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Related Experiment Video

Updated: Jul 13, 2025

Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection

Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection

Published on: May 24, 2020

An efficient, scarless, selection-free technology for phage engineering.

Moran G Goren¹, Tridib Mahata¹, Udi Qimron¹

¹Department of Clinical Microbiology and Immunology, School of Medicine, Tel Aviv University, Tel Aviv, Israel.

|October 17, 2023

Summary

This summary is machine-generated.

A novel non-CRISPR phage engineering method adapts bacterial genome engineering tools. This efficient technique allows genetic modification of Escherichia coli phages, enabling new research avenues.

Keywords:

Bacteriophages DNA engineering pORTMAGE

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Area of Science:

Molecular Biology
Microbiology
Genetics

Background:

CRISPR-Cas systems dominate current phage engineering.
A need exists for alternative, non-CRISPR phage modification methods.

Purpose of the Study:

To develop and demonstrate a non-CRISPR-based genetic engineering technology for Escherichia coli phages.
To adapt the pORTMAGE technology, originally for bacterial genomes, for phage engineering applications.

Main Methods:

Utilized Escherichia coli strains harboring a plasmid with a potent recombinase and a transient repair system suppressor.
Electroporated synthetic oligonucleotides encoding desired phage mutations into E. coli.
Infected engineered E. coli with target bacteriophages (T5, T7, P1, λ) to facilitate recombination.

Main Results:

Achieved high efficiency (1-14%) in generating desired phage mutants.
Successfully demonstrated single-base substitutions, deletions (50-201 bp), and insertions (20 bp).
Validated the method across four different Escherichia coli phage types.

Conclusions:

The developed non-CRISPR method provides an efficient alternative for engineering Escherichia coli phages.
The technology's versatility allows for various genetic modifications and has potential for broad applicability to other phage and bacterial strains.