iGEM 2025 — Team gems-taiwan

{% extends "layout.html" %} {% block title %}Notebook{% endblock %} {% block
lead %}This serves as a chronological record of your team's progress throughout
the season. It documents your daily activities, experiments, discussions, and
decisions.{% endblock %} {% block page\_content %}
![](https://static.igem.wiki/teams/5729/banners/notebook1.webp)

![Bookshelf](https://static.igem.wiki/teams/5729/notebook/bs.webp)

April

May

June

July

August

September

October

📚

### Select a Month

Click any bookshelf to view the corresponding PDF document

{% endblock %}

# Multi-Project DBTL

### Project Cycles

### References

* Dimarogona, M., Topakas, E., & Christakopoulos, P.
  (2012). Cellulose Degradation By Oxidative Enzymes. Computational
  and Structural Biotechnology Journal, 2(3).
  https://doi.org/10.5936/csbj.201209015
* Furukawa, M., Kawakami, N., Tomizawa, A., & Miyamoto,
  K. (2019). Efficient Degradation of Poly(ethylene terephthalate)
  with Thermobifida fusca Cutinase Exhibiting Improved Catalytic
  Activity Generated using Mutagenesis and Additive-based
  Approaches. Scientific Reports, 9(1), 16038.
  https://doi.org/10.1038/s41598-019-52379-z
* Liu, M., & Yu, H. (2012). Co-production of a whole
  cellulase system in Escherichia coli. Biochemical Engineering
  Journal, 69, 204–210. https://doi.org/10.1016/j.bej.2012.09.011
* Shilling, P. J., Mirzadeh, K., Cumming, A. J.,
  Widesheim, M., Köck, Z., & Daley, D. O. (2020). Improved designs
  for pET expression plasmids increase protein production yield in
  Escherichia coli. Communications Biology, 3(1), 214.
  https://doi.org/10.1038/s42003-020-0939-8
* Tang, Z., Li, P., Sun, R., Liu, M., Jin, W., Gou, L.,
  Chen, H., Wu, Q., Bu, T., & Li, C. (2018). Optimum mixture and
  synergy analysis of three main cellulases. International Journal
  of Agriculture and Biology, 20(2), 255–262.
  https://doi.org/10.17957/IJAB/15.0485
* Twist Bioscience. (n.d.). Gene synthesis [Clonal].
  Twist Bioscience. Retrieved September 24, 2025, from
  https://www.twistbioscience.com/products/genes/gene-synthesis?tab=clonal

{% extends "layout.html" %} {% block title %}Experiments{% endblock %} {% block
lead %}Describe the research, experiments, and protocols you used in your
project. It is designed to provide sufficient information for other teams to
replicate our work.{% endblock %} {% block page\_content %}

![](https://static.igem.wiki/teams/5729/banners/experiment1.webp)

## Overview

The experiment section includes the procedure for cloning, expression and
functional evaluation of our plasmid construct and target proteins.
After the target genes were amplified using PCR and inserted into the
pET28a vector using NEBuilder HiFi and Golden Gate Assembly, plasmids
were then propagated in DH5α *E.coli* cells. The colonies were then
screened by colony PCR and confirmed by sequencing before transformation
into BL21 *E.coli* for protein expression.

Protein expression was induced with IPTG, followed by large-scale
extraction and purification. Dialysis was then performed to remove the
impurities. Protein expression and purity were analyzed using SDS-PAGE
and Western blot. Enzyme activity was assessed by using p-nitrophenyl
butyrate (pNPB) hydrolysis assay under different pH and temperature
conditions.

![](https://static.igem.wiki/teams/5729/experiments/introduction.webp)

![Button 1](https://static.igem.wiki/teams/5729/experiments/molecular-cloning.webp)

## Molecular Cloning

PCR Amplification, Double Restriction Enzyme Digestion, Gel Electrophoresis, DNA Assembly, E. coli Competent Cell
Transformation, Colony PCR, Liquid Bacterial Culture, DNA Isolation

![Button 2](https://static.igem.wiki/teams/5729/experiments/recombinant-protein-production.webp)

## Recombinant Protein Production

IPTG induction, Bradford Assay, Trial Scale Protein Extraction, Large Scale Protein Extraction, Protein
Purification & Dialysis, SDS-PAGE, Western Blotting

![Button 3](https://static.igem.wiki/teams/5729/experiments/functional-assay.webp)

## Functional Assay

pNPB Assay, HTS PET film degradation, PET Film Degradation, Pretreatment, Cotton Degradation in Textile, DNS Assay,
PET Degradation in Textile, HPLC Sample Preparation

# Molecular Cloning

## PCR Amplification

##### PCR Amplification

PCR amplification is used for amplifying the desired sequences from the synthetic template. Therefore, the
sequence
accuracy is an important issue in this experiment. In this section, we used two different proofreading DNA
polymerases
to obtain desired sequences in this project: Ex Taq DNA polymerase (Takara) and Q5 High-Fidelity DNA
polymerase (NEB).
Also, to avoid random mutation and ensure the sequence accuracy, we limited the thermal cycles of PCR within
25 cycles.
The following sections show more details about our experiments.

**Ex Taq DNA polymerase (Takara)**

1. Materials

* TaKaRa Ex Taq™ (5 units/μl)
* 10X Ex Taq Buffer
* dNTP Mixture (2.5 mM each)
* DNA Template
* Forward Primer
* Reverse Primer
* PCR-grade water

2. Procedure

1. Master mix preparation by adding all the following components for each sample.

   | Reagent | 1 Reaction (50 μL) |
   | --- | --- |
   | 10× ExTaq buffer | 5 μL |
   | 2.5 mM dNTP mix | 4 μL |
   | 10 μM Forward primer | 1 μL |
   | 10 μM Reverse primer | 1 μL |
   | Template (10 ng/μL) | 2 μL |
   | TaKara Ex Taq  TM | 0.25 μL |
   | PCR-grade water | 36.75 μL |
2. Incubate in the PCR machine and run the following thermal cycle.

   | Step | | Temperature | Duration |
   | --- | --- | --- | --- |
   | Initial Denaturation | | 98 °C | 30 sec |
   | 25 Cycles | Denature | 98 °C | 10 sec |
   | Annealing | 50–72 °C | 30 sec |
   | Extension | 72 °C | 1 min/kb |
   | Final Extension | | 72 °C | 2 min |

**Q5 High-Fidelity DNA polymerase (NEB)**

1. Materials
   * 5x Q5 Reaction buffer
   * 10 mM dNTP
   * Forward primer
   * Reverse primer
   * Template DNA
   * Q5 High-Fidelity DNA Polymerase
   * PCR-grade water

2. Procedure
   1. Master mix preparation by adding all the following components for each sample.

      | Reagent | 1 Reaction (50 μL) |
      | --- | --- |
      | 5x Q5 Reaction buffer | 10 μL |
      | 10 mM dNTP mix | 1 μL |
      | 10 μM Forward primer | 2.5 μL |
      | 10 μM Reverse primer | 2.5 μL |
      | Template DNA (10 ng/μL) | 2 μL |
      | Q5 High-Fidelity DNA Polymerase | 0.5 μL |
      | PCR-grade water | 31.5 μL |
   2. Incubate in the PCR machine and run the following thermal cycle.

      | Step | | Temperature | Duration |
      | --- | --- | --- | --- |
      | Initial Denaturation | | 98 °C | 30 sec |
      | 25 Cycles | Denature | 98 °C | 10 sec |
      | Annealing | 50–72 °C | 30 sec |
      | Extension | 72 °C | 30 sec/kb |
      | Final Extension | | 72 °C | 2 min |

![PCR program diagram](https://static.igem.wiki/teams/5729/experiments/pcr-amplification.webp)

## Double Restriction Enzyme Digestion

##### **Double Restriction Enzyme Digestion**

Double restriction enzyme digestion is where the vector backbone is digested using two specific
restriction
enzymes that
depend on the restriction sites for DNA assembly.

1. Materials

* 10X CutSmart Buffer (NEB)
* DNA Template
* ddH2O
* Enzyme List: EcoRI-HF, XhoI, HindIII-HF, SalI-HF, BamHI-HF.
  All enzymes at concentrations of 20,000 units/mL (NEB)

2. Procedure
   1. Calculate the reaction formula

      | Reagent | Notes | Volume |
      | --- | --- | --- |
      | 10× CutSmart buffer | 1X | 2 μL |
      | Enzyme 1 | 20 units/μL | 0.5 μL |
      | Enzyme 2 | 20 units/μL | 0.5 μL |
      | DNA Template | 500 ng | = 500 (ng) / Plasmid conc. |
      | ddH2O | Up to 20 μL | = 20 − (sum of everything else) |
   2. Prepare the reaction mixture in a microcentrifuge tube by adding ddH2O, DNA, buffer, with the
      restriction enzymes added
      last.
   3. Incubate the reaction in a water bath or a thermocycler at 37°C for at least 4 hours or overnight. If
      incubating in a
      water bath, seal the microtube with parafilm to prevent evaporation.

   ![Restriction digestion result](https://static.igem.wiki/teams/5729/experiments/2srestriction.webp)

## Gel Electrophoresis

##### **DNA Gel Electrophoresis**

Gel electrophoresis is used to separate DNA fragments by size, allowing for size-based separation and
analysis.

1. Materials
   * Agarose Powder (Bionovas)
   * 1X TAE Buffer (Omics Bio)
   * DNA Safety Dye (Bioman scientific)
   * 6X DNA Loading Dye (NEB)

2. Procedure

   1. Assemble the gel tray and comb.
   2. Decide the concentration of gel and weigh the appropriate amount of agarose powder.
   3. Add 1X TAE buffer to the agarose powder in a conical flask.
   4. Cover the flask with plastic wrap, and use a tip to puncture a hole through it.
   5. Microwave for 30 sec and agitate carefully with heat-resistant gloves.
   6. Repeat a short microwave for 5 seconds with agitation until all particles dissolve.
   7. Cool the solution until warm and not smoking.
   8. Add the DNA safety dye at 20,000X concentration and mix gently.
   9. Pour the solution into the gel tray, insert the comb, and let it solidify.
   10. Remove the comb and place the gel in the electrophoresis tank.
   11. Mix 1 μL 6X DNA loading dye with 5 μL sample.
   12. Load 6 μL into the well.
   13. Close the lid, set the power supply to 100V.
   14. Press ‘START’.
   15. After separation, visualize the DNA bands under UV light.

   ![](https://static.igem.wiki/teams/5729/experiments/gelelectrophoresis3.webp)

## DNA Assembly

##### **NEBuilder HiFi DNA Assembly**

NEBuilder HiFi DNA Assembly is a seamless cloning method that enables the precise and efficient joining of
multiple DNA
fragments into a vector. It is ideal for constructing complex constructs like multi-gene operons or tagged
fusion
proteins.

1. Materials
   * 2X NEBuilder HiFi Assembly Master Mix (NEB)
   * Vector: pET28a vector
   * Inserts: TfCut2 5ZOA, Variant 1, Variant 2, Variant 3, Variant 3, Variant 4, Variant 5,
     Variant 6, BsEglS, BhBglA or CbhA
   * ddH2O

2. Procedure
   1. Calculate the reaction formula. For 2-3 Fragment Assembly:

      | Reagent | Volume |
      | --- | --- |
      | 2X NEBuilder HiFi Assembly Master Mix | 10 μL |
      | Total amount of fragments (insert + vector) | X μL (0.03–0.2 pmols) |
      | ddH2O | (10 − X) μL |
      | Total Volume | 20 μL |

      \*vector:insert = 1:2
   2. Prepare reagents above for the reaction.
   3. Use a thermocycler to incubate the reaction at 50°C for 15 minutes.
   4. Following incubation, store samples on ice or at -20°C for subsequent transformation.

##### **Golden Gate Assembly**

Golden Gate Assembly is a one-step, one-tube molecular cloning method that uses a Type IIS restriction
enzyme and DNA
ligase to seamlessly and directionally assemble multiple DNA fragments into a single, scarless DNA
construct.

1. Materials

   | Vectors | Inserts |
   | --- | --- |
   | pJUMP29-1A | Promoter BBa\_J23100  RBS BBa\_J428032  CIAP  Double terminator BBa\_B0015 |
   | pJUMP29-1B | Promoter BBa\_J23100  RBS BBa\_J428032  β-lactamase  Double terminator BBa\_B0015 |
   | pJUMP29-1C | TphC  TpiB  TpiA |
   | pJUMP29-1D | Promoter BBa\_J23100  RBS BBa\_J428032  TphR  Double terminator BBa\_B0015 |

   * T4 DNA Ligase (Promega or NEB)
   * T4 DNA Ligase 10X Buffer (Promega or NEB)
   * Enzymes List: BsaI-HF (20,000 units/mL), BsmBI-v2 (10,000 units/mL) (NEB)
   * ddH2O

2. Procedure

   | Reagent | Volume |
   | --- | --- |
   | Vector | X μL (10 fmol) |
   | Inserts | X μL (20 fmol) |
   | T4 DNA ligase | 1 μL |
   | T4 DNA ligase 10X buffer | 2 μL |
   | Enzyme | 1 μL |
   | ddH2O | 20 - (sum of everything else) |
   | Total Volume | 20 μL |

   Formula for calculating vector and insert volume:

   $$\text{Vector: } 0.02 = \left( \frac{x}{\text{sequence length (bp)} \times 650} \right) \div 1000
   \div
   \text{DNA concentration}$$

   $$\text{Insert: } 0.01 = \left( \frac{x}{\text{sequence length (bp)} \times 650} \right) \div 1000
   \div
   \text{DNA concentration}$$

   1. Prepare the reagents above for the reaction. \*Enzyme added last.
   2. Use the thermocycler to run the program below.

   | Temperature | Time | Cycles |
   | --- | --- | --- |
   | 37 °C (Restriction enzyme digestion) | 1.5 minutes | 25-45 cycles |
   | 16 °C (Ligation) | 3 minutes |
   | 37 °C (Restriction enzyme digestion) | 5 minutes | 1 cycle |
   | 60 °C (Heat inactivation of BsaI restriction enzyme) | 10 minutes | 1 cycle |

![](https://static.igem.wiki/teams/5729/experiments/dna-assembly.webp)

## E. coli Competent Cells Transformation

##### **E.coli Competent Cell Transformation**

E. coli competent cell transformation is a method used to introduce foreign DNA, usually plasmids, into
bacterial cells
for cloning or protein expression.

