Experiments

1. Primer Design

Retrieve the target gene sequence from databases such as NCBI. Follow these principles for primer design:

1. Primer length is typically 20–30 bp, with 25 bp recommended.
2. Primer GC content should be maintained between 40 and 60%, with a Tm value around 60°C.
3. For genes with high GC content (>60%), use a DNA polymerase with GC buffer.
4. The Tm values of forward and reverse primers should be as close as possible.
5. Avoid complementarity of more than 4 bases between forward and reverse primers to prevent primer dimer formation.
6. Include protective bases (GGGAAA) and a restriction enzyme site at the 5′ end.
7. When designing the reverse primer, replace the stop codons TAG or TGA with TAA to reduce methylation effects (G and C are prone to methylation).
8. Select restriction enzyme sites that are not present in the target gene. Prefer sites with sticky ends for easier ligation (e.g., KpnI, BamHI, XbaI, NdeI, SalI, HindIII, NheI). Avoid isoschizomers.
9. When using the pETduet-1 vector for plasmid construction, ensure the start codon (AT) is removed from the forward primer to prevent frameshift mutations.

2. Recombinant Plasmid Construction

Plasmid Extraction

1. Streak the glycerol-preserved bacterial strain containing the plasmid onto a plate, pick a single fresh colony, and inoculate into 4 mL of LB medium with the appropriate antibiotic. Incubate at 37°C for 12–16 hours.
2. Column equilibration: Add 500 μL of equilibration buffer BL to the CP3 adsorption column (placed in a collection tube). Centrifuge at 12,000 rpm for 1 minute, discard the flow-through, and return the column to the collection tube.
3. Transfer 4 mL of overnight culture to a centrifuge tube. Centrifuge at 12,000 rpm for 1 minute and carefully remove the supernatant.
4. Resuspend the bacterial pellet in 250 μL of solution P1 (containing RNase A) using a pipette or vortex mixer.
5. Add 250 μL of solution P2 and mix gently by inverting the tube 6–8 times for complete lysis. The solution should become clear and viscous. Do not exceed 5 minutes.
6. Add 350 μL of solution P3 and mix immediately by inverting 6–8 times. Centrifuge at 12,000 rpm for 10 minutes.
7. Transfer the supernatant to the CP3 adsorption column. Centrifuge at 12,000 rpm for 30–60 seconds, discard the flow-through, and return the column to the collection tube.
8. Add 600 μL of wash buffer PW (containing ethanol) to the column. Centrifuge at 12,000 rpm for 30–60 seconds, discard the flow-through, and return the column to the collection tube.
9. Repeat the wash step.
10. Place the column back into the collection tube and centrifuge at 12,000 rpm for 2 minutes. Open the column and air-dry the membrane using a cold blow-dryer to remove residual ethanol.
11. Transfer the column to a clean collection tube. Apply 30 μL of ddH₂O (preheated to 50°C) to the center of the membrane. Incubate at 50°C for 5 minutes, then centrifuge at 12,000 rpm for 2 minutes to collect the plasmid DNA.
12. Measure the DNA concentration using a spectrophotometer.

PCR

Prepare a 50 μL reaction mixture in a PCR tube:

Component	Volume
ddH₂O	20 μL
Upstream primer	2 μL
Downstream primer	2 μL
Template	1 μL
Prime STAR Max	25 μL

PCR Thermal Cycling Condition:

Step	Temperature	Duration	Cycles
Initial denaturation	98°C	30 s	1
Denaturation	98°C	10 s	32
Annealing	56°C	15 s	32
Extension	72°C	1 kb /5 s	32
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Agarose Gel Electrophoresis

50X TAE buffer:

Component	Weight or volume
Tris	242 g
EDTA	18.6 g
Acetic acid	57.1 ml
Deionized water	800 ml

After adjusting the pH to 8.3 with NaOH, add deionized water to make the volume up to 1L and store it at room temperature. Dilute with deionized water to 1X before use.

1. Prepare 50X electrophoresis buffer and dilute to 1X with deionized water.
2. Weigh 1 g of agarose into a conical flask, add 100 mL of 1X electrophoresis buffer, and mix well. Heat in a microwave until clear and transparent (approximately 3 minutes).
3. Pour the solution into a gel tray, add 1 μL of EB solution, mix thoroughly, and allow to solidify for 20 minutes.
4. Place the gel in the electrophoresis tank and add 1X buffer until the gel is submerged. Gently shake the gel to remove bubbles from the wells.
5. Mix the samples with an appropriate volume of 6X loading buffer and carefully load into the wells.
6. Select a suitable DNA ladder based on fragment size. For standard wells (50 μL), load 5 μL of ladder; for small wells (10 μL), load 2 μL. Use one lane for small gels and two lanes for large gels.
7. Set the voltage to 180 V and start electrophoresis.
8. After approximately 15 minutes (when the bromophenol blue band reaches the middle of the gel), stop electrophoresis and analyze the gel using a gel imaging system.

