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Nucleic Acids Research
Article . 2017 . Peer-reviewed
License: CC BY NC
Data sources: Crossref
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Nucleic Acids Research
Article
License: CC BY NC
Data sources: UnpayWall
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PubMed Central
Article . 2017
License: CC BY NC
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Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA

Authors: Lau, Yu Heng; Stirling, Finn; Kuo, James; Karrenbelt, Michiel A. P.; Chan, Yujia A.; Riesselman, Adam; Horton, Connor A.; +6 Authors

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

Abstract

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.

Keywords

DNA, Bacterial, Salmonella typhimurium, Microbial Viability, Reproducibility of Results, Genes, Bacterial, Leucine, Genes, Synthetic, Synthetic Biology and Bioengineering, Codon, Genetic Engineering, Genome, Bacterial

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    influence
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    This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
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selected citations
These citations are derived from selected sources.
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
65
Top 10%
Top 10%
Top 1%
Green
gold