Genomically Recoded Organisms Expand Biological Functions

Lajoie, Marc J.; Rovner, Alexis J.; Goodman, Daniel B.; Aerni, Hans‐Rudolf; Haimovich, Adrian D.; Kuznetsov, Gleb; Mercer, Jaron A. M.; Wang, Harris H.; Carr, Peter A.; Mosberg, Joshua A.; Rohland, Nadin; Schultz, Peter G.; Jacobson, Joseph M.; Rinehart, Jesse; Church, George M.; Isaacs, Farren J.

doi:10.1126/science.1241459

Cited by 753 publications

(943 citation statements)

References 71 publications

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“…Multiplex Automatable Genome Engineering (MAGE) entails the delivery of synthetic DNA oligonucleotides into growing cells to mutate specific genomic sequences during DNA replication 70 . It works efficiently in E. coli and was used impressively to recode all 321 TAG stop codons in the E. coli genome to TAA stop codons in order to provide a genomically-recoded organism capable of utilising non-standard amino acids 71 . Other genome editing methods rely on homologous recombination (recombineering), and, in E. coli, this can be used to insert or delete DNA at almost any desired locus via the lambda Red method which utilises a phage recombinase to recombine long regions of homology 72 .…”

Section: Box 1: Genome Editingmentioning

confidence: 99%

Bricks and blueprints: methods and standards for DNA assembly

Casini

Storch

Baldwin

et al. 2015

Nat Rev Mol Cell Biol

247

186

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PrefaceDNA assembly is a key part of constructing gene expression systems and even whole chromosomes. In the past decade a plethora of powerful new DNA assembly methods including Gibson assembly, Golden Gate and LCR have been developed. In this Innovation article we discuss these methods and standards such as MoClo, GoldenBraid, MODAL and PaperClip, which have been developed to facilitate a streamlined assembly workflow, aid material exchange, and the creation of modular, reusable DNA parts. IntroductionOur capacity to cut and paste DNA from different sources and to assemble it into gene constructs has been one of the key drivers of biological research and biotechnology over the past four decades. However, despite countless advances in molecular biology, the assembly of DNA parts into new constructs remains a craft that is both time consuming and unpredictable. The decreasing costs of gene synthesis promises to alleviate these limitations by providing custom-made double-stranded DNA fragments typically between 200 and 2000 bp in length 1 . Nonetheless, gene synthesis does not eliminate the need for DNA assembly, which remains necessary for the production of constructs beyond one kilobase in size, both in research labs and at gene synthesis companies. DNA assembly also enables carrying out projects with more complex experimental needs and is especially valuable for building diverse plasmid libraries and creating multicomponent systems, and has even been used to construct synthetic cells 2 .Addressing the limitations of DNA assembly methods has been one of the key goals of synthetic biology, a scientific discipline focused on the construction and testing of new or redesigned versions of genes, gene networks, pathways and cells 3,4 . In order to tackle projects of increasing scale and complexity, researchers have invested significant effort into developing new tools for DNA assembly and into matching them with improved, lower-cost gene synthesis (for reviewes on gene synthesis see REFS. 1,5 1,5 ), as well as a suite of important new tools for genome editing (Box 1). With these combined advances the field is now at a point where gene synthesis and DNA assembly can empower even undergraduate students to construct entire eukaryotic chromosomes 6 . This acceleration in the scale of DNA assembly enables construction projects too complex to be drawn out on the back of an envelope, which instead require an engineering approach. In the past decade important 1 assembly methods such as Gibson assembly and Golden Gate have been developed 7,8 , which define new protocols for joining together DNA parts. Alongside these methods, researchers have also developed various physical standards such as MODAL (Modular Overlap-Directed Assembly with Linkers) 9 and MoClo (Modular Cloning system) 12 that define rules for the format of DNA parts that can be used with them. These physical standards facilitate the re-use of parts between experiments, exchange of parts between research groups and importantly provide modularity in construction. ...

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Section: Box 1: Genome Editingmentioning

confidence: 99%

Bricks and blueprints: methods and standards for DNA assembly

Casini

Storch

Baldwin

et al. 2015

Nat Rev Mol Cell Biol

247

186

View full text Add to dashboard Cite

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“…from Escherichia coli (23), no genome-wide replacement of a sense codon has been reported. Although the translation function of the AGG codon has been shown to permit efficient suppression with nonstandard amino acids (24)(25)(26), AGG necessarily remains translated as Arg in each of these studies.…”

Section: Significancementioning

confidence: 99%

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

Napolitano

Landon

Gregg

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as noncoding information for gene regulation and genome maintenance. The rare arginine codons AGA and AGG (AGR) present a case study in codon choice, with AGRs encoding important transcriptional and translational properties distinct from the other synonymous alternatives (CGN). We created a strain of Escherichia coli with all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU codons, but the remaining 13 "recalcitrant" AGRs required diversification to identify viable alternatives. Successful replacement codons tended to conserve local ribosomal binding site-like motifs and local mRNA secondary structure, sometimes at the expense of amino acid identity. Based on these observations, we empirically defined metrics for a multidimensional "safe replacement zone" (SRZ) within which alternative codons are more likely to be viable. To evaluate synonymous and nonsynonymous alternatives to essential AGRs further, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild-type allele, allowing us to evaluate exhaustively the fitness impact of all 64 codon alternatives. Using this method, we confirmed the relevance of the SRZ by tracking codon fitness over time in 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. Our unbiased and systematic strategy for identifying unpredicted design flaws in synthetic genomes and for elucidating rules governing codon choice will be crucial for designing genomes exhibiting radically altered genetic codes.codon choice | genome editing | recoded genomes

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“…Two years ago, Church and his team (which included Isaacs) reported the synthesis of a strain of Escherichia coli that had a reprogrammed genetic code 3 . Instead of recognizing a particular DNA triplet known as the amber stop codon as an order to terminate protein synthesis, the recoded bacterium read the same instruction as a directive to incorporate a new kind of amino acid into its proteins.…”

Section: By E L I E D O L G I Nmentioning

confidence: 99%

Crunch time for pet theory on dark matter

Castelvecchi¹

2015

Nature

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Genomically Recoded Organisms Expand Biological Functions

Cited by 753 publications

References 71 publications

Bricks and blueprints: methods and standards for DNA assembly

Bricks and blueprints: methods and standards for DNA assembly

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

Crunch time for pet theory on dark matter

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