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- W2020478051 abstract "Abstract Genome engineering can be used to produce bacterial strains with a wide range of desired phenotypes. However, the incorporation of gene-sized DNA fragments is often challenging due to the intricacy of the procedure, off-target effects and low insertion efficiency. Here we report a genome engineering method enabling the continuous incorporation of gene-sized double-stranded DNAs (dsDNAs) into the Escherichia coli genome. DNA substrates are inserted without introducing additional marker genes, by synchronously turning an endogenous counter-selectable marker gene ON and OFF. To accomplish this, we utilized λ Red protein-mediated recombination to insert dsDNAs within the promoter region of a counter-selectable marker gene, tolC . By repeatedly switching the marker gene ON and OFF, a number of desired gene-sized dsDNAs can be inserted consecutively. With this method, we successfully inserted approximately 13 kb gene clusters to generate engineered E. coli strains producing 1,4-butanediol (1,4-BDO)." @default.
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- W2020478051 date "2015-03-04" @default.
- W2020478051 modified "2023-09-26" @default.
- W2020478051 title "Repetitive genomic insertion of gene-sized dsDNAs by targeting the promoter region of a counter-selectable marker" @default.
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- W2020478051 doi "https://doi.org/10.1038/srep08712" @default.
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