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 Medium

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

1.4Kana⁺Amp⁺Glucose LB:

The LB medium was configured as follows. To this solution, glucose was added to achieve a final concentration of 20 mM in the 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 → Save

Detailed steps:

• Calculation of substance requirements based on the volume of medium required.
• Weigh the substances according to the calculation.
• Add ddH₂ O to fix the volume and stir well to dissolve it.
• Autoclave the conical vials after closing the mouth with a breathable parafilm and a leather band.
• Kanamycin and benzylpenicillin should be added to the medium at a concentration of 1:1000 at a temperature of approximately 65°C, and the solution should be thoroughly agitated.
• 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.
• Sign the signal and 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.

1.5 Kana⁺Amp⁺L-arabinose LB:

The LB medium was configured as follows. 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 → Save

Detailed steps:

1. 1. Calculation of substance requirements based on the volume of medium required.
2. 2. Weigh the substances according to the calculation.
3. 3. Add ddH₂O to fix the volume and stir well to dissolve it.
4. 4. Autoclave the conical vials after closing the mouth with a breathable parafilm and a leather band.
5. 5. Kanamycin, benzylpenicillin and L-arabinose should be added to the medium at a concentration of 1:1000 at a temperature of approximately 65°C, and the solution should be thoroughly agitated.
6. 6. 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.
7. 7. Sign the signal and 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.

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

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. 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 1M 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

Breed Preservation

1. Glycerol Stock

1. Prepare 50% Glycerol Solution:Mix equal volumes of 100% glycerol and dH₂O, then 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, 0.5 µl upstream primer, 0.5 µl downstream primer, 1 µl template, 10.5 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.

Y is the extension time, determined by the length of the PCR fragment, usually 30s/1Kb. For example, a 6300 bp (6.3 Kb) fragment should be set to 3:30.

2. Nucleic Acid Gel Electrophoresis

Prepare the nucleic acid gel: Remember the ratio 1:10:100 (0.8g agarose: 8 µl nucleic acid dye: 80 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 5 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 xg) 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 xg) 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 xg) 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 xg) 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

If it is simply to delete a certain site, add 5 µl gel recovery product: 5 µl homologous recombination enzyme. If it is to connect the target gene with the vector, adjust the system according to the concentration to make the concentrations of the two the same, for example, if the vector and gene concentrations are the same, prepare 2.5 µl vector gel recovery product: 2.5 µl target gene gel recovery product: 5 µl homologous recombination enzyme. Set the PCR program to react at 50°C for 30 minutes, then save at 4°C indefinitely. Note that this is a 10 µl system, remember to select the 10 µl system when setting up the PCR.

5. Transformation

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

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

1. Cultivate the original MC1061 bacterial liquid, use a toothpick to pick a single colony from the biosafety cabinet into 20 ml LB (without antibiotics), culture at 37°C at 180 rpm for 5 hours until it becomes turbid (measure the OD value to be 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 1 µl of plasmid (constructed plasmid, such as the sample returned by the company) to the competent cells, if it is a PCR product after homologous recombination, add 10 µl, ice bath for 30 minutes (let the competent cells melt on ice, operate on ice throughout the process, the pipette tip should be below the liquid surface but not touch the wall).
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 900 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 900 µl, gently mix.
10. Take 20-30 µl 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: Pick a single colony from the transformation plate into 20 mL of liquid medium with kanamycin, incubate overnight at 37°C.
2. Expression:Add 400 µL kanamycin to 400 mL TB medium, then inoculate with 6-8 mL (2%) bacterial culture. Incubate for 3-4 hours until OD600 reaches 0.8.
3. Induction:Add 400 µL (1%) of 0.5 M IPTG and incubate at 16°C, 180-200 rpm for 24 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 4500 rpm 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. SDS-PAGE

Polyacrylamide Gel Electrophoresis (PAGE) is a method of electrophoresis in which a polyacrylamide gel is used as a support medium for the separation of proteins or nucleic acids. Polyacrylamide gel is made of acrylamide monomer (ACR) and cross-linking agent N,N-methylene bisacrylsmide (N,N-methylene bisacrylsmide abbreviated as BIS) in the role of catalyst polymerization and cross-linking of three-dimensional mesh structure of the gel. By varying the ratio of monomer concentration to cross-linking agent, gels with different pore sizes can be obtained, which can be used to separate substances with different molecular weights. We used SDS-PAGE to verify the expression of the imported proteins.

2. 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 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. Mix it and inject it into the upper end of the separation gel, insert the comb and be careful to avoid air bubbles.
4. While the gel concentrate is being polymerized, mix the protein sample with an equal volume of 4X sample buffer, denature in a water or metal bath at 95°C for 10 min, and cool to room temperature for use.
5. 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 1X 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.

3. Anchor Peptide Purification

3.1 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/3s off, for 40 minutes (avoid excessive agitation or foaming).
4. Stop when the liquid turns clear and less viscous; if not, extend by 5-10 minutes.

