Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis | Nucleic Acids Research | Oxford Academic

Christopher Cozens, Vitor B Pinheiro, Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis, Nucleic Acids Research, Volume 46, Issue 8, 4 May 2018, Page e51, https://doi.org/10.1093/nar/gky067 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 mutations – particularly for generating large, high quality libraries for directed evolution. Here, we present Darwin Assembly, which can deliver high quality libraries of >108 transformants, targeting multiple (>10) distal sites with minimal wild-type contamination (<0.25% of total population) and which takes a single working day from purified plasmid to library transformation. We demonstrate its efficacy with whole gene codon reassignment of chloramphenicol acety

Christopher Cozens, Vitor B Pinheiro, Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis, Nucleic Acids Research, Volume 46, Issue 8, 4 May 2018, Page e51, https://doi.org/10.1093/nar/gky067 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 mutations – particularly for generating large, high quality libraries