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| | + | <title>K4263008</title> |
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| | + | <h2 style="font-weight: bold;">PTS</h2> |
| | + | <h2>Introduction</h2> |
| | + | <p>It codes the key enzyme which catalyst FPP (farnesyl pyrophosphate) transform into patchoulol<sup>[1]</sup>.</p> |
| | + | <h2>Characterization</h2> |
| | + | <p>In order to test the function of PTS (Patchoulol synthase), we construct "Promoter-PTS-terminator" (Figure 1). If <em>PTS</em> is functional, we can test the product of patchoulol after fermentation of recombinant <em>P.pastoris</em> not only in the static system but also dynamic regulation.</p> |
| | + | <img src="https://static.igem.wiki/teams/4263/wiki/parts/image/008-1-min.png" alt=""> |
| | + | <h4>Figure 1 Gene circuit of <em>PTS</em> (static system) </h4> |
| | + | <img src="https://static.igem.wiki/teams/4263/wiki/parts/image/008-2-min.png" alt="" style="width: 30%;margin-bottom:5px;"> |
| | + | <h4>Figure 2 Gene circuit of light-control system of <em>PTS</em> (dynamic system)</h4> |
| | + | <h2 style="text-indent: 40px;">Analysis of Terpenoids</h2> |
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| − | __NOTOC__
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| − | <partinfo>BBa_K4263008 short</partinfo>
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| − | coding the key enzyme which catalyst FPP(farnesyl pyrophosphate) transform into patchoulol.
| + | <p>The quantification of patchoulol was performed on a GC system (Agilent 7820 a, USA) equipped with HP-5 column (30 m × 0.25 mm, 0.25μM U.M film thickness) and a flame ionization detector (FID). The oven temperature shall be kept at 90℃ for 0.5 min, then gradually increased to 120℃ at the speed of 20℃/ min, then increased to 160℃ at the speed of 3℃/ min for 5 min, and finally increased to 250℃ at the speed of 20℃/ min for 10 min. Patchoulol was quantified by external standard method (figure 3,4,5).</p> |
| − | | + | <img src="https://static.igem.wiki/teams/4263/wiki/parts/image/008-3-min.jpg" alt=""> |
| − | <!-- Add more about the biology of this part here | + | <h4>Figure 3 The blue bars and the vertical coordinates on the right show the yield of patchoulol for different promoters. </h4> |
| − | ===Usage and Biology=== | + | <img src="https://static.igem.wiki/teams/4263/wiki/parts/image/008-4-min.png" alt=""> |
| − | | + | <h4>Figure 4 The blue bars and the vertical coordinates on the right show the yield of patchoulol for different light condition.</h4> |
| − | <!-- --> | + | <img src="https://static.igem.wiki/teams/4263/wiki/parts/image/008-5-min.jpg" alt=""> |
| − | <span class='h3bb'>Sequence and Features</span> | + | <h4>Figure 5 Gas Chromatogram of Patchoulol</h4> |
| − | <partinfo>BBa_K4263008 SequenceAndFeatures</partinfo> | + | <p>Weigh 0.5mg of patchoulol standard with an analytical balance and dissolve it in 5ml of n-dodecane. Volume it to 5ml with a volumetric flask. The concentration of the standard mother liquor is 1g/L; Dilute the standard mother liquor to 0.75g/l, 0.75g/l, 0.5g/l, 0.25g/l and 0.1g/l, and then determine the relationship between the absorption peak of the standard and the concentration of the standard according to the GC method. Fit the linear regression equation y=3×10<sup>7</sup> - 556362, R<sup>2</sup> = 0.9995, Y represents the peak area, X represents the concentration of the standard patchoulol (figure 6).</p> |
| − | | + | <img src="https://static.igem.wiki/teams/4263/wiki/parts/image/008-6-min.png" alt="" style="margin-bottom:5px;"> |
| − | | + | <h4>Figure 6 The standard curve of patchoulol</h4> |
| − | <!-- Uncomment this to enable Functional Parameter display | + | <h2>Reference</h2> |
| − | ===Functional Parameters===
| + | <p>[1] Xu, H.; Goldfuss, B.; Schnakenburg, G.; Dickschat, J. S. Beilstein J. Org. Chem. 2022, 18, 13-24. doi:10.3762/bjoc.18.2</p> |
| − | <partinfo>BBa_K4263008 parameters</partinfo> | + | </article> |
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PTS
Introduction
It codes the key enzyme which catalyst FPP (farnesyl pyrophosphate) transform into patchoulol[1].
Characterization
In order to test the function of PTS (Patchoulol synthase), we construct "Promoter-PTS-terminator" (Figure 1). If PTS is functional, we can test the product of patchoulol after fermentation of recombinant P.pastoris not only in the static system but also dynamic regulation.
Figure 1 Gene circuit of PTS (static system)
Figure 2 Gene circuit of light-control system of PTS (dynamic system)
Analysis of Terpenoids
The quantification of patchoulol was performed on a GC system (Agilent 7820 a, USA) equipped with HP-5 column (30 m × 0.25 mm, 0.25μM U.M film thickness) and a flame ionization detector (FID). The oven temperature shall be kept at 90℃ for 0.5 min, then gradually increased to 120℃ at the speed of 20℃/ min, then increased to 160℃ at the speed of 3℃/ min for 5 min, and finally increased to 250℃ at the speed of 20℃/ min for 10 min. Patchoulol was quantified by external standard method (figure 3,4,5).
Figure 3 The blue bars and the vertical coordinates on the right show the yield of patchoulol for different promoters.
Figure 4 The blue bars and the vertical coordinates on the right show the yield of patchoulol for different light condition.
Figure 5 Gas Chromatogram of Patchoulol
Weigh 0.5mg of patchoulol standard with an analytical balance and dissolve it in 5ml of n-dodecane. Volume it to 5ml with a volumetric flask. The concentration of the standard mother liquor is 1g/L; Dilute the standard mother liquor to 0.75g/l, 0.75g/l, 0.5g/l, 0.25g/l and 0.1g/l, and then determine the relationship between the absorption peak of the standard and the concentration of the standard according to the GC method. Fit the linear regression equation y=3×107 - 556362, R2 = 0.9995, Y represents the peak area, X represents the concentration of the standard patchoulol (figure 6).
Figure 6 The standard curve of patchoulol
Reference
[1] Xu, H.; Goldfuss, B.; Schnakenburg, G.; Dickschat, J. S. Beilstein J. Org. Chem. 2022, 18, 13-24. doi:10.3762/bjoc.18.2
