# Protocol for end-design-free rebooting of terminally redundant Pseudomonas phages in clinical isolates of Pseudomonas aeruginosa

## TL;DR

This paper provides a detailed protocol for rebooting Pseudomonas phages with a specific genome structure using clinical isolates of Pseudomonas aeruginosa.

## Contribution

A novel protocol for rebooting Pseudomonas phages with a terminally redundant, circularly permuted genome using clinical isolates.

## Key findings

- Steps for constructing a 65 kbp Pseudomonas phage DNA genome through seamless in vitro assembly.
- Phage DNA purification and electroporation-based rebooting using clinical isolates of P. aeruginosa.
- Successful synthesis of phages with terminally redundant and circularly permuted genomes.

## Abstract

Synthetic phage platforms are robust microbiology tools with therapeutic potential against antimicrobial-resistant bacteria. Here, we present a protocol for rebooting Pseudomonas phages with a terminally redundant, circularly permuted 65 kbp genome. We describe steps for designing PCR primers to generate DNA fragments, reconstituting the complete linear phage genome, performing seamless in vitro assembly, and finally, purifying and electroporating the DNA using a P. aeruginosa clinical isolate.

•Steps for constructing 65 kbp Pseudomonas phage DNA by genome assembly•Preparation of competent cells for phage rebooting with P. aeruginosa clinical isolates•Phage DNA purification and electroporation-based rebooting of linear phage genomes•Synthesis of phages with terminally redundant and circularly permuted genomes

Steps for constructing 65 kbp Pseudomonas phage DNA by genome assembly

Preparation of competent cells for phage rebooting with P. aeruginosa clinical isolates

Phage DNA purification and electroporation-based rebooting of linear phage genomes

Synthesis of phages with terminally redundant and circularly permuted genomes

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Synthetic phage platforms are robust microbiology tools with therapeutic potential against antimicrobial-resistant bacteria. Here, we present a protocol for rebooting Pseudomonas phages with a terminally redundant, circularly permuted 65 kbp genome. We describe steps for designing PCR primers to generate DNA fragments, reconstituting the complete linear phage genome, performing seamless in vitro assembly, and finally, purifying and electroporating the DNA using a P. aeruginosa clinical isolate.