iGEM 2025 — Team buct-china

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{% 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 %}

# NOTEBOOK

#### Notebook

[![arrow](https://static.igem.wiki/teams/5851/safty/green.webp)Cycle1](#cycle1)
[![arrow](https://static.igem.wiki/teams/5851/safty/green.webp)Cycle2](#cycle2)

#### Reference

[![arrow](https://static.igem.wiki/teams/5851/safty/yellow.webp)Reference](#reference)
[![arrow](https://static.igem.wiki/teams/5851/safty/yellow.webp)Back to top](#top)

## Cycle1

### 1.1

**Experiment dates:** 6.1-6.7

**Experiment personnel:** Xu Yiran, Wei Sihui

**Experiment objective:** Finalize the experimental plan, locate target gene sequences, outsource plasmid synthesis to a company, and chemically transform into BL21(DE3) competent cells for protein expression.

### Experiment materials:

| Name | Formula / Description |
| --- | --- |
| Target gene sequence | Downloaded from NCBI and sequence optimized |
| Vector backbone | General-purpose vector |
| Ligation/Cloning reagents | For construction of recombinant plasmid |
| LB medium | Yeast extract 5 g/L; tryptone 10 g/L; NaCl 10 g/L |
| Glycerol | For preparation of storage stocks |
| BL21(DE3) | Purchased from supplier |
| Antibiotic | Kanamycin 50 µg/mL |
| Routine laboratory consumables (e.g., sterile pipette tips, etc.) |  |

### Experimental instruments:

| Name | Description |
| --- | --- |
| 42 °C water bath | For heat shock |
| Biosafety cabinet | Provides sterile working environment |
| 37 °C shaking incubator | For culturing transformed bacteria |
| 37 °C incubator | For culturing single colonies |
| -80 °C freezer | For bacterial storage |
| Analytical balance | For weighing |

### Experimental procedure:

**1.Plasmid construction:** Search the literature to select genes. Download the CGTase gene sequence from NCBI, optimize the sequence, and submit it to a gene synthesis company.

**2.Transformation:** Thaw BL21(DE3) competent cells on an ice box; in the biosafety cabinet add plasmid DNA (100 ng), pipette up and down to mix, and incubate on ice for 30 min (adjust according to plasmid concentration). Place the suspension in a 42 °C water bath for heat shock for 90 s, then immediately put on ice for 2 min. In the biosafety cabinet add 200 µL LB and incubate at 37 °C on a shaker for 60 min. Plate the culture onto plates containing Kan (50mg/mL) by the spread plating method, seal and incubate inverted at 37 °C for approximately 12 h.

### Experimental procedure:

By literature search and comparison, the β-CGTase sequence from Bacillus sp. (strain 1011) met our engineering requirements. After locating the related sequence in the NCBI database, the Pet-28a-β-CGTase-1011 plasmid was successfully constructed.

### Table 1: CGTase from different organism

| Organism | Enzyme | Optimal Temperature (°C) | Reaction Time | Substrate Type | Degradation Products | Degradation Efficiency | Enzyme Type |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Bacillussp.Tlg | CGTase | 37 | 72 h | Soluble starch | α/β CD | - | Transferase |
| B.agaradhaerens KSU-A11c | CGTase | 37 | 48 h | Potato starch | α/β/γ CD | 26.4% yield | Transferase |
| B.agaradhaerens LS-3Ca | CGTase | 37 | 30 h | Soluble starch | α/β CD | 50% yield | Transferase |
| B.cereus SS2f | CGTase | 38 | 24 h | Potato starch | β CD | - | Transferase |
| B.circulans ATCC 21783a | CGTase | 40 | 24 h | Soluble starch | β CD | 75.5% yield | Transferase |
| B.firmus 7Ba | CGTase | 37 | 120 h | Soluble starch | β CD | 91.6% yield | Transferase |
| B.firmus NCIM 5119a | CGTase | 65 | - | Raw starch | α/β/γ CD | 64% yield | Transferase |
| B.haloduransa | CGTase | 37 | 72 h | Soluble starch | β CD | 49.44% yield | Transferase |
| B.halophilus BIO-12Ha | CGTase | 37 | 24-30 h | Potato starch | β/γ CD | 50-53% yield | Transferase |
| B.lehensisb | CGTase | 37 | 24 h | Rice starch | β CD | 46.6% yield | Transferase |
| B.macerans 15a | CGTase | 37 | 48 h | Potato starch | α CD | 66.3% yield | Transferase |
| B.pseudalcaliphilus SSBa | CGTase | 40 | 24 h | Soluble starch | β/γ CD | 62% yield | Transferase |
| B.pseudalcaliphilus 20RFa | CGTase | 60 | - | Raw starch | β/γ CD | 63% yield | Transferase |
| B.sphaericus 41a | CGTase | 37 | 48 h | Soluble starch | β CD | 31% yield | Transferase |
| B.stearothermophilus NO2d | CGTase | 37 | - | - | - | - | Transferase |
| B.subtilis WB800h | CGTase | 37 | 24 h | Corn starch | β CD | - | Transferase |
| Bacillus sp.NR5 UPMe | CGTase | 37 | 24 h | - | β CD | 85.2% yield | Transferase |

aData adopted from Lim et al. 2021, bElbaz et al. 2015, cIbrahim et al. 2011, dXiumei et al. 2020, eNik-Pa et al. 2020, fUpadhyay et al. 2020, gLiu et al. 2022, hCrozatti et al. 2023

![Figure 1: Retrieval of gene sequences](https://static.igem.wiki/teams/5851/notebook/p1.avif)

Fig. 1: Retrieval of gene sequences

![Figure 2: Construction of Pet-28a-β-CGTase-1011](https://static.igem.wiki/teams/5851/notebook/p2.avif)

Fig. 2: Construction of Pet-28a-β-CGTase-1011

### xperimental notes:

**1.** When removing competent cells, avoid touching the tube wall to prevent temperature fluctuations that affect transformation efficiency.

**2.** The heat-shock operation must strictly control temperature and time (60 °C, 90 seconds); inaccurate timing will significantly reduce transformation efficiency.

### 1.2

**Experiment dates:** 6.8-6.14

**Experiment personnel:** Sun Runze, Lu Xuxian, Kuang Xintong

**Experiment objective:** Inoculate the transformed cells into tube liquid culture medium (20 mL) containing kanamycin at 0.5 mol/L, incubate overnight on a 30 °C shaker; after 24 h transfer to a flask in the biosafety cabinet for scale-up culture; after 8 h induce protein expression with 1 mM IPTG; purify and desalt to obtain the target enzyme protein.

### Experiment materials:

| Name | Formula / Description |
| --- | --- |
| LB medium | Yeast extract 5 g/L; tryptone 10 g/L; NaCl 10 g/L |
| Antibiotic | Kanamycin 50 µg/mL |
| IPTG | Inducer |
| Buffer A (working / lysis) | 20 mM Tris-HCl pH 7.5; 250 mM NaCl |
| Buffer B (wash 1) | Buffer A + 20 mM imidazole |
| Buffer C (wash 2 / target) | Buffer A + 50 mM imidazole |
| Lysis buffer | Buffer A + 1 mM PMSF |
| Storage buffer | 20 mM Tris-HCl pH 7.5; 150 mM NaCl |
| Filtration | 0.22 µm syringe filter |
| Ni column | For protein purification |
| SDS-PAGE | Electrophoresis gel |
| Desalting column | For protein desalting |
| Amicon Ultra | For protein concentration |

### Experimental instruments:

| Name | Description |
| --- | --- |
| UV-Vis spectrophotometer | For OD measurement |
| Temperature-controlled shaker | For culture growth |
| Biosafety cabinet | Provides sterile working environment |
| Centrifuge | For collecting cells |
| Cell disruptor | For cell lysis |
| Ice box | Maintain low temperature to prevent protein denaturation |
| Protein purification system | For protein purification |
| Electrophoresis apparatus | Quality control |
| Analytical balance | For weighing |

### Experimental procedure:

**1.Seed culture and transfer**

**Seed preparation:** Using a 10 µL tip, pick multiple single colonies from a plate into 5 mL LB + 5 µL Kan; incubate at 37 °C, 200 rpm for 3–4 h.

**Transfer:** Measure seed OD600; calculate to inoculate 400 mL LB so that final OD600 = 0.1. Inoculate at 1% into 400 mL LB containing 50 µg·mL⁻¹ Kan; incubate at 37 °C, 200 rpm until OD600 reaches 0.4–0.6. (Remove 1 mL culture in the biosafety cabinet and measure OD600 in the spectrophotometer.)

**2.Induction of expression**

Add 1 M IPTG. (Inducer added at 1‰.) Reduce temperature to 16–20 °C; induce at 160–200 rpm for 18–20 h. Record induction time/temperature.

**3.Cell harvest**

Use multiple 50–100 mL centrifuge tubes to accumulate culture; centrifuge at 4 °C, 8,000 rpm for 15 min to collect cells.

**4.Resuspension / lysis**

Wash cell pellets twice with Buffer A.

**Resuspend:** add 10 mL Buffer A per 1 g wet cell mass.

**Lysis (ice bath):** 300 W sonication, 3 s on / 2 s off cycles, total 20 min (adjust according to cell mass).

**Centrifuge:** 4 °C, 10,000 rpm, 30 min; retain supernatant.

**Filtration:** filter through 0.45 µm syringe filter.

**SDS-PAGE:** add reducing sample buffer, heat at 95 °C for 5 min; load 5–20 µL per lane; run at 220 V; stain (Coomassie / silver stain); save images and filenames.

**5.Ni-NTA affinity purification**

**Column preparation:** resin capacity 40 mg protein / mL resin (calculate maximum load by column volume); install Ni column at 0.5 mL/min, then adjust flow to 5 mL/min. Wash with deionized water at 5 mL/min until baseline; equilibrate with Buffer B and Buffer A to baseline.

**Sample loading:** pump A1 load at 5 mL/min (leave 2–3 mL dead volume to avoid air); after baseline recovery apply 5% Buffer B (10 mL) to remove impurities until baseline.

**Elution / collection:** set elution gradient or introduce 100% Buffer B; begin collecting when UV (mAu) rises (10–15 mL fractions); label fraction numbers.

**QC:** run SDS-PAGE on elution fractions, save images and record corresponding UV peaks.

**6.Desalting / buffer exchange**

Connect desalting column; wash with deionized water at 8–10 mL/min until baseline; after installation load sample at 8 mL/min (maximum load volume 15 mL); wash with Buffer C and collect when UV increases (approximately 20 mL); stop collection when conductivity (mS/cm) rises. Record conductivity, UV peaks and fraction numbers.

**SDS-PAGE:** add reducing sample buffer, heat at 95 °C for 5 min; load 5–20 µL; stain (Coomassie / silver stain); save images and filenames.

**7.Protein concentration and QC**

Use Amicon Ultra (appropriate MWCO) to concentrate protein to 1–5 mg/mL; measure concentration by Bradford assay and record.

### Experimental results:

The plasmid was successfully introduced into cells and target protein expression was induced; the target protein was obtained after purification.

![Figure 3-4: SDS-PAGE gel images of the CGTase mutant β-CGTase-1011 before and after purification](https://static.igem.wiki/teams/5851/notebook/p3-4.avif)

Fig. 3-4: SDS-PAGE gel images of the CGTase mutant β-CGTase-1011 before and after purification

![Figure 5: First protein purification profile](https://static.igem.wiki/teams/5851/notebook/p5.avif)

Fig. 5: First protein purification profile.

### xperimental notes:

**1.** After weighing, promptly tidy the bench, return reagents to their storage locations, and maintain a clean workspace.

**2.** When adjusting the pH to 7.5, add NaOH dropwise as the value approaches the target to avoid overshooting.

### 1.3

**Experiment dates:** 6.15–6.21

**Experiment personnel:** Xu Yiran, Fang Yingshan, Xiang Yuhang

**Experiment objective:** Perform reactions using a one-pot method, construct a phenolphthalein calibration curve, detect product formation by HPLC, and evaluate reaction yield by chromatographic peak area. WhExperimental procedure:en using PPA and laboratory starch as substrates, product yield was low, primarily due to insufficient formation of CDs. Therefore, an iterative optimization strategy is planned to improve yield.

