Methods

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1. Materials

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1.1 Buffers & Media

1×TAE Buffer (g/L):
Tris: 4.84 g.
EDTA: 0.744 g.
pH: Adjust the pH to 8.5 using glacial acetic acid.
Bring the volume to 1 L with ddH₂O.

LB Medium (g/L):
NaCl: 10.0 g.
Yeast Extract: 5.0 g.
Tryptone: 10.0 g.
Agar: 15.0 g.
pH: Adjust the pH to 7.0.
Autoclave at 121°C for 20 minutes.

LBS (Sucrose Selection) (g/L):
LB + Sucrose: 120.0 g.
pH: Adjust the pH to 7.0.
Autoclave at 121°C for 20 minutes.

TSB Medium (g/L):
Tryptone Soya Broth: 30.0 g.
Agar: 15.0 g.
pH: Adjust the pH to 7.0.
Autoclave at 121°C for 20 minutes.

100 mM MgCl₂ Buffer (g/L):
MgCl₂: 9.521 g.
Dissolve in ultrapure water, then sterilize at 121°C for 20 minutes using an autoclave.

100 mM CaCl₂ Buffer (g/L):
CaCl₂: 11.0984 g.
Dissolve in ultrapure water, then sterilize at 121°C for 20 minutes using an autoclave.

100 mM Glycerol–CaCl₂ Buffer (g/L):
CaCl₂: 11.0984 g.
Glycerol: 100 mL.
Dissolve in ultrapure water, then sterilize at 121°C for 20 minutes using an autoclave.

Sucrose Buffer (g/L):
Sucrose: 102.69 g.
Dissolve in ultrapure water, then sterilize at 121°C for 20 minutes using an autoclave.

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1.2 Antibiotics

E. coli: Km 30 μg/mL; Cm 30 μg/mL; Tc 20 μg/mL; Gm 10 μg/mL; Amp 100 μg/mL.

P. aeruginosa: Km 30 μg/mL; Cm 30 μg/mL; Gm 100 μg/mL; Amp 100 μg/mL; Tc 200 μg/mL (solid)/100 μg/mL (liquid).

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1.3 Culture Conditions

37°C, 220 rpm unless stated otherwise.

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2. Mutant Strain Construction

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2.1 PCR amplification of DNA Fragments

PCR was used to obtain the target DNA fragments. The PCR amplification system is shown in Table 1-1, and the PCR program is shown in Table 1-2.

Table 1-1 PCR Reaction System Table 1-2 PCR Program

Note: The annealing temperature is determined by the F/R primers; the extension time is determined by the length of the DNA fragment and the type of DNA polymerase used.

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2.2 Overlap PCR

Two pairs of primers, up F/up R and low F/low R, were designed to amplify the upstream and downstream sequences of the target DNA fragment, respectively. Using Escherichia coli EC1000 genomic DNA as the template, the upstream (UP) and downstream (LOW) sequences were amplified with the up F/up R and low F/low R primer pairs, respectively. These two amplicons were then used as templates for overlapping PCR with the up F/low R primer pair. The resulting PCR product was analyzed by agarose gel electrophoresis, and the target band was excised and purified to obtain the successfully fused UP–LOW fragment.

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2.3 Gel Extraction

DNA Gel Purification and Recovery. The DNA gel extraction procedure was performed according to the manufacturer's instructions for the DNA purification kit, with the following steps:

1. Activate the DNA adsorption column: Place the column into a collection tube, add 500 μL of BL buffer, centrifuge at 12,000 rpm for 1 min, and discard the flow-through.

2. Weigh the DNA gel slice: Add PC buffer at a ratio of 100 μL per 0.1 g of gel. Incubate the tube in a 50°C water bath until the gel is completely dissolved, gently inverting the tube periodically to accelerate dissolution.

3. Load the sample: Transfer the entire solution to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the flow-through.

4. Wash the column: Add 600 μL of ethanol–PW buffer, centrifuge at 12,000 rpm for 1 min, discard the flow-through, and repeat this step.

5. Dry the column: Centrifuge the empty column at 12,000 rpm for 2 mins, then place it into a new 1.5 mL tube and air-dry at room temperature for approximately 10 mins until all ethanol has evaporated.

6. Elute the DNA: Add an appropriate volume of pre-warmed (70°C) EB buffer to the column, centrifuge at 12,000 rpm for 2 mins, and collect the eluted target DNA fragment.

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2.4 Large-Scale Double Digestion

Table 1-3 Double enzyme digestion reaction system

Component	Volume
10× digestion buffer	R µL
DNA fragment(plasmid)	40 µL
Restriction endonuclease A	4 µL
Restriction endonuclease B	4 µL
ddH2O	Up to 200 µL

R was determined by the type of restriction endonuclease. The condition of enzyme digestion is 37 ℃ reaction for 8-16h.

2.5 Digestion Clean-up

Similar to gel extraction but add equal volume PC buffer to digestion mix before loading.

