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Robust and Reproducible Protocol for Phage Genome "Rebooting" Using Transformation-Associated Recombination (TAR)
Ivan Baykov1,2, Olga Kurchenko3,4, Ekaterina Mikhaylova3,4
1Laboratory of Molecular Microbiology, Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia. ivan_baykov@mail.ru.
Methods in Molecular Biology (Clifton, N.J.)
|December 8, 2023
Summary
Rebooting bacteriophage genomes is crucial for phage editing and synthetic biology. This chapter details methods for producing infectious phages from their genomes using native or intermediate hosts.
Area of Science:
- Microbiology
- Synthetic Biology
- Virology
Background:
- Phage genome manipulation is essential for advancing genetic engineering and synthetic phage creation.
- The process of reconstituting infectious phages from their genetic material is termed 'genome rebooting'.
- Standardized protocols are needed for efficient phage genome reconstruction.
Purpose of the Study:
- To provide a detailed protocol for bacteriophage genome "rebooting".
- To enable the production of infectious phages from engineered or synthetic genomes.
- To facilitate research in phage therapy and synthetic virology.
Main Methods:
- Genome "rebooting" using a native host.
- Genome "rebooting" using an intermediate host.
- Detailed protocols for T7 bacteriophage (Escherichia coli host) and KP32_192 bacteriophage (Klebsiella pneumoniae host).
Main Results:
- Successful "rebooting" of T7 bacteriophage genome.
- Successful "rebooting" of KP32_192 bacteriophage genome.
- Demonstration of a viable method for producing infectious phages from their genomes.
Conclusions:
- The described "rebooting" protocols are effective for producing infectious bacteriophages.
- This methodology supports the creation of synthetic bacteriophages and genome editing applications.
- The protocols are applicable to different bacteriophage-host systems, including T7 and KP32_192.
Keywords:
Gap repair cloningGenome rebootingPodovirusSynthetic bacteriophageTransformation-associated recombination cloning
