Optimized Transformation of Newly Constructed Escherichia coli-Clostridia Shuttle Vectors into Clostridium beijerinckii
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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.
KeywordsClostridium beijerinckii E. coli-Clostridia shuttle vector Electroporation
This work was supported by the R & D Program of MOTIE/KEIT (10049674) and the Technology Development Program to Solve Climate Changes (Systems Metabolic Engineering for Biorefineries) (NRF-2012-C1AAA001-2012M1A2A2026556) from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea. Further support from the Energy Efficiency & Resources Core Technology Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) granted financial resource from the Ministry of Trade, Industry & Energy (MOTIE), Republic of Korea (No. 20132020000170) is appreciated.
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