PT  - JOURNAL ARTICLE
AU  - Matthew S. Faber
AU  - James T. Van Leuven
AU  - Martina M. Ederer
AU  - Yesol Sapozhnikov
AU  - Zoë L. Wilson
AU  - Holly A. Wichman
AU  - Timothy A. Whitehead
AU  - Craig R. Miller
TI  - Saturation mutagenesis genome engineering of infective ΦX174 bacteriophage via unamplified oligo pools and golden gate assembly
AID  - 10.1101/798546
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 798546
4099  - http://biorxiv.org/content/early/2019/10/09/798546.short
4100  - http://biorxiv.org/content/early/2019/10/09/798546.full
AB  - Here we present a novel protocol for the construction of saturation single-site—and massive multi-site—mutant libraries of a bacteriophage. We segmented the ΦX174 genome into 14 non-toxic and non-replicative fragments compatible with golden gate assembly. We next used nicking mutagenesis with oligonucleotides prepared from unamplified oligo pools with individual segments as templates to prepare near-comprehensive single-site mutagenesis libraries of genes encoding the F capsid protein (421 amino acids scanned) and G spike protein (172 amino acids scanned). Libraries possessed greater than 99% of all 11,860 programmed mutations. Golden Gate cloning was then used to assemble the complete ΦX174 mutant genome and generate libraries of infective viruses. This protocol will enable reverse genetics experiments for studying viral evolution and, with some modifications, can be applied for engineering of therapeutically relevant bacteriophages with larger genomes.