Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

Abstract

Engineering proteins for designer functions and biotechnological applications almost invariably requires (or at least benefits from) multiple mutations to non-contiguous residues. Several methods for multiple site-directed mutagenesis exist, but there remains a need for fast and simple methods to efficiently introduce such mutationsparticularly for generating large, high quality libraries for directed evolution.Here, we present Darwin Assembly, which can deliver high quality libraries of over 10 8 transformants, targeting multiple (> 10) distal sites with minimal wild-type contamination (lower than 0.25% of total population) and which takes a single working day from purified plasmid to library transformation. DarwinAssembly uses commercially available enzymes, can be readily automated, and offers a cost-effective route to highly complex and customizable library generation.

Methods for enzyme library creation: Which one will you choose?

“…It is noteworthy as it represents a relatively simple, fast, and low‐cost solution to the introduction of multiple mutations in distal sites of a protein. [ 104 ] Among its strengths (Table 4), it allows for flexibility in the evolutionary pathway that the researcher uncovers: non‐contiguous mutations are introduced simultaneously and not sequentially so the evolutionary pathway chosen does not rely on the very first mutation introduced, as per other methods. The method lends itself to automation, for instance by using the Antha software of Synthace.…”