After identifying the genes and vectors required for our project, we constructed different plasmids—pET21a-Mfp3 and pET21a-Mfp5—by designing specific primers and selecting homologous recombination strategies for each. We amplified relevant fragments via PCR and ligated them into linearized plasmid vectors. Positive clones were identified and selected using colony PCR and Sanger sequencing. Following liquid culture and induction of target protein expression, we assessed hydroxylation reactions using the NBT staining method, adhesion via Coomassie Brilliant Blue staining, and performed pipette tip adhesion tests to determine the final adhesive proteins.

For antimicrobial peptide screening, we synthesized corresponding peptides directly based on their amino acid sequences and carried out subsequent antimicrobial activity assays. Additionally, to accommodate codon usage preferences in the E. coli expression system, we designed and optimized the nucleic acid sequences.

For the expression of fusion proteins, we constructed plasmid DNA using homologous recombination, induced expression and purified the fusion proteins, then conducted functional assays including adhesion tests (Coomassie Brilliant Blue staining and pipette tip adhesion assays), antifouling tests, and antimicrobial tests.

1. Label the centrifuge tube with the correctname, Transfer the cultured bacterial into 2 ml EP，Place the labelled tubes into a centrifuge. Set the centrifuge at appropriate parameters (12000 rpm, 1 min)
2. Remove the supernatant using a 1ml volume pipette leaving a bacterial precipitation inside the labelled tubes. Repeat the above steps 1 and 2, and continue until all the bacterial solutions have been centrifuged and collected.
3. Add 250 μL of buffer P1 into labelled tubes using a 500μL volume pipette. And fully suspense the bacterial precipitate in buffer P1 until uniform turbid solution (bacterial suspension).
4. Using a 500μL volume pipette, add 250 μL of buffer P2 reagent into each labelled tubes. Gently and attentively rotate the tubes perpendicularly 7-10 times and 3-4 minutes.
5. Using a 500μL volume pipette, add 350μL of P3 reagent into labelled tubes. Gently and attentively rotate the tubes perpendicularly 7-10 times.
6. Place the labelled tubes into a centrifuge, set at appropriate parameter (12000 rpm 10 min). After centrifuge, using a 1ml volume pipette, transfer all the supernatant in the labelled tubes into a Spin Column, then placed inside a collection tube. Place the collection tube with a spin column in the centrifuge, set at appropriate parameters (12000 rpm, 1 min)
7. After centrifugation, discard the waste liquid in the collection tube. Using a 1mL volume pipette, add 600μL of PW2 Wash buffer into the spin column. Centrifugation, at 12,000 rpm, for 1 minute. Repeat this operation once.
8. Centrifuge the centrifugal column tube and collection tube at 12000 revolutions per minute for 1 minute to completely remove the corresponding residual liquid. After centrifugation, transfer the column tube to a new 1.5-milliliter centrifuge tube, open the cap and let it stand at room temperature for 2 minutes to fully evaporate the residual liquid.
9. Using a 200μL volume pipette, add 65 μL of Elution buffer on the surface of filtering film of the spin column. Centrifuge at 12000 rpm for 2 minutes, and obtain the final plasmid DNA solution.
10. Through Nanodrop analysis, the purity and concentration of the plasmid DNA were determined.

1. Take out the required reagents: plamid sample, forward primer, reverse primer, mix dNTP, and ddH₂O. Place all reagents in an ice box and operate at low temperature.
2. Use a pipette to add samples in the following order: total PCR reaction volume is 50 uL

20 μL ddH₂O

25 μL mix dNTP

2 μL forward primer

2 μL reverse primer

3 μL sample plasmid

3. After adding each component, gently pipette up and down to mix thoroughly without creating bubbles. Put the PCR tube into the PCR instrument, setting up PCR amplifying condition.