1. Materials

   * Recombinant Plasmids
   * Competent Cells
     + Value 108 HIT-DH5α competent cell (RBC)
     + HIT-21 BL21(DE3) competent cell (RBC)
     + ECOS-101 (DH5a) competent cell (Yeastern Biotech)
   * Terrific Broth (TB) Medium (Bionovas)
   * Antibiotics
     + Ampicillin sodium salt (Bionovas)
     + Kanamycin sulfate (Omics Bio)
     + Chloramphenicol (BioShop)
     + Spectinomycin (Kadin tech)

   ![cell transformation](https://static.igem.wiki/teams/5729/experiments/transformation2.avif)
2. Procedure
   1. Mix plasmid with competent cells in the ratio of 1:15 (If the DNA is assembly product, the ratio is
      2:100 to
      10:100).
   2. Incubate on ice for 20 minutes.
   3. Heatshock at 42°C for 60 seconds.
   4. Incubate on ice for 3 minutes.
   5. If the DNA is the assembly product, add 1 mL TB medium and incubate at 37°C for 1 hour. If not, skip
      to step 7.
   6. After incubation, centrifuge at 16,000 g for 30 seconds. Remove 1 mL medium and resuspend the pellet
      in the
      remaining medium.
   7. Use a Drigalski spatula to spread the medium on the plate with 100 μL antibiotics.

      | Antibiotics | Stock Concentration (mg/mL) |
      | --- | --- |
      | Ampicillin | 50 mg/mL |
      | Chloramphenicol | 25 mg/mL |
      | Kanamycin | 50 mg/mL |
      | Spectinomycin | 50 mg/mL |
   8. Incubate at 37°C for 16 hours.

## Colony PCR

##### **Colony PCR**

Colony PCR is used to quickly screen bacterial colonies for the presence of a specific DNA insert or
plasmid, without
needing to isolate plasmid DNA first.

- Materials
  * Q-Amp 2X ScreeningFire Taq Master Mix (Bio Genesis Tech)
  * Forward Primer (10 μM)
  * Reverse Primer (10 μM)
  * Deionized water (ddH2O)
  * Agar Plate
  * Antibiotics
    + Ampicillin sodium salt (Bionovas)
    + Kanamycin sulfate (Omics Bio)
    + Chloramphenicol (BioShop)
    + Spectinomycin (Kadin tech)
  * MiniAmp Thermal Cyclers (Thermo Fisher Scientific)
    Incubator

![colonyPCR](https://static.igem.wiki/teams/5729/experiments/colonypcr.webp)

1. Procedure

   |  | 1 Reaction | Final Volume |
   | --- | --- | --- |
   | Q-Amp 2X ScreeningFire Taq Master Mix | 1X | 10μL |
   | Forward primer (10μM) | 0.5μM | 1μL |
   | Reverse primer (10μM) | 0.5μM | 1μL |
   | ddH2O | - | 8μL |
   | Total Volume | - | 20μL |

   1. Choose well-isolated colonies from your agar plate. Circle and number them.
   2. Draw the square grids on a new agar plate and label with numbers according to the colony's
      numbers.
   3. Add 100 μL antibiotics and spread over the surface until absorbed.

      | Antibiotics | Stock Concentration (mg/mL) |
      | --- | --- |
      | Ampicillin | 50 mg/mL |
      | Chloramphenicol | 25 mg/mL |
      | Kanamycin | 50 mg/mL |
      | Spectinomycin | 50 mg/mL |
   4. Prepare and label PCR tubes with the number corresponding to the agar plate.
   5. Prepare PCR master mix by adding the reagents above in sterile 1.5 mL microcentrifuge
      tubes.
   6. Aliquot the PCR mixture into PCR tubes.
   7. Pick a colony by using a sterile micropipette tip and streak on the gridded plate.
   8. Transfer and briefly stir the remaining cell on tip into the PCR master mix.
   9. Repeat steps 7 to 8 for the remaining colonies.
   10. Load the PCR tubes into the thermocycler and run the program below.

   | Temperature | Time | Cycles |
   | --- | --- | --- |
   | 95 °C | 2 min | 1 cycle |
   | 95 °C | 30 sec | 30 cycles |
   | 65 °C | 40 sec |
   | 72 °C | 1 kb/min |
   | 72 °C | 1 min | 1 cycle |

## Liquid Bacterial Culture

##### **Liquid Bacterial Culture**

Liquid culture is a method to grow bacteria such as E. coli in nutrient-rich broth, allowing rapid
multiplication in a
suspended, aerated environment.

1. Materials
   * Antibiotics
     + Ampicillin sodium salt (Bionovas)
     + Kanamycin sulfate (Omics Bio)
     + Chloramphenicol (BioShop)
     + Spectinomycin (Kadin tech)
   * Terrific Broth (TB) Medium (Bionovas)
   * Pipette Tip
   * Bacterial Colonies
   * Incubator with orbital shaker

2. Procedure
   1. Label the tube with bacteria strain, sample name, type of antibiotics and date.
   2. Light and work near the Bunsen burner to maintain sterile conditions.
   3. Pipette 3 mL Terrific Broth (TB) medium to the labeled tube.
   4. Add the antibiotics by the following table. Using a sterile pipette tip, pick a single colony from
      agar plate and
      swirl the tip into the TB media with antibiotics.

      | Antibiotics | Stock Concentration (mg/mL) | Volume per 3 mL | Final Concentration (µg/mL) |
      | --- | --- | --- | --- |
      | Ampicillin | 50 mg/mL | 3 µL | 50 µg/mL |
      | Chloramphenicol | 25 mg/mL | 3 µL | 25 µg/mL |
      | Kanamycin | 50 mg/mL | 1.5 µL | 25 µg/mL |
      | Spectinomycin | 50 mg/mL | 3 µL | 50 µg/mL |
   5. Using a sterile pipette tip, pick a single colony from agar plate and swirl the tip into the TB
      media with antibiotics.
   6. Loosely cover the tube without airtight to allow oxygen exchange for bacterial growth.
   7. Incubate bacterial culture at 37°C, 200 rpm for 12–18 hr in a shaking incubator.

![SDS-PAGE Setup or Result](https://static.igem.wiki/teams/5729/experiments/liquid-bacterial-culture2.webp)

## DNA Isolation

##### **DNA Isolation**

DNA isolation is the process of isolating DNA from other components like RNA and proteins. The process
involves cell
lysis using a buffer containing detergents, followed by removal of proteins and other contaminants using
enzymatic
treatment or organic solvents.

1. Materials
   * Bacterial Culture
   * Mini Plus Kit (Viogene)
   * Centrigure

2. Procedure
   1. Transfer 1 mL of overnight bacterial culture to 1.5 mL microcentrifuge tube.
   2. Centrifuge at 16,000 x g for 1 minute.
   3. Discard supernatants.
   4. Repeat steps 1–3 until all culture medium has been processed and pellets collected.
   5. Use Viogene mini plus kit for DNA isolation.
   6. Add 200 μL of MX1 buffer.
   7. Resuspend pellets by pipette mixing.
   8. Add 250 μL of MX2 buffer.
   9. Gently mix buffer well (do not vortex) and invert 8–10 times.
   10. Incubate the mixture at room temperature for 2 minutes.
   11. Add 350 μL MX3 buffer.
   12. Immediately mix and invert the solution.
   13. Centrifuge at 16,000 x g for 5 minutes.
   14. While centrifuging, assemble column collection tube and collect supernatant.
   15. Transfer supernatant (~500 μL) to column and centrifuge at 7,000 x g for 30 seconds.
   16. Discard flow-through.
   17. Add 500 μL WN buffer and centrifuge at 7,000 x g for 30 seconds.
   18. Discard flow-through.
   19. Add 700 μL WS buffer and centrifuge at 7,000 x g for 30 seconds.
   20. Discard flow-through.
   21. Centrifuge empty column at 10,000 x g for 3 minutes to remove residual ethanol.
   22. Place column into new 1.5 mL microcentrifuge tube.
   23. Add 20–50 μL elution buffer onto the center of the membrane and incubate the column at room
       temperature for 3
       minutes.
   24. Centrifuge at 10,000 x g for 3 minutes.
   25. Turn on Biodrops and open ‘Apps’.
   26. Clean micro-volume sample port with ddH2O, EtOH, then ddH2O.
   27. Measure the elution buffer as blank.
   28. Measure the concentration, A260/230, A260/280 of the samples and record.
   29. Clean micro-volume port with ddH2O, EtOH, then ddH2O.

![DNA Isolation Setup](https://static.igem.wiki/teams/5729/experiments/dna-isolation3.webp)

# Recombinant protein production

## IPTG Induction

##### **IPTG Induction**

IPTG is a stable, non-metabolizable analog of allolactose that induces the lac operon in E. coli. By binding
and
inactivating the lac repressor, it allows the transcription from lac operon–controlled promoters such as
lac, tac, T7
promoters, enabling control over the timing and level of recombinant protein production, including enzymes,
fluorescent
proteins, and pharmaceuticals proteins.

- Materials
  * Antibiotics
    + Ampicillin sodium salt (Bionovas)
    + Kanamycin sulfate (Omics Bio)
    + Chloramphenicol (BioShop)
    + Spectinomycin (Kadin tech)
  * Terrific Broth (TB) Medium (Bionovas)
  * Overnight Liquid Culture
  * IPTG (Bionovas)
  * Incubator with orbital shaker

![IPTG Induction procedure](https://static.igem.wiki/teams/5729/experiments/iptg5.avif)

1. Procedure
   1. Prepare 250 mL Terrific Broth (TB) medium in a 500 mL flask.
   2. Add the antibiotics by the following table.

      | Antibiotics | Stock Concentration (mg/mL) | Volume per 3 mL | Final Concentration (µg/mL) |
      | --- | --- | --- | --- |
      | Ampicillin | 50 mg/mL | 3 µL | 50 µg/mL |
      | Chloramphenicol | 25 mg/mL | 3 µL | 25 µg/mL |
      | Kanamycin | 50 mg/mL | 1.5 µL | 25 µg/mL |
      | Spectinomycin | 50 mg/mL | 3 µL | 50 µg/mL |
   3. Add 10 mL of overnight liquid culture to 250 mL TB medium with antibiotics (ratio 1:25).
   4. Incubate at 37°C on the shaker at 200 rpm.
   5. Measure OD600 after 2 hours. Then, measure every hour when OD600 is under 0.2,
      every 30 minutes when it is between 0.2–0.3, and every 15 minutes when it is between 0.3–0.5.
   6. Add 1 M IPTG (final concentration of 1 mM) when OD600 is between 0.5–0.8.
   7. Incubate at 16°C and 200 rpm for 20 hours.

## Bradford Protein Assay

##### **Bradford Protein Assay**

The Bradford protein assay is a colorimetric method used to measure protein concentration, where the binding
of
Coomassie Brilliant Blue dye to proteins produces a color change that can be quantified using a
spectrophotometer.

1. Materials
   * Albumin Standard (2.0 mg/mL) (T-Pro Biotech)
   * Bradford assay reagent (Bio-Rad)
   * Deionized water (ddH 2O)
   * BioDrop Duo+ Microvolume Spectrophotometer (Harvard Bioscience)

2. Procedure
   1. Prepare Albumin standards from 2 mg/mL stock:

   |  | 600 μg/ml | 500 μg/ml | 400 μg/ml | 300 μg/ml | 200 μg/ml | 100 μg/ml |
   | --- | --- | --- | --- | --- | --- | --- |
   | BSA | 30 | 25 | 20 | 15 | 10 | 5 |
   | ddH2O | 70 | 75 | 80 | 85 | 90 | 95 |

   2. Prepare 1X Bradford reagent by diluting 1 part reagent concentrate with 4 parts distilled water.
   3. Aliquot 980 μL of 1X Bradford reagent into labeled tubes.
   4. Add 20 μL of each standard or diluted protein sample into the corresponding tubes.
   5. Vortex gently and incubate at room temperature for 5 minutes.
   6. Measure and record absorbance at 595nm using a spectrophotometer.

![Bradford Protein Assay](https://static.igem.wiki/teams/5729/experiments/bradfordprotein6.avif)

## Trial Scale Protein Extraction

##### **Trial Scale Protein Extraction**

The trial-scale protein extraction from E. coli using CelLytic B is to test different lysis conditions.

1. Materials
   * 10X CelLytic B Solution (Sigma Aldrich)
   * Deionized water (ddH2O)
   * Bacterial Culture

2. Procedure
   1. Centrifuge 1 mL liquid culture at 15800 xg for 2 minutes to pellet the cells, OD600: 0.5-1.0
   2. Prepare CelLytic B solution at different dilutions (5, 10, 20, 25, and 30-fold dilutions) with a total
      volume of 400 μL
      for each dilution.
   3. Resuspend cell pellet in 400 µL of each different CelLytic B dilution.
   4. Briefly vortex the solutions to resuspend the cell pellets and mix for 5-10 minutes to ensure full
      extraction of the
      soluble proteins.
   5. Centrifuge the cell lysate at 15800 xg for 5 minutes to pellet any insoluble material.
   6. Carefully remove the soluble protein fraction from the cell debris.
   7. Analyze the supernatant and the insoluble fraction by SDS-PAGE and/or Western blot to determine which
      fraction contains
      the protein of interest. For SDS-PAGE, it is recommended that 5-15 µL of each sample be applied to the
      gel.

![Trial Protein Extraction](https://static.igem.wiki/teams/5729/experiments/trial-scale-protein-extraction2.avif)

## Large-Scale Protein Extraction

##### **Large-Scale Protein Extraction**

Large-scale protein extraction is used to isolate proteins from high-volume cell cultures for purification
or analysis.

1. Materials
   * Liquid Culture* 1X CelLytic B (Sigma Aldrich)
     * DNase I (20 mg/mL) (Bionovas)
     * 100X Protease Inhibitors (TargetMol)
     * Lysozyme (10 mg/mL) (RBC)

2. Procedure
   1. Centrifuge total 250 mL liquid culture at 1967 x g for 10 minutes to pellet the cells when
      OD600 ≈ 2.0.
      Repeat until all bacterial culture has been processed and the pellets collected.
   2. Prepare 1X CelLytic B by diluting 1 mL 10X CelLytic B with 9 mL H2O.
   3. Resuspend pellets in 10 mL 1X CelLytic B and mix thoroughly.
   4. Add reagents in the following order:
      1. DNase I (final concentration of 25 µg/mL) to decrease the viscosity of the solution.
      2. Protease inhibitors (final 1X) to prevent proteolytic degradation.
      3. Lysozyme (final concentration of 0.2 mg/mL) to enhance cell lysis.
   5. Incubate the suspension with shaking at room temperature for 15 minutes to fully extract the soluble
      proteins from
      the cells.
   6. Centrifuge at maximum speed for 15 minutes to pellet the insoluble material.
   7. Carefully transfer the supernatant to a new 1.5 mL tube. Another round of extraction will yield more
      soluble
      protein if required; however, this will result in a more dilute soluble protein sample.
   8. Analyze the supernatant by SDS-PAGE and/or Western blot. For SDS-PAGE, it is recommended that 5–15
      µL of each
      sample be applied to the gel.

