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.
Type: | Article |
---|---|
Title: | Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis |
Location: | England |
Open access status: | An open access version is available from UCL Discovery |
DOI: | 10.1093/nar/gky067 |
Publisher version: | https://doi.org/10.1093/nar/gky067 |
Language: | English |
Additional information: | Copyright © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology |
URI: | https://discovery.ucl.ac.uk/id/eprint/10043295 |
Archive Staff Only
![]() |
View Item |