EXPERIMENTS

1.Preparation and Dispensing of LB Solid Medium

LB fluid medium	LB solid medium
Tryptone (10 g/L)	Tryptone (10 g/L)
Yeast extract (5 g/L)	Yeast extract (5 g/L)
NaCl (10 g/L)	NaCl (10 g/L)
-	Agar (15 g/L)
ddH2O: Make up to 1000 mL

Apparatus and materials:
Electronic balance, Weighing paper, Erlenmeyer flask, Measuring cylinder, Magnetic stirrer. Autoclave, Sterile petri dishes, Sealing film and plastic wrap, rubber bands, clean bench, Heat-resistant gloves.
Medium Preparation Procedure
(1)Turn on the electronic balance and weigh: 10 g Tryptone, 5 g Yeast extract, 10 g NaCl, (If solid medium is needed) 15 g Agar
(2)Add the weighed solid reagents into the 1000 mL Erlenmeyer flask.
(3)Add approximately 800 mL of ddH₂O and gently swirl or use the magnetic stirrer until the reagents dissolve completely.
(4)Add ddH₂O to bring the total volume up to 1000 mL and mix well.
Sterilization
(1)Seal the Erlenmeyer flask with aluminum foil or sealing film and secure it with a rubber band.
(2)Place the flask into the autoclave, set at 121 °C, and sterilize for 20 min.
(3)After sterilization, allow the pressure to return to normal before carefully removing the flask.
Dispensing into Petri Dishes (Performing under sterile conditions)
(1)Cool the sterilized medium to around 55-60 °C (it should feel warm to the touch but not too hot).
(2)Place sterile petri dishes on the sterile workbench.
(3)Open the petri dishes and, using the alcohol lamp for sterilization, pour the medium into the dishes to fill them to about half their volume.
(4)Gently shake the petri dishes to distribute the medium evenly.
(5)Once the medium solidifies, cover the petri dishes and seal them (using sealing film or tape).
(6)Label the petri dishes (including information such as medium type, date, and the person who prepared them).
(7)Store the sealed petri dishes upside down (to prevent condensation on the surface) in the refrigerator at 4 °C (It is recommended to use them within 1-2 weeks).

2.Cultivation of E. coli with Plasmids in Liquid Medium
Apparatus and materials:
Plasmid-carrying E. coli DH5α: pET28a, pUC57-BSCBD, pUC57-gp17, pUC57-TFP, Kanamycin, LB medium, Pipette and sterile pipettes.

Preparation of Bacterial Cultures
(1)Retrieve the prepared E. coli plates carrying the plasmids pET28a, pUC57-BSCBD, pUC57-gp17, and pUC57-TFP from the refrigerator.
(2)Ensure that these plates are stored at 4°C and are not older than a week for optimal bacterial viability.
(3)Take 8 sterile culture tubes and add 2 mL of LB medium to each container using a pipette.
(4)Using a separate pipette, add kanamycin to each container to achieve a final concentration of 0.2 μg/mL.
(5)Using a pipette, carefully pick a single colony from the plate of E. coli strain carrying the plasmid of interest (pET28a, pUC57-BSCBD, pUC57-gp17, or pUC57-TFP).
(6)Gently dip the pipette tip into the colony and transfer the bacterial sample into the appropriate 2 mL medium container.
(7)Repeat the process for each plasmid-containing strain, making sure to keep the strains separated and correctly labeled.
(8)Incubate overnight in a culture incubator at 37°C and 220 rpm.

3.Plasmid extraction
Apparatus and materials:
Column tube CP3, Collection tube, 1.5 mL centrifuge tube, 2 mL centrifuge tube, Centrifuge, Vortex mixer, Dry bath incubator, Buffer P1, Buffer P2, Buffer P3, Buffer PWT, Deionized distilled water, Inoculated bacterial culture (2 mL, containing target plasmid)
Procedure
(1)Harvest Bacterial Cells: Transfer 2 mL of the target plasmid-containing bacterial culture into a 2 mL centrifuge tube. Centrifuge at 8000 × g for 2 min. Discard the supernatant completely.
(2)Resuspension: Add 250 μL Buffer SP1 to the cell pellet. Resuspend thoroughly by vortexing or pipetting until the pellet is completely suspended.
(3)Lysis: Add 250 μL Buffer SP2. Immediately invert the tube gently 8 times to mix completely. Incubate at room temperature for 2-4 min (do not exceed 5 min).
(4)Neutralization: Add 350 μL Buffer SP3. Gently invert the tube up and down until a white flocculent precipitate forms and the solution below becomes clear.
(5)Supernatant Transfer: Centrifuge at 12000 × g for 3 min. Carefully transfer the clear supernatant to a CP3 column tube (adsorption column) placed in a collection tube.
(6)Binding: Centrifuge the column at 12000 × g for 1 min. Discard the flow-through from the collection tube.
(7)Wash: Add 500 μL Buffer PWT to the column. Centrifuge at 9000 × g for 1 min. Discard the flow-through. Repeat this wash step once more for a total of two washes.
(8)Drying the Column: Centrifuge the empty column at 9000 × g for 1 min to remove residual buffer.
(9)Elution: Place the CP3 column into a clean 1.5 mL centrifuge tube. Add 50-100 μL elution buffer or deionized distilled water to the center of the adsorption membrane. Incubate at room temperature for 1 min.
(10)Collection of Plasmid DNA: Centrifuge at 12000 × g for 1 min. The eluted plasmid DNA is now in the 1.5 mL tube. Store the plasmid DNA at -20 °C.

