Engineering

Phage Engineering

Overview

Following an exhaustive review of the literature, we initially targeted four genes, wspF , yhjH , aiiA , and ytnP , for editing. Employing the introduction of high-copy-number plasmids for indirect validation, coupled with the elimination of the pf4 sequence for verification, we demonstrated the feasibility of all four genes as biofilm repressing elements. However, due to the high failure rate of homologous recombination, successful integration into the genome was achieved only for aiiA and yhjH .

1、Electroporation-mediated Transfer of wspF , yhjH , aiiA , and ytnP Gene into pHERD20T Plasmid of P. aeruginosa PAO1

a) Preparation of Competent Cells:

• Grow a fresh overnight culture of P. aeruginosa PAO1 in LB broth at 37°C with shaking at 200 rpm.
• Harvest the cells by centrifugation at 4000 x g for 10 minutes at 4°C.

b) Restriction Digestion and Gel Purification:

• Digest the pHERD20T vector and aiiA /yhjH /wspF /ytnP genes PCR product with suitable restriction enzymes.
• Run the digestion products on an agarose gel and extract the desired fragments using a gel extraction kit.

c) Ligation:

• Ligate the aiiA /yhjH /wspF /ytnP gene fragment into the pHERD20T vector using T4 DNA ligase at 16°C overnight.

d) Transformation into E. coli :

• Transform the ligation mixture into competent E. coli cells (DH5α) via heat-shock method, and select for transformants on LB agar plates.

e) Preparation of pHERD20T-aiiA /yhjH /ytnP /wspF Plasmid for Electroporation:

• • Extract the pHERD20T-aiiA /yhjH /ytnP /wspF plasmid from positive E. coli colonies.

f) Electroporation into P. aeruginosa PAO1:

• Mix 1-2 µl of the pHERD20T-aiiA /yhjH /ytnP /wspF plasmid with 50 µl of competent P. aeruginosa PAO1 cells.
• Transfer the mixture into a pre-chilled electroporation cuvette and electroporate at 2.5 kV, 200 Ω, 25 µF.
• Immediately add 1 ml of SOC medium and incubate at 37°C for 1 hour with shaking.
• Spread the cells onto LB agar plates supplemented with carbenicillin and incubate at 37°C overnight.

g) Confirmation of electroporation:

We herein report the successful electroporation-mediated transfer of wspF , yhjH , aiiA , and ytnP genes into the pHERD20T plasmid, as evidenced by PCR analysis (Figure 1).

2. Deletion of pf4 Phage Sequence and Plasmid Integration in Pseudomonas aeruginosa PAO1

a) Deletion of pf4 Phage Sequence:

Use a SacB system for homologous recombination to facilitate the deletion of the pf4 sequence. Perform PCR to obtain the flanking regions, purify the PCR products and ligate them to form a deletion construct. Transform the deletion construct into competent P. aeruginosa PAO1 cells and select for successful recombinants on LB agar plates.

b) Preparation of pHERD20T-aiiA /wspF Plasmid:

• Clone aiiA and wspF genes into pHERD20T vector using restriction enzymes and T4 DNA ligase.
• Transform the ligated plasmid into competent E. coli cells and select for successful transformants on LB agar plates supplemented with carbenicillin.
• Extract the recombinant plasmid from positive E. coli colonies using a plasmid extraction kit.

c) Electroporation into P. aeruginosa PAO1:

• Prepare competent P. aeruginosa PAO1 cells as described in the prior protocol.
• Mix 1-2 µl of the pHERD20T recombinant plasmid with 50 µl of competent P. aeruginosa PAO1 cells.
• Electroporate the mixture and recover the cells in SOC medium, followed by selection on LB agar plates supplemented with carbenicillin.

d) Verification of Gene Incorporation:

• Perform colony PCR to confirm the integration of aiiA and wspF genes into the P. aeruginosa PAO1 genome.

Here, we have proficiently engineered the pf4 mutant strains with the introduction of high-copy number plasmids pHERD20T-aiiA /wspF (Figure 3).

