2005 Fiscal Year Final Research Report Summary
Genome Manipulation of Bacillus subtilis
| Project/Area Number |
15580070
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied microbiology
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| Research Institution | Sojo University |
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Principal Investigator |
TAGUCHI Hisataka Sojo University, Faculty of Biotechnology and Life Science, associate professor, 生物生命学部, 助教授 (90212018)
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| Co-Investigator(Kenkyū-buntansha) |
AKAMATSU Takashi Sojo University, Faculty of Biotechnology and Life Science, professor, 生物生命学部, 教授 (50133567)
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| Project Period (FY) |
2003 – 2005
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| Keywords | High transformation efficiency / Bacillus subtilis / protoplast / competent cell / pBluescript II / pC194 / DNA-binding-protein / inverted repeat |
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| Research Abstract |
pCB1 is a chimeric plasmid of pC194, replicating in Bacillus subtilis, and pBluescript II, a cloning vector for Escherichia coli. The transformation efficiency of B.subtilis by μg DNA of pCB1 (native DNA : 4×10^8, purified DNA : 7×10^4) is higher than that by pC194 (native DNA : 2×10^2, purified DNA : 7×10^3). The native DNA means the DNA in lysed protoplasts. The mechanism causing the high transformation efficiency was studied.
(1)Detection of the nucleotide sequence of pBluescript II causing the high transformation efficiency
pCB4, which is pC194 harboring a fragment of pBluescript II(270 b, f1-lac), showed the high transformation efficiency. The efficiency by pCB4 is 30,000 times higher than that by pCl94, corresponding to 20% of that by pCB1. Two inverted repeats exist in the 270 b fragment. It was suggested that interactions between these repeats and DNA binding proteins were involved the transformation efficiency.
(2)Purification of DNA binding protein
Protoplast solution of B.subtilis harboring pUB110 was directly applied to the top of Sephacryl S500-HR column (1.5×17cm), and gel filtration chromatography was done. The purpose of the chromatography was separation of pUB110 from chromosomal DNA and cellular proteins without removing DNA binding proteins. Fractions containing pUB110 with transformation activity was separated and pooled. The electrophoretic mobility of the separated pUB110 on a agarose gel was smaller than that of pUB110 purified with conventional method. DNA binding proteins of the separated pUB110 were analyzed by SDS-PAGE. Several protein bands were observed with the molecular weight of 143,109,78,58,54,50,48,42, and 38 kd.
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