4.PCR Amplification
Apparatus and Materials:
PCR thermocycler, PCR tubes (0.2 mL), Vortex mixer, Microcentrifuge, 2 × Hieff PCR Master Mix, Template DNA, Forward and reverse primers, ddH₂O
Procedure
(1)Preparation of PCR Reaction Mixture

Reagent	Volume (μL)
2 × Hieff PCR Master Mix	25
Primer F (10 μM)	2
Primer R (10 μM)	2
DNA template	1 (10 ng)
ddH2O	20
Total	50

(2)PCR Thermocycler Setup

Step	Temperature	Time
1	94 ℃	5 min
2	94 ℃	30 s
3	55 ℃	30 s
4	72 ℃	30 s/Kb
5	Go to step 2	30 Cycles
6	72 ℃	4 min
7	4 ℃	infinite hold

(3)Place the PCR tubes in the thermocycler.
(4)Start the PCR program with the optimized annealing temperature and extension time.
(5)After completion, store samples at 4°C for immediate analysis or at -20°C for long-term storage.

5.Agarose Gel Electrophoresis
Apparatus and materials:
Casting/gel tray, casting stand, well combs, voltage source, gel box, microwave oven, pipette and pipette tips, 1 × TAE buffer, agarose, 10,000 × nucleic acid dye (YeaGreen), UV transilluminator.
Procedure
(1)Gel Preparation: Prepare agarose solution: Add 1 g agarose to 100 mL of 1 × TAE buffer in an Erlenmeyer flask (1:100 ratio). Gently swirl the flask to mix the agarose and buffer.
(2)Dissolving Agarose: Heat the Erlenmeyer flask in a microwave oven until the solution begins to boil. Swirl the flask to mix the solution thoroughly. Repeat heating and swirling until the solution is completely clear with no visible undissolved agarose particles.
(3)Adding DNA Stain: Allow the agarose solution to cool slightly. Add 10 μL of 10,000 × YeaGreen nucleic acid dye to the flask (final dilution 1:10,000). Swirl the flask gently to ensure thorough mixing of the dye.
(4)Casting the Gel: Pour the agarose solution into the prepared gel casting tray fitted with well combs.
(5)Remove any air bubbles by gently tapping the tray. Allow the gel to solidify at room temperature for approximately 20 min.
(6)Setting up Electrophoresis: Once solidified, carefully remove the well combs. Place the gel casting tray into the electrophoresis tank. Add 1× TAE buffer to the tank until the gel surface is completely submerged (approximately 2-3 mm above the gel).
(7)Sample Loading: Starting from the left well, load 5 μL of DNA molecular weight marker. Load 10 μL DNA samples into the remaining wells.
(8)Running Electrophoresis: Place the lid on the electrophoresis tank and ensure proper electrode connections. Set the power supply parameters (Voltage: 180 V, Current: 300 mA, Time: 30 min). Press the start button to begin electrophoresis.
(9)Visualization: After the run is complete (approximately 20-30 min), turn off the power supply. Carefully remove the gel from the electrophoresis tank. Visualize DNA bands using UV light. Document results by photography if needed.

6.Gel Extraction
Apparatus and Materials:
Analytical balance, dry bath incubator (50°C), centrifuge, razor blade, Buffer B2, Wash Solution, Elution Buffer
Procedure
(1)Gel Slice Excision: Using a clean scalpel or razor blade, carefully excise the agarose gel slice containing the DNA fragment of interest under UV or blue light illumination. Transfer the gel slice to a pre-weighed 1.5 mL microcentrifuge tube. Weigh the tube with the gel slice and calculate the gel weight by subtraction.
(2)Gel Solubilization: Add Buffer B2 to the gel slice at a ratio of 3 times the gel weight. Incubate the mixture at 50°C in a dry bath incubator. Vortex the tube every 2-3 min to facilitate dissolution. Continue heating until the gel slice is completely dissolved.
(3)DNA Binding: Transfer the entire mixture to a spin column placed in a collection tube. Centrifuge at 8,000 × g for 30 s. Discard the flow-through from the collection tube. Place the column back into the same collection tube.
(4)Washing: Add 500 μL Wash Solution to the column. Centrifuge at 9,000 × g for 30 s. (5)Discard the flow-through from the collection tube. Repeat this washing step once more for a total of two washes.
(6)Column Drying: Place the column into a new collection tube. Centrifuge at 9,000 × g for 1 min to remove residual wash solution.
(7)DNA Elution: Place the column into a new 1.5 mL microcentrifuge tube. Add 50 μL of Elution Buffer to the center of the column membrane. Incubate at room temperature for 1 min to allow the buffer to penetrate the membrane. Centrifuge at 12,000 × g for 1 min. The purified DNA is now in the 1.5 mL tube and ready for downstream applications.
(8)Storage: Store the purified DNA at 4°C for short-term use or at -20°C for long-term storage.

