title: Rapid editing and evolution of bacterial genomes using libraries of synthetic DNA.
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.
authors.affiliation: 1] Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA. [2] Systems Biology Institute, Yale University, West Haven, Connecticut, USA. [3].
authors.forename: Ryan R
authors.initials: RR
authors.lastname: Gallagher
authors.affiliation: 1] Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA. [2] Systems Biology Institute, Yale University, West Haven, Connecticut, USA. [3].
authors.forename: Zhe
authors.initials: Z
authors.lastname: Li
authors.affiliation: 1] Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA. [2] Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.
authors.forename: Aaron O
authors.identifier: 0000000188181763
authors.initials: AO
authors.lastname: Lewis
authors.affiliation: 1] Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA. [2] Systems Biology Institute, Yale University, West Haven, Connecticut, USA.
authors.forename: Farren J
authors.initials: FJ
authors.lastname: Isaacs
mesh_terms: D000483:Alleles; D004277:DNA, Single-Stranded; D004926:Escherichia coli / Q000235:genetics; D048168:Escherichia coli K12 / Q000235:genetics; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods; D014644:Genetic Variation; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods*; D016297:Mutagenesis, Site-Directed
chemical_list: D004277:DNA, Single-Stranded