Gel DNA Extraction

1. After electrophoresis, excise the target DNA band under UV light. Minimize the gel volume and transfer to a 2 mL EP tube.
2. Add an appropriate volume of Binding Buffer XP2 (typically 100 μL per 0.1 g gel) to dissolve the gel and enhance DNA binding to the column.
3. Incubate the tube at 65°C, shaking every 2 minutes until the gel is completely dissolved.
4. Transfer the solution to a HiBind DNA column placed in a 2 mL collection tube. Let stand for 3 minutes, then centrifuge at 12,000 rpm for 1 minute.
5. Reload the flow-through into the column, incubate for 1 minute, and centrifuge at 12,000 rpm for 1 minute. Discard the flow-through.
6. Add 700 μL of SPW Wash Buffer to the column and centrifuge at 12,000 rpm for 1 minute. Discard the flow-through.
7. Repeat the wash step.
8. Centrifuge the column at 12,000 rpm for 3 minutes to dry. Discard the collection tube and air-dry the column with a cold blow-dryer until no ethanol smell remains.
9. Add 30 μL of preheated ddH₂O to the center of the membrane. Place the column in a 1.5 mL EP tube and incubate for 3–5 minutes. Centrifuge at 12,000 rpm for 5 minutes to collect the eluate.
10. Verify the extraction by running 2 μL of the eluate on a gel.

Recombinant Plasmid and Chemical Transformation Method

1. Prepare a 10 μL ligation reaction system. The insert-to-vector ratio is determined by calculation: the insert volume is calculated as insert length × 0.04 / c, and the vector volume as insert length × 0.02 / c. The combined volume of insert and vector should be 5 μL (adjusted with ddH₂O if necessary). Add 5 μL of ligase to reach a final reaction volume of 10 μL.
2. React in a 50°C water bath for 30 minutes, then place on ice for 2 minutes.
3. In a sterile environment, add 10 μL of the recombinant plasmid to 50 μL of DH5α competent cells. Incubate on ice for 30 minutes.
4. Heat-shock at 42°C for 90 seconds, then immediately place on ice for 2 minutes. Add 600 μL of sterile LB medium and incubate at 37°C with 200 rpm for 1 hour.
5. Centrifuge at 6,000 rpm for 1 minutes, remove most of the supernatant leaving approximately 100 μL, and resuspend the pellet to concentrate. Spread onto LB solid medium plates with the appropriate antibiotic. Incubate at 37°C for 12 hours.

Electroporation Method

Preparation of electrocompetent cells

1. Streak the preserved bacterial culture from -80°C onto an LB plate without antibiotics using a sterile loop. Incubate at 37°C overnight.
2. Pick a single colony and inoculate it into 4 mL of LB medium without antibiotics. Incubate at 37°C with shaking at 220 rpm for 8 hours.
3. Transfer 1 mL of the culture to 50 mL of LB medium. Incubate at 37°C with shaking at 220 rpm for 1.5 – 2 hours until OD600 reaches approximately 0.6.
4. Pour the culture into a sterile 50 mL centrifuge tube. Centrifuge at 6,000 rpm at 4°C for 5 minutes. For a 100 mL culture, use two tubes.
5. Discard the supernatant and add 20 mL of sterile 10% glycerol (W/V). Resuspend and centrifuge at 6,000 rpm at 4°C for 10 minutes. Repeat twice.
6. Resuspend each pellet in 300 μL of 10% glycerol. Adjust the volume based on bacterial density.
7. Aliquot 90 μL into 1.5 mL centrifuge tubes.

Use a BIO-RAD electroporator to transfer the verified recombinant plasmid into competent cells:

1. Place a cleaned and UV-sterilized 1 mm electroporation cuvette on ice.
2. Thaw the prepared competent cells on ice, add 1–5 μL of recombinant plasmid, mix gently, and transfer to the cuvette.
3. Set the electroporation parameters, ensure the cuvette is dry, and deliver the pulse.
4. Immediately add 600 μL of sterile LB medium to the cuvette, mix thoroughly, and transfer to a 1.5 mL centrifuge tube. Incubate at 37°C for 45–60 minutes for recovery.
5. Spread 200 μL of the culture onto a selective solid medium with the appropriate antibiotic. Incubate inverted at 37°C for approximately 10 hours.
6. Pick 3 single colonies of appropriate size from the selective medium for subsequent experiments.
7. Mix the bacterial culture with an equal volume of glycerol in a cryovial and store at -20°C.

