期刊
VIROLOGY
卷 434, 期 2, 页码 278-284出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.virol.2012.09.020
关键词
Refactoring; empty setX174; Phage; Yeast; Recombination; Synthetic biology; Genome engineering; Synthetic genomics; DNA assembly; Irreducible complexity; Overprinting; Overlapping genes
类别
资金
- NIGMS
- NSF SynBERC
- Stanford
- NSERC
The 5386 nucleotide bacteriophage empty setX174 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, empty setX174.1, that fully separates all primary phage protein coding sequences along with cognate translation control elements. To specify empty setX174.1f, a decompressed genome the same length as wild type, we truncated the gene F coding sequence. We synthesized DNA encoding fragments of empty setX174.1f 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 empty setX174 particles containing the empty setX174.1f 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. (C) 2012 Elsevier Inc. All rights reserved.
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