7.DNA Concentration Measurement
Apparatus and Materials:
NanoDrop spectrophotometer, DNA samples, ddH₂O, Pipettes and pipette tips, Lint-free wipes.
Procedure
(1)Instrument Preparation: Turn on the NanoDrop spectrophotometer and allow it to warm up according to manufacturer's instructions. Launch the NanoDrop software on the connected computer. Select "Nucleic Acid" measurement mode.
(2)Blanking: Clean pedestal with ddH₂O. Pipette 2 μL ddH₂O onto pedestal. Click "Blank" to establish the baseline measurement. Lift the sampling arm and clean pedestal with a lint-free wipe.
(3)Sample Measurement: Pipette 2 μL of the linearized vector sample onto the lower pedestal. Lower the sampling arm and click "Measure". Record the concentration (ng/μL). Clean the pedestals after measurement. Repeat the same process for the insert fragment sample.

8.Gibson Assembly for Seamless DNA Cloning
Apparatus and Materials:
Thermalcycler, Microcentrifuge, 2× CloneExpress Mix, Insert DNA fragment, Linearized vector DNA, ddH₂O, 0.2 mL PCR tubes, Pipettes and pipette tips
Procedure
(1)Prepare the Gibson Assembly reaction in a 0.2 mL PCR tube (Gibson Assembly Master Mix, Insert DNA, Vector DNA, ddH₂O up to 10 μL).
(2)Mix gently by pipetting and briefly centrifuge to collect contents at the bottom.

Reagent	Volume (μL)
2 × Clonexpress mix	5
Target Gene Insert	0.02 × Insert bp*
pET28a Vector	0.04 × Vector bp*
ddH2O	Up to 10
Total	10

(3)Place the reaction tube in a heating block. Incubate at 50℃ for 10 min.

Step	Temperature	Time
1	50 ℃	30 min
2	10 ℃	infinite hold

9.Transformation of DNA into E. coli DH5α Using the Heat Shock Method
Apparatus and Materials:
Ice bucket, Water bath, Shaking incubator, centrifuge, Competent E. coli DH5α cells, Gibson Assembly product, LB liquid medium, LB agar plates with antibiotic, 1.5 mL microcentrifuge tubes, Pipettes and sterile pipette tips, Sterile spreader.
Procedure
(1)Thawing and Sample Addition: Remove E. coli DH5α competent cells from the -80°C freezer. Thaw the competent cells on ice for 5 min. Add 10 μL of the Gibson Assembly product to the thawed competent cells. Mix gently by tapping the tube. Incubate the mixture on ice for 30 min to allow DNA uptake preparation.
(2)Heat Shock and Recovery: Quickly transfer the tubes to a 42°C water bath incubate for 90 s. Immediately transfer the tubes back to ice and incubate for 3-5 min. Add 700 μL of sterile LB liquid medium to the transformation mixture. Transfer to a shaking incubator set at 37°C, 200 rpm, incubate for 60 min.
(3)Centrifugation and Plating: Centrifuge the tubes at 5,000 rpm for 2 min to pellet the cells. Carefully discard the supernatant, leaving approximately 100 μL. Gently resuspend the cell pellet. Spread the entire cell suspension evenly onto LB agar plates containing the appropriate antibiotic. Use a sterile spreader for even distribution. Allow the liquid to absorb into the agar.
(4)Incubation: Incubate the plates upside down at 37°C overnight (12-16 h). Check for colony growth the following day.

10.Colony PCR for Screening Recombinant Clones
Apparatus and materials:
Pipettes and pipette tips, PCR tubes, Thermocycler, Recombinant clones from transformation plates, Forward primer (10 μM), Reverse primer (10 μM), 2× Rapid Taq Master Mix, ddH₂O.
Procedure
(1)Using pipette tip, gently touch a single colony from the transformation plate.
(2)Transfer the colony material to a fresh LB agar plate containing the appropriate antibiotic by gently streaking to create a backup colony.
(3)Dip the same pipette tip (with remaining bacterial material) into a PCR tube containing the prepared reaction mixture.
(4)Mix gently by pipetting up and down 2-3 times to release bacterial cells into the buffer.
(5)PCR Reaction Setup:

Reagent	Volume (μL)
2 × Rapid Taq Master Mix	10
Primer F (10 μM)	2
Primer R (10 μM)	2
ddH2O	6
Total	20

(6)PCR Thermocycling Program:

Step	Temperature	Time
1	94 ℃	5 min
2	94 ℃	10 s
3	50 ℃	20 s
4	72 ℃	2-3 s/Kb
5	Go to step 2	30 Cycles
6	72 ℃	4 min
7	4 ℃	infinite hold

(7)Place the prepared PCR tubes into the thermocycler.
(8)Start the PCR program and wait for completion (approximately 2-3 h).
(9)Store completed reactions at 4°C if immediate analysis is not possible.

