TY - JOUR
T1 - Development of a novel plasmid vector pTIO-1 adapted for electrotransformation of Porphyromonas gingivalis
AU - Tagawa, Junpei
AU - Inoue, Tetsuyoshi
AU - Naito, Mariko
AU - Sato, Keiko
AU - Kuwahara, Tomomi
AU - Nakayama, Masaaki
AU - Nakayama, Koji
AU - Yamashiro, Takashi
AU - Ohara, Naoya
N1 - Funding Information:
This work was supported by grants-in-aid from the Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan ( 22390342 , 26293401 ) and by the Association for Research on Lactic Acid Bacteria .
PY - 2014/10
Y1 - 2014/10
N2 - We report here the construction of a plasmid vector designed for the efficient electrotransformation of the periodontal pathogen Porphyromonas gingivalis. The novel Escherichia coli-. Bacteroides/. P. gingivalis shuttle vector, designated pTIO-1, is based on the 11.0-kb E. coli-. Bacteroides conjugative shuttle vector, pVAL-1 (a pB8-51 derivative). To construct pTIO-1, the pB8-51 origin of replication and erythromycin resistance determinant of pVAL-1 were cloned into the E. coli cloning vector pBluescript II SK(-) and non-functional regions were deleted. pTIO-1 has an almost complete multiple cloning site from pBluescript II SK(-). The size of pTIO-1 is 4.5. kb, which is convenient for routine gene manipulation. pTIO-1 was introduced into P. gingivalis via electroporation, and erythromycin-resistant transformants carrying pTIO-1 were obtained. We characterized the transformation efficiency, copy number, host range, stability, and insert size capacity of pTIO-1. An efficient plasmid electrotransformation of P. gingivalis will facilitate functional analysis and expression of P. gingivalis genes, including the virulence factors of this bacterium.
AB - We report here the construction of a plasmid vector designed for the efficient electrotransformation of the periodontal pathogen Porphyromonas gingivalis. The novel Escherichia coli-. Bacteroides/. P. gingivalis shuttle vector, designated pTIO-1, is based on the 11.0-kb E. coli-. Bacteroides conjugative shuttle vector, pVAL-1 (a pB8-51 derivative). To construct pTIO-1, the pB8-51 origin of replication and erythromycin resistance determinant of pVAL-1 were cloned into the E. coli cloning vector pBluescript II SK(-) and non-functional regions were deleted. pTIO-1 has an almost complete multiple cloning site from pBluescript II SK(-). The size of pTIO-1 is 4.5. kb, which is convenient for routine gene manipulation. pTIO-1 was introduced into P. gingivalis via electroporation, and erythromycin-resistant transformants carrying pTIO-1 were obtained. We characterized the transformation efficiency, copy number, host range, stability, and insert size capacity of pTIO-1. An efficient plasmid electrotransformation of P. gingivalis will facilitate functional analysis and expression of P. gingivalis genes, including the virulence factors of this bacterium.
KW - Electroporation
KW - Periodontal disease
KW - Plasmid
KW - Porphyromonas
KW - Transformation
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U2 - 10.1016/j.mimet.2014.07.032
DO - 10.1016/j.mimet.2014.07.032
M3 - Article
C2 - 25102110
AN - SCOPUS:84907343214
SN - 0167-7012
VL - 105
SP - 174
EP - 179
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
ER -