A fully decompressed synthetic bacteriophage øX174 genome assembled and archived in yeast.
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.
Department of Bioengineering, Y2E2-269B, 473 Via Ortega, Stanford, CA 94305-4201, USA.
Paul R
PR
Jaschke
Erica K
EK
Lieberman
Jon
J
Rodriguez
Adrian
A
Sierra
Drew
D
Endy
D010584:Bacteriophage phi X 174 / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D005818:Genetic Engineering / Q000379:methods*; D016679:Genome, Viral; D008967:Molecular Biology / Q000379:methods; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics
D004279:DNA, Viral