Saturation mutagenesis genome engineering of infective FX174 bacteriophage via unamplified oligo pools and golden gate assembly

Matthew S. Faber; James T. Van Leuven; Martina M. Ederer; Yesol Sapozhnikov; Zoë L. Wilson; Holly A. Wichman; Timothy A. Whitehead; Craig R. Miller

Based on the above considerations, adapting this method to larger viruses will require modifications for the genome assembly steps. There are a variety of methods available for attempting to improve genome library assembly and amplification. For improving genome assembly, we speculate that a combination of hierarchical assembly (Lee et al. 2015) with other yeast-based assembly methods (Ando et al. 2015) will allow for large viral genomes to be efficiently assembled. Yehl et al. 2019 build large libraries at targeted epitope regions by combining methods; plasmid-based mutagenesis and cloning followed by recombination with wild-type phage during infection. Finally, we speculate that the Tx.Tl cell free expression system (Garamella et al. 2016) will be able to produce the viral libraries with less bias than in vivo amplification in E. coli. In summary, we have presented a method for efficient deep mutational scanning of the bacteriophage ΦX174. We anticipate that this method will find utility in fundamental molecular evolution studies as well as translate to potential medicinal applications.