Coding

Part:BBa_K3733006

Designed by: Zhenhao Han   Group: iGEM21_HZAU-China   (2021-09-28)
Revision as of 17:36, 13 October 2021 by Zzh-HZAU (Talk | contribs)


ScGS: Streptomyces coelicolor geosmin synthase

Gesomin synthase from Streptomyces coelicolor A3(2) (ScGS) is a single 726-amino acid protein which catalyze the Mg2+ dependent conversion of farnesyl diphosphate to a mixture including geosmin. ScGS is a bifunctional enzyme in which the N-terminal domain catalyze the cyclization of FPP to form germacradienol, while the C-terminal domain then convert this sesquiterpenoid product to geosmin.


Usage and Biology

The ScGS is a bifunctional sesquiterpene cyclase, with the presence of Mg2+, the N-terminal half of this protein catalyzes the ionization and cyclization of farnesyl diphosphate to form germacradienol and inorganic pyrophosphate(PPi). Then the C-terminal domain, highly homologous with the former, catalyzes the protonation, cyclization, and fragmentation of germacradienol to form geosmin and acetone.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Functional Parameters

To obtain ScGS, pET-28a(+)-ScGS(with His-tag) was transferred into E.coli BL21(DE3), and the cells were inoculated in 25 mL cultures of LB medium with 10 μg/mL kanamycin. These cultures were grown at 37℃ with 250 rpm shaking until the OD600 reached 0.5-0.8, then 0.3 mM isopropyl β-D-1-thiogalactopyranoside(IPTG) were added, following by an overnight cultivation at 16℃ with 250 rpm shaking to induce protein expression. The washed and harvested cells were resuspended with a Binding Buffer, and then the cells were lysed by ultrasonication. Purification was performed according to the protocol of Ni-NTA SefinoseTM Resin (Sangon Biotech, Shanghai, China). As it shows in the following figure(figure 1.), the existence of ScGS in our chasis is clearly proved by SDS-PAGE analysis.

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Categories
//cds
//chassis/prokaryote/ecoli
Parameters
biologyEscherichia coli
proteinScGS