Cell-free synthesis and characterization of Salmonella, Escherichia coli, and Shigella-specific bacteriophages

BACKGROUND: Cell-free gene expression (CFE) systems provide a rapid and modular platform for synthesizing bacteriophages without the need for living host cells. However, the generalizability of CFE-based phage synthesis across diverse phages, as well as the functional comparability of in vitro-synthesized phages to host-derived counterparts, has not yet been fully explored. In this study, we evaluated the ability of an Escherichia coli-based CFE platform to synthesize diverse bacteriophages relevant to food safety and synthetic biology applications. RESULTS: Using an E. coli-based CFE system, we synthesized seven phages from four different families, achieving infectious titers ranging from 10⁶ to 10¹¹ plaque-forming units per milliliter (PFU/mL). Of these seven phages, five phages, vB_SalM-LPST153 (LPST153), vB_SenM-S16 (S16), SP6, vB_Sens_Jbel (Jbel), and vB_EcoM_Alf5 (Alf5), are reported here for the first time as successfully synthesized and characterized in a CFE system. CFE-synthesized phages remained capable of infecting host strains; however, significant differences in efficiency of plating (EOP) were observed across multiple Salmonella, E. coli, and Shigella hosts. Long-term storage tests for LPST153 showed that phage buffer (PB) stably maintained CFE titers above 10⁹ PFU/mL for 12 weeks, while purified CFE-synthesized and host-derived LPST153 lysates exhibited broadly comparable thermal stability profiles across the tested temperatures. Using the PHEIGES workflow, we also demonstrated complete in vitro assembly and expression of LPST153 from PCR fragments. In addition, phages were successfully synthesized using CFE lysates derived from the endotoxin-free E. coli strain ClearColi™, demonstrating the synthesis of infectious phages in an endotoxin-free system. CONCLUSIONS: These findings expand the diversity of bacteriophages compatible with cell-free synthesis (CFS) and demonstrate that CFE-synthesized phages remain infectious and retain several functional properties, although some differences in host-dependent infectivity were observed. The ability to synthesize infectious phages in endotoxin-free systems and assemble phage genomes entirely in vitro highlights the potential of CFE platforms for scalable, decentralized, and cold-chain-independent manufacturing of phage-based therapeutics and food safety interventions.