Colony PCR

Pick 4–6 single colonies from the antibiotic plate and resuspend in 10 μL of ultra-pure water as a template.

Prepare a 20 μL reaction system in a PCR tube:

Component	Volume
ddH₂O	7 μL
Upstream primer	1 μL
Downstream primer	1 μL
Template	1 μL
Taq	10 μL

PCR Run Protocol:

Step	Temperature	Duration	Cycles
Initial denaturation	98°C	5 min	1
Denaturation	98°C	10 s	29
Annealing	56°C	30 s	29
Extension	72°C	1 kb/5 s	29
Final extension	72°C	10 min	1
Hold	4°C	∞	1

Verify by agarose gel electrophoresis and send the correct plasmid for sequencing.

3. Expression of the Target Protein

LB Medium Preparation

2.5 g/100ml LB Medium (per liter)

• LB Medium Powder 25 g
• For solid medium, add 2g agar per 100 ml

Protein Expression

1. Inoculate the verified bacterial culture into 5 mL of LB medium, culture, and extract the plasmid.
2. Thaw BL21(DE3) competent cells on ice.
3. Add 10 μL of plasmid to 100 μL of competent cells in a sterile environment and incubate on ice for 30 minutes.
4. Heat-shock at 42°C for 45 seconds, then place on ice for 2 minutes.
5. Add 500 μL of sterile LB medium and recover at 37°C with shaking for 1 hour.
6. Centrifuge at 6,000 rpm for 5 minutes.
7. Remove most supernatant, leaving approximately 100 μL, resuspend to concentrate, and spread onto LB solid medium with the appropriate antibiotic (ampicillin sodium for FastPETase, kanamycin sulfate for HotPETase, and ampicillin sodium for FastPETase-M4). Incubate at 37°C for 12 hours.
8. Pick a single colony and inoculate it into 10 mL of sterile LB medium with the appropriate antibiotic. Incubate at 37°C for 12 hours until OD600 reaches approximately 0.6.
9. Transfer the entire culture to 1 L of sterile LB medium with the appropriate antibiotic. Incubate at 37°C with shaking until OD600 reaches the appropriate density (approximately 6–8 hours).
10. Add 1 mL of IPTG solution and induce at 18°C with shaking for 18–20 hours.

4. Protein Purification

Lysis Buffer (1L)

Component	Weight or volume
Tris	6.06 g
NaCl	17.53 g
Imidazole	0.68 g
ddH₂O	900 ml

Dilute with HCl to pH 8.0, then make up to 1 liter with distilled water.

Wash Buffer System (1L)

Different imidazole concentrations of Wash Buffer	30 mM	50 mM	300 mM	500 mM
NaCl	17.53 g	17.53 g	17.53 g	17.53 g
Tris	6.06 g	6.06 g	6.06 g	6.06 g
Imidazole	2.06 g	3.44 g	20.58 g	34.38 g
ddH₂O	Dilute to a total volume of 1 liter

1. Centrifuge the induced culture at 4,000 rpm for 40 minutes and discard the supernatant.
2. Resuspend the pellet in a small volume of 10 mM imidazole buffer and disrupt the cells using a high-pressure homogenizer (800 MPa for 10 minutes) to release the target protein.
3. Centrifuge the lysate at 12,000 rpm for 40 minutes, filter the supernatant, and apply to a Ni-NTA agarose 6FF column. Rotate at 4°C for 30 minutes for binding.
4. Wash the column with 50 mM imidazole buffer until the flow-through no longer changes the color of Coomassie Brilliant Blue G-250.
5. Elute the target protein with 300 mM imidazole buffer until the eluate no longer changes the color of Coomassie Brilliant Blue G-250.
6. Clean the column with 500 mM imidazole buffer and store in 20% ethanol at 4°C.
7. Concentrate the eluate using a 10 kDa ultrafiltration tube at 4°C and 4,000 rpm. Exchange the buffer and concentrate again.
8. Prepare an SDS-PAGE gel using a 12% pre-cast gel kit. Prepare the sample by mixing 4 μL of 5X SDS loading buffer with 16 μL of concentrated protein and heating at 100°C for denaturation. Load the sample and an 180 kDa Plus Prestained Protein Marker. Run at 150 V for 1 hour.
9. Stain the gel with Feto SDS-PAGE staining buffer for 3 minutes with shaking, destain with deionized water for 5 minutes, and image using a gel documentation system. Verify the molecular weight and purity by SDS-PAGE.
10. Measure the absorbance at 280 nm using a NanoDrop One. Calculate the extinction coefficient by entering the amino acid sequence into the NovoPro online tool (https://www.novopro.cn/tools/) and determine the protein concentration using the Beer-Lambert law.