Pre-denaturation: 95 ℃, 5 min;

Denaturation: 95 ℃, 15 s;

Annealing: 55 ℃, 15 s;

Extend: 72 ℃, 1 min

Over extension: 72 ℃， 5min

Incubation/ Store: 4 ℃

4. Start the PCR program and allow it to run until completion

1. Preparing 1% agarose gel:
2. Weigh out the 2g agarose powder, dissolve into 200mL 1*TAE buffer in a flask.
3. Heat the misxture in a microwave about 3 minutes until the agarose is completely dissolved. Pause mintues to make it cool.
4. After cooling, add 20μL of nucleic acid dye (1:10000) into the cooled agarose solution and gentlely mix.
5. Pour the agarose solution into the gel casting tray with the comb, and wait for it to solidify about 20 minutes.
6. Take each of PCR product and mix with 6× DNA loading buffer, gently mix well.
7. Slowly pipette each sample mixture into the small holes of the gel.
8. Add DNA marker into the leftmost hole as a reference.
9. Set the electrophoresis condition (120V, 30min).
10. After electrophoresis, observe the gel by using an ultraviolet imaging system.

1. Use pipette to transfer the PCR product to a 1.5mL centrifuge tube. Add ddH₂O to make the volume reach 100μL.
2. Add 300μL of buffer GDP, then invert the mixture several times to ensure thorough mixing.
3. Transfer the entire mixture to the spin column.
4. Centrifuge at 12000 rpm for 1minutes.
5. Pour out the waste liquid, keep the column and put it back into the collection tube.
6. Add 700μL wash buffer GW to the column.
7. Centrifuge at 12000 rpm for 1minutes.
8. Pour out the effluent and repeat the washing steps once.
9. Centrifuge again for 2 minutes to ensure that all residual ethanol is removed. Let it stand at room temperature for 1minute to fully evaporate the residual ethanol.
10. Transfer the purification column to a new 1.5 mL centrifuge tube.
11. Slowly add 20μL of elution buffer to the column center. Let it stand at room temperature for 2minutes.
12. Centrifuge at 12000 rpm for 1minute and collect the eluent (the purified PCR product).

1. The DNA seamless cloning technology enables the rapid and precise insertion of fragments into any position on any vector. By linearizing the vector in any way, the 5' ends of the primers used for reverse/forward amplification of the insertion fragment are introduced into the terminal sequence of the linearized vector, ensuring that the 5' and 3' ends of the PCR product respectively carry sequences (15 - 20 bp) consistent with the two terminal sequences of the linearized vector. After mixing the PCR product with the linearized vector in a certain proportion and catalyzed by the recombinase, the transformation can be completed within 5 - 15 minutes at 50°C, thus achieving the targeted cloning.
• Prepare the following reaction system on the ice:
• 2* ClonExpress Mix: 5uL
• Linear vector: 0.03 pmol
• Insert fragment: 0.06pmpl
• ddH₂O: to 10 uL
2. Reaction codition:

Use a pipette to gently draw and mix (do not shake for mixing), and then perform a short centrifugation to collect the reaction solution at the bottom of the tube.

3. Single fragment recombination reaction, at 50°C for 5 minutes; then cooled to 4°C or immediately placed on ice for cooling.
4. Recombinant product transformation：

Place the chemical competent cells used for cloning on ice to thaw (e.g: DH5α competent cell or BL21(DE3) Competent cell)

5. Add 10 μl of the recombinant product to 100 μl of the competent cells, gently tap the wall of the tube to mix (do not shake for mixing), and let it stand on ice for 30 minutes.
6. After incubation at 42°C for 45 seconds in a water bath, it was immediately placed on ice for cooling for 2 to 3 minutes.
7. Add 900 μl LB liquid medium (without antibiotics), and shake the bacteria at 37°C for 1 hour (rotation speed 220 rpm).
8. Preheat the corresponding resistant LB solid culture medium plates in a 37°C incubator.
9. Centrifuge at 5,000 rpm (2,500 × g) for 5 minutes. Discard 900 μl of the supernatant. Resuspend the bacteria in the remaining culture medium and gently spread it evenly on a plate containing the appropriate resistance using a sterile spreader.
10. In a 37°C incubator, perform inverted culture for 12 - 16 hours.

