Composite

Part:BBa_K1950010

Designed by: Kyohei Takekawa   Group: iGEM16_Nagahama   (2016-10-14)


trc promoter + ispC-ribB(G108S) fusion

We want to make Escherichia coli synthesize a lot of farnesol, which is one kind of terpenes. We can achieve this purpose by fusing two genes, ispC and ribB(G108S). We tried to make the fusion genes which are spliced/jointed ispC and ribB(G108S). Expression of a Dxr-RibB(G108S) fusion improved terpene titers more than 4-fold and alleviated accumulation of intracellular DXP.We tested whether the fusing gene was made by measuring ispC and ribB(G108S) transcription. The amounts of transcription were quantified by RT-RCR. In the fusion gene, ispC and ribB(G108S) are transferred from a single promoter. If two genes are fused successfully, two genes are expected to be higher in the recombinant than in WT.


fig.1:Linker sequence between ispC and ribBG108S
Above sequence is reference data of ispC-ribBG108S, and blue colored sequence is linker G2. Blow sequence is this part data. Two sequences are matched. This proved that ispC and ribBG108S are linked by linker G2.


fig.2:Gel electrophoresis of colony pcr products
Lane 1 is BBa_K1950010. Lane 2 is 1 kb step DNA Ladder (Promega). BBa_1950010 is amplified by VF2 primer and VR primer. PCR product length expected 2254bp.


fig.3:Gel electrophoresis of RT-PCR products.
Lane1:100bp DNA ladder Lane2:ispC(WT) Lane3:ispC(recombinant) Lane4:m-ribB(WT) Lane5:m-ribB(recombinant) Lane6:gapA(WT) Lane7:gapA(recombinant) Lane8:ispC(WT) Negative control Lane9:ispC(recombinant) Negative control Lane10:m-ribB(WT) Negative control Lane11:m-ribB(recombinant)Negative control Lane12:gapA(WT) Negative control Lane13:gapA(recombinant)Negative control




Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1679
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 521
    Illegal AgeI site found at 1569
  • 1000
    COMPATIBLE WITH RFC[1000]



reference
Kirby J, Nishimoto M, Chow W. N.R, Baidoo E.E., Wang G, Martin J, … & Keasling J D.(2015).Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate.Applied and Environmental Microbiology,81(1), 130-138.

[edit]
Categories
Parameters
None