BCA Protein Quantification Method

1. Prepare the BCA working solution by mixing reagents A and B at a 50:1 ratio based on the number of samples (200 μL per sample). For example, for 10 samples, mix 2000 μL of A with 40 μL of B.
2. Prepare protein standards by diluting a 5 g/L stock solution 10-fold with PBS to obtain 0.5 g/L standards. Prepare a dilution series for the standard curve.
3. Add PBS and standards to a 96-well plate according to the table. For samples, dilute accordingly (e.g., for 10-fold dilution, add 18 μL PBS and 2 μL sample; for 20-fold, add 19 μL PBS and 1 μL sample). Add 200 μL of BCA working solution to each well (prepare fresh).
4. Incubate the plate at 37°C for 45 minutes or at room temperature for 2 hours. A purple color will develop, intensifying with protein concentration.
5. Measure the absorbance at 562 nm using a microplate reader. Plot the standard curve with protein concentration on the x-axis and absorbance on the y-axis. Calculate the sample protein concentration.

BCA protein concentration Standard curve:

Number	PBS/μL	Protein standard /μL	Concentration(g/L)
A	20	0	0
B	19	1	0.025
C	18	2	0.05
D	16	4	0.1
E	12	8	0.2
F	8	12	0.3
G	4	16	0.4
H	0	20	0.5

SDS-Polyacrylamide Gel Electrophoresis

10% SDS preparation system:

Component	Weight
SDS	10 g
ddH₂O	100 ml

Dilute ddH₂O to 100ml and adjust the pH to 7.2 with concentrated hydrochloric acid.

10% APS preparation system:

Component	Weight
APS	0.1 g
ddH₂O	1 ml

Dilute ddH₂O to 1 ml, store at 4°C, and prepare as needed.

10X electrophoresis buffer preparation system:

Component	Weight
Tris	30.3 g
Glycine	144.2 g
SDS	10 g

Dissolve H₂O to a total volume of 1000 ml, and store at room temperature.

Dyeing solution preparation system:

Component	Weight
Coomassie Brilliant Blue R-250	1 g
Methanol	450 ml
ddH₂O	100 ml
Glacial acetic acid	450 ml

Bleaching liquid configuration system:

Component	Weight
Methanol	100 ml
ddH₂O	100 ml
Acetic acid	800 ml

A liquid preparation system:

Component	Weight
Monomer acrylamide	29 g
Methyl acrylate dimethacrylamide	0.8 g
ddH₂O	100 ml

After filtration, store at 4°C in the dark.

B liquid configuration system:

Component	Weight
Tris	18.17 g
10% SDS	4 ml
ddH₂O	100 ml

Adjust the pH to 8.8 with concentrated hydrochloric acid and store it in a dark place at 4°C.

C liquid configuration system:

Component	Weight
Tris	6.06 g
10% SDS	4 ml
ddH₂O	100 ml

Adjust the pH to 6.8 with concentrated hydrochloric acid and store it in a dark place at 4°C.

Gel configuration system:

Component	8%	9%	10%	12%	15%	Concentrated glue
ddH₂O/ml	2.4	2.25	2.1	1.75	1.25	1.45
B solution/ml	1.25	1.25	1.25	1.25	1.25	C solution 0.625
A solution/ml	1.35	1.5	1.65	2	2.5	0.42
10%APS/ul	50	50	50	50	50	30
TEMED/ul	8	8	8	8	8	5
Separation range/Kda	30-120	30-90	18-75	15-60	15-45

1. Prepare glass plates by cleaning and assembling them (ensure the frosted sides and bottoms are aligned). Test for leaks by filling with water and waiting 3–10 minutes.
2. Prepare samples by mixing with 5X loading buffer (containing mercaptoethanol). Denature by boiling for 10 minutes, cool, and centrifuge to collect supernatant.
3. Prepare separating gel based on the target protein size (higher concentration for smaller proteins). Quickly pour into the gel assembly (approximately 3.2 mL). Overlay with isopropanol and let polymerize for 10–30 minutes. Remove isopropanol and dry with filter paper.
4. Prepare stacking gel and pour over the separating gel. Insert a comb without bubbles and add excess gel around the comb. Polymerize for 20–30 minutes.
5. After polymerization, place the gel in the electrophoresis tank and fill with buffer. Carefully remove the comb and load 10–20 μL of sample per well. Include a protein ladder in one well.
6. Connect the tank to the power supply (red to red, black to black). Run at 220 V until the bromophenol blue reaches approximately 1 cm from the bottom (approximately 45 minutes).
7. Carefully disassemble the gel, remove the stacking gel, and place the gel in a staining box. Cover with Coomassie Brilliant Blue stain.
8. Stain at room temperature with gentle shaking (<80 rpm) for 20 minutes. Destain with destaining solution until the background is clear. Image the gel using a pre-warmed gel documentation system.

