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

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 engineerin...

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Autores principales: Jeong, Jaehwan, Seo, Han Na, Jung, Yu Kyung, Lee, Jeewon, Ryu, Gyuri, Lee, Wookjae, Kwon, Euijin, Ryoo, Keunsoo, Kim, Jungyeon, Cho, Hwa-Young, Cho, Kwang Myung, Park, Jin Hwan, Bang, Duhee
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4348660/
https://www.ncbi.nlm.nih.gov/pubmed/25736821
http://dx.doi.org/10.1038/srep08712
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author Jeong, Jaehwan
Seo, Han Na
Jung, Yu Kyung
Lee, Jeewon
Ryu, Gyuri
Lee, Wookjae
Kwon, Euijin
Ryoo, Keunsoo
Kim, Jungyeon
Cho, Hwa-Young
Cho, Kwang Myung
Park, Jin Hwan
Bang, Duhee
author_facet Jeong, Jaehwan
Seo, Han Na
Jung, Yu Kyung
Lee, Jeewon
Ryu, Gyuri
Lee, Wookjae
Kwon, Euijin
Ryoo, Keunsoo
Kim, Jungyeon
Cho, Hwa-Young
Cho, Kwang Myung
Park, Jin Hwan
Bang, Duhee
author_sort Jeong, Jaehwan
collection PubMed
description 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).
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spelling pubmed-43486602015-03-10 Repetitive genomic insertion of gene-sized dsDNAs by targeting the promoter region of a counter-selectable marker Jeong, Jaehwan Seo, Han Na Jung, Yu Kyung Lee, Jeewon Ryu, Gyuri Lee, Wookjae Kwon, Euijin Ryoo, Keunsoo Kim, Jungyeon Cho, Hwa-Young Cho, Kwang Myung Park, Jin Hwan Bang, Duhee Sci Rep Article 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). Nature Publishing Group 2015-03-04 /pmc/articles/PMC4348660/ /pubmed/25736821 http://dx.doi.org/10.1038/srep08712 Text en Copyright © 2015, Macmillan Publishers Limited. All rights reserved http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Jeong, Jaehwan
Seo, Han Na
Jung, Yu Kyung
Lee, Jeewon
Ryu, Gyuri
Lee, Wookjae
Kwon, Euijin
Ryoo, Keunsoo
Kim, Jungyeon
Cho, Hwa-Young
Cho, Kwang Myung
Park, Jin Hwan
Bang, Duhee
Repetitive genomic insertion of gene-sized dsDNAs by targeting the promoter region of a counter-selectable marker
title Repetitive genomic insertion of gene-sized dsDNAs by targeting the promoter region of a counter-selectable marker
title_full Repetitive genomic insertion of gene-sized dsDNAs by targeting the promoter region of a counter-selectable marker
title_fullStr Repetitive genomic insertion of gene-sized dsDNAs by targeting the promoter region of a counter-selectable marker
title_full_unstemmed Repetitive genomic insertion of gene-sized dsDNAs by targeting the promoter region of a counter-selectable marker
title_short Repetitive genomic insertion of gene-sized dsDNAs by targeting the promoter region of a counter-selectable marker
title_sort repetitive genomic insertion of gene-sized dsdnas by targeting the promoter region of a counter-selectable marker
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4348660/
https://www.ncbi.nlm.nih.gov/pubmed/25736821
http://dx.doi.org/10.1038/srep08712
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