(228ck) Nicking Mutagenesis: A Plasmid-Based Single-Pot Saturation Mutagenesis Method

The coupling of synthetic biology to protein mutational studies (i.e. directed evolution or deep mutational scanning) has greatly expanded the utility of these methods^1,2. A core component of these experiments is the generation of a mutational library. Error prone PCR and cassette mutagenesis are the most common strategies but suffer from limitations in scalability, deficient codon sampling, and imprecise control over the number of mutations introduced. Based on these limitations, we developed Nicking Mutagenesis, a robust and accessible method for the construction of high quality, user-defined mutational libraries. Nicking Mutagenesis is a single day, single pot method using routinely prepped plasmid dsDNA as an input substrate. In this talk I will describe the overall method and demonstrate the efficacy on multiple systems. I will present data for saturation mutagenesis at multiple positions and single-site saturation mutagenesis libraries containing all possible single codon substitutions for full-length genes. Efficacy of the method will be presented by assessing library coverage using deep sequencing, revealing 100% coverage of all possible single non-synonymous mutations (2840 total) and 100% of all possible programmed codon mutations (8946 total), with over 60% of the library containing exactly one mutation. Resources for portability of the method, including plasmid sharing and protocol capture will also be discussed.

1. Currin, A., Swainston, N., Day, P. J. & Kell, D. B. Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently. Chem. Soc. Rev. 44, 1172â??1239 (2015).

2. Fowler, D. M. & Fields, S. Deep mutational scanning: a new style of protein science. Nat. Methods 11, 801â??807 (2014).