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 sought a reverse genetics system for ΦX174 wherein the virus chromosome was segmented and encoded on individual vector plasmids. Our method of construction closely followed that for the assembly of human coronavirus NL63 (Donaldson et al. 2008) , where each plasmid contained unique BsmB1 type IIS restriction endonuclease sites flanking unique five nucleotide overlaps of wild-type (WT) ΦX174. This architecture allows for faithful and easy assembly of the complete genome via Golden Gate cloning. The ΦX174 genome was separated into 14 separate nontoxic fragments by PCR and cloned into vector plasmids where genes were separated from their promoters and larger genes were segmented (full sequences for all plasmid inserts are given in Supplemental Note S1). The segments ranged from 129 to 634 base pairs in length, with necessary additional truncations of genes D and A. ΦX174 phage could be reconstituted by digesting pooled plasmids with BsmB1, ligating inserts overnight, and transforming into electrocompetent E. coli C Figure 2 . Structural view of potential epistatic interactions between F and G libraries. A. Pentameric complex between F and G shown in surface view. For both F and G, one monomer is shown as a ribbon color-coded according to the saturation mutagenesis library prepared in this work. B. Close-up of interaction surface between the F3 sub-library of F protein (green ribbons) and G2 sub-library of G protein (purple spheres). C. Distance mapping between alpha carbons in F and G crystal structure. Residues within 10 angstrom are colors red, 10-20 are black, and >20 are blue. Euclidian distances calculated from F-G crystal structure. Heatmap shows that many opportunities exist for close proximity (interactions) both within and between fragments.