration relationship.

## **5.4 Glucose Oxidase Activity Assay**

### 1. Formula of 1.5% Glucose solution

Weight 5 g of glucose and dissolve it into 100 mL of deionized water.

### 2. Experimental procedure

Add 150 µL of 5% glucose solution, 150µL of o-dianisidine solution, and 10µL of horseradish peroxidase
solution to the 96 well in sequent. Put the system in water bath at 25˚C to let it react for 10 min, and
add 10 µL of sample (Test solution) into the system. After the reaction, test the mixture using a
microplate reader at 460 nm wave length. Test the sample 1 time per 1 min, and test for 5 times. Under
the conditions of 25 ˚C and PBS buffer (pH = 6.5), we define that 1 GOD unit is needed to convert 1µmol
of glucose to gluconic acid and hydrogen peroxide per minute. People can also use spectrophotometer or
some kit instead of microplate reader.

{% endcall %} {% endblock %}

{% extends "layout.html" %} {% block title %}Results{% endblock %} {% block lead
%}Present the results of your project, along with a detailed analysis and
discussion of their significance. Also outline future plans and reflections on
the impact of your project.{% endblock %} {% block page\_content %} {% from
"/components/article-content.html" import articleContent %} {% call
articleContent("Results", [
{"content": "Overview","anchor": "#title\_1"},
{"content": "The Construction of Plasmid","anchor": "#title\_2", "children": [
{"content": "PCR amplification of β-T-Gal and GOD","anchor": "#level2\_1"},
{"content": "Linearization of pPICZαA","anchor": "#level2\_2"},
{"content": "Homologous Recombination of pPICZαA-β-T-Gal and pPICZαA-GOD","anchor": "#level2\_3"}
]},
{"content": "Protein Expression","anchor": "#title\_3", "children": [
{"content": "Linearization of Recombinant Plasmids","anchor": "#level2\_4"},
{"content": "Plasmid Transformation","anchor": "#level2\_5"},
{"content": "Detection of Recombinant Plasmids in Pichia pastoris","anchor": "#level2\_6"},
{"content": "SDS-PAGE Analysis of GOD and β-T-Gal","anchor": "#level2\_7"}
]},
{"content": "Function Test","anchor": "#title\_4", "children": [
{"content": "β-T-Gal Hydrolysis Activity Test","anchor": "#level2\_8"},
{"content": "GOD Activity Test","anchor": "#level2\_9"},
{"content": "TLC Test for β-T-Gal Activity","anchor": "#level2\_10"},
{"content": "TLC Analysis of Dual-Enzyme Synergy","anchor": "#level2\_11"},
{"content": "Quantitative Analysis of Dual-Enzyme Synergistic Products by HPLC","anchor": "#level2\_12"}
]},
{"content": "Discussion","anchor": "#title\_5"},
{"content": "References","anchor": "#title\_6"}
]) %}

# **Overview**

Lactose is composed of
β-D-glucose and β-D-galactose. For lactose to be absorbed by the human body, it must be hydrolyzed into
monosaccharides with the help of lactase and β-T-galactosidase. These enzymes are abundant in newborns;
however, their activity gradually decreases with age, a phenomenon known as lactase non-persistence
(LNP). Lactose intolerance refers to a condition where individuals experience digestive symptoms—such as
bloating, diarrhea, and flatulence—after consuming lactose-containing foods or beverages. According to
data from the National Institutes of Health (NIH), approximately 68% of the global population suffers
from lactose malabsorption (NIDDK, 2018) [1]. To address this issue, this project developed a
dairy product tailored for lactose-intolerant individuals. Specifically,
β-T-Galactosidase (β-T-Gal) and glucose oxidase
(GOD) were used to decompose
dissolved lactose in milk into prebiotics, including galactooligosaccharide (GOS) and glucuronic
acid.

