title: Genome engineering using a synthetic gene circuit in Bacillus subtilis.
abstract: Genome engineering without leaving foreign DNA behind requires an efficient counter-selectable marker system. Here, we developed a genome engineering method in Bacillus subtilis using a synthetic gene circuit as a counter-selectable marker system. The system contained two repressible promoters (B. subtilis xylA (Pxyl) and spac (Pspac)) and two repressor genes (lacI and xylR). Pxyl-lacI was integrated into the B. subtilis genome with a target gene containing a desired mutation. The xylR and Pspac-chloramphenicol resistant genes (cat) were located on a helper plasmid. In the presence of xylose, repression of XylR by xylose induced LacI expression, the LacIs repressed the Pspac promoter and the cells become chloramphenicol sensitive. Thus, to survive in the presence of chloramphenicol, the cell must delete Pxyl-lacI by recombination between the wild-type and mutated target genes. The recombination leads to mutation of the target gene. The remaining helper plasmid was removed easily under the chloramphenicol absent condition. In this study, we showed base insertion, deletion and point mutation of the B. subtilis genome without leaving any foreign DNA behind. Additionally, we successfully deleted a 2-kb gene (amyE) and a 38-kb operon (ppsABCDE). This method will be useful to construct designer Bacillus strains for various industrial applications.
authors.affiliation: Super-Bacteria Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea.
authors.forename: Da-Eun
authors.initials: DE
authors.lastname: Jeong
authors.affiliation: Super-Bacteria Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea Biosystems and Bioengineering Program, University of Science and Technology (UST), 217 Gajung-ro, Yuseong-gu, Daejeon 305-350, Republic of Korea.
authors.forename: Seung-Hwan
authors.initials: SH
authors.lastname: Park
authors.affiliation: Super-Bacteria Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea.
authors.forename: Jae-Gu
authors.initials: JG
authors.lastname: Pan
authors.affiliation: Genofocus Inc., 533-1 Yongsan-dong, Yuseong-gu, Daejeon 305-500, Republic of Korea.
authors.forename: Eui-Joong
authors.initials: EJ
authors.lastname: Kim
authors.affiliation: Super-Bacteria Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea Biosystems and Bioengineering Program, University of Science and Technology (UST), 217 Gajung-ro, Yuseong-gu, Daejeon 305-350, Republic of Korea sookeun@kribb.re.kr.
authors.forename: Soo-Keun
authors.initials: SK
authors.lastname: Choi
mesh_terms: D001412:Bacillus subtilis / Q000187:drug effects / Q000235:genetics*; D001483:Base Sequence; D002702:Chloramphenicol Resistance / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D053263:Gene Regulatory Networks; D005813:Genes, Synthetic; D005818:Genetic Engineering / Q000379:methods*; D005819:Genetic Markers; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009876:Operon; D010957:Plasmids / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D005819:Genetic Markers