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Nucleic Acids Res. 2017 Jun 20;45(11):6971-6980. doi: 10.1093/nar/gkx415.

Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA.

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Wyss Institute for Biologically Inspired Engineering, Harvard University, 3 Blackfan Circle, 5th Floor, Boston, MA 02115, USA.
Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Alpert 536, Boston, MA 02115, USA.
Systems and Synthetic Biology, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands.
Program in Biomedical Informatics, Harvard Medical School, Boston, MA 02115, USA.
Synthetic Genomics, Inc., 11149 North Torrey Pines Road, La Jolla, CA 92037, USA.
Synthetic Biology and Bioenergy Group, J. Craig Venter Institute, 4120 Capricorn Lane, La Jolla, CA 92037, USA.


The ability to rewrite large stretches of genomic DNA enables the creation of new organisms with customized functions. However, few methods currently exist for accumulating such widespread genomic changes in a single organism. In this study, we demonstrate a rapid approach for rewriting bacterial genomes with modified synthetic DNA. We recode 200 kb of the Salmonella typhimurium LT2 genome through a process we term SIRCAS (stepwise integration of rolling circle amplified segments), towards constructing an attenuated and genetically isolated bacterial chassis. The SIRCAS process involves direct iterative recombineering of 10-25 kb synthetic DNA constructs which are assembled in yeast and amplified by rolling circle amplification. Using SIRCAS, we create a Salmonella with 1557 synonymous leucine codon replacements across 176 genes, the largest number of cumulative recoding changes in a single bacterial strain to date. We demonstrate reproducibility over sixteen two-day cycles of integration and parallelization for hierarchical construction of a synthetic genome by conjugation. The resulting recoded strain grows at a similar rate to the wild-type strain and does not exhibit any major growth defects. This work is the first instance of synthetic bacterial recoding beyond the Escherichia coli genome, and reveals that Salmonella is remarkably amenable to genome-scale modification.

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