Culture

1. Culture medium

1.1 TB

Cultivation of E. coli using TB medium while expressing proteins in experiments.

The TB medium was configured as follows.

The preparation process is as follows.

Calculate → Weigh → Dissolve → Sterilize → Save

Detailed steps:

1. Calculation of substance requirements based on the volume of medium required.
2. Weigh the substances according to the calculation.
3. Add ddH₂O to fix the volume and stir well to dissolve it.
4. Autoclave the conical vials after closing the mouth with a breathable parafilm and a leather band.
5. Store the liquid medium in a 4 ℃ refrigerator and use it as soon as possible. Close the solid medium with a parafilm and store it in a refrigerator at 4℃ and use it as soon as possible.

1.2 LB

Cultivation of E.coli in experiments using LB medium (with appropriate antibiotics if needed).

The LB medium was configured as follows.

If formulated as a solid medium, add an additional 15 g of agar powder (1.5%) before sterilization, then autoclave the configured medium.

The preparation process is as follows.

Calculate → Weigh → Dissolve → Sterilize → Pour Plate (if needed) → Save

Detailed steps:

1. Calculation of substance requirements based on the volume of medium required.
2. Weigh the substances according to the calculation.
3. Add ddH₂O to fix the volume and stir well to dissolve it.
4. Autoclave the conical vials after closing the mouth with a breathable parafilm and a leather band.
5. In case of LB agar the inverted plate operation is carried out when the medium is cooled to about 45 ℃. The petri dish lid can be tilted to minimize condensate contamination, and the inverted plate operation is carried out in an ultra-clean bench next to an alcohol lamp.
6. Store the liquid medium in a 4 ℃ refrigerator and use it as soon as possible. Close the solid medium with a parafilm and store it in a refrigerator at 4 ℃ and use it as soon as possible.

1.3 M9

1. Configure of 1 mol/L MgSO₄: weigh 2.46g of MgSO₄-7H₂O and dissolve it in 10ml of ddH₂O, autoclaved it.
2. Configure 1 mol/L CaCl₂: weigh 2.191g of CaCl₂-6H₂O and dissolve it in 10ml of ddH₂O, autoclave it.
3. Configure 5×M9 salt solution:

   Ingredient	Quantity
   Na2HPO4・H2O	12.8g
   KH2PO4	3g
   NACL	0.5g
   NH4CL	1 g
   ddH2O	To 200ml

   Autoclave standby.

4. Configure 20% glucose solution: weigh 4g glucose and dissolve it in 20ml of ddH₂O, then filter with 0.22μm filter to remove bacteria.
5. Configure M9 culture medium

   Ingredient	Quantity
   5×M9	200ml
   1M MgSO4	2ml
   20% Glucose solution	20ml
   1M CaCl2	0.1ml
   ddH2O	To 1L

1.4 MR

In the experiment, E. coli was cultured using MR medium (with appropriate antibiotics or inducers added as necessary) for fermentation studies.

The preparation method for MR medium is as follows:

Note that glucose and certain heat-sensitive supplements (e.g., MOPS, MgSO₄) must be sterilized by filtration and added only after the base medium has cooled. They must not undergo autoclaving.

The preparation process is as follows:

Calculation → Weighing → Dissolution → Aliquoting and Sterilization → Addition of Sterile Components → Storage

Detailed steps:

1. Calculate reagent quantities based on required medium volume.
2. Weigh reagents according to calculations. Heat-sensitive components like 3-(N-morpholinyl)propane sulfonic acid (MOPS), glucose, magnesium sulfate, and trace elements require separate filter sterilization.
3. Add deionized water to final volume and stir thoroughly until dissolved.
4. Seal flasks with breathable plastic wrap and leather bands, then sterilize in an autoclave.
5. Prepare 0.5 g/ml MOPS solution: Weigh 20.926 g MOPS, dissolve in 42 ml deionized water, and filter through a 0.22 μm membrane to remove bacteria.
6. Prepare a 0.5 g/ml glucose solution: Weigh 20 g glucose, dissolve in 40 ml deionized water by heating until completely dissolved, then filter through a 0.22 μm membrane filter to remove bacteria.
7. Prepare a 0.5 g/ml MgSO₄·7H₂O solution: Weigh 0.8 g of magnesium sulfate heptahydrate, dissolve in 1.6 ml of deionized water, and filter through a 0.22-micron membrane filter to remove bacteria.
8. Prepare trace element solution: Weigh 0.5 g ferrous sulfate heptahydrate (FeSO₄·7H₂O), 0.1 g calcium chloride (CaCl₂), 0.11 g zinc sulfate heptahydrate (ZnSO₄·7H₂O), 0.025 g manganese sulfate tetrahydrate (MnSO₄·4H₂O), 0.025g copper sulfate pentahydrate (CuSO₄·5H₂O), 0.05g ammonium molybdate tetrahydrate ((NH₄)₆Mo₇O₂₄·4H₂O), 0.001g sodium tetraborate decahydrate (Na₂B₄O₇·10H₂O), dissolved in 0.5M HCl solution, stirred until completely dissolved, then sterilized by filtration through a 0.22μm membrane filter. (Note: This formula yields 20mL of trace element stock solution; scale proportionally as needed. Add 5mL of this stock solution per liter of MR medium.)
9. Sterile addition of heat-sensitive components: After autoclaving the base medium, cool it below 60°C and perform sterile operations in a laminar flow hood. Add sterile-filtered glucose stock solution to a final concentration of 20 g/L; add sterile-filtered MgSO₄·7H₂O solution to a final concentration of 0.8 g/L; add sterile-filtered MOPS solution to a final concentration of 100 mM; add 5 mL of sterile-filtered trace element solution.
10. After thorough mixing, the liquid medium can be stored at 4°C and used promptly.

