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Updated: Apr 24, 2026

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
Published on: April 1, 2016
Rapid editing and evolution of bacterial genomes using libraries of synthetic DNA
Ryan R Gallagher1, Zhe Li1, Aaron O Lewis2
11] Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA. [2] Systems Biology Institute, Yale University, West Haven, Connecticut, USA. [3].
Abstract:
Multiplex automated genome engineering (MAGE) is a powerful technology for in vivo genome editing that uses synthetic single-stranded DNA (ssDNA) to introduce targeted modifications directly into the Escherichia coli chromosome. MAGE is a cyclical process that involves transformation of ssDNA (by electroporation) followed by outgrowth, during which bacteriophage homologous recombination proteins mediate annealing of ssDNAs to their genomic targets. By iteratively introducing libraries of mutagenic ssDNAs targeting multiple sites, MAGE can generate combinatorial genetic diversity in a cell population. Alternatively, MAGE can introduce precise mutant alleles at many loci for genome-wide editing or for recoding projects that are not possible with other methods. In recent technological advances, MAGE has been improved by strain modifications and selection techniques that enhance allelic replacement. This protocol describes the manual execution of MAGE wherein each cycle takes ≈ 2.5 h, which, if carried out by two people, allows ≈ 10 continuous cycles of MAGE-based mutagenesis per day.
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