Isaacs, F.J. et al. Science 333, 348–353 (2011).
Isaacs et al. achieve large-scale genome editing in E. coli by stitching together smaller-scale viable intermediates. They started by using multiplex automated engineering to produce strains with mutations in different sections of the chromosome. Through a clever combination of integrated resistance markers and a conjugation-initiating gene, their hierarchical conjugative assembly genome engineering approach allowed them to seamlessly convert all stop codons from TAG to TAA.
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Large-scale genome editing in Escherichia coli. Nat Methods 8, 709 (2011). https://doi.org/10.1038/nmeth.1689
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DOI: https://doi.org/10.1038/nmeth.1689