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 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).
Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 120-749, Korea.
Jaehwan
J
Jeong
Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 120-749, Korea.
Han Na
HN
Seo
Samsung Advanced Institute of Technology, 130 Samsung-ro, Yeongtong-gu, Suwon-si, Gyeonggi-do, 443-803, Korea.
Yu Kyung
YK
Jung
Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 120-749, Korea.
Jeewon
J
Lee
Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 120-749, Korea.
Gyuri
G
Ryu
Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 120-749, Korea.
Wookjae
W
Lee
Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 120-749, Korea.
Euijin
E
Kwon
Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 120-749, Korea.
Keunsoo
K
Ryoo
Department of Chemistry, University College London, 20 Gordon Street, London, WC1H 0AJ, U.K.
Jungyeon
J
Kim
Samsung Advanced Institute of Technology, 130 Samsung-ro, Yeongtong-gu, Suwon-si, Gyeonggi-do, 443-803, Korea.
Hwa-Young
HY
Cho
Samsung Advanced Institute of Technology, 130 Samsung-ro, Yeongtong-gu, Suwon-si, Gyeonggi-do, 443-803, Korea.
Kwang Myung
KM
Cho
Samsung Advanced Institute of Technology, 130 Samsung-ro, Yeongtong-gu, Suwon-si, Gyeonggi-do, 443-803, Korea.
Jin Hwan
JH
Park
Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 120-749, Korea.
Duhee
D
Bang
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D053898:Biosynthetic Pathways / Q000235:genetics; D002072:Butylene Glycols / Q000378:metabolism; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics; D005819:Genetic Markers; D016680:Genome, Bacterial / Q000235:genetics*; D026901:Membrane Transport Proteins / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods*; D008957:Models, Genetic; D005810:Multigene Family / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011995:Recombination, Genetic; D015203:Reproducibility of Results
D001425:Bacterial Outer Membrane Proteins; D002072:Butylene Glycols; D029968:Escherichia coli Proteins; D005819:Genetic Markers; D026901:Membrane Transport Proteins; C073126:tolC protein, E coli; C039681:1,4-butanediol; D004247:DNA