About: Saturation mutagenesis genome engineering of infective ΦX174 bacteriophage via unamplified oligo pools and golden gate assembly

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About: Saturation mutagenesis genome engineering of infective ΦX174 bacteriophage via unamplified oligo pools and golden gate assembly Goto Sponge NotDistinct Permalink

An Entity of Type : schema:ScholarlyArticle, within Data Space : wasabi.inria.fr associated with source document(s)

Attributes	Values
type	Academic Article research paper schema:ScholarlyArticle
isDefinedBy	Covid-on-the-Web dataset
title	Saturation mutagenesis genome engineering of infective ΦX174 bacteriophage via unamplified oligo pools and golden gate assembly
Creator	Miller, Craig Ederer, Martina Faber, Matthew Sapozhnikov, Yesol Van Leuven, James Whitehead, Timothy Wichman, Holly Wilson, Zoë
source	BioRxiv; MedRxiv
abstract	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.
has issue date	2019-10-09(xsd:dateTime)
bibo:doi	10.1101/798546
has license	medrxiv
sha1sum (hex)	f0f3f4ab41fdd2d12c3fa45dc53a17d7c58a4068
schema:url	https://doi.org/10.1101/798546
resource representing a document's title	Saturation mutagenesis genome engineering of infective ΦX174 bacteriophage via unamplified oligo pools and golden gate assembly
schema:publication	bioRxiv
resource representing a document's body	covid:f0f3f4ab41fdd2d12c3fa45dc53a17d7c58a4068#body_text
is schema:about of	named entity 'Libraries' named entity 'individual' named entity 'encoding' named entity 'bacteriophage' named entity 'possessed' named entity 'spike' named entity 'reverse genetics' named entity 'massive' named entity 'saturation' named entity 'libraries' named entity 'complete' named entity 'genomes' named entity 'genome' named entity 'amino acids' named entity 'amino acids' named entity 'ΦX174' named entity 'purifying selection' named entity 'bioRxiv' named entity 'ΦX174' named entity 'capsid' named entity 'molecular modeling' named entity 'mutagenesis' named entity 'oligo' named entity 'ΦX174' named entity 'chromosome' named entity 'reverse genetics' named entity 'host cell' named entity 'cell lysates' named entity 'mutation' named entity 'plasmid' named entity 'plasmid' named entity 'XL1' named entity 'microviridae' named entity 'E. coli' named entity 'mutagenesis' named entity 'Sanger sequenced' named entity 'experimental evolution' named entity 'Viral' named entity 'plasmids' named entity 'ΦX174' named entity 'plasmids' named entity 'dsDNA' named entity 'saturation mutagenesis' named entity 'mutagenic' named entity 'bioassay' named entity 'capsid' named entity 'peer-reviewed' named entity 'angstrom' named entity 'E. coli' named entity 'plasmids' named entity 'phage' named entity 'epistasis' named entity 'recombineering' named entity 'plasmids' named entity 'genome' named entity 'plasmid' named entity 'titer' named entity 'competent cells' named entity 'protein coding' named entity 'ΦX174' named entity 'gene' named entity 'Golden Gate cloning' named entity 'peer-reviewed' named entity 'epitope' named entity 'restriction enzyme' named entity 'plasmid' named entity 'mutagenesis' named entity 'oligo' named entity 'evolutionary studies' named entity 'plasmids' named entity 'recombinant' named entity 'oligonucleotides' named entity 'genome assembly' named entity 'kanamycin' named entity 'Grand Island, NY' named entity 'bacteriophage' named entity 'missense'

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