11.Plasmid extraction and sequencing
Apparatus and materials:
Column tube CP3, Collection tube, 1.5 mL centrifuge tube, 2 mL centrifuge tube, Centrifuge, Vortex mixer, Dry bath incubator, Buffer P1, Buffer P2, Buffer P3, Buffer PWT, Deionized distilled water, Inoculated bacterial culture (2 mL, containing target plasmid)
Procedure
(1)Harvest Bacterial Cells: Transfer 2 mL of the target plasmid-containing bacterial culture into a 2 mL centrifuge tube. Centrifuge at 8000 × g for 2 min. Discard the supernatant completely.
(2)Resuspension: Add 250 μL Buffer SP1 to the cell pellet. Resuspend thoroughly by vortexing or pipetting until the pellet is completely suspended.
(3)Lysis: Add 250 μL Buffer SP2. Immediately invert the tube gently 8 times to mix completely. Incubate at room temperature for 2-4 min (do not exceed 5 min).
(4)Neutralization: Add 350 μL Buffer SP3. Gently invert the tube up and down until a white flocculent precipitate forms and the solution below becomes clear.
(5)Supernatant Transfer: Centrifuge at 12000 × g for 3 min. Carefully transfer the clear supernatant to a CP3 column tube (adsorption column) placed in a collection tube.
(6)Binding: Centrifuge the column at 12000 × g for 1 min. Discard the flow-through from the collection tube.
(7)Wash: Add 500 μL Buffer PWT to the column. Centrifuge at 9000 × g for 1 min. Discard the flow-through. Repeat this wash step once more for a total of two washes.
(8)Drying the Column: Centrifuge the empty column at 9000 × g for 1 min to remove residual buffer.
(9)Elution: Place the CP3 column into a clean 1.5 mL centrifuge tube. Add 50-100 μL elution buffer or deionized distilled water to the center of the adsorption membrane. Incubate at room temperature for 1 min.
(10)Collection of Plasmid DNA: Centrifuge at 12000 × g for 1 min. The eluted plasmid DNA is now in the 1.5 mL tube. Store the plasmid DNA at -20 °C.
(11)Send the sample to the company for sequencing.

12.Transformation of DNA into E. coli BL21(DE3)
Apparatus and Materials:
Ice bucket, Water bath, Shaking incubator, centrifuge, Competent E. coli BL21(DE3) cells, Sequence-verified plasmids (pET28a-BSCBD, pET28a-gp17, pET28a-TFP), LB liquid medium, LB agar plates with antibiotic, 1.5 mL microcentrifuge tubes, Pipettes and sterile pipette tips, Sterile spreader.
Procedure
(1)Thawing and Sample Addition: Remove E. coli BL21(DE3) competent cells from the -80°C freezer. Thaw the competent cells on ice for 5 min. Add 10 μL of sequence-verified plasmids (pET28a-BSCBD, pET28a-gp17, pET28a-TFP) to the thawed competent cells. Mix gently by tapping the tube. Incubate the mixture on ice for 30 min to allow DNA uptake preparation.
(2)Heat Shock and Recovery: Quickly transfer the tubes to a 42°C water bath incubate for 90 s. Immediately transfer the tubes back to ice and incubate for 3-5 min. Add 700 μL of sterile LB liquid medium to the transformation mixture. Transfer to a shaking incubator set at 37°C, 200 rpm, incubate for 60 min.
(3)Centrifugation and Plating: Centrifuge the tubes at 5,000 rpm for 2 min to pellet the cells. Carefully discard the supernatant, leaving approximately 100 μL. Gently resuspend the cell pellet. Spread the entire cell suspension evenly onto LB agar plates containing the appropriate antibiotic. Use a sterile spreader for even distribution. Allow the liquid to absorb into the agar.
(4)Incubation: Incubate the plates upside down at 37°C overnight (12-16 h). Check for colony growth the following day.

13.Cultivation of E. coli BL21(DE3) with Plasmids
Apparatus and materials:
Plasmid-carrying E. coli BL21(DE3): pET28a-BSCBD, pET28a-gp17, pET28a-TFP, Kanamycin, LB medium, Pipette and sterile pipettes.
Preparation of Bacterial Cultures
(1)Retrieve the prepared E. coli BL21(DE3) carrying the plasmids pET28a-BSCBD, pET28a-gp17, pET28a-TFP from the refrigerator.
(2)Ensure that these plates are stored at 4°C and are not older than a week for optimal bacterial viability.
(3)Take 8 sterile culture tubes and add 2 mL of LB medium to each container using a pipette.
(4)Using a separate pipette , add kanamycin to each container to achieve a final concentration of 0.2 μg/mL.
(5)Using a pipette, carefully pick a single colony from the plate of E. coli BL21(DE3) carrying the plasmid of interest (pET28a-BSCBD, pET28a-gp17, pET28a-TFP).
(6)Gently dip the pipette tip into the colony and transfer the bacterial sample into the appropriate 2 mL medium container.
(7)Repeat the process for each plasmid-containing strain, making sure to keep the strains separated and correctly labeled.
(8)Incubate overnight in a culture incubator at 37°C and 220 rpm.