3. Integration of aiiA and yhjH Target Genes into the PAO1 Genome through Homologous Recombination

Initially, we employed PCR to selectively amplify our designated genetic targets, yhjH and wspF , followed by purification of the amplified products (Figure 5). Custom synthesis of the genetic constructs for aiiA and ytnP , inclusive of their respective homologous arms, was performed by Sangon Biotech. Subsequent steps involved the assembly of genetic material through the Gibson assembly technique and its introduction into DH5α competent cells utilizing the heat shock method. Verification of successful construct integration was conducted using PCR and electrophoresis assessments (Figure 6). The culmination of the process saw the successful conjugation of yhjH and aiiA in the PAO1 genome, confirmed through further PCR validations (Figure 7).

Experiments

Results

1. Phage Engineering

Please refer to the Wet Lab-Engineering page for details.

2. Phage Characterization

a) Initially, we performed polymerase chain reaction (PCR) analyses to ascertain the precise construction of our engineered strains (Figure 3.1). The acquired data compellingly delineated the successful integration of all target genes into the prophage pf4 locus within the P. aeruginosa PAO1 genome.

b) Subsequently, we aimed to ascertain the liberation efficacy of the engineered phages. The phage plaque assay showed that plaques were manifested by the yhjH and aiiA integrated engineered strains, as well as in strain carrying yhjH gene in the pHERD20T plasmid (Figure 3.2). This observation confirmed that the phage engineering endeavor did not impair the phage viability of the strains, and maintained the same release efficacy of the engineered phages.

3. Gene expression Analysis

a) qPCR

Initially, our objective was to ascertain the successful transcription of genes aiiA , ytnP , yhjH and wspF , either integrated in pf4 prophage or expressed in high-copy plasmids. We isolated mRNA, reverse transcribed it into cDNA, and employed quantitative PCR. Figure 4.1 demonstrated that the mRNA of all our engineered genes were successfully transcribed.

b) C-di-GMP Assay

The c-di-GMP assay is aimed to quantify cyclic-di-GMP (c-di-GMP) levels within bacterial cells, for functional validation of the DGC and PDE that integrated into the pf4 prophage. The observed reduction in c-di-GMP level, upon the genomic insertion or plasmid-borne carriage of yhjH , which is in accordance with this gene's role in degrading c-di-GMP. Moreover, the reduction in c-di-GMP level following the overexpression of wspF also confirmed its role as a repressor of the wspF DGC, which played critical roles in c-di-GMP synthesis in P. aeruginosa (Figure 4.2). These results show that both wspF and yhjH are potential candidates for prophage engineering and targeting the c-di-GMP mediated biofilm formation.2

c) Impact of engineered phages on quorum sensing inhibition

Addition of engineered pf phage carrying aiiA and ytnP , which encodes a quorum quenching enzyme, was found to supress the pqs, las and rhl quorum sensing in a dose-dependent manner (Figure 4.3-4.6). This result highlighted that prophage carried aiiA is able to encode a functional enzyme product for degrading the quorum sensing signal molecules.

4. Biofilm formation Assessment

We further examined whether biofilm formation is affected by our engineered strategies. Firstly, the PAO1-aiiA strain was found to produce much less biofilms compared to the wild-type PAO1 strain, while the PAO1-yhjH strain did not show significant change in biofilm amount (Figure 5A). Interestingly, engineered strains which overexpress aiiA , ytnP , and wspF genes harbored in pHERD20T plasmids formed significant less amount of biofilms compared to the wild-type PAO1 strain (Figure 5B). Intriguingly, the outcomes of yhjH expressing strain even showed an increasing in biofilm formation, which suggests that yhjH may not serve as a ideal candidate for biofilm reduction endeavors. Moreover, a marked attenuation in the biofilm formation capability was observed for aiiA and wspF genes amplified on pHERD20T plasmids following pf4 phage knockout (Figure 5C). Hence, we propose that aiiA , ytnP , and wspF emerge as promising candidates for curtailing biofilm formation.

