1. LB liquid medium

Make 500 mL LB medium by adding 5 g tryptone, 2.5 g yeast powder, 5 g NaCl, and water until LB broth is 500 mL and mixing thoroughly. Sterilize the two LB mediums at 121 °C for 20 min.

2. LB solid medium

Make 200 mL solid LB medium by adding 2 g tryptone, 1 g yeast powder, 2 g NaCl, 3 g agar powder, and water until solid LB is 200 mL and mixing thoroughly. Sterilize the two LB mediums at 121 °C for 20 min. Allow medium to cool to 50-60 °C after autoclaving. Aseptically pour plates in a laminar flow hood. Allow to solidify.

3. His Buffer A:

Add 1.78 g of Na₂HPO₄·2H₂O, 14.6 g of NaCl, and 1.02 g of imidazole. Adjust the pH to 7.4 by adding concentrated hydrochloric acid.

4. His Buffer B:

Add 0.89 g of Na₂HPO₄·2H₂O, 7.3 g of NaCl, and 8.5 g of imidazole. Adjust the pH to 7.4 by adding concentrated hydrochloric acid.

5. PBS Buffer:

Add 16 g of NaCl, 0.4 g of KCl, 2.84 g of Na₂HPO₄, 0.48 g of KH₂PO₄, and 2 L of water.

6. SDS-PAGE gel

1) Prepare resolving gel by mixing 4.9 mL water, 6.0 mL 30% acrylamide, 3.8 mL 1.5 M Tris pH 8.8, 0.15 mL 10% SDS, 0.15 mL 10% ammonium persulfate (APS), and 6 μL TEMED in a tube.

2) Pour the resolving gel solution into the assembled cassette.

3) Prepare stacking gel by mixing 6.8 mL water, 1.7 mL 30% acrylamide, 1.25 mL 1.5 M Tris pH 6.8, 0.1 mL 10% SDS, 0.1 mL 10% APS, and 0.01 mL TEMED.

4) Pour stacking gel solution on top of polymerized resolving gel. Insert comb and allow stacking gel to polymerize.

 

1. Equilibrate the adsorption column

1) Add 500 μL of Buffer S to the adsorption column and place a collection tube below it.

2) Centrifuge at 12,000 ×g for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

2. Transfer the E. coli culture into a 2 mL centrifuge tube, centrifuge at 12,000 ×g for 2 min to collect the bacteria. Discard the supernatant and retain the precipitate.

3. Add 250 μL of Buffer SP1 to the 2 mL centrifuge tube, and thoroughly suspend the precipitate.

4. Add 250 μL of Buffer SP2, and gently invert the tube 5-10 times to mix the solution.

5. Add 350 μL of Buffer SP3 and mix the solution by gently inverting the tube 5-10 times.

6. Centrifuge at 12,000 ×g for 5-10 min. Transfer 700 μL of the supernatant to the adsorption column, centrifuge at 8,000 ×g for 30 sec, and discard the filtrate in the collection tube.

7. Add 500 μL of Wash Solution, centrifuge at 9,000 ×g for 30 sec, and discard the filtrate in the collection tube.

8. Repeat wash step.

9. Centrifuge the empty column at 9,000 ×g for 1 min.

10. Place the column into a clean 1.5 mL centrifuge tube, and add 25 μL of Elution Buffer to the center of the adsorption membrane. Incubate for 1 min at room temperature, centrifuge at 9,000 ×g, and keep the DNA solution in the tube.

 

To construct the pET28a-His-autolysinCBD and pET28a-His-MS18 plasmids, we used the synthesized plasmids containing the His-autolysinCBD or His-MS18 fragments as templates for PCR amplification of the target fragments.

The reaction system for PCR amplification is shown in the table below:

 

The reaction procedure for PCR amplification is shown in the table below:

Notice: Repeat step 2 to step 4 for 30 times.

 

1. The digestion system of the pET28a vector (for autolysinCBD) is shown in the table below:

Reagent	Volume (μL)
pET28a vector (for autolysinCBD)	20
NdeI	1
XhoI	1
Buffer (CutSmart)	5
ddH2O	23
Total	50

The enzyme digestion systems are incubated at 37 ℃ for 3 h.

