1.DNA Synthesis

Primer synthesis service is provided by Sangon Biotech (Shanghai) Co., Ltd. Gene synthesis service is provided by Genscript.

2.PCR

Preparation: The simple fragment amplification system is generally 50 μL, and the components of the non-50 μL system can be increased or decreased in proportion. Using different DNA polymerases, the optimal amount of template and primer may vary. 2X Phanta Max Master Mix is commonly used in our laboratory. The following is the general reaction system for PCR using this enzyme.

When the fragment size is upon 8,000 bps, a two-steps-PCR is applied in experiment, The PCR programme is as followed.

3. Homologous Recombination

Two-fragment recombination is performed using ClonExpression® II One Step Cloning Kit from Vazyme biotech co., ltd and following the provided protocol.

4. Plasmid Extraction

Plasmid Extraction is performed using Plasmid Mini Kit I from Omega Bio-Tek biotech co., ltd and following the provided protocol.

5. Gel Extraction

Gel Extraction is performed using SanPrep Column DNA Gel Extraction Kit from Sangon Biotech (Shanghai) Co., Ltd and following the provided protocol.

1. Bacillus subtilis was cultured in LB medium in a shaker at 200 rpm at 37°C overnight;

2. Transfer 1/100 to growth medium for culture at 200 rpm on a shaking table at 37°C;

3. Culture to OD₆₀₀ about 0.5;

4. Ice cooling for 20-30 mins (to stop the growth of bacteria, try not to be too long);

5. Centrifugal force 5000 rpm (large centrifuge) at 4°C for 10 min;

6. Wash three times with wash buffer;

7. Resuspension in the washing buffer at 1/100 of the original culture volume;

8. Pack 120 μL per centrifuge tube into 1.5 mL centrifuge tube, freeze liquid nitrogen and store in -80℃ refrigerator.

Wash buffer:

Heat shock method

1. Remove the competent cells from -80℃ and immediately melt them in an ice water bath for 5 min.

2. Add the transposition DNA into 100 μL competent cells, gently stir well, and incubate on ice for 30 min.

3. Place the centrifuge tube in the 42℃ water bath without shaking. After the heat shock of 90 s, immediately place it in the ice water bath for 2-3 min.

4. Add 900 μL LB medium preheated at room temperature or 37℃ to the centrifuge tube, and then put it in a 37℃ shaker for 45 min.

5. Take different volumes of transformation products, spread them on the correct resistant plates with sterile coating sticks, and incubate them overnight at 37℃ in an incubator.

Electric shock method

1.Choose a 1 mm shock cup.Remove the cup from the ethanol, wash it with pure water, and dry it, and pre-cool it in a -20℃ refrigerator for 20 mins.

2.Remove the competent B.subtilis 168 from the -80℃ ultra-low temperature refrigerator and put on the ice to thaw.

3.Add the 40 ng transformation plasmid into the competent cells, gently tap and mix, then remove the receptive state night and add it into the narrow gap of the shock cup.

4.Turn on the BIO-RAD electroshock converter, place electric shock cup in the slot of the electrocardiograph, and the set voltage to 1250V for electric shock conversion.

5.Add 500 μL resuscitation medium to the shock cup, transfer the bacterial solution to the EP tube, and resuscitated for 3 hrs on a shaking table at 37℃, 200 rpm.

Resuscitation medium:

SDS-PAGE

1. Sample processing: Labeled, 200 μL culture medium supernatant was removed and added with 50ul 5x Loading Buffer, the rest was discarded, and the precipitate was resuspended with 200 μL 1xLoading Buffer. The supernatant was vortexed and mixed, and the precipitate was blown and mixed. They were divided into whole bacteria, supernatant and precipitate. The samples were boiled at 95-100° C for 10 min, and stored at -20°C for a short time. The samples were removed before protein electrophoresis and centrifuged at 12000rpm and 25°C for 5-10 min.

