Difference between revisions of "Part:BBa K517002"
| Line 5: | Line 5: | ||
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 | 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 Alpha-Pinene Synthase function by British Columbia iGEM 2011=== | ||
| + | |||
| + | [[Image:Ubcigem2011alphapingcms.jpg | frame | center | '''Gas Chromatography Mass Spectrum of Alpha-pinene Synthase Products:''' We expressed the alpha-pinene synthase (<partinfo>BBa_K517002</partinfo>) 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 alpha-pinene synthase. The purified synthase was used in an ''in vitro'' enzymatic assay to produce alpha-pinene from geranyl diphosphate (GPP) substrate. (A) '''Monoterpene product spectrum of our alpha-pinene synthase.''' Through gas chromatography mass spectrometry, we analysed the products of the alpha-pinene synthase and found alpha-pinene, beta-pinene and small quantities of other monoterpenes. This product spectrum agrees with prior findings (Keeling et al. 2011 BMC Plant Biology). (B) '''Identification of alpha-pinene as a product.''' Cross referencing with standards and a compound library revealed that the alpha-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. ]] | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
Revision as of 22:09, 2 October 2011
alpha-pinene synthase
The (-)-a-pinene synthase (PsTPS-Pin) synthase is a monoterpene synthase isolated from Sitka spruce converts geranyl pyrophosphate to 83.4%(-)-alpha-pinene, 12.6%(-)-beta-pinene, 2.1% Linalool, 1.0% (-)-beta-phellandrene, 0.4% Camphene, and 0.4% myrcene. (-)-a-pinene synthase has been previously characterized by expression in C41 DE3 E. coli followed by a His-tag purification.
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 Alpha-Pinene Synthase function by British Columbia iGEM 2011
Gas Chromatography Mass Spectrum of Alpha-pinene Synthase Products: We expressed the alpha-pinene synthase (BBa_K517002) 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 alpha-pinene synthase. The purified synthase was used in an in vitro enzymatic assay to produce alpha-pinene from geranyl diphosphate (GPP) substrate. (A) Monoterpene product spectrum of our alpha-pinene synthase. Through gas chromatography mass spectrometry, we analysed the products of the alpha-pinene synthase and found alpha-pinene, beta-pinene and small quantities of other monoterpenes. This product spectrum agrees with prior findings (Keeling et al. 2011 BMC Plant Biology). (B) Identification of alpha-pinene as a product. Cross referencing with standards and a compound library revealed that the alpha-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:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1200
Illegal XhoI site found at 1280 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]