2. Antibiotic

The following table shows the concentration of antibiotics and working solution used, which should be diluted 1:1000 at the time of use. Note that solid media need to be added after sterilization when cooled to approximately 45 ℃ to avoid inactivation of antibiotics.

The preparation process is as follows.

Calculation → Weighing → Dissolving → Dispense

Detailed steps:

1. Calculation: according to the concentration and the required amount to calculate the antibiotic mass and sterile water volume to be weighed.
2. Weighing: according to the calculation results.
3. Dissolution: use a pipette gun ( the gun’s head needs to be sterilized ) to suck sterile water (ddH2O is sterilized by high-pressure steam ) and dilute to the corresponding volume.
4. Dissolution: use a pipette gun ( the gun’s head needs to be sterilized ) to suck sterile water (ddH2O is sterilized by high-pressure steam ) and dilute to the corresponding volume.
5. Sub-packaging: by using disposable syringe and 0.22 μM sterile syringe filter (bioshrap, catalog number: bs-pes-22 ) to filter and sub-pack the antibiotic into several 1.5/2 ml EP tubes and then store them in a - 20℃ refrigerator, and use it as soon as possible.

3. IPTG\L-arabinose

3.1 Preparation of 0.5M IPTG Stock Solution

1. Personal Protective Equipment (PPE):Put on appropriate PPE including lab coat, gloves, and safety goggles.
2. Weighing IPTG: In a fume hood, accurately weigh the required amount of IPTG powder to prepare a 0.5M solution. The amount needed can be calculated using the molecular weight of IPTG and the desired volume of the stock solution.
3. Dissolution:Add the weighed IPTG to a suitable volume of sterile water in a clean container. Stir gently until the IPTG is completely dissolved. Ensure that the solution is at room temperature to facilitate dissolution.
4. Sterilization:Filter the solution through a 0.22 µm sterile filtration system to remove any potential contaminants. This step should be done in a laminar flow hood to maintain sterility.
5. Aliquoting: Using aseptic technique, aliquot the filtered IPTG solution into 1 mL centrifuge tubes. Label each tube with the solution name, concentration, date of preparation, and any other relevant information.
6. Storage: Store the aliquoted IPTG stock solution at -20°C. Avoid repeated freeze-thaw cycles to maintain the integrity of the solution.

3.2 Preparation of L-arabinose

1. Personal Protective Equipment (PPE):Put on appropriate PPE including lab coat, gloves, and safety goggles.
2. Weighing L-arabinose: In a fume hood, accurately weigh the required amount of L-arabinose powder to prepare a 200mg/ml solution. The amount needed can be calculated using the molecular weight of L-arabinose and the desired volume of the stock solution.
3. Dissolution:Add the weighed L-arabinose to a suitable volume of sterile water in a clean container. Stir gently until the L-arabinose is completely dissolved. Ensure that the solution is at room temperature to facilitate dissolution.
4. Sterilization: Filter the solution through a 0.22 µm sterile filtration system to remove any potential contaminants. This step should be done in a laminar flow hood to maintain sterility.
5. Aliquoting:Using aseptic technique, aliquot the filtered L-arabinose solution into 1.5 mL centrifuge tubes. Label each tube with the solution name, concentration, date of preparation, and any other relevant information.
6. Storage:Store the aliquoted L-arabinose stock solution at -20°C. Avoid repeated freeze-thaw cycles to maintain the integrity of the solution.

All of the aforementioned operations must be conducted within a biological safety cabinet in order to prevent contamination.

4. Thiamine\RS-3-Hydroxybutyric Acid Sodium Salt\3-Aminopropionic Acid Solution

4.1 Preparation of Thiamine

1. Personal Protective Equipment (PPE): Wear appropriate PPE, including a lab coat, nitrile gloves, and safety goggles. All weighing and dispensing operations must be conducted within a fume hood to prevent powder inhalation and contamination.
2. Weighing Thiamine: Precisely weigh the required amount of thiamine powder in a fume hood to prepare a 200 mg/mL solution. For example, to prepare 10 mL of stock solution, weigh: 200 mg/mL × 10 mL = 2000 mg, i.e., 2.0 g.
3. Dissolution: Add the weighed thiamine powder to an appropriate volume of sterile deionized water in a clean container. Vortex or gently stir until completely dissolved. Thiamine is highly soluble in water and dissolves rapidly at room temperature.
4. Sterilization: Filter the solution through a 0.22-micron sterile syringe filter into a sterile receiving tube to remove potential contaminants. This step should be performed within a biosafety cabinet or laminar flow hood to maintain sterility.
5. Aliquoting: Using aseptic technique, immediately aliquot the filtered thiamine solution into sterile 1.5 mL centrifuge tubes in single-use volumes (e.g., 1.0 mL/tube). Clearly label each tube with the solution name, concentration (200 mg/mL), preparation date, and expiration date.
6. Storage: Store aliquoted thiamine stock solution protected from light at -20°C. Avoid repeated freeze-thaw cycles to maintain solution stability and biological activity.