 

2. The digestion system of the pET28a vector (for MS18) is shown in the table below:

Reagent	Volume (μL)
pET28a vector (for MS18)	20
BamHI	1
XhoI	1
Buffer (CutSmart)	5
ddH2O	23
Total	50

 

1. Preparation of agarose gel

1) Add 50 mL 1× TAE buffer and 0.5 g agarose to a flask, and place it in a microwave oven on high for 2 min.

2) Take it out after the solution turns transparent and shake it evenly.

3) Add 2 μL nucleic acid dye GelRed to the molten agarose.

4) Assemble the gel tray and insert the comb into the mold.

5) Pour the liquid agarose into the mold, avoiding air bubbles.

6) Wait for the solidification of the gel at room temperature.

7) Carefully remove the comb and transfer the solidified gel to the electrophoresis tank.

8) Completely submerge the gel in 1´ TAE buffer.

2. Loading samples

1) Add DNA loading buffer into samples to dye them blue, then load into wells sequentially.

2) Add 10 μL of Trans15K DNA Marker into a lane.

3. Electrophoresis

1) Place wells at the negative electrode and cover the tank.

2) Turn on the power, and run the agarose gel at 120 V for 15 min.

4. Visualizing DNA bands

1) Turn off the power, and remove the gel.

2) Place the gel under the Ultra Slim LED Illuminator to visualize the results.

3) Take out the gel, and put it under the gel-imaging system to save data.

 

1. Excise the DNA band of interest under UV light using a clean scalpel.

2. Transfer the gel slices into centrifuge tubes, and measure the weight of the gel slices.

3. Add 3 times of sample volume of Buffer B2 into the centrifuge tubes, and mix until the gel completely dissolved.

4. Transfer the solution into the absorption column, and centrifuge at 9000 ×g for 30 sec. Pour off the filtrate into the collection tube.

5. Add 500 μL of Wash Solution and centrifuge at 9000 ×g for 30 sec. Pour off the filtrate into the collection tube.

6. Repeat wash step.

7. Centrifuge the empty column at 9000 ×g for 1 min.

8. Place the absorption column into a clean 1.5 mL centrifuge tube, add 15-40 μL Elution Buffer to the center of the absorption membrane, and centrifuge at 9000 ×g for 1 min. Store the DNA solution in the tube.

 

To recombine the pET28a-His-autolysinCBD and pET28a-His-MS18 plasmids, we homologous recombined the target fragment (autolysinCBD or MS18) after gel recovery with the corresponding pET28a vector. We used the ClonExpress Ultra One Step Cloning Kit, and the reaction system is listed below:

Incubate at 50 °C for 5 min; cool to 4 °C or place on ice immediately.

 

1. Add 5 μL of plasmids into 50 μL of E. coli BL21(DE3) competent cells, respectively. Incubate the mixture on ice for 30 min.

2. Heat shock at 42 °C for 45 sec, and put the mixture to the ice for 2 min.

3. Add the LB culture into the tubes to 0.7 mL, and shake the tubes at 37 ℃, 200 rpm for 60 min.

4. Centrifuge it at 5000 rpm for 1 min, remove 600 μL of supernatant, and resuspend the cells in the remaining LB culture.

5. Spread 50 μL cells onto LB agar plates containing kanamycin and incubate overnight at 37 ℃.

 

1. Place 50 μL of Lysis Buffer for Microorganism to Direct PCR in a sterilized microtube. Pick a single colony with a sterilized toothpick or lance tip, place it in the microtube, and stir a few times before removing it.

2. After heat denaturation at 80 °C for 15 min, centrifuge at low speed and take 1~5 μL of supernatant after lysis as PCR template.