**Outline**

**1.****The Construction
of****Pl****asmid**

1. 1.  PCRamplificationof
β-T-Gal
andGOD

1.2.
LinearizationofpPICZαA

1.3.
HomologousrecombinationofpPICZαA-β-T-Gal
andpPICZαA-GOD

**2.****Protein
Expression**

2.1.  Linearized
recombinant
plasmid

2.2.  Plasmidtransformation

2.3.  Detection of
RecombinantPlasmidin*Pichia**pastoris*

2.4.
SDS-PAGEanalysisofGODandβ-T-Gal

**3.****Function
Test**

3. 1.  β-T-Gal hydrolysis
activity test

3.2.  GODactivity test

3.3.  TLC test for β-T-Gal
activity

3.4.  TLC Analysis of
Dual-Enzyme Synergy

3.5.
Quantitativeanalysisofdual-enzymesynergisticproductsbyHPLC

# **1.****The****Construction****of****Plasmid**

## **1.1 PCR amplification of β-T-Gal and GOD**

PCR
amplification was performed to obtain the target genes encoding β-T-Galactosidase (β-T-Gal) and glucose
oxidase (GOD), with lengths of 2209 bp and 1294 bp, respectively. As shown in Fig. 1, distinct and sharp
bands were observed, which matched the expected sizes of the two target genes. This result confirms the
successful amplification of β-T-Gal and GOD.

![](https://static.igem.wiki/teams/5923/images/result-fig1.webp)

**Fig 1.****Gel Electrophoresis Validation of
PCR-Amplified
β-T-Gal and GOD**

## **1.2 Linearization of pPICZαA**

After
double digestion, linearized pPICZαA vector fragments were obtained. Gel electrophoresis (Fig. 2) showed
clear bands at the expected size, verifying both the successful linearization of the vector and the
correctness of the fragment length.

![](https://static.igem.wiki/teams/5923/images/result-fig2.webp)

**Fig 2. Gel
Electrophoresis Validation of Double-Digested pPICZαA**

## **1.3 Homologous Recombination of pPICZαA-β-T-Gal and pPICZαA-GOD**

Recombinant plasmids (after
homologous recombination) were transformed into competent *E. coli* DH5α cells using a standard
heat-shock transformation protocol. As shown in Fig. 3, zeocin-resistant colonies were clearly observed,
indicating that the recombinant plasmids had been successfully introduced into *E. coli* DH5α.
Since the LB solid medium was supplemented with zeocin, and the original *E. coli* DH5α strain is
inherently sensitive to zeocin, only *E. coli* cells harboring the recombinant plasmids (which
confer zeocin resistance) could survive and form colonies on this selective medium.

![](https://static.igem.wiki/teams/5923/images/result-fig3.webp)

**Fig 3. Heat-Shock
Transformation of** ***E. coli*** **DH5α**

Plasmids were extracted from cultured
*E. coli* DH5α cells. Gel electrophoresis (Fig. 4A) showed clear bands for the recombinant
plasmids pPICZαA-β-T-Gal and pPICZαA-GOD, with lengths significantly longer than that of the empty
pPICZαA vector—confirming the successful homologous recombination of the target genes into the vector.
To rule out the presence of random mutations that might impair protein function, the purified
recombinant plasmids were subjected to full-length sequencing by Beijing Tsingke Biotech Co., Ltd. The
sequencing results (Fig. 4B for pPICZαA-GOD and Fig. 4C for pPICZαA-β-T-Gal) confirmed that both
expression vectors were correctly constructed without mutations.

![](https://static.igem.wiki/teams/5923/images/result-fig4.webp)

**Fig 4. Colony PCR and Sequencing results of*****E.
coli*** **DH5α**

# **2. Protein Expression**

## **2.1 Linearization of Recombinant Plasmids**

Gel
electrophoresis of the treated plasmids showed distinct and sharp bands (Fig. 5A for pPICZαA-GOD and
Fig. 5B for pPICZαA-β-T-Gal). The lengths of these bands were consistent with the predicted sizes in the
plasmid maps (Fig. 5C for pPICZαA-β-T-Gal and Fig. 5D for pPICZαA-GOD), providing clear evidence of
successful plasmid linearization.

