Difference between revisions of "Part:BBa K1448000"
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* Origin of the enzyme: ''Citrus unshiu'' | * Origin of the enzyme: ''Citrus unshiu'' | ||
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'''Introduction''' | '''Introduction''' | ||
− | Since | + | Since ɤ-terpinene is a volatile material, we expected that an amount of ɤ-terpinene were detected at the headspace of the culture in which the transformant with ɤ-terpinene generator is cultivated and tried the detection by GC analysis. |
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== '''Reference''' == | == '''Reference''' == | ||
− | * | + | *[[http://www.sciencedirect.com/science/article/pii/S0168945203003777 Shimada et al, 2003]]Takehiko Shimada Tomoko Endoa Hiroshi Fujiia Masakazu Harab Takanori Uedaa Masayuki Kitaa Mitsuo Omura (2003) Molecular cloning and functional characterization of four monoterpene synthase genes from Citrus unshiu Marc. |
+ | *[[http://pubs.acs.org/doi/abs/10.1021/jf020993f MARIO C. FOTI et al, 2003]]MARIO C. FOTI*,† AND K. U. INGOLD‡ (2003) Mechanism of Inhibition of Lipid Peroxidation by γ-Terpinene, an Unusual and Potentially Useful Hydrocarbon Antioxidant | ||
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Revision as of 23:07, 17 October 2014
gamma-terpinene synthase
This part contains ɤ-terpinene synthase region of Citrus unshiu and was isolated by Mr.Shimada from Citrus unshiu.http://www.sciencedirect.com/science/article/pii/S0168945203003777 Shimada et al, 2003
Further information:
- DDBJ entry: http://getentry.ddbj.nig.ac.jp/getentry/na/AB110639/?format=flatfile&filetype=html&trace=true&show_suppressed=false&limit=10 AB110639
- Origin of the enzyme: Citrus unshiu
Background and principles
Characterization
Western-bloting analysis
Yeast cells carrying plasmids of p427-TEF-GST-monoterpenoid synthase were cultivated overnight in SD medium (0.67% yeast nitrogen base and 2% glucose with G418) 50mL at 28°C. Cells were collected and kept at -30˚C. Western blot analysis was carried out using anti-GST antibody and 12.5% polyacrylamide gel with SDS.
<Results>
Western blot data (Fig.2) showed clear bands around 90kDa in the lanes of p427-TEF- monoterpenoid synthase but not in the lane of empty vector.
In vivo detection of ɤ-terpinene using GC
Introduction
Since ɤ-terpinene is a volatile material, we expected that an amount of ɤ-terpinene were detected at the headspace of the culture in which the transformant with ɤ-terpinene generator is cultivated and tried the detection by GC analysis.
GC analysis ofɤ-terpinene in the culture
Materials and Methods
We cultivated the transformants containing ɤ-terpinene generator in p427-TEF or only empty p427-TEF in 50 mL SD medium (0.67% yeast nitrogen base and 2% glucose with G418) at 29˚C, 120 rpm for 24 h.
Analytic instrument: Shimadzu GC14A
Column: GLScience InertCap1 (15m Length,0.25 mm I.D., 0.25mm film thickness)
Injection port 200C, Detector port 210 C
Detector: Flame Ionization Detector
Column Oven Temperature 40C/5min- 10C/min-200C/5min
Carrier Gas: Helium
Result
we uesd SPME (SOLID PHASE MICROEXTRACTION) to catch ɤ-terpinene at the headspace of the culture. However, any amounts of ɤ-terpinene were not detected at the headspace of the culture (the exposure time is 30min).
Next we tried to catch ɤ-terpinene in the liquid phase of the culture using monotrap (the exposure time is 1h) and an amount of ɤ-terpinene was detected in the liquid phase of the culture.
Reference
- http://www.sciencedirect.com/science/article/pii/S0168945203003777 Shimada et al, 2003Takehiko Shimada Tomoko Endoa Hiroshi Fujiia Masakazu Harab Takanori Uedaa Masayuki Kitaa Mitsuo Omura (2003) Molecular cloning and functional characterization of four monoterpene synthase genes from Citrus unshiu Marc.
- http://pubs.acs.org/doi/abs/10.1021/jf020993f MARIO C. FOTI et al, 2003MARIO C. FOTI*,† AND K. U. INGOLD‡ (2003) Mechanism of Inhibition of Lipid Peroxidation by γ-Terpinene, an Unusual and Potentially Useful Hydrocarbon Antioxidant
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 204
Illegal XhoI site found at 715 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]