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

The fragment-based system provides an ideal way to generate libraries for the study of epistasis and multi-mutational step regions of the neighborhood around wild type. When a gene is in multiple fragments, the number of mutational variants we expect from the assembled mutant fragments is the product of their individual variant number. Hence, since G1 contains 2640 variants and G2 contains 800 variants, they can be combined to generate 2.1 million double mutation variants. Further, nothing requires the fragments to be from the same gene. For example, since the F and G proteins bind to each other to form the capsid of phiX174 ( Fig. 2) , libraries combining mutations in F with mutations in G will capture mutations that can readily interact in the protein and have the potential to generate interesting instances of epistasis (Fig 2) . The central challenge then becomes how to survey the vast size of these combinatorial plasmid libraries. Right now, our transformational efficiencies vary, but even at the upper end of hundreds of thousands of transformants per reaction, only a fraction of the total diversity in the library can be surveyed.