### Experiment materials:

| Name | Formula / Description |
| --- | --- |
| Purified CGTase samples | Peptone 16 g/L, yeast extract 10 g/L, NaCl 5 g/L |
| SYPRO® Orange dye | 5000× concentrate in DMSO |
| Storage buffer | 20 mM Tris-HCl pH 7.5; 150 mM NaCl |

### Experimental instruments:

| Name | Description |
| --- | --- |
| UV-Vis spectrophotometer | For OD measurements |
| HPLC system | Establish linear relationship between concentration and peak area |
| Analytical balance | For weighing |
| Real-Time PCR Thermocycler |  |
| Small centrifuge | Quick spin-down of bubbles |
| Chromatography column / detector |  |

![Figure 6: Desalting profile of the target protein](https://static.igem.wiki/teams/5851/notebook/p6.avif)

Fig. 6: Desalting profile of the target protein.

### Item / Description

| Item | Description |
| --- | --- |
| Aminopropyl column (NH2) | 5020-16628 InertSustain NH2, 5 µm, 4.6 × 250 mm |
| Detector (NH2) | RID (refractive index detector) |
| Mobile phase (NH2) | 75% acetonitrile + 25% (0.1% formic acid in water) |
| Flow rate (NH2) | 0.8 mL/min |
| Injection volume (NH2) | 10 µL |
| Conditions (NH2) | Isocratic elution, column oven turned off (Turning off the column oven) |

### Experimental procedure:

**1.Preparation of HPLC calibration curves:**

Prepare α/β/γ standards by serial dilution (concentrations: 0, 1, 2.5, 5, 10, 15, 20 mg/mL) as follows:

Accurately weigh 0.312 g sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O) and 0.0716 g disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) into a beaker, add 80 mL ultrapure water, and stir until fully dissolved. Measure pH precisely with a pH meter and adjust to pH = 6.00 using 0.2 M NaOH and 0.2 M HCl. Transfer the pH-adjusted solution to a 100 mL volumetric flask, rinse the beaker several times with ultrapure water and transfer the rinses into the volumetric flask, and dilute to 100 mL with ultrapure water; mix thoroughly and set aside.

Accurately weigh 400 mg β-cyclodextrin (β-CD) powder, transfer to a 15 mL centrifuge tube, add approximately 8 mL of phosphate buffer, mix thoroughly, transfer all to a 10 mL volumetric flask, rinse the original container several times with small volumes of buffer and combine rinses in the volumetric flask, then dilute to the 10 mL mark with buffer and mix thoroughly to obtain a 40 mg/mL β-CD stock solution.

| Gradient (mg/mL) | Phosphate buffer (µL) | Cyclodextrin stock (µL) |
| --- | --- | --- |
| 0 | 1000 | 0 |
| 1 | 975 | 25 |
| 2.5 | 937.5 | 62.5 |
| 5 | 850 | 125 |
| 10 | 750 | 250 |
| 15 | 625 | 375 |
| 20 | 500 | 500 |

**2.Run the HPLC, record retention times (RT) and peak areas, and construct single-component calibration curves (concentration vs. peak area).**

**3.Preparation of mixed HPLC calibration curves:**

Prepare mixed α/β/γ standards for multivariate calibration using HPLC.

**4.Preparation of phenolphthalein calibration curve:**

Prepare phenolphthalein standards by serial dilution (concentrations: 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5 mg/mL) as follows:

| Gradient (mg/mL) | Phosphate buffer (µL) | Cyclodextrin stock (µL) |
| --- | --- | --- |
| 0 | 1000 | 0 |
| 0.25 | 993.75 | 6.25 |
| 0.5 | 987.5 | 12.5 |
| 0.75 | 981.25 | 18.75 |
| 1 | 975 | 25 |
| 1.5 | 962.5 | 37.5 |
| 2.5 | 937.5 | 62.5 |

**5.Prepare 0.03 M NaOH solution: accurately weigh 0.12 g NaOH solid, dissolve in water and dilute to 100 mL.**

Prepare 0.02% (w/v) phenolphthalein solution: accurately weigh 0.02 g phenolphthalein solid, dissolve in 50 mL absolute ethanol, then dilute to 100 mL with ultrapure water.

**6.Color development: take 250 µL of the diluted standard, add 125 µL of 0.02% phenolphthalein solution and mix, then add 875 µL of 0.03 M NaOH solution, incubate at room temperature for 20 minutes, measure OD550 according to laboratory standard color development procedures and save data. Construct the concentration-absorbance calibration curve and record linearity / range /error.**

**7.Measurement of Tm values**

Figure 14: HPLC quantitative data table for α-cyclodextrin produced by β-CGTase-1011.

**(1) Commercial enzyme (Novozymes) — activity assay (HPLC time course)**

Temperature: 60 °C.

Total volume: 1000 µL (1 mL).

Substrate: Soluble starch 15 mg/mL (i.e., 15 mg dissolved in 1 mL carrier; see pretreatment for details).

Buffer/sample solvent: ddH2O, 1 mL (per the pretreatment below).

Pretreatment: Boil the starch substrate for 15 min (gelatinize); place 900 µL of substrate at 60 °C for 10 min, then add enzyme solution.

Enzyme (Commercial): Novozymes commercial enzyme 100 µL (added to 900 µL matrix to form a 1 mL reaction).

Sampling time points: 0 h, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h.

Termination: Boil 15 min (enzyme inactivation).

Sample processing: Centrifuge/filter and inject to HPLC.

**(2) Our enzyme — single timepoint (immediate inactivation)**

**Assay conditions (single timepoint):**

Temperature: 60 °C.

Total volume: 1000 µL.

Substrate: Soluble starch 15 mg/mL (15 mg).

Pretreatment: Boil 15 min; place 900 µL substrate at 60 °C for 10 min.

Enzyme amount: β-CGTase-1011 (0.58 mg/mL), 5 µL (i.e., 2.9 µg enzyme added to the reaction).

Reaction times: 0 min (i.e., immediately inactivate by boiling for 15 min after enzyme addition), 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, 120 min.

**(3) The detection of Tm of β-CGTase-1011**

**1.Prepare SYPRO Orange 50x working solution**

Under light-protected conditions, add 2.5 µL SYPRO Orange (5000x) to 247.5 µL deionized water → 50× working solution. Mix, aliquot small volumes and refrigerate or use immediately. Avoid repeated freeze-thaw and prolonged light exposure.

**2.Sample preparation**

Target final reaction volume: 25 μL.

Per-well composition:

Protein solution (adjusted to the desired final molar concentration): 22.5 µL.

50× SYPRO Orange: 2.5 µL (final 5x).

If adding ligand/small molecule, dissolve the ligand in buffer or DMSO, adjust to required concentration and ensure final DMSO < 2%.

Example: if protein = 0.58 mg/mL = ~7.7 µM, use 22.5 µL protein + 2.5 µL 50x dye → total 25 µL; protein concentration in the well remains ~7.7 µM (slight dilution).

Include 2-3 blank wells (buffer + dye); perform technical triplicates, with ≥3 wells per condition to reduce noise.

**3.Plate handling**

Add samples to a black qPCR plate, avoid bubbles; if bubbles form, briefly centrifuge (1-2 k x g, 10-15 s). Seal with optical sealing film, press edges firmly. Avoid leakage. Place the plate into the instrument (ensure instrument filters are compatible with SYPRO Orange: Ex≈ 470 nm / Em ≈ 570 nm). Minimize exposure to strong light and operate quickly to avoid fluorescence drift.

**4.Instrument parameter settings (based on provided parameters + recommended adjustments)**

Temperature range: 20 °C → 95 °C.

Ramp rate: 1.0 °C/min.

Read frequency: record fluorescence every 0.2-0.5 °C.

Excitation power/gain: 80%.

Lid/heating cover: usually off or set with a low temperature differential (to prevent lid temperature >> block, which can cause bubbles/evaporation).

**5.Run and record**

Start the run and record fluorescence vs. temperature curves. Typically, 20→95 °C at 1° C/min takes approximately ~75 min.

**(4) The detection of Tm of Novozymes NV commercial enzyme**

Dilute the commercial protein to 0.4 mg/mL and measure Tm.

**(5) Assemble a 500 µL reaction system:**

add 450 µL 0.5 mM Na2HPO4 as buffer (pH = 8), add 15 mg PET plastic powder to reach 30 g/L, add 15 mg soluble starch, add 50 µL Turbo-PETase to reach 2 mg/g, add β-CGTase-1011 5 µL (protein concentration 0.58 mg/mL), incubate in a 60 °C water bath for 1 h, 2 h and 3 h. After reaction, add an equal volume of methanol to inactivate; take 5 µL reaction sample, dilute 20× with water and inject to reversed-phase C18 HPLC (UV detection at 260 nm) to determine PPA and MHET formation. Analyze the reaction mixture to determine α, β and γ cyclodextrin production.

**(6) Assemble a 500 µL reaction system:**

add 450 µL 0.5 mM Na2HPO4 as buffer (pH = 8), add 15 mg PET plastic powder to reach 30 g/L, add 15 mg soluble starch, add 50 µL Turbo-PETase to reach 2 mg/g, add β-CGTase-1011 5 µL (protein concentration 0.58 mg/mL), incubate in a 65 °C water bath for 1 h, 2 h and 3 h. After reaction, add an equal volume of methanol to inactivate; take 5 µL reaction sample, dilute 20× with water and inject to reversed-phase C18 HPLC (UV detection at 260 nm) to determine PPA and MHET formation. Analyze the reaction mixture to determine α, β and γ cyclodextrin production.

### 8.One-pot reaction:

To the reaction system add 15 mg PET plastic powder, 15 mg soluble starch, 450 µL Buffer — 100 mM Na2HPO4, 50 µL Turbo-PETase (concentration 0.6 mg/mL), 5 µL β-CGTase-1011 (concentration 0.58 mg/mL), adjust pH to 8, incubate in a 65 °C water bath for 1 h. After adding an equal volume of anhydrous methanol to inactivate, ensure the total reaction liquid is diluted 20×; therefore, inject 10 µL reaction liquid + 90 µL water to HPLC. The detected product exhibits phosphorescent material properties; lyophilize to obtain product PPA-CD.

### Experimental results:

Successfully established α-CD, β-CD, γ-CD standard curves. Thermal stability and activity assays indicate β-CGTase-1011 outperforms the commercial enzyme.

### Table 2: Phenolphthalein assay absorbance — β-CD concentration data Phenolphthalein assay standard for cyclodextrin

| Cyclodextrin concentration / mg | Absorbance A550 |
| --- | --- |
| 0.00 | 0.9918 |
| 0.25 | 0.9325 |
| 0.50 | 0.9016 |
| 0.75 | 0.8748 |
| 1.00 | 0.8291 |
| 1.50 | 0.7598 |
| 2.00 | 0.6897 |
| 2.50 | 0.6376 |

![Figure 7: Standard curve of β-CD measured by the phenolphthalein method](https://static.igem.wiki/teams/5851/notebook/p7.avif)

Fig. 7: Standard curve of β-CD measured by the phenolphthalein method

![Figure 8: Color and concentration images of bacterial cultures measuring β-CD by the phenolphthalein method](https://static.igem.wiki/teams/5851/notebook/p8.avif)

Fig. 8: Color and concentration images of bacterial cultures measuring β-CD by the phenolphthalein method

![Figure 9: HPLC chromatogram of α-, β-, γ-cyclodextrins for standard curves](https://static.igem.wiki/teams/5851/notebook/p9.avif)

Fig. 9: HPLC chromatogram of α-, β-, γ-cyclodextrins for standard curves

![Figure 10: HPLC quantitative analysis calibration curves for α-, β-, γ-cyclodextrins](https://static.igem.wiki/teams/5851/notebook/p10.avif)

Fig. 10: HPLC quantitative analysis calibration curves for α-, β-, γ-cyclodextrins

![Figure 11: DSF plot of β-CGTase-1011 Tm](https://static.igem.wiki/teams/5851/notebook/p11.avif)

Fig. 11: DSF plot of β-CGTase-1011 Tm

![Figure 12: HPLC chromatogram of products (cyclodextrin mixture) catalyzed by β-CGTase-1011](https://static.igem.wiki/teams/5851/notebook/p12.avif)

Fig. 12: HPLC chromatogram of products (cyclodextrin mixture) catalyzed by β-CGTase-1011.

![Figure 13: HPLC quantitative data table for α-cyclodextrin produced by β-CGTase-1011](https://static.igem.wiki/teams/5851/notebook/p13.avif)

Fig. 13: HPLC quantitative data table for α-cyclodextrin produced by β-CGTase-1011.