![](https://static.igem.wiki/teams/5923/images/result-fig5.webp)

**Fig 5. Linearization
of** **pPICZαA-GOD and** **pPICZαA-β-T-Gal**

## **2.2 Plasmid Transformation**

YPD
petri dishes were supplemented with zeocin, which specifically inhibits the growth of wild-type
*Pichia pastoris*. Thus, only *Pichia pastoris* cells that successfully took up the
recombinant plasmids (pPICZαA-GOD or pPICZαA-β-T-Gal, which carry zeocin resistance genes) could
survive. As shown in Fig. 6 (Fig. 6A for pPICZαA-β-T-Gal and Fig. 6B for pPICZαA-GOD), dense colonies
were observed on the YPD plates, confirming the successful transformation of recombinant plasmids
into *Pichia pastoris* host cells.

![](https://static.igem.wiki/teams/5923/images/result-fig6.webp)

**Fig 6.*****Pichia pastoris*** **Cultured on YPD Solid Medium**

## **2.3 Detection of Recombinant Plasmids in Pichia pastoris**

Plasmids were extracted from
*Pichia pastoris* colonies and subjected to PCR amplification. Gel electrophoresis (Fig. 7) was
used to verify the integrity of the recombinant plasmids (pPICZαA-GOD and pPICZαA-β-T-Gal) in the host
cells. The results showed that the lengths of the amplified fragments were consistent with the expected
sizes of the recombinant plasmids, indicating that the plasmid structure remained unchanged after
transformation into *Pichia pastoris*.

![](https://static.igem.wiki/teams/5923/images/result-fig7.webp)

**Fig 7. Yeast Colony
PCR Results**

## **2.4 SDS-PAGE Analysis of pPICZαA-β-T-Gal and pPICZαA-GOD**

β-T-Gal
and GOD have molecular weights of ~80 kDa and ~48 kDa, respectively. SDS-PAGE analysis was performed to
verify the expression of these two proteins. As shown in Fig. 8A (for GOD) and Fig. 8B (for β-T-Gal),
clear bands were observed at the expected molecular weights, confirming the successful expression of
both recombinant proteins.

![](https://static.igem.wiki/teams/5923/images/result-fig8.webp)

**Fig 8. SDS-PAGE
Analysis of GOD and β-T-Gal**

# **3****. Function Test**

## **3.1 β-T-Gal Hydrolysis Activity Test**

To
evaluate the catalytic activity of the purified β-T-galactosidase (β-T-Gal), this study utilized its
specific hydrolysis of o-nitrophenyl-β-D-galactoside (ONPG) to produce the yellow product o-nitrophenol
(ONP). Enzyme activity was quantified by measuring the characteristic absorbance of ONP at 420 nm. The
experimental method is briefly described as follows: Enzyme solutions of β-T-Gal at different
concentrations were reacted with ONPG substrate at 40°C and pH 7.0 for a specific duration. The reaction
was terminated by adding sodium carbonate solution, followed by immediate absorbance measurement using a
microplate reader. As shown in **Table 1**, three parallel experiments were conducted at each
concentration to ensure data reliability.

**Table
1. Absorbance measurement at 420 nm**

|  |  |  |  |
| --- | --- | --- | --- |
| **mg/ml** | **A420** | | |
| **0** | 0.032 | 0.034 | 0.031 |
| **1** | 0.071 | 0.078 | 0.077 |
| **2** | 0.084 | 0.089 | 0.091 |
| **5** | 0.126 | 0.133 | 0.132 |
| **10** | 0.232 | 0.239 | 0.239 |
| **15** | 0.305 | 0.289 | 0.295 |
| **20** | 0.296 | 0.299 | 0.298 |

 Results demonstrated that the
purified β-T-Gal exhibited significant catalytic activity. As shown in Fig. 9, the absorbance value of
the reaction system increased significantly with rising enzyme concentration, exhibiting a good linear
relationship between the two. This linear trend clearly confirmed that ONP production is proportional to
enzyme concentration, indicating the successful purification of β-T-Gal with highly efficient and
concentration-dependent catalytic activity.

