title: Optimized Transformation of Newly Constructed Escherichia coli-Clostridia Shuttle Vectors into Clostridium beijerinckii.
abstract: Three Escherichia coli-Clostridia shuttle vectors, pKBA411-MCS, pKBE411-MCS, and pKBM411-MCS, which contain p15A, ColE1, and pMB1 origins for replication in E. coli, respectively, along with the pAMB origin for replication in C. beijerinckii, were constructed and examined for their transformation efficiencies into Clostridium beijerinckii NCIMB8052. The transformation condition of pKBM411-MCS, which was optimized by varying resistance, buffer composition, and DNA concentration, was further employed for the transformation of the other plasmids, pKBA411-MCS and pKBE411-MCS into C. beijerinckii. It was found out that transformation efficiency is highly dependent on the origin of replication. The highest transformation efficiency of 7.44 × 10(3) colony-forming units per microgram of DNA was obtained at 5.0 kV cm(-1) field strength, 200 Ω resistance, 270 mM sucrose concentration, 150 ng μg(-1), and 3.0 μg DNA using pKBM411-MCS having pMB1 and pAMB origins of replication. The application of the newly constructed vector system was also investigated by introducing the putative alcohol dehydrogenase gene of C. beijerinckii.
authors.affiliation: Center for Bio-based Chemistry, Green Chemistry & Engineering Division, Korea Research Institute of Chemical Technology, P.O. Box 107, 141 Gajeong-ro, Yuseong-gu, Daejeon, 305-600, Republic of Korea.
authors.forename: Young Hoon
authors.initials: YH
authors.lastname: Oh
authors.forename: Gyeong Tae
authors.initials: GT
authors.lastname: Eom
authors.forename: Kyoung Hee
authors.initials: KH
authors.lastname: Kang
authors.forename: Jae Woo
authors.initials: JW
authors.lastname: Choi
authors.forename: Bong Keun
authors.initials: BK
authors.lastname: Song
authors.forename: Seung Hwan
authors.initials: SH
authors.lastname: Lee
authors.forename: Si Jae
authors.initials: SJ
authors.lastname: Park
mesh_terms: D000096:Acetone / Q000378:metabolism; D000440:Butanols / Q000378:metabolism; D046970:Clostridium beijerinckii / Q000187:drug effects / Q000378:metabolism*; D018274:Electroporation; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation / Q000187:drug effects; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics; D018741:Replication Origin / Q000235:genetics; D013395:Sucrose / Q000494:pharmacology; D014169:Transformation, Bacterial / Q000187:drug effects
chemical_list: D000440:Butanols; D000096:Acetone; D000431:Ethanol; D013395:Sucrose