Team:UNebraska-Lincoln/demonstrate
UNL 2018 Improving Early Detection of The Emerald Ash Borer
Demonstrate
The data strongly suggests that our part works well in collaboration with mevalonate-dependent pathway enzymes encoded on plasmid 100167. This is likely due to amorphadiene synthase and 7-epi-sequithujene synthase both utilizing farnesyl pyrophosphate as a precursor. We first confirmed production of farnesyl pyrophosphate by running previously reported assays with biosynthesis of amorphadiene. Prominent amorphadiene concentrations from GC-MS were used to conclude that plasmid 100167 ultimately contained necessary genes for the biosynthesis of the FPP precursor. We then ran assays with our 7-epi-sesquithujene synthase gene cloned into the plasmid 100169 backbone in place of the amorphadiene synthase gene. By comparing our GC-MS results from each assay to GC-MS results obtained from the NIST library, we unmistakably showed the successful biosynthesis of 7-epi-sesquithujene. Further detail about the different procedures can be found in the Experiments and Results pages.
Fig. 5: Standard mass spectrum of 7-epi-sesquithujene from the NIST library
Fig. 6: Mass Spectrum of produced 7-epi-sesquithujene
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