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

Our strategy for generating large user-defined mutagenesis libraries of bacteriophage ΦX174 is shown in Figure 1 . The circular 5386 nucleotide genome compactly encodes 11 genes where the F and G genes encode the capsid and spike proteins, respectively (Fig. 1A) . The genome was segmented onto 14 separate plasmids, including 3 plasmids for the F gene and 2 for the G gene (Fig. 1B) . Fragment boundaries were first engineered to divide large genes, separate regulatory regions from protein coding genes, and result in fragments of similar size. Initial attempts to clone some fragments resulted in low plasmid yield, presumably due to gene toxicity. These genes were further divided and tested empirically, resulting in the final set of 14 fragments. User-defined comprehensive mutations on F and G were encoded using . Cartoon structure of the ΦX174 virion. F and G proteins are organized into pentamers and are the only exposed proteins in the mature virion. B. A linear schematic of the circular ΦX174 genome showing the size and location of the synthetic genome fragments. The location of mutated residues in genes F and G are indicated. C. An oligo pool containing all mutagenic oligos for genes F and G was generated and used in Nicking Scanning Mutagenesis for the generation of saturation mutant libraries for genes F and G. D. Golden Gate cloning was used to assemble the mutant genomes, which were transformed into XL1-Blue competent cells (Agilent) and plated on susceptible E. coli C cells. The resulting plaques were sequenced. E. Linear schematics depicting some of the possible mutant libraries that can be generated using the workflow and existing libraries.