Composite

Part:BBa_K118025:Experience

Designed by: Chris French   Group: iGEM08_Edinburgh   (2008-10-23)
Revision as of 20:54, 23 September 2011 by Ayjchan (Talk | contribs) (→‎User Reviews)

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K118025

Limonene synthase expression with pRARE2 in C41 DE3 E.coli that can be used to synthesize limonene terpenes.

User Reviews

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British Columbia iGEM 2011

This composite part was expressed in our C41 E. coli that contained a pRARE plasmid encoding rare tRNA synthases. However, when we tried to express the limonene synthase in yeast, we were not able to obtain evidence for protein expression or function. So, we recommend that this part only be introduced to organisms capable of expressing foreign DNA.

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Characterization by British Columbia iGEM 2011

GC-MS analysis of Limonene Synthase function by British Columbia iGEM 2011

The limonene generator with lac promoter (BBa_K118025) includes the limonene generator (BBa_K118024) under the control of a lac promoter (BBa_J33207). This composite part's purpose is to express the limonene synthase (BBa_I742110), which is also available with a ribosome site (BBa_I742111).

Gas Chromatography Mass Spectrum of (+)-Limonene: We expressed the limonene generator with lac promoter (BBa_K118025) in C41 DE3 E. coli and lysed the cell culture to obtain unpurified protein extract. The crude extract was used in an in vitro enzymatic assay to produce limonene from geranyl diphosphate (GPP) substrate. The samples were prepared and analyzed by gas chromatography mass spectrometry. (A) The gas chromatography retention time of 9.9 minutes and (B) the mass spectrometry base peak at 93m/z (Figure B) are characteristic of limonene. (A) We analysed a positive limonene-containing control that yielded a strong peak as indicated by the black line as well as a negative control where no GPP was added to the assay, which yielded no peak as indicated by the red line. In comparison, our expressed limonene synthase that was incubated with GPP (indicated by the blue line) shows the indicative limonene peak at 9.9 minutes. (B) Cross reference with a compound library revealed that the limonene synthase sample's peak chromatography at 9.9 minutes (top panel) matches the library's d-limonene peak chromatography (bottom panel).