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Precise manipulation of bacterial chromosomes by conjugative assembly genome engineering

Nature Protocols volume 9, pages 2285–2300 (2014)Cite this article

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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.

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Figure 1: Comparison of conjugation in plasmids, Hfr strains and CAGE.
Figure 2: Overview of CAGE.
Figure 3: Placement of oriT-kan and P/N markers to ensure integrity of the desired genomic regions.
Figure 4: Overview of the conjugation procedure in CAGE.
Figure 5: Overview of the selection procedure in CAGE.
Figure 6: Genotyping by MASC-PCR.

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Acknowledgements

The authors acknowledge support from the 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 (F.J.I.), Gruber Foundation (N.J.M.), the National Institute of Health Cellular and Molecular Biology Training Grant (N.J.M.), the National Institute of Health Genetics Training Grant (N.J.M.), and the National Institute of Health Biophysics Training Grant (D.W.M.) and the Yale Chemical Biology Institute Training Grant (D.W.M.).

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Authors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA

    Natalie J Ma & Farren J Isaacs

  2. Systems Biology Institute, Yale University, West Haven, Connecticut, USA

    Natalie J Ma, Daniel W Moonan & Farren J Isaacs

  3. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA

    Daniel W Moonan

Authors
  1. Natalie J Ma
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  2. Daniel W Moonan
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Contributions

F.J.I. developed the CAGE methodology in collaboration with A. Tolonen, M. Lajoie and G. Church. N.J.M. finalized the CAGE protocol, wrote the manuscript and created figures. D.W.M. provided feedback on manuscript and created figures.

Corresponding author

Correspondence to Farren J Isaacs.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Safe Insertion Regions (SIRs) in E. coli MG1655. (PDF 167 kb)

Supplementary Data

CAGE universal cassettes. (PDF 132 kb)

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Ma, N., Moonan, D. & Isaacs, F. Precise manipulation of bacterial chromosomes by conjugative assembly genome engineering. Nat Protoc 9, 2285–2300 (2014). https://doi.org/10.1038/nprot.2014.081

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