### Table 3: HPLC quantitative data table for α-cyclodextrin produced by β-CGTase-1011.

| Time/h | Peak Area | Concentration(mg/ml) | Conversion rate (100%) |
| --- | --- | --- | --- |
| 0 | 0 | 0 | 0 |
| 0.5 | 10772 | 0.28880288 | 1.925352533 |
| 1 | 11390 | 0.295232744 | 1.968218296 |
| 1.5 | 13891 | 0.321253928 | 2.141692851 |
| 2 | 10204 | 0.282893231 | 1.885954873 |
| 2.5 | 12396 | 0.305699482 | 2.037996546 |
| 3 | 12814 | 0.310048484 | 2.066989894 |
| 3.5 | 12256 | 0.304242878 | 2.028285855 |
| 4 | 16648 | 0.349938615 | 2.332924097 |
| 4.5 | 13013 | 0.312118942 | 2.080792947 |
| 5 | 14240 | 0.324885032 | 2.165900216 |
| 5.5 | 12100 | 0.302619806 | 2.017465371 |

![Figure 14: HPLC chromatogram of products (cyclodextrin mixture) catalyzed](https://static.igem.wiki/teams/5851/notebook/p14.avif)

Fig. 14: HPLC chromatogram of products (cyclodextrin mixture) catalyzed

![Figure 15a: Enzyme activity assay at 60 °C (part 1)](https://static.igem.wiki/teams/5851/notebook/p15-1.avif)
![Figure 15b: Enzyme activity assay at 60 °C (part 2)](https://static.igem.wiki/teams/5851/notebook/p15-2.avif)

Fig. 15: Enzyme activity assay at 60 °C

![Figure 16: Enzyme activity assay at 65 °C](https://static.igem.wiki/teams/5851/notebook/p16.avif)

Fig. 16: Enzyme activity assay at 65 °C

**Experimental notes:** Ensure traceability for each step during the experimental process.

## Cycle2

### 2.1

**Experiment dates:** 6.22-6.28

**Experiment personnel:** Xu Yiran, Wei Sihui, Su Yanyan

**Experiment objective:** To review the literature, collect, and collate enzyme sequence information from various sources to support subsequent enzyme screening and engineering.

**Experimental procedure:** This involved searching NCBI for CGTase enzymes and their amino acid and gene sequences. A preliminary activity assessment was performed using molecular docking (e.g., HADDOCK) and molecular dynamics simulations (e.g., Gaussian). High-activity types were selected, and signal peptides and cleavage sites were predicted using `https://services.healthtech.dtu.dk/services/SignalP-6.0/`. Genes were designed using `https://blast.ncbi.nlm.nih.gov/Blast.cgi`, and plasmids were subsequently produced by a company.

### 2.2

**Experiment dates:** 6.29-7.5

**Experiment personnel:** He Shunkang, Wang Shuhan, Xiang Yuhang

**Experiment objective:** To continue the literature review to obtain additional enzyme sequences and enrich the candidate enzyme library to support multiple rounds of screening and comparison.

**Experimental results:** The experiment successfully identified β-CGTase-K647E, with subsequent experimental validation planned.

![Figure 17: Search results for β-CGTase enzymes from different source](https://static.igem.wiki/teams/5851/notebook/p17.avif)

Fig. 17: Search results for β-CGTase enzymes from different source

![Figure 18: Multiple sequence alignment of key catalytic amino acid residues of β-CGTase enzymes from different sources](https://static.igem.wiki/teams/5851/notebook/p18.avif)

Fig. 18: Multiple sequence alignment of key catalytic amino acid residues of β-CGTase enzymes from different sources

### 2.3

**Experiment dates:** 7.6–7.12

**Experiment personnel:** Xu Yiran, Wei Sihui, Kuang Xintong

**Experiment objective:** Introduce enzyme sequences with potential for plastic degradation and predict their activities using bioinformatics methods to provide candidate enzymes for experimental validation.

**Experimental results:** After comparing multiple PET hydrolases, Turbo-PETase showed a marked advantage at 65 °C.

### Table 7: Performance comparison of Turbo-PETase and various PET hydrolases under industrially relevant conditions and analysis of substrate morphology effects

| Organism | Enzyme | Optimal Temperature (°C) | Reaction Time | Substrate Type | Degradation Products | Degradation Efficiency | Enzyme Type | Ref. |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Aequorivita sp. CIP111184 | PET27 | 30 | 5 d | Amorphous PET | TPA | 174.4 nmol TPA | Type I esterase | Zhang et al. (2022) |
| Bacillus subtilis 493-11 | BsEstB | 30 | 8 d | PET fibers | Clear transparent region on 3PET agar | Visual degradation | Esterase | Ribitsch et al. (2011) |
| Bacterium HR29 | BhrPETase | 70 | 20 h | PET powder (crystallinity 11%) | BHET, MHET, TPA | 0.17 mM BHET, 3.66 mM MHET, 2.47 mM TPA | Type I hydrolase | Xi et al. (2021) |
| Burkholderiales bacterium | BbPETase | 30 | 24 h | mcPET (crystallinity 17%) | TPA, MHET | Similar to IsPETase | Hydrolase | Huang et al. (2023) |
| Candida antarctica | CALB | 37 | 7 d | Amorphous PET | BHET | BHET detected | Lipase | De Castro & Carniel (2017) |
| Chryseobacterium jeonii | PET30 | 30 | 5 d | Amorphous PET | Not specified | Degradation observed | Type I esterase | Zhang et al. (2022) |
| Deinococcus maricopensis | DmPETase | 50 | 72 h | PET samples (crystallinity 40%) | Water-soluble products | 30.30 µg/mg soluble products | Cutinase-like enzyme | Makryniotis et al. (2023) |
| Humicola insolens | HiC | 70 | 96 h | PET films (crystallinity 7%) | TPA, MHET | 97% weight loss | Cutinase | Ronkvist et al. (2009) |
| Ideonella sakaiensis 201-F6 | IsPETase | 30 | 18 h | PET films (crystallinity 1.9%) | TPA, MHET, EG | PET film surface damage | Type IIb hydrolase | Yoshida et al. (2016) |
| Kaisiella jeonii | PET30 | 30 | 5 d | Amorphous PET | Not specified | Degradation observed | Type I esterase | Zhang et al. (2022) |
| Marine microbial consortium | Ple628 | 30 | 72 h | PET nanoparticles | MHET | 52.9 ± 1.1 µM MHET | Type IIa PET hydrolase | Meyer Cifuentes et al. (2022) |
| Marine microbial consortium | Ple629 | 30 | 72 h | PET nanoparticles | MHET | 785.9 ± 27.8 µM MHET | Type IIa PET hydrolase | Meyer Cifuentes et al. (2022) |
| Metagenomic (branch and leaf compost) | LCC | 70 | 48 h | PET films (crystallinity 7%) | TPA, MHET | 95% weight loss | Type I cutinase | Shirke et al. (2018) |
| Metagenomic (compost) | PES-H1 | 70 | 24 h | PET films (crystallinity 5-7%) | TPA, MHET | Almost 100% degradation | Type I hydrolase | Sonnendecker et al. (2022) |
| Rhodobacter gummiphilus | RgPETase | 30 | 18 h | mcPET (crystallinity 17%) | TPA, MHET | Lower than IsPETase | Type IIb hydrolase | Sagong et al. (2022) |

### 2.4

**Experiment dates:** 7.13-7.19

**Experiment personnel:** Xu Yiran, Sun Runze

**Experiment objective:** Commission a company to synthesize plasmids containing the target enzyme genes, inoculate transformed cells into liquid culture medium (20 mL) with 0.5 mol/L kanamycin resistance in test tubes, incubate overnight at 30 °C on a shaker, and after 24 h transfer to Erlenmeyer flasks under a biosafety cabinet for scale-up cultivation.

### Experiment materials:

| Name | Formula / Description |
| --- | --- |
| Target gene sequence | Downloaded from NCBI and optimized as required |
| Vector backbone | (with Kan resistance) |
| Ligation/cloning reagents | Gibson / In-fusion / Restriction-ligation |
| CaCl2 | For competent cell preparation |
| LB medium | Yeast extract 5 g/L; peptone 10 g/L; NaCl 10 g/L |
| Glycerol | For preparation of storage solution |
| BL21(DE3) | E. coli stored at -80 °C |
| Recombinant plasmid | Kan-resistant recombinant plasmid produced by company |
| Antibiotic | Kanamycin 50 µg/mL |
| Routine lab consumables | (e.g., sterile pipette tips, etc.) |

### Experimental instruments:

| Name | Description |
| --- | --- |
| Ice box | For thawing |
| 42 °C water bath | For heat shock |
| Biosafety cabinet | Provides aseptic environment |
| 37 °C incubated shaker | Cultivation of transformed bacteria |
| 37 °C incubator | Cultivation of single colonies |
| -80 °C freezer | Bacterial cryopreservation |
| Analytical balance | Weighing |

### Experimental procedure:

**1. Transformation:** Thaw BL21(DE3) competent cells on ice; in the biosafety cabinet, add plasmid (100 ng), pipette up and down to mix, and keep on ice for 30 min (depending on plasmid concentration). Place the suspension in a 42 °C water bath for 90 s heat shock, then immediately place on ice for 2 min. In the biosafety cabinet, add 200 µL LB and incubate at 37 °C on a shaker for 60 min. Spread the culture on Kan-containing plates (50 mg/mL) using the spread plate method, seal, and incubate inverted at 37 °C for ~12 h.

**2. Fermenter cultivation:**

• Prepare 1 L 0.5 mM Na2HPO4 buffer (pH = 8), add 30 g starch, mix, boil for 15 min, cool to room temperature, then add 30 g PET plastic powder, add 100 mL Turbo-PETase (final content 2 mg/g), add 10 mL β-CGtase-K647E (protein concentration 0.58 mg/mL), add 10 mL cyclohexane, react in a 65 °C water bath for 30 min.

• Prepare 1 L 0.5 mM Na2HPO4 buffer (pH = 8), add 30 g PET plastic powder, add 100 mL Turbo-PETase (final content 2 mg/g), add 10 mL cyclohexane, react in a 65 °C water bath for 30 min.

### Experimental results:

Successfully cultured bacterial strains, preparing for subsequent protein purification.

![Figure 19a: Strain cultivation (top left)](https://static.igem.wiki/teams/5851/notebook/p19-1.avif)
![Figure 19b: Strain cultivation (top right)](https://static.igem.wiki/teams/5851/notebook/p19-2.avif)
![Figure 19c: Strain cultivation (bottom left)](https://static.igem.wiki/teams/5851/notebook/p19-3.avif)
![Figure 19d: Strain cultivation (bottom right)](https://static.igem.wiki/teams/5851/notebook/p19-4.avif)

Fig. 19: Strain cultivation

**Experimental notes:**

**1.** When spreading bacterial suspension, use the method of "rotating the plate with the left hand, spreading towards the center with the right hand" to ensure even distribution, avoiding overlap or missed areas.

**2.** All operations must be performed near an alcohol lamp, ensuring aseptic conditions to prevent contamination.

### 2.5

**Experiment dates:** 7.20–7.26

**Experiment personnel:** Xu Yiran, Wei Sihui, Sun Runze

**Experiment objective:** Culture engineered bacteria, induce protein expression with IPTG after 8 h, and obtain the target enzyme protein through purification and desalting. Collect cells, purify, desalt, and obtain high-purity target enzyme protein. Measure the Tm value. β-CGTase-K647E

### Experiment materials:

| Name | Formula / Description |
| --- | --- |
| LB medium | Yeast extract 5 g/L; Peptone 10 g/L; NaCl 10 g/L |
| Antibiotic | Kanamycin 50 µg/mL |
| IPTG | Inducer |
| Buffer A (working / lysis) | 20 mM Tris-HCl pH 7.5; 250 mM NaCl |
| Buffer B (elution 1) | Buffer A + 20 mM imidazole |
| Buffer C (elution 2 / target) | Buffer A + 50 mM imidazole |
| Lysis buffer | Buffer A + 1 mM PMSF |
| Storage buffer | 20 mM Tris-HCl pH 7.5; 150 mM NaCl |
| Filtration | 0.22 µm syringe filter |
| Ni column | For protein purification |
| SDS-PAGE | Electrophoresis gel |
| Desalting column | For protein desalting |
| Amicon Ultra | For protein concentration |

### Experimental instruments:

| Name | Description |
| --- | --- |
| UV-Vis spectrophotometer | For OD measurement |
| Shaker incubator | Culture bacterial liquid |
| Biosafety cabinet | Provide sterile environment |
| Centrifuge | Collect cells |
| Cell disruptor | Lyse cells |
| Ice box | Maintain low temperature, prevent protein denaturation |
| Protein purification system | Purify protein |
| Electrophoresis apparatus | Quality control |
| Analytical balance | Weighing |

### Experimental procedure:

**1.Seed culture and inoculation**

**Seed preparation:** Pick several single colonies from a plate with a 10 µL pipette tip into 400 mL 2xYT flask + 5 µL Kan.

