Part:BBa_K517003

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]

Functional Parameters: Austin_UTexas

BBa_K517003 parameters

Burden Imposed by this Part:

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.