2. According to the instructions of the electrophoresis device, install a clean and dry glass plate, and detect leakage.

3. The separation gel ( 12% ) was prepared, the ingredients were quickly vortexed and mixed after addition, and carefully injected into the prepared glass plate gap (about 5 mL) with a pipettor, leaving enough space for the concentrated gel. A thin layer of isopropanol is gently added to the top layer to remove air bubbles, flatten the separation gel, and prevent the oxygen in the air from inhibiting the polymerization of the gel. After about 1 h, the gel polymerization was completed, the isopropyl alcohol was poured off, the residual isopropyl alcohol at the top was blotted by filter paper, and the glass plate was placed upside down on the paper to drain. Prepare the comb.

4. Prepare concentrated gel ( 5% ), the pH of buffer used in the two gel is different, and each component is quickly vortexed and mixed after adding, and it is infused into the separation gel with a pipettor (about 2 mL). After filling, it is quickly and carefully inserted into the sampling comb, first inserted on one side and then on the other side to avoid bubbles as much as possible.

5. After the concentrated gel solidifies (about 30min), carefully pull out the comb.

6. The gel was fixed on the electrophoresis device, and enough electrophoresis buffer was added, and 10 μL of each sample was added to the sampling well

8. When the sample is electrophorezed in the concentrated gel, the voltage of 80 V can be used and the time can be set to 6 h in order to stop and pause manually. Then the power supply was turned off and the electrophoresis buffer was recovered.

9. Remove the gel, immerse it in at least 5 times the volume of Coomassie Brilliant Blue R-250 staining solution, place it on a horizontal shaker for staining at room temperature for at least 4 h, then remove the stained gel and withdraw the staining solution for re-use, immerse the gel in Coomassie Brilliant blue decolorization solution, decolorize it for 4-8 h on a horizontal shaker, and remove the dye solution for further use. During this period, the decolorization solution was changed 3-4 times until the gel was decolorized until the band was clear. The results were observed and recorded and photographed.

Western Blot

1. Sample processing: Labeled, 200 μL supernatant was removed and added to 50 μL of 5x Loading Buffer, the rest was discarded, and the precipitate was resuspended in 200 μL of 1xLoading Buffer. The supernatant was vortexed to mix and the precipitate was blown to mix. They were divided into whole bacteria, supernatant and precipitate. The sample was boiled at 95-100 ℃ for 10 min. It could be stored at -20℃ for a short time, removed before protein electrophoresis, and centrifuged at 12000 rpm and 25℃ for 5-10 min.

2. According to the instructions of the electrophoresis device, install a clean and dry glass plate to detect leakage.

3. The separation gel ( 12% ) was prepared, the ingredients were quickly vortexed and mixed after addition, and carefully injected into the prepared glass plate gap (about 5 mL) with a pipettor, leaving enough space for the concentrated gel. A thin layer of isopropanol is gently added to the top layer to remove air bubbles, flatten the separation gel, and prevent the oxygen in the air from inhibiting the polymerization of the gel. After about 1 h, the gel polymerization was completed, the isopropyl alcohol was poured off, the residual isopropyl alcohol at the top was blotted by filter paper, and the glass plate was placed upside down on the paper to drain. Prepare comb.

4. Prepare concentrated gel ( 5% ), the buffer pH used in the two gel is different, and the components are quickly vortexed and mixed after adding, and are infused into the separation gel with a pipet (about 2 mL). After filling, quickly and carefully insert the sampling comb, first insert one side and then insert the other side to avoid bubbles as much as possible.

5. The gel was fixed on the electrophoresis device, and enough 1*Gly electrophoresis buffer was added, and 10 μL of each sample was added to the sampling well.

6. Load sample.

7. Prepare the membrane transfer buffer, prepare the transfer tank, enamel plate, sponge pad, etc.

8. After turning off the power, cut off the concentrated gel, pry open the glass, cut off the unpointed belt, and keep the gel moist with the membrane buffer during the process.

