Team:Valencia UPV/Project/results/pheromone analysis

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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/>
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<p><h3 class="hook" align="left"><a>Project</a> > <a href="https://2014.igem.org/Team:Valencia_UPV/Project/results">Results</a> > <a>Pheromone Analysis</a></h3></p><br/><br/>
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<div align="center"><img width="100%" src="https://static.igem.org/mediawiki/2014/8/86/VUPVProceso-1.png" alt="process"></img></div><br/>
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<div align="center"><span class="coda"><roja>P</roja>heromone <roja>A</roja>nalysis</span> </div><br/><br/>
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<p>It was a long way to get here but, after our first results, future looked promising. This is what we got so far: the different parts needed to build the <a class="normal-link-page" href="https://2014.igem.org/Team:Valencia_UPV/Project/results/constructs#biosyn">pheromone biosynthesis device</a> were domesticated and assembled with the help of <a class="normal-link-page" href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/gb">GoldenBraid 2.0</a>. 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>. Now, the moment of truth has arrived: do our plants actually produce the target pheromones?</p><br/><br/>
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<div align="center"><img width="850px" src="https://static.igem.org/mediawiki/2014/8/86/VUPVProceso-1.png"></img></div><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/>
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<p>To answer this question, we analysed the volatiles produced by our "Sexy Plants" using <a class="normal-link-page" href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/sample_preparation">HS-SPME</a> coupled to <a class="normal-link-page" href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/sample_analysis">GC-MS</a>. We co-infiltrated our pheromone biosynthesis device together with a construct carrying the silencing suppressor P19(*),  as well as the P19 construct alone  as negative control. Below you can see a representative full scan chromatogram of each of them.</p><br/><br/>
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<div align="center"><img width="750px" src="https://static.igem.org/mediawiki/2014/e/e4/VUPVFigura_Cromatogramas.png"></img></div><br/>
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<div align="center"><p style="text-align: justify; font-size: 0.8em; width: 750px;"><b>Figure 1</b>. GC-MS analysis of the volatile organic compounds from a genetically engineered and  control <i>N. benthamiana</i>  plants. On the right, an overlay chromatogram (control/sexy plant) of the two pheromone peaks from a SIM mode acquisition for pheromone representative ions.</p></div><br/>
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<p>With just one glance, we could identify two peaks in the genetically engineered <i>N. benthamiana</i> plants that were not present in the control.<br/>
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Could those peaks be our pheromones? A comparison of their mass spectra with the NIST mass spectrum library retrieved <span class="red-bold">(Z)-11-hexadecen-1-ol</span> and <span class="blue-bold">(Z)-11-hexadecenyl acetate</span> as best matches. Furthermore, this putative identification was confirmed by comparing their retention time and mass spectra with those of pure <span class="red-bold">(Z)-11-hexadecen-1-ol</span> and <span class="blue-bold">(Z)-11-hexadecenyl acetate</span> synthesised at the CEQA (UPV, Spain) and analysed under identical GC-MS conditions. So, yes, <b>our genetically engineered <i>N. benthamiana</i> is sexy!!!!</b>  
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<td><img width="50%" src="https://static.igem.org/mediawiki/2014/a/a2/VUPVN._benthamiana_RECORTADA.png" alt="nicotiana"></img></td>
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<td><img width="50%" src="https://static.igem.org/mediawiki/2014/c/c3/VUPVDiapositiva2.jpg" alt="diap_1"></img></td>
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<div align="center"><img width="600px" src="https://static.igem.org/mediawiki/2014/8/86/VUPVProceso-1.png" alt="process"></img></div><br/>
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<div align="center"><p style="font-size: 0.8em; width: 70%;">Biosynthetic pathway of pheromone production. A continuous line shows the substrate and a discontinuous line shows the resulting pheromones.</p></div><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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<p>More good news. Not only were the pheromones being produced, but they were also <b>two of the most abundant volatiles</b> in the plant. In addition, the ratio between the abundance of <a class="red-bold">(Z)-11-hexadecen-1-ol</a> and <a class="blue-bold">(Z)-11-hexadecenyl acetate</a> was approximately 4 to 1. This means that unprecedented conversion rates were achieved, compared to previous publications (<a class="blue-bold">Ding et al. 2014</a>). Such superior conversion rate  can be attributed to the  use of a single multigene construction (<a class="normal-link-page" href="https://2014.igem.org/Team:Valencia_UPV/Project/results/constructs#biosyn">see Results- Constructs- Biosynthesis</a>) comprising  all three genes needed to produce the pheromones in a single plasmid and thus, facilitating the simultaneous expression of the three enzymes in the plant cells (<a class="normal-link-page" href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/biosynthesis">see Biosynthesis</a>).</p><br/><br/>
