The target DNA fragments were amplified using Polymerase Chain Reaction (PCR). The specific procedure is as follows:

1. All reaction components were thawed on ice prior to use. The components were added to a PCR tube in the specific volumes listed in Table 1 to constitute the reaction mixture.
2. The reaction mixture was mixed thoroughly by gentle pipetting and then briefly centrifuged to collect the contents at the bottom of the tube.
3. The PCR tube was placed into a thermal cycle, and the amplification program was initiated using the parameters detailed in Table 2.
4. All reagents were stored at -20℃.

1. A 1% (w/v) agarose gel was prepared by dissolving agarose powder in 1× TAE buffer. The solution was heated in a microwave oven until the agarose was completely dissolved.
2. After the solution had cooled slightly, 4S Green Plus nucleic acid stain (10,000×) was added at a ratio of 1 µL of stain per 20 mL of agarose solution.
3. The molten agarose solution was poured into a gel casting tray fitted with a comb of the appropriate well number.
4. The gel was allowed to cool and solidify completely. The comb was then carefully removed, and the gel was placed into an electrophoresis chamber submerged in 1× TAE buffer.
5. Prior to loading, 10× DNA loading buffer was added to each sample and mixed. The sample mixtures were subsequently loaded into the wells of the agarose gel using a micropipette.
6. Electrophoresis was conducted at a constant voltage of 105 V for 30 minutes.

Following electrophoresis, the gel was visualized under UV illumination. The DNA bands of the correct size were excised from the gel. The DNA fragments were then purified from the agarose slice using a Vazyme Gel Extraction Kit, following the manufacturer's protocol.

1. The required mass of DNA for the recombination reaction was calculated based on the following formulas:
   • For single-fragment homologous recombination:
     Optimal mass of vector (ng) = 0.02 × (vector length in bp)
     (This corresponds to an amount of 0.03 pmol.)
     Optimal mass of insert (ng) = 0.04 × (insert length in bp)
     (This corresponds to an amount of 0.06 pmol.)
   • For multi-fragment homologous recombination:
     Optimal mass of vector (ng) = 0.02 × (vector length in bp)
     (This corresponds to an amount of 0.03 pmol.)
     Optimal mass of each insert (ng) = 0.02 × (insert length in bp)
     (This corresponds to an amount of 0.03 pmol for each insert.)

   The reaction was assembled on ice as detailed in Table 3.

2. The reaction components were mixed thoroughly by gentle pipetting; vortexing was avoided. The mixture was then briefly centrifuged to collect the solution at the bottom of the tube.
3. Recombination Reaction:
   • For single-fragment assembly, the reaction was incubated at 50℃ for 5 minutes.
   • For 2-3 fragment assembly, the reaction was incubated at 50℃ for 15 minutes.
   • Following incubation, the reaction was terminated by cooling to 4℃ or by placing the tube immediately on ice. The assembled product could be stored at -20℃ for up to one week prior to transformation.

1. Chemically competent E. coli cells and the plasmid DNA were retrieved from storage at -80°C and -20°C, respectively, and thawed on ice.
2. A volume of 10 µL of plasmid DNA was added to 100 µL of thawed competent cells in a microcentrifuge tube. The mixture was gently agitated by flicking the tube and then incubated on ice for 30 minutes.
3. The cell-plasmid mixture was subjected to a heat shock by incubation at 42°C for 90 seconds, followed by immediate transfer to ice for 2-3 minutes.
4. For cell recovery, 700 µL of antibiotic-free LB medium was added to the tube. The culture was then incubated in a shaking incubator at 37°C for 1 hour.
5. Following recovery, a 100 µL aliquot of the cell suspension was spread onto an LB agar plate supplemented with the appropriate antibiotic.
6. The plate was inverted and incubated overnight at 37°C.

Colony PCR was performed to screen for positive transformants.

1. The PCR reaction mixture was prepared on ice according to the volumes specified in Table 4.
2. Individual colonies were selected from the overnight culture plate using a sterile pipette tip. Each colony was then dipped directly into a corresponding PCR tube containing the reaction mixture, thereby introducing the template DNA.
3. The PCR products were analyzed by agarose gel electrophoresis. Colonies that yielded a DNA band of the expected size were considered positive. These positive colonies were subsequently streaked onto new selective agar plates and also inoculated into liquid medium for further cultivation. A portion of the culture was preserved as a glycerol stock (prepared by mixing the cell culture and sterile glycerol in a 1:1 ratio), while the remainder was processed for plasmid extraction and subsequent DNA sequencing verification.

