Faber, M. S. | bioRxiv Science

bioRxiv2019

Saturation mutagenesis genome engineering of infective {Phi}X174 bacteriophage via unamplified oligo pools and golden gate assembly

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 {Phi}X174 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 {Phi}X174 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.

genomics↗

bioRxiv2019

Impact of initial protein stability on local fitness landscapes

It is incompletely understood how biophysical properties like protein stability impact molecular evolution and epistasis. Epistasis is defined as specific when a mutation exclusively influences the phenotypic effect of another mutation, often at physically interacting residues. By contrast, nonspecific epistasis results when a mutation is influenced by a large number of non-local mutations. As most mutations are pleiotropic, the in vivo folding probability - governed by basal protein stability - is thought to determine activity-enhancing mutational tolerance, which implies that nonspecific epistasis is dominant. However, evidence exists for both specific and nonspecific epistasis as the prevalent factor, with limited comprehensive datasets to validate either claim. Using deep mutational scanning we probe how in vivo enzyme folding probability impacts local fitness landscapes. We computationally designed two different variants of the amidase AmiE in which catalytic efficiencies are statistically indistinguishable but the enzyme variants have lower probabilities of folding in vivo. Local fitness landscapes show only slight alterations among variants, with essentially the same global distribution of fitness effects. However, specific epistasis was predominant for the subset of mutations exhibiting positive sign epistasis. These mutations mapped to spatially distinct locations on AmiE near the initial mutation or proximal to the active site. Intriguingly, the majority of specific epistatic mutations were codon-dependent, with different synonymous codons resulting in fitness sign reversals. Together, these results offer a nuanced view of how protein folding probability impacts local fitness landscapes, and suggest that transcriptional-translational effects are an equally important determinant as stability in determining evolutionary outcomes.

evolutionary biology↗