Difference between revisions of "Part:BBa K1448000"
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− | [[File:westernblot of terpinene.png|thumb| | + | [[File:westernblot of terpinene.png|thumb|550px|right|(Fig.2 Western-bloting analysis of GST and d-limonene synthase (Lane2), ɤ-terpinene synthase (Lane3) and β-pinene synthase(Lane4) fusion proteins expressed in yeast. (A) The gel picture of SDS-PAGE and western-blot; Lanes: 1, p427-TEF; 2, p427-TEF-LS; 3, p427-TEF-ɤTPNS; 4, p427-TEF-βPINS. (B) vector map of plasmids. (C) The names and molecular weights of each mono-terpene synthase.)]] |
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− | < | + | 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. |
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+ | <Results> | ||
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+ | 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. | ||
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Revision as of 06:08, 14 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-MS
Reference
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]