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

Young Hoon Oh; Gyeong Tae Eom; Kyoung Hee Kang; Jae Woo Choi; Bong Keun Song; Seung Hwan Lee; Si Jae Park

doi:10.1007/s12010-015-1740-x

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

Young Hoon Oh
, Gyeong Tae Eom
, Kyoung Hee Kang
, Jae Woo Choi
, Bong Keun Song
, Seung Hwan Lee
, Si Jae Park

Department of Chemical Engineering & Materials Science

Research output: Contribution to journal › Article › peer-review

6 Scopus citations

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 × 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.

Original language	English
Pages (from-to)	226-236
Number of pages	11
Journal	Applied Biochemistry and Biotechnology
Volume	177
Issue number	1
DOIs	https://doi.org/10.1007/s12010-015-1740-x
State	Published - 28 Sep 2015

Bibliographical note

Publisher Copyright:
© 2015, Springer Science+Business Media New York.

Keywords

Clostridium beijerinckii
E. coli-Clostridia shuttle vector
Electroporation

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10.1007/s12010-015-1740-x

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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 × 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.",

keywords = "Clostridium beijerinckii, E. coli-Clostridia shuttle vector, Electroporation",

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doi = "10.1007/s12010-015-1740-x",

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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

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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 - https://www.scopus.com/pages/publications/84940449523

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

Abstract

Bibliographical note

Keywords

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