![](https://static.igem.wiki/teams/5923/images/result-fig9.webp)

**Fig 9. Results of
β-T-Gal Hydrolysis Activity Test**

## **3.2 GOD Activity Test**

The
activity of purified glucose oxidase (GOD) was determined using a glucose oxidase (GOD)-peroxidase (POD)
coupled reaction system. The principle is as follows: GOD catalyzes the oxidation of glucose to produce
H₂O₂. Subsequently, POD catalyzes the decomposition of H₂O₂ in the presence of oxygen. The resulting
reactive oxygen species oxidize colorless o-anisidine to form a brownish-yellow product. The absorbance
of this product at a specific wavelength is positively correlated with GOD activity. A series of GOD
dilutions (Table 2) was measured for reaction absorbance using a Multiskan™ FC microplate
reader.

**Table 2. Absorbance
measurement at 500 nm**

|  |  |  |  |
| --- | --- | --- | --- |
| **mg/ml** | **A500** | | |
| **0** | 0.076 | 0.074 | 0.075 |
| **0.5** | 0.178 | 0.179 | 0.178 |
| **1** | 0.184 | 0.183 | 0.183 |
| **2.5** | 0.469 | 0.467 | 0.465 |
| **5** | 0.725 | 0.723 | 0.721 |
| **10** | 1.251 | 1.249 | 1.247 |
| **20** | 1.508 | 1.503 | 1.5 |

 Results demonstrated (Fig. 10)
that although the absorbance curve was not strictly linear, its overall trend of significant increase
with rising enzyme concentration clearly confirmed that the purified GOD retained potent catalytic
activity.

![](https://static.igem.wiki/teams/5923/images/result-fig10.webp)

**Fig 10. Results of the
GOD Hydrolysis Activity Test**

## **3.3 TLC Test for β-T-Gal Activity**

Thin-layer chromatography (TLC) was
used to assess β-T-Gal activity under two experimental conditions: (1) fixed β-T-Gal concentration with
gradient lactose concentrations; (2) fixed lactose concentration with gradient time. Fig 11 shows the
results of the lactose-gradient experiment: the migration distance of samples increased with increasing
lactose concentration. This indicates that when the substrate is insufficient and β-T-Gal is not
saturated, the lactose concentration acts as the limiting factor for the hydrolysis reaction, and the
amount of product increases with the increase in substrate. As the reaction time extends from 0 min to
60 min, there is little difference in the intensity of the "lactose substrate spot" on the TLC plate,
and no obvious distinction in the resolution of spots between adjacent time points. This may be because
β-T-Gal has completed the hydrolysis of most lactose within 15 min; the change in the amount of
substrate is minimal in the subsequent time, resulting in insignificant differences between spots at
adjacent time points and making it impossible to clearly distinguish the degree of hydrolysis. The time
gradient needs to be adjusted in subsequent experiments to enable such a distinction.

![](https://static.igem.wiki/teams/5923/images/result-fig11.webp)

**Fig 11. TLC Analysis
of β-T-Gal Activity**

At a
fixed enzyme concentration, increasing lactose concentration not only significantly elevated the yield
of the primary hydrolysis product glucose. Notably, in the high-concentration lactose system (5%),
galacto-oligosaccharides (GOS) were also observed to form alongside glucose. Moreover, GOS production
increased with rising lactose concentration, indicating that high substrate concentration promotes
β-T-Gal-catalyzed glycosylation reactions, thereby synthesizing GOS.

## **3.4 TLC Analysis of Dual-Enzyme Synergy**

To
investigate the efficiency of lactose hydrolysis under the synergistic action of β-T-Gal and GOD, we
comparatively analyzed the hydrolysis effects in both pure lactose solution and fresh milk. Thin Layer
Chromatography (TLC) results demonstrated that, under the synergistic action of the two enzymes, lactose
was hydrolyzed into glucose and galactooligosaccharides (GOS). Moreover, the hydrolysis effect in fresh
milk was identical to that in the pure lactose solution (Fig. 12). This confirms that the synergistic
effect of β-T-Gal and GOD is feasible in fresh milk.

