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

We therefore suspected that large userdefined libraries of phage could be generated by combining the advantages of replicative plasmids with advances in DNA synthesis. The bacteriophage ΦX174 is an excellent system to test this approach. High resolution X-ray crystallography structures of the capsid and spike proteins (McKenna et al. 1992 , Dokland et al. 1997 are available for ΦX174, making it amenable to compare empirical mutagenesis results and molecular modeling predictions, it has a very small genome (Sanger et al. 1978) of 5,386 nucleotides that is readily sequenced, and it is easily grown on a variety of lab E. coli strains. These attractive features of the ΦX174 experimental system have led to proof-of-principle demonstrations from other groups for synthetic genome assembly from oligonucleotides (Smith et al. 2003) and complete refactoring of the phage (Jaschke et al. 2012 , Jaschke et al. 2018 ).