3.2 Obtaining GFP-Anchor Peptide

1. Centrifuge lysate at 12,000 rpm for 1.5 hours in 2 mL tubes (ensure balance).
2. Collect supernatant into a 50 mL centrifuge tube.

Nickel Column Purification:

1. Remove nickel column from 4°C, drain ethanol solution.
2. Equilibrate column with 1x PB 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 1x PBS, store in 20% ethanol at 4°C.

3.3 Anchoring peptide aquisition

Enzymatic Cleavage with TEV Protease:

Use the TEV protease kit (P2308, Beyotime Inc.) and prepare the cleavage system according to the specified ratios.

- Place the reaction system in a 4°C refrigerator for 24 hours.

Ni²⁺-NTA Column Purification:

1. Remove Ni²⁺-NTA Column from 4°C, secure vertically, and drain ethanol.
2. Wash column with 1x PB buffer three times to equilibrate.
3. Fill with 10 mM imidazole solution, drain to remove non-specific bindings.
4. Close ports, add cleavage solution, invert to mix, incubate on ice for 10 minutes.
5. Open ports, collect eluted target protein.
6. Add 15 mL of 1 M imidazole, invert to dissociate residual proteins, then drain.

Ni²⁺-NTA Column Storage:

- Wash with 1x PB buffer, add 20% ethanol, and store at 4°C.

3.4 Coomassie Brilliant Blue Assay for Protein Concentration:

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.

3.5 Determination of Protein Concentration Using BCA Assay Kit

Dilution of Standards:

Dilute the BSA standards with the same buffer system as the samples according to the following table:

Preparation of BCA Working Solution

Based on the number of samples, prepare an appropriate amount of BCA working solution by mixing reagent A and reagent B in a volume ratio of 50:1, and mix well.

Note: Before preparing the BCA working solution, please shake reagent A well to mix.

Standard Colorimetric Cup Method:

1. Take 0.1 ml of each standard and sample in appropriate tubes.
2. Add 2.0 ml of BCA working solution, mix thoroughly.
3. Cap and incubate at 37°C for 30 minutes, then cool to room temperature or place at room temperature for 2 hours.
4. Measure the absorbance at 562 nm using a UV spectrophotometer.
5. Calculate the protein concentration in the samples based on the standard curve.

Microplate Method:

1. Take 25 μl of freshly prepared BSA standard solution and sample from the table and add to a 96-well plate.
2. Add 200 μl of BCA working solution to each well and mix well.
3. Cap and incubate at 37°C for 30 minutes, then cool to room temperature or place at room temperature for 2 hours.
4. Measure the absorbance at 562 nm using a UV spectrophotometer.
5. Calculate the protein concentration in the samples based on the standard curve

4. Affinity validation of the anchored peptide

1. Measure the concentration of the four purified EGFP-anchor peptides using the Coomassie Brilliant Blue method and dilute them to the same level.
2. Mix 200 µL of EGFP-anchor peptide solution with 200 µL of 100 µg/mL PS/PLA/PET/PP solution (plastic particle size 8-13 µm, solvent is pure water).
3. Incubate at 4°C for 15 minutes.
4. Centrifuge at 12,000g for 2 minutes and discard the supernatant to remove unreacted anchor peptides.
5. Resuspend the pellet in 100 µL of PB buffer.
6. Prepare slides and observe under an inverted fluorescence microscope, capturing both fluorescence and bright-field images.

5. Colorimetric identification of plastics

5.1 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. Aliquot into 50 mL tubes, centrifuge at 10,000 rpm for 1 hour, discard supernatant, and resuspend gold nanoparticles in glycine buffer (pH 8.5).
5. Store at 4°C.

5.2 Gold Nanoparticle Modification

1. Mix 1 mL of gold nanoparticles with 30 µL of anchor peptide and stir. Incubate at room temperature for 10 minutes to prepare the anchor peptide-gold nanoparticle solution.

Note: For larger volumes, adjust proportions accordingly. Prepare fresh and use immediately.

5.3 Microplastic Detection

1. Prepare a plastic gradient solution (2, 4, 6, 8, 9, 10 µg/mL) using glass test tubes for each concentration.
2. Mix 100 µL of the prepared plastic gradient solution with 1 mL of anchor peptide-gold nanoparticle solution. Incubate at room temperature for 10 minutes.
3. Record the color and spectral data using an RGB sensor and UV-Vis spectrophotometer.

6. High-Performance Liquid Chromatography Analysis

6.1 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), PCA and Vanillin were utilized in determining the quantity of product from each PET hydrolysis reaction.

6.2 Standard Curve of HPLC analysis

Prepare stock solutions of tetradecanoylphorbol acetate (TPA), p-coumaric acid (PCA), vanillin 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.

