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Updated: May 10, 2026

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
Improved bacterial mutagenesis by high-frequency allele exchange, demonstrated in Clostridium difficile and
Alexandra Faulds-Pain1, Brendan W Wren
1Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom.
None:
Here we show that the frequency of mutant isolation by two-step allele exchange can be improved by increasing the length of homologous DNA and the opportunity for recombination, obviating the need for counterselection markers. These principles are demonstrated in Clostridium difficile and Streptococcus suis but are likely to be generally applicable.
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