14.Scale-up Culture Protocol
Apparatus and Materials:
Shaking incubator, Spectrophotometer, Sterile Erlenmeyer flasks, LB medium, IPTG (Isopropyl β-D-1-thiogalactopyranoside), Kanamycin, Pipette and sterile pipettes.
Procedure
(1)Inoculate the primary seed culture into 100 mL of fresh LB liquid medium in sterile Erlenmeyer flasks (1:50). Add kanamycin at 50 μg/mL for plasmid selection. Incubate in a culture incubator at 37°C and 220 rpm.
(2)Approximately 2 h or until log phase growth (OD600 until it reaches 0.4-0.6), add IPTG to a final concentration of 0.1 mM. Mix gently to ensure even distribution of IPTG.
(3)Incubate in a culture incubator at 25°C and 220 rpm.
(4)After 18-20 h post-induction, harvest the cells by centrifugation. Proceed with protein purification or store cell pellets at -80°C for future use.

15.SDS-PAGE Gel Preparation
Apparatus and Materials:
Gel casting apparatus with glass plates, Gel combs, Pipettes and pipette tips, Gel preparation cups, Lower-layer gel solution (2×), Lower-layer gel buffer (2×), Upper-layer gel solution (2×), Colored upper-layer gel buffer (2×), Modified coagulant, ddH₂O, SDS-PAGE running buffer.
Procedure
(1)Gel Casting Assembly: Assemble the gel casting apparatus according to manufacturer's instructions.
(2)Ensure glass plates are clean and properly sealed to prevent leakage.
(3)Lower Layer Gel Preparation (for 1.00 mm thick mini gel): Take equal volumes (2.7 mL each) of Lower-layer gel solution (2×), Lower-layer gel buffer (2×). Mix the solutions thoroughly in a gel preparation cup.
(4)Lower Layer Gel Polymerization: Add 55 μL of modified coagulant to the mixed solution from step 2. Stir gently to mix well, avoiding the formation of air bubbles. Pour the mixed lower-layer gel solution into the gel casting mold, filling to a level 1.5 cm below the upper edge of the glass plate. Cover the gel surface with a thin layer of distilled water or isopropanol to ensure a flat surface and prevent air bubble formation.
(5)Lower Layer Gel Solidification: Allow the gel to stand at room temperature for 6-10 min. Check for solidification: A clear boundary between the gel and the overlay phase indicates complete polymerization.
(6)Upper Layer Gel Preparation (for 1.00 mm thick mini gel): Carefully pour off the water/alcohol overlay from the solidified lower gel. Take equal volumes (0.75 mL each) of Upper-layer gel solution (2×), Colored upper-layer gel buffer (2×). Mix thoroughly in a fresh gel preparation cup.
(7)Upper Layer Gel Polymerization: Add polymerization initiator: Add 15 μL of modified coagulant to the mixed solution from step 5. Mix carefully: Stir gently to ensure uniform mixing while avoiding air bubbles.
(8)Upper Layer Gel Casting: Add the upper-layer gel solution on top of the solidified lower gel until the solution reaches the top of the glass plate. Slowly insert the sample comb into the gel, ensuring no air bubbles are trapped beneath the comb teeth.
(9)Final Polymerization and Well Preparation: Allow the gel to stand for 10-15 min until the upper layer is completely solidified. Carefully remove the comb by pulling it straight up to avoid damaging the wells. Use a pipette tip or syringe filled with SDS-PAGE running buffer to rinse the sample wells and remove any unpolymerized gel material.
(10)Final Preparation: The gel is now ready for SDS-PAGE electrophoresis. Ensure all wells are clean and free of debris before sample loading.

16.Bacterial Cell Sonication and His-tag Protein Purification
Apparatus and Materials:
Centrifuge, Sonicator with probe, Ice bath, shaker, lysis Buffer, wash Buffer, elution Buffer, BeyoGold™ His-tag Purification Resin (50% slurry), Large centrifuge tubes, 2 mL microcentrifuge tubes, Pipettes and tips.
Procedure
(1)Cell Harvesting: Transfer bacterial cells from the shake flask to large centrifuge tubes. Centrifuge at 4,000 × g for 10 min at 8°C. Discard the supernatant completely and note the wet weight of the bacterial pellet. Add lysis buffer at 4 mL per gram of wet bacterial pellet and resuspend completely.
(2)Place the sample tube in an ice bath throughout the sonication process. Sonication parameters (Power: 200-300W, Pulse duration: 3 s on, Interval: 3 s off, Total times: 10 min). Keep the sample on ice continuously to prevent protein denaturation.
(3)Clarification of Lysate: Centrifuge the sonicated sample at 10,000 × g for 20-30 min at 4°C. Carefully collect the supernatant (soluble protein fraction) and place it in an ice-water bath. Reserve 20 μL of supernatant for subsequent protein analysis.
(4)Resin Preparation: Take 1 mL of well-mixed 50% BeyoGold™ His-tag Purification Resin. Centrifuge at 1,000 × g for 10 s at 4°C and discard the storage solution. Add 0.5 mL non-denaturing lysis buffer to the resin. Mix gently to equilibrate. Centrifuge at 1,000 × g for 10 s at 4°C. Discard the supernatant. Repeat equilibration 1-2 times.
(5)Protein binding: Add approximately 4 mL of bacterial lysate supernatant to the equilibrated resin. Incubate on a swing shaker or horizontal shaker at 4°C for 60 min.
(6)Column Loading: Load the mixture of lysate and BeyoGold™ His-tag Purification Resin into the empty column tube of the affinity chromatography column provided in the kit.
(7)Flow-through Collection: Open the cap at the bottom of the purification column. Allow the liquid to flow out under gravity. Collect approximately 20 μL of the flow-through for subsequent analysis.
(8)Column Washing: Wash the column 5 times with non-denaturing washing solution. Add 0.5-1 mL of washing solution each time. Collect approximately 20 μL of each wash fraction for subsequent analysis and detection.
(9)Protein Elution: Elute the target protein 2 times using elution buffer. Use 1 mL of elution buffer each time. Collect each elution fraction separately for analysis. Analyze elution fractions by SDS-PAGE to confirm protein purity and yield.

