Buffer S

Buffer SP1

Buffer SP2

Buffer SP3

Buffer DW1

Wash solution

Elution buffer

Absorption column

Collection tube

Centrifuge

EP tube

Agarose Powder

TAE or TBE Buffer

Loading Dye

GelRed (Beyotime- D0139)

Trans15K DNA Marker

Microwave oven

Gel Casting Tray and Comb

UV Transilluminator

Electrophoresis Apparatus

Wash Solution

Buffer B2

Elution Buffer

Column with collection tube

Centrifuge

Razor Blade

Microcentrifuge Tubes

Pipettes and Tips

2× ClonExpress Mix

Linearized Vector

Gene fragment

PCR Tube

Pipettes and Tips

PCR Thermal Cycler

1) Thaw the 2× ClonExpress Mix on ice.
2) Prepare the Cloning Reaction in a PCR tube, set up the reaction as follows:

Reagent	Volume (µl)
Linearized Vector	[0.02 × length bp] ng
Gene fragment	[0.04 × length bp] ng
2× ClonExpress Mix	5
ddH2O	-
Total	10

3) Gently mix the reaction by pipetting up and down. Incubate at 50°C for 5 min.

Competent Cells

SOC Medium

LB Agar Plates with Antibiotics

Water Bath

Incubator

Pipettes and Tips

1) Thaw competent cells on ice. 2) Add 10 μL of cloning reaction to 50 μL of competent cells. Gently mix by flicking the tube. 3) Incubate the mixture on ice for 30 min. 4) Heat-shock the cells at 42°C for 45 sec. 5) Recover Cells: Immediately transfer the cells back to ice for 2 min. 6) Add 700 μL of pre-warmed SOC medium to the cells. 7) Incubate the cells at 37°C with shaking (200-250 rpm) for 1 hour. 8) Spread 200 μL of the transformed cells onto the LB agar plate with kanamycin. 9) Incubate the plates at 37°C overnight.

PCR Master Mix

Primers

PCR Tube

Sterile Pipette Tips

PCR Thermal Cycler

1) Using a sterile toothpick or pipette tip, gently pick a single bacterial colony from the LB agar plate.
2) Suspend the picked colony in 100 μL LB medium with kanamycin in a microcentrifuge tube. Incubate at 37°C for 1 hour.
3) Use 1-2 μL of this bacterial suspension as the template for the PCR reaction.
4) PCR reaction setup as follows

Reagent	Volume (µl)
PCR Master Mix (2×)	5
Primer F	1
Primer R	1
DNA template	1
ddH2O	2
Total	10

5) Mix gently and briefly centrifuge to ensure all components are at the bottom of the tube.
6) Place the PCR tubes in the PCR thermal cycler and set the following parameters:

Process	Temperature	Time	Cycle
Pre-Denaturation	95℃	2 min	1
Denaturation	95℃	10 sec	30
Annealing	70℃	30 sec
Extension	72℃	2.5 min
Final Extension	72℃	10 min	1
Hold	4℃	until samples are removed	1

IPTG

LB medium

Kanamycin

Shaker Incubator

1) Inoculate a single colony of BL21 E. coli containing the plasmid into 10 mL of LB medium with the 50 μg/mL kanamycin.
2) Incubate overnight at 37°C with shaking (200-250 rpm).
3) Dilute the overnight culture 1:100 into 50 mL fresh LB medium containing the 50 μg/mL kanamycin.
4) Incubate at 37°C with shaking until the OD600 reaches 0.4-0.6.
5) Add IPTG to a final concentration of 1 mM to induce protein expression.
6) Incubate at 16°C for 18 hours with shaking.
7) Harvesting Cells: centrifuge the culture at 4,000 ×g for 15 min at 4°C to pellet the cells. Discard the supernatant and proceed to protein purification.

IPTG

Beyotime His-tag Protein Purification Kit (Denaturant-resistant), P2229S

Sonicator

50 mL Centrifuge Tubes

Centrifuge Tubes

Pipettes and Tips

1) Cell Lysis a.Resuspend the cell pellet in 10 mL lysis buffer provided in the Purification Kit. Use non-denaturing condition for tailspike protein and denaturing condition for plyV12 and gp17 protein.
b.(Optional) For non-denaturing conditions: Add lysozyme to a final concentration of 1 mg/mL and gently mix, avoiding the formation of bubbles as much as possible. Place in an ice-water bath or on ice for 30 min.
c.Lyse the cells by sonication on ice (30 sec on, 30 sec off, for a total of 15 min).
d.Centrifuge the lysate at 12,000 ×g for 30 min at 4°C to remove cell debris. Take 100 µL of supernatant as a sample for subsequent analysis, and transfer the remaining supernatant to a new, clean centrifuge tube.
2) Protein Binding
a.Take 1 mL of well-mixed 50% BeyoGold™ His-tag Purification Resin and add it to the column. Add 0.5 mL of lysis buffer, mix to equilibrate, and discard the liquid. Repeat the equilibration 1-2 times and discard the liquid.
b.Add the cell lysis supernatant to a column containing Purification Resin
c.Incubate the mixture at 4℃ with gentle shaking for 1 hour to allow binding.
3) Protein Wash
a.Open the cap at the bottom of the purification column and let the liquid flow out by gravity. Collect approximately 100 µL of the flow-through for subsequent analysis.
d.Wash the resin 5 times, each time adding 0.5-1 mL wash buffer. Collect approximately 100 µL of the flow-through wash buffer each time for subsequent analysis.
4) Protein Elution: Elute the target protein 6-10 times, each time using 0.5 mL elution buffer. Collect each elution fraction into different centrifuge tubes. The collected elution fractions are the purified His-tagged protein samples.

