Optimized Transformation of Newly Constructed Escherichia coli-Clostridia Shuttle Vectors into Clostridium beijerinckii

• Published in: Applied biochemistry and biotechnology Vol. 177; no. 1; pp. 226 - 236
• Main Authors: Oh, Young Hoon, Eom, Gyeong Tae, Kang, Kyoung Hee, Choi, Jae Woo, Song, Bong Keun, Lee, Seung Hwan, Park, Si Jae
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.09.2015; Springer Nature B.V
• Subjects: Acetone - metabolism; alcohol dehydrogenase; Bacteria; Biochemistry; Biotechnology; Butanols - metabolism; Chemistry; Chemistry and Materials Science; Clostridium beijerinckii; Clostridium beijerinckii - drug effects; Clostridium beijerinckii - metabolism; Deoxyribonucleic acid; DNA; E coli; Electroporation; Enzymes; Escherichia; Escherichia coli; Escherichia coli - drug effects; Escherichia coli - metabolism; Ethanol - metabolism; Fermentation - drug effects; Gene expression; genes; Genes, Bacterial; genetic vectors; Genetic Vectors - metabolism; plasmids; Recombination, Genetic - genetics; replication origin; Replication Origin - genetics; sucrose; Sucrose - pharmacology; transformation; Transformation, Bacterial - drug effects; Electroporation; Clostridia shuttle vector
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-015-1740-x

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 × 10³ 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.