5. Various Parameter Tests

Enzyme Activity Assay

Enzyme - substrate reaction

1. Add 5 mg of PLA or PBAT plastic powder to 5 mL EP tubes. Add 1 mL of seawater or PB buffer and an appropriate concentration of free enzyme. Incubate at a temperature gradient of 30–70°C. Include triplicates and a blank (70 tubes total).
2. After 10 and 20 hours, measure PBAT degradation using a NanoDrop and PLA degradation using HPLC.

UV-Vis Spectrophotometer:

1. Measure the concentration of PBAT hydrolysis products at 240 nm using a UV-Vis spectrophotometer.
2. The products are identified as TPA analogs with a molar extinction coefficient of 17,000 M⁻¹cm⁻¹ and expressed as TPA equivalents (TPAeq).

TPA Standard Curve

1. Mix 700 μL of gradient TPA solutions, 100 μL of 100 mM PB (pH 6.15), 100 μL of 1 mM ammonium ferrous sulfate, and finally 100 μL of 3% hydrogen peroxide. Record data 3 minutes after adding hydrogen peroxide using a fluorescence spectrophotometer.
2. Plot the TPA standard curve using Origin software.

6. Calcium Carbonate-Silicon Dioxide Composite Immobilization

1. Prepare 0.15 M calcium chloride and sodium carbonate solutions.
2. Calculate the volume of enzyme solution to add for a final protein concentration of 1 mg/mL.
3. Add the enzyme solution to 20 mL of calcium chloride solution. Rapidly add 20 mL of sodium carbonate solution with vigorous stirring. Continue stirring for 20–30 minutes.
4. Let stand at 4°C for 1–2 hours. Centrifuge at 8,000 rpm for 10 minutes and collect the supernatant.
5. Wash the precipitate 2–3 times with pre-chilled Tris-HCl.
6. Prepare a solution with Tris-HCl buffer and ammonia water (approximately 0.4 M, pH 8–9).
7. Resuspend the calcium carbonate in the above solution. Slowly add tetraethyl orthosilicate (TEOS) solution with gentle stirring. Use two groups: one with 1X and the other with 3X TEOS relative to calcium carbonate.
8. React at room temperature with gentle stirring for 6–24 hours.
9. After the reaction, let it age for 1 hour. Wash 3 times with Tris-HCl buffer by centrifugation.
10. Lyophilization.

Thermal Stability Testing

1. Place 10 mg of freeze-dried powder in glass tubes, flush with nitrogen, seal, and heat at 160°C, 180°C, 200°C, 220°C, 240°C, 260°C, 280°C, and 300°C for 3 minutes.
2. Add 2.5 mL of 50 mM PB and 1667 μL of 200 mM EDTA for chelation. Shake and let stand at room temperature until the precipitate dissolves.
3. Prepare reaction buffer: 1.4 mL PB + 1 mL surfactant for a 1.5% concentration. Prepare substrate: 1 mL acetone + 4 μL PNPB.
4. Clean cuvettes with alcohol and water 3 times. Add 900 μL reaction buffer and 10 μL chelated enzyme solution. Finally, add 100 μL of substrate and measure using a spectrophotometer.
5. Determine the enzyme concentration in the chelated solution using the BCA method. Calculate the reaction rate per unit enzyme concentration based on spectrophotometric data.

Film Pressing and Degradation

Film Pressing

1. Weigh 1 g of dried PBAT and PLA at ratios of 1:1 and 3:7. Also weigh 1 g of pure PBAT. Add immobilized enzyme at 5% or 3% to each group.
2. Preheat the powder in the film press for 1 minute. Press at 30 kPa for 1.5 minutes. Heat PBAT to 160°C and the PLA/PBAT mixture to 180°C.

Degradation Demonstration

Place 3 cm × 3 cm pressed plastic films in seawater and simulated seawater (50 mM PB pH 8 with NaCl added to seawater salinity). Record changes daily.