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.

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.
   

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.

   

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

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)

   
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 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



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 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.

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.

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

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

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.

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.

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.

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 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.

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

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.

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.

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.

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.

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)

   

   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.

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

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.

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.

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.

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.

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)

   


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

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.