1. Activation (Starter Culture Preparation): A starter culture was prepared by inoculating 50 µL of the glycerol stock into 5 mL of LB medium containing the appropriate antibiotic. The culture was incubated for 12 hours at 37°C with shaking at 200 rpm.
2. Inoculation: The starter culture was used to inoculate 50 mL of fresh LB medium (a 1:1000 dilution, corresponding to a 2% v/v inoculum based on the provided text, but typically 1:100 is standard) in an Erlenmeyer flask. The culture was grown at 37°C with shaking at 200 rpm until the optical density at 600 nm (OD₆₀₀) reached a value of approximately 0.6.
3. Induction: Protein expression was induced by the addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. The culture was subsequently incubated for 12-16 hours at a reduced temperature of 20°C with shaking at 200 rpm.

1. Cell Harvesting and Washing: The bacterial culture was harvested by centrifugation at 8,000 rpm for 5 minutes at 4°C. To process the entire culture volume, this step was repeated by decanting the supernatant and adding the remaining culture to the same centrifuge tube. The resulting cell pellet was washed once with RO water and subsequently with 5 mL of PBS (Phosphate-Buffered Saline), with a centrifugation step following each wash to collect the pellet.
2. Cell Resuspension: After the final wash, the supernatant was completely discarded, and the tube was inverted on absorbent paper to remove any residual liquid. The cell pellet was then thoroughly resuspended in 2 mL of fresh PBS and transferred to a new 5 mL tube. To ensure complete transfer, the original centrifuge tube was rinsed with an additional 1 mL of PBS, which was then pooled with the cell suspension.
3. Cell Lysis by Sonication: The tube containing the cell suspension was placed in an ice-water bath to maintain a low temperature. The cells were lysed using an ultrasonic processor operating at approximately 300 W. The sonication program consisted of cycles of 3 seconds on followed by a 5-second pause, for a total duration of 10 minutes.
4. Separation of Soluble and Insoluble Fractions: The resulting cell lysate was clarified by centrifugation at 5,000 rpm for 20 minutes at 4°C.
5. Sample Collection: The supernatant, containing the soluble protein fraction, was carefully collected for subsequent purification and stored at -20°C. The pellet, containing insoluble proteins and cell debris, was resuspended in 2 mL of PBS, and an aliquot was retained for analysis by SDS-PAGE.

1. Column Preparation: A Ni-NTA (Nickel-Nitriloacetic Acid) affinity column, previously stored in 20% ethanol, was prepared for use. The storage solution was drained, and the column was equilibrated by washing sequentially with 5 mL of Buffer A (Binding/Wash Buffer), 3 mL of Buffer B (Elution Buffer), and finally 5 mL of Buffer A.
2. Protein Binding and Elution: The clarified cell lysate (soluble fraction) was loaded onto the equilibrated column. The column was then washed twice with 5 mL of Buffer A to remove non-specifically bound proteins. The His-tagged target protein was subsequently eluted with 3 mL of Buffer B, and the eluate was collected.
3. Column Regeneration and Storage: Following elution, the column was regenerated by washing with 3 mL of Buffer B and 5 mL of Buffer A. It was then prepared for storage by flushing with 20% ethanol.
4. Protein Concentration and Buffer Exchange: The collected eluate was transferred to an ultrafiltration centrifugal device (e.g., a spin concentrator). The protein was concentrated by centrifugation at 6,500 rpm for 30 minutes at 4°C. To perform a buffer exchange, 2 mL of ultrapure water was added to the concentrated protein, and the centrifugation was repeated. This washing step was performed once more. If the final volume of the concentrated protein solution exceeded 500-700 µL, an additional centrifugation step (5-15 minutes) was performed to achieve the desired concentration.
5. Storage: The final purified and concentrated protein solution was stored at -20°C.

Protein samples were analyzed by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE).