![](https://static.igem.wiki/teams/5923/images/result-fig12.webp)

**Fig 12. TLC Results of
Dual-Enzyme (β-T-Gal + GOD) Synergistic Activity**

## **3.5 Quantitative Analysis of Dual-Enzyme Synergistic Products by HPLC**

To
quantitatively compare the decomposition efficiency of lactose in milk under the synergistic action of
dual enzymes (β-T-Gal and GOD), we employed High-Performance Liquid Chromatography (HPLC) to dynamically
monitor the decrease in lactose concentration and the increase in glucose concentration in milk during
the reaction. The experimental setup was as follows: β-T-Gal and GOD were mixed at a 1:1 ratio and added
to the milk system; the reaction was initiated under three temperature conditions (25°C, 37°C, and
42°C), and samples were taken at 10-minute, 20-minute, and 60-minute intervals after the start of the
reaction. The concentrations of lactose and glucose at each time point were determined by HPLC (Table 3
and Table 4), and the relevant results are shown in Fig. 13.

**Table 3. Lactose
Concentration Detected by HPLC**

|  |  |  |  |
| --- | --- | --- | --- |
| **Time (min)** | **25℃** | **37℃** | **42℃** |
| **0** | 3.5 | 2.7 | 2.4 |
| **10** | 2.6 | 2.1 | 2.5 |
| **20** | 2.5 | 1.7 | 1.4 |
| **60** | 2.1 | 2.1 | 1.2 |

**Table 4. Glucose
Concentration Detected by HPLC**

|  |  |  |  |
| --- | --- | --- | --- |
| **Time (min)** | **25℃** | **37℃** | **42℃** |
| **0** | 0 | 0 | 0 |
| **10** | 0 | 2 | 3.5 |
| **20** | 0 | 3.4 | 6.1 |
| **60** | 3.6 | 5.6 | 6.7 |

In terms of the effect of temperature
on reaction efficiency, both the lactose decomposition rate and glucose production rate at 42°C were
significantly higher than those at 25°C and 37°C. **This indicates that 42°C is the optimal reaction
temperature for the dual-enzyme synergistic system to catalyze lactose decomposition in milk.** A
further analysis of the reaction kinetic characteristics at the optimal temperature (42°C) revealed that
the first 20 minutes of the reaction were the stage with the fastest glucose production rate. This
phenomenon suggests that during the initial stage of dual-enzyme synergy (the first 20 minutes), the
binding efficiency between the enzymes and the substrate (lactose) is the highest, and the catalytic
activity is at its peak, allowing lactose to be rapidly decomposed into glucose. However, as the
reaction proceeds, the glucose production rate gradually slows down, which may be attributed to the
decrease in substrate (lactose) concentration, the feedback inhibition caused by the accumulation of the
product (glucose), or a slight decline in enzyme activity over the reaction time.

![](https://static.igem.wiki/teams/5923/images/result-13-1.webp)

**Fig 13. Quantitative
Analysis of Dual-Enzyme Synergistic Products by HPLC**

In summary, this study
determined that the optimal reaction conditions for dual-enzyme synergistic lactose decomposition are
42°C, identifying the initial 20 minutes as the critical phase for high-efficiency conversion. During
this period, enzyme-substrate binding is highly efficient, catalytic activity reaches its peak, and
rapid lactose degradation is achievable. Therefore, controlling the reaction at 42°C for 20 minutes
represents an optimized strategy balancing efficiency and energy consumption.

# **Discussion**

This
study successfully constructed the recombinant expression plasmids pPICZαA-β-T-Gal and pPICZαA-GOD, and
achieved the recombinant expression and purification of both enzymes in *Pichia pastoris*.
Functional validation results demonstrated that the purified β-T-Gal and GOD possess high catalytic
activity. Furthermore, the dual-enzyme system exhibited significant synergistic effects within a milk
matrix, efficiently decomposing lactose into glucose and generating galacto-oligosaccharides (GOS) with
prebiotic potential. This research provides crucial enzyme resources and process parameters for
developing functional dairy products tailored for lactose-intolerant individuals.

