2002 Fiscal Year Final Research Report Summary
Development of double stranded DNA sensing fluorescent proteins and the application for highly sensitive Salmonella detection
| Project/Area Number |
13650849
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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 |
生物・生体工学
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| Research Institution | Tokyo University of Agriculture and Technology |
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Principal Investigator |
NARITA Mitsuaki Faculty of Technology, Department of Biotechnology, Professor, 工学部, 教授 (40015102)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHIDA Hiromi Marine Biotechnology Institute Co., ltd, PD researcher, 釜石研究所, PD研究員 (10313305)
NARITA Mitsuaki Faculty of Technology, Department of Biotechnology, Professor (40015102)
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| Project Period (FY) |
2001 – 2002
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| Keywords | DnaA / oriC / PCR / fluorescence resonance energy transfer / Salmonella / Site directed mutagenesis |
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| Research Abstract |
In recent years, salmonellosis is widespread throughout the world. For the rapid and sensitive detection of Salmonella have been paid Increasing demand.
We have started research to develop a novel method for the detection of a double -stranded DNA with a specific sequence using an fluorescent-labeled engineered DNA-binding protein, DnaA.. DnaA is known to bind to bacterial oriC region. The DNA fragment detection system is based on fluorescence resonance energy transfer (FRET) between fluorescent -〓beled oriC and DnaA .
First, we constructed fusion proteins between green fluorescent protein (GFP) and DnaA (GFP-DnaA). GFP-DnaAs showed fluorescence in recombinant E. coli in vivo. However, fusion proteins were produced as an inclusion body and the refolding of the target fusion protein was not achieved. Second, we constructed DnaAIV, a fusion protein of the DNA-binding domain of DnaA and glutathione S- transferase. DnaAIV was produced mainly in inclusion body fraction in recombinant E. coli however the refold of this fusion protein was successfully achieved.
Fluorescein -5- maleimide (F5M) -labeled DnaAIV bound to Salmonella's oriC region which was labeled by 6-carboxytetramethylrhodamine (TAMRA) or hexachloro -6- carboxy - fluoresceine (HEX). F5M derived fluorescent intensity was reduced when TAMRA or HEX labeled oriC was added. No fluorescent change was observed when control DNA was added. These results show FRET, based on DnaA-oriC specific binding is a useful technique to detect Salmonella.
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