Engineering

1. 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).