λ Recombineering Used to Engineer the Genome of Phage T7.

Jordan D Jensen,Alison J Rattray,Donald L Court,Adam R Parks,Sankar Adhya

doi:10.3390/antibiotics9110805

Abstract

Bacteriophage T7 and T7-like bacteriophages are valuable genetic models for lytic phage biology that have heretofore been intractable with in vivo genetic engineering methods. This manuscript describes that the presence of λ Red recombination proteins makes in vivo recombineering of T7 possible, so that single base changes and whole gene replacements on the T7 genome can be made. Red recombination functions also increase the efficiency of T7 genome DNA transfection of cells by ~100-fold. Likewise, Red function enables two other T7-like bacteriophages that do not normally propagate in E. coli to be recovered following genome transfection. These results constitute major technical advances in the speed and efficiency of bacteriophage T7 engineering and will aid in the rapid development of new phage variants for a variety of applications.

Highlights

Interest has grown in rapid, efficient, and scalable engineering of bacteriophages, virulent phages like T7 [1]
We demonstrate that the efficiency of phage recovery following electroporation of T7 and other T7-like phage genomes is enhanced by λ Red recombination functions
T7 and other T7-like bacteriophages are valuable for their innate virulence and bacteria-killing properties

Summary

Introduction

Interest has grown in rapid, efficient, and scalable engineering of bacteriophages, virulent phages like T7 [1]. Increased interest in bacteriophage therapy has focused attention on T7 [2] due to its rapid growth, large burst size (~250 particles per infection), general resistance to restriction by the bacterial host, and rapid degradation of the host genome [3]. Phage T7 has been studied intensively for more than fifty years. When T7 infects E. coli strains, rapid cell death and lysis occurs, usually within twenty minutes, with the release of progeny phage particles that spread the infection. The time period from phage infection to host cell lysis is less than 20 min at 37 ◦ C [4,5]

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Results

Conclusion