Journal
NATURE PROTOCOLS
Volume 9, Issue 10, Pages 2285-2300Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2014.081
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Funding
- U.S. Department of Energy [DE-FG02-02ER63445]
- Defense Advanced Research Projects Agency [N66001-12-C-4020, N66001-12-C-4211]
- Arnold and Mabel Beckman Foundation
- Gruber Foundation
- National Institute of Health Cellular and Molecular Biology Training Grant
- National Institute of Health Genetics Training Grant
- National Institute of Health Biophysics Training Grant
- Yale Chemical Biology Institute Training Grant
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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.
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