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Cell-free transcription-translation (TXTL) enables achieving an ever-growing number of applications, ranging from the rapid characterization of DNA parts to the production of biologics. As TXTL systems gain in versatility and efficacy, larger DNAs can be expressed in vitro extending the scope of cell-free biomanufacturing to new territories. The demonstration that complex entities such as infectious bacteriophages can be synthesized from their genomes in TXTL reactions opens new opportunities, especially for biomedical applications. Over the last century, phages have been instrumental in the discovery of many ground-breaking biotechnologies including CRISPR. The primary function of phages is to infect bacteria. In that capacity, phages are considered an alternative approach to tackling current societal problems such as the rise of antibiotic-resistant microbes. TXTL provides alternative means to produce phages and with several advantages over in vivo synthesis methods. In this chapter, we describe the basic procedures to purify phage genomes, cell-free synthesize phages, and quantitate them using an all-E. coli TXTL system. 

Abstract Bacteriophages constitute an invaluable biological reservoir for biotechnology and medicine. The ability to exploit such vast resources is hampered by the lack of methods to rapidly engineer, assemble, package genomes, and select phages. Cell-free transcription-translation (TXTL) offers experimental settings to address such a limitation. Here, we describe PHage Engineering by In vitro Gene Expression and Selection (PHEIGES) using T7 phage genome and Escherichia coli TXTL. Phage genomes are assembled in vitro from PCR-amplified fragments and directly expressed in batch TXTL reactions to produce up to 10¹¹PFU/ml engineered phages within one day. We further demonstrate a significant genotype-phenotype linkage of phage assembly in bulk TXTL. This enables rapid selection of phages with altered rough lipopolysaccharides specificity from phage genomes incorporating tail fiber mutant libraries. We establish the scalability of PHEIGES by one pot assembly of such mutants with fluorescent gene integration and 10% length-reduced genome.