Cell-free synthesis of bacteriophages

Abstract

Antimicrobial resistance is a global health crisis, for which alternative therapeutics to antibiotic drugs are urgently needed. One promising alternative, phage therapy, uses bacteriophages to kill bacteria. However, production of phages for therapy requires purification of phages from pathogenic organisms, risking introduction of immunogenic proteins and dangerous chemicals into phage preparations. Moreover, some phages are not suitable for therapy, encoding unwanted genes which are toxic to humans or benefit bacteria. Phage genome engineering can remove unwanted genes and enhance antibacterial efficacy of phages. However, current in vivo phage engineering methods are time intensive and restricted in flexibility. Cell-free synthesis enables in vitro synthesis of phages solely from their genomes (phage ‘rebooting’). This open-system technology removes pathogenic bacteria from phage production, enables synthesis of fully synthetic phage from chemically synthesised genomes, and can be accomplished with minimal expertise and common lab equipment. In this thesis, we developed and optimised a homebrewed cell-free system capable of rebooting Escherichia coli phage T7 and a novel non-E. coli phage (xenophage) targeting a clinical isolate of Klebsiella pneumoniae isolated from the Citizen Phage Library. A pipeline to phenotypically enhance phages with protein decorations to phage capsids was tested, using decoration with fluorescent protein as a proof of concept. This supports the growing body of cell-free phage rebooting applications and advances our capacity to rapidly reboot enhanced phages for therapy.