4.2 Preparation of Sodium RS-3-Hydroxybutyrate and 3-Aminopropionic Acid Solutions

1. Personal Protective Equipment (PPE): Wear appropriate PPE, including a lab coat, nitrile gloves, and safety goggles. All weighing and aliquoting operations must be performed in a fume hood to prevent powder inhalation and contamination.
2. Weighing RS-3-Hydroxybutyric Acid Sodium Salt and 3-Aminopropionic Acid Powders: Precisely weigh the required amounts of RS-3-hydroxybutyric acid sodium salt and 3-aminopropionic acid powders in a fume hood to prepare a 400 mg/mL solution. For example, to prepare a 10 mL stock solution, weigh 400 mg/mL × 10 mL = 4000 mg of each powder, equivalent to 4.0 g.
3. Dissolution: Add the weighed sodium RS-3-hydroxybutyrate and 3-aminopropionic acid powders to an appropriate volume of sterile deionized water in a clean container. Vortex or gently stir until completely dissolved, forming a clear solution.
4. Sterilization: Filter the solution through a 0.22-micron sterile filtration system into a sterile receiving tube to remove potential contaminants. This step should be performed within a biosafety cabinet or laminar flow hood to maintain sterility.
5. Aliquoting: Using aseptic technique, aliquot the filtered sodium RS-3-hydroxybutyrate and 3-aminopropionic acid solution into sterile 1.5 mL centrifuge tubes in single-use volumes (e.g., 1.0 mL/tube). Each tube must be clearly labeled with the solution name, concentration (400 mg/mL), preparation date, and expiration date.
6. Storage: Store the aliquoted sodium RS-3-hydroxybutyrate and 3-aminopropionic acid stock solutions at -20°C. Avoid repeated freeze-thaw cycles to maintain solution stability.

Breed Preservation

1. Glycerol Stock

1. Prepare a 50% glycerol solution: Mix equal volumes of 100% glycerol and ultrapure water, and store at room temperature.
2. Inoculate Glycerol Stock: Transfer 500 μL of bacterial culture to a 2 mL tube, add 500 μL of 50% glycerol, and mix gently.
3. Storage: Use screw top tubes, label with strain and date, and store at -80°C to prevent unexpected opening.
4. Stability:Maintain stocks at -80°C for long-term stability; avoid repeated freeze-thaw cycles.

2. Recovery

1. Recovery of Bacteria:Under sterile conditions, open the glycerol stock tube and use a sterile inoculation loop or pipette tip to scrape an appropriate amount of frozen bacteria, then streak onto an LB agar plate with or without the appropriate antibiotic.
2. Overnight Cultivation:Incubate the streaked LB agar plate at the optimal temperature for the bacterial strain to allow for growth.
3. Isolation of Single Colonies: After overnight incubation, pick individual colonies for further processing or analysis.

Plasmid construction and Transformation

1. PCR

Prepare a 25 µl reaction system using PCR tubes (ignite an alcohol lamp, no need to operate inside a laminar flow hood). 12.5 µl PCR MIX, 1 µl upstream primer, 1 µl downstream primer, 0.5 µl template, 10 µl ddH2O. Gently centrifuge to mix the reaction system.

Set up the PCR program: (Usually use high-fidelity enzymes from Shenggong, or set according to the instructions for different enzymes)

X should be set lower than the lowest Tm value of the two primers by 5-6. For example, if the Tm values of the two primers are 80 and 72, it should be set to 65.

2. Nucleic Acid Gel Electrophoresis

Prepare the nucleic acid gel: Remember the ratio 1:10:100 (0.6g agarose: 6 µl nucleic acid dye: 60 ml 1x TAE buffer).

After adding agarose and TAE buffer, mix well and heat in a microwave until the solution is clear and transparent without any agarose lumps (use a larger conical flask to prevent boiling over). After heating, add the nucleic acid dye (highly toxic, handle with care), mix well. Place the plastic plate in the groove, then pour the solution, insert the comb (remove it vertically without damaging the wells). Do not add all at once to prevent the gel from being too thick.

Add 7 µl marker, 25 µl PCR product. Electrophoresis at 150V for 20 minutes.

3. Gel Recovery

Remove the electrophoresed nucleic acid gel from the glass plate, place it under ultraviolet light, and remember to take protective measures. After taking a photo for preservation, cut off a small piece of the gel and place it into a 1.5 mL EP tube.