1. Prepare the PCR system:
   • Prepare the standard PCR reaction system (20 μL), which includes:
   • 2*PCR Mix buffer: 10 uL
   • Primers (forward and reverse primers): each 1 uL
   • DNA template: 2 uL
   • ddH2O: 6 uL
2. Pick the colony:
3. Gently pick the target colony using a sterile pipette. Stir gently in the PCR tube (containing 10 ul of ddH2O) to dissolve it into the reaction mixture, or gently drop the colony onto the bottom of the PCR tube to ensure that a small amount of bacterial cells enter the system.
4. Perform the PCR reaction:
5. Conduct the PCR directly (heating at 95°C for 5-10 minutes) to facilitate the lysis of the bacteria and release the template DNA. Place it in the PCR machine and run the amplification. The program and cycle conditions are the same as those of the conventional PCR, and are set according to the target fragment and primers (such as pre-denaturation, denaturation, annealing, extension, number of cycles, etc.).
6. PCR product detection:
7. Take the reaction solution and analyze it in an agarose gel electrophoresis to observe whether there is a target band. The detection results are used to identify the positive clones.

All operations were performed in a laminar flow cabinet to ensure aseptic conditions.

Pick the target single positive bacterial colony using a sterile pipette or toothpick. Inoculate it onto 4 mL of fresh LB liquid medium containing antibiotics (in a culture tube), and incubate at 37°C, 180 - 220 rpm for 6 - 16 hours (usually overnight) to obtain the seed culture.

For expanding cultivation

Add 100μL Amp+ to each of 100mL LB liquid medium (final concentration is 50ug/mL). Ensure contactless addition by holding the pipette perpendicular to the bottom of the conical flask during inoculation.

• For each of the bacterial seed cultures (prepared culture of the positive colony), inoculate 1mL from both samples and transfer into 100mL LB-Amp. Maintain the upper portion of the conical flask empty when the bottleneck is exposed.

• It was cultured under shaking at 37°C and at a speed of 220 rpm until the OD600 reached approximately 0.4 - 0.8 (for the expression-type strain)

*Notes: Antibiotics will be denatured in high temperature of steam sterilization, hence adding before preparation of culture instead of preparation of LB.

• Gently swirl the conical flask. Using a pipette, transfer 300μL seed culture into one well of a 96-well microplate.

• Send the microplate to the microplate reader to test OD600 every 2 hours to monitor the growth curve of E. coli by measuring OD600 until the culture reaches the logarithmic phase (OD600 ≈ 0.4–0.8).

IPTG induction

1. Weigh 0.61g of isopropyl-β-D-thiogalactoside (IPTG) (with a molar mass Mr of 238.3g/mol).
2. Add 2.5ml of water and the above-weighed 0.61g IPTG (final concentration is 1M) into a centrifuge tube, and mix well to dissolve.
3. Solution filtration: Draw the above solution into a syringe, replace the needle with a filter membrane (usually with a pore size of 0.22μm), and slowly inject it into a clean small tube to obtain a 1 M IPTG stock solution.
4. Use a pipette with a range of 100-1000 μL to take 0.1 mL of IPTG solution from the stock solution.
5. Add the IPTG solution to the bacterial cultures respectively (1:1000)
6. Place the cultures in a constant temperature shaker at 25°C for overnight (20 hours).

1. After the induction expression is completed, the bacterial culture solution is transferred from the culture bottle to a 50-milliliter centrifuge tube that has been pre-cooled in advance.
2. The PBS solution of 500 ml is pre-cooled on ice in advance.
3. The centrifuge is pre-cooled to 4 degrees Celsius in advance to maintain the low-temperature condition during the centrifugal collection process.
4. The bacterial cultures were taken from shake flasks and each transferred into two 50 mL collection tubes.
5. The cultures were centrifuged at 6000 rpm for 10 minutes to collect the bacterial pellets.
6. After centrifugation, discard the supernatant. Resuspend the bacterial precipitate in pre-cooled PBS solution and centrifuge again at 6000 rpm for 10 minutes. Wash once.

