Difference between revisions of "Part:BBa K517002:Experience"
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===GC-MS analysis of Alpha-Pinene Synthase function 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 | + | [[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) 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) 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) 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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Revision as of 21:25, 23 September 2011
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Applications of BBa_K517002
Alpha-pinene synthase expression with pRARE2 in C41 DE3 E.coli that can be used to synthesize alpha-pinene.
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UNIQ0e55d30c4f2d8791-partinfo-00000000-QINU UNIQ0e55d30c4f2d8791-partinfo-00000001-QINU
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) 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) 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) 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.