9. Measure the size of the gel, cut out a PVDF membrane slightly larger than the gel, and put the membrane into the methyl alcohol 20 s.

10. Successively add film, gel, add another sponge pad buckle, clamp the black face groove black. face, clamp the white face groove red face into the transfer tank, add the transfer buffer.

11. Electrophoresis at 60 V (400 mA) for 1.5h and put ice box ice bath in the tank.

12. Block with bovine serum albumin (BSA) for 1h by shaking on a shaker.

13. Incubate with primary antibodies and eluted three times with EZ-Buffers H 10X TBST Buffer.

14. Incubate with secondary antibodies and eluted three times with EZ-Buffers H 10X TBST Buffer.

15. Developing.

Iodixanol density gradient centrifugation was performed according to OptiPrep^TM kit steps

1. Prepare a solution in PBS buffer containing 1 mM magnesium chloride and 2.5 mM potassium chloride, referred to as PBS-MK;

2. Prepare a 54% (W/V) iodixanol working solution in PBS-MK, referred to as Solution E;

3. Prepare a 15% iodixanol solution in PBS-MK solution supplemented with 1 M sodium chloride, and prepare 25%, 40%, and 50% (W/V) iodixanol solutions in PBS-MK solution;

4. Sequentially layer 15%, 25%, 40%, and 50% iodixanol solutions into a centrifuge tube in a 1:3:2:1 ratio, with a total volume of 7 mL. Subsequently, add 5 mL of the liquid to be centrifuged on the top layer.

5. Use a Thermo WX 90+ ultracentrifuge equipped with a TH-641 rotor, centrifuge at 4°C and 36,000 rpm for 4 hrs. Collect the band between the 25% and 40% layers, resuspend the collected sample, and then store it at -80°C.

Staining and Observation by Transmission Electron Microscope at 120 kV

1. Pipette 10 μL of the sample onto a copper grid and let it sit for 30 s to allow the sample to fully settle into the grid;

2. Use filter paper to blot dry the sample liquid, then add 10 μL of negative staining solution, stain for 30 s, and subsequently use filter paper to blot dry the staining solution;

3. Secure the stained copper grid on a holder, place it on the specimen stage, and operate the transmission electron microscope to observe the sample.

1. Protein Quantification and Preparation: The purified protein sample containing SpyCatcher-VDAL-CPPs was quantified using the Bradford assay to a concentration of 100 μg/mL.

2. Plant Material Preparation: Three healthy Arabidopsis thaliana Columbia wild-type plants of similar size and growth stage were selected. Leaves of comparable size and shape were excised and washed three times with ddH₂O, then blotted dry. Three leaves were grouped together, and a total of four groups were prepared.

3. Treatment: Each group of leaves was incubated for 36 h at 28°C in one of the following solutions: ddH₂O, 100 μM salicylic acid (SA) solution, 50 μg/mL SpyCatcher-VDAL-CPPs protein solution, or a mixture of 100 μM SA solution and 50 μg/mL SpyCatcher-VDAL-CPPs protein solution.

4. DAB Staining: After incubation, the leaves were washed three times with ddH₂O and blotted dry. Samples were immersed in DAB staining solution and subjected to negative pressure (-0.1 MPa) for 30 mins, followed by room temperature incubation in the dark for 12 hrs until positive sites appeared dark brown. Samples were then rinsed three times with ddH₂O and blotted dry.

5. Decolorization: Samples were immersed in tissue decolorization solution and incubated at 75°C for 30 mins until the background color was completely removed.

6. The samples were then rinsed three times with distilled water and blotted dry. Subsequently, they were immersed in a tissue preservation solution for 30 mins before being imaged. (DAB staining kit and procedure were obtained from Beijing Solarbio Science & Technology Co., Ltd.)

7. All leaves were arranged on a white background and flattened to ensure complete expansion.(Note that the ddH₂O and SA groups in this experiment served as negative and positive controls, respectively, and were identical to those used in the BBa_K5335026 element verification.)