Protocol

1. Primer design

2. PCR amplification

Using NEB Q5® High-Fidelity DNA Polymerase with 5% DMSO for the amplification of upstream P1&P2, as well as downstream P3&P4.

Extension:

The recommended extension temperature is 72°C. Extension times are generally 20–30 seconds per kb for complex, genomic samples, but can be reduced to 10 seconds per kb for simple templates (plasmid, E. coli , etc.) or complex templates < 1 kb. Extension time can be increased to 40 seconds per kb for cDNA or long, complex templates, if necessary.

A final extension of 2 minutes at 72°C is recommended.

2-step PCR:

When primers with annealing temperatures ≥ 72°C are used, a 2-step thermocycling protocol (combining annealing and extension into one step) is possible.

Proceed to 1% Agarose Gel Electrophoresis, Gel Staining, Gel imaging, PCR purification, Nanodrop.

3. Gibson assembly

1) Set up the following reaction on ice:

PK18 digested vector with BamHI and HindIII: 50ng

Upstream PCR product: 75-78ng

Downstream PCR product: 75-78ng

2)* Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.

** Control reagents are provided for 5 experiments.

*** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.

3)Incubate samples in a thermocycler at 50°C for 60 minutes when 2 or 3 fragments are being assembled. Following incubation, store samples on ice or at -20°C for subsequent transformation.

Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases.

4. Transformation with DH5α competent cell using heat shock method

Obtain DH5α competent cell from the –80°C ultra-freezer and thaw on ice. Add 10μl of the Gibson reaction to a 1.5ml microcentrifuge tube containing the competent cell respectively.

1) Incubate the microcentrifuge tube on ice for 30 minutes.

2) Heat-shock the cells for 30 seconds in a 42°C water bath.

3) Quickly place the microcentrifuge tubes on ice for 5 minutes.

4) Add 900μl of SOC medium was added to each microcentrifuge tubes.

5) Place the microcentrifuge tubes in a 50 ml falcon tube and incubate for 2 hours at 37°C in the incubator shaker at 200 rpm.

6) Plate 50μl and 100μl from each transformation and spread on a pre-warmed selective plate iptg xgal Gm60 LBL plate. The remaining transformation was centrifuged at 12,000 rpm for 2 mins, remove the supernatant, resuspend the cell with the 100μl of SOC medium and spread all of it on a pre-warmed selective. Incubate the selective plate overnight in a 37°C incubator.

7) Select single colonies from their respective selective plate and tick/streak on a LBL agar plate containing Gm60 antibiotic to store the strains and for PCR verification.

8) Check with PCR using Taq polymerase with 5% DMSO, use PK18 F & R primers.

9) After PCR, run a gel electrophoresis with 1% agarose gel to check for the correct insert. Inoculate the correct transformants in LBL Gm30, culture overnight in 37°C incubator shaker at 200 rpm for conjugation/making frozen stock the next day.

5. Conjugation

Day 1: Inoculation and Overnight Culture

Inoculate the following strains in 2 mL LB and incubate overnight at 37°C with shaking:

a) Strain #35 - Wildtype Pseudomonas aeruginosa PAO1

b) Strain #21 - E. coli + RK600 (Gm 6 μg/mL)

c) Donor strains PK18+insert (Gm 60μg/mL)

Day 2: Preparation for Gene Transfer

9:00 am

- Dilute the overnight culture of Recipient strains 1:5 to 1:10 into 2 mL LB medium. Incubate the diluted culture at 42°C without shaking.

- Dilute the overnight culture of Donor strains 1:5 to 1:10 into 2 mL LB medium and add antibiotics. Incubate at 37°C with shaking.

12:00 pm

- Dilute the overnight cultures of helper strains (1:5 to 1:10) in 2 mL or more LB medium, adding antibiotics. Incubate at 37°C with shaking.