![Large Scale Protein Extraction](https://static.igem.wiki/teams/5729/experiments/proteinextraction5.avif)

## Protein Purification & Dialysis

##### **Protein Purification**

Protein purification is the process of isolating a specific protein from a complex mixture, such as a cell
lysate, using
techniques that separate proteins based on properties like size, charge, solubility, or binding affinity
(His-tag).

1. Materials
   * HisPur Ni-NTA Purification Kit (Thermo Fisher Scientific)
     + 2 M Imidazole
     + 10X PBS
     + Ni-NTA column

2. Procedure
   1. Prepare the following buffer (50 mL).

      | Buffer | Imidazole Final Concentration (mM) | 10X PBS (mL) | 2M Imidazole (µM) | Water (mL) |
      | --- | --- | --- | --- | --- |
      | Equilibrate Buffer | 10 | 5 | 250 | 44.75 |
      | Wash Buffer | 25 | 5 | 625 | 44.375 |
      | Elute Buffer | 150 | 5 | 3750 | 41.25 |
   2. Centrifuge the Ni-NTA column at 175 x g for 2 minutes to remove the storage buffer.
   3. Add 6 mL equilibrate buffer.
   4. Add all protein lysate and incubate for 30 minutes at room temperature.
   5. Add 6 mL of wash buffer and remove the flow-through. Repeat this step 3 times.
   6. Centrifuge at 175 x g for 2 minutes.
   7. Collect the flow-through from the last wash buffer step.
   8. Add 3 mL of elute buffer.
   9. Centrifuge at 175 x g for 2 minutes.
   10. Collect the flow-through as the purified protein.

![Protein Purification](https://static.igem.wiki/teams/5729/experiments/proteinpurification6.avif)

##### **Protein Dialysis**

Dialysis removes undesired chemicals, which may interfere further experiment, from protein extracts. In
our experiment,
we removed imidazole which we use to elute his-tag protein from the Ni-NTA column. Since the molecular
weight of TfCut2
is 32.5 kDa, we choose 10 kDa dialysis cassettes to remove imidazole.

1. Materials
   * 4.8 L 1X PBS pH 8
   * Slide-A-Lyzer G3 Dialysis Cassettes, 10K MWCO, 3 mL (Thermo Fisher Scientific)
   * 2 L Beaker × 3
   * Plastic wrap
   * Ice bag
   * Stir plate
   * Stir bar

2. Procedure
   1. Dilute 10X PBS: Mix 160 mL 10X PBS and 1.3 L deionized water.
   2. Adjust pH to 8.
   3. Top up H2O to 1.6 L, wrap opening of beaker with plastic wrap.
   4. Prepare three beakers of 1.6 L PBS.
   5. Open the dialysis cassette by gently twisting the cap counterclockwise.
   6. Add the protein lysate to the cassette, slowly withdrawing the pipette while dispensing. Do not
      overload the
      cassette.
       *Note:* To load the cassette, insert the serological pipette fully into the
      device
      and slowly
      remove the
      pipette while filling. Repeat as needed.
   7. Remove the excess air in the cassette by simultaneously pressing the membrane gently on both sides
      using your
      gloved thumb and forefinger and insert the cap (Figure 4).
       *Note:* The minimum sample
      volume
      required for the cassettes is approximately ½ of the
      cassette’s maximum
      volume.
   8. Insert cap and lock by gently twisting it clockwise until the arrows align and a firm stop is felt.
   9. Place dialysis cassette in 2 L beaker with 1.6 L 1X PBS and stir bar. Stir on ice for 2 hours.
   10. Replace 1X PBS and stir on ice for another 2 hours.
   11. Replace 1X PBS and stir on ice overnight.
   12. Transfer lysate containing recombinant protein from dialysis cassette to 1.5 mL microtubes.
   13. The recombinant protein can be used for further experiments or stored at -80°C.

![Protein Dialysis](https://static.igem.wiki/teams/5729/experiments/protein-dialysis1.avif)

**Ni-NTA column Regeneration**

After purification, we regenerate the Ni-NTA column for the next round of purification. We wash Ni-NTA
column with a MES
(2-(N-morpholine)ethanesulfonic acid) buffer containing 0.1 M NaCl to remove residual imidazole and
nonspecifically
bound proteins, followed by washing with ultrapure water and then storing it in a solution of 20% ethanol in
water to
prevent microbial growth. This process returns the resin to a condition where it can be reused for protein
purification
without significant loss of binding capacity.

1. Materials
   * 1M MES buffer (pH 5.0) (Thermo Fisher Scientific)
   * NaCl
   * Deionized water
   * 20% ethanol

2. Procedure
   1. Prepare 50 mL regeneration buffer: Add 1 mL 1M MES buffer and 0.2922 g NaCl. Add deionized water to
      50 mL.
   2. Add 10 volumes (30 mL) of regeneration buffer to the Ni-NTA column.
   3. Discard the flow-through.
   4. Add 10 volumes (30 mL) of deionized water to the Ni-NTA column.
   5. Discard the flow-through.
   6. Add 15 mL 20% ethanol and seal the Ni-NTA column.
   7. Store the Ni-NTA column at 4 °C.

## SDS-PAGE and Coomassie Blue Staining

##### **SDS-PAGE**

SDS-PAGE, which stands for Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, is used to separate
proteins by
size. Smaller proteins will move faster and travel farther than larger ones, enabling size-based separation.

1. Material
   * TGX Stain-Free FastCast Acrylamide Kit, 10% (Bio-Rad)
   * TEMED (Bio Basic)
   * 10% APS (Bio Basic)
   * ddH2O
   * 6X Laemmli Sample Buffer (Omics Bio)
   * 1X Running Buffer (Omics Bio)

2. Procedure
   1. Assemble casting frames with two 1.5 mm glass plates.
   2. Prepare solutions for the stacking (top) and resolving (bottom) gels:
      * Casting preparation volumes (n = number of gel)

        | Component | Stacker (top gel) | Resolver (bottom gel) |
        | --- | --- | --- |
        | Resolver A | — | 4 mL × n |
        | Resolver B | — | 4 mL × n |
        | Stacker A | 1.5 mL × n | — |
        | Stacker B | 1.5 mL × n | — |
        | Total Volume | 3 mL × n | 8 mL × n |
        | TEMED | 3 μL × n | 4 μL × n |
        | 10% APS | 15 μL × n | 40 μL × n |

        **Note:** TEMED is stored at room temperature. 10%
        APS is stored at −20°C.
   3. Transfer the resolving gel between the glass plates using a 1000 μL pipette.
   4. Overlay with 1 mL ddH2O to level the surface.
   5. Allow the gel to polymerize fully for around 20–30 minutes.
   6. Discard the overlay water gently.
   7. Add the stacking gel until it overflows.
   8. Insert comb to form wells and allow it to set (~10–15 minutes).
   9. Remove the comb and mount the gel into the electrophoresis tank.
   10. Mix 5–15 μL of protein sample with 6X Laemmli Sample Buffer to make a total of 20 μL.
   11. Mix 5 μL ladder with 1 μL 6X Laemmli Sample Buffer.
   12. Heat samples at 95–100°C for 5–10 minutes.
   13. Briefly spin down.
   14. Fill the inner and outer chambers of the gel tank with 1X running buffer.
   15. Load the protein ladder in the first lane.
   16. Load prepared protein samples into the remaining wells.
   17. Close the lid and connect the electrodes.
   18. Run at constant voltage 200 V for 10 minutes until the dye reaches the bottom of the stacking
       gel, then
       continue electrophoresis at a constant voltage (100–150 V) until the dye reaches near the bottom
       of the gel.
   19. Carefully disassemble the glass plates and collect the gel for subsequentanalysis.

![SDS-PAGE](https://static.igem.wiki/teams/5729/experiments/sdspage5.avif)

##### **Coomassie Blue Staining**

Coomassie blue staining is used to visualize proteins in polyacrylamide gels after electrophoresis.

1. Materials
   * Coomassie blue R250
   * Methanol
   * Acetic Acid

2. Procedure

   | Coomassie Blue Solution Recipe (50 mL) | |
   | --- | --- |
   | Methanol | 50% |
   | Acetic acid | 10% |

   | Coomassie Blue Destain Buffer (50 mL) | |
   | --- | --- |
   | Methanol | 30% |
   | Acetic acid | 10% |

   1. Transfer the gel to a staining tray.
   2. Wash gel with ddH2O for 5 minutes, repeat 3 times.
   3. Stain the gel with 0.05% Coomassie Blue R250 for 1 hour on the shaker at 50 rpm.
   4. Destain the gel by soaking in Coomassie Blue destain buffer for at least 1 hour until protein bands
      are clearly
      visible against a transparent background.
   5. Document the gel image and compare the protein bands to the ladder.

![Coomassie Blue Staining](https://static.igem.wiki/teams/5729/experiments/coomassie-blue-staining.webp)

## Western Blotting

##### **Western Blotting**

Western blotting is a technique used to detect a specific protein in a sample. Also known as immunoblotting,
it uses
specific antibodies to detect a target protein and provides information on its presence, quantity, and
characteristics
within a complex protein mixture.

1. Materials
   * PVDF membrane (Millipore)
   * 3M papers
   * Sponge pad
   * Filter paper
   * Acrylamide gel
   * Cassette
   * 1X Transferring buffer
   * His-Tag Antibody (AD1.1.10) (sc-53073, Santa Cruz Biotechnology)
   * Peroxidase AffiniPure® Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch)
   * T-Pro LumiDura Chemiluminescent Substrate (ECL) Kit (T-Pro Biotech)

2. Procedure

    **Gel collection**1. After SDS-PAGE, carefully collect the gel for transfer.
    **Transferring** 2. Soak the PVDF membrane in methanol for 1 minute.
   3. Equilibrate the membrane in 1X transfer buffer for 3 minutes.
   4. Equilibrate the gel in 1X transfer buffer for 5 minutes.
   5. Wet 3M papers in 1X transfer buffer.
   6. Assemble the transfer sandwich in the cassette in the following order: sponge pad → filter paper →
      gel → PVDF
      membrane → filter paper → sponge pad. Ensure there are no air bubbles between the gel and membrane.
   7. Place the cassette into the tank filled with cold 1X transfer buffer and an ice block.
   8. Transfer at 100 V for 90 minutes.
    **Blocking** 9. Block PVDF membrane in 5% non-fat milk for 1 h at room temperature.
    **Primary antibody incubation** 10. Incubate with primary antibody (1°Ab) at 4 °C overnight with agitation.
   11. Wash 10 times with 1X PBST for 5 minutes each.
   12. Incubate with secondary antibody (2°Ab) 1 h at room temperature with agitation.
   13. Wash 10 times with 1X PBST for 5 minutes each.
    **Detection** 14. Prepare chemiluminescent reagent (ECL) by mixing 1:1 (reagent A and reagent B).
   15. Expose the membrane to ECL reagent for 1–2 minutes.
   16. Detect the chemiluminescent signal using an imaging system and save the image for later analysis.

![Western Blot](https://static.igem.wiki/teams/5729/experiments/westernblot4.webp)

# Functional assay

## pNPB Assay

##### **pNPB Assay**

The pNPB (para-nitrophenyl butyrate) assay is a colorimetric test used to measure the esterase or
cutinase-like activity
of enzymes such as TfCut2 (from Thermobifida fusca) by monitoring the hydrolysis of the ester bond in pNPB.
The
intensity of the yellow coloration is proportional to the catalytic ability of enzymes.

1. Materials
   * pNPB Powder (Kadin Tech)
   * 200 mM Tris-HCl (pH 7, 7.5, 8, 8.5)
   * 100 mM HEPES Buffer (pH 7, 7.5, 8, 8.5)
   * TfCut2 Proteins
   * PBS Buffer
   * PCR Strips

2. Procedure
   1. Thaw pNPB completely and prepare freshly in DMSO.
   2. Prepare reaction mixtures for each of the pH conditions (pH 7, 7.5, 8, 8.5) by:
      1. Adding 3.5 mL 200 mM Tris-HCl with pNPB (final concentration of 0.25 mM or 2 mM) or
      2. Adding 3.5 mL 100 mM HEPES buffer and pNPB (final concentration of 2 mM) into reagent
         reservoirs.
   3. Set up a PCR plate.
   4. Using an 8-channel pipette, dispense 90 μL of the buffer with pNPB solution into each PCR tube,
      ensuring each pH
      condition is represented.
   5. Repeat the dispensing for all pH conditions as needed.
   6. Thaw TfCut2 proteins on ice.
   7. Aliquot 10 μL PBS buffer or Tris-HCl to designated rows for blank controls.
   8. Aliquot 10 μL of the TfCut2 proteins to designated rows for the test reactions.
   9. Place the PCR strips into the thermal cycler and incubate at three different temperatures (e.g.,
      55 °C,
      60 °C, 65 °C) for 30 minutes.
   10. Measure the reaction at 405 nm using the ELISA plate reader.

![pNPB Assay](https://static.igem.wiki/teams/5729/experiments/pnpbassay3.webp)

## High Throughput Screening PET Film Degradation

##### **High Throughput Screening PET Film Degradation**

The pNPB (para-nitrophenyl butyrate) assay is a colorimetric test used to measure the esterase or
cutinase-like activity
of enzymes such as TfCut2 (from Thermobifida fusca) by monitoring the hydrolysis of the ester bond in pNPB.
The
intensity of the yellow coloration is proportional to the catalytic ability of enzymes.

1. Materials

   * 0.25 × 0.8 cm PET Films
   * 100 mM or 500 mM HEPES Buffer (pH 7, 7.5, 8, 8.5)
   * 10 mM CaCl2
   * TfCut2 Proteins
   * 1X PBS
   * 8-Strips PCR tube
   * 96-well UV microplate (Basic Life)
   * FlexA-200 UV-Vis ELISA Reader (Clubio)

   ![PET Degradation layout](https://static.igem.wiki/teams/5729/experiments/pet-degradation-layout.webp)

   Figure 1. PET Degradation sample layout.

2. Procedure
   1. Put a 0.25 × 0.8 cm PET film inside each of the 8-strip PCR tubes for 12 rows using a sample layout
      based on
      Figure 1.
   2. Prepare 4500 µL of HEPES buffer with 10 mM CaCl2 for each of the 4 pH conditions (7,
      7.5, 8, 8.5)
      inside reagent reservoirs.
   3. Using an 8-channel pipette, aliquot 180 µL of buffer to each tube of the respective pH.
   4. Add 120 µL of each TfCut2 variant to a separate 8-strip PCR tube. Add 120 µL of PBS buffer
      to one tube
      for the blank.
   5. Aliquot 20 µL from each tube of the 8-strip PCR tube to the 12 rows of 8-strip PCR tubes.
   6. Spin down in the mini-centrifuge and incubate at the designated temperature in the thermoshaker at
      800 rpm for 48
      hours.
   7. Transfer to a 96-well UV microplate and analyze OD260 in an ELISA reader.