1. Lysis buffer preparation: 40 mM Tris-HCl, 80 mM NaCl and 60 mM imidazole, pH = 7.0, with 8 M urea
2. A total of 45 mL binding buffer, 5 mL lysozyme solution (final concentration: 1 mg/mL), and 500 μg protease inhibitor cocktail were prepared.
3. The wet weight of the bacterial pellet was measured by weighing the tube before and after adding the pellet. Buffer was added at a 1 mg: 15mL ( weight-to-volume ratio). Use the mixer oscillator to thoroughly mix.
4. At a temperature of 4 degrees Celsius, the bacterial suspension was placed in a rotary mixing instrument and incubated under rotation for 2 hours.
5. The bacterial suspension was placed on ice to maintain low temperature and prevent protein denaturation.
6. Ultrasonication was performed until the solution became clear and non-viscous, indicating complete lysis.
7. After the liquid becomes clear for 30 minutes, set the power to 40%. Start the ultrasonic disruption for 2 seconds, then stop for 3 seconds. Continue this cycle until the disruption is complete.
8. 1 mL of whole cell lysate was collected for later analysis (e.g., SDS-PAGE).
9. The remaining lysate was centrifuged at 12000 rpm, 4°C, 20 minutes to remove cell debris. The supernatant, containing soluble proteins including the target fusion protein, was collected for purification.

• The purification reagent we use is HisSep Ni-NTA Agarose Resin (Yeasen). This resin is based on cross-linked 6% agarose gel as the matrix. It is chemically coupled with tetra-coordinated nickel tri-acetate (NTA) as the ligand. After chelating with nickel ions (Ni2+), a very stable octahedral structure is formed, with the nickel ions at the center of the octahedron. Such a structure can protect the nickel ions from being attacked by small molecules, making it more stable and capable of withstanding certain concentrations of reducing agents, denaturants, or coupling agents and other harsh conditions.
• Based on the amount of the purified sample, add 1 mL of HisSep Ni-NTA Agarose Resin to the centrifuge tube. Centrifuge at 1000 rpm for 1 minute, then discard the supernatant.
• Add 5 times the volume of Lysis Buffer to the centrifuge tube to wash the filler. Centrifuge at 1000 rpm for 1 minute, then discard the supernatant; repeat this process twice or more.
• Add the sample, seal the centrifuge tube, and incubate at 4℃ for 3 hours or at 37℃ for 1 hour.
• After incubation, perform a 1000 rpm centrifugation for 1 minute, then aspirate the supernatant. The supernatant is retained and used as the flow-through for electrophoretic analysis.
• Use 5 times the volume of Wash Buffer to wash the filler, centrifuge at 1000 rpm for 1 minute to remove the supernatant (be careful not to suck up the filler), repeat this process 3-5 times. During the process, replace the new centrifuge tube to collect different washing solutions. At the same time, adjust the concentration of imidazole for impurity removal (10 mM, 20 mM, 50 mM, 100 mM, 150 mM, 500 mM).
• Add 3 times the column volume of Elution Buffer (500 mM imidazole) for elution, incubate at room temperature for 10-15 minutes, centrifuge at 1000 rpm for 1 minute each time (1 ml), repeat 3 times.

1. Create a buffer that blending 0.34g sodium acetate with 0.38g sodium borate and 0.2g ascorbic acid. Maintain the pH level around 5.5.
2. Combine the elution liquid of target proteins with 1ml buffer, add 50 U tyrosinase from mushroom, leave the solution in 37 degrees Celsius for 30min, then add 5% acetic acid to finish the reaction. Repeat the process on all proteins.

