title: Design, synthesis, and testing toward a 57-codon genome.
abstract: Recoding--the repurposing of genetic codons--is a powerful strategy for enhancing genomes with functions not commonly found in nature. Here, we report computational design, synthesis, and progress toward assembly of a 3.97-megabase, 57-codon Escherichia coli genome in which all 62,214 instances of seven codons were replaced with synonymous alternatives across all protein-coding genes. We have validated 63% of recoded genes by individually testing 55 segments of 50 kilobases each. We observed that 91% of tested essential genes retained functionality with limited fitness effect. We demonstrate identification and correction of lethal design exceptions, only 13 of which were found in 2229 genes. This work underscores the feasibility of rewriting genomes and establishes a framework for large-scale design, assembly, troubleshooting, and phenotypic analysis of synthetic organisms.
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Nili
authors.initials: N
authors.lastname: Ostrov
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Program in Systems Biology, Harvard University, Cambridge, MA 02138, USA. Ecole des Mines de Paris, Mines Paristech, Paris 75272, France.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Landon
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Wyss Institute for Biologically Inspired Engineering, Boston, MA 02115, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Guell
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Program in Biophysics, Harvard University, Boston, MA 02115, USA.
authors.forename: Gleb
authors.initials: G
authors.lastname: Kuznetsov
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Teramoto
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Natalie
authors.initials: N
authors.lastname: Cervantes
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Minerva
authors.initials: M
authors.lastname: Zhou
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Kerry
authors.initials: K
authors.lastname: Singh
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Program in Biological and Biomedical Sciences, Harvard University, Cambridge, MA 02138, USA.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Napolitano
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Moosburner
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Ellen
authors.initials: E
authors.lastname: Shrock
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, Boston, MA 02115, USA.
authors.forename: Benjamin W
authors.initials: BW
authors.lastname: Pruitt
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, Boston, MA 02115, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Conway
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Wyss Institute for Biologically Inspired Engineering, Boston, MA 02115, USA.
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Goodman
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Cameron L
authors.initials: CL
authors.lastname: Gardner
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Gary
authors.initials: G
authors.lastname: Tyree
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Gonzales
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Barry L
authors.initials: BL
authors.lastname: Wanner
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Julie E
authors.initials: JE
authors.lastname: Norville
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. mlajoie@uw.edu gchurch@genetics.med.harvard.edu.
authors.forename: Marc J
authors.initials: MJ
authors.lastname: Lajoie
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Wyss Institute for Biologically Inspired Engineering, Boston, MA 02115, USA. mlajoie@uw.edu gchurch@genetics.med.harvard.edu.
authors.forename: George M
authors.initials: GM
authors.lastname: Church
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D020043:Genes, Essential; D005804:Genes, Lethal; D005813:Genes, Synthetic; D005815:Genetic Code / Q000235:genetics / Q000502:physiology*; D005818:Genetic Engineering; D016680:Genome, Bacterial; D010641:Phenotype; D014176:Protein Biosynthesis / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins