Difference between revisions of "Part:BBa K517003:Experience"

Revision as of 22:10, 28 September 2011

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Applications of BBa_K517003

Beta-pinene synthase expression with pRARE2 in C41 DE3 E.coli that can be used to synthesize alpha- and beta-pinene.

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 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, 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 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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UNIQc106cd99a3770739-partinfo-00000001-QINU UNIQc106cd99a3770739-partinfo-00000002-QINU

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). (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.

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 ===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 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 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. ]]
+[[Image:Ubcigem2011alphapingcms.jpg | frame | center | '''Gas Chromatography Mass Spectrum of Alpha-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, 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 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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