Repetitive genomic insertion of gene-sized dsDNAs by targeting the promoter region of a counter-selectable marker

Jaehwan Jeong, Han Na Seo, Yu Kyung Jung, Jeewon Lee, Gyuri Ryu, Wookjae Lee, Euijin Kwon, Keunsoo Ryoo, Jungyeon Kim, Hwa Young Cho, Kwang Myung Cho, Jin Hwan Park, Duhee Bang

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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).

Original languageEnglish
Article number8712
JournalScientific reports
Volume5
DOIs
Publication statusPublished - 2015 Jan 1

All Science Journal Classification (ASJC) codes

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    Jeong, J., Seo, H. N., Jung, Y. K., Lee, J., Ryu, G., Lee, W., Kwon, E., Ryoo, K., Kim, J., Cho, H. Y., Cho, K. M., Park, J. H., & Bang, D. (2015). Repetitive genomic insertion of gene-sized dsDNAs by targeting the promoter region of a counter-selectable marker. Scientific reports, 5, [8712]. https://doi.org/10.1038/srep08712