The PCR reaction system is shown in the table below:

Reagent	Volume (μL)
Lysis supernatant	1
Premix Taq (Ex Taq Version 2.0)	10
Forward Primer (10 μM)	1
Reverse Primer (10 μM)	1
sterilized water	7

 

The PCR reaction condition is shown in the table below:

Step	Temperature (˚C)	Time (sec)
1	94	600
2	94	30
3	55	30
4	72	120
5	72	300
6	4	¥

Notice: Repeat step 2 to step 4 for 28 times.

 

3. Take positive transformants and add 3 mL LB culture medium containing kanamycin, shake bacteria, and incubate at 37 ℃ at 220 rpm for 12-16 h.

 

1. Inoculate overnight cultures 1: 100 into 200 mL fresh LB culture medium containing kanamycin in conical flasks.

2. Incubate at 37°C, 200 rpm until OD₆₀₀ reaches 0.6.

3. After the temperature of the LB bacterial medium is reduced to 16 ℃, add IPTG to a final concentration of 1 mM.

4. Induction at 16 ℃, 200 rpm for 24 h.

5. Harvest cells by centrifugation at 10000 rpm for 5 min at 4 °C. Discard supernatant.

6. Resuspend the pellet in lysis buffer by adding lysis buffer (containing Tris-HCl with 25 mM imidazole).

7. Sonication of the bacterial resuspension is performed using φ6 probes at 50% power for 50 min.

 

1. Equilibrate 1 mL nickel column with 10 column volumes of ultrapure water, followed by 10 column volumes of His Buffer A at ~1 mL/min flow rate.

2. Load filtered cell lysate supernatant containing target proteins onto the column at 1 mL/min.

3. Wash loaded column with 10 column volumes of His Buffer A at 1 mL/min.

4. Elute proteins with imidazole gradient buffer at 1 mL/min while collecting 1 mL fractions. Store eluates temporarily at 4 °C.

5. Wash the column with His Buffer B, His Buffer A, water, and 20% ethanol sequentially. Store the column in 20% ethanol.

 

1. SDS-PAGE

1) Combine 4 μL loading buffer with 16 μL each purified protein fraction in tubes.

2) Assemble the gel cassette and apparatus.

3) Perform a leak test by adding water to the cassette gap and observing for leaks.

4) Load protein samples into wells of polymerized gel.

5) Run the gel at 80 V for 30 min, then 120 V for 1 h.

6) Stain gel with Coomassie blue. Destain with water and visualize protein bands.

2. BCA Protein Quantification

1) Mix BCA Reagent A with BCA Reagent B at a 50:1 ratio to create the working reagent.

2) Add 25 μL each for all samples and standards into the microplate wells.

3) Add 200 μL of the working reagent into each well.

4) Tap the microplate to mix each well.

5) Cover the microplate with foil to minimize light exposure.

6) Incubate the mixture at 37 °C for 30 min.

7) Measure the absorbance at 562 nm with the microplate reader.

8) Make a graph plotting the concentrations and absorbances

 

1. Prepare 10⁶ CFU/mL Bacillus cereus and Vibrio parahaemolyticus suspensions (i.e. OD₆₀₀=1 and then dilute 1000 times).

2. Test different TMB concentrations from 0.01-0.05% (w/v) as substrate.

3. Select enzyme reaction times of 0.5 h, 1 h, 1.5 h and 2 h.

 

1. Dilute overnight cultures of Bacillus cereus and Vibrio parahaemolyticus in PBS from 10² to 10⁷ CFU/mL.

2. Add 1 mL of each bacterial dilution or PBS control to tubes.

3. Add equal volumes of autolysinCBD or MS18 binding proteins and 1 mg/mL anti-His HRP conjugated antibody.

4. Incubate at 37 °C for 30 min with shaking.

5. Centrifuge at 8000 rpm for 5 min. Wash the pellet with PBS.

6. Resuspend labeled bacteria in 50 μL TMB substrate and transfer to a 96-well plate.

7. React at 37 °C for optimized time based on modeling.

8. Scan colorimetric results with a smartphone app and analyze RGB values.

9. Construct a standard curve of R/B and G/B ratios. Import into the app.

10. App scan of test samples directly outputs bacterial concentration.