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| | <div align="center"><span class="coda"><roja>M</roja>ethodology</span> </div><br/><br/> | | <div align="center"><span class="coda"><roja>M</roja>ethodology</span> </div><br/><br/> |
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| - | <li class="active"><a href="#tab1">Clonning</a></li>
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| - | <li><a href="#tab3">Expression analysis</a></li>
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| - | <p>A plant life cycle spans months or even years. This makes stable genetic transformation of plants an unaffordable lengthy process for our project. Fortunately, there is a nice shortcut available: <b>Agrobacterium-mediated transient gene expression</b>. Using this technique, large gene constructs can be transferred to somatic cells in the plant and their expression tested and measured in a few days. When transiently transformed, the plant expresses the transgene during a few days or weeks, but they are not transferred to the offspring.</p></br/>
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| - | <p>On this project, the transient expression of our constructs in <i>N. benthamiana</i> leaves was the key technique used (1) to produce pheromones on the plant, (2) to check the specificity of the the trichomes promoter, (3) to test the switch regulated activity and to (4) assess the colour production of the biosafety module.</p></br/> | + | <h3>Clonning</h3> |
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| - | <p><i>Agrobacterium tumefaciens</i> is a bacterium from the soil that infects plants causing tumor development. The disease is caused because of the transference of a T-DNA (transfer DNA) that contains genes encoding enzymes synthesizing opines and phytohormones from the Ti Agrobacterium plasmid (see Fig. 1) to the plant genome. The synthesis of plant hormones enables the cell to grow uncontrollably forming the typical crown gall tumors (Fig. 2).</p></br/>
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| | + | In order to assemble the necessary BioBricks (BB) to create the Sexy Plant, we employed a modular DNA cloning method called <a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/gb" class="normal-link-page">GoldenBraid </a>(GB). The GB constructs were assembled following this procedure <a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/flowchart" class="normal-link-page">Flowchart</a>. To convert GoldenBraid assemblies to the BioBricks standards, we followed the conversion <a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/flowchart" class="normal-link-page">from GB to BB procedure.</a> </p> |
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| | + | <h3>Expression analysis</h3> |
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| | + | <p>As plants are complex organisms they require the use of more sophisticated transformation techniques than the ones used with bacteria. In order to introduce a given construct into the plant cells and insert it in the genome, soil bacteria called Agrobacterium tumefaciens/Rhizobium radiobacter are used. By injecting these bacteria in the plant leaves, they can induce <a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/expression" class="normal-link-page">Transient gene expression</a> in the host plant.</p> |
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| - | <div align="center"><img width="300px" src="https://static.igem.org/mediawiki/2014/d/de/Ivllobel_pathway.png" alt="" title="Pheromone Pathway"></img> | + | <h3>Pheromone analysis</h3><br/> |
| - | <img width="300px" src="https://static.igem.org/mediawiki/2014/d/de/Ivllobel_pathway.png" alt="pheromone_pathway" title="Pheromone Pathway"></img></div><br/> | + | <p>Such a complex project as the Sexy Plant, requires many different measurement techniques. </p> |
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| | + | <p>In order to analyse the pheromone production in the plant, we collected transformed Nicotiana benthamiana leaf samples and performed a <a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/sample_preparation" class="normal-link-page">Headspace SPME</a>, a technique that traps the volatile organic compounds produced in the sample. Then, the volatiles were analysed and identified by<a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/sample_analysis">Gas Chromatography-Mass Spectrometry.</a></p> |
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| - | <p>Figure 1 shows the schema of the Ti plasmid being the genes encoded in the T-DNA, the ones are transferred to the plant genome. The T-DNA is defined and recognized by conserved, flanking T-DNA borders known as the Left (LB) and Right Border (RB). These polarized border sequences serve as the target for the Vir endonucleases which subsequently assist in integration of the T-DNA in the plant genome.</p></br/> | + | <p>Willing to test if the plants efficiently released the pheromone, we also performed a <a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/dynamic_headspace"> Dynamic Headspace sampling technique.</a></p> |
