Cozens, C. and Pinheiro, Vitor B. (2018) Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis. Nucleic Acids Research 46 (8), e51. ISSN 0305-1048.
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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.
Metadata
| Item Type: | Article |
|---|---|
| School: | Birkbeck Faculties and Schools > Faculty of Science > School of Natural Sciences |
| Research Centres and Institutes: | Structural Molecular Biology, Institute of (ISMB) |
| Depositing User: | Vitor Bernardes pinheiro |
| Date Deposited: | 21 Feb 2018 10:16 |
| Last Modified: | 02 Aug 2023 17:39 |
| URI: | https://eprints.bbk.ac.uk/id/eprint/21244 |
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