Abstract
The λ phage Red proteins greatly enhance homologous recombination in Escherichia coli. Red-mediated recombination or “recombineering” can be used to construct targeted gene deletions as well as to introduce point mutations into the genome. Here, we describe our method for scanning mutagenesis using recombineered oligonucleotide libraries. This approach entails randomization of specific codons within a target gene, followed by functional selection to isolate mutants. Oligonucleotide library mutagenesis has generated hundreds of novel antibiotic resistance mutations in genes encoding ribosomal proteins, and should be applicable to other systems for which functional selections exist.
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Acknowledgment
This work was supported by grant R01 GM078634 from the National Institutes of Health.
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Diner, E.J., Garza-Sánchez, F., Hayes, C.S. (2011). Genome Engineering Using Targeted Oligonucleotide Libraries and Functional Selection. In: Williams, J. (eds) Strain Engineering. Methods in Molecular Biology, vol 765. Humana Press. https://doi.org/10.1007/978-1-61779-197-0_5
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DOI: https://doi.org/10.1007/978-1-61779-197-0_5
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