![HTS Degradation layout](https://static.igem.wiki/teams/5729/experiments/petdegradation3.webp)

## PET Film Degradation

##### **PET Film Degradation**

1. Materials
   * 3 × 0.5 cm PET Films
   * 1 M HEPES Buffer (pH 7.5)
   * 1 M CaCl2
   * Sterile water
   * TfCut2 Proteins
   * PBS Buffer
   * 2 mL microtubes
   * 96-well UV microplate (Basic Life)
   * FlexA-200 UV-Vis ELISA Reader (Clubio)
   * 1% SDS
   * Anhydrous ethanol

2. Procedure

   **B-1. Enzyme Degradation and UV absorbance Measurement**

   1. Put 8 pieces of 3 × 0.5 cm PET film inside a 2 mL microtube.
   2. Add 900 μL 1 M HEPES (pH 7.5) and 18 μL 1 M CaCl2.
   3. Add TfCut2 enzyme and sterile water to reach a final concentration of 27.8 μg/mL.
   4. Seal the microtubes with parafilm.
   5. Incubate in the thermoshaker at 51.8 °C and 800 rpm for 48 hours.
   6. Transfer 100 μL reaction buffer to a 96-well UV microplate and analyze OD260 in
      an
      ELISA reader.
   7. Keep the reaction buffer in a −20 °C refrigerator for further experiments.

   **B-2. PET film Wash**

   1. Transfer PET film to another 2 mL microtube.
   2. Add 1.8 mL 1% SDS and incubate in a 50 °C oven for 30 minutes.
   3. Add 1.8 mL anhydrous ethanol and incubate in a 50 °C oven for 5 minutes.
   4. Rinse PET film with deionized water.
   5. Open the lid and dry the PET film in a 50 °C oven overnight.

## Pretreatment

##### **Pretreatment**

Alkaline-thermal pretreatment is performed to reduce the crystallinity of PET textiles.

1. Materials
   * PET or blend textile
   * 15% NaOH
   * Strainer
   * Deionized water

2. Procedure
   1. Weigh the 0.1 × 0.5 cm textiles (~2 g) and place them into 50 mL tubes.
   2. Add 20 mL 15% NaOH to the tubes.
   3. Textiles in 15% NaOH will be treated under the following conditions:
      1. Autoclave for 15 min or 30 min
      2. 80 °C for 4 hr, 8 hr, or overnight
      1. Autoclaving Method
         1. For the autoclaving method, place the tubes in a 500 mL beaker, cover with aluminum foil,
            and autoclave.
            After autoclaving, transfer the tube to a −80 °C freezer.
         2. Allow the treated textiles to return to room temperature.
      2. Dry Bath Heating Method
         1. Seal the 50 mL centrifuge tube with parafilm.
         2. Incubate in the dry bath for 4 hr, 8 hr, or overnight.
   4. Place pretreated textiles on the strainer and wash with tap water for 5 min.
   5. Rinse textiles with deionized water.
   6. Dry the textiles in a 55 °C oven overnight.

![Textile Pretreatment](https://static.igem.wiki/teams/5729/experiments/textilepretreatment3.webp)

## Cotton Degradation in Textile

##### **Cotton Degradation in Textile**

1. Materials
   * 500 mM Sodium citrate pH 4.8 (Cheng Yi Chemical)
   * Sterile water
   * Cellulase from *Trichoderma reesei* (Sigma Aldrich, C2730)
   * Dry bath or water bath
   * 50 mL centrifuge tubes

2. Procedure
   1. Prepare cellulase reaction mixture.

      | Reagents | Volume (mL) |
      | --- | --- |
      | 500 mM Sodium citrate pH 4.8 | 2 |
      | Cellulase (>700 unit/g) | 0.8 |
      | Sterile water | 17.2 |
      | Total | 20 |
   2. Add 20 mL cellulase reaction mixture into each 50mL microtube.
   3. Cut each composition type of PET-cotton blend textile into 5 pieces of 5 cm × 2.5 cm.
   4. Cut each of the 5 pieces into smaller pieces, approximately 0.5 cm × 0.1 cm.
   5. Measure the mass of the five pieces of textile for each composition type of blend textile.
   6. Add each textile type into a microtube and label accordingly.
   7. Incubate at 50 °C for 72–96 hours.
   8. Preserve the cellulase reaction mixture at −20 °C for the DNS assay.

![Cellulase Degradation](https://static.igem.wiki/teams/5729/experiments/cellulasedegradation3.webp)

## DNS Assay

##### **DNS Assay**

**Prepare DNS Reagent**

1. Materials
   * 15 mL centrifuge tubes
   * 3,5-Dinitrosalicylic acid (DNS) (Kadin tech)
   * Potassium sodium tartrate (Duksan Pure Chemicals)
   * 2M NaOH
   * 10 mg/mL Sodium metabisulfite: freshly prepared (Duksan Pure Chemicals)
   * Deionized water

2. Procedure
   1. Prepare fresh sodium metabisulfite: 0.1 g sodium metabisulfite in 10 mL water.
   2. Add 3 g potassium sodium tartrate and 0.1 g DNS to the tube.
   3. Add deionized water to 6 mL and vortex.
   4. Add 1.25 mL 2M NaOH and vortex.
   5. Add 380 µL 10 mg/mL sodium metabisulfite.
   6. Vortex until all powder is dissolved in the mixture.
   7. Add deionized water until the final volume is 10 mL.
   8. Avoid light and store at room temperature. This reagent can be stored for 6 months.

![](https://static.igem.wiki/teams/5729/experiments/dnsreagent3.webp)

**DNS Assay**

1. Materials
   * DNS reagent
   * D-Glucose standard stock (1 mg/mL) (LGC Labor GmbH)
   * Sterile water
   * 8-strip PCR tubes
   * PCR plate
   * Sealing film
   * PCR machine
   * Micro Plates 96-Well (Paul Boettger GmbH)
   * FlexA-200 UV-Vis ELISA Reader (Clubio)

2. Procedure
   1. Prepare glucose standard with 0, 0.2, 0.4, 0.6, 0.8, 1.0 mg/mL and load 25 μL to the PCR plate.
   2. Dilute the sample to 1/10 of the original concentration.
   3. Transfer 25 μL of the diluted sample to the PCR plate.
   4. Add 25 μL DNS reagent to the diluted sample and standard.
   5. Seal with film.
   6. Use a PCR machine to incubate the reaction at 100 °C for 5 min.
   7. Add 50 μL sterile water to each well.
   8. Transfer reaction to the 96-well plate.
   9. Measure OD540 with an ELISA reader.

![DNS Assay](https://static.igem.wiki/teams/5729/experiments/dnsassay3.webp)

## PET Degradation in Textile

##### **PET Degradation in Textile**

1. Materials

   * Pretreated Textile
     + 100% PET fiber
     + 100% PET textile A
     + 100% PET textile B
     + 35% PET 65% cotton blend textile
     + 45% PET 55% cotton blend textile
     + 65% PET 35% cotton blend textile
   * 1M HEPES Buffer (pH 7.5)
   * 1M CaCl2
   * Sterile water
   * TfCut2 Proteins
   * PBS Buffer
   * 2 mL microtubes
   * 96-well UV microplate (Basic Life)
   * FlexA-200 UV-Vis ELISA Reader (Clubio)

   ![PET Textile Degradation](https://static.igem.wiki/teams/5729/experiments/pet-textile-degradation.webp)

2. Procedure
   1. Put 0.05–0.1 g textile into the 2 mL microtube.
   2. Add 900 μL 1M HEPES (pH 7.5) and 18 μL 1M CaCl2.
   3. Add *TfCut2* enzyme and sterile water. The final concentration should be 27.8 μg/mL.
   4. Seal the microtubes with parafilm.
   5. Incubate in the thermoshaker at 51.8 ℃ and 800 rpm for 48 hours.
   6. Transfer 100 μL reaction buffer to a 96-well UV microplate and analyze OD260 in ELISA
      reader.
   7. Keep the reaction buffer in the −20 ℃ refrigerator for further experiments.

## HPLC Sample Preparation

##### **HPLC Sample Preparation**

1. Materials
   * Standard stock: 10 mg/mL in DMSO.
   * Standard concentration: 25, 50, 100, 200 µg/mL, diluted in MeOH.
   * Terephthalic acid (TPA): Final product.
   * 4-((2-Hydroxyethoxy)carbonyl)benzoic acid: Intermediate product.
   * Enzyme buffer: 5 µg/mL enzyme, 500 mM HEPES, 10 mM CaCl2, pH 7.5.

2. Procedure
   1. Transfer 100 μL supernatant to a new microtube. Seal with parafilm and incubate at
      95 °C
      for
      10 minutes (enzyme heat inactivation).
   2. Cool down to room temperature and add 100 μL DMSO.
   3. Sonicate for 5 minutes.
   4. Centrifuge at maximum speed (23,444 × g) for 5 minutes.
   5. Transfer 100 μL supernatant to a new microtube.
   6. Add 900 μL MeOH (dilute 1:10).
   7. Load the mixture into a syringe and pass it through a 0.22 µm nylon filter. Collect the
      flow-through in an
      HPLC sample vial.

![HPLC Sample Prep](https://static.igem.wiki/teams/5729/experiments/hplc-sample-prep.webp)

{% endblock %}

### Content

* [Introduction](#section0)
* [pNPB Assays](#section1)
* [PET film degradation](#section2)
* [Pretreatment](#section3)
* [PET textile degradation](#section4)
* [Cotton PET blend degradation](#section5)
* [Discussion](#section6)
* [References](#referencesAccordion)

# Introduction

To determine whether our engineered TfCut2 and its variants can effectively degrade PET–cotton blended
textiles, we employed a primary enzymatic assay, pNPB, along with several multi-step degradation experiments.

Catalytic activity was first confirmed using the pNPB assay, which showed that the enzymes did indeed
hydrolyze ester bonds under various pH and temperature conditions.

Afterwards, we conducted a series of rigorous experiments to test for degradation of PET, by measuring
soluble degradation products through UV absorbance at 260 nm. High-throughput PET film degradation was
performed to determine the optimal experimental conditions. We then formally degraded PET film at a larger
scale for direct evidence of PET breakdown.

Pretreatments were employed to reduce the crystallinity of the textile samples before degradation. The
effect of the pretreatment is verified with FT-IR and DSC.

We then test our TfCut2 variant’s ability to degrade on pure PET textile. Similar to PET film, UV
absorbance was also used to assess degradation of pure PET textiles. Moreover, techniques like SEM and HPLC
were used to confirm degradation at the microscopic and molecular levels.

Lastly, we tested degradation on cotton-PET blended textiles. Prior to degradation we applied commercial
cellulase to degrade cotton fibers, in order to improve degradation efficiency. DSC was then used to
determine cotton fibers degradation. Afterwards, we evaluate degradation with the same methods as used in
pure PET textile.

# pNPB Assays

Before applying our TfCut2 variants in PET degradation, we first aimed to verify their general hydrolytic
activity and determine optimal working conditions using the model substrate pNPB. The experiments below
trace this validation process from baseline wild-type characterization to comparative variant testing
across buffer systems.

To confirm our TfCut2- 5ZOA (Wild-type) and its six variants (Variants 1 to 6) are functional after
protein production and purification, enzyme activity was measured using the pNPB assay, which is a widely
used method to quantify enzymatic hydrolytic activity. When the enzyme hydrolyzes pNPB to p-nitrophenol,
we can measure absorbance at 405 nm using a microplate reader. In the beginning, we used Tris-HCl as a
reaction buffer based on literature review (Chen et al., 2010; Dong et al., 2020). Further research
directs us into using different buffers since we found that Tris-HCl buffer, although utilized in most
studies, is not the most ideal buffer due to its inhibitory effects on TfCut2 (Schmidt et al., 2016). In
the end, there are three types of buffers that we utilized: Tris-HCl, Calcium-induced Tris-HCl, and
Calcium-induced HEPES (Then et al., 2015). The experimental conditions, including pH and
temperature, also followed the papers used before.

## Preliminary Tests

To assess the optimal reaction conditions for each enzyme, we first tested TfCut2 wild-type (PDB ID:
5ZOA) at pH 6–9 and temperatures ranging from 55°C to 70°C. This was to ensure that wild-type enzyme (PDB
ID: 5ZOA) was functional under these conditions, allowing us to proceed with testing the six variants. The
results showed that enzyme activity had an increasing trend at pH 6–8 under temperatures of 55°C to 65°C
(Figure 1). We observed decreasing OD405 values at pH 9, which is consistent with the description in the
paper that the substrate undergoes spontaneous hydrolysis at elevated temperatures and pH values of 9 or
above (Furukawa et al., 2019). Overall, our enzyme was most active around pH 7–8, with an over 90%
decrease at pH 9. Temperature also had a great impact, since we observed that enzymes were most active
under 55°C and least active under 70°C. Hence, subsequent experiments will be conducted at lower
temperature to achieve better enzymatic activity.

![](https://static.igem.wiki/teams/5729/results/enzymeactivityassay1-00.webp)

Figure 1. Initial pNPB assay of TfCut2 wild type 5ZOA.

In addition to using 2D plots for visualizing temperature and pH effects, we employed Response Surface
Methodology (RSM) graphs to determine the most optimal pH and temperature conditions. RSM is a collection
of statistical and mathematical techniques to model and analyze how a response is influenced by several
quantitative input variables. The primary objective of RSM is to optimize this response by identifying the
optimal levels of the input factors (Mushtaq et al., 2015). 3D surface plots and 2D contour plots are
commonly used to visualize the relationships and interactions between the independent variables and the
response variable. The RSM predictions were based on experimental data collected on June 4 and 6, with
five replicates at each condition (Figure 2).

![](https://static.igem.wiki/teams/5729/results/enzymeactivityassay2.webp)

Figure 2. The optimal condition of TfCut2 estimated by RSM methodology

The optimal condition was identified at pH 7.5 and temperature around 50°C. The
temperature obtained from RSM would be referenced in further experiments. Hence, the conditions that will
be tested in later experiments would still be focusing on pH 7-8 and temperatures below 55°C.

