Difference between revisions of "Part:BBa K3733038"

Latest revision as of 10:05, 21 October 2021

ScGS with a His-tag

Geosmin synthase from Streptomyces coelicolor A3(2) (ScGS) is a single 726-amino acid protein which catalyzes the Mg²⁺ dependent conversion of farnesyl diphosphate to a mixture including geosmin. A 6×His tag is added in its C-terminal to make it accessible for Ni-NTA purification.

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(PP_i). 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 the protein, 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 Sefinose^TM 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 proved by SDS-PAGE analysis.

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Figure 1. SDS-PAGE analysis of ScGS with 6×His tag expression

References

[1] 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.

@@ Line 1: / Line 1: @@
 __NOTOC__
-<partinfo>BBa_K3733006 short</partinfo>
+<partinfo>BBa_K3733038 short</partinfo>
-<p>Gesomin synthase from <i>Streptomyces coelicolor</i> A3(2) (<b>ScGS</b>) is a single 726-amino acid protein which catalyze the Mg<sup>2+</sup>-dependent terpenoid cyclization and fragmentation reaction sequence to produce <b>geosmin</b>.</p>
+<p>Geosmin synthase from <i>Streptomyces coelicolor</i> A3(2) (<b>ScGS</b>) is a single 726-amino acid protein which catalyzes the Mg<sup>2+</sup> dependent conversion of farnesyl diphosphate to a mixture including geosmin. A 6×His tag is added in its C-terminal to make it accessible for Ni-NTA purification.</p>
 ===Usage and Biology===
 <p>The ScGS is a bifunctional sesquiterpene cyclase, with the presence of Mg<sup>2+</sup>, the N-terminal half of this protein catalyzes the ionization and cyclization of farnesyl diphosphate to form germacradienol and inorganic pyrophosphate(PP<sub>i</sub>). Then the C-terminal domain, highly homologous with the former, catalyzes the protonation, cyclization, and fragmentation of germacradienol to form geosmin and acetone.</p>
@@ Line 15: / Line 16: @@
 ===Functional Parameters===
-<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>
-===References===
+<p>To obtain the protein, pET-28a(+)-ScGS(with 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 proved by SDS-PAGE analysis.</p>
-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.
+<html>
+<head>
+<meta charset="utf-8">
+<title>无标题文档</title>
+</head>
+<body>
+<center><img src="https://static.igem.org/mediawiki/parts/3/35/T--HZAU-China--ScGS-1.png
+" style="width:389px;height:405px"></center>
+<center><b>Figure 1. </b>SDS-PAGE analysis of ScGS with 6×His tag expression </center>
+<br>
+</body>
+</html>
+<h3>References</h3>
+<P>
+[1] 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.
+</P>