17.SDS-PAGE for Protein Analysis
Apparatus and Materials:
Gel electrophoresis apparatus, Power supply, SDS-PAGE running buffer, Protein samples (treated with SDS loading buffer), Protein molecular weight marker, Prepared polyacrylamide gel, Pipettes and tips.
Procedure
(1)Gel Set up and Sample Loading: Load the prepared polyacrylamide gel into the electrophoresis tank . Add SDS-PAGE running buffer to the tank, ensuring both upper and lower chambers are properly filled. Using a micropipette, carefully load 5 μL of treated protein sample into each well of the gel.
(2)Electrophoresis: Connect the electrophoresis apparatus to the power supply, ensuring correct polarity (proteins migrate toward the positive electrode). Initial voltage: 80-100V for sample stacking. Running voltage: 120-150V for separation.
(3)Electrophoresis Completion: Monitor the progress by observing the dye front (tracking dye) movement. When the indicator dye reaches the bottom of the gel, turn off the power supply.
(4)Gel Removal: Carefully remove the gel cassette from the electrophoresis tank. Use appropriate tools to pry open the glass plates. Carefully peel off the polyacrylamide gel completely. Handle gently to avoid tearing or damaging the gel.
(5)Protein Staining: Place the gel into a clean plastic container or petri dish. Pour blue nucleic acid dye solution over the gel, ensuring the solution completely submerges the entire gel. Gently shake the container to ensure the dye solution makes full contact with the gel surface.
(6)Imaging: Turn off room lights and activate the lamp. Stained protein bands will emit bright and can be directly observed and photographed for documentation.

18.Bradford method for protein concentration determination
Apparatus and Materials:
Microplate reader, 96-well microplate, Micropipettes and tips, 1.5 mL microcentrifuge tubes, BSA (Bovine Serum Albumin) standard solution (0.2 mg/mL), 1× PBS (Phosphate Buffered Saline), Bradford working solution.
Procedure
(1)BSA Standard Preparation: Prepare BSA stock solution at 0.2 mg/mL concentration in 1× PBS.
(2)Standard Curve Preparation: Take 8 centrifuge tubes and label them 0-7. Prepare standard dilutions according to the following table. Vortex each tube to ensure complete mixing of the standard solutions.

Tube No.	1× PBS (μL)	0.2 mg/mL BSA (μL)	Final BSA Concentration (μg/mL)
0 (Blank)	300	0	0
1	285	15	10
2	270	30	20
3	240	60	40
4	210	90	60
5	180	120	80
6	150	150	100
7	75	225	150

(3)Microplate Assay Setup: Add 20 μL of each BSA standard solution to designated wells in the microplate. Add 20 μL of appropriately diluted protein samples to separate wells. Add 200 μL of Bradford working solution to each well containing standards and samples.
(4)Incubation and Measurement: Incubate the microplate at room temperature for 5 min. Using a microplate reader, measure the A595 value of each well. Use tube 0 (blank control) to zero the instrument before measurements.
(5)Data Analysis: Plot a standard curve. Fit a linear trendline to determine the equation of the standard curve.

19.AuPt Nanoparticle Solution Preparation
Apparatus and Materials:
Magnetic stirrer with heating capability, Analytical balance, ddH₂O, Chloroplatinic acid (H₂PtCl₆), Chloroauric acid (HAuCl₄), Trisodium citrate (C₆H₅Na₃O₇), Calcium chloride (CaCl₂), Potassium Carbonate (K₂CO₃), 10 × PBS.
Procedure
(1)AuPt Nanoparticle Synthesis: In a 100 mL flask, combine: ddH₂O: 45 mL, Chloroplatinic acid: 0.38 mL (10 mg/mL), Chloroauric acid: 0.3 mL (10 mg/mL).
(2)Heating and mixing: Stir the mixture continuously using a magnetic stirrer. Heat to boiling and maintain for 10 min.
(3)Reduction step: Add 0.2 mL of trisodium citrate solution (114.1 mg/mL) to the boiling mixture. Allow the reaction to proceed under condensation reflux for 30 min.
(4)Cooling step: Gradually cool the resulting AuPt nanoparticle solution to ambient temperature. Store at 4°C for future use.
(5)Supporting Reagent Preparation: Prepare the following reagents according to the specified concentrations and volumes.