## pNPB assay with Tris-HCl buffer

We conducted the assays using 200mM Tris-HCl buffer and pNPB (2mM final concentration) with the addition
of TfCut2 (10 mM final concentration). The conditions we obtained from the preliminary tests were used in
our engineered enzyme. Based on our RSM graph, the peak appeared to be around pH 8. To obtain more precise
results, we narrowed down the pH range to 7, 7.5, 8, and 8.5 and broadened the temperature range from 35°C
to 70°C in 5°C increments. We observed that only the relative activity of Variant 1 (G62A/P193H/S197D),
Variant 2 (H77R/D204C/E253C) and Variant 3 (A65H/L90A/I213S) were higher than TfCut2-5ZOA (wild-type) at
pH 7 across the temperature range of 35°C to 60°C (Figure 3A). Evidently, the optimal working conditions
of these three enzymes were at pH 7 and at a temperature between 55°C to 60°C. Variant 4 (D12S) and
Variant 5 (T234L) expressed a similar trend and showed higher hydrolyzing activity—up to three times
greater than the wild type—at a pH of 7 and under 65°C (Figure 3B). However, Variant 6 (Full MutCompute)
showed little to no enzyme activity under each condition.This finding aligns with the drylab design, as
Variant 6 (Full MutCompute) was primarily a machine-learning-guided enzyme in which all original mutation
sites were replaced with machine-predicted ones. As a result, its structure differs drastically from the
other Variants, making it a far-riskier construct. For more detailed information, explore the [drylab](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D#section5)
page.

#### A

![](https://static.igem.wiki/teams/5729/results/enzymeactivityassay3.webp)

#### B

![](https://static.igem.wiki/teams/5729/results/enzymeactivityassay4.webp)

Figure 3. Residual activity of (A) Variant 1 (G62A/P193H/S197D), Variant 2 (H77R/D204C/E253C),
and Variant 3 (A65H/L90A/I213S) (B) Variant 4 (D12S), Variant 5 (T234L), and Variant 6 (Full MutCompute)
relative to the wild-type TfCut2 in Tris-HCl buffer. Error bars indicate the standard error of triplicate
determinations.

We also used RSM to graph out the results of our Tris-HCl buffer pNPB assays (Figure 4). On the graphs we
mapped out, pH and temperature values are used on the x and y-axes, respectively. The z-axis shows the
OD405 readings, which represents the activities of our enzymes. We also marked out the optimal point, which
was predicted based on the surface.

Table 1.The Optimized Temperature and pH for pNPB Assay in Tris-HCl Buffer.

![](https://static.igem.wiki/teams/5729/results/tristable1.webp)
![](https://static.igem.wiki/teams/5729/results/figure4-1.avif)

Figure 4. RSM graphs of 5ZOA (labeled as WT), Variant 1 (G62A/P193H/S197D), Variant 2
(H77R/D204C/E253C), Variant 3 (A65H/L90A/I213S), Variant 4 (D12S), Variant 5 (T234L), and Variant 6 (Full
MutCompute).The red star of each graph represents the optimal condition. Error bars indicate the standard
error from experiments.

With the exception of V6 (Figure 4.G), RSM graphs of the variants are identical. In terms of the pH axis,
we see a parabola-shaped trend that peaks at pH range 7.5~8. The trend observed among temperature is also
presented here, as we see an increased OD reading with lower temperatures. The optimal conditions (marked
with a red star) in every graph are located at a temperature range of 35°C-40°C and pH 7.5-8.

The Tris-HCl results informed us that Variant 1-3 are outperforming wild type under moderate temperature
and slightly alkaline pH. These patterns indicate that our enzyme exhibits stability under conditions
typical for polyester degradation. However, the performance of Variant 6 identified that machine-predicted
mutants may compromise efficiency.

## pNPB assay with HEPES buffer

In a study regarding effects of various buffers on polyester hydrolase activity, Schmidt et al. (2016)
concluded that Tris competitively inhibits aminopeptidases, cholinesterases, and
hydrolases such as TfCut2 when used as a buffer. The activity of TfCut2 decreased when placed in the
Tris-HCl buffer, especially when the molarity of Tris was increased, meaning that Tris-HCl inhibits TfCut2
activity. Hence, we changed our experimental approach from using Tris-HCl buffer for pNPB assays to using
HEPES buffer to prevent inaccurate results and to improve the activity of TfCut2. Moreover, Then et al.
(2015) revealed that TfCut2 is not sufficiently thermostable at high temperatures required for efficient
PET degradation. According to literature, the addition Ca²⁺ cations to the enzymes and enhances
thermostability, thereby increasing the melting point of the enzymes up to 14.1 °C from the usual 71.2 °C
(Then et al, 2015). Therefore, we added CaCl₂ to our HEPES buffer to improve enzyme activity and more efficient
PET degradation at elevated temperatures. The pNPB assay was conducted using the same protocol as
previously described but with our new buffer and calcium ion addition. When the buffer system changed to
HEPES, we observed a change in our results.

#### A

![](https://static.igem.wiki/teams/5729/results/enzymeactivityassay6.webp)

#### B

![](https://static.igem.wiki/teams/5729/results/enzymeactivityassay7.webp)

Figure 6. Residual activity of (A) Variant 1 (G62A/P193H/S197D), Variant 2 (H77R/D204C/E253C),
and Variant 3(A65H/L90A/I213S) (B) Variant 4 (D12S), Variant 5 (T234L), and Variant 6 (Full MutCompute)
relative the wild-type TfCut2 in HEPES buffer with Ca²⁺. Error bars indicate the standard error of
triplicate determinations.

Under most conditions, we see Variants 1-3 (G62A/P193H/S197D, H77R/D204C/E253C, and A65H/L90A/I213S
respectively) performing almost as well as, if not better than, the 5ZOA wild type. A trend was observed
among pH levels, with most variants exhibiting the highest level of activity relative to the wild type at
pH 7.5. However, at elevated temperatures from around 60°C to 70°C, Variant 3 performed substantially
better than other variants and relative to the wild type, especially at a pH of 7, suggesting that its
efficacy will be the highest in degrading PET. The effect of temperature was not as pronounced across all
data values, suggesting the addition of calcium ions may have allowed the enzyme activity to resume at a
similar rate to lower temperatures even under elevated temperatures.

Across all tested conditions, Variants 4 (A65H/L90A/I213S), 5 (D12S), and 6 (Full MutCompute Variant) had lower levels of activity than the wild type, with Variant
5 performing better than Variants 4 and 6 at all data points. Notably, Variant 6 exhibited generally
little to no activity relative to the wild type at all tested conditions, similar to its performance under
Tris-HCl buffer, suggesting that the Variant 6 is not suitable for our following degradation process.

Table 2.The Optimize Temperature and pH for pNPB Assay in CaCl₂ Buffer.

![](https://static.igem.wiki/teams/5729/results/cacltable2.webp)
![](https://static.igem.wiki/teams/5729/results/figure7-1.avif)
Figure 7. RSM graphs of 5ZOA (WT) and Variant 1 (G62A/P193H/S197D) Variant 2
(H77R/D204C/E253C) to Variant 3 (A65H/L90A/I213S) Variants 4 (D12S), Variant 5 (T234L) and Variant 6 (Full
MutCompute) inside calcium-induced HEPES buffer. The red star of each graph represents the optimal
condition. Error bars indicate the standard error from experiments.

Similar to the RSM of the Tris-HCl buffer, the RSM of the HEPES buffer also reflected trends among pH and
temperature. With all variants except Variant 6 (Figure 7.G), temperature and activity are negatively
correlated: an increase of temperature will decrease the activity. Conversely to the Tris-HCl buffer
trend, the Figure 7 trend among pH levels seems to suggest a peak at pH level 7-7.5, which is a bit lower
than the pH 7.5-8 of the Tris buffer.

## Conclusion

In conclusion, we conducted pNPB assays to assess the activity of our engineered enzymes. Our results
show that the majority of engineered variants, excluding Variant 6 (Full MutCompute), demonstrate
comparable activity to wild type 5ZOA, indicating successful retention of enzyme function in ester bond
cleavage. While these conditions cannot be directly applied to PET degradation, the data obtained and
conditions tested serve as a reference for subsequent PET degradation experiments.

# PET Film Degradation

Before using TfCut2 and its mutant to degrade textile, we must ensure they can digest PET film whose
crystallinity is lower than the PET in textile and more easily to be degraded in theory. Thus, we used PET
film to optimize the enzyme degrade condition and ensure its hydrolysis activity toward PET.

The results of our project are divided into four phases: preliminary PET film degradation, high
throughput PET film degradation, expanding experimental conditions, and formal PET film degradation.
During preliminary PET film degradation, because we never digested PET in our laboratory before, we used
the condition from published research to test our enzyme and the method to evaluate enzyme activity. In
this phase, we establish UV absorbance, which represents the PET degrading product terephthalic acid at
260 nm as a method by testing PET film degradation at pH 8 and 60 °C for 48 h. Secondly, during our high
throughput PET film degradation, used reduced volumes and standardized PET film sizes (0.8 × 0.25 cm)
across 96 samples, testing in 100 mM HEPES buffer under conditions matched to the pNPB assay, measuring UV
absorbance across pH 7.0–8.5 and 55–70 °C, with no-enzyme blanks included as negative controls. Third,
shifting to 500 mM HEPES to replicate literature conditions, expanding temperature testing to 35–70 °C,
and visualizing results through surface plots while maintaining the same control setup. Fourth, formal PET
film degradation. We increased the reaction scale and confirmed enzymatic PET degradation directly by UV
absorbance and HPLC and calculated the PET film weight loss after 48 h at 51.8 °C.

Across these phases, Variant 3 (A65H, L90A, I213S) and 4 (D12S) consistently showed significantly higher activity, wild-type TfCut2
5ZOA performed moderately, and the remaining Variants either showed weak or negligible activity. Activity
was strongest at 51.8 °C, while the surface plots provided a clear visualization of how pH and temperature
influenced enzyme performance. Direct degradation assays confirmed that UV absorbance measurements
reflected true PET hydrolysis.

## Testing the Feasibility of PET Degradation

Unlike pNPB assay, the products of PET degradation such as terephthalic acid (TPA), MHET, BHET, and
ethylene glycol (EG) are not able to be detected by the visible spectrophotometer. Although high
performance liquid chromatography (HPLC) can precisely quantify the concentration of PET degradation
products, it is impractical to use HPLC to optimalize the pH value and temperature because of its slow
analysis time, high operating and maintenance cost. However, our ELISA reader can detect UV absorbance,
and since PET degradation products can absorb UV on 254 nm, we can use UV absorbance to do the high
throughput screening for the optimal reaction conditions for TfCut2 to degrade PET.

To validate our concept, we first performed PET degradation using the previously published reaction
conditions (Wei et al., 2020) for TfCut2 and measured whether there was a significant change in the UV
absorbance. We degraded one 0.25 x 0.5 cm PET film in 100 μL 200 mM Tris-HCl (pH 8) with 5 μg/mL enzyme at
60℃ 800 rpm for 48 hours. The results showed that UV absorbance at 260 nm can measure enzyme activity
toward PET (Figure 1). Although UV absorbance at 240 nm can also be used for detecting PET degraded
products, our UV microplate has a strong background below 250 nm. Thus, we used UV absorbance at 260 nm to
do the further experiments.

![](https://static.igem.wiki/teams/5729/results/petfilmdegradation1.webp)

Figure 1. PET degradation preliminary test on UV absorbance. PET films (0.25 x 0.5 cm) were
degraded by 5 ug/mL TfCut2 and its variants in 200 mM Tris-HCl (pH 8) at 60℃ for 48 hours. UV absorbance
at 260 nm and 240 nm were detected by FlexA-200 ELISA reader. Data are presented as the mean ± standard
deviation (SD) from three replicates.

## High-Throughput Screening for Optimalizing PET Film Degradation

In order to optimize enzyme reaction conditions, we scaled down the enzymatic reaction and measured UV
absorbance for simultaneous high-throughput screening to obtain the best reaction parameters.

In the first part of the experiment, we tested PET hydrolysis in 100 mM HEPES with 10 mM CaCl₂ to align
with the conditions used in our pNPB assay. The addition of Ca²⁺ was informed by the previous study (Then
et al., 2015), who showed that Ca²⁺/Mg²⁺ binding enhances polyester hydrolase stability and activity.
Activity was measured across pH 7.0–8.5 and 55–70 °C. Results again showed Variant 3 as the most active
enzyme, followed by the wild type, while other variants remained weak.

![](https://static.igem.wiki/teams/5729/results/petfilmdegradation2.webp)

Figure 2. The surface graph of the first high-throughput screening PET film degradation. A 0.25
x 0.5 cm PET film was degraded by TfCut2 or its mutant in 100 mM HEPES with 10 mM CaCl₂ for 48 hours.
Variant 3 (A65H/L90A/I213S triple mutant) is the most active enzyme, followed by the wild type, while
other variants perform with low levels of activity. UV absorbance at 260 nm was detected by FlexA-200
ELISA reader.

In the previous study, Then et al. used 500 mM HEPES as a reaction buffer for TfCut2 to degrade PET (Then
et al., 2016). In order to obtain a better result, we changed the reaction buffer to 500 mM HEPES with 10
mM CaCl₂. PET films were incubated for 48 h across multiple temperatures (35°C–70°C) and pH values
(7.0–8.5). All TfCut2 and its variants except Variant 6 (Full MutCompute) demonstrate activity in
degrading PET films. Variant 3 (A65H/L90A/I213S) exhibits the highest activity at 50°C pH 8, followed by
Variant 4 at the same temperature but pH 7.5. Variant 5 (T234L) also exhibits elevated activity compared
to the wild type, while Variant 2 (H77R/D204C/E253C) is relatively close to the wild type.

![](https://static.igem.wiki/teams/5729/results/petfilmdegradation3.webp)

Figure 3. The surface graph of the second high-throughput screening PET film degradation. A 0.25
x 0.5 cm PET film was degraded by TfCut2 or its mutant in 500 mM HEPES with 10 mM CaCl₂ for 48 hours. Data
visualization highlights activity peaks for Variant 3 (A65H/L90A/I213S) and Variant 4, while the wild type
showed moderate activity and the other variants displayed minimal or negligible responses. UV absorbance
at 260 nm was detected by FlexA-200 ELISA reader.

## Response Surface Methodology on High-Throughput Screening PET Film Degradation

In the previous section, we did the high-throughput screening and obtained the surface graph for TfCut2
wild-type (5ZOA) and its mutants. However, the optimal reaction conditions may lie between the conditions
we tested. To obtain precise optimal reaction parameters, we used Response Surface Methodology to
determine the best reaction conditions for TfCut2, along with its predicted UV absorbance (Table 1), and
the response surface graphs for each enzyme (Figure 4).

For more information on RSM please check our [drylab RSM](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D?tab=modeling#section8) page

Table 1.The Optimized Temperature and pH for PET Degradation.