3:00 pm

- Preheat LB agar plates (without antibiotics) to 37°C.

3:45 pm

- Wash an appropriate volume (1 mL) of culture from helper and Donor strains (cultures containing antibiotics) with fresh LB medium twice (centrifuge at 10,000g for 3 minutes).

- Resuspend the washed cells in equal volumes.

- Prepare 2 mL new Eppendorf tubes and add the re-suspended cells as follows: 300 μL Recipient + 300 μL RK600 + 300 μL Donor. Ensure that the OD of the cultures is similar.

- Mix well and centrifuge the mixed culture at 10,000g for 2 minutes. Discard the supernatant and spot the pellet (approximately 50-80 μL) on LB agar plates. Incubate at 37°C.

Day 3: Selection

1. Prepare selective plates containing antibiotic (Gm 60) according to the following recipe:

2. Wash down the pellet from LB agar plates with 1 mL 0.9% NaCl or ABTC medium. Centrifuge the microcentrifuge tube at 12,000 rpm for 2 minutes, remove the supernatant, resuspend the cells with 100 μL of NaCl/ABTC, and spread all of it on ABTC+Gm60 plates without dilution.

Day 4: Isolation of Mutants

1. Prepare 10% Sucrose+ABTC agar plates without antibiotic.

2. Pick 2 colonies and streak them on individual 10S+ABTC plates.

3. Incubate the plates overnight at 37°C.

Day 5: Verification and Picking

1. Prepare LBL and LBL+Gm60 media.

2. Patch/cross-streak 28-32 colonies onto LBL+Gm60 plates first and then onto LBL plates. Label each colony clearly with numbers for identification.

3. Incubate the plates overnight at 37°C.

Day 6: Mutant Verification

1. Check if mutants are sensitive to Gm.

2. Perform PCR with Taq polymerase and 5% DMSO, using F&R primers to screen for true mutants.

3. After PCR, run gel electrophoresis with 1% agarose gel to verify mutants.

4. Restreak the correct mutants on LBL plates. Incubate overnight at 37°C.

Day 7: Confirmation and Culturing

1. Inoculate 4-16 colonies into 3 mL ABTC medium. Culture overnight at 37°C with shaking at 200 rpm for rechecking with PCR.

2. Prepare frozen stocks the next day.

（1） Primers Configuring

I. Materials

1. Primer powder

2. Miniature centrifuge

II. Methods

1. Process primer powder

1) The primer powder was first centrifuged with a small centrifuge at 4000rpm for 60s

2) Slowly add ddH2O sterilized by pressure filtration to ensure the final concentration is 10nM

3) Mix with a vortex

4) -20℃ frozen storage (can be divided to avoid multiple freeze-thaw)

（2）PCR

I. Materials

1. Q5 High-Fidelity 2× Master Mix

2. Forward primers and reverse primers

3. ddH2O

4. bacterial suspension

II. Methods

1. Prepare the reaction components according to Table 1.

2. Gently mix the reaction. Transfer PCR tubes from ice to a PCR machine.

3. Run PCR and the thermocycling conditions are set as Table 2.

I. Materials

1. TAE Buffer; 2. Agarose; 3. DNA marker; 4. double-stranded DNA intercalating dye

II. Methods

1. Preparation of Agarose Gel: 50ml 1X TAE buffer and 0.5g agarose, microwave solution for 1min and then heat intermittently for 3-5 times until there are no particles. Add 5uL of nucleic acid dye, shake well and pour into the gel tray for solidification.

2. Place the gel tray into the cassette and pour the solution into the tray. Insert the comb into the top of the gel and allow the gel to solidify for 30 min. Avoid bubbles in the gel.

3. Once the gel has solidified, carefully remove the comb by pulling straight up.

4. Ensure the gel is in the correct orientation, with the negative/black electrode above the wells so that the DNA runs toward the positive/red electrode.