8. The acquired images were imported into ImageJ software and converted to 8-bit grayscale images. Subsequently, the images were inverted. Given that DAB staining results in the deposition of brown-colored precipitates in regions with reactive oxygen species (ROS), with the intensity of the color directly correlating with ROS levels, the inverted images displayed lighter regions corresponding to higher ROS content.

9. Using the grayscale measurement tool in ImageJ, the contours of each leaf were outlined, and the average grayscale value for each leaf was calculated. After data normalization and analysis.

96-Well Microplate Assay for Bacterial Biofilms

1. Add 100 μL of culture medium to each well of a 96-well polystyrene microplate, inoculate with 10 μL of overnight culture, and incubate at 37°C for 36 h;

2. Aspirate the culture medium, and wash each well with 200 μL of sterile PBS buffer three times;

3. Add 100 μL of 1% crystal violet solution to each well and stain at room temperature for 5 min;

4. After aspirating the crystal violet staining solution from the culture wells, rinse off the excess dye with running water;

5. Invert the microplate on filter paper to remove residual water, and dry in a 37°C oven or at room temperature;

6. After complete drying, add 100 μL of 33% glacial acetic acid to each well, and let it act in a 37°C incubator for 30 min to dissolve the crystal violet;

7. Measure the OD value of the solution in the culture wells under 590 nm conditions using an enzyme-linked immunosorbent assay (ELISA) reader;

8. For each strain in each experiment, perform multiple wells of repetition, and take the OD average value of three intact biofilm random samples (D value);

9. Use the uninoculated culture medium as a negative control, and twice the negative value as the cutoff value (Dc).

Based on the D value, strains can be categorized into three types

1. Strong biofilm-forming strains (D > 2xDc);

2. Weak biofilm-forming strains (Dc < D ≤ 2xDc);

3. Non-biofilm-forming strains (D ≤ Dc).

Aerobic induced lysis experiment

1. The B.subtilis 168 liquid containing the recombinant plasmid was cultured in 5 mL LB medium until OD₆₀₀ was about 1.0

2. Divide the bacterial solution into two parts, adding 60 mM/L D-xylose to one part and leaving the other part untreated.

3. After mixing, determine the OD₆₀₀ value of two bacterial solutions. Each bacterial solution was sampled at 100 μL each time.Place bacterial solution in a 37℃ shaker and cultured at 120 rpm. Determine with an enzyme marker every 40 min for 8 consecutive times.

Hypoxia induced lysis experiment

1. B.subtilis 168 bacterial solution containing recombinant plasmid and without recombinant plasmid was added into 10 mL bacterial bottle containing 5 mL LB medium, and No. 1-8, No. 1, 2, 3 and 4 were common strains; 5, 6, 7 and 8 were recombinant strains.

2. Add 60 mM/L D-xylose to No. 2 and No. 6, add 120mM/L D-xylose to No. 3 and No. 7, add 180 mM/L D-xylose to No. 4 and No. 8.

3. Eight bacterial vials were placed in a 37℃ shaker and cultured at 120rpm for 12 hrs. After that, the OD₆₀₀ value of each sample was measured, and three samples of each bacterial solution were taken, each of which was 100 μL.

Xylose induced bacterial expression experiment

1. The glycerol bacteria were resuscitated by the bacteria bottle, placed on the oscillator, cultured the engineered bacteria at 37°C for 4-6 hrs until the OD₆₀₀ of the bacterial solution reached about 0.5;

2. Adding 60 mM/L xylose to induce the expression of KillerRed in engineered bacteria. If a control group is required, an equal amount of sterile water can be added for comparison;

3. Continue to culture the engineered bacteria on the oscillator at 37°C for 10-12 h, and the bacterial solution in the induction group is significantly redder than that in the control group, which can be used for the subsequent bacterial suicide experiment.

