Translational_Unit

Part:BBa_K517003

Designed by: Marianne Park   Group: iGEM11_British_Columbia   (2011-09-21)
Revision as of 22:11, 2 October 2011 by Ayjchan (Talk | contribs)

beta-pinene synthase

The (-)-b-pinene synthase (PsTPS-Pin2) synthase is a monoterpene synthase isolated from Sitka spruce converts geranyl pyrophosphate to 70.5%(-)-beta-pinene, 29.5%(-)-alpha-pinene. (-)-b-pinene synthase has been previously characterized by expression in C41 DE3 E. coli followed by a His-tag purification. The purified enzymes were assayed in vitro with GPP and sent for GC-MS.

Reference: Keeling, CI, Weisshar S, Ralph SG, Jancsik S, Hamberger B, Dullat HK, and Joerg Bohlmann. Transcriptome mining, functional characterization, and phylogeny of a large terpene synthase gene family in spruce (Picea spp.) BMC. 2011 March 7 11:43

Characterization by British Columbia iGEM 2011

GC-MS analysis of Beta-Pinene Synthase function by British Columbia iGEM 2011

Gas Chromatography Mass Spectrum of Beta-pinene Synthase Products: We expressed the beta-pinene synthase (BBa_K517003) in C41 DE3 E. coli and lysed the cell culture to obtain protein extract which was run through a nickel column to purify the HIS-tagged beta-pinene synthase. The purified synthase was used in an in vitro enzymatic assay to produce beta-pinene from geranyl diphosphate (GPP) substrate. (A) Monoterpene product spectrum of our beta-pinene synthase. Through gas chromatography mass spectrometry, we analysed the products of the beta-pinene synthase and found alpha-pinene and beta-pinene. This product spectrum agrees with prior findings (Keeling et al. 2011 BMC Plant Biology). In comparison, the control without GPP indicated by the dark blue link did not produce any monoterpenes. (B) Identification of alpha-pinene as a product. Cross referencing with standards and a compound library revealed that the beta-pinene product had the characteristic base peaks and retention time of 3.6 minutes. Top panel displays peak spectrum for our sample. Bottom panel displays peak spectrum for library standard. (C) Identification of beta-pinene as a product. Cross referencing with standards and a compound library revealed that the beta-pinene product had the characteristic base peaks and retention time of 5.6 minutes. Top panel displays peak spectrum for our sample. Bottom panel displays peak spectrum for library standard.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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