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| − | | + | ===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 RNA with special structures can form stem loops autonomously. Complete with RNA Thermometer(HZAU-China 2021:BBa_K3733011) was used in this study.</p> |
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| | ===Usage and Biology=== | | ===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> | + | <p>The RNA thermometer has a special sequence capable of forming stem-loop structures at 27 ° C and below and opening at 37 ° C. At the same time, there is a specific binding site for RNase E on the sequence that can form the secondary structure. At the transcriptional level, if this sequence is present on mRNA, it is able to bind RNase E and thus degrade the entire RNA strand. Therefore, we introduce the thermometer into the suicide module to achieve temperature sensitive suicide. |
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| | ===Sequence and Features=== | | ===Sequence and Features=== |
| − | <partinfo>BBa_K3733006 SequenceAndFeatures</partinfo> | + | <partinfo>BBa_K4169005 SequenceAndFeatures</partinfo> |
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| − | ===Functional Parameters===
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| − | <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 proved by SDS-PAGE analysis.</p>
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| − | <center><img src="https://static.igem.org/mediawiki/parts/3/35/T--HZAU-China--ScGS-1.png
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| − | <center><b>Figure 1. </b>SDS-PAGE analysis of ScGS with His-tag expression </center>
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| − | <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>
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| − | <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 proves the feasibility of the part.</p>
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| − | <center><img src="https://static.igem.org/mediawiki/parts/a/ae/T--HZAU-China-gcms.png" style="width:869px;height:433px"></center>
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| − | <b>Figure 2. </b>Identification of geosmin by GC-MS. <b>A.</b> Total ion current chromatogram of geosmin standard(<b>Red Line</b>) and extracted product(<b>Blue line</b>). <b>B.</b> Mass spectrum of geosmin standard. <b>C.</b> Mass spectrum of the extracted product.
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| | <h3>References</h3> | | <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> | + | <p>Hoynes-O'Connor A, Hinman K, Kirchner L,et al. De novo design of heat-repressible RNA thermosensors in E. coli.[J] Nucleic Acids Research. 2015 Jul 13;43(12):6166-79.</p> |
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| − | <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>
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Under natural conditions, some RNA with special structures can form stem loops autonomously. Complete with RNA Thermometer(HZAU-China 2021:BBa_K3733011) was used in this study.
The RNA thermometer has a special sequence capable of forming stem-loop structures at 27 ° C and below and opening at 37 ° C. At the same time, there is a specific binding site for RNase E on the sequence that can form the secondary structure. At the transcriptional level, if this sequence is present on mRNA, it is able to bind RNase E and thus degrade the entire RNA strand. Therefore, we introduce the thermometer into the suicide module to achieve temperature sensitive suicide.
Hoynes-O'Connor A, Hinman K, Kirchner L,et al. De novo design of heat-repressible RNA thermosensors in E. coli.[J] Nucleic Acids Research. 2015 Jul 13;43(12):6166-79.
