Difference between revisions of "Part:BBa K4169005"

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	<partinfo>BBa_K4169005</partinfo>		<partinfo>BBa_K4169005</partinfo>
	===RNA Thermometer===		===RNA Thermometer===
−	<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. ScGS is a bifunctional enzyme whose N-terminal domain catalyzes the cyclization of FPP to form germacradienol, while C-terminal domain then converts this sesquiterpenoid product to <b>geosmin</b>.</p>	+	<p>Under natural conditions, some Rnas with special structures can form stem loops autonomously. Complete with RNA Gloria (HZAU-China 2021) was used in this study.</p>
	===Usage and Biology===		===Usage and Biology===

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Revision as of 05:16, 10 October 2022

BBa_K4169005

RNA Thermometer

Under natural conditions, some Rnas with special structures can form stem loops autonomously. Complete with RNA Gloria (HZAU-China 2021) was used in this study.

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.1111

Sequence and Features

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 Sefinose^TM Resin (Sangon Biotech, Shanghai, China). As it shows in the following figure(Figure 1.), the existence of ScGS in our chasis was proved by SDS-PAGE analysis.

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

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 OD₆₀₀ 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.

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(Figure 2.), thus proves the feasibility of the part.

Figure 2. Identification of geosmin by GC-MS. A. Total ion current chromatogram of geosmin standard(Red Line) and extracted product(Blue line). B. Mass spectrum of geosmin standard. C. Mass spectrum of the extracted product.

References

Hoynes-O'Connor A, Hinman K, Kirchner L, Moon TS. De novo design of heat-repressible RNA thermosensors in E. coli. Nucleic Acids Res. 2015 Jul 13;43(12):6166-79.