1. Cut the single DNA band from the agarose gel (try to remove excess parts) and place it into a clean centrifuge tube, weigh it.
2. Note: When using a gel cutter (OSE-GC) to cut the gel, align the cutter with the DNA band in the agarose gel and press down to cut. After cutting, push the center rod to push the gel piece into a clean centrifuge tube. Depending on the width of the gel well, you can perform single or continuous cutting.
3. Add 3 times the volume of gel dissolution liquid PE (if the gel weight is 0.1g, its volume can be considered as 100 µl, then add 300 µl gel dissolution liquid PE. When using a gel cutter to cut 1% agarose gel, the weight of a single piece is about 0.06 g, the actual weight of the gel piece is related to the gel concentration and thickness), dissolve at room temperature (15-25°C) for 5-10 minutes, during which gently invert the centrifuge tube up and down to ensure the gel piece is fully dissolved. (If the volume of the gel piece is too large, it can be cut into smaller pieces beforehand).
4. Note: For large fragments greater than 5 kb or gels with a concentration greater than 1.5%, it is recommended to heat dissolve at 50°C for 5-10 minutes; after the gel piece is completely dissolved, it is best to lower the solution temperature to room temperature before applying it to the column, as the adsorption column has a stronger DNA binding capacity at room temperature.
5. Add the solution from step 2 to an adsorption column CA5 (place the adsorption column into a collection tube), let it sit at room temperature for 2 minutes, centrifuge at 12,000 rpm (approximately 13,400 ×g) for 30-60 seconds, discard the waste liquid in the collection tube, and place the adsorption column CA5 back into the collection tube.
6. Note: The volume of the adsorption column is 800 µl, if the sample volume is greater than 800 µl, it can be added in batches.
7. Add 600 µl wash solution PW (check if anhydrous ethanol has been added before use) to the adsorption column CA5, centrifuge at 12,000 rpm (approximately 13,400 ×g) for 30-60 seconds, discard the waste liquid in the collection tube, and place the adsorption column CA5 back into the collection tube. Note: If the DNA being recovered is for salt-sensitive experiments, such as blunt-end ligation experiments or direct sequencing, it is recommended to let the PW sit for 2-5 minutes before centrifugation.
8. Repeat step 4.
9. Place the adsorption column CA5 back into the collection tube, centrifuge at 12,000 rpm (approximately 13,400 ×g) for 2 minutes to remove as much wash solution as possible. Let the adsorption column CA5 sit at room temperature for a few minutes to thoroughly dry to prevent residual wash solution from affecting the next step of the experiment.
10. Note: Residual ethanol in the wash solution can affect subsequent enzymatic reactions (restriction enzyme digestion, PCR, etc.).
11. Place the adsorption column CA5 into a clean centrifuge tube, add an appropriate amount of elution buffer TB to the center of the adsorption membrane and let it sit at room temperature for 2 minutes. Centrifuge at 12,000 rpm (approximately 13,400 ×g) for 2 minutes to collect the DNA solution. Note: The elution volume should not be less than 30 µl, as a smaller volume can affect the recovery efficiency. The pH value of the elution buffer has a significant impact on the elution efficiency. If sequencing is to be performed later, ddH2O should be used as the elution buffer, and its pH value should be maintained between 7.0-8.5. A pH value below 7.0 will reduce the elution efficiency; and the DNA product should be stored at -20°C to prevent DNA degradation. DNA can also be eluted with a buffer (10 mM Tris-Cl, pH 8.0).

4. Homologous Recombination

To ligate the target gene to the vector, adjust the reaction system to ensure both components are at the same concentration. For example, when the vector and gene concentrations are equal, prepare the system as follows: maintain a 1:2 molar ratio of linearized vector to insert fragment, dilute with ddH₂O to 5 µl, then add 5 µl of homologous recombination enzyme. Incubate at 50°C for 30 minutes.

5. Transformation

For general verification of whether the vector is successfully constructed, use DH5α, and for induced expression, use BL21(DE3) competent cells.

If commercial ones are not available, they can be prepared on-site:

1. Cultivate the original E. coli stock culture. Using a toothpick, transfer a single colony from the biosafety cabinet into 20 mL of LB medium (without antibiotics). Incubate at 37°C and 180 rpm for 5 hours until the liquid phase becomes turbid (measured OD value reaches 0.3–0.6).
2. Transfer 1 ml of the bacterial liquid into a 1.5 ml EP tube, centrifuge at 12,000 rpm for 1 minute at 4°C (use a refrigerated centrifuge), discard the supernatant (prepare as many centrifuged bacterial liquids as needed for the competent cells).
3. Add 1 ml of ice-bathed CaCl2 (calcium chloride concentration of 0.1 mol/l, i.e., dissolve 1.11 g of calcium chloride in 100 ml of sterile water) to each tube, gently mix, and ice bath for 30 minutes. (Be gentle, handle the bacterial liquid on ice)
4. Centrifuge at 12,000 rpm for 1 minute, invert to remove residual liquid, then add 100 µl of 0.1 mol/l calcium chloride, the competent cells are prepared, store at -80°C.
5. Add 50 ng of plasmid to the competent cells. If using PCR products, add 10 µl after homologous recombination. Ice-bathe for 30 minutes (thaw the competent cells on ice; maintain ice-bathed conditions throughout the procedure; ensure pipette tips remain below the liquid surface without touching the walls).
6. (Preheat the water bath in advance) Heat shock at 42°C for 60-90 seconds.
7. Quickly ice bath for 2-3 minutes (must be fast and the ice bath time should not be too long, usually take 2 and a half minutes as a compromise).
8. Add 1000 µl of LB medium, culture at 37°C at 180 rpm for 1 hour to recover.
9. Centrifuge at 12,000 rpm for 1 minute, discard the supernatant 800 µl, gently mix.
10. Take the entire resuspended bacterial suspension and spread it on LB medium with the corresponding antibiotics, spread evenly with a glass rod, seal (to prevent water loss), and culture upside down for 12-16 hours. (If it is a plasmid obtained through the PCR process, spread it all).

The entire transformation process must be carried out in a biosafety cabinet and an alcohol lamp must be ignited, the pipette tips, culture medium, and EP tubes must be sterilized.

Expression

1. Bacterial Culture and Target Protein Expression

1. Colony Picking: Inoculate 100ul glycerol stock culture into 20 mL of LB liquid medium with the appropriate antibiotic, incubate overnight at 37°C.
2. Expression:Add 500 µL antibiotic to 500 mL MR medium, then inoculate with 5 mL (1%) bacterial culture. Incubate at 37°C,for 2-3 hours until OD600 reaches 0.6.
3. Induction:Once the OD600 reaches the target range, induce protein expression according to the medium used:
   • For MR Medium: Add 400 µL of 0.5M IPTG (to a final concentration of 0.4 mM).Add 3.75 mL of 200 mg/mL L-arabinose (to a final concentration of 1.5 mg/mL). Incubate at 30°C, 200 rpm for 20 hours.
   • For TB Medium: Add 5400 µL of 0.5M IPTG (to a final concentration of 0.54 mM). Incubate at 20°C, 200 rpm for 20 hours.

Synthesis of PEA

1. Colony Picking: Inoculate 100ul glycerol stock culture into 20 mL of LB liquid medium with the appropriate antibiotic, incubate overnight at 37°C.
2. Expression:Add 500 µL antibiotic to 500 mL MR medium, then inoculate with 5 mL (1%) bacterial culture. Incubate at 37°C,for 2-3 hours until OD600 reaches 0.6.
3. Induction:Add 400 µL(final concentration:0.4mM) of 0.5M IPTG(IPTG supplementation was required only for the PEA02-BL2112 (DE3) group), 3.75ml (final concentration:1.5mg/ml) of 200mg/ml L-arabinose, 25ul of 200 mg/mL Thiamine, 2.5 ml of a 400mg/mL solution of each monomer(sodium(RS)-3-hydroxybutyrate and 3-Aminopropanoic acid, monomer supplementation was required only for the PEA01-BL2112 (DE3) group), and incubate at 30°C, 200 rpm for 96 hours.

2. Bacterial Culture Harvesting and Pellet Resuspension

1. Take four 50 mL centrifuge tubes, and add 50 mL of bacterial culture to each tube, balancing the tubes appropriately.
2. Centrifuge at 3500 rpm, 4℃ for 15 minutes.
3. Discard the supernatant, and resuspend the bacterial pellet in each tube with 5-10 mL of PBS buffer. Combine the resuspended cultures into a single centrifuge tube, and store at -20°C.
4. Depending on the total volume of the bacterial culture (e.g., 400 mL), repeat the above steps as necessary.

Verification

1. Bacterial Lysis

1. Transfer 20-50 mL bacterial culture to a 50 mL beaker.
2. Place the beaker in a larger beaker filled with ice, keeping the ice 1-2 cm below the small beaker's rim to prevent water backflow.
3. Sonicate at 170-190W, 1s on/1.5s off, for 15 minutes (avoid excessive agitation or foaming).
4. Stop when the liquid turns clear and less viscous; if not, extend by 5-10 minutes.
5. Centrifuge lysate at 12,000 rpm for 1.5 hours.
6. Collect supernatant into a 50 mL centrifuge tube.

2. Nickel Column Purification

1. Remove nickel column from 4°C, drain ethanol solution.
2. Equilibrate column with 1× PBS buffer three times.
3. Equilibrate with 10 mM imidazole solution.
4. Add 10-15 mL supernatant to column, invert to mix, incubate on ice for 45 minutes.
5. Attach tubing, secure column, and collect flow-through.
6. Wash with 30 mM imidazole buffer at 2-3 drops/sec.
7. Test impurities with Coomassie Brilliant Blue; continue washing if blue.
8. Elute protein with 300 mM imidazole, incubate 5 minutes, and collect in 2 mL tubes.
9. Repeat elution if strong fluorescence; collect 15-20 mL, store at 4°C.
10. Wash column with 1× PBS, store in 20% ethanol at 4°C.