Reagent	Concentration	Volume (mL)	Preparation Notes
K₂CO₃	0.1 M	100	Dissolve 1.38 g in ddH2O
Trisodium Citrate	114.1 mg/mL	100	Dissolve 11.41 g in ddH2O
CaCl₂	200 mM	100	Dissolve 2.94 g in ddH2O
0.5 × PBS	0.5 ×	500	Dilute 10× PBS stock 1:20 with ddH2O
0.5 × PBS (0.05% Tween-20)	contain 0.05% Tween-20	200	Add 100 μL Tween-20 to 200 mL 0.5× PBS
Chloroauric acid	10 mg/mL	3.8	Dissolve 38 mg in ddH2O
Chloroplatinic acid	10 mg/mL	3	Dissolve 30 mg in ddH2O

(6)Storage: AuPt nanoflowers store at 4°C, protected from light. Buffer solutions store at 4°C, check for precipitation before use. Metal salt solutions store at room temperature.

20.Synthesis of HRP-PBP-CaHPO₄@AuPt Nanoflowers
Apparatus and Materials:
2 mL microcentrifuge tubes, Centrifuge, Vortex mixer, pH meter, HRP (Horseradish Peroxidase), PBP (Phage-derived Binding Proteins, include gp17, TFP), 0.5 × PBS, CaCl₂ solution (200 mM), AuPt nanoparticle solution (prepared as per previous protocol), K₂CO₃ solution (0.1 M), Pipettes and sterile tips
Procedure
(1)HRP-PBP-CaHPO₄ Nanoflower Synthesis: In a 2 mL microcentrifuge tube, combine HRP solution 250 μL (2 mg/mL), PBP solution 100 μL (1 mg/mL), 0.5 × PBS 1 mL (5 mM, pH 7.4). Add 20 μL of CaCl₂ solution (200 mM) to the mixture. Mix gently by pipetting to ensure uniform distribution. Incubate at room temperature for 18 h to allow biomineralization and nanoflower formation.
(2)Purification of nanoflowers: Centrifuge the reaction mixture to pellet the nanoflowers. Wash five times with PBS to remove unreacted components. After each wash, gently vortex to resuspend the pellet. Resuspend the purified HRP-PBP-CaHPO₄ nanoflowers in 200 μL PBS.
(3)AuPt Nanoparticle Coupling: pH adjustment for electrostatic coupling, take 500 μL of AuPt nanoparticle solution (from previous synthesis). Adjust pH to 8.0 using K₂CO₃ solution to optimize electrostatic interactions. Add 200 μL of HRP-PBP-CaHPO₄ nanoflowers (1 mg/mL) to the pH-adjusted AuPt solution
(4)Incubate at room temperature for 30 min under static conditions to allow electrostatic adsorption
(5)Purification of hybrid nanoflowers: Centrifuge at 12,000 rpm for 2 min at 4°C to pellet the hybrid nanoflowers. Wash the pellet with PBS to remove unbound AuPt nanoparticles. Resuspend the HRP-PBP-CaHPO₄@AuPt nanoflowers in 200 μL PBS.
(6)Storage: Store the final hybrid nanoflowers at 4°C for future use. Label with preparation date and concentration.

21.Transmission Electron Microscopy
Apparatus and Materials:
Nanoflower sample, Deionized (DI) water, TEM Grids, Transmission Electron Microscope: Talos F200x TEM (Thermo Fisher Scientific, USA) or an equivalent analytical TEM, Sonicator, Fine-tipped tweezers for handling TEM grids.
Procedure
•Sample preparation
(1)Sample Dilution: Dilute the raw nanoflower solution with deionized (DI) water at a 1:2 ratio.
(2)Dispersion: Briefly sonicate the diluted sample for 30-60 seconds in a bath sonicator to ensure a homogeneous suspension and break up large aggregates before deposition.
(3)Sample Deposition: Using fine tweezers, hold a TEM grid (carbon film facing up). Pipette 5 µL of the diluted and sonicated nanoflower solution and carefully drop it onto the center of the carbon film.
(4)Drying: Place the grid inside a covered petri dish to protect it from dust and allow it to dry completely at room temperature for at least 1-2 hours or until all solvent has evaporated.
•TEM Imaging:
(1)Grid Loading: Once completely dry, carefully load the prepared TEM grid into a suitable TEM holder according to the instrument’s specific operating manual.
(2)Insertion: Insert the holder into the TEM column and allow the system to achieve acceptable vacuum.
(3)Microscope Setup: Align the microscope according to standard operating procedures. Begin observation at a low magnification to locate a suitable area with well-dispersed nanoparticles.
(4)Image Acquisition: Increase magnification to desired levels for detailed morphological analysis.

