Cozens, C; Pinheiro, VB; (2018) Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis. Nucleic Acids Research , 46 (8) e51. 10.1093/nar/gky067.

Preview

Text gky067.pdf - Published Version Download (1MB) | Preview

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 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 acetyl transferase, mutating 19 codons in a single reaction in KOD DNA polymerase and generating high quality, multiple-site libraries in T7 RNA polymerase and Tgo DNA polymerase. Darwin Assembly uses commercially available enzymes, can be readily automated, and offers a cost-effective route to highly complex and customizable library generation.

Download activity - last month

Export as CSVJSONXML

Download activity - last 12 months

Export as XMLJSONCSV

Downloads by country - last 12 months

Export as CSVXMLJSON

Archive Staff Only

View Item