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Rapid editing and evolution of bacterial genomes using libraries of synthetic DNA

Nature Protocols volume 9, pages 2301–2316 (2014)Cite this article

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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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Figure 1: Multiplex automated genome engineering (MAGE) processes and applications.
Figure 2: Proposed mechanism and categories of MAGE-based mutagenesis.
Figure 4: Model-guided MAGE experimental design.
Figure 5: The MAGE cycle.
Figure 3: The MAGE workflow.
Figure 6: Mathematical calculations for functional diversity of mutagenized protein-coding sequences.
Figure 7: Oligo design guidelines for target loci on different strands and different replichores (chromosome halves that are bidirectionally replicated).
Figure 8: Oligo design parameters affecting allelic replacement frequency.
Figure 9: Genotype assays by MASC-PCR.
Figure 10: Mechanism for coselection MAGE.

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Acknowledgements

Funding was provided by the US Department of Energy (DE-FG02-02ER63445), Defense Advanced Research Projects Agency (N66001-12-C-4020, N66001-12-C-4211), and the Arnold and Mabel Beckman Foundation (F.J.I.).

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Author notes

  1. Ryan R Gallagher and Zhe Li: These authors contributed equally to this work.

Authors and Affiliations

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

    Ryan R Gallagher, Zhe Li, Aaron O Lewis & Farren J Isaacs

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

    Ryan R Gallagher, Zhe Li & Farren J Isaacs

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

    Aaron O Lewis

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  1. Ryan R Gallagher
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  2. Zhe Li
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Contributions

R.R.G., Z.L. and F.J.I. wrote the manuscript with contributions on mathematical modeling from A.O.L.

Corresponding author

Correspondence to Farren J Isaacs.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1: Safe insertion regions in E. coli MG1655

(Numbering according to NCBI's RefSeq accession number PRJNA57779). (PDF 227 kb)

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Gallagher, R., Li, Z., Lewis, A. et al. Rapid editing and evolution of bacterial genomes using libraries of synthetic DNA. Nat Protoc 9, 2301–2316 (2014). https://doi.org/10.1038/nprot.2014.082

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