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- Title
Optimized Transformation of Newly Constructed Escherichia coli-Clostridia Shuttle Vectors into Clostridium beijerinckii.
- Authors
Oh, Young; Eom, Gyeong; Kang, Kyoung; Choi, Jae; Song, Bong; Lee, Seung; Park, Si
- 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 colony-forming units per microgram of DNA was obtained at 5.0 kV cm field strength, 200 Ω resistance, 270 mM sucrose concentration, 150 ng μg, 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.
- Subjects
GENETIC vectors; CLOSTRIDIUM beijerinckii; ESCHERICHIA coli; ELECTROPORATION; GENETIC transformation
- Publication
Applied Biochemistry & Biotechnology, 2015, Vol 177, Issue 1, p226
- ISSN
0273-2289
- Publication type
Academic Journal
- DOI
10.1007/s12010-015-1740-x