![](https://static.igem.wiki/teams/5729/results/petfilmdegradation4.webp)
![](https://static.igem.wiki/teams/5729/results/petfilmdegradation5.webp)

Figure 4. The response surface graph of TfCut2 and its mutants. The graphs show the response
surface of (A) TfCut2-5ZOA wild-type, (B) TfCut2-Variant 1 G62A/P193H/S197D triple mutant, (C)
TfCut2-Variant 2 (H77R/D204C/E253C) triple mutant, (D) TfCut2-Variant 3 (A65H/L90A/I213S) triple mutant, (E)
TfCut2-Variant 4 (D12S) single mutant, (F) TfCut2-Variant 5 (T234L) single mutant, (G) TfCut2-Variant 6 full
MutCompute mutant. All response surface graphs are merged in (H). The response surface graphs were generated
by R (4.5.1) using response surface methodology.

In order to obtain the best reaction conditions for enzymes, we adopted three strategies using
response surface methodology: considering all TfCut2 and its mutants regardless of their activity, excluding
the mutant without activity, and only considering the mutants whose activity is better than wild-type. Next,
we merged the datasets of the individual enzymes into three groups according to three strategies. Then
determined their respective response surfaces and optimal reaction pH and temperature (Table 2). Due to the
limitation of our instrument, we decided to use 51.8℃ pH 7.5 as the optimal reaction condition for the
further experiments.

Table 2. Strategies to obtain the optimized reaction condition.

![](https://static.igem.wiki/teams/5729/results/petfilmdegradation6.webp)

## PET-degrading activity of six variants

After confirming assay conditions across buffers, pH values, and temperatures, we selected the most
promising conditions for direct product quantification. In this section, we scaled up the reaction: used 8
pieces of 3 x 0.5 cm PET film (surface area around 12 cm2) in a 1.8 mL 500 mM HEPES with 10 mM CaCl₂ (pH
7.5) reaction buffer with 27.8 μg/mL enzyme. After incubating TfCut2 and its mutants in a thermoshaker at
51.8°C, 800 rpm for 48 hours, we utilized the ELISA reader to measure UV absorbance at 260 nm. The results
were consistent with the absorbance data: Variant 3 (A65H/L90A/I213S) released the most TPA and produced the
greatest weight loss, followed by Variant 4 (D12S)and the TfCut2-5ZOA (wild type) (Figure 5A).

To make the data results more intuitive, we roughly converted UV absorbance data into a mass of TPA
produced during PET degradation. We established a standard curve of TPA (Figure 5B) and converted the UV
absorbance at 260 nm to the concentration of TPA:
![](https://static.igem.wiki/teams/5729/results/latex1.avif)
Then we multiply the concentration of TPA by total reaction volume 1.8 mL to calculate the total mass of TPA
in reaction buffer:
![](https://static.igem.wiki/teams/5729/results/latex2.avif)
However, the total mass of TPA is not equivalent to the PET film weight loss. One PET molecule can be
degraded to one TPA molecule and one ethylene glycol (EG) molecule. To calculate the PET weight loss, total
mass of TPA must multiply the ratio of PET monomer molecular weight (Mw of PET monomer) and TPA molecular
weight (Mw of TPA).
![](https://static.igem.wiki/teams/5729/results/latex3.avif)
Through the calculations above, we were able to determine the PET weight loss (Figure 5C). However, this
approximation is a rough estimate. PET degradation actually produces intermediate compounds MHET and BHET,
which will absorb UV absorbance at 260 nm. This rough calculation can only combine all of these intermediate
compounds as the final TPA.

![](https://static.igem.wiki/teams/5729/results/petfilmdegradation7.webp)

Figure 5. Quantification of PET film degradation by engineered variants. (A) Absorbance at 260 nm
(A260) of the reaction supernatant after incubation of PET with different variants. (B) Standard curve for
terephthalic acid (TPA) showing a linear relationship between TPA concentration (μg/mL) and A260. The linear
regression equation and the coefficient of determination (R2) are indicated. (C) Calculated mass of released
TPA (dark gray bars) and the corresponding equivalent mass of degraded PET (light gray bars) in miligrams
(mg). These values were calculated from the A260 measurements in (A) using the TPA standard curve from (B).
Data are presented as the mean ± standard error (SEM) from three independent replicates.

As mentioned above, because UV absorbance counts all chemicals which can absorb UV light, it overestimates
the concentration of TPA around 15 to 40% (Zhong-Johnson et al., 2021). To obtain more accurate data, we
choose one set of samples from three PET film degradation and utilize high performance liquid chromatography
(HPLC) to analyze the PET degraded product in the reaction buffer.

In order to quantify the intermediate product of PET degradation, we made a
4-((2-hydroxyethoxy)carbonyl)benzoic acid (MHET) standard curve and measured the concentration of MHET in
the reaction buffer. Then we used the following equation to calculate these MHET weights equal to how much
PET degraded.
![](https://static.igem.wiki/teams/5729/results/latex4.avif)
Besides UV absorbance and HPLC, we did gravimetric analysis by measuring the weight of PET films before and
after TfCut2 digestion. After PET film degradation, we washed PET film by 1% SDS for 30 minutes and
anhydrous ethanol for 5 minutes at 50℃. And rinse with deionized water and dry in the 50℃ oven for at least
48 hours. Then we measured the weight of PET films and the results show in table 3.

Table 3. The PET film weight loss after degradation by TfCut2 and its variants.

![](https://static.igem.wiki/teams/5729/results/petfilmdegradation8.avif)

Each of the three methods for estimating PET weight loss presents distinct limitations. As reported by the
previous study (Zhong-Johnson et al., 2021), the PET weight loss calculated from UV absorbance was greater
than that derived from HPLC, in some instances even surpassing the mass determined by gravimetric analysis.
In contrast, for the high A260 groups, the HPLC estimated mass exceeded the mass estimated from UV
absorbance. This discrepancy can be attributed to the A260 values of these groups exceeding the upper
detection limit of the ELISA reader, thereby precluding an accurate quantification of PET degradation
products. With HPLC, the absence of a standard for the intermediate product, Bis(2-hydroxyethyl)
terephthalate (BHET), prevents its quantification, potentially leading to an underestimation of the extent
of PET degradation. Meanwhile, in gravimetric analysis, measuring the change in PET mass poses a significant
challenge. Inadequate washing of the PET film can leave residual buffers or enzymes, which introduces
artifacts into the measurement. This often results in a measured mass greater than that prior to
degradation. Therefore, obtaining a BHET standard for HPLC-based quantification would likely yield a more
precise estimation of PET weight loss.

To prove the TfCut2 and its mutants can degrade PET films, we used scanning electron microscopy (SEM) to
observe the PET film surfaces (Figure 6). SEM images of the PET film surface also corroborate the enzyme
degradation of PET. In the SEM images, mutants except Variant 6 can penetrate the PET film surface and
create pore structures during degradation (Figure 6E-N).

![](https://static.igem.wiki/teams/5729/results/petfilmdegradation9.webp)

Figure 6. Scanning electron microscopy (SEM) imaging of PET film surfaces after enzyme
degradation. SEM photos of PET films after incubation for 48 hours at 51.8°C by different enzyme variants.
(A, B) PET film incubated without enzyme as negative control, showing a smooth and unaltered surface. (C, D)
PET film incubated with TfCut2-5ZOA wild type. (E, F) TfCut2-Variant 1 (G62A/P193H/S197D) triple mutant. (G,
H) TfCut2-Variant 2 (H77R/D204C/E253C) triple mutant. (I, J) TfCut2-Variant 3 (A65H/L90A/I213S) triple
mutant. (K, L) TfCut2-Variant 4 D12S single mutant. (M, N) TfCut2-Variant 5 (T234L) single mutant. (O, P)
TfCut2-Variant 6 Full MutCompute mutant. Panels in the first and third columns (A, C, E, G, I, K, M, O) were
captured at 5,000× magnification. Panels in the second and fourth columns (B, D, F, H, J, L, N, P) show a
magnified view of the same samples at 20,000× magnification. Extensive surface erosion and pitting are
visible on films treated with active variants, indicating enzymatic degradation. All scale bars are
indicated in the panels.

In conclusion, the collective data from UV absorbance, HPLC, and gravimetric analysis demonstrate that
TfCut2-Variant 3 (A65H/L90A/I213S) triple mutant and TfCut2-Variant 4 (D12S) single mutant exhibit enhanced
PET degradation capabilities relative to the wild type TfCut2-5ZOA.

# Pretreatment

PET textile often possesses exceedingly high tensile strength and thermal stability—both attributed to
their higher-than-normal crystallinity, where the polymers in the crystalline regions are then densely
packed, more rigid, less permeable, and melt at higher temperatures, thus resisting the effectiveness of
enzymatic attack. These crystalline regions limit the available polymer surface for enzymes to penetrate the
dense polymer matrix (Dewi et al., 2025). Therefore, to overcome these limitations, we incorporated a
thermal-alkaline pretreatment method to decrease the crystallinity of PET and enhance the subsequent enzyme
degradation of PET textiles. We applied the pretreatment method described by previous research (Boondaeng et
al., 2023), which involves applying 15% NaOH to textiles at high temperatures. Mechanistically, the high
temperature damages the PET matrix, allowing NaOH to depolymerize the ester bonds in PET, thereby forming
terephthalate in its sodium form. In our study, we tested pretreatment under different conditions: 15% NaOH
with autoclaving at 127°C for 15 and 30 minutes, and 15% NaOH using drybath for 4, 8, and 20 hours. This
treatment effectively hydrolyzes the ester bonds at the surface while also modifying the polymer's structure to
increase its accessibility and susceptibility to enzymatic hydrolysis (Dewi et al., 2025).

## Alkaline Pretreatment of PET Using Autoclaving

After pretreatment with 15% NaOH and autoclaving for 15 minutes, we observed visual changes of the 100% PET
textile and cotton-PET blends (55:45 and 65:35) (Figure 1). The treated 100% PET textile (Figure 1A, left
tube) became fibrous and powdery, compared to the untreated sample (Figure 1A, right tube), which retained a
smooth and tightly woven appearance. The untreated 55:45 cotton-PET blends (Figure 1B, left tube) were
smooth and dense, whereas the treated textiles (Figure 1B, right tube) showed loosened fibers. Untreated
65:35 cotton-PET blend (Figure 1C, right) appeared compact, while treated textiles (Figure 1C, left)
exhibited rugged fibers and a dull appearance.

![](https://static.igem.wiki/teams/5729/results/pretreatment1.webp)

Figure 1. Visual appearance of (A) 100% PET textile, (B) 55:45 cotton-PET blends, (C) 65:35
cotton-PET blend after pretreatment with 15% NaOH and autoclaving. In each panel, the left tube is
pretreated and the right tube is untreated.

Scanning electron microscopy (SEM) imaging further revealed the morphological changes in the fibers at
magnification of 500x and 10,000x (Figure 2). The 100% PET textile (Figure 2A and 2B) showed fibers with
rough and disrupted surfaces (Figure 2A) and the presence of surface ruptures in higher magnification
(Figure 2B). The 65:35 cotton-PET blends exhibited uneven and loosened structure (Figure 2C), and roughened
surfaces with grooves and peeling (Figure 2D). These observations confirmed the NaOH with autoclaving,
effectively disrupting the fiber surface and structure of the PET fibers.

![](https://static.igem.wiki/teams/5729/results/pretreatment2.webp)

Figure 2. Scanning electron microscopy (SEM) image of textiles with pretreatment of 15% NaOH and
autoclaving. (A, B) 100% PET and (C, D) 65:35 cotton-PET blends at (A,C) 500x and (B, D) 10,000x
magnifications. All scale bars are indicated in the panels.

In addition, Fourier Transform Infrared Spectroscopy (FT-IR) analysis was used to determine the chemical
modifications of pretreated textiles. Control spectra established the diagnostic peaks for cotton (–OH
stretching at~3300 cm⁻¹ and C–O–C glycosidic bonds at 1159–995 cm⁻¹) and PET (ester carbonyl at 1712 cm⁻¹
and aromatic C–H at 724 cm⁻¹) (Figure 3A). The absorbance peak of ester carbonyl (–COO stretch) at 1712 cm⁻¹
were identified, and the reduction rate was calculated relative to the untreated control (Table 1).

After pretreatment with 15% NaOH and autoclaving for 15 minutes, the intensity of the PET ester carbonyl
peak at 1712 cm⁻¹ decreased by 13% in pure PET textile (Figure 3B), confirming partial hydrolysis of ester
bonds. In blended textiles, the 55:45 cotton-PET textile showed the highest reduction of 15% (Figure 3C),
indicating that blends with less PET are more chemically susceptible, whereas the 65:35 cotton-PET blend
displayed only a 4.7% reduction (Figure 3D), reflecting the greater resistance of PET-rich textiles.
Overall, FT-IR results showed reduction of ester carbonyl after pretreatment, indicating partial disruption
of ester linkages in PET matrix.

![](https://static.igem.wiki/teams/5729/results/pretreatment3.webp)

Figure 3. Fourier Transform Infrared Spectroscopy (FT-IR) spectrogram of textiles before and after
pretreatment of 15% NaOH and autoclaving. (A) Overlay of 100% PET control and 100% cotton control. (B) 100%
PET pretreated (red) overlaid with untreated (black). (C) 55:45 cotton-PET blends pretreated (red) overlaid
with untreated (black). (D) 65:35 cotton-PET blends pretreated (red) overlaid with untreated (black).

Table 1. Absorbance peak of ester carbonyl (–COO stretch) at 1712 cm⁻¹ in untreated and pretreated
100% PET textile and cotton-PET blended textiles with 15% NaOH and autoclaving for 15 minutes.

![](https://static.igem.wiki/teams/5729/results/pretreatment4.webp)

## Alkaline Pretreatment of PET Using Dry Bath

Due to the safety risk and high energy consumption of autoclaving, we sought alternative methods to reduce
PET crystallinity. Building on the evidence of physical and chemical disruption of PET fibers by 15% NaOH
with autoclaving, we tested milder and more accessible pretreatments using 15% NaOH at 80°C for 4, 8, and 20
hours on the dry bath, as an alternative to harsher autoclaving conditions (Figure 4). Two types of 100% PET
textiles (Textile A and B) and PET fibers were pretreated under these conditions to compare the
crystallinity reduction achieved by each condition. After pretreatment, the samples were sent for SEM and
DSC analysis to assess the morphology and crystallinity of PET samples.

![](https://static.igem.wiki/teams/5729/results/pretreatment5.webp)

Figure 4. Visual appearance of 100% PET fiber before and after 80℃ dry bath pretreatments. The
tubes from left to right are untreated fiber, fiber pretreated for 4, 8, and 20 hours.

