PT - JOURNAL ARTICLE AU - Julie E. Norville AU - Cameron L. Gardner AU - Eduardo Aponte AU - Conor K. Camplisson AU - Alexandra Gonzales AU - David K. Barclay AU - Katerina A. Turner AU - Victoria Longe AU - Maria Mincheva AU - Jun Teramoto AU - Kento Tominaga AU - Ryota Sugimoto AU - James E. DiCarlo AU - Marc Guell AU - Eriona Hysolli AU - John Aach AU - Christopher J. Gregg AU - Barry L. Wanner AU - George M. Church TI - Assembly of Radically Recoded <em>E. coli</em> Genome Segments AID - 10.1101/070417 DP - 2016 Jan 01 TA - bioRxiv PG - 070417 4099 - http://biorxiv.org/content/early/2016/08/31/070417.short 4100 - http://biorxiv.org/content/early/2016/08/31/070417.full AB - 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).