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

Research output: Contribution to journalArticlepeer-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 languageEnglish
Pages (from-to)226-236
Number of pages11
JournalApplied Biochemistry and Biotechnology
Volume177
Issue number1
DOIs
StatePublished - 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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