Firstly, protein expression and
purification form the foundation for studying enzymatic properties. SDS-PAGE analysis revealed clear
bands at the expected molecular weights (Fig. 8), confirming the successful acquisition of highly pure
β-T-Gal and GOD. Subsequent enzyme activity assays further verified the functional integrity of the
purified proteins. The hydrolysis activity curve of β-T-Gal (Fig. 9) showed a good linear relationship
between its catalytic activity and enzyme concentration, which is consistent with classical enzyme
kinetics theory, indicating the enzyme's stability under the tested conditions without significant
aggregation or inactivation. Similarly, the GOD activity test (Fig. 10), while not perfectly linear,
demonstrated a significant increase in absorbance with rising enzyme concentration, unequivocally
confirming its ability to oxidize glucose. This non-linear relationship might stem from substrate
diffusion limitations or slight product inhibition effects in high-concentration enzyme solutions, which
are common phenomena in coupled enzyme reaction systems.

Secondly, TLC analysis revealed the
diversity in β-T-Gal's catalytic behavior, demonstrating both hydrolytic and transglycosylase
activities. At low lactose concentrations, hydrolysis dominated, yielding primarily glucose and
galactose (Fig. 11). However, when the lactose concentration increased to 5%, the generation of GOS was
clearly observed (Fig. 11), and its yield increased with increasing substrate concentration. This
phenomenon aligns with the transglycosylation reaction mechanism of β-T-galactosidase: under high
substrate concentrations, the glycosylated intermediate formed by the enzyme and lactose is more likely
to be attacked by another lactose molecule rather than a water molecule, leading to GOS formation. This
finding holds significant application value, as GOS is a recognized prebiotic that can selectively
promote the proliferation of beneficial bacteria in the gut. This implies that our dual-enzyme system
can not only eliminate lactose but also enrich the product with beneficial components *in situ*,
thereby enhancing its added value.

Most
crucially, HPLC quantitative analysis confirmed the synergistic action of β-T-Gal and GOD in the milk
system and identified the optimal reaction conditions. The catalytic efficiency of the dual-enzyme
system at 42°C was significantly higher than at 25°C and 37°C (Fig. 13), indicating that 42°C is the
ideal temperature point balancing the optimal activities of both enzymes. The reaction kinetic curve
showed a rapid increase in glucose production within the first 20 minutes, followed by a slowed rate.
This typical biphasic kinetic profile can be attributed to several factors: the high initial rate is due
to abundant lactose substrate and enzymes being at their peak activity; as the reaction progresses,
substrate depletion and product (glucose) accumulation may cause feedback inhibition on both GOD and
β-T-Gal, while the enzyme molecules themselves might also experience slight inactivation due to
prolonged thermal incubation. Therefore, controlling the reaction at 42°C for 20 minutes represents an
ideal process window for achieving high-efficiency lactose decomposition while balancing energy
consumption.

Although this study yielded
encouraging results, certain limitations exist, pointing the way for future research. The current TLC
analysis allows only qualitative/semi-quantitative assessment of products. Subsequent studies should
include authentic standards of GOS, glucose, and glucuronic acid as internal references for precise
quantification of the target prebiotics. Furthermore, this study tested the synergy of β-T-Gal and GOD
only at a 1:1 ratio. Optimizing the enzyme ratio (e.g., 2:1, 1:2) could potentially further enhance the
overall system efficiency and reduce costs. Simultaneously, refining the temperature gradient (e.g.,
35-50°C) would help pinpoint the optimal temperature more accurately. Finally, evaluating the stability
and shelf-life of the developed dairy product and ultimately validating its efficacy in alleviating
lactose intolerance symptoms through clinical trials are critical steps for translating these laboratory
findings into a practical product.

# **References**

[1]  U.S. Department of
Health and Human Services. (2018, February). *Definition & facts for lactose
intolerance*. National Institute of Diabetes and Digestive and Kidney Diseases
(NIDDK)

{% endcall %} {% endblock %}