铜绿假单胞菌临床分离株中末端冗余假单胞菌噬菌体无末端设计重启的方案

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

作者信息

Yokoyama Daigo, Kimura Nana, Yamamoto Haruka, Sakata Yoshiaki, Fujiki Jumpei, Iwano Hidetomo

机构信息

Laboratory of Veterinary Biochemistry, Department of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido, Japan.

出版信息

STAR Protoc. 2025 Aug 4;6(3):104012. doi: 10.1016/j.xpro.2025.104012.

DOI:10.1016/j.xpro.2025.104012

PMID:40763034

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12345254/

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.

摘要

合成噬菌体平台是强大的微生物学工具，具有对抗耐抗菌细菌的治疗潜力。在此，我们展示了一种用于重启具有末端冗余、环状排列的65 kbp基因组的铜绿假单胞菌噬菌体的方案。我们描述了设计PCR引物以生成DNA片段、重建完整线性噬菌体基因组、进行无缝体外组装的步骤，最后，使用铜绿假单胞菌临床分离株纯化并电穿孔DNA。