Note: leave one sample of each protein as contract in testing

1. Prepare the NBT staining solution using deionized water, with a concentration of 10 mg/ml. Store it as a stock solution at -20 degrees Celsius in a dark place.
2. Dip 50 microliters of the adhesion protein sample onto a nitrocellulose membrane and let it dry overnight. Wash the membrane with PBS buffer to remove impurities.
3. The membrane of the sample protein solution was incubated in 20 milliliters of freshly prepared 1 milligram per milliliter NBT solution at room temperature in the dark (covered with aluminum foil) for 45 minutes. Then, the membrane was washed twice with 10 milliliters of 0.16M sodium borate solution and soaked in another 20 milliliters of sodium borate solution overnight. The colored film is recorded as an image through photography.

1. Add 10μl protein on PVDF layer.
2. Wait until the liquid is dried, then add another round of 10μl protein.
3. After the new round is dried, add 30μl protein and dry the sample for an entire night.
4. Drop 50μl onto each of the five different substrate surfaces: PE, PV, PS, stainless steel, and silicone
5. Repeat the process on all proteins.
6. The next day, place differerent materials in different tubes containing purified water, and shake in the shaking incubator for 2 hours to wash off all of the unbound proteins.
7. Then dry at room temperature until no more liquids are left on the surface.
8. After that is done, stain the material with Coomassie Brilliant Blue for 30 minutes .
9. Upon completion, use ImageJ software to analyze the intensity of the stained spots.

1. Another way we used to test the adhesive of the proteins is by using PTFE (polytetrafluoroethylene) pipette tips. Where PTFE (polytetrafluoroethylene) pipette tips of the same specifications are dipped into 1 mg/mL solutions of modified and unmodified recombinant proteins, BSA, and Cell-Tak™ separately.
2. Subsequently, attach each tip to the polypropylene centrifuge tube caps. Remember to be extra careful during this part, as you don’t want to knock over any pipette tips. Let the tips stand at room temperature for 1 hour to allow them to stick to the caps.
3. Finally, observe the macroscopic adhesion of the sample proteins by turning the centrifuge tube caps upside down and seeing if the pipette tips fall off.

1. We selected the model organisms Escherichia coli and Bacillus subtilis as the objects for the antibacterial peptide activity test.

2. The growing and active Escherichia coli and Bacillus subtilis were re-inoculated into fresh culture medium. Corresponding amounts of antibacterial peptide stock solution were added to the freshly inoculated bacterial solution according to different antibacterial peptide concentrations (0 ug/mL, 25 ug/mL, 50 ug/mL, 100 ug/mL, 400 ug/mL).

3. The OD600 value was measured every 1 hour and the detection was conducted for 24 hours.

4. Based on the measured OD600 absorbance, the bacterial growth curves of different experimental groups were plotted.

1. To investigate the antibacterial effect of the fusion protein coating, the bacterial solutions of Escherichia coli and Bacillus subtilis were diluted to 1×106 CFU/mL.
2. Glass slides and stainless steel plates coated with the protein were added to a 12-well plate, with each well containing 1 mL of the bacterial solution.
3. The plates were incubated at 37°C with a speed of 220 rpm for 20 hours.

1. Firstly, cultivate E. coli and B. subtilis in a 48-well plate until their OD600 reaches 0.6.
2. Then, place the coated glass sheet (a 10mm glass sheet covering 18 micrograms of recombinant fusion protein) into the plate and incubate it at a speed of 60 rpm for 120 minutes.
3. After that, remove the glass sheet, wash the coating 3 times with 1 mL of sterile 0.9% NaCl solution.
4. Next, use an ultrasonic cleaning machine (for 5 minutes) to wash off the bacteria on the coating in 1 mL of sterile 0.9% sodium chloride solution.
5. Finally, spread 100 microliters of the bacterial suspension on the LB plate, incubate it at 37°C for 24 hours, and observe the growth of the colonies.