Cell-Free Bacteriophage Genome Synthesis Using Low-Cost Sequence-Verified Array-Synthesized Oligonucleotides

Abstract

Synthesizing engineered bacteriophages (phages) for human use has potential in various applications ranging from drug screening using a phage display to clinical use using phage therapy. However, the engineering of phages conventionally involves the use of an in vivo system that has low production efficiency because of high virulence against the host and low transformation efficiency. To circumvent these issues, de novo phage genome synthesis using chemically synthesized oligonucleotides (oligos) has increased the potential for engineering phages in a cell-free system. Here, we present a cell-free, low-cost, de novo gene synthesis technology called Sniper assembly for phage genome construction. With massively parallel sequencing of microarray-synthesized oligos, we generated and identified approximately 100 000 clonal DNA clusters in vitro and 5000 error-free ones in a cell-free environment. To demonstrate its practical application, we synthesized the Acinetobacter phage AP205 genome (4268 bp) using 65 sequence-verified DNA clones. Compared to previous reports, Sniper assembly lowered the genome synthesis cost ($0.0137/bp) by producing low-cost sequence-verified DNA.

The Synthesis and Assembly of a Truncated Cyanophage Genome and Its Expression in a Heterogenous Host

“…Using rational design, DNA synthesis and assembly as well as non-natural functions such as degenerate codons and truncated genomes have been introduced into modified microorganisms, providing new insights into the design and synthesis of artificial cyanophages with a broad spectrum. Although artificial synthesis of the ø X174, T7 and AP205 phages has been achieved [ 35 , 36 , 37 , 38 ], the synthesis of the full-length cyanophage genome has not yet been reported.…”

Renaissance for Phage-Based Bacterial Control

“…Through the application of engineering principles to biological systems, synthetic biology stands to expand phage-based technology by facilitating the creation of novel phage chassis and modular genetic components. Several phage genomes have been completely assembled using only synthetic DNA oligonucleotides, , allowing for rapid and large-scale genome modification and refactoring while simultaneously circumventing low recombination efficiency associated with in vivo genome engineering. Moreover, synthetic phage genomes can be “rebooted” in nonhost or cell-free systems, which suggests the potential for phage production against unculturable hosts.…”

Synthesis and assembly of full-length cyanophage A-4L genome

“…As synthetic A-4L genome cannot be existed in E. coli , the resurrection of artificial cyanophage through conjugation is unfeasible. Electroporation [ 37 ], protoplast fusion [ 38 ] and cell-free system [ 7 ] were attempted but all failed in rebooting, which robbed us of verifying the impact of mutations on the synthetic genome.…”

“…Large assemblies may be unstably maintained in E. coli but can be easily completed in yeast [ 5 ]. Viral genomes [ 6 ] have also been constructed, including poliovirus, simian immunodeficiency virus, coxsackievirus, adenovirus, tobacco mosaic virus, human endogenous retrovirus, coronavirus, as well as bacteriophage ɸ X174, T7, AP205 and G4 [ 7 ]. The cloning of many phage genomes in bacteria is stalled by possible gene virulence [ 8 ].…”