Color light sensitivity experiment

1. The enzyme label plate is partitioned for color processing, with nine holes as a color block and the most central hole as the experimental hole, so as to avoid interference from other colors;

2. Add 50 μL of bacterial solution to each experimental hole, and place an artificial light source directly on the enzyme-label plate, so that the bacterial solution is exposed to different colors of light;

3. After lighting, let it stand for 3 hrs. Then measure the OD₆₀₀ of the bacterial solution in an automatic microplate reader (Synergy^H1 hybrid multimodal reader), compare the difference in growth values.

Photoinduced bacterial suicide experiment

1. The bacterial solution was placed under different experimental conditions with a bacterial bottle. The Control group was treated with Light protection, the Green Light group was irradiated with artificial green light (light intensity =3000 lux), and the Natural Light group was irradiated with natural light (light intensity = 3000 lux).

2. The bacterial liquid was sampled every half hr and repeated for three groups. OD₆₀₀ was measured by using automatic microplate reader (Synergy^H1 hybrid multimodal reader), and the results were analyzed by drawing.

1. Inoculate single colonies with successful plasmid construction into LB medium.

2. After 12 h of cultivation, inoculate the bacterial liquid into fresh LB liquid medium at a ratio of 100:1, and add SA to make the concentration of SA in the medium 0 μM, 0.1 μM, 1 μM, 10 μM, 100 μM, and 1000 μM respectively. Use the engineered bacteria without the P_sal promoter as a blank control.

3. Take 200 μL and add it to a 96-well plate, set up five parallel replicates for each concentration, and run the plate reader under conditions of 37°C and 200 rpm for 4 hrs, measuring the fluorescence intensity at 486 nm every 10 mins.

Crude extraction of TAA

1. Pick a single colony of successfully recombined Bacillus subtilis 168 from a plate and inoculate it into a PA bottle containing 5 mL of LB liquid medium (composition: 10 g tryptone, 5 g yeast extract, 10 g NaCl, distilled water up to 1000 mL; adjust pH to 7.0-7.2).
2. Incubate with shaking at 28°C, 200 rpm, for 12 hrs. Then, transfer to 50 mL of fresh LB liquid medium at an inoculation volume ratio of 1/100 (v/v) and ferment at 28°C, 200 rpm, for 24 hrs.
3. Transfer 20 mL of the bacterial culture into a 50 mL EP tube and centrifuge at 10,000 rpm for 5 mins. Collect the supernatant for further use.
4. Transfer 2 mL of the supernatant to a new 50 mL EP tube, then add 18 mL of acetone to achieve a final acetone concentration of 90%.
5. Vortex vigorously for 1 min, then centrifuge at 12,000 rpm for 6 mins. Discard the supernatant and retain the brown precipitate. Place the centrifuge tube containing the precipitate in a 37°C oven and dry completely.
6. Dissolve the precipitate in 3 mL of deionized water, then add 2 mL of acetonitrile to achieve a final acetonitrile concentration of 40%. Vortex to mix thoroughly, and centrifuge at 12,000 rpm for 6 mins.
7. Transfer 2 mL of the supernatant into a new centrifuge tube, add 18 mL of acetonitrile to achieve a final acetonitrile concentration of 90%, vortex thoroughly, and centrifuge at 12,000 rpm for 6 mins. Discard the supernatant, retain the brown precipitate, and air dry it naturally. The precipitate is the crude TAA extract.

HPLC detection conditions

1. HPLC Instrument: Prominence LC-20A
2. Column: Agilent TC-C18 column (25 cm × 4.6 mm, 5 μm)
3. Injection Volume: 10 μL
4. Detection Wavelength: 260 nm
5. Flow Rate: 1 mL/min
6. Mobile Phase: 4.65 g/L ammonium acetate + 10% methanol, adjusted to pH 3.5 with acetic acid, degassed by ultrasound for 10 mins before use.
7. Samples for Detection: TAA standards at concentrations of 0.1 μM, 1 μM, 10 μM, 100 μM, and 1 mM, as well as the TAA crude extract dissolved in 1 mL of mobile phase.