3. Coomassie Brilliant Blue Assay for Protein Concentration

1. Standard Curve Preparation:Mix 1 mL Coomassie Brilliant Blue solution with 20 µL protein standard. After 1.5 minutes, measure absorbance at OD595 to create the standard curve.
2. Sample Measurement:Mix 1 mL Coomassie Brilliant Blue solution with 20 µL protein sample. After 1.5 minutes, measure OD595, and use the standard curve to determine protein concentration.

4. SDS-PAGE

4.1 Preparation of polyacrylamide gels

1. Installation of the gel board model: Wash the glass board, dry it and set it aside. When making glue, choose the right glass plate, assemble the glue plate model, put the glass plate into the rack after alignment, and the clamps on both sides are tightened, so that the short glass is leaning out and the long glass is leaning in.
2. According to the following table formula configuration of the separation of the glue solution, mixing with a light hand shaking, carefully inject the mixture into the prepared glass plate gap, leaving enough space for the concentration of the glue (~ 2.5cm), gently in the top layer of the addition of 0.5ml of deionized water to cover the (liquid sealing should be slow, to avoid deformation of the glue is washed out), in order to stop the inhibition of the air oxygen on the coagulation. Just add water can be seen between the water and the gel liquid interface, and then gradually disappear, soon appeared again interface, which indicates that the gel has been polymerized. And then leave a few moments to make the polymerization complete, the whole process takes about 30 minutes (25 ℃ room temperature).

Separation gel concentration	Gel volume	Volume of each component required(in ml)
		H2O	30%Acr-Bis（29：1）	SDS-PAGE Separating Gel Buffer(4×）	10%APS	TEMED
6%	5ml	2.75	1.0	1.25	0.05	0.004
8%	5ml	2.42	1.33	1.25	0.05	0.003
10%	5ml	2.08	1.67	1.25	0.05	0.004
12%	5ml	1.75	2.0	1.25	0.05	0.002
15%	5ml	1.25	2.5	1.25	0.05	0.002

3. The preparation of 5% concentrated gel: the first has been polymerized to separate the upper layer of the gel water suction, and then use filter paper to absorb the residual water. Prepare the concentrated gel solution according to the following formula(Take preparing a piece of glue, about 2 mL, as an example). Mix it and inject it into the upper end of the separation gel, insert the comb and be careful to avoid air bubbles.

Reagent	Volume(mL)	Purpose
H₂0	1.4	Solvent
30% Acrylamide/Bis Solution (29:1)	0.33	Gel matrix formation
0.5 M Tris-HCI (pH 6.8)	0.25	Provides stacking pH environment
10% (w/v) SDS	0.02	Denaturant and charge provider
10% (w/v) Ammonium Persulfate (APS)	0.02	Polymerization initiator (free-radical source)
TEMED	0.002	Polymerization catalyst

4. After the polymerization of concentrated gel is complete, put the gel template into the electrophoresis tank and fix it, with the small glass plate facing inward and the large glass plate facing outward, add 1× electrophoresis buffer to both the upper and lower tanks, and carefully pull out the comb, check for leakage, and remove away air bubbles at the bottom of the gel between the two glass plates.

4.2 Sample Preparation and Loading

1. While the gel concentrate is being polymerized, mix the protein sample with an equal volume of 4× sample buffer, denature in a water or metal bath at 95°C for 10 min, and cool to room temperature for use.
2. Using a micro-syringe, add the standard solution and denatured protein sample (typically 10–20 μL) to the bottom of the sample well.

4.3 Electrophoresis and Staining

1. Connect the power supply. Use a constant voltage of 80-100V during the concentration gel stage. When the bromophenol blue band enters the separation gel, increase the voltage to 120-150V.
2. When the bromophenol blue band migrates to approximately 0.5 cm from the bottom of the gel, turn off the power supply to stop electrophoresis.
3. Carefully pry open the glass plate, remove the gel, and trim off the concentrating gel. Place the gel into a container, add sufficient Coomassie Brilliant Blue staining solution, and slowly agitate on a shaker for 1-2 hours.
4. Drain the staining solution (reusable).
5. Add sufficient decolorizing solution and decolorize on a shaker, replacing the solution several times until the background is transparent and protein bands are clearly visible.

5. Preparation of Bacterial Lyophilized Powder Using a Freeze Dryer

1. After 96 hours of induction, centrifuge to collect the bacterial cells. Discard the supernatant completely. Resuspend the bacterial pellet in pre-chilled 1x PBS buffer, gently pipette to ensure uniformity, and prepare a high-concentration bacterial suspension.
2. In a laminar flow hood, aliquot the bacterial suspension into sterile lyophilization vials at 0.5–1.0 mL per vial. Avoid touching the vial walls during aliquoting.
3. Immediately transfer the aliquoted vials (opening facing upward) to a -80°C ultra-low temperature freezer. Freeze for at least 4 hours or overnight to ensure complete solidification of the samples.
4. Pre-cool the freeze dryer to below -50°C.
5. Swiftly remove pre-frozen samples from the freezer and immediately transfer them to the freeze dryer's cold trap shelf. This process must be rapid to prevent sample thawing.
6. Cover with the glass dome and activate the vacuum pump. Freeze dry at -50°C for 24 to 36 hours.
7. Upon completion of freeze-drying, promptly remove the samples from the freeze dryer. Immediately seal the vial opening with a sterile rubber stopper in a laminar flow hood, then firmly press the aluminum cap to achieve a vacuum seal. Store the sealed bacterial lyophilized powder sample tubes at 4°C in the dark and label them accordingly.