1. Gel Preparation: A polyacrylamide gel was prepared using the SDS-PAGE Gel Preparation Kit (Solarbio, Beijing). The gel consisted of a 5% stacking gel and a separating gel of an appropriate acrylamide concentration, selected based on the target protein's molecular weight. The gel casting apparatus was assembled, and the separating gel solution was poured between the glass plates. A layer of water was added to ensure a flat surface. After polymerization, the water was removed, and the stacking gel solution was poured on top. A comb was inserted to create loading wells. The gel was allowed to polymerize completely and was stored at 4°C until use.
2. Sample Preparation: For each sample, 9 µL of protein solution was mixed with 3 µL of 4× SDS-PAGE loading buffer. The mixture was denatured by heating at 100°C for 10 minutes, then cooled to room temperature and briefly centrifuged.
3. Electrophoresis: The prepared gel cassette was installed in the electrophoresis tank, which was filled with 1× Tris-glycine-SDS running buffer. After removing the comb, a protein molecular weight marker and the prepared protein samples were loaded into the wells. Electrophoresis was conducted at a constant voltage of 120 V until the bromophenol blue dye front reached the bottom of the gel.
4. Visualization: Following electrophoresis, the gel was stained with a Coomassie Brilliant Blue staining solution for approximately 40 minutes and subsequently destained in a destaining solution with gentle agitation until the protein bands were clearly visible.

Plasmid DNA was extracted and purified from the bacterial cultures using the FastPure® Plasmid Mini Kit (Vazyme) according to the manufacturer's instructions.

A single colony of E. coli was selected and inoculated into LB liquid medium. After 20 hours of cultivation, the culture was diluted to an optical density at 600 nm (OD₆₀₀) of 0.1. Following an additional incubation period of 2-3 hours at 37°C, protein expression was induced by the addition of IPTG to a final concentration of 0.2 mM. After 12 hours of induction, a 1.5 mL aliquot of the culture was harvested by centrifugation at 12,000 rpm for 2 minutes. The supernatant was discarded, and the cell pellet was washed with 1 mL of 1× PBS buffer. A wet mount was prepared from the cell suspension on a microscope slide. The fluorescence intensity of the Bs2 protein was observed under a fluorescence microscope using an excitation wavelength of 447 nm.

Bacterial growth in the 96-well plates was quantified by measuring the optical density at 600 nm (OD₆₀₀) using a microplate reader. Prior to measurement, the plate layout and measurement parameters were defined in the instrument's control software. Upon completion of the reading cycle, the resulting data was exported and saved for further analysis.

The Minimum Inhibitory Concentration (MIC) of the test samples against an indicator bacterial strain was determined using a broth microdilution method.

1. Preparation: All materials, including culture media, PBS buffer, and pipette tips, were sterilized by autoclaving at 121°C for 25 minutes. Sterile 96-well microtiter plates were sterilized under UV light in a laminar flow hood for 30 minutes.
2. Serial Dilution: A two-fold serial dilution of the test sample was prepared directly in the plate. First, 100 µL of culture medium was added to each well. Then, 100 µL of the sample was added to the first column (A1-H1) and mixed. Subsequently, 100 µL was transferred from the first column to the second, and this process was repeated sequentially up to the tenth column.
3. Inoculation and Controls: An inoculum of the indicator bacteria, adjusted to a concentration of approximately 10⁸ CFU/mL, was prepared. A volume of 100 µL of this bacterial suspension was then added to all wells from column 1 to column 11.
   • Test Wells: Columns 1-10 contained serially diluted samples and bacteria.
   • Positive Control: Column 11 contained culture medium and bacteria only, serving as the positive growth control.
   • Blank Control: Column 12 contained culture medium and the undiluted sample (without bacteria), serving as the blank control to correct for sample absorbance.
4. Incubation and Analysis: The plate was incubated at 37°C for 16-20 hours. The assay was considered valid only if significant bacterial growth was observed in the positive control wells. The MIC was defined as the lowest concentration of the sample that inhibited bacterial growth by 50% or more, determined by comparing the absorbance of the test wells to that of the positive growth control.

The antimicrobial activity of the samples was also evaluated using an agar well diffusion method.

1. Preparation: LB agar medium and all necessary equipment were sterilized by autoclaving at 121°C for 25 minutes.
2. Plate and Well Preparation: Under aseptic conditions, molten LB agar was poured into sterile Petri dishes to a uniform depth of approximately 4 mm. After the agar solidified, wells of a consistent diameter were created using a sterile cork borer.
3. Inoculation: An inoculum of the indicator strain was prepared at a concentration of approximately 10⁸ CFU/mL. A sterile cotton swab was used to evenly inoculate the entire surface of the agar plates, creating a bacterial lawn.
4. Sample Application: After allowing the surface of the agar to dry, a defined volume of the test sample was added to the wells. An appropriate antibiotic and sterile water were used as the positive and negative controls, respectively.
5. Incubation and Observation: The plates were sealed and incubated at 37°C for 12-16 hours. The antimicrobial effect was assessed by measuring the diameter of the zone of inhibition (the clear area around the well where bacterial growth was prevented).