5. Prepare the samples by adding 6X loading buffer to each. Combine 5 μL of DNA with 1 μL of 6X loading buffer in order to load 5μL. Load samples into wells. Avoid bubbles. Lid on cassette and ensure the red and black wires are connected to the matching red and black electrodes on the cassette.

6. Electrophoresis 180V, 20min.

7. Remove the tray with the gel and image with UV.

I. Materials

1) Phage stock solution

2) Bacterial broth culture

3) Sterile microcentrifuge tube, micropipetters and sterile tips

II. Methods:

1) Configuration of Bacterial broth culture

2) Phage extraction

1. Centrifuge 1.5-2ml of bacterial solution at 10,000 rpm for 1 minute.

2. Extract the supernatant with a syringe, remove the needle, and filter with a 0.22um filter head to obtain the phage stock solution.

3. Perform gradient dilution using PCR tubes. Mix 180 µl of LB with 20 µl of bacterial solution for each dilution step. The initial tube represents a 10-1 concentration. Then, transfer 20 µl from the first tube to subsequent tubes (up to the eighth), each time creating a tenfold dilution, resulting in a dilution series ranging from 10-1 to 10-8 in concentration.

4. Deposit 2-3 µl of the diluted bacterial solution onto a plate.

5. Incubate the plates at 37°C and check for results after 4-6 hours of culture. Observe and record the growth of bacterial colonies or any other relevant observations.

Ⅰ. Materials

RNApure Bacteria Kit（DNase I）; RNase free pipettes and tips; Bacterial suspension

Ⅱ. Methods

1、Centrifuge at 12,000 rpm at 4℃ for 2 min to collect bacteria (the maximum volume of bacteria should not exceed 1× 109), and carefully remove all supernatants.

2、The bacteria were thoroughly suspended with 100 μl TE buffer containing Lysozyme （400 μg/ml） and incubated at room temperature for 5 min.

3、Add 350 μl Buffer RL (Contain β-mercaptoethanol), swirl and mix well, add the solution and precipitation into the Spin Columns FL installed in the collection tube, and centrifugate at 12,000 rpm for 2 minutes.

4、Add 250 μl of anhydrous ethanol to the filtrate obtained in the previous step and mix well (precipitation may occur at this time). The obtained solution and precipitation were transferred into the Spin Columns RM which had been loaded into the collection tube, centrifuged at 12,000 rpm for 1 minute, discarded the waste liquid, and put the adsorption column back into the collection tube.

5、Add 350μl Buffer RW1 into the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube.

6、Add 500 μl Buffer RW2 to the adsorption column (check whether anhydrous ethanol is added before use), centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

7、Repeat step 6.

8、Return the adsorption column to the collection tube and centrifuge at 12,000 rpm for 2 min.

9、The adsorption column was loaded into a new RNase-Free collection tube, and 30-50 μl RNase-Free Water was added to the middle of the adsorption film, and the RNA solution was collected at room temperature for 1 min, centrifuged at 12,000 rpm for 1 min, and stored at -80℃.

10、The RNA was measured to determine whether the RNA contained more impurities and concentrations.

Ⅰ. Materials

PCR amplification apparatus, PCR tubes, pipettes and tips, gDNA Clean Reaction Mix Ver.2, 5X EVo M-MLVRT Reaction Mix Ver.2, Total RNA（Eight RNA samples taken on RNA extraction）, RNase free water

Ⅱ. Methods

1、 Prepare the reaction liquid on the ice according to the table below

2、Set the reaction conditions in the instrument: 37 ℃ for 15 minutes, 85 ℃ for 5 seconds, 4 ℃ forever.

3、After the reaction, the cDNA product was stored at -80℃.

