Total synthesis of Escherichia coli with a recoded genome

@article{Fredens2019TotalSO,
  title={Total synthesis of Escherichia coli with a recoded genome},
  author={Julius Fredens and Kaihang Wang and Daniel de la Torre and Louise F. H. Funke and Wesley E. Robertson and Yonka Christova and Tiongsun Chia and Wolfgang Hellmut Schmied and Daniel L. Dunkelmann and V{\'a}clav Ber{\'a}nek and Chayasith Uttamapinant and Andres Gonzalez Llamazares and Thomas S. Elliott and Jason W. Chin},
  journal={Nature},
  year={2019},
  volume={569},
  pages={514-518}
}
Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon—out of up to 6 synonyms—to encode each amino acid. [] Key Method We create a variant of Escherichia coli with a four-megabase synthetic genome through a high-fidelity convergent total synthesis. Our synthetic genome implements a defined recoding and refactoring scheme—with simple corrections at just seven positions—to replace every known occurrence of two sense codons and a stop codon in the genome. Thus, we…

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References

SHOWING 1-10 OF 55 REFERENCES

Design, synthesis, and testing toward a 57-codon genome

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 are reported.

Defining synonymous codon compression schemes by genome recoding

This work endow E. coli with a system for efficient, programmable replacement of genomic DNA with long synthetic DNA, through the in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda-red-mediated recombination and simultaneous positive and negative selection.

Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

Significance This work presents the genome-wide replacement of all rare AGR (AGA and AGG) arginine codons in the essential genes of Escherichia coli with synonymous CGN alternatives. Synonymous codon

Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli

The in vivo reassignment of the AGG sense codon from arginine to l-homoarginine showed the feasibility of breaking the degeneracy of sense codons to enhance the amino-acid diversity in the genetic code.

Codon reassignment in the Escherichia coli genetic code

Only a few genetic modifications of E. coli were needed to circumvent the lethality of codon reassignment; erasing all UAG triplets from the genome was unnecessary, and UAG was assigned unambiguously to a natural or non-natural amino acid, according to the specificity of the UAG-decoding tRNA.

Genomically Recoded Organisms Expand Biological Functions

The construction and characterization of a genomically recoded organism (GRO) is described, which exhibited improved properties for incorporation of nonstandard amino acids that expand the chemical diversity of proteins in vivo and exhibited increased resistance to T7 bacteriophage, demonstrating that new genetic codes could enable increased viral resistance.

Highly reproductive Escherichia coli cells with no specific assignment to the UAG codon

This study developed E. coli strains in which the UAG codon was reserved for non-natural amino acids, without compromising the reproductive strength of the host cells, and showed superior productivities for hirudin molecules sulfonated on a particular tyrosine residue, and the Fab fragments of Herceptin containing multiple azido groups.

Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement

H hierarchical conjugative assembly genome engineering (CAGE) was developed to merge these sets of codon modifications into genomes with 80 precise changes, which demonstrate that these synonymous codon substitutions can be combined into higher-order strains without synthetic lethal effects.

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

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.
...