A fully decompressed synthetic bacteriophage øX174 genome assembled and archived in yeast

Abstract

The 5386 nucleotide bacteriophage øX174 genome has a complicated architecture that encodes 11 gene products via overlapping protein coding sequences spanning multiple reading frames. We designed a 6302 nucleotide synthetic surrogate, øX174.1, that fully separates all primary phage protein coding sequences along with cognate translation control elements. To specify øX174.1f, a decompressed genome the same length as wild type, we truncated the gene F coding sequence. We synthesized DNA encoding fragments of øX174.1f and used a combination of in vitro- and yeast-based assembly to produce yeast vectors encoding natural or designer bacteriophage genomes. We isolated clonal preparations of yeast plasmid DNA and transfected E. coli C strains. We recovered viable øX174 particles containing the øX174.1f genome from E. coli C strains that independently express full-length gene F. We expect that yeast can serve as a genomic 'drydock' within which to maintain and manipulate clonal lineages of other obligate lytic phage.

Engineering Modular Viral Scaffolds for Targeted Bacterial Population Editing

“…We used an efficient yeast-based platform (Jaschke et al, 2012; Lu et al, 2013) to create phages with novel host ranges based on common viral scaffolds. Inspired by gap-repair cloning in yeast and the work of Gibson and co-workers (Gibson et al, 2008), we captured phage genomes into Saccharomyces cerevisiae , thus enabling facile genetic manipulation of modified genomes that can be subsequently re-activated or “rebooted” into functional phages after transformation of genomic DNA into bacteria (Figure 1A).…”

“…Previously, a scheme for engineering phage T4 through electroporation of PCR products was devised (Pouillot et al, 2010), but it is based on a particular feature of the genetic regulation of T4 and cannot easily be applied to other phage families. Recently, the 5.4 kb filamentous coliphage ϕX174 was assembled in yeast in order to stably store the genome and aid in phage refactoring (Jaschke et al, 2012). In this approach, the majority of the genome assembly was performed in vitro and the YAC cloning was mostly used to store the resulting genome, whereas the majority of the genome engineering in our approach stems from the actual gap-repair cloning process in yeast.…”

Omics approaches in food safety: fulfilling the promise?

“…In addition, for biocontrol of E. coli O157:H7, non-lytic phages have been engineered to encode proteins that are lethal for the host cell (lethal transcriptional regulator); this engineered phage can kill E. coli without releasing a phage progeny, and therefore, without potential ecological disturbance [69]. As viable synthetic phages have already been constructed [70,71], it is likely that we will see use of synthetic phages to control and detect foodborne pathogens in the not too distant future. The potential of combining omics methods and synthetic biology is also illustrated by new approaches that have been taken to address food contamination by aflatoxin, a cancer-inducing mycotoxin produced by Aspergillus flavus [72–75].…”

Genetically Engineered Phages: a Review of Advances over the Last Decade

“…This technique has been used to capture and genetically modify the genomes of the coliphages T3 (38,208 bp) and T7 (39,937 bp) (84,85) as well as the Klebsiella phage K11 (41,181 bp) (85). It was further used to capture and archive the genome of fully refactored phage ⌽X174 (6,302 bp) (86). This strategy requires the extraction of the phage genome from yeast and its introduction into bacteria, and thus its efficiency is restricted by bacterial transformation efficiencies.…”