Synthetic engineering and biological containment of bacteriophages

Abstract

The serious threats posed by drug-resistant bacterial infections and recent developments in synthetic biology have fueled a growing interest in genetically engineered phages with therapeutic potential. To date, many investigations on engineered phages have been limited to proof of concept or fundamental studies using phages with relatively small genomes or commercially available "phage display kits". Moreover, safeguards supporting efficient translation for practical use have not been implemented. Here, we developed a cell-free phage engineering and rebooting platform. We successfully assembled natural, designer, and chemically synthesized genomes and rebooted functional phages infecting gram-negative bacteria and acid-fast mycobacteria. Furthermore, we demonstrated the creation of biologically contained phages for the treatment of bacterial infections. These synthetic biocontained phages exhibited similar properties to those of a parent phage against lethal sepsis in vivo. This efficient, flexible, and rational approach will serve to accelerate phage biology studies and can be used for many practical applications, including phage therapy.

Keywords: bacteriophage; biological containment; cell-free genome engineering; phage therapy; synthetic biology.

Fig. 1.

Rebooting of WT phages from genomes assembled in vitro. (A) Schematic illustrating the workflow for rebooting phages from genomes assembled in vitro via electroporation. DNA fragments exhibiting homology with adjacent fragments were amplified from the phage genome or from chemically synthesized DNA. PCR fragments were assembled using the Gibson Assembly and then electroporated into host bacteria to reboot phages. (B–J) Rebooting of various phages from genomes assembled in vitro by electroporating into host bacteria. (K) Rebooting of phage TM4 from chemically synthesized DNA fragments by electroporating into M. smegmatis mc²155. White arrows indicate plaques. M, Quick-Load 1 kb Plus DNA Ladder (NEB, Japan).

Fig. 2.

Rebooting of engineered phages from designer genomes assembled in vitro. (A–D) Rebooting of engineered P22 from in vitro–assembled genomes. (A) Overview of P22 genome engineering. (B) DNA fragments amplified from the P22 genome were assembled and installed into Salmonella LT2. White arrows indicate plaques. (C) Confirmation of P22∆c2 genome structure using PCR. (D) Plaque formation assay of WT P22 and P22_∆c2. (E–G) Rebooting of synthetic T3 with T7 tail fiber, T3_T7(gp17), from genomes assembled in vitro. (E) Overview of T3 genome engineering. (F) DNA fragments amplified from the T3 and T7 genomes were assembled and installed into E. coli 10G. (G) Plaque formation assays to confirm the host range expansion of T3_T7(gp17). (H–K) Rebooting of engineered mycophage D29 from genomes assembled in vitro. (H) Overview of D29 genome engineering. (I) PCR fragments amplified from the D29 and P_hsp60-Nluc fragment were assembled and installed into M. smegmatis mc²155. (J) Confirmation of D29_{∆72-73::Nluc} genome structure using PCR. (K) Evaluation of Nluc expression from D29_{∆72-73::Nluc} (n = 3). M, Quick-Load 1 kb Plus DNA Ladder (NEB, Japan).

Fig. 3.

Cell-free engineering and cell-free rebooting of phages. (A) Workflow for the cell-free rebooting of phages from genomes assembled in vitro. Phage genome fragments were amplified and then assembled via the Gibson Assembly. Phages were produced in a cell-free TXTL system from a genome assembled in vitro. (B) Design of synthetic T7_lacZ genome. (C) Rebooting of synthetic T7_lacZ from a genome assembled in vitro in a cell-free TXTL system. (D) Plaque formation assay on an X-gal plate to confirm lacZ expression from synthetic T7_lacZ genome. M, Quick-Load 1 kb Plus DNA Ladder (NEB, Japan).

Fig. 4.

Creation of biocontained phages. (A) Schematic illustrating the workflow for creating biocontained phages. A phage genome without a virion gene was assembled and introduced into host bacteria harboring a plasmid carrying the corresponding gene. A synthetic phage genome lacking the virion gene produced progeny phages because the virion protein was supplied from the plasmid. Biocontained progeny phages were released from lysed cells. (B–I) Creation of T7_∆head and SP6_∆head. (B, F) Overview of phage genome engineering. (C, G) PCR-amplified DNA fragments were assembled and installed into host bacteria expressing the capsid gene. (D, H) Confirmation of the deletion of head gene(s) in biocontained phages using PCR. (E, I) Plaque formation assays. M, Quick-Load 1 kb Plus DNA Ladder (NEB, Japan).

Fig. 5.

Evaluation of containment and bactericidal activity of biocontained phages in vitro and in vivo. (A) Killing curves of Salmonella LT2 treated with WT SP6 or SP6_∆head, and (B) changes in the amount of WT SP6 or SP6_∆head. PFU was calculated from the number of plaques on a lawn of LT2 harboring pMW-SP6-g31. The data are presented as the mean of three independent experiments; error bars represent SD. * and ** indicate P values of < 0.05 and < 0.01, respectively, as calculated using the Student t test between the control and MOI = 1, 10, or 100. (C) Phage therapy experiment. Treatment of murine sepsis model using WT SP6 or SP6_∆head. P values were calculated by performing a log-rank test between control and WT SP6 or SP6_∆head.