Difference between revisions of "Part:BBa K3733038"

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===Functional Parameters===
 
===Functional Parameters===
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<p>To obtain ScGS, pET-28a(+)-ScGS with a 6×His-tag was transferred into <i>E.coli</i> 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 OD<sub>600</sub> reached 0.5-0.8, then 0.3 mM isopropyl <i>β</i>-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 Sefinose<sup><sup>TM</sup></sup> Resin (Sangon Biotech, Shanghai, China). As it shows in the following figure(<b>Figure 1</b>), the existence of ScGS with a 6×His-tag in our chasis was clearly proved by SDS-PAGE analysis.</p>
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<p>To obtain ScGS, pET-28a(+)-ScGS with a 6×His-tag was transferred into <i>E.coli</i> 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 OD<sub>600</sub> reached 0.5-0.8, then 0.3 mM isopropyl <i>β</i>-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 Sefinose<sup>TM</sup> Resin (Sangon Biotech, Shanghai, China). As it shows in the following figure(<b>Figure 1</b>), the existence of ScGS with a 6×His-tag in our chasis was clearly proved by SDS-PAGE analysis.</p>
  
 
===References===
 
===References===
 
Harris G G, Lombardi P M, Pemberton T A, et al. Structural Studies of Geosmin Synthase, a Bifunctional Sesquiterpene Synthase with αα Domain Architecture That Catalyzes a Unique Cyclization–Fragmentation Reaction Sequence[J]. Biochemistry, 2015, 54(48): 7142-7155.
 
Harris G G, Lombardi P M, Pemberton T A, et al. Structural Studies of Geosmin Synthase, a Bifunctional Sesquiterpene Synthase with αα Domain Architecture That Catalyzes a Unique Cyclization–Fragmentation Reaction Sequence[J]. Biochemistry, 2015, 54(48): 7142-7155.

Revision as of 18:04, 17 October 2021


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 terpenoid cyclization and fragmentation reaction sequence to produce 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 a 6×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 with a 6×His-tag in our chasis was clearly proved by SDS-PAGE analysis.

References

Harris G G, Lombardi P M, Pemberton T A, et al. Structural Studies of Geosmin Synthase, a Bifunctional Sesquiterpene Synthase with αα Domain Architecture That Catalyzes a Unique Cyclization–Fragmentation Reaction Sequence[J]. Biochemistry, 2015, 54(48): 7142-7155.