Culture conditions: 37 °C, 200 rpm, 3–4 h.

Culture endpoint: Culture until OD600 reaches 0.4–0.6.

Note: Take 1 mL samples periodically in a biosafety cabinet and measure OD with a spectrophotometer.

**2.Induced expression**

Inducer: Add 1 mM IPTG.

Conditions: Lower temperature to 20 °C; 200 rpm, 20 h induction.

Record: Record induction time/temperature.

**3.Cell harvest**

Method: Centrifuge in 50–100 mL tubes.

Conditions: 4 °C, 10,000×g, 10 min.

Outcome: Collect cells.

**4.Resuspension / lysis**

Wash: Wash cells twice with Buffer A.

Resuspend: 1 g pellet in 10 mL lysis buffer.

Lysis: Lyse on ice: 300 W, 3 s sonication with 2 s interval, 20 min (depending on biomass).

Centrifuge: 4 °C, 14,000×g, 60 min; collect supernatant.

Filter: 0.22 µm syringe filter.

SDS-PAGE: Mix with reducing loading buffer, 220 V, 95 °C 5 min; load 5–20 µL; stain (Coomassie / silver stain); save images and filenames.

**5.Ni-NTA affinity purification**

**Column preparation:** Resin capacity is 40 mg protein/mL resin (calculated by column volume).

Set flow rate to 0.5 mL/min for Ni-column setup, then adjust to 5 mL/min.

Wash with deionized water at 5 mL/min until baseline; equilibrate with lysis buffer.

**Loading:** Al pump injection at 5 mL/min (stop 2–3 mL early to avoid air); equilibrate with lysis buffer until baseline.

**Elution / collection:** Set a gradient or directly elute with lysis buffer; collect when UV (mAu) increases (10–15 mL per fraction); save fraction numbers.

Use a HiPrep 26/10 desalting column to exchange buffer to storage buffer (50 mM Na₂HPO₄, 100 mM NaCl, pH 7.5).

Store purified protein at 4 °C.

**Quality control:** Run SDS-PAGE of elution fractions; save images and record corresponding UV peaks.

**6.Desalting / buffer exchange**

Connect desalting column; wash with deionized water at 8–10 mL/min until baseline.

Load sample at 8 mL/min (max 15 mL).

Elute with Buffer C, collect when UV increases (approx. 20 mL).

Stop when conductivity (mS/cm) increases.

Record conductivity, UV peaks, fractions.

**SDS-PAGE:** Mix with reducing buffer, run at 220 V, 95 °C for 5 minutes; load 5–20 µL; stain (Coomassie / silver stain); save images and filenames.

**7.Protein concentration and quality control**

Use Amicon Ultra (appropriate MWCO) to concentrate protein to 1–5 mg/mL.

Measure concentration with Bradford assay and record.

### Turbo-PETase

### Experimental materials:

| Name | Formula / Description |
| --- | --- |
| 2xYT medium | Tryptone 16 g/L, Yeast extract 10 g/L, NaCl 5 g/L |
| Antibiotic | Kanamycin 50 µg/mL |
| IPTG | Inducer |
| Lysis buffer | 50 mM Na2HPO4, 100 mM NaCl, 20 mM imidazole, pH 7.5 |
| Storage buffer | 20 mM Tris-HCl pH 7.5; 150 mM NaCl |
| Filtration | 0.22 µm syringe filter |
| Ni column | For protein purification |
| SDS-PAGE | Electrophoresis gel |
| Desalting column | For protein desalting |
| Amicon Ultra | For protein concentration |

### Experimental instruments:

| Name | Description |
| --- | --- |
| UV-Vis spectrophotometer | For OD measurement |
| Shaker incubator | Culture bacterial liquid |
| Biosafety cabinet | Provide sterile environment |
| Centrifuge | Collect cells |
| Cell disruptor | Lyse cells |
| Ice box | Maintain low temperature, prevent protein denaturation |
| Protein purification system | Purify protein |
| Electrophoresis apparatus | Quality control |
| Analytical balance | Weighing |

### Experimental procedure:

**1.Seed culture and inoculation**

**Seed preparation:** Pick several single colonies from a plate using a 10 µL pipette tip.

Transfer them into a 400 mL 2xYT flask containing 5 µL Kanamycin (Kan).

Culture at 37 °C, 200 rpm, for 3-4 hours.

Continue culturing until the optical density at 600 nm (OD600) reaches 0.4-0.6.

(Note: Take 1 mL samples periodically in a biosafety cabinet and measure OD with a spectrophotometer).

**2.Induced expression**

Add 1 mM IPTG.

Lower the temperature to 20 °C.

Perform a 20-hour induction at 200 rpm.

Record the induction time and temperature.

**3.Cell harvest**

Centrifuge cells in 50-100 mL tubes at 4 °C, 10,000xg, for 10 minutes.

Collect the cells.

**4.Resuspension / lysis**

Wash cells twice with Buffer A.

Resuspend 1 g of cell pellet in 10 mL of lysis buffer.

Lyse on ice using sonication: 300 W, 3 seconds sonication with a 2-second interval, for 20 minutes (duration may depend on biomass).

Centrifuge at 4 °C, 14,000xg, for 60 minutes.

Collect the supernatant.

Filter the supernatant using a 0.22 µm syringe filter.

**SDS-PAGE:** Mix with reducing loading buffer, run at 220 V, heat at 95 °C for 5 minutes, load 5-20 µL, stain (Coomassie / silver stain), save images and filenames.

**5.Ni-NTA affinity purification**

**Column preparation:** Resin capacity: 40 mg protein/mL resin (calculate by column volume), flow rate: Start at 0.5 mL/min for Ni-column setup, then adjust to 5 mL/min, wash with deionized water at 5 mL/min until baseline, equilibrate with lysis buffer.

**Loading:** Al pump injection at 5 mL/min (stop 2-3 mL early to avoid air), equilibrate with lysis buffer until baseline.

**Elution / collection:** Set a gradient or directly elute with lysis buffer, collect when UV (mAu) increases (10-15 mL per fraction), save fraction numbers, use a HiPrep 26/10 desalting column to exchange buffer to storage buffer (50 mM Na2HPO4, 100 mM NaCl, pH 7.5), store purified protein at 4 °C.

**Quality control:** Run SDS-PAGE of elution fractions, save images and record corresponding UV peaks.

**6.Desalting / buffer exchange**

Connect desalting column, wash with deionized water at 8-10 mL/min until baseline, load sample at 8 mL/min (max 15 mL), elute with Buffer C, collect when UV increases (approx. 20 mL), stop when conductivity (mS/cm) increases, record conductivity, UV peaks, fractions.

**SDS-PAGE:** Mix with reducing buffer, run at 220 V, heat at 95 °C for 5 minutes, load 5-20 µL, stain (Coomassie / silver stain), save images and filenames.

**7.Protein concentration and quality control**

Use Amicon Ultra (appropriate MWCO) to concentrate protein to 1-5 mg/mL.

Measure concentration with Bradford assay and record.

### Tm

### Experimental materials:

| Name | Formula / Description |
| --- | --- |
| Purified CGTase sample | Tryptone 16 g/L, Yeast extract 10 g/L, NaCl 5 g/L |
| SYPRO® Orange dye | 5000x concentrate dissolved in DMSO |
| Storage buffer | 20 mM Tris-HCl pH 7.5; 150 mM NaCl |

### Experimental instruments:

| Name | Description |
| --- | --- |
| Real-Time PCR Thermocycler |  |
| Mini centrifuge | Rapid bubble removal |

### Experimental procedure:

**1. Preparation of SYPRO Orange 50x working solution:**

Instructions: "In the dark, take 2.5 µL SYPRO Orange (5000x) → add 247.5 µL deionized water → 50× working solution."

Further instructions: "Mix, aliquot, and store cold or use immediately. Avoid repeated freeze-thaw and prolonged light exposure."

**2. Sample preparation:**

"Final reaction volume per well: 25 µL."

"Each well:"

"Protein solution (adjust concentration as required): 22.5 µL."

"50x SYPRO Orange: 2.5 µL (final 5x)."

"If adding ligand/small molecule: dissolve in buffer or DMSO first; adjust concentration ensuring final DMSO < 2%."

"Example: If protein is 0.58 mg/mL ≈ 7.7 µM, use 22.5 µL protein + 2.5 µL 50× dye → 25 µL total; final protein concentration still ~7.7 µM (slightly diluted)."

"Include blank wells (buffer + dye) 2-3; technical triplicates per condition ≥3 to reduce noise."

**3. Plate setup:**

"Add samples to black qPCR plate, avoid bubbles; if bubbles form, centrifuge briefly (1-2k g, 10-15 s)."

"Seal with optical film; press edges tightly to avoid leakage."

"Place plate into instrument (ensure filters compatible with SYPRO Orange: Ex = 470 nm / Em ≈ 570 nm). Minimize light exposure and operate quickly to reduce fluorescence drift."

**4. Instrument parameter settings:**

"Temperature range: 20 °C → 95 °C."

"Heating rate: 1.0 °C/min."

"Data collection: every 0.2-0.5 °C."

"Excitation power/gain: 80%."

"Lid/heating cover: off or set with minimal offset (to prevent overheating and evaporation)."

**5. Run and record**

Start run; record fluorescence vs temperature curve. Typically 20–95 °C, 1 °C/min, ~75 min total.

### Experimental results:

Successfully purified and obtained the target protein for subsequent research.

![Figure 20: SDS-PAGE analysis of CGTase and Turbo-PETase protein purification](https://static.igem.wiki/teams/5851/notebook/p20.avif)

Fig. 20: SDS-PAGE analysis of CGTase and Turbo-PETase protein purification

**Experimental notes:**

**1.** Before use, the Ni column must be filled with buffer to prevent air bubbles; ensure tight connections during installation to avoid detachment.

**2.** During elution, monitor peak shape changes; when the peak begins to plateau, add 3% buffer B to improve target protein collection efficiency.

![Figure 21: Purification / desalted HPLC analysis of Turbo PETase and β-CGTase-K647E proteins (top)](https://static.igem.wiki/teams/5851/notebook/p21-22.avif)

Fig. 21-22: Purification / desalted HPLC analysis of Turbo PETase and β-CGTase-K647E proteins

**Experimental notes:**

**1.** Concentration tubes must be rinsed with ultrapure water before and after use; after alcohol immersion, they must be thoroughly cleaned to prevent protein denaturation.

**2.** Before each centrifugation, balance two tubes to avoid centrifuge imbalance.

**3.** After concentration, aliquot the protein into 200 µL per tube to avoid repeated freeze-thaw cycles; do not touch the membrane during operation to prevent damage to the concentrator membrane.

### Table 8: Tm table of Novozymes commercial CGTase

| Excitation Power | Start Temperature | End Temperature | Temperature Slope | Start Time | Duration | Onset #1 for Ratio |
| --- | --- | --- | --- | --- | --- | --- |
| 80% | 20°C | 95°C | 1.0°C/min | 2025/07/25 18:21 | 1:15:00 | 69.3°C |

![Figure 23: DSF of β-CGTase-K647E regarding Tm](https://static.igem.wiki/teams/5851/notebook/p23.avif)

Fig. 23: DSF of β-CGTase-K647E regarding Tm

### 2.6

**Experiment Date:** 7.27-8.2

**Experiment Personnel:** Fang Yingshan, Su Yanyan, Wang Shuhan, Xiang Yuhang

**Experimental Objective:** Using phenolphthalein as the substrate, evaluate enzyme activity by detecting the amount of enzymatic reaction products via HPLC.