Ⅰ. Materials

Reagents and consumables: Primer, PCR grade water, quantitative PCR tube; RNase free pipettes and tips; 2X SYBR Green Pro Taq HS Premix; (Roche) LightCycler® 2.0, 480, 96; Bacterial suspension

Ⅱ. Methods

1) Preparation of PCR reaction solution

2) qPCR reaction conditions

Ⅰ. Materials

1. Centrifuge, 1.5 ml centrifuge tube, pipettes and tips

2. ELISA Kit: c-di-GMP Sensor, c-di-GMP, DFHB1-1TFluorophore, 4X c-di-GMP Assay (CA) Buffer, 4X Bacterial Compatibility(BC) Reagent, RNase-Free Water

Ⅱ. Methods

1. Standard Samples (Refer Table 1): Add 20 μl of the 10X c-di-GMP standards (Standard 5~8) to the appropriate standard wells in a black bottom 96-well assay plate. Add up to 5 μl of appropriate bacterial culture media to each of the standard wells (Refer Table 2). Make up the total volume to 50 μl with RNase-free water.

2. Samples: Dilute the culture media 1:5 RNase-free water. Up to 50 μl of the diluted bacteria in culture media can be tested using the c-di-GMP assay. Make up the volume in the well to 70 μl with RNase-free water (Refer Table 3). Do not use culture media to make up the volume.

3. Reagent Addition: Add 50 μl of 4X c-di-GMP assay (CA) buffer, 50ul of bacterial compatibility (BC) reagent, 20 μl of 10X fluorophore, and 10 μl of sensor to the each of the wells (standard and unknown samples)

4. Incubation: Incubate the plate for 30 minutes at room temperature in a dark place. The incubation time the m be extended up to 24h without affecting the performance of the assay.

5. Reading: Read the plate in a fluorescence plate reader with a GFP/FITC filter set or with excitation set at 482nm and emission at 535nm.

6. Calculation: The standard curve was fitted to calculate the concentration of c-di-GMP in each sample

Ⅰ. Materials

1) Strains for QSI assay: PAO1-gfp; lasB-gfp; pqsA-gfp; rhlA-gfp

2) Centrifuge; 1.5 ml centrifuge tube; pipettes and tips; 96-well plates

Ⅱ. Methods

1) Preparation of compound stock solution and dilution range:

a) Prepare phage according to phage plaque assay and is dissolved in DMSO.

b) Serial dilute the phage.

c) Prepare the final concentration testing for 1st well and serial dilute with ABTGC medium, so final concentration after adding bacteria is desired highest concentration.

• Add 100 µl of the medium to all 96 wells except for column 12 using a multi-channel pipette.
• To column 12, add 200 µl of the medium that contains compounds at 2x the final concentration that the titration will start at.
• Perform 3 technical replicates for each condition.

2) Preparation of inoculum:

a) Dissolve a single colony picked from LB streak plate in 2ml LB broth and incubate overnight at 37oC, 200rpm.

b) Check OD600 with a spectrophotometer.

c) Dilute the bacterial solution with ABTGC medium to final OD600 0.02

3) Inoculation and incubation:

a) Aliquot 100ul of the bacterial solution to all wells, mix well. Final volume per well: 200ul (100ul compound + 100ul bacteria)

b) Parafilm plate and incubate microplate reader at 37oC for 12 hours. Measure the following parameters at every 15 minutes interval for at least 16 hours: OD600, GFP signal (excitation 488nm, emission 535nm).

4) Results analysis:

a) Calculate the average (OD600 and GFP) for each condition at all time points.

b) Calculate GFP/OD600 for each condition at all time points.

c) Plot OD600 and GFP/OD600 graphs against time.

Ⅰ. Materials

Luria-Bertani (LB) broth; Bacteria Suspension

Ⅱ. Methods

1. Incubate the bacterial suspension overnight.

2. Dilute 50 µL of the bacterial solution into 950 µL of LB broth.

3. Utilizing LB broth as a blank, allocate 500 µL of the bacterial solution for Optical Density (OD) value measurement. Proceed to dilute the bacterial suspension until an OD value of 0.01 is attained.

4. Dispense the prepared samples into the culture plate with 150 µL of the solution allocated per well. Utilize LB broth in the first column to serve as the control. For each group, replicate the samples thrice to ensure reliability in the data acquired.

Notebook