Coding

Part:BBa_K1448000

Designed by: yuma kobayashi   Group: iGEM14_KIT-Kyoto   (2014-09-22)
Revision as of 06:08, 14 October 2014 by UMBABA (Talk | contribs) (Western-bloting analysis)

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:

Image of Citrus unshiu


Background and principles

Fig.2 Reaction catalyzed by ɤ-terpinene synthase.

















Characterization

Western-bloting analysis

(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.)

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 204
    Illegal XhoI site found at 715
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
n/agamma-terpinene synthase