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<p>But wait, there's more! Our sexy plants had one surprise in store for us. Even though we did not succeeded in cloning FAO1, the enzyme catalyzing the the biosynthesis of <span class="green-bold">(Z)-11-hexadecenal</span> from <span class="red-bold">(Z)-11-hexadecen-1-ol</span> (<a class="normal-link-page" href="https://2014.igem.org/Team:Valencia_UPV/Project/notebook">see Notebook: FAO1 obtention</a>), we could identify a small peak in our engineered plants chromatograms that was absent in the controls and had a mass spectrum that matched that of <span class="green-bold">(Z)-11-hexadecenal</span>. The analysis of a pure <span class="green-bold">(Z)-11-hexadecenal</span> standard (synthesised at the CEQA) confirmed our expectations, <b>our sexy plants produce a small amount of</b> <span class="green-bold">(Z)-11-hexadecenal</span>. How? We think that the conversion of <span class="red-bold">(Z)-11-hexadecen-1-ol</span> into <span class="green-bold">(Z)-11–hexadecenal</span> is probably performed by an endogenous alcohol oxidase from the plant, present in the genome of <i>N. benthamiana</i> but with not apparent physiological relevance (<a class="normal-link-page" href="http://solcyc.solgenomics.net/BENTH/NEW-IMAGE?type=GENE&object=GJZM-8047&detail-level=2">Sol Genomics Network</a>). As the conversion rate was low, this opened a field for futher studies on pheromone plant-production.</p><br/><br/>
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<div align="center"><img width="450px" src="https://static.igem.org/mediawiki/2014/1/1e/VUPVAldehyde-1.png" alt="aldehyde_peak"></img></div><br/>
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<div align="center"><p style="text-align: center; font-size: 0.8em; width: 750px;"><b>Figure 2</b>. GC-MS detection of <span class="green-bold">(Z)-11-hexadecenal</span> in <i>N. benthamiana</i> leaves .</p></div><br/>
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<p>At the end of the journey <a class="black-bold">we have achieved a plant able to produce three insect sexual pheromones</a>:</p><br/>
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<li><span class="red-bold">(Z)-11-hexadecen-1-ol</span> as the most abundant plant volatile.</li>
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<li><span class="blue-bold">(Z)-11-hexadecenyl acetate</span> as one of the most abundant volatile molecules in the plant, with an unprecedented conversion yield.</li>
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<p>As it can be observed, not only the production of the pheromones was achieved, but also they were among the most abundant volatile compounds in the plant. In addition, as it can be appreciated, the ratio between the abundance of <a class="red-bold">(Z)-11-hexadecen-1-ol</a> and <a class="blue-bold">(Z)-11-hexadecenyl acetate</a> was approximately 4 to 1. This means that unprecedented conversion rates were achieved, compared to previous publications (<a class="red-bold">Ding et al. 2014</a>). Such improved conversion rate can be attributed??? to the  use of a single multigene construction (<a class="red-bold">see Results- Constructs- Biosynthesis</a>) comprising  all three genes needed to produce the pheromones in a single plasmid and thus, facilitating the simultaneous expression of the three enzymes in the plant cells. (<a class="red-bold">see Biosynthesis</a>).</p><br/><br/>
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<li><span class="green-bold">(Z)-11-hexadecenal</span>, produced  by an endogenous plant enzyme at low yield that could be improved in future studies.</li>
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<p>Finally, even though the obtention of the enzyme necessary for the biosynthesis of <a class="green-bold">(Z)-11-hexadecenal</a> was not possible (<a class="blue-bold">see notebook: FAO1 obtention</a>), the possible presence of this pheromone was also checked. Therefore, the standard was analysed and the corresponding molecule was looked for in the transformed N. benthamiana leaf chromatogram. Surprisingly, a small peak was identified, that was not present in the control and corresponded to <a class="green-bold">(Z)-11-hexadecenal</a>. The conversion of <a class="red-bold">(Z)-11-hexadecen-1-ol</a> into <a class="green-bold">(Z)-11–hexadecenal</a> was probably performed by an endogenous alcohol oxidase from the plant, which was documented as not having significant in vivo activity (<a class="blue-bold">Sol Genomics Network</a>). As the conversion rate was low, this opened a field for futher studies on pheromone plant-production.</p><br/><br/>
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</ul><br/><br/>
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<p>(*)P19 is a silencing suppressor that inhibits the plant silencing mechanism.</p><br/><br/><br/><br/>
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Latest revision as of 00:29, 18 October 2014