2.6 Ligation

Table 1-4Ligation system

Component	Volume
10×T4 DNA Ligase Buffer	2 µL
Vector	2 µL
T4 DNA Ligase	1 µL
DNA fragment after enzyme digestion	15 µL

Incubate 16°C 8–16 h.

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2.7 Preparation of Competent E. coliont> Cells

A single colony was picked from a freshly streaked E. coliont> growth plate after 12 h of incubation, inoculated into fresh LB liquid medium without antibiotics, and cultured with shaking for 12 hours.

The bacterial culture was then transferred at a 1% ratio into 50 mL of fresh LB liquid medium and grown with shaking until the logarithmic phase (OD₆₀₀ ≈ 0.4).

The bacterial suspension was transferred into a pre‑chilled 100 mL centrifuge tube, placed on ice for 30 mins, centrifuged at 4,100 rpm for 10 mins at 4°C, and the supernatant was discarded.

Pre‑chilled 100 mM MgCl₂ buffer (10% of the original culture volume) was added to resuspend the cells, followed by incubation on ice for 10 mins. The suspension was centrifuged at 4,100 rpm for 10 mins at 4°C, and the supernatant was discarded.

Pre‑chilled 100 mM CaCl₂ buffer (4% of the original culture volume) was added to resuspend the cells, followed by incubation on ice for 30 mins. The suspension was centrifuged at 4,100 rpm for 10 mins at 4°C, and the supernatant was discarded.

Finally, pre‑chilled 100 mM glycerol–CaCl₂ buffer (4% of the original culture volume) was added to resuspend the cells. Aliquots of 100 μL were transferred into pre‑chilled 1.5 mL centrifuge tubes and stored at −80°C.

Heat Shock Transformation of Competent E. coliont> Cells

3 μL of plasmid (20 μL from the DNA ligation reaction described in section 2.2.8) was added to the competent E. coliont> cells prepared above.

The transformation mixture was incubated on ice for 30 mins, immediately heat‑shocked in a 42°C water bath for 90 s, and then returned to ice for 5 mins (temperature changes during this process should be rapid).

Approximately 0.8 mL of fresh LB medium was added to the transformation mixture, which was then incubated with shaking at 37°C for 60 mins (90 mins for ligation reaction mixtures).

Subsequently, about 100 μL of the bacterial suspension was evenly spread onto agar plates containing the appropriate antibiotic (for ligation reaction mixtures, the cells were collected by centrifugation before plating). The plates were incubated upside‑down at 37°C.

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2.8 Recombinant Vector (or Plasmid) Extraction

The recombinant vector (or plasmid) extraction procedure was performed according to the manufacturer's instructions for the Plasmid Mini‑Prep Kit (Tiangen Biotech Co., Ltd.), with the following steps:

Transfer 5 mL of bacterial culture into a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 1 min to collect the cells.

Add 250 μL of RnaseA‑P1 buffer to the tube and resuspend the cells completely.

Add 250 μL of P2 buffer, gently invert to mix, and incubate to ensure complete cell lysis.

Add 350 μL of P3 buffer, gently invert to mix, and centrifuge at 12,000 rpm for 10 mins at 4°C.

Activate the adsorption column: Place the column into a collection tube, add 500 μL of BL buffer, and centrifuge at 12,000 rpm for 1 min.

Transfer the entire supernatant into the adsorption column (avoiding the white precipitate), centrifuge at 12,000 rpm for 1 min, and discard the flow‑through.

Add 500 μL of PD buffer, centrifuge at 12,000 rpm for 1 minute to remove proteins and discard the flow‑through.

Add 600 μL of ethanol–PW buffer, centrifuge at 12,000 rpm for 1 min, discard the flow‑through, and repeat this step.

Centrifuge the empty column at 12,000 rpm for 2 mins, then place it into a new 1.5 mL tube and air‑dry at room temperature for approximately 10 mins until all ethanol has evaporated.

Add an appropriate volume of pre‑warmed (70°C) EB buffer to the column, centrifuge at 12,000 rpm for 2 mins, and collect the eluted recombinant vector (or plasmid).

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2.9 Small‑Scale Double Digestion

The reaction system for the small‑scale double digestion used in this study is shown in Table 1‑3.

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2.10 Conjugation

The conjugative plasmid from section 2.8 was transformed into Escherichia coli S17‑1 (hereinafter referred to as S17‑1) to serve as the donor strain, with Pseudomonas aeruginosa as the recipient strain.

Culture both the donor and recipient strains with shaking at 37°C and 220 rpm until reaching the logarithmic phase. Mix the donor and recipient cultures at volume ratios of 3:1 and 5:1 in sterile 1.5 mL centrifuge tubes, centrifuge at 5,500 rpm for 5 mins, and discard the supernatant.

Add 1 mL of fresh TSB liquid medium to the donor pellet, resuspend, transfer to the recipient pellet, mix thoroughly, centrifuge at 5,500 rpm for 5 mins, and discard the supernatant.

Add another 1 mL of fresh TSB liquid medium to the mixture, resuspend, centrifuge at 5,500 rpm for 5 mins, and discard the supernatant.

