Part:BBa_K1653024
Terpene precursor mass-production device
We want to make the E. coli produces farnesol and geraniol which are one of the terpenes. To produce great quantity of terpenes they need many terpene precursor. E. coli produces a small amount of the terpene precursor in MEP pathway. In MEP pathway, there are four enzymes (ispD, ispF, idi, dxs) which are speed limiting enzyme for terpenes precursors produce in E. coli. In order to create a high-yield strains producing IPP and DMAPP, we exogenously engineer to superimpose these genes into E. coli to create strains overproducing IPP and DMAPP in a MEP pathway.
[[http://2015.igem.org/Team:Nagahama/Medal_Parts#Terpene_precursor_mass-production_device.28BBa_K1653024.29 here] is our result page.]
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3609
Illegal BamHI site found at 2937 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 811
To confirm increased production of terpene precursors by Terpene precursor mass-production device. We put attention on ubiquinone. Ubiquinone 8 is made from Farnesyl diphosphate (FPP) which is one of the terpene precursors. quinone is one of the electron carrier present in the cell membrane of prokaryotes. And also they glow when exposed to UV rays. In the measurement of production of quinone it was measured by thin-layer chromatography. (TLC silica gel)
Result:
The figure on the left is analysis of ubiquinone 8 by thin-layer chromatography. Right lane is JM109 /Terpene precursor mass-production device with IPTG Left lane is JM109/Terpene precursor mass-production device IPTG minus . Both it was 2μl spot.
The right of the figure,Estimation of ubiquinone-8 content instead Each intensity of spots indicating the content of ubiquinone-8
From two figures, those which are overexpressed in reintroduced to E. coli four genes, it is better to have overexpressed were many production of ubiquinone 8 as compared with those that do not overexpress
Discussion:
From this result, the amount of ubiquinone 8 of the final material by the increased amount of terpene precursors is increased by re-introducing the four genes are over-expressed in E. coli.
Therefore, it considered could strengthen the MEP pathway.
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Fig. 1 Analysis of Ubiquinone-8 synthesized by
E. coli JM109/BBa_K1653025 (terpene precursor production device) by TLC.
Right lane: IPTG plus, Left lane: IPTG minus
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