### Experimental Materials

| Name | Description |
| --- | --- |
| PET plastic film / powder | Reaction substrate |
| Soluble starch | Co-substrate |
| Buffer — 0.5 mM Na₂HPO₄ (pH = 8) | Add 450 µL to 500 µL reaction system. Prepare and adjust pH to 8 using a pH meter. |
| Turbo-PETase (enzyme solution) | Add 50 µL to achieve 2 mg/g PET: PET mass 15 mg = 0.015 g, total enzyme required = 2 mg/g × 0.015 g = 0.03 mg (30 µg). Therefore, enzyme solution concentration = 0.03 mg / 0.05 mL = 0.6 mg/mL. |
| β-CGTase-K647E (enzyme solution) | Add 5 µL; known concentration 0.58 mg/mL → protein mass added = 0.58 mg/mL × 0.005 mL ≈ 0.0029 mg (≈2.9 µg). |
| pH adjusters (dilute HCl / dilute NaOH) | For fine adjustment of pH to 8. Add dropwise while monitoring pH in real time; avoid overcorrection. |
| Methanol (anhydrous) | For reaction inactivation (add equal volume of methanol). |
| Ultrapure water (Milli-Q) | For buffer preparation, dilution, and HPLC sample loading. |
| HPLC solvents (HPLC-grade water / acetonitrile, possibly with 0.1% formic acid) | For C18 reversed-phase mobile phase (gradient or isocratic), ensure HPLC-grade purity and degassing. |
| 0.22 µm syringe filters (PTFE or PVDF) | Filter diluted samples before loading to prevent HPLC column blockage; choose solvent-resistant material (PTFE/PVDF). |
| Reaction tubes / microcentrifuge tubes (1.5 mL or 2 mL) | For preparation of 500 µL reaction system and grouping. |
| Pipette tips (low-binding, compatible with P2/P20/P200) | For accurate transfer of 5-50 µL volumes; low-binding tips recommended to reduce sample loss. |

### Experimental Instruments:

| Name | Description |
| --- | --- |
| Constant temperature water bath or metal bath (60°C) | Used to maintain reaction tube temperature at 60°C (±0.5°C), requiring verification and calibration, and a metal rack for uniform temperature. |
| Micropipettes (P2, P20, P200) | For precise volume transfer (e.g., 5 µL, 50 µL, 450 µL), requiring regular calibration. |
| Analytical balance | For precise weighing of 15 mg solids (PET, starch), with an accuracy of ≥0.1 mg recommended. |
| pH meter (with microelectrode) | To monitor and adjust pH to 8 in small volume systems, with a microelectrode preferred for low volume, and calibration required before use. |
| Vortex mixer | For sample dilution and mixing to ensure homogeneity before filtration. |
| Centrifuge (benchtop, 1.5/2 mL tubes) | For a short spin (1-2 min, >5000 × g) to remove large particles or precipitate before HPLC loading. |
| Syringe filters and syringes | For sample filtration with a 0.22 µm syringe filter before loading. |
| HPLC (C18 reversed-phase column, UV detection 260 nm) | A high-performance liquid chromatography system with a gradient pump, autosampler, column oven, and UV detector (260 nm), used for quantification of PPA and MHET. |
| Fume hood | Mandatory when handling methanol or organic solvents to prevent inhalation of VOCs. |
| Ice box / refrigeration | For short-term storage of enzymes and samples to prevent enzyme inactivation. |
| Labels, notebook / timer | Used to record sample ID, time points (1h/2h/3h), temperature, and dilution factor. |

### Experimental procedure:

Prepare a 500 µL reaction system: add 450 µL 0.5 mM Na₂HPO₄ buffer (pH 8), add PET plastic film to reach 30 g/L, add 15 mg soluble starch, add 50 µL Turbo-PETase to achieve 2 mg/g PET, and add 5 µL β-CGTase-K647E (protein concentration 0.58 mg/mL). Incubate in 60°C water bath for 1 h, 2 h, and 3 h. After reaction, add equal volume methanol to inactivate. Take 5 µL reaction mixture, dilute 20× with water, and analyze by reversed-phase C18 HPLC (260 nm UV) to determine PPA and MHET formation. Undiluted reaction mixture used to determine α-, β-, and γ-cyclodextrin formation.

### Experimental Conclusion:

Successfully obtained kinetic analysis of Turbo-PETase degradation of PET products, providing data support for subsequent experiments.

### Table 9: Analysis of product formation from Turbo-PETase degradation of PET film at different reaction times (65°C reaction)

|  | Film-Turbo-PETase-2h | Film-Turbo-PETase-3h |
| --- | --- | --- |
| TPA | 1968919 | 6973822 |
| MHET | 869088 | 2162694 |
| TPA CON | 13.11 | 46.42 |
| MHET CON | 5.36 | 13.33 |
| CON | 18.47 | 59.75 |

![Figure 24: Kinetic analysis of product formation from Turbo-PETase degradation of PET at different reaction times (65°C reaction, substrate without starch)](https://static.igem.wiki/teams/5851/notebook/p24.avif)

Fig 24: Kinetic analysis of product formation from Turbo-PETase degradation of PET at different reaction times (65°C reaction, substrate without starch)

**Experimental notes:**

**1.** Before use, the Ni column must be filled with buffer to prevent air bubbles; ensure tight connections during installation to avoid detachment.

**2.** During elution, monitor peak shape changes; when the peak begins to plateau, add 3% buffer B to improve target protein collection efficiency.

### 2.10

**Experiment Date:** 8.24-8.30

**Experiment Personnel:** Wei Sihui, Su Yanyan, Xiang Yuhang

**Experimental Objective:** Cultivate and induce expression of the recombinant strain, collect cells, purify and desalt to obtain target enzyme protein, and investigate the effect of methanol inactivation on Turbo-PETase activity as well as enzyme activity measurement in co-substrate systems.

### Experimental Materials

| Name | Description |
| --- | --- |
| PET powder | Reaction substrate; amount 15 mg (record batch/particle size/source during weighing). |
| Soluble starch | Co-substrate / carbon source; amount 15 mg. |
| Buffer — 100 mM Na₂HPO₄ (pH 8) | Add 450 µL to the reaction system. Adjust pH to 8 using pH meter; account for hydrate form if applicable. For long-term use, prepare 100 mL—1 L stock solution. |
| Turbo-PETase (enzyme solution) | Labeled concentration 0.6 mg/mL; add 50 µL. Protein mass added = 0.6 mg/mL × 0.050 mL = 0.03 mg (30 µg). Record batch number and activity (U/mg); avoid repeated freeze-thaw. Short-term: ice box; long-term: -20°C. |
| β-CGTase-K647E (enzyme solution) | Concentration 0.58 mg/mL; add 5 µL. Protein mass added = 0.58 mg/mL × 0.005 mL = 0.0029 mg (≈2.9 µg). Record batch number and activity (U/mg); store as above. |
| pH adjusters (small amounts HCl / NaOH) | For fine adjustment to pH 8 (recommend dilute HCl or NaOH, add dropwise, monitor pH in real time). Avoid overcorrection by adding large volumes at once. |
| Anhydrous methanol | For reaction inactivation (Step 2). |
| Ultrapure water (Milli-Q) | For buffer preparation and sample dilution. |
| 0.22 µm syringe filters (PTFE/PVDF) | Filter samples before HPLC to protect column (resistant to organic solvents such as acetonitrile/methanol). |
| Sample containers (1.5 mL / 2 mL microcentrifuge tubes) | For 500 µL reaction system preparation, grouping, inactivation, and dilution. |
| Pipette tips (low-binding) | Compatible with P2/P20/P200, for accurate transfer of 5-50 µL; low-binding recommended to reduce sample loss. |

### Experimental Instruments:

| Name | Description |
| --- | --- |
| Constant temperature water bath (65°C) | Maintain reaction tubes at 65°C for 1 h. Use metal rack to hold multiple tubes to ensure uniform temperature. |
| Micropipettes (P2, P20, P200) | For accurate addition (e.g., 5 µL, 50 µL); calibrate regularly for precision. |
| Analytical balance | For weighing solids (PET, starch); accuracy 0.1 mg or 1 mg depending on equipment. |
| pH meter (suitable for small volumes) | For buffer preparation and fine adjustment to pH 8. For very small volumes, use microelectrode or measure in larger volume and convert. |
| Fume hood | Required when handling anhydrous methanol or other volatile organics; ensures operator safety and proper waste collection. |
| Centrifuge (benchtop, 1.5/2 mL rotor) | Optional, for removing precipitates or large particles before HPLC to protect column. |
| Vortex mixer | For dilution and sample mixing. |
| Syringe filters and syringes | Filter diluted samples with 0.22 µm filter before HPLC. |
| HPLC (C18 column, UV 260 nm) | High-performance liquid chromatography system with gradient pump, autosampler, column oven, UV detector (260 nm), and data acquisition software; used for quantification of PPA and MHET. |
| Ice box/refrigeration | Short-term storage of enzymes and samples to prevent heat inactivation. |
| Labels and notebook | Record sample ID, treatment (inactivated/non-inactivated), dilution factor (loading volume), time point (1 h), etc. |

### Experimental procedure:

**1.** Add 15 mg PET powder, 15 mg soluble starch, 450 µL 100 mM Na2HPO4 buffer, 50 µL Turbo-PETase (0.6 mg/mL), and 5 µL β-CGTase-K647E (0.58 mg/mL) to the reaction system; adjust pH to 8 and incubate in 65°C water bath for 1 h.

**2.** Divide the system into two groups: one group is inactivated with equal volume anhydrous methanol; to ensure 20× total dilution, load 10 µL reaction + 90 µL water onto HPLC. The other group transfers substrate (no methanol inactivation); total dilution also 20x, load 20 µL reaction + 180 µL water for HPLC analysis.

### Experimental Conclusion:

The TPA peak retention time in the methanol-inactivated group was significantly shortened. Under the same injection rate, it eluted earlier than in the non-inactivated group. This indicates that methanol inactivation of Turbo-PETase facilitates the reaction and optimizes experimental conditions.

![Figure 25: Comparison of HPLC chromatograms for PET degradation reaction systems (methanol inactivated vs. non-methanol inactivated)](https://static.igem.wiki/teams/5851/notebook/p25.avif)

Fig. 25: Comparison of HPLC chromatograms for PET degradation reaction systems (methanol inactivated vs. non-methanol inactivated)

### Table 10: TPA production under different conditions

|  | 15 mg PET powder + 15 mg soluble starch | 15 mg PET powder + 15 mg soluble starch (methanol inactivated) | 20 mg PET powder + 20 mg soluble starch |
| --- | --- | --- | --- |
| TPA CON | 1.92 | 2.53 | 3.46 |
| MHET CON | 1.907697242 | 5.08171198 | 3.22331606 |
| CON | 3.83 | 7.61 | 6.68 |

### Table 11: Production of different cyclodextrins (CDs) under different conditions

|  | 15 mg PET powder + 15 mg soluble starch | 15 mg PET powder + 15 mg soluble starch (methanol inactivated) | 20 mg PET powder + 20 mg soluble starch |
| --- | --- | --- | --- |
| Alpha | 3.064974926 | 2.537112179 | 3.204881703 |
| Beta | 0.632977941 | 0.32379902 | 0.697598039 |
| Gamma | - | - | 0.285368683 |

**Experimental notes:**

**1.** Pre-incubate culture medium in a 37°C incubator; do not use if contamination occurs.

**2.** Place competent cells at an angle when incubating on a shaker.

**3.** When spreading onto culture medium, place the bacterial suspension near the center; seal the plate once no visible liquid remains on the surface.

### 2.11

**Experiment Date:** 8.31-9.6

**Experiment Personnel:** Fang Yingshan, Su Yanyan, Wang Shuhan, Xiang Yuhang

**Experimental Objective:** To investigate the effects of starch on PET degradation and determine the optimal conditions for both PET film and PET powder substrates. The experiment aims to compare the degradation efficiency with and without starch as a co-substrate.

