@article {Norville070417, author = {Julie E. Norville and Cameron L. Gardner and Eduardo Aponte and Conor K. Camplisson and Alexandra Gonzales and David K. Barclay and Katerina A. Turner and Victoria Longe and Maria Mincheva and Jun Teramoto and Kento Tominaga and Ryota Sugimoto and James E. DiCarlo and Marc Guell and Eriona Hysolli and John Aach and Christopher J. Gregg and Barry L. Wanner and George M. Church}, title = {Assembly of Radically Recoded E. coli Genome Segments}, elocation-id = {070417}, year = {2016}, doi = {10.1101/070417}, publisher = {Cold Spring Harbor Laboratory}, 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).}, URL = {https://www.biorxiv.org/content/early/2016/08/31/070417}, eprint = {https://www.biorxiv.org/content/early/2016/08/31/070417.full.pdf}, journal = {bioRxiv} }