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Team:Valencia UPV/Project/results/pheromone analysis
From 2014.igem.org
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<p>It has been a hard way to obtain the first results. Summarizing: the different parts needed to build the <a class="blue-bold">pheromone biosynthesis device</a> were domesticated and assembled with the help of <a class="blue-bold">GoldenBraid 2.0</a> following the <a class="blue-bold">flowchart</a> shown in the Mehtodology section. Once the constructs were obtained, they were transiently transformed by <a class="blue-bold">agroinfiltration</a> into our plant chassis, <a class="italic">N. benthamiana</a>. At this point, it was time to check if the plant was actually producing the target pheromones.</p><br/><br/> | <p>It has been a hard way to obtain the first results. Summarizing: the different parts needed to build the <a class="blue-bold">pheromone biosynthesis device</a> were domesticated and assembled with the help of <a class="blue-bold">GoldenBraid 2.0</a> following the <a class="blue-bold">flowchart</a> shown in the Mehtodology section. Once the constructs were obtained, they were transiently transformed by <a class="blue-bold">agroinfiltration</a> into our plant chassis, <a class="italic">N. benthamiana</a>. At this point, it was time to check if the plant was actually producing the target pheromones.</p><br/><br/> | ||
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<p>With this objective, the agroinfiltated plants were analysed for pheromone production using <a class="blue-bold">HS-SPME</a> coupled to <a class="blue-bold">GC-MS</a>. After the analysis we obtained different chromatograms to prove it. This is the volatile profile of a normal Nicotiana benthamiana leaf, the control of the experiment (Figure 1):</p><br/><br/> | <p>With this objective, the agroinfiltated plants were analysed for pheromone production using <a class="blue-bold">HS-SPME</a> coupled to <a class="blue-bold">GC-MS</a>. After the analysis we obtained different chromatograms to prove it. This is the volatile profile of a normal Nicotiana benthamiana leaf, the control of the experiment (Figure 1):</p><br/><br/> | ||
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| + | <p>On the other hand, Figure 2 shows the volatile profile of the modified one. As it can be appreciated at first sight, there are two additional peaks in the transformed plants which are not present in the control. The mass spectrum of the molecules corresponding to each peak was compared with the NIST database, which confirmed that both molecules were the desired pheromones, <a class="red-bold">(Z)-11-hexadecen-1-ol</a> and <a class="blue-bold">(Z)-11-hexadecenyl acetate</a> (<a class="blue-bold">see biosynthesis</a>). In addition, to have an unquestionable validation we performed a GC-MS analysis of the standards, chemically synthetized pheromones provided by the C.E.Q.A. They corresponded with the obtained peaks, both the retention time and the mass spectrum.</p><br/><br/> | ||
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Revision as of 22:18, 12 October 2014
It has been a hard way to obtain the first results. Summarizing: the different parts needed to build the pheromone biosynthesis device were domesticated and assembled with the help of GoldenBraid 2.0 following the flowchart shown in the Mehtodology section. Once the constructs were obtained, they were transiently transformed by agroinfiltration into our plant chassis, N. benthamiana. At this point, it was time to check if the plant was actually producing the target pheromones.
With this objective, the agroinfiltated plants were analysed for pheromone production using HS-SPME coupled to GC-MS. After the analysis we obtained different chromatograms to prove it. This is the volatile profile of a normal Nicotiana benthamiana leaf, the control of the experiment (Figure 1):
On the other hand, Figure 2 shows the volatile profile of the modified one. As it can be appreciated at first sight, there are two additional peaks in the transformed plants which are not present in the control. The mass spectrum of the molecules corresponding to each peak was compared with the NIST database, which confirmed that both molecules were the desired pheromones, (Z)-11-hexadecen-1-ol and (Z)-11-hexadecenyl acetate (see biosynthesis). In addition, to have an unquestionable validation we performed a GC-MS analysis of the standards, chemically synthetized pheromones provided by the C.E.Q.A. They corresponded with the obtained peaks, both the retention time and the mass spectrum.