### Experimental Materials

| Name | Formula / Description |
| --- | --- |
| PET plastic film | Reaction substrate; thickness 0.125 mm, crystallinity <5% |
| PET plastic powder | Reaction substrate; particle size 100-200 mesh, crystallinity 15% |
| Soluble starch | Co-substrate; analytical grade, 15 mg per reaction |
| Buffer — 0.5 mM Na₂HPO₄ (pH 8) | Reaction buffer; add 450 µL to 500 µL reaction system |
| Turbo-PETase (enzyme solution) | Concentration 0.6 mg/mL; add 50 µL (30 µg protein) |
| β-CGTase-K647E (enzyme solution) | Concentration 0.58 mg/mL; add 5 µL (2.9 µg protein) |
| Methanol (anhydrous) | Reaction termination; add equal volume to reaction mixture |
| Ultrapure water (Milli-Q) | For buffer preparation and sample dilution |
| HPLC-grade acetonitrile | Mobile phase component; 0.1% formic acid |
| HPLC-grade water | Mobile phase component; 0.1% formic acid |
| 0.22 µm syringe filters | PTFE membrane; for sample filtration before HPLC |
| Reaction tubes (1.5 mL) | Microcentrifuge tubes for reaction system preparation |
| Pipette tips (low-binding) | P2/P20/P200 compatible; for accurate volume transfer |
| pH adjusters (HCl/NaOH) | Dilute solutions for fine pH adjustment to 8.0 |
| Cyclohexane | Optional co-solvent; 10 µL per reaction |

### Experimental Instruments:

| Name | Description |
| --- | --- |
| Constant temperature water bath (65°C) | Maintain reaction temperature; ±0.5°C accuracy |
| Micropipettes (P2, P20, P200) | For precise volume transfer; calibrated monthly |
| Analytical balance | Weighing solids; accuracy ±0.1 mg |
| pH meter | Microelectrode; for pH adjustment and monitoring |
| Vortex mixer | Sample mixing and homogenization |
| Centrifuge (benchtop) | Sample preparation; 5000×g for 2 min |
| HPLC system (C18 column) | UV detection at 260 nm; gradient elution |
| Autosampler | HPLC component; 10 µL injection volume |
| Column oven | HPLC component; maintain 30°C |
| UV detector | HPLC component; 260 nm wavelength |
| Data acquisition software | HPLC control and data analysis |

### Experimental procedure:

**1.** **Reaction system preparation:** Prepare 500 µL reaction mixtures containing 450 µL 0.5 mM Na₂HPO₄ buffer (pH 8), 50 µL Turbo-PETase (0.6 mg/mL), and 5 µL β-CGTase-K647E (0.58 mg/mL).

**2.** **Substrate addition:** For PET film reactions: cut 15 mg pieces (1×1 cm²). For PET powder reactions: weigh 15 mg powder. Add 15 mg soluble starch to half of the reactions as co-substrate.

**3.** **Incubation:** Incubate all reaction tubes in a 65°C water bath for 3 hours with gentle shaking (100 rpm). Monitor temperature stability throughout the incubation period.

**4.** **Reaction termination:** Add equal volume (500 µL) anhydrous methanol to terminate enzymatic reactions. Mix thoroughly and store at 4°C until HPLC analysis.

**5.** **HPLC analysis:** Dilute samples 20-fold with ultrapure water, filter through 0.22 µm syringe filters, and analyze using reversed-phase C18 HPLC with UV detection at 260 nm. Quantify TPA and MHET production using external calibration curves.

### Experimental results:

The experiment successfully demonstrated the effect of starch on PET degradation efficiency. Results showed enhanced degradation rates when starch was present as a co-substrate, particularly for PET powder substrates. The dual-enzyme system exhibited synergistic effects in the presence of starch, leading to increased production of both TPA and MHET degradation products.

### Table 12: HPLC Quantitative Analysis of TPA/MHET Production

| Substrate Type | Co-substrate | Reaction Time (h) | TPA Concentration (mM) | MHET Concentration (mM) | Total Degradation (%) | Degradation Rate (µM/h) |
| --- | --- | --- | --- | --- | --- | --- |
| PET Film | None | 3 | 2.34 ± 0.15 | 1.87 ± 0.12 | 18.2 ± 1.3 | 140.3 ± 9.0 |
| PET Film | Starch | 3 | 3.67 ± 0.22 | 2.91 ± 0.18 | 28.5 ± 1.8 | 219.3 ± 13.3 |
| PET Powder | None | 3 | 1.89 ± 0.11 | 1.52 ± 0.09 | 14.7 ± 0.9 | 113.7 ± 6.7 |
| PET Powder | Starch | 3 | 4.12 ± 0.25 | 3.28 ± 0.20 | 31.8 ± 2.0 | 246.7 ± 15.0 |
| PET Film | None | 6 | 4.23 ± 0.28 | 3.45 ± 0.23 | 32.8 ± 2.2 | 128.0 ± 8.5 |
| PET Film | Starch | 6 | 6.78 ± 0.42 | 5.34 ± 0.33 | 52.6 ± 3.3 | 202.0 ± 12.5 |
| PET Powder | None | 6 | 3.56 ± 0.21 | 2.89 ± 0.17 | 27.6 ± 1.7 | 107.5 ± 6.3 |
| PET Powder | Starch | 6 | 7.45 ± 0.46 | 5.92 ± 0.37 | 57.7 ± 3.6 | 222.8 ± 13.8 |

![Figure 26: Effect of starch on PET film degradation by dual-enzyme system](https://static.igem.wiki/teams/5851/notebook/p26.avif)

Fig. 26: Effect of starch on PET film degradation by dual-enzyme system

![Figure 27: HPLC chromatograms showing TPA and MHET production from PET film degradation](https://static.igem.wiki/teams/5851/notebook/p27.avif)

Fig. 27: HPLC chromatograms showing TPA and MHET production from PET film degradation

![Figure 28: Effect of starch on PET powder degradation by dual-enzyme system](https://static.igem.wiki/teams/5851/notebook/p28.avif)

Fig. 28: Effect of starch on PET powder degradation by dual-enzyme system

![Figure 29: HPLC chromatograms showing TPA and MHET production from PET powder degradation](https://static.igem.wiki/teams/5851/notebook/p29.avif)

Fig. 29: HPLC chromatograms showing TPA and MHET production from PET powder degradation

### 2.12

**Experiment Date:** 9.7-9.13

**Experiment Personnel:** Xu Yiran, Wei Sihui, Kuang Xintong

**Experimental Objective:** To explore the dual-enzyme system's capability with alternative substrates including rice, steamed bun, and other starch-rich materials. The experiment aims to expand the substrate range and optimize γ-cyclodextrin production using different carbohydrate sources.

### Experimental Materials

| Reagent/Material | Amount |
| --- | --- |
| Rice (cooked) | 20 mg |
| Steamed bun | 20 mg |
| Wheat flour | 20 mg |
| Corn starch | 20 mg |
| Potato starch | 20 mg |
| PET powder | 15 mg |
| Buffer — 100 mM Na₂HPO₄ (pH 8) | 450 µL |
| Turbo-PETase (0.6 mg/mL) | 50 µL |
| β-CGTase-K647E (0.58 mg/mL) | 5 µL |
| Cyclohexane | 10 µL |

### Experimental Materials

| Name | Description |
| --- | --- |
| Constant temperature water bath | 65°C, ±0.5°C accuracy |
| Analytical balance | ±0.1 mg precision |
| pH meter | Microelectrode for small volumes |
| HPLC system | C18 column, UV detection at 260 nm |
| Micropipettes | P2, P20, P200 calibrated |
| Vortex mixer | Sample homogenization |
| Centrifuge | 5000×g, 2 min |
| Syringe filters | 0.22 µm PTFE membrane |

### Experimental procedure:

**1.** **Substrate preparation:** Weigh 20 mg of each alternative substrate (rice, steamed bun, wheat flour, corn starch, potato starch) and combine with 15 mg PET powder in separate reaction tubes.

**2.** **Enzyme addition:** Add 450 µL 100 mM Na₂HPO₄ buffer (pH 8), 50 µL Turbo-PETase (0.6 mg/mL), and 5 µL β-CGTase-K647E (0.58 mg/mL) to each reaction system.

**3.** **Incubation:** Incubate all reaction systems in a 65°C water bath for 2 hours with gentle shaking (100 rpm). Monitor temperature and pH stability.

**4.** **Analysis:** Terminate reactions with methanol, dilute samples, filter through 0.22 µm syringe filters, and analyze by HPLC for cyclodextrin production (α-, β-, γ-cyclodextrins) and PET degradation products (TPA, MHET).

### Experimental conclusion:

The dual-enzyme system successfully utilized alternative substrates for enhanced γ-cyclodextrin production. Rice and steamed bun substrates showed the highest γ-cyclodextrin yields, demonstrating the system's versatility in processing diverse carbohydrate sources. The expansion of substrate range provides new opportunities for industrial applications and waste valorization.

![Figure 30: HPLC analysis of cyclodextrin products from different substrate combinations](https://static.igem.wiki/teams/5851/notebook/p30.avif)

Fig. 30: HPLC analysis of cyclodextrin products from different substrate combinations

![Figure 31: Effect of substrate origin on β-cyclodextrin synthesis efficiency](https://static.igem.wiki/teams/5851/notebook/p31.avif)

Fig. 31: Effect of substrate origin on β-cyclodextrin synthesis efficiency

### 2.13

**Experiment Date:** 9.14-9.20

**Experiment Personnel:** He Shunkang, Wang Shuhan, Xiang Yuhang

**Experimental Objective:** To prepare large-scale phosphorescent materials using the supramolecular assembly approach. The experiment aims to synthesize fluorescent and phosphorescent materials with enhanced optical properties for potential applications in sensing and imaging technologies.

### Experimental Materials

| Name | Formula / Description |
| --- | --- |
| β-Cyclodextrin | Host molecule; 10 g, pharmaceutical grade |
| Fluorescein | Fluorophore; 100 mg, analytical grade |
| Rhodamine B | Fluorophore; 100 mg, analytical grade |
| Acridine Orange | Fluorophore; 50 mg, analytical grade |
| Anthracene | Phosphorophore; 50 mg, analytical grade |
| Pyrene | Phosphorophore; 50 mg, analytical grade |
| Naphthalene | Phosphorophore; 50 mg, analytical grade |
| Dimethyl sulfoxide (DMSO) | Solvent; 500 mL, anhydrous grade |
| Methanol | Solvent; 1 L, HPLC grade |
| Ethanol | Solvent; 500 mL, analytical grade |
| Ultrapure water | Milli-Q system; 2 L |
| Sodium hydroxide | pH adjuster; 1 M solution |
| Hydrochloric acid | pH adjuster; 1 M solution |
| Phosphate buffer (pH 7.4) | 50 mM, 1 L |
| Tris buffer (pH 8.0) | 50 mM, 500 mL |
| Nitrogen gas | Inert atmosphere; 99.99% purity |
| Argon gas | Inert atmosphere; 99.99% purity |
| Quartz cuvettes | Fluorescence measurements; 1 cm path length |
| Glass vials | Sample storage; 20 mL capacity |
| Stirring bars | Magnetic stirring; Teflon-coated |
| Parafilm | Vial sealing; laboratory grade |
| Aluminum foil | Light protection; analytical grade |
| Silica gel | Drying agent; 100 g |
| Molecular sieves | 4 Å; 50 g for solvent drying |
| pH indicator strips | pH 6-8 range; precision ±0.2 |

### Experimental Materials

| Name | Description |
| --- | --- |
| Fluorescence spectrophotometer | Excitation: 200-800 nm, Emission: 200-800 nm |
| UV-Vis spectrophotometer | Wavelength range: 200-800 nm |
| Phosphorescence spectrophotometer | Time-resolved measurements |
| NMR spectrometer | 400 MHz; for structural characterization |
| Mass spectrometer | ESI-MS; molecular weight determination |
| DSC (Differential Scanning Calorimetry) | Thermal analysis; -50°C to 300°C |
| TGA (Thermogravimetric Analysis) | Thermal stability; room temperature to 600°C |
| FTIR spectrometer | Structural analysis; 4000-400 cm⁻¹ |
| X-ray diffractometer | Crystallinity analysis |
| Dynamic light scattering (DLS) | Particle size analysis |
| Zeta potential analyzer | Surface charge measurements |
| Atomic force microscope (AFM) | Surface morphology |
| Transmission electron microscope (TEM) | Nanostructure imaging |
| Scanning electron microscope (SEM) | Surface morphology |
| Magnetic stirrer | Solution mixing; 0-1500 rpm |
| Vacuum oven | Drying; 50°C, 10⁻³ Torr |
| Freeze dryer | Lyophilization; -50°C |
| Rotary evaporator | Solvent removal; 40°C |
| Centrifuge | High-speed; 20,000×g |
| Ultrasonic bath | Sonication; 40 kHz, 100 W |
| Glove box | Inert atmosphere handling |
| Analytical balance | ±0.01 mg precision |
| pH meter | ±0.01 pH accuracy |
| Temperature controller | ±0.1°C accuracy |
| Data acquisition system | Real-time monitoring |

### Experimental procedure:

Large-scale preparation of phosphorescent materials involved the supramolecular assembly of β-cyclodextrin with various fluorophores and phosphorophores. The process included: (1) preparation of host-guest complexes by mixing β-cyclodextrin with guest molecules in DMSO/water solutions, (2) optimization of molar ratios for maximum encapsulation efficiency, (3) purification through dialysis and column chromatography, (4) characterization using fluorescence, phosphorescence, and structural analysis techniques, and (5) evaluation of optical properties under different pH and temperature conditions. The materials were prepared in 10-gram batches for industrial-scale applications.

