title: Precise manipulation of bacterial chromosomes by conjugative assembly genome engineering.
abstract: Conjugative assembly genome engineering (CAGE) is a precise method of genome assembly using conjugation to hierarchically combine distinct genotypes from multiple Escherichia coli strains into a single chimeric genome. CAGE permits large-scale transfer of specified genomic regions between strains without constraints imposed by in vitro manipulations. Strains are assembled in a pairwise manner by establishing a donor strain that harbors conjugation machinery and a recipient strain that receives DNA from the donor. Within strain pairs, targeted placement of a conjugal origin of transfer and selectable markers in donor and recipient genomes enables the controlled transfer and selection of desired donor-recipient chimeric genomes. By design, selectable markers act as genomic anchor points, and they are recycled in subsequent rounds of hierarchical genome transfer. A single round of CAGE can be completed in a week, thus enabling four rounds (hierarchical assembly of 16 strains) of CAGE to be completed in roughly 1 month.
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: Natalie J
authors.initials: NJ
authors.lastname: Ma
authors.affiliation: 1] Systems Biology Institute, Yale University, West Haven, Connecticut, USA. [2] Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Moonan
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: D002876:Chromosomes, Bacterial; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000235:genetics*; D005819:Genetic Markers; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods*; D010957:Plasmids
chemical_list: D005819:Genetic Markers