Add 100 µL of fresh TSB liquid medium to resuspend the cells, then spread the entire suspension onto a non‑selective TSB agar plate. Incubate upright at 37°C for 2 days.

Resuspend the bacterial lawn with an appropriate amount of fresh TSB liquid medium, dilute, and spread onto selective agar plates containing the appropriate antibiotics. After approximately 2 days of incubation at 37°C, scrape the bacterial lawn, dilute appropriately, and spread onto the corresponding selective plates.

Heat Shock TransformaE. coli

3 μL of plasmid (20 μL from the DNA ligation reaction described in section 2.2.8) was added to the competent E. coli cells prepared above.

The transformation mixture was incubated on ice for 30 mins, immediately heat‑shocked in a 42°C water bath for 90 s, and then returned to ice for 5 mins (temperature changes during this process should be rapid).

Approximately 0.8 mL of fresh LB medium was added to the transformation mixture, which was then incubated with shaking at 37°C for 60 mins (90 mins for ligation reaction mixtures).

Subsequently, about 100 μL of the bacterial suspension was evenly spread onto agar plates containing the appropriate antibiotic (for ligation reaction mixtures, the cells were collected by centrifugation before plating). The plates were incubated upside‑down at 37°C.

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3. P. aeruginosa Detection (National Standard Method)

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3.1 Water Sample Filtration

All operations were performed in a Class 100 clean bench.

A 250 mL water sample was filtered through a 0.45 μm membrane filter.

The filter membrane was placed onto a CN agar plate, ensuring no air bubbles were trapped.

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3.2 Cultivation

The plates were inverted and incubated at 36°C ± 1°C for 24–48 h.

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3.3 Result Observation and Colony Counting

Colonies were observed at 20–24 h and again at 40–48 h.

Blue/green colonies (pyocyanin-producing) were counted and subjected to the pyocyanin test.

Fluorescent non-blue-green colonies were counted and subjected to the acetamide broth test.

Reddish-brown colonies were subjected to oxidase, acetamide, and King’s B tests.

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3.4 Confirmatory Tests

Nutrient agar: Purification of suspected colonies.

Oxidase test: A purple color change within 10 s was considered positive.

King’s B medium: Fluorescence was examined under UV light.

Pyocyanin test: Extraction with chloroform followed by the addition of HCl; pink color was considered positive.

Acetamide broth test: Nessler’s reagent was added; The color change from yellow to brick-red was considered positive.

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3.5 Counting and Reporting

The count was calculated using the formula:

N=P+F(cFnF)+R(cRnR)N=P+F(nFcF)+R(nRcR)

The results were reported in CFU/250 mL.

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4. Medium Optimization

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4.1 PB/MMO Mixed Medium

Optimization included:

Ratio adjustment； Ion substitution；Carbon and nitrogen source combinations；Sodium bicarbonate/sodium nitrate gradients；Oxygenation treatments；Hydrogen peroxide addition；Oxygen-releasing tablet tests

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4.2 LB/TSB/BHI Comparison

Addition of H₂O₂ and oxygen-releasing tablets; BHI was ultimately selected as the optimal medium.

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4.3 BHI Optimization

UV-treated oxygen-releasing tablets；HEPES addition；Reporter gene plasmid introduction；Potassium nitrate gradients

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5. Experimental Validation

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5.1 Fluorescence Validation

After constructing the target plasmid, it was transformed into pre-prepared E. coli via heat shock. The transformed bacterial suspension was spread onto LB plates containing the appropriate antibiotic and incubated at 37°C overnight. Single colonies were picked and inoculated into 5 mL liquid LB medium. The appropriate signaling molecule PQS and specific substrate ONPG, or the virulence factor PYO and specific substrate MUG, were added. The cultures were sealed in quantitative plates and incubated at 37°C for 12–16 hours. After incubation, color changes were observed, and fluorescence was examined under UV light.

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5.2 Enzyme Activity Validation

A single colony of the Pseudomonas aeruginosa strain to be tested was inoculated into 5 mL liquid LB medium and cultured overnight at 37°C with shaking until reaching the stationary phase. OD₆₀₀ was measured. A 50 μL aliquot of the bacterial suspension was transferred to a 1.5 mL centrifuge tube, followed by the sequential addition of 420 μL Z Buffer, 20 μL chloroform, and 10 μL 0.1% SDS. After mixing, the tube was incubated in a 30°C incubator for at least 1 hour. Meanwhile, a 0.004 g/mL ONPG substrate buffer was prepared as required, fully dissolved, and also incubated at 30°C for 30 minutes. After incubation, 100 μL of the prepared ONPG substrate buffer was added to each tube, timing was started immediately, and the tubes were returned to the 30°C incubator for 30–90 minutes. The reaction was terminated by adding 250 μL of 1 mol/L Na₂CO₃, and the reaction time was recorded. Finally, OD₄₂₀ and OD₅₅₀ of the reaction solution were measured.

β-galactosidase activity was calculated using the formula:

Miller units =1000×（OD420–1.75×OD550）/[（culture OD600）×（Volume of culture）×（Reaction time）]

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References

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