Assembly of Radically Recoded E. coli Genome Segments

Julie E. Norville; Cameron L. Gardner; Eduardo Aponte; Conor K. Camplisson; Alexandra Gonzales; David K. Barclay; Katerina A. Turner; Victoria Longe; Maria Mincheva; Jun Teramoto; Kento Tominaga; Ryota Sugimoto; James E. DiCarlo; Marc Guell; Eriona Hysolli; John Aach; Christopher J. Gregg; Barry L. Wanner; George M. Church

doi:10.1101/070417

bioRxiv · 10.1101/070417

Assembly of Radically Recoded E. coli Genome Segments

Julie E. Norville·Cameron L. Gardner·Eduardo Aponte·Conor K. Camplisson·Alexandra Gonzales·David K. Barclay·Katerina A. Turner·Victoria Longe·Maria Mincheva·Jun Teramoto·Kento Tominaga·Ryota Sugimoto·James E. DiCarlo·Marc Guell·Eriona Hysolli·John Aach·Christopher J. Gregg·Barry L. Wanner·George M. Church

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Abstract

The large potential of radically recoded organisms (RROs) in medicine and industry depends on improved technologies for efficient assembly and testing of recoded genomes for biosafety and functionality. Here we describe a next generation platform for conjugative assembly genome engineering, termed CAGE 2.0, that enables the scarless integration of large synthetically recoded E. coli segments at isogenic and adjacent genomic loci. A stable tdk dual selective marker is employed to facilitate cyclical assembly and removal of attachment sites used for targeted segment delivery by sitespecific recombination. Bypassing the need for vector transformation harnesses the multi Mb capacity of CAGE, while minimizing artifacts associated with RecA-mediated homologous recombination. Our method expands the genome engineering toolkit for radical modification across many organisms and recombinase-mediated cassette exchange (RMCE).

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Julie E. Norville, Cameron L. Gardner, Eduardo Aponte, Conor K. Camplisson, Alexandra Gonzales, David K. Barclay, Katerina A. Turner, Victoria Longe, Maria Mincheva, Jun Teramoto, Kento Tominaga, Ryota Sugimoto, James E. DiCarlo, Marc Guell, Eriona Hysolli, John Aach, Christopher J. Gregg, Barry L. Wanner, George M. Church. 2016-08-19. Assembly of Radically Recoded E. coli Genome Segments. https://doi.org/10.1101/070417

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