Difference between revisions of "Part:BBa K3733038"
Line 1: Line 1:
  
 
__NOTOC__
 
__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 catalyzes the Mg<sup>2+</sup> 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 <b>geosmin</b>.</p>
+
<p>Gesomin 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>
  
  
Line 17: Line 17:
 
===Functional Parameters===
 
===Functional Parameters===
  
−
<p>To obtain ScGS, 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 in our chasis was clearly proved by SDS-PAGE analysis.</p>
+
<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 with a 6×His tag in our chasis was clearly proved by SDS-PAGE analysis.</p>
  
 
<html>
 
<html>
Line 26: Line 26:
 
<body>
 
<body>
 
<center><img src="（这里是图片链接）" style="width:793px;height:360px"></center>
 
<center><img src="（这里是图片链接）" style="width:793px;height:360px"></center>
−
<center><b>Figure 1. </b>SDS-PAGE analysis of ScGS with His-tag expression </center>
+
<center><b>Figure 1. </b>SDS-PAGE analysis of ScGS with 6×His tag expression </center>
 
<br>
 
<br>
 
</body>
 
</body>
 
</html>
 
</html>
−
 
−
<p>In order to identify the synthesis of geosmin, engineered bacteria in TB medium containing 5% glycerol were first induced ScGS expression with 0.7mM IPTG when OD<sub>600</sub> reached about 0.7, following by an overnight culture at 18℃ and continuing cultivation for next 72h at 25℃. From this way we could smell a strong and unusual odor from the culture comparing to the control.</p>
 
−
<p>For further demonstration, we prepared the sample via headspace liguid-phase microextraction(HS-LPME) and a gas chromatography-mass spectrometry(GC-MS) test was conducted. The results given by GC-MS fairly shows the existence of geosmin in our culture(<b>figure 2.</b>), thus prove the feasibility of the part.</p>
 
−
 
−
<html>
 
−
<head>
 
−
<meta charset="utf-8">
 
−
<title>无标题文档</title>
 
−
</head>
 
−
<body>
 
−
<center><img src="（这里是图片链接）" style="width:793px;height:360px"></center>
 
−
<center><b>Figure *. </b>GC-MS analysis of geosmin extracted from the culture </center>
 
−
<br>
 
−
</body>
 
  
  
Line 51: Line 37:
 
[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.
 
[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>
 
</P>
−
 
−
<P>[2] Xie Y, He J, Huang J, et al. Determination of 2-methylisoborneol and geosmin produced by Streptomyces sp. and Anabaena PCC7120[J]. Journal of agricultural and food chemistry, 2007, 55(17): 6823-6828.</P>
 

Revision as of 15:57, 18 October 2021


ScGS with a His-tag

Gesomin 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(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 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 SefinoseTM Resin (Sangon Biotech, Shanghai, China). As it shows in the following figure(figure 1.), the existence of with a 6×His tag in our chasis was clearly proved by SDS-PAGE analysis.

无标题文档

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.