15% SDS-PAGE Color Preparation kit, C671104

Tris-Glycine-SDS buffer

Protein Ladder

Loading Buffer

SDS-PAGE Gel Casting System

1) Prepare a 15% SDS-PAGE gel according to the instructions provided with the 15% SDS-PAGE Color Preparation kit, C671104
2) Mix protein samples with 2× loading buffer and heat at 95°C for 10 min.
3) Load the samples and protein ladder into the wells of the SDS-PAGE gel.
4) Place the gel in the electrophoresis tank and add 1×Tris-Glycine-SDS running buffer.
5) Run the gel at 100 V for the stacking gel and then increase to150 V for the resolving gel until the dye front reaches the bottom.

Coomassie Brilliant Blue R-250 Stain Solution

Destaining Solution

Gel Imaging Systems

1) After electrophoresis, carefully remove the gel from the glass plates and place it in a staining tray.
2) Cover the gel with the staining solution and gently agitate on a shaker for 30 min at room temperature
3) Replace the staining solution with the destaining solution and gently agitate on a shaker.
4) Change the destaining solution periodically until the background is clear and protein bands are distinctly visible.
5) Once the gel is adequately destained, place it in distilled water to remove any residual destaining solution. Visualize the gel using a gel imaging system.

Aurochlorohydric acid (HAuCl4) Solution: 10 mg/mL

Sodium Citrate Solution: 10 mg/mL

Potassium Carbonate Solution: 100 mM

1% Bovine Serum Albumin (BSA)

Phosphate Buffered Saline (PBS)

Round-Bottom Flask

Magnetic Stirrer and Stir Bar

Shaker

Spectrophotometer

Microplate

1) Preparation of Gold Nanoparticles (AuNPs)
a.Add 100 mL of ultra-pure water to a round-bottom flask.
b.Add 1 mL of 10 mg/mL HAuCl4 solution to the flask. And place the flask on a magnetic stirrer and heat the mixture to boiling, allowing it to reflux.
c.After the mixture reaches boiling, add 1 mL of 10 mg/mL sodium citrate solution to the flask.
d.Continue stirring and heating until the solution turns a deep purple-red color, indicating the formation of gold nanoparticles.
e.Continue heating and stirring for an additional 10 min to ensure complete reaction.
f.Remove the flask from the heat and allow the solution to cool to room temperature. Store the solution at 4°C in the dark for future use.
2) Protein Conjugation with AuNPs
a.Adjusting pH of AuNPs Solution: Take 1 mL of the AuNPs solution prepared in the previous steps. Then gradually add 7-8 μL of 100 mM potassium carbonate solution to adjust the pH to 8.0. Monitor pH carefully.
b.Add 100 μg of PBP protein to the pH-adjusted AuNPs solution. Incubate the mixture on a shaker at room temperature for 30 min to allow protein binding.
c.Centrifuge the mixture at 12,000 rpm for 5 minutes at 4°C. Then carefully discard the supernatant to remove unbound protein.
d.Add 1% BSA solution to resuspend the pellet and incubate at room temperature for 30 min to block any remaining binding sites on the AuNPs.
e.Centrifuge again at 12,000 rpm for 5 min at 4°C and discard the supernatant.
f.Resuspend the AuNPs@PBP pellet in 333 μL of PBS buffer.
g.Store the conjugated AuNPs@PBP solution at 4°C for future use.
3) Measurement of AuNPs@PBP Absorbance
a.Scan the full wavelength range to determine the maximum absorption peak of the AuNPs@PBP solution using a spectrophotometer.
b.Note that AuNPs typically have a maximum absorption peak at 530 nm. Successful conjugation with PBP protein should result in a red shift of 2-3 nm in the absorption peak.

Phosphate Buffered Saline (PBS)

Bacterial culture

Smartphone with camera

RGB analysis software or app

Fresh lettuce and apples from a local supermarket

Part 1: AuNPs @ PBP and Smartphone-Based Colorimetric Detection of Bacteria
1) Preparation of Bacterial Suspension:
a.Cultivate the target bacteria to achieve an OD600 of 1 (approximately 10^9 CFU/mL).
b.Collect the bacterial cells by centrifuging the culture and discard the supernatant.
c.Resuspend the bacterial pellet in PBS and adjust the suspension to an OD600 of 1 (approximately 10^9 CFU/mL).
d.Perform serial dilutions in PBS to achieve concentrations ranging from 10^3 to 10^9 CFU/mL.
2) Add 200 μL of each bacterial dilution to 100 μL of AuNPs @ PBP solution and incubate the mixtures at room temperature for 30 min. Include appropriate controls, such as pET28a empty vector controls, to validate the assay specificity and accuracy.
3) After incubation, Transfer 200 μL of the supernatant into new 1.5 mL centrifuge tubes for imaging.
4) Imaging and Data Collection:
a.Assemble the smartphone and lightbox setup. Ensure the lightbox is equipped with a white LED light tube and black curtains to minimize external light interference.
b.Place the centrifuge tubes containing the supernatant in the lightbox. Capture images of the samples using the smartphone camera.
5) Data Analysis
a.Extract the RGB values from the captured images using the RGB analysis software or app.
b.Generate a standard curve based on the RGB values corresponding to known bacterial concentrations.
c.Import the standard curve into the app for subsequent analysis.