SEM imaging of Textile A showed the changes in its morphology under different pretreatment conditions
(Figure 5). Pretreatment with 15% NaOH at 80°C resulted in disruption of PET fibers progressively over time.
After 4 hours of incubation, the fibers showed roughened surface (Figure 5A) and prolonged treatment for 8
hr exhibited thinning of the fibers and roughened edges (Figure 5B). Extended incubation for 20 hr further
caused severe erosion and breakdown of the fiber (Figure 5C), showing effective PET structural disruption
over time under 15% NaOH at 80°C pretreatment.

![](https://static.igem.wiki/teams/5729/results/pretreatment6.webp)

Figure 5. Scanning electron microscopy (SEM) images of pure PET textile with 80°C for (A) 4 hours,
(B) 8 hours and (C) 20 hours on the dry bath. Images were captured at 20,000× magnification. All scale bars
are indicated in the panels.

Differential scanning calorimetry (DSC) analysis was performed to further quantify the crystallinity of the
PET textiles and fibers among all different pretreatment conditions. DSC results provided values for
enthalpy of fusion ΔHm enthalpy of crystallization ΔHc as illustrated in Figure 5 for Textile A..
This two variable can be compared to the enthalpy of fusion theoretical value for 100% crystalline PET
ΔHf, which is equal to 140 J/g, to calculate the sample’s crystallinity using the formula:
![](https://static.igem.wiki/teams/5729/results/latex5.avif)

![](https://static.igem.wiki/teams/5729/results/pretreatment7.webp)

Figure 5. DSC thermogram of Textile A comparing crystallinity for untreated (Red), 15% NaOH at
80°C for 4 hours (Brown) and 15% NaOH at 80°C for 8 hours (Blue).

We summarized the degree of crystallinity of the samples across all tested conditions (Table 2). The
untreated samples showed the crystallinities of 47.9%, 44.1% and 36.5% for Textile A, Textile B and PET
fiber, respectively. Textile A and B both show a significant reduction in crystallinity under 15% NaOH at
127 °C for 15 and 30 minutes compared to untreated results. Consistent with the SEM results, pretreatment
with 15% NaOH at 80°C showed time-dependent reduction in crystallinity with the 4 hr incubation decreased
about half of the original crystallinity in all samples (22.1% for Textile A, 22.7% for Textile B and 11.7%
for PET fiber), and dropped to as low as 1.5% for Textile A, 1.4% for Textile B and 5.6% for PET fiber when
incubated 20 hr at 80°C. These results further indicate disruption in crystalline domains after
pretreatment. Surprisingly, autoclaving for 15 minutes showed a lower crystallinity reduction compared to
pretreatment at 80°C, indicating greater crystallinity reduction in moderate temperature for longer periods
than short exposure to autoclaving temperature.

Table 2. Crystallinity of 100% PET textiles and PET fiber under various pretreatment conditions,
compared to untreated control. Textile A is 100% PET textile A in weft-rib weave while Textile B is 100% PET
textile B in warp-knit mesh.

![](https://static.igem.wiki/teams/5729/results/pretreatment8.webp)

In summary, these results validated that our alkaline-thermal pretreatment strategies successfully altered
fiber morphology, disrupted the PET matrix and reduced the crystallinity in PET textiles, establishing
pretreatment as a critical first step to enhance the enzyme accessibility and enable efficient textile
degradation. Considering only moderate crystallinity reduction is required for enzymatic degradation and
ensuring the safety and practical conditions, we selected 15% NaOH at 80°C as the pretreatment conditions
for subsequent degradation experiments. Although an 80°C dry bath for more than 8 hours achieves better
decrystallization, the textile would be too fragile to wash by tap water since the textile became powder and
escaped from the strainer. Therefore, we used 15% NaOH at 80°C for 4 hours as the pretreatment for the
further experiments.

# Pure PET textile degradation

After verifying the enzyme degradation on PET film and confirming the pretreatment conditions, we performed
the enzyme degradation experiment on 100% PET textiles before moving on to cotton-PET blend textiles. 100%
PET textiles have simpler components compared to blended textiles, making them ideal for testing enzyme
performance on textile substrates. Success in high crystallinity pure PET textiles degradation indicated the
viability of our process and its potential to degrade more complex cotton-PET blends.

## Preliminary Tests on Pure PET Fiber Degradation

In the first round of pure PET textile degradation, we chose PET fiber because of its low crystallinity
compared to textile A and B. Pretreatment was done by incubating the fibers in 15% NaOH at 80℃ dry bath for
8 hours. Since the PET fiber was too fragile to wash with tap water, we used 1 M HEPES buffer (pH 3.5 to 4)
to neutralize the residual strong alkali for 3 volumes of NaOH. Around 30-80 mg of pretreated PET fibers
were incubated in a reaction buffer containing 500 mM HEPES (pH 7.5) and 10 mM CaCl₂ with 27.8 μg/mL enzyme
at 51.8℃ for 48 hours. However, we failed to detect the UV absorbance at 260 nm because all of the reading
values from pretreated fiber were above the maximum limit of detection, including the negative control
without enzyme. Even though we diluted the reaction buffer to 1/128, there is no significant difference
among the groups (Figure 1). That may be because the PET degraded products still remain on the fiber,
suppressing the enzymes or masking the signal caused by the enzymes.

![](https://static.igem.wiki/teams/5729/results/purepettextiledegradation1.webp)

Figure 1. The preliminary tests on untreated and pretreated pure PET fiber degradation. Absorbance
at 260 nm (A260) of the reaction supernatant after incubation of PET fiber with different variants.

## Pure PET Textile Degradation of TfCut2 and Its Mutants

In order to investigate the characteristics of enzymatic degradation on 100% PET textiles, we utilized pure
PET textiles from three different origins for degradation experiments: 100% PET fiber (Figure 2A), 100% PET
textile A in weft-rib weave (Figure 2B), and 100% PET textile B in warp-knit mesh (Figure 2C).

![](https://static.igem.wiki/teams/5729/results/purepettextiledegradation2.webp)

Figure 2. Pure PET textile used for TfCut2 and its mutant degradation. 100% PET fiber (A), 100%
PET textile A in weft-rib weave (B), and 100% PET textile B in warp-knit mesh (C).

To address the issue of excessive degradation products from pretreatment interfering with subsequent UV
absorbance measurements, we shortened the pretreatment time from 8 hours to 4 hours. Instead of using HEPES
buffer to neutralize the pretreated textile, we rinsed the pretreated textile with tap water for five
minutes, followed by a rinse with deionized water. Finally, they were dried in an oven at 50°C for at least
48 hours before proceeding with enzymatic degradation.

We used approximately 30-80 mg of textiles in a 1.8 mL 500 mM HEPES with 10 mM CaCl₂ (pH 7.5) reaction
buffer with 27.8 μg/mL enzyme. After incubating TfCut2 and its mutants in a thermoshaker at 51.8°C, 800 rpm
for 48 hours, we utilized the ELISA reader to measure UV absorbance at 260 nm. Pretreated PET fibers
released significantly more TPA than untreated samples. Variant 3 exhibited the highest activity, with
absorbance at 260 nm nearly three-fold greater than the wild type. The wild-type 5ZOA and Variants 1, 2, 4,
and 5 also showed increased TPA release for the pretreated samples, whereas Variant 6 displayed negligible
activity. These results demonstrate that pretreatment significantly improves degradation efficiency and
identify Variant 3 as the most effective enzyme for degrading pure PET textile.

Furthermore, the degradation efficiency of the enzyme was also observed to be dependent on the textile
type. With the exception of Variant 6, all mutants and the wild type TfCut2 effectively degraded the
pretreated PET fiber, and to a lesser extent, the pretreated textile B. The higher degradability of the pure
PET fiber can be attributed to its loose structure and lower crystallinity. Notably, despite having
comparable crystallinities, textile B showed higher susceptibility to degradation than textile A, implying
that the textile's weaving pattern could be another contributing factor.

![](https://static.igem.wiki/teams/5729/results/purepettextiledegradation3.webp)

Figure 3. Comparison of enzymatic degradation of pure PET textiles. The degradation of 100% PET
textile A, textile B, and PET fiber, in both untreated and pretreated forms was carried out using the
TfCut2-5ZOA wild type and its six variants. Degradation efficiency was quantified by measuring the absorbance
of the supernatant at 260 nm (A260), which corresponds to the concentration of soluble degradation products
released. Each reaction was incubated at 51.8°C for 48 hours and contained 30-80 mg of PET material and 27.8
µg/mL of enzyme in 500 mM HEPES with 10 mM CaCl₂ pH 7.5. Data shown are representative of a single
experiment.

Next, we choose pretreated PET fiber, which is the most susceptible to our enzymes, to do the further HPLC
analysis and SEM imaging. The HPLC results were consistent with the UV absorbance data, showing that Variant
3 (A65H/L90A/I213S) exhibited the highest degradation efficiency, degrading nearly 0.1 mg of PET (Table 1).
This finding was further supported by SEM imaging. Control samples without enzyme treatment exhibited smooth
fiber surfaces (Figure A and B). Fibers incubated with TfCut2 5ZOA wild type (Figure C and D), Variant
2 (H77R/D204C/E253C) (Figure G and H), Variant 4 (D12S) (Figure K and L), Variant 5 (T234L)(Figure M and N)
and Variant 6 (Variant O and P) showed no noticeable surface alterations, whereas fibers treated with
Variant 1(G62A/P193H/S197D) displayed uneven surface (Figure E and F). In contrast, Variant 3
(A65H/L90A/I213S) caused pronounced surface disruption, with visible grooves observed at higher
magnification (Figure I and J), which were consistent with previous analyses, confirming the superior
effectiveness of Variant 3(A65H/L90A/I213S) in PET degradation.

Table 1. Quantification of PET fiber degradation products after hydrolysis by TfCut2-5ZOA and its
mutants.

![](https://static.igem.wiki/teams/5729/results/purepettextiledegradation4.webp)
![](https://static.igem.wiki/teams/5729/results/purepettextiledegradation5.webp)

Figure 4. Scanning electron microscopy (SEM) imaging of pure PET fiber surfaces after enzyme
degradation. SEM photos of PET fibers after incubation for 48 hours at 51.8°C by different enzyme variants.
(A, B) PET fiber incubated without enzyme as negative control, showing a smooth and unaltered surface. (C,
D) PET fiber incubated with TfCut2-5ZOA wild type. (E, F) TfCut2-Variant 1 G62A/P193H/S197D triple mutant.
(G, H) TfCut2-Variant 2 H77R/D204C/E253C triple mutant. (I, J) TfCut2-Variant 3 A65H/L90A/I213S triple
mutant. (K, L) TfCut2-Variant 4 D12S single mutant. (M, N) TfCut2-Variant 5 T234L single mutant. (O, P)
TfCut2-Variant 6 Full MutCompute mutant. Panels in the first and third columns (A, C, E, G, I, K, M, O) were
captured at 5,000× magnification. Panels in the second and fourth columns (B, D, F, H, J, L, N, P) show a
magnified view of the same samples at 20,000× magnification. All scale bars are indicated in the panels.

In conclusion, our results confirm that alkaline pretreatment significantly enhances enzymatic degradation
in pure PET textiles, TfCut2-Variant 3 A65H/L90A/I213S triple mutant. outperformed TfCut2-5ZOA wild type by
nearly two-fold, demonstrating clear potential for PET breakdown. These findings prove that our engineered
enzymes are capable of degrading pure PET substrates and the foundation for more complex Cotton/PET blend
textile degradation.

# Cotton PET blend degradation

Our previous work established that TfCut2 mutants degrade PET, including alkali-pretreated,
high-crystallinity textiles, more efficiently than the wild type. In this phase, we investigate their
efficacy on the PET component of blend textiles. We will blend textiles with TfCut2 and its mutants and
examine their ability to work in concert with cellulase. The ultimate goal is to develop a method for
completely decomposing blended textiles, enabling their recycling and reuse within the textile industry to
support a circular economy.

To assess the feasibility of our cotton-PET blend textile enzymatic degradation system, we prepared samples
from PET Cotton blends (surface area ~ 62.5 cm^2) at three different PET contents: 35% cotton 65% PET
blend
textile (35:65 cotton-PET Blends) in plain weave (Figure 1A), 55% cotton 45% PET blend textile (55:45
cotton-PET Blends) in tricot warp knit (Figure 1B), and 65% cotton 35% PET blend textile (65:35 cotton-PET
Blends) in single Jersey knit (Figure 1C).

![](https://static.igem.wiki/teams/5729/results/cottonpetblend1.webp)

Figure 1. Blend textile used for TfCut2 and its mutant degradation. 35% cotton 65% PET blend
textile in in plain weave (A), 55% cotton 45% PET blend textile in tricot warp knit(B), and 65% cotton 35%
PET blend textile in single Jersey knit (C).

In order to improve the efficiency of alkali pretreatment and enzymatic degradation, the textiles were
cut into 0.1 x 0.5 cm pieces. Alkali pretreatment was performed according to the conditions described in
the previous section. The purpose of this treatment was not only to reduce the crystallinity of the PET
polymer but also to make the cotton component more susceptible to degradation by cellulase.

## Cotton Degradation of Blend Textiles

After pretreatment, blend textiles were treated with or without commercial cellulase (Cellulase from
*Trichoderma reesei*). Then we evaluated the cotton degradation via DNS assay (Table 1). DNS
Analysis is used
to provide quantitative data on the degradation rate within different cotton-PET textile blends, where it
aids us in analyzing the amount of glucose released after enzymatic degradation. The glucose concentration
determined from OD540 was converted into glucose mass and then multiplied by the initial common mass to
calculate the overall degradation rate for each textile blend. The following equations were used to
determine each measurement:
![](https://static.igem.wiki/teams/5729/results/latex6.avif)

The results indicated that the 55:45 cotton-PET blend shows the highest degradation rate of 94.9% among
the three textile ratios tested, where the 65:35 blend also showed strong enzymatic hydrolysis (81.7%),
while the 35:65 blend resulted in a lower degradation rate (61.0%). These results successfully quantified
reducing sugar release from different cotton-PET blend ratios and further demonstrate that moderate cotton
contents provide an optimal condition for enzymatic attack.

Table 1. DNS Assay for 35:65 cotton-PET Blends, 55:45 cotton-PET Blends, and 65:35 cotton-PET
Blends

![]()

## PET Degradation of Blend Textiles

In this section, we conducted and evaluated PET degradation on cotton PET blend textiles with or without
cotton degradation. After alkaline pretreatment and cotton degradation, we washed textiles with tap water
for 5 minutes and rinsed with deionized water then dried in a 50℃ oven for at least 48 hours. Well-prepared
blend textiles were aliquoted evenly into eight 2 mL microtubes, each containing 1.8 mL of 500 mM HEPES
buffer (pH 7.5) with 10 mM CaCl₂ and 27.8 μg/mL enzymes. In other words, each reaction contained a total of
50 μg enzyme in a reaction volume of 1.8ml. After incubation on a thermoshaker at 51.8℃ 800 rpm for 48
hours, PET degraded products were measured by UV absorbance at 260 nm via ELISA reader.