6. Purification of PEA

1. Mix the freeze-dried bacterial cells with an appropriate amount of chloroform. Incubate at 45°C with shaking for 3-4 hours to fully extract PEA from the cells.
2. Filter the chloroform extract through a 0.45 μm membrane filter to remove bacterial debris and other insoluble impurities. Collect the clear filtrate.
3. Place the filtered, clear chloroform extract in a fume hood. Allow the chloroform to evaporate naturally under airflow until the solution concentrates to approximately 1/10 of its original volume or until viscous material precipitates.
4. Add 100 μl of chloroform to resuspend the precipitate, then add 1 ml of methanol. Centrifuge at 12,000 rpm for 10 minutes. Discard the supernatant. After the methanol has completely evaporated, resuspend the pellet in 500μl of PBS buffer.

7. Nile Red Staining

1. Thoroughly resuspend the purified PEA precipitate in 1 mL of PBS buffer by pipetting. Perform a serial dilution of this PEA suspension using PBS buffer to prepare sample suspensions with optical densities (OD₆₀₀) of 1.0, 0.5, and 0.3 at 600 nm.
2. Transfer 1 mL of each PEA sample suspension (OD=1.0, 0.5, 0.3) into separate 1.5 mL centrifuge tubes.
3. Precisely add 3 μL of Nile Red stock solution to each tube.
4. Immediately vortex to mix thoroughly, maintaining complete protection from light throughout (e.g., wrap tubes in aluminum foil).
5. Place the light-protected tubes on a shaker and incubate at 25°C under dark conditions at 200 rpm for 20 minutes to ensure complete dye binding to PEA.
6. After incubation, centrifuge tubes at 12,000 rpm for 1 minute to precipitate PEA. Carefully discard the supernatant (containing unbound free dye).
7. Add 1 mL of pre-chilled PBS buffer to each tube containing the precipitate. Gently resuspend the pellet by pipetting or vortexing to ensure complete and uniform dispersion.
8. Transfer the resuspended sample to a cuvette for detection using a fluorometer. Set the excitation wavelength (Ex) to 530 nm and measure the fluorescence emission intensity at 610 nm (emission wavelength Em). Simultaneously measure the fluorescence value of 1 mL PBS buffer alone as a blank control.

8. FTIR

Sample Preparation: Take approximately 1–2 mg of purified and dried PEA sample (white powder or film form). Ensure the sample is dry with no moisture or solvent residues.

9. TEM

Sample Preparation Before Submission:

1. Take a sample approximately the size of a mung bean and place it in a 1.5 mL centrifuge tube.
2. Add an appropriate amount of 2.5% glutaraldehyde solution and gently pipette to mix.
3. Take 500 μL of the above preliminarily fixed sample suspension and add it to a centrifuge tube containing 9.5 mL of PBS. Securely cap the tube and gently invert it 10 times to ensure thorough mixing.
4. Tightly wrap the tube cap and body junction with sealing film to prevent leakage during transport.
5. Place the sealed tube upright in a dedicated foam box or sturdy cardboard box. Surround the box with pre-frozen ice packs to maintain a transport temperature of approximately 4°C.

10. Purification of K5C via Inverse Transition Cycle (ITC)

1. After sonication of cells, centrifuge the lysate at 4°C and 10,000 rpm for 30 minutes. Collect the supernatant.
2. Add NaCl to the supernatant to a final concentration of 2 M.
3. Incubate the solution at 35°C for 1 hour to induce the K5C phase transition.
4. Centrifuge at 10,000 rpm, 35°C for 15 minutes. Retain the supernatant for analysis and collect the K5C-containing pellet.
5. Resuspend the pellet in pre-chilled buffer (200 mM PB buffer, pH 7.0) and incubate on ice for 30 minutes to solubilize K5C.
6. Incubate the solubilized mixture at 35°C for 1 hour, then centrifuge at 10,000 rpm and 35°C for 15 minutes. Resuspend the pellet in pre-chilled buffer and incubate on ice for 1 hour to dissolve.
7. After final dissolution, centrifuge at 4°C and 10,000 rpm for 15 minutes. Collect the supernatant containing purified K5C; the pellet may be retained for subsequent analysis.