22.Catalytic Performance Determination
Apparatus and Materials:
Microplate reader, 96-well microplates, Incubators, Timer, AuPt nanoparticles, HRP-PBP-CaHPO₄ nanoflowers, HRP-PBP-CaHPO₄@AuPt nanoflowers, TMB (3,3',5,5'-tetramethylbenzidine) solution, H₂O₂ (hydrogen peroxide) solution, pipettes and tips, Microcentrifuge tubes.
Procedure
(1)Comparative Catalytic Activity Assessment
Replicates: All experiments performed in triplicate
Test samples: Three different catalyst systems
a.AuPt nanoparticles alone
b.HRP-PBP-CaHPO₄ nanoflowers
c.HRP-PBP-CaHPO₄@AuPt hybrid nanoflowers
Catalytic Activity Assay: Reaction mixture preparation: For each well, add the following components in order 10 μL of respective nanoflower suspension. 50 μL TMB solution. 150 μL H₂O₂ solution.
Reaction conditions: Room temperature (25°C). Reaction 30 min. Gently mix by pipetting or plate shaking.
Detection and documentation: Measure absorbance at 650 nm using a microplate reader. Take photos with smartphone to document color development. Record all measurements in triplicate.
(2)Reaction Condition Optimization
a.Temperature Optimization: Test catalytic activity at 4°C, 25°C, 30°C, 37°C, 50°C
Use the same reaction system as Part 1. Reaction time: 30 min at each temperature. Place reaction plates in appropriate temperature-controlled environments.
b.Time Course Optimization: Monitor catalytic activity at 5, 10, 15, 20, 25, 30 min.
Use the same reaction system with optimal temperature from previous experiment. Take absorbance readings at each time point. Document color development progression.

23.Bacterial Detection
Apparatus and Materials:
96-well microplates, Microplate reader, Incubator, PBP solution (10 μg/mL in PBS), HRP-PBP-CaHPO₄@AuPt nanoflowers (1 mg/mL), 0.5 × PBS (0.05% Tween-20), Blocking solution (5% BSA in PBS), TMB solution, H₂O₂ solution, Bacterial samples
Procedure
(1)PBP Immobilization in 96-Well Plates
a.Protein coating: Add 100 μL of PBP solution (10 μg/mL in PBS) to each well of the 96-well plate.
b.Incubation: Cover the plate and incubate overnight at 4°C to allow protein adsorption to the plate surface.
c.Washing: Remove unbound PBP by washing with 200 μL washing solution. Perform 3-5 wash cycles. After each wash, gently tap the plate and invert on absorbent paper to remove residual liquid.
d.Blocking: Add 200 μL blocking solution (5% BSA in PBS) to each well. Incubate at room temperature for 1 h to ensure effective blocking. Store at 4°C if not used immediately (avoid freeze-thaw cycles).
(2)Bacterial Detection Assay
a.Sample Preparation: Prepare bacterial samples at concentrations of 10¹-10⁷ CFU/mL in PBS. Use PBS as negative control. Perform 5 independent experiments, each with triplicate wells.
b.Detection Protocol: Add 200 μL of diluted bacterial sample to each test well. Add 10 μL of HRP-PBP-CaHPO₄@AuPt nanoflowers (1 mg/mL),
c.Incubation: Place in 37°C incubator for 20 min to allow bacterial binding and nanoflower attachment.
d.Washing and Signal Development: Remove liquid and tap dry on absorbent paper. Add 200 μL washing solution to each well. Let stand for 1 minute, then discard washing solution. Tap dry on absorbent paper. Repeat washing 5 times,
e.Colorimetric detection: Add 50 μL TMB substrate and 50 μL H₂O₂ solution to each well. For enhanced color reaction, use H₂O₂ at 0.01%-1% concentration. Incubate at 37°C in the dark for 10 min.
f.Measurement and documentation: Measure optical density at 650 nm using microplate reader. Photograph results with smartphone for visual documentation.
(3)Specificity Test
Test nanoflowers functionalized with TFP/gp17 against:
•E. coli
•B. subtilis
•Mixed culture of E. coli and B. subtilis
- Compare RGB values with controls.
(4)Storage Stability
Store HRP, HRP-TFP-CaHPO₄, and HRP-TFP-CaHPO₄-AuPt NPs at room temperature (25°C) for 4 weeks, measuring their catalytic activity weekly using the same detection method as in point 21.
(5)Real Sample Detection
- Dilute milk/tea with PBS (1:10, w/v); mix thoroughly.
- Spike with bacteria (10⁰, 10², 10⁴ CFU/mL);
- Detect as above.
(6)Data Analysis
a.RGB Analysis Protocol: Use an online tool Image Color Picker to analyze smartphone photographs. Extract Red (R), Green (G), and Blue (B) values from each well. Calculate B/R ratios for enhanced discrimination.
b.Statistical Analysis: Plot bacterial concentration vs. RGB values and B/R ratios. Establish linear relationship equations.
Calculate Limit of Detection (LOD) using the formula:
                                                            LOD = 3 × (SD/K)
Calculate Relative Standard Deviation (RSD) using the formula
                                                            RSD = (SD/mean) × 100%
(Where LOD = Limit of Detection, the lowest concentration or amount that the method can reliably detect. 3 = Confidence level constant representing 99% confidence. SD = standard deviation of blank response. K = slope of calibration curve. Mean = Arithmetic mean value of the dataset).