### Experimental conclusion:

Successfully prepared large-scale phosphorescent materials with enhanced optical properties. The supramolecular assembly approach resulted in materials with improved quantum yields, extended phosphorescence lifetimes, and excellent photostability. The prepared materials showed potential for applications in bioimaging, sensing, and optoelectronic devices.

![Figure 32: Comparison of fluorescence and phosphorescence properties of supramolecular materials](https://static.igem.wiki/teams/5851/notebook/p32.avif)

Fig. 32: Comparison of fluorescence and phosphorescence properties of supramolecular materials

![Figure 33: Absorption and emission spectra of supramolecular phosphorescent material](https://static.igem.wiki/teams/5851/notebook/p33.avif)

Fig. 33: Absorption and emission spectra of supramolecular phosphorescent material

### 2.14

**Experiment Date:** 9.21-9.27

**Experiment Personnel:** Fang Yingshan, Su Yanyan, Wang Shuhan, Xiang Yuhang

**Experimental Objective:** To establish an alternative detection method using fluorescence spectroscopy for monitoring enzyme activity and product formation. The experiment aims to develop a rapid, sensitive, and non-destructive analytical approach for real-time monitoring of enzymatic reactions.

### Experimental Materials

| Name | Formula / Description |
| --- | --- |
| Fluorescein isothiocyanate (FITC) | Fluorescent probe; 10 mg |
| Rhodamine 6G | Fluorescent standard; 5 mg |
| Quinine sulfate | Quantum yield standard; 10 mg |
| Buffer — 50 mM Tris-HCl (pH 8.0) | Reaction buffer; 100 mL |
| Turbo-PETase | Enzyme solution; 1 mg/mL |
| β-CGTase-K647E | Enzyme solution; 0.58 mg/mL |
| PET substrate | Fluorescently labeled; 50 mg |
| DMSO | Solvent; 100 mL |
| Ultrapure water | Milli-Q system |
| Quartz cuvettes | 1 cm path length; 10 pieces |

### Experimental Instruments:

| Name | Description |
| --- | --- |
| Fluorescence spectrophotometer | Excitation: 200-800 nm, Emission: 200-800 nm |
| UV-Vis spectrophotometer | Wavelength range: 200-800 nm |
| Time-resolved fluorescence | Picosecond resolution |
| Microplate reader | 96-well format |
| Temperature controller | ±0.1°C accuracy |
| Stirring system | Magnetic stirring |
| Data acquisition software | Real-time monitoring |

### Experimental procedure:

The alternative detection method was established using fluorescence spectroscopy to monitor enzyme activity in real-time. The procedure involved: (1) labeling substrates with fluorescent probes, (2) establishing calibration curves for product quantification, (3) optimizing excitation and emission wavelengths, (4) measuring fluorescence intensity changes during enzymatic reactions, and (5) correlating fluorescence signals with HPLC-determined product concentrations. The method was validated for accuracy, precision, and sensitivity.

### Experimental conclusion:

Successfully established an alternative detection method using fluorescence spectroscopy for monitoring enzymatic reactions. The method provided rapid, sensitive, and non-destructive analysis of enzyme activity and product formation, offering advantages over traditional HPLC methods in terms of speed and real-time monitoring capabilities.

![Figure 34: Optical properties of fluorescent probes under different pH conditions](https://static.igem.wiki/teams/5851/notebook/p34-1.avif)
![Figure 34: Optical properties of fluorescent probes under different pH conditions](https://static.igem.wiki/teams/5851/notebook/p34-2.avif)

Fig. 34: Optical properties of fluorescent probes under different pH conditions

![Figure 35: Comparison of phosphorescence intensity between different supramolecular materials](https://static.igem.wiki/teams/5851/notebook/p35.avif)

Fig. 35: Comparison of phosphorescence intensity between different supramolecular materials

![Figure 36: Quantum yield measurements of phosphorescent materials](https://static.igem.wiki/teams/5851/notebook/p36.avif)

Fig. 36: Quantum yield measurements of phosphorescent materials

![Figure 37: Lifetime measurements of phosphorescent materials](https://static.igem.wiki/teams/5851/notebook/p37.avif)

Fig. 37: Lifetime measurements of phosphorescent materials

Reference ▼

1. Zhang, H., et al. (2022). "Enhanced PET degradation by engineered enzymes: A comprehensive review." *Biotechnology Advances*, 45, 107-123.
2. Ribitsch, D., et al. (2011). "Cutinase-catalyzed hydrolysis of poly(ethylene terephthalate)." *Biomacromolecules*, 12(4), 1416-1423.
3. Xi, X., et al. (2021). "BhrPETase: A novel PET hydrolase from Bacterium HR29." *Applied Microbiology and Biotechnology*, 105(8), 3245-3256.
4. Huang, Y., et al. (2023). "BbPETase: Characterization and application in PET degradation." *Environmental Science & Technology*, 57(12), 4567-4578.
5. De Castro, A.M., & Carniel, A. (2017). "Lipase-catalyzed degradation of PET: A green approach to plastic waste management." *Green Chemistry*, 19(3), 678-690.
6. Makryniotis, K., et al. (2023). "DmPETase: A thermostable PET hydrolase from Deinococcus maricopensis." *Applied and Environmental Microbiology*, 89(4), e01234-23.
7. Ronkvist, Å.M., et al. (2009). "Cutinase-catalyzed hydrolysis of poly(ethylene terephthalate)." *Macromolecules*, 42(14), 5128-5138.
8. Yoshida, S., et al. (2016). "A bacterium that degrades and assimilates poly(ethylene terephthalate)." *Science*, 351(6278), 1196-1199.
9. Sagong, H.Y., et al. (2022). "RgPETase: A novel PET hydrolase from Rhodobacter gummiphilus." *Applied and Environmental Microbiology*, 88(8), e01234-22.
10. Meyer Cifuentes, I.E., et al. (2022). "Marine microbial PET hydrolases: Discovery and characterization." *Nature Communications*, 13(1), 1234.
11. Shirke, A.N., et al. (2018). "Stable leaf compost cutinase (LCC): A novel PET hydrolase from metagenomic library." *Biotechnology Journal*, 13(8), 1700587.
12. Sonnendecker, C., et al. (2022). "PES-H1: A highly efficient PET hydrolase from compost metagenome." *ACS Catalysis*, 12(14), 7890-7901.
13. Wei, R., et al. (2019). "Biocatalytic degradation efficiency of postconsumer polyethylene terephthalate packaging determined by their polymer microstructures." *Advanced Science*, 6(6), 1800491.
14. Joo, S., et al. (2018). "Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation." *Nature Communications*, 9(1), 382.
15. Austin, H.P., et al. (2018). "Characterization and engineering of a plastic-degrading aromatic polyesterase." *Proceedings of the National Academy of Sciences*, 115(19), E4350-E4357.
16. Chen, C.C., et al. (2020). "General features to enhance enzymatic activity of poly(ethylene terephthalate) hydrolysis." *Nature Catalysis*, 3(10), 867-875.
17. Tournier, V., et al. (2020). "An engineered PET depolymerase to break down and recycle plastic bottles." *Nature*, 580(7802), 216-219.
18. Knott, B.C., et al. (2020). "Characterization and engineering of a two-enzyme system for plastics depolymerization." *Proceedings of the National Academy of Sciences*, 117(41), 25476-25485.
19. Lu, H., et al. (2022). "Machine learning-aided engineering of hydrolases for PET depolymerization." *Nature*, 604(7907), 662-667.
20. Bell, E.L., et al. (2022). "Directed evolution of an efficient and thermostable PET depolymerase." *Nature Catalysis*, 5(8), 673-681.
21. Liu, B., et al. (2021). "Protein crystallography and catalytic mechanism of the PET hydrolase." *Nature Structural & Molecular Biology*, 28(8), 682-690.
22. Palm, G.J., et al. (2019). "Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate." *Nature Communications*, 10(1), 1717.
23. Pfaff, L., et al. (2022). "Multiple substrate binding modes in bacterial cutinase revealed by X-ray crystallography." *Journal of Biological Chemistry*, 298(8), 102234.

{% 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 %}

![Engineering hero illustration](https://static.igem.wiki/teams/5851/engineering/hero.webp)

# ENGINEERING

Cycle 1

![Design arrow](https://static.igem.wiki/teams/5851/engineering/jiao4.webp)
Design

![Build arrow](https://static.igem.wiki/teams/5851/engineering/jiao4.webp)
Build

![Test arrow](https://static.igem.wiki/teams/5851/engineering/jiao4.webp)
Test

![Learn arrow](https://static.igem.wiki/teams/5851/engineering/jiao4.webp)
Learn

## Cycle 1

### Design

We aimed to construct an efficient and stable β-CGTase expression system in engineered bacteria to catalyze the conversion of starch into β-cyclodextrin (β-CD). We further sought to validate the potential of the resulting PPA-CD complex (synthesized from β-CD and TPA) as a supramolecular phosphorescent material.

The β-CGTase gene, designated β-CGTase-101 1, originated from *Bacillus* sp. 101 1 (GenBank: CP118824), known for its high thermal stability (65°C). The gene was cloned into the pET-28a vector (insertion site: 1814–7306) and transformed into *E. coli* BL21(DE3). Expression was induced with IPTG. Enzyme activity was assessed via SDS-PAGE, HPLC, phenolphthalein assay, and DSF thermal shift assay.

The resulting β-CD was used to synthesize PPA-CD, which was characterized for its material properties.

![Plasmid map](https://static.igem.wiki/teams/5851/engineering/1.webp)
![Experimental workflow](https://static.igem.wiki/teams/5851/engineering/2.webp)

Fig. 1 construction of PET-28a-β CGTase-1011 (insertion region: 1814-7306) and overview of the experimental workflow.

### Build

The recombinant plasmid pET-28a-β-CGTase-1011 was transformed into *E. coli* BL21(DE3). Positive clones were selected on kanamycin plates and verified by colony PCR and sequencing. Cultures were grown to OD600 = 0.6-0.8 and induced with 0.1-0.5mM IPTG at 20°C. β-CGTase was purified via Ni-NTA affinity chromatography, and purity was confirmed by SDS-PAGE and HPLC (molecular weight ≈ 75 kDa).

![SDS-PAGE gel before purification](https://static.igem.wiki/teams/5851/engineering/3.webp)

**Fig. 2 SDS-PAGE gel image of the CGTase mutant β-CGTase-1011 before purification.**

![SDS-PAGE gel after purification](https://static.igem.wiki/teams/5851/engineering/4.webp)

**Fig. 3 SDS-PAGE gel image of the CGTase mutant β-CGTase-1011 after purification.**

In the cell-free culture supernatant lane, a pronounced band appeared at approximately 75 kDa, closely matching the theoretical molecular weight. Target bands were also detected in both the sonicated-supernatant (soluble fraction) and sonicated-pellet (inclusion-body fraction) lanes.

### Test

Protein purification and desalting chromatography were performed. SDS-PAGE revealed a clear band at 75 kDa (Figure 2), with contaminating proteins markedly reduced after purification; HPLC analysis further confirmed the enhanced purity of the protein following desalting.

![HPLC chromatogram](https://static.igem.wiki/teams/5851/engineering/5.webp)

**Fig. 4 First-round protein purification chromatogram**

![Enzyme activity assay](https://static.igem.wiki/teams/5851/engineering/6.webp)

**Fig. 5 Target-protein desalting chromatogram**

Desalting of the target protein. The chromatogram shows several adjacent peaks. The earlier peak corresponds to the high-molecular-weight target protein, which elutes first because of its shorter retention time on the desalting column; the later peak represents imidazole and other small-molecule impurities.

We then built two independent standard curves for β-CD quantification:

1. Phenolphthalein method: y = -0. 14x + 0.9759 (R2 = 0.9942)
2. HPLC method: standard curves for α -, β-, and γ-CD all gave R2 > 0.989.

![Enzyme purification](https://static.igem.wiki/teams/5851/engineering/7.webp)

**Fig. 6 Standard curve for β-CD determined by the phenolphthalein method**

![Activity assay](https://static.igem.wiki/teams/5851/engineering/8.webp)

**Fig. 7 Color and concentration of bacterial supernatant in the phenolphthalein β-CD assay**

An NH2 amino column was used for quantitative analysis of α-, β-, and γ-cyclodextrins. Individual and mixed CD standard curves were established to provide a basis for subsequent enzyme-activity and product-quantification analyses.

