Difference between revisions of "Part:BBa K517003"
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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 | ||
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| + | ==Characterization by British Columbia iGEM 2011== | ||
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| + | ===GC-MS analysis of Beta-Pinene Synthase function by British Columbia iGEM 2011=== | ||
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| + | [[Image:Ubcigem2011betapingcms.jpg | frame | center |'''Gas Chromatography Mass Spectrum of Beta-pinene Synthase Products:''' We expressed the beta-pinene synthase (<partinfo>BBa_K517003</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 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. ]] | ||
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| + | ==Functional Parameters: Austin_UTexas== | ||
| + | <html> | ||
| + | <body> | ||
<partinfo>BBa_K517003 parameters</partinfo> | <partinfo>BBa_K517003 parameters</partinfo> | ||
| − | < | + | <h3><center>Burden Imposed by this Part:</center></h3> |
| + | <figure> | ||
| + | <div class = "center"> | ||
| + | <center><img src = "https://static.igem.org/mediawiki/parts/f/fa/T--Austin_Utexas--no_burden_icon.png" style = "width:160px;height:120px"></center> | ||
| + | </div> | ||
| + | <figcaption><center><b>Burden Value: 3.9 ± 4.2% </b></center></figcaption> | ||
| + | </figure> | ||
| + | <p> Burden is the percent reduction in the growth rate of <i>E. coli</i> 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 | ||
| + | <a href="https://parts.igem.org/Part:BBa_K3174002">BBa_K3174002</a> - <a href="https://parts.igem.org/Part:BBa_K3174007">BBa_K3174007</a> 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 <a href="http://2019.igem.org/Team:Austin_UTexas">2019 Austin_UTexas team</a>.</p> | ||
| + | <p>This functional parameter was added by the <a href="https://2020.igem.org/Team:Austin_UTexas/Contribution">2020 Austin_UTexas team.</a></p> | ||
| + | </body> | ||
| + | </html> | ||
Latest revision as of 22:15, 3 September 2020
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:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Functional Parameters: Austin_UTexas
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.