TY - JOUR
T1 - Optimized Transformation of Newly Constructed Escherichia coli-Clostridia Shuttle Vectors into Clostridium beijerinckii
AU - Oh, Young Hoon
AU - Eom, Gyeong Tae
AU - Kang, Kyoung Hee
AU - Choi, Jae Woo
AU - Song, Bong Keun
AU - Lee, Seung Hwan
AU - Park, Si Jae
N1 - Publisher Copyright:
© 2015, Springer Science+Business Media New York.
PY - 2015/9/28
Y1 - 2015/9/28
N2 - 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.
AB - 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.
KW - Clostridium beijerinckii
KW - E. coli-Clostridia shuttle vector
KW - Electroporation
UR - http://www.scopus.com/inward/record.url?scp=84940449523&partnerID=8YFLogxK
U2 - 10.1007/s12010-015-1740-x
DO - 10.1007/s12010-015-1740-x
M3 - Article
C2 - 26152821
AN - SCOPUS:84940449523
SN - 0273-2289
VL - 177
SP - 226
EP - 236
JO - Applied Biochemistry and Biotechnology
JF - Applied Biochemistry and Biotechnology
IS - 1
ER -