As shown in Figure 2, TfCut2-Variant 3 A65H/L90A/I213S triple mutant (Variant 3) and TfCut2-Variant 4 D12S
single mutant (Variant 4) exhibited the highest overall activity in degrading PET directly from the blend
textiles, far exceeding the benchmark wild type enzyme TfCut2-5ZOA and other Variants. In contrast, other
mutants showed little to no activity.

![](https://static.igem.wiki/teams/5729/results/cottonpetblend3.webp)

Figure 3. Enzymatic degradation of PET from cotton-polyester blended fabrics with and without
prior removal of cotton. It shows the absorbance at 260 nm (A260), indicating the concentration of
terephthalic acid (TPA) released from various blended textiles by TfCut2-5ZOA wild type and its mutants.
Three types of textile with distinct cotton-to-PET ratios were used: 35:65 cotton-PET Blends, 55:45
cotton-PET Blends, and 65:35 cotton-PET Blends. For each fabric type, two conditions were compared: Direct
enzymatic treatment on the pretreated blend textiles (darker bars: blue, gray, light blue). Two-step
enzymatic treatment, where the cotton component was first removed by cellulase hydrolysis, followed by the
degradation of the remaining PET (lighter bars: orange, yellow, green). All degradation reactions were
performed at 51.8°C for 48 hours.

Interestingly, PET degradation activity was substantially lower after cotton removal compared to the direct
treatment of the blended textiles. The most unexpected finding in this study was that the PET degradation
efficiency was decreased, rather than increased, after the prior removal of the cotton component. We propose
a hypothesis to explain this phenomenon based on the experimental protocol: the issue may stem from an
incompatibility between the buffer systems used for the sequential enzymatic reaction. Another hypothesis is
that the cotton fibers within the blend's structure may play a role in increasing the accessibility of PET
fibers. The removal of cotton might lead to a collapse or tightening of the remaining PET structure, thereby
reducing degradation efficiency.

Furthermore, our study revealed a key finding: the degradation efficiency of PET in blended textile does
not have a direct relationship with its chemical composition—that is, the ratio of cotton to PET. For
instance, although the 55:45 cotton PET blend yielded a relatively high concentration of degradation
products, textiles with either higher or lower PET content did not exhibit a predictable corresponding trend
of increase or decrease in degradation.

This lack of correlation strongly suggests that the dominant factor influencing enzymatic degradation may
not be the chemical composition, but rather the physical architecture of the textile. Our data clearly
indicate that the textile manufacturing method has a significant impact on the catalytic efficiency of
TfCut2. Specifically, the two samples with a woven structure—the 35:65 cotton PET blend and the 100% PET
Textile A—showed markedly lower degradation rates compared to the other samples, which possess a knitted
structure. We hypothesize that the tightly interlaced warp and weft yarns in woven fabrics may limit enzyme
penetration and diffusion, thus reducing the accessible surface area of the PET fibers. In contrast, the
typically looser and more flexible loop structure of knitted fabrics may provide greater access to reaction
sites for the enzyme, thereby facilitating a more effective degradation process.

To further validate the initial findings from the UV absorbance measurement, we selected the top-performing
condition—the 55:45 cotton PET blend textile under direct enzymatic treatment—for subsequent experiments.
The degradation products were then quantified more precisely using High-Performance Liquid Chromatography
(HPLC) (Table 2) and supported by SEM imaging (Figure 4).

The HPLC analysis confirmed that Variant 3(A65H/L90A/I213S) was the most effective enzyme, liberating the
highest concentration of degradation products (TPA and MHET) from the 55:45 cotton PET blend. In contrast,
Variant 4 (D12S), which also appeared highly active in the initial UV screen, demonstrated only a marginal
improvement in performance over the wild type enzyme in this more rigorous assay. These results underscore
that while UV absorbance is a useful tool for rapid preliminary screening, HPLC analysis provides a more
accurate differentiation of the true catalytic efficiencies among enzyme Variants.

Table 2. Quantification of 55:45 cotton PET blend textile degradation products after hydrolysis by
TfCut2-5ZOA and its mutants.

![](https://static.igem.wiki/teams/5729/results/cottonpetblend4.webp)
![](https://static.igem.wiki/teams/5729/results/cottonpetblend5.webp)

Figure 4. SEM imaging of alkali pretreated 55% Cotton 45% PET blended textile after TfCut2
enzymatic degradation. (A, B) Control without treatment, (C, D) wild type TfCut-5ZOA, (E, F) TfCut2 Variant
1 (G62A/P193H/S197D), (G, H) TfCut2 Variant 2 (H77R/D204C/E253C), (I, J) TfCut2 Variant 3 (A65H/L90A/I213S) ,
(K, L) TfCut2 Variant 4 (D12S), (M, N) TfCut2 Variant 5 (T234L), (O, P) TfCut2 Variant 6. Panels in the first
and third columns (A, C, E, G, I, K, M, O) were captured at 2,000× magnification. Panels in the second and
fourth columns (B, D, F, H, J, L, N, P) show a magnified view of the same samples at 20,000× magnification.
All scale bars are indicated in the panels.

In conclusion, we validated the use of TfCut2 Variants for the enzymatic degradation of PET from
cotton-polyester blended textiles, identifying Variant 3(A65H/L90A/I213S) as the most effective catalyst.
Our key finding is that the textile's physical architecture, specifically its knitted or woven structure, is
a more critical determinant of degradation efficiency than the chemical ratio of cotton to PET.
Unexpectedly, the prior removal of cotton inhibited the most active enzymes, likely due to experimental
artifacts such as buffer incompatibility or structural collapse of the remaining PET fibers. These results
collectively underscore the necessity of a holistic approach that considers both enzyme performance and the
physical characteristics of textile waste to develop robust and efficient biorecycling strategies.

# Discussion

## Key Findings:

**Enzyme activity assay**

1. The optimal condition for pNPB assays are within pH ranges 7~8, and at temperature 35℃.
2. The conditions for Tris buffer and HEPES buffer are slightly different in terms of pH ranges, where the
   optimal condition for Tris buffer is between 7.5~8, the pH range for HEPES buffer is between 7~7.5.
3. All variants are proven to have activity, but Variant 6 exhibit sufficiently lower activities.

**PET film degradation**

1. The optimal condition for TfCut2 and its mutants is 51.8℃ pH 7.5.
2. PET film degradation shows different optimal conditions from pNPB assay.
3. Variants 3, 4 show better PET film degradation activity than 5ZOA.
4. Variant 3 can degrade PET in a lower concentration of buffer (100 mM HEPES) while other mutants and
   wild type cannot.

**PET textile degradation**

1. Alkaline pretreatment is necessary for high crystallinity PET degradation.
2. Variants 3 show better PET textile degradation activity than 5ZOA.
3. TfCut2 and its mutants show better degradation activity in knitted structure (textile B) rather than
   woven structure (textile A).

**Blend textile degradation**

1. PET degradation efficiency was decreased in cotton degraded blend textiles.
2. TfCut2 and its mutants show better degradation activity in knitted structure (55:45 cotton PET and 65:35
   cotton PET) rather than woven structure (35:65 cotton PET).
3. Variants 3 show better PET degradation activity in blend textile than 5ZOA.

This study successfully characterized and engineered the wild-type TfCut2 enzyme (5ZOA) and a series of
its variants, systematically evaluating their potential to degrade various forms of poly(ethylene
terephthalate) (PET). By integrating rational design, literature-based mutations, and computational
predictions, we aimed to enhance the enzyme's catalytic activity, stability, and substrate interaction.

Our findings reveal a complex interplay between mutation strategy and actual enzymatic performance.
Variant 3 (A65H/L90A/I213S) consistently emerged as the most effective enzyme across all tested
substrates, from PET film to complex blend textiles. Its design, which combined mutations inspired by the
highly efficient LCC enzyme (L90A/I213S) with a novel mutation to increase PET docking sites (A65H),
proved exceptionally successful. The superior performance of Variant 3 validates the hypothesis that
reducing product inhibition by MHET through the creation of alternative substrate binding positions is a
highly effective strategy for engineering PET-degrading enzymes.

Furthermore, the MutCompute-driven Variant 4 (D12S) also demonstrated better degradation activity than
the wild type, showing that data-driven predictions can successfully identify beneficial single-point
mutations. Conversely, Variant 1 (G62A/P193H/S197D), designed to incorporate a catalytic triad, did not
yield a significant improvement over the wild type, indicating that theoretical catalytic enhancements do
not always translate to effective degradation of a solid polymer substrate. Meanwhile, Variant 6, which
incorporated all non-conserved stabilizing mutations, exhibited a drastic loss of activity, suggesting
that excessive structural stabilization may restrict the conformational flexibility required for
catalysis—a critical insight for future enzyme engineering.

A key finding of this study is the profound influence of textile physical structure on enzymatic
degradation efficiency. All effective variants showed a clear preference for the knitted structure over
the woven structure. This highlights that macroscopic physical accessibility is as critical as biochemical
activity. Furthermore, we also observed that PET degradation was suppressed during the degradation of
cotton, revealing the challenges of processing blended textiles.

Looking ahead, this study opens several avenues for future investigation. The discrepancy in optimal
conditions between the pNPB model substrate and the actual PET substrate warrants further exploration; we
hypothesize that the PBS buffer used during purification may interfere with calcium ions, and future work
should investigate using an alternative such as borate buffer. The preference for knitted structures and
the inhibitory mechanism of cotton on PET degradation require deeper investigation, possibly by testing
different cellulases in concert with our variants.

To advance these enzymes toward practical application, long-term degradation experiments are necessary to
assess their thermostability and improve total PET conversion, which may require specialized hardware and
computational support. Finally, to overcome the challenges of quantifying reaction kinetics on a solid
substrate, we propose the development of a TPA dual reporter system for more precise quantification.

In summary, this research not only identifies a highly efficient TfCut2 mutant (Variant 3) but also
reveals critical factors influencing enzymatic efficiency, from substrate accessibility at the molecular
level to textile structure at the macroscopic level. These findings lay a crucial foundation for
developing robust enzymatic cocktails for the recycling of complex textiles, bringing the vision of a
circular economy for the textile industry one step closer to reality.

## Future Works:

1. Our optimal degradation condition differs from prior studies, potentially due to PBS used during protein
   purification interacting with calcium ions; therefore, future tests should explore borate buffers to
   enhance
   enzyme stability and activity.
2. Because TfCut2 and its mutants performed better on knitted structures than woven ones, further work
   should
   test different cellulases and textile structures to verify this pattern
3. PET degradation declined after cotton breakdown; future studies should determine whether this results
   from
   buffer issues, fiber collapse, or enzyme interactions.
4. Extended degradation experiments are needed to assess thermostability and improve PET hydrolysis with
   better temperature control and hardware.
5. A TPA dual-reporter system could enable real-time, quantitative tracking of PET degradation and kinetic
   modeling of TfCut2 variants.

## References

* Boondaeng, A., Keabpimai, J., Srichola, P., Vaithanomsat, P., Trakunjae, C., & Niyomvong, N.
  (2023). Optimization of Textile Waste Blends of Cotton and PET by Enzymatic Hydrolysis with Reusable
  Chemical Pretreatment. *Polymers, 15*(8), 1964. https://doi.org/10.3390/polym15081964
* Chen, S., Su, L., Billig, S., Zimmermann, W., Chen, J., & Wu, J. (2010). Biochemical
  characterization of the cutinases from *Thermobifida fusca*. *Journal of Molecular Catalysis
  B: Enzymatic, 63*(3–4), 121–127. https://doi.org/10.1016/j.molcatb.2010.01.001
* Dewi, S., Horbach, T., Neumann, T., Jaeger, K., Streit, W., & Hartmann, L. (2025). Evaluation of
  Pretreatment Strategies and Combinations of Enzymes for Degradation of Polyethylene Terephthalate.
  *Journal of Polymer Science, 63*(13), 2770–2781. https://doi.org/10.1002/pol.20250254
* Dong, Q., Yuan, S., Wu, L., Su, L., Zhao, Q., Wu, J., Huang, W., & Zhou, J. (2020).
  Structure-guided engineering of a *Thermobifida fusca* cutinase for enhanced hydrolysis on
  natural polyester substrate. *Bioresources and Bioprocessing, 7*(1), 37.
  https://doi.org/10.1186/s40643-020-00324-8
* Furukawa, M., Kawakami, N., Tomizawa, A., & Miyamoto, K. (2019). Efficient Degradation of
  Poly(ethylene terephthalate) with *Thermobifida fusca* Cutinase Exhibiting Improved Catalytic
  Activity Generated using Mutagenesis and Additive-based Approaches. *Scientific Reports, 9*(1),
  16038. https://doi.org/10.1038/s41598-019-52379-z
* Mushtaq, M., Sultana, B., Bhatti, H. N., & Asghar, M. (2015). RSM based optimized enzyme-assisted
  extraction of antioxidant phenolics from underutilized watermelon (Citrullus lanatus Thunb.) rind.
  *Journal of Food Science and Technology, 52*(8), 5048–5056.
  https://doi.org/10.1007/s13197-014-1562-9
* Schmidt, J., Wei, R., Oeser, T., Belisário‐Ferrari, M. R., Barth, M., Then, J., & Zimmermann, W.
  (2016). Effect of Tris, MOPS, and phosphate buffers on the hydrolysis of polyethylene terephthalate
  films by polyester hydrolases. *FEBS Open Bio, 6*(9), 919–927.
  https://doi.org/10.1002/2211-5463.12097
* Then, J., Wei, R., Oeser, T., Barth, M., Belisário‐Ferrari, M. R., Schmidt, J., & Zimmermann, W.
  (2015). Ca2+ and Mg2+ binding site engineering increases the degradation of polyethylene
  terephthalate films by polyester hydrolases from *Thermobifida fusca*. *Biotechnology
  Journal, 10*(4), 592–598. https://doi.org/10.1002/biot.201400620
* Then, J., Wei, R., Oeser, T., Gerdts, A., Schmidt, J., Barth, M., & Zimmermann, W. (2016). A
  disulfide bridge in the calcium binding site of a polyester hydrolase increases its thermal
  stability and activity against polyethylene terephthalate. *FEBS Open Bio, 6*(5), 425–432.
  https://doi.org/10.1002/2211-5463.12053
* Zhong-Johnson, E. Z. L., Voigt, C. A., & Sinskey, A. J. (2021). An absorbance method for analysis
  of enzymatic degradation kinetics of poly(ethylene terephthalate) films. *Scientific Reports,
  11*(1), 928. https://doi.org/10.1038/s41598-020-79031-5