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

doi:10.1038/srep08712

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

Department of Chemistry

Research output: Contribution to journal › Article › peer-review

1 Citation (Scopus)

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 language	English
Article number	8712
Journal	Scientific reports
Volume	5
DOIs	https://doi.org/10.1038/srep08712
Publication status	Published - 2015

Bibliographical note

Funding Information:
This research was supported by the Korea Health Technology R&D Project through Korea Health Industry Development Institute (KHIDI) [HI13C2163]; Intelligent Synthetic Biology Center of Global Frontier Project [NRF-2012M3A6A8053632]; Pioneer Research Center Program [NRF-2012-0009557] through National Research Foundation of Korea; Coperative Research Program for Agriculture Science & Technology Development, Next-Generation BioGreen 21 [PJ009034012014] through Rural Development Administration, Republic of Korea. Funding for open access charge was supported by Pioneer Research Center Program [NRF-2012-0009557] through National Research Foundation of Korea. Jaehwan Jeong is supported by a National Junior Research Fellowship from the National Research Foundation of Korea [NRF-2011-0012414]. This research was supported by the Samsung Advanced Institute of Technology. We thank members of Duhee Bang Lab for their critical comments.

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General

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10.1038/srep08712

Cite this

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

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).",

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

note = "Funding Information: This research was supported by the Korea Health Technology R&D Project through Korea Health Industry Development Institute (KHIDI) [HI13C2163]; Intelligent Synthetic Biology Center of Global Frontier Project [NRF-2012M3A6A8053632]; Pioneer Research Center Program [NRF-2012-0009557] through National Research Foundation of Korea; Coperative Research Program for Agriculture Science & Technology Development, Next-Generation BioGreen 21 [PJ009034012014] through Rural Development Administration, Republic of Korea. Funding for open access charge was supported by Pioneer Research Center Program [NRF-2012-0009557] through National Research Foundation of Korea. Jaehwan Jeong is supported by a National Junior Research Fellowship from the National Research Foundation of Korea [NRF-2011-0012414]. This research was supported by the Samsung Advanced Institute of Technology. We thank members of Duhee Bang Lab for their critical comments.",

year = "2015",

doi = "10.1038/srep08712",

language = "English",

volume = "5",

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AU - Jeong, Jaehwan

AU - Seo, Han Na

AU - Jung, Yu Kyung

AU - Lee, Jeewon

AU - Ryu, Gyuri

AU - Lee, Wookjae

AU - Kwon, Euijin

AU - Ryoo, Keunsoo

AU - Kim, Jungyeon

AU - Cho, Hwa Young

AU - Cho, Kwang Myung

AU - Park, Jin Hwan

AU - Bang, Duhee

N1 - Funding Information: This research was supported by the Korea Health Technology R&D Project through Korea Health Industry Development Institute (KHIDI) [HI13C2163]; Intelligent Synthetic Biology Center of Global Frontier Project [NRF-2012M3A6A8053632]; Pioneer Research Center Program [NRF-2012-0009557] through National Research Foundation of Korea; Coperative Research Program for Agriculture Science & Technology Development, Next-Generation BioGreen 21 [PJ009034012014] through Rural Development Administration, Republic of Korea. Funding for open access charge was supported by Pioneer Research Center Program [NRF-2012-0009557] through National Research Foundation of Korea. Jaehwan Jeong is supported by a National Junior Research Fellowship from the National Research Foundation of Korea [NRF-2011-0012414]. This research was supported by the Samsung Advanced Institute of Technology. We thank members of Duhee Bang Lab for their critical comments.

PY - 2015

Y1 - 2015

N2 - 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).

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

Abstract

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