Project > Results > Pheromone Analysis



Pheromone Analysis


It was a long way to get here but, after our first results, future looked promising. This is what we got so far: the different parts needed to build the pheromone biosynthesis device were domesticated and assembled with the help of GoldenBraid 2.0. Once the constructs were obtained, they were transiently transformed by agroinfiltration into our plant chassis, N. benthamiana. Now, the moment of truth has arrived: do our plants actually produce the target pheromones?




To answer this question, we analysed the volatiles produced by our "Sexy Plants" using HS-SPME coupled to GC-MS. We co-infiltrated our pheromone biosynthesis device together with a construct carrying the silencing suppressor P19(*), as well as the P19 construct alone as negative control. Below you can see a representative full scan chromatogram of each of them.




Figure 1. GC-MS analysis of the volatile organic compounds from a genetically engineered and control N. benthamiana plants. On the right, an overlay chromatogram (control/sexy plant) of the two pheromone peaks from a SIM mode acquisition for pheromone representative ions.


With just one glance, we could identify two peaks in the genetically engineered N. benthamiana plants that were not present in the control.
Could those peaks be our pheromones? A comparison of their mass spectra with the NIST mass spectrum library retrieved (Z)-11-hexadecen-1-ol and (Z)-11-hexadecenyl acetate as best matches. Furthermore, this putative identification was confirmed by comparing their retention time and mass spectra with those of pure (Z)-11-hexadecen-1-ol and (Z)-11-hexadecenyl acetate synthesised at the CEQA (UPV, Spain) and analysed under identical GC-MS conditions. So, yes, our genetically engineered N. benthamiana is sexy!!!!



More good news. Not only were the pheromones being produced, but they were also two of the most abundant volatiles in the plant. In addition, the ratio between the abundance of (Z)-11-hexadecen-1-ol and (Z)-11-hexadecenyl acetate was approximately 4 to 1. This means that unprecedented conversion rates were achieved, compared to previous publications (Ding et al. 2014). Such superior conversion rate can be attributed to the use of a single multigene construction (see Results- Constructs- Biosynthesis) comprising all three genes needed to produce the pheromones in a single plasmid and thus, facilitating the simultaneous expression of the three enzymes in the plant cells (see Biosynthesis).



But wait, there's more! Our sexy plants had one surprise in store for us. Even though we did not succeeded in cloning FAO1, the enzyme catalyzing the the biosynthesis of (Z)-11-hexadecenal from (Z)-11-hexadecen-1-ol (see Notebook: FAO1 obtention), we could identify a small peak in our engineered plants chromatograms that was absent in the controls and had a mass spectrum that matched that of (Z)-11-hexadecenal. The analysis of a pure (Z)-11-hexadecenal standard (synthesised at the CEQA) confirmed our expectations, our sexy plants produce a small amount of (Z)-11-hexadecenal. How? We think that the conversion of (Z)-11-hexadecen-1-ol into (Z)-11–hexadecenal is probably performed by an endogenous alcohol oxidase from the plant, present in the genome of N. benthamiana but with not apparent physiological relevance (Sol Genomics Network). As the conversion rate was low, this opened a field for futher studies on pheromone plant-production.



aldehyde_peak

Figure 2. GC-MS detection of (Z)-11-hexadecenal in N. benthamiana leaves .


At the end of the journey we have achieved a plant able to produce three insect sexual pheromones:


  • (Z)-11-hexadecen-1-ol as the most abundant plant volatile.
  • (Z)-11-hexadecenyl acetate as one of the most abundant volatile molecules in the plant, with an unprecedented conversion yield.
  • (Z)-11-hexadecenal, produced by an endogenous plant enzyme at low yield that could be improved in future studies.


(*)P19 is a silencing suppressor that inhibits the plant silencing mechanism.