11. Bionic Mineralization for Preparing K5C@Fe₃O₄ Carrier

1. Prepare precursor solution A (2 mL): Dissolve 0.16 g FeCl₃ and 0.65 g FeCl₂·4H₂O in ultrapure water, then mix with purified K5C protein to a final concentration of 900 μg/mL.
2. Prepare precursor solution B (5 mL): 3.0 M NaOH solution.
3. Slowly add solution B to solution A under gentle shaking (100 rpm), ensuring the pipette tip remains below the liquid surface to avoid localized high pH.
4. Incubate the mixture by shaking at 35°C and 180 rpm for 30 minutes. Observe the solution darkening, indicating Fe₃O₄ nanoparticle formation.
5. Attach a strong magnet to the outer wall of the reaction vessel and separate the K5C@Fe₃O₄ nanoparticles via magnetic force.
6. Discard the supernatant and wash the nanoparticles with an excess of buffer (200 mM PB buffer, pH 7.0). Repeat the magnetic separation and washing steps at least twice to remove unbound components.
7. Suspend the final K5C@Fe₃O₄ nanoparticles in suitable buffer (200 mM PB buffer, pH 7.0) and store at 4°C for later use.

12. Covalent Immobilization of Enzymes and PET Degradation Detection

1. In a 1 mL reaction system, add 250 μL of SpyTag-TfCa-SnoopCatcher enzyme solution to 500 μL of pre-prepared bioactive magnetic bead suspension. Incubate at 37°C with shaking at 180 rpm for 4 hours to achieve covalent immobilization of TfCa.
2. After immobilization, wash with PB buffer (200 mM, pH 7.0). Use a strong magnet to adhere the beads to the tube wall, then carefully discard the supernatant. Wash the beads three times to remove unbound enzyme.
3. Add 250 μL of SnoopCatcher-Tfh enzyme solution to the magnetic beads and incubate for 4 hours under the same conditions (37°C, 180 rpm) to immobilize the second enzyme.
4. After the second immobilization, wash the beads three times with PB buffer. Centrifuge at 12,000 rpm for 1 minute and carefully discard the supernatant.
5. Resuspend the final immobilized enzyme complex in glycine buffer (pH 9.0) to a final volume of 1 mL.
6. Add 10 mg of solid PET microplastics to the reaction mixture and incubate with shaking at 40°C for 24 hours.
7. After incubation, centrifuge at 12,000 rpm for 2 minutes and collect the supernatant for subsequent product analysis.

13. HPLC

Prior to HPLC analysis, samples require thermal treatment. Heat the sample solution at 90°C for 15 minutes to promote compound dissolution and reaction. Following thermal treatment, centrifuge at 12,000 rpm for 20 minutes to precipitate insoluble material. Carefully collect the supernatant and perform high-performance liquid chromatography (HPLC) analysis using the Waters E2695 system to quantitatively determine compound concentrations.

13.1 HPLC analysis

A Waters E2695 chromatographic system, equipped with a Shim-pack GIS column, was employed in this study for HPLC analysis. The mobile phase was comprised of a mixture of methanol and acetonitrile, flowing at a rate of 0.5 mL/min, with detection at a wavelength of 254 nm. The elution protocol commenced with a gradient elution using 95% formic acid and 5% from 0 to 2 minutes. This was followed by a linear gradient elution, increasing the acetonitrile concentration to 20% while maintaining 80% formic acid from 2 to 14 minutes. The system was then re-equilibrated to the initial conditions of 95% formic acid and 5% acetonitrile over the 14 to 15-minute interval, employing a gradient pump mode throughout the entire chromatographic run. The total peak areas of MHET, terephthalic acid (TPA) and PCA were utilized in determining the quantity of product from each PET hydrolysis reaction.

13.2 Standard Curve of HPLC analysis

Prepare stock solutions of tetradecanoylphorbol acetate (TPA), p-coumaric acid (PCA) and methyl 4-hydroxy-3-methoxyphenyl ether (MHET) at a concentration of 10 mg/mL, with TPA specifically dissolved in dimethyl sulfoxide (DMSO). For the construction of a standard curve, dilute these stock solutions to prepare a series of mixed solutions with final concentrations of 0.05, 0.1, 0.25, 0.5, 1, and 2 mM. Subject the mixed solutions to thermal treatment at 90°C for 15 minutes to facilitate compound solubilization and reaction. Post-thermal treatment, centrifuge the solutions at 12,000 rpm for 20 minutes to pellet any insoluble material. Carefully collect the supernatant and proceed with high-performance liquid chromatography (HPLC) analysis using a Waters E2695 system to quantify the compounds and establish the standard curve.

14. Ethylene glycol（EG）detection

14.1 Preparation of Gold Nanoparticles

1. Mix 1 mL of chloroauric acid with 49 mL water, then heat to boiling or until bubbles form.
2. Add 2 mL of 1% trisodium citrate (pre-prepared) all at once. Insert a stirring bar and heat for 30 minutes.
3. Turn off heat and stir for 15 minutes.
4. Store at 4°C.

14.2 EG Detection

1. Prepare a series of EG solutions from the 1 M stock with final concentrations of 0, 10, 20, 30, 40, 50, 100, and 150 mM.
2. Mix 0.8 mL of the gold nanoparticle solution with each prepared EG solution.
3. Record the color and spectral data using a sensor and a UV-vis spectrophotometer.