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Updated: Sep 11, 2025

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System
Published on: August 17, 2017
Synthetic and Functional Engineering of Bacteriophages: Approaches for Tailored Bactericidal, Diagnostic, and
Ola Alessa1, Yoshifumi Aiba1, Mahmoud Arbaah1
1Division of Bacteriology, Department of Infection and Immunity, School of Medicine, Jichi Medical University, Shimotsuke 329-0498, Japan.
Abstract:
Bacteriophages (phages), the most abundant biological entities on Earth, have long served as both model systems and therapeutic tools. Recent advances in synthetic biology and genetic engineering have revolutionized the capacity to tailor phages with enhanced functionality beyond their natural capabilities. This review outlines the current landscape of synthetic and functional engineering of phages, encompassing both in-vivo and in-vitro strategies. We describe in-vivo approaches such as phage recombineering systems, CRISPR-Cas-assisted editing, and bacterial retron-based methods, as well as synthetic assembly platforms including yeast-based artificial chromosomes, Gibson, Golden Gate, and iPac assemblies. In addition, we explore in-vitro rebooting using TXTL (transcription-translation) systems, which offer a flexible alternative to cell-based rebooting but are less effective for large genomes or structurally complex phages. Special focus is given to the design of customized phages for targeted applications, including host range expansion via receptor-binding protein modifications, delivery of antimicrobial proteins or CRISPR payloads, and the construction of biocontained, non-replicative capsid systems for safe clinical use. Through illustrative examples, we highlight how these technologies enable the transformation of phages into programmable bactericidal agents, precision diagnostic tools, and drug delivery vehicles. Together, these advances establish a powerful foundation for next-generation antimicrobial platforms and synthetic microbiology.
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