Degradation

6.3 PET Degradation:

PET powder (1000 mesh, granular form) was sourced from a commercial supplier. Precisely 0.4 grams of this PET powder was measured and transferred into a 50 mL conical flask. Thereafter, 10 mL of bacterial culture was inoculated into the flask. The culture was derived from a homogenous batch of bacteria that had been expressed and subjected to centrifugation at 4500 rpm for a period of 20 minutes to pellet the cells. Post-centrifugation, the bacterial pellet was resuspended in a series of buffer solutions with varying pH levels (pH = 7 - 10) to adjust the optical density (OD600) to the desired values. The flask containing the mixture was then sealed with a sterile membrane to maintain sterility and integrity of the reaction environment. The sealed flask was placed on a constant temperature shaker, which was set to oscillate at a speed of 180 rpm for a continuous period of 24 hours. The enzymatic reaction was terminated by subjecting the flask to a thermal treatment at 95°C for a duration of 15 minutes.

6.4 TPA Degradation

Resuspend the E. coli BL21 (DE3) strain, which has undergone triple plasmid co-transformation and is induced with 0.4 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 24 hours, in 3 mL of M9 medium to achieve an optical density at 600 nm (OD600) of 65. Then, incorporate 100 µL of n-butanol and 100 µL of a 0.12 mol/L terephthalic acid (TPA) standard solution in dimethyl sulfoxide (DMSO). Subsequently, adjust the volume to 10 mL with a 50 mM glycine buffer prepared at a pH of 8.5. Cultivate the assay mixture at a temperature of 20°C by shaking at a speed of 180 revolutions per minute (rpm) for a continuous period of 24 hours. Finally, terminate the enzymatic reaction by thermal inactivation at 95°C for 15 minutes.

Gene editing

1. Plasmid Construction Workflow for PHCY-yahK-KO(piGEM24_09)

The sgRNA sequence designed on the Benchling platform was introduced into the N20 site of the PHCY-26D plasmid through PCR and seamless ligation. The constructed plasmid was subsequently verified by sequencing. The verified plasmid was then linearized and seamlessly ligated with the HA1/2 fragments, which were obtained via colony PCR and overlap PCR, to generate the PHCY-yahK-KO(piGEM24_09) plasmid. The successful construction was confirmed by sequencing.

2. YahK Gene Knockout Procedure

For the YahK gene knockout, PHCY-25A and piGEM24_09 plasmids are used, and the strain employed for editing is Escherichia coli BL21(DE3).

1. Preparation of Competent Cells and Transformation with PHCY-25A Plasmid:Prepare chemically competent cells of E. coli BL21(DE3) and transform them with the PHCY-25A plasmid. The resulting strain is E. coli BL21(DE3)/PHCY-25A.
2. Preparation of Competent Cells of the Transformed Strain:The E. coli BL21(DE3)/PHCY-25A strain is prepared as chemically competent cells. The medium should contain kanamycin (Kan) and glucose.
3. Transformation with piGEM24_09 Plasmid:Transform the piGEM24_09 plasmid into the E. coli BL21(DE3)/PHCY-25A competent cells. Plate the transformants on LB agar plates containing ampicillin (Amp), kanamycin (Kan), and glucose, and incubate at 30°C.
4. Colony Growth and Induction:Once colonies have grown, select 3-4 colonies from the plate and inoculate them into 2 ml of LB medium. Add 2 μl of ampicillin (100 mg/ml) and 2 μl of kanamycin (50 mg/ml), and incubate at 30°C for 2 hours. Then, induce with 20 μl of IPTG (100 mM) and incubate at 30°C for 1 hour, followed by 40 μl of L-arabinose (1 M), incubating for an additional 3 hours at 30°C.
5. Dilution and Plating for Colony Screening:Dilute the culture 100-fold and 1000-fold. Plate 100 μl of the diluted cultures on LB agar plates containing ampicillin (Amp), kanamycin (Kan), and L-arabinose. Incubate at 30°C. After colonies grow, confirm successful knockout by PCR verification.

PCR validation was performed using the primers Test-KO-F and Test-KO-R, as shown in Figures 1 and 2. In the wild-type (WT) group, the PCR amplification product before knockout was 2219 bp, while the knockout (KO) group showed a PCR product of 1169 bp after successful gene deletion. Therefore, the success of the knockout can be determined based on the length of the PCR products.

3. Plasmid Curing Procedure

Inoculate a colony confirmed to have a successful knockout into 2 ml of LB medium without antibiotics, and incubate overnight at 37°C. Then, streak 2 μl of the culture onto an LB plate containing 1% sucrose, and incubate at 37°C. Most of the colonies that grow on the plate will have lost the pHCY-25A and piGEM24_09 plasmids.

To verify plasmid curing, select a colony and inoculate it into three different LB media: one without antibiotics, one with ampicillin (Amp⁺), and one with kanamycin (Kan⁺). Incubate all three tubes at 37°C. If the colony grows in the antibiotic-free medium but not in the Amp⁺ or Kan⁺ media, it confirms the loss of both the pHCY-25A and piGEM24_09 plasmids. The culture in the antibiotic-free medium can then be used for subsequent experiments.