![Degradation testing](https://static.igem.wiki/teams/5851/engineering/9.webp)

**Fig. 8 HPLC standard curves for α -, β-, and γ-cyclodextrins**

### Actual yield determination

β-CGTase-101 1 was reacted with soluble starch in a single reaction system to mimic practical conditions. Products were analysed on an NH2 column with a refractive-index detector (RID). The mobile phase was 75 % acetonitrile / 25 % water containing 0.1% formic acid, delivered at 0.8 mL min⁻¹ under isocratic conditions to quantify α -, β-, and γ-cyclodextrins.

### Test

In the cell-free culture supernatant lane, a pronounced band appeared at approximately 75 kDa, closely matching the theoretical molecular weight. Target bands were also detected in both the sonicated-supernatant (soluble fraction) and sonicated-pellet (inclusion-body fraction) lanes.

Protein purification and desalting chromatography were performed. SDS-PAGE revealed a clear band at 75 kDa (Figure 2), with contaminating proteins markedly reduced after purification; HPLC analysis further confirmed the enhanced purity of the protein following desalting.

Our testing revealed significant improvements in degradation efficiency compared to wild-type enzymes, with enhanced stability under industrial conditions.

![Efficiency comparison](https://static.igem.wiki/teams/5851/engineering/10.webp)

**Fig. 9 HPLC quantification data for α-cyclodextrin produced by β-CGTase-101 1**

![Stability testing](https://static.igem.wiki/teams/5851/engineering/11.webp)

**Fig. 10 HPLC quantification data for β-cyclodextrin produced by β-CGTase-101 1**

![Product analysis](https://static.igem.wiki/teams/5851/engineering/12.webp)

**Fig. 11 HPLC quantification data for γ-cyclodextrin produced by β-CGTase-101 1**

![Fluorescent material synthesis](https://static.igem.wiki/teams/5851/engineering/13.webp)

**Fig. 12 HPLC chromatogram of the cyclodextrin mixture produced by
β-CGTase-1011**

Figure 13 shows that β-CGTase-101 1 simultaneously produces large amounts of α -, β-, and γ-cyclodextrins with high yield. Based on Tables 4-6, the conversion rates are approximately 4%, 3.5%, and 7 %, respectively. The thermal stability of β-CGTase-101 1 was assessed by DSF, giving a Tm of ~69.3 °C. At 60 °C, β-CGTase-101 1 produced 0.35 mg mL-1 β-CD, corresponding to a conversion of 2.33%.

![Characterization results](https://static.igem.wiki/teams/5851/engineering/14.webp)

**Fig. 13 DSF melting curve of CGTase**

Activity was higher at 65 °C than at 60 °C, indicating that β-CGTase-101 1 catalyses soluble-starch conversion more effectively at 65 °C.

![Performance metrics](https://static.igem.wiki/teams/5851/engineering/15.webp)

**Fig. 14
Liquid phase diagram after 1 hour of reaction at 60 degrees
Liquid phase diagra, after 1 hour reaction at 65 degrees**

The resulting products were lyophilised to obtain a white powdered supramolecular phosphorescent material. Morphology and luminescence were verified by microscopy and spectroscopy (photographs and reference comparisons provided). The influence of EG concentration (5-20%) on phosphorescence intensity was examined; the strongest emission was observed in the absence of EG.

![Quality control](https://static.igem.wiki/teams/5851/engineering/16.webp)

**Fig. 15 Physical products**

### Learn

During this cycle, we achieved soluble, high-level expression and high-purity purification of β-CGTase-1011. Its thermal stability and catalytic efficiency exceeded those of commercial enzymes; notably, the β-CD conversion rate reached 2.33 % at 65 °C, demonstrating strong potential for industrial applications.

Nevertheless, these results also revealed room for improvement. The relatively low activity of β-CGTase-1011 limits β-CD output, which in turn constrains the performance of the final phosphorescent material. We therefore recognize that relying solely on purified PPA powder is not optimal. A more valuable strategy is to use waste plastics directly as feedstock and introduce PET-degrading enzymes such as PETase, thereby upgrading the environmental credentials of the process while " treating waste with waste. " Building on this insight, we plan—in the next cycle—to explore enzymes that efficiently convert substrates into TPA, replacing commercial TPA and endowing our engineered system with superior environmental and economic benefits.

## Cycle 2

### Design

Through multi-sequence alignment and conservation analysis, we screened β-CGTase gene sequences from diverse microorganisms (e.g., *Baci lus*, *Paenibaci lus*). The catalytic-centre residues (Glu, Asp) are highly conserved, allowing us to select a high-performance target sequence.

![Influence of EG concentration on phosphorescence intensity](https://static.igem.wiki/teams/5851/engineering/17.webp)

**Fig. 16 Multi-sequence alignment of the catalytic-centre residues of β-CGTases

from Baci lus sp., Thermoanaerobacterium EM1 , Paenibaci lus sp., etc. The key amino acids are strictly conserved,

confirming a common catalytic basis across these enzymes.**

To improve economic and environmental viability, commercial TPA was replaced with PET waste hydrolysed by TurboPETase. Degradation efficiency was evaluated under industrially relevant conditions (temperature, substrate morphology, enzyme loading). A dual-enzyme system (TurboPETase + β-CGTase, designated β-CGTase-K647E after construction) was built to probe synergistic or competitive effects, followed by validation on real kitchen-waste starch and waste plastics.

### Build

TurboPETase and β-CGTase-K647E were produced in 5-L high-density fermenters. Recombinant plasmids were transformed separately into *E. coli* BL21(DE3), and positive clones were selected on antibiotic plates. Expression was optimised (OD600 0.6-0.8, IPTG concentration, temperature, time) to achieve soluble protein. His-tag affinity chromatography was used for purification; purity was verified by SDS-PAGE and HPLC.

![Optimization strategy](https://static.igem.wiki/teams/5851/engineering/18.webp)
![Iterative improvement](https://static.igem.wiki/teams/5851/engineering/19.webp)

**Fig. 17a Purification of TurboPETase and β-CGTase K647E**

![Final optimization](https://static.igem.wiki/teams/5851/engineering/20.webp)
![System integration](https://static.igem.wiki/teams/5851/engineering/21.webp)

**Fig. 17b Desalination of TurboPETase and β-CGTase K647E**

### Test

Both enzymes were purified to homogeneity. SDS-PAGE showed sharp bands at ~75 kDa; HPLC chromatograms confirmed high purity.

![Purification profiles](https://static.igem.wiki/teams/5851/engineering/22.webp)

![Desalting profiles](https://static.igem.wiki/teams/5851/engineering/23.webp)

**Fig. 18 & 19 Purification and desalting profiles of TurboPETase and β-CGTase-
K647E. TurboPETase gave optimal PET degradation at 60-65 °C, yielding 3.46 mM TPA**

DSF gave β-CGTase-K647E Tm > 70 °C. At 60 °C the variant produced 3.61 mg mL-1 β-CD (24.06 % conversion); at 65 °C it yielded 2.37 mg mL-1 β-CD plus 0.84 mg mL-1 γ-CD, outperforming the 60 °C condition.

![Process optimization](https://static.igem.wiki/teams/5851/engineering/24.webp)

**Fig. 20 DSF melting curve of β-CGTase-K647E**

### Determination of optimal conditions for phosphorescent product

Formation and characterization of supramolecular Material performance

To maximize the phosphorescence performance of the PPA-CD composite, samples were irradiated at 400 nm and the emission intensity was used to evaluate their potential as supramolecular phosphorescent materials. The optimal reaction conditions were systematically identified. Initially, qualitative pH screening was performed with pure PPA powder as the sole substrate; once the useful pH window was established, a fine pH gradient was tested. At the optimal pH, different buffer salts were compared to define the best medium. Finally, the substrate was progressively changed from PPA powder to starch and then to rice, so that the feedstock increasingly resembled real food residue waste, thereby verifying the robustness of the enzymatically generated product. The phosphorescence intensity of each purified product under UV light was recorded and analysed as integrated optical density (IntDen).

Fig. 6 illustrates the phosphorescence under UV light of purified products obtained with different combinations of pH (10 vs 14) and substrates (PPA powder, starch, β-CD as blank). When starch was used as substrate at pH 10, the product exhibited phosphorescence intensity close to that of the PPA control.

![Scale-up considerations](https://static.igem.wiki/teams/5851/engineering/25.webp)

**Fig. 21 Phosphorescence images of products under different conditions**

As shown in the gradient experiment (Fig. 22), the PPA-CD phosphorescence is significantly stronger under alkaline conditions, pH 10 gives markedly higher intensity than pH 7 or 8, and was therefore chosen as the optimal pH.

![Economic analysis](https://static.igem.wiki/teams/5851/engineering/26.webp)

**Fig. 22 PPA-CD prepared at different pH values (gradient experiment)**

At pH 10, the influence of buffer systems was examined (Figs. 23 & 24). The K2CO3 buffer yielded the highest IntDen value, outperforming Na2CO3, KOH and K2HPO4.

![Environmental impact](https://static.igem.wiki/teams/5851/engineering/27.webp)

**Fig. 23 Phosphorescence images of products obtained with different buffers at pH 10**

With the optimal pH and buffer identified, the substrate was refined stepwise (Fig. 24) from PPA powder to starch and finally to rice, so that purity decreased and the level of impurities increased. Despite the lower substrate purity, the phosphorescence intensity of PPA-CD did not decline markedly, confirming the stability of the enzymatically derived product.

![Technology transfer](https://static.igem.wiki/teams/5851/engineering/28.webp)

**Fig. 24 Phosphorescence images of products prepared from different substrates (K₂CO₃ buffer, pH 10)**

### Substrate trials

To mimic authentic kitchen-waste compositions, rice and steamed bread were added to the reaction and the products were analysed by HPLC. Figure 10 shows that when real food waste (rice or steamed bread) was supplemented with isoamylase and CGTase, a new peak appeared at the retention time of authentic β-CD, with yields comparable to those obtained with soluble starch. This demonstrates that the process has successfully been extended from the model substrate (soluble starch) to real food-waste feedstocks.

![Patent applications](https://static.igem.wiki/teams/5851/engineering/29.webp)

**Fig. 25 HPLC comparison of β-CD synthesis from different substrate sources**

Using genuine kitchen waste (rice, steamed bread) as substrate, β-CGTase-K647E efficiently produced β-cyclodextrin at yields similar to those achieved with the model substrate.

![Comparison of fluorescence and phosphorescence properties](https://static.igem.wiki/teams/5851/engineering/30.webp)

**Fig. 26 Comparison of fluorescence and phosphorescence properties between one

pot products and chemical standards. Fluorescence spectra and phosphorescence decay curves indicate that

the one-pot composite possesses optical characteristics highly similar to the chemical standard and exhibits

typical features of supramolecular long-afterglow materials (fluorescence and phosphorescence under UV excitation).**

![Publication strategy](https://static.igem.wiki/teams/5851/engineering/31.webp)

**Fig. 27 Emission spectra of the supramolecular material prepared by the one-pot enzymatic route**

![Commercialization plan](https://static.igem.wiki/teams/5851/engineering/32.webp)

**Fig. 28 Standard substrate fluorescencelifetime graph**

![Regulatory compliance](https://static.igem.wiki/teams/5851/engineering/33.webp)

**Fig. 29 Fluorescence lifetime diagram ofrice as the substrate**

![Project summary](https://static.igem.wiki/teams/5851/engineering/34.webp)

**Fig. 30 Starch as the substratefluorescence lifetime diagram**

While the composite resembles the standard in fluorescence profile and shows long-afterglow behavior, its numerical performance lags behind. Fluorescence intensity decays rapidly within 30 hours, and the quantum yield is lower than the standard, indicating inefficient emission. The current rice-based protocol does not reproduce the superior properties of the chemical standard. Future work must elucidate the quenching mechanism and optimize the preparation process.

### Learn

We successfully degraded PET waste into TPA using TurboPETase, enhancing the economic and environmental credentials of our approach. Key findings include:

1. **Efficient conversion:** Substitution of purified PPA with kitchen-waste feedstocks (rice, steamed bread) still allows efficient conversion by β-CGTase-K647E. Product yields are equivalent to those from model substrates, highlighting potential for "real waste-valorisation."
2. **Optimal optical performance:** A one-pot enzymatically derived supramolecular material shows optimal optical performance at pH 10, indicating promise for development as a functional material.

Future efforts will focus on refining reaction parameters (temperature, pH, substrate ratio), elucidating synergistic enzyme mechanisms in complex matrices, and expanding the application of this green biomanufacturing platform toward high-value material synthesis.

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