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| - | <p>Genes naturally encoded in the T-DNA can be replaced with the gene or genes of interest so <i>Agrobacterium</i> can be used as a vector to deliver them to the plant. After cloning the genes of interest in a single plasmid [see cloning section], this plasmid is transferred to <i>Agrobacterium</i>.</p></br/> | + | <p>We also wanted to study moth’s response to pheromones produced by our genetically engineered plants. Therefore we performed an <a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/EAG">Electroantennography</a> to test the antennae detection and signal transmission upon stimulation with our plant samples. In addition, we performed a <a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/windtunnel">Wind tunnel assay</a> to observe male moths behaviour under stimulation with our pheromones.</p> |
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| - | <p>LB medium with the appropriate antibiotics is inoculated with a single <i>Agrobacterium</i> colony and grown to saturation. The saturated culture is used to inoculate fresh LB medium that is grown overnight. After that, cells are pelleted and resuspended into a suitable buffer solution to an optical density of 0.2 at 600nm. This buffer contains acetosyringone, a phenolic compound that induces the expression of the vir genes responsible of the transfer of the T-DNA from the plasmid to the plant genome.</p></br/> | + | <p>Finally, to test the induction of gene expression triggered by our cupper-activated switch, we performed a <a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/windtunnel">Luciferase expression assay</a>. </p> |
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| - | <p>After incubation of the cultures for 2 hours, <i>N. benthamiana</i> leaves of 4-weeks old plants are inoculated with one of the methods described below. In order to check the constructs we used the syringe-agroinfiltration, while for the production of high amounts of pheromone we chose the vacuum method.</p></br/>
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| - | <li>On syringe-agroinfiltration, the <i>Agrobacterium</i> solution is placed in a syringe (without needle) and it is injected into the airspaces inside the leaf by pressing the tip of the syringe against the underside of the leaf while simultaneously applying a counterpressure on the other side.</li><br/>
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| - | <li>On vacuum infiltration the whole plants are submerged in the solution, and placed into a vacuum chamber. After that, vacuum is applied forcing air out of the stomata. When the vacuum is released, the pressure difference forces solution through the stomata.<br/>
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| - | Once inside the leaf the <i>Agrobacterium</i> transfers the genes of interest in high copy numbers into the plant cells. After 4-7 days depending on the objective leaf samples are collected.</li><br/>
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| - | <p>The implementation of a genetic switch was key in the development of the Sexy Plant. Therefore, we needed an accurate expression analysis technique to test its performance.</p></br/>
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| - | <p>Remember that we introduced an <span class="blue-bold">Inducible genetic switch</span> with the objective of activating the pheromone production upon the insect mating season. The switch is activated with the addition of Copper sulphate to the plant leaves.</p></br/>
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| - | <p>In order to test the expression induced by the switch, we decided to perform a Luciferase assay (Dual-Glo ® Luciferase Assay system, Promega). This assay is based on the use of two luminescent proteins, one from an insect, the Firefly Luciferase and the other one from the Sea pansy, the <i>Renilla</i> luciferase. The first one is used as a reporter, being expressed when the switch is activated, and the other one is used as an internal standard, which allows differentiating if changes in the expression of the reporter are due to the switch induction or other factors.</p></br/>
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| - | <div align="center"><img width="400px" style="margin-right: 30px;" src="https://static.igem.org/mediawiki/2014/c/c0/VUPV397px-Firefly_composite.png" alt="Bioluminescent_firefly" title="Bioluminescent firefly"></img><img width="250px" style="margin-left: 30px;" src="https://static.igem.org/mediawiki/2014/6/6d/VUPVRenilla.jpg" alt="sea_pansy" title="Sea Pansy"></img><br/>
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| - | <p style="text-align: justify; font-style: italic; font-size: 0.8em; width: 700px;"><span class="black-bold">Figure 1</span>. Bioluminescent firefly
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| - | Source: Emmanuelm at en.wikipedia.</p>
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| - | <p style="text-align: right; font-style: italic; font-size: 0.8em; width: 700px;"><span class="black-bold">Figure 2</span>. Sea pansy, Renilla reniformis
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| - | Source: http://www.jaxshells.org/</p></div>
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| - | <p><br/><br/>Therefore, we collected leaf samples from transformed <i>N. benthamiana</i> with our <span class="blue-bold">construct</span>, which was comprised of the switch, the Firefly luciferase reporter, and the <i>Renilla</i> luciferase internal standard. We cut small discs from the leaves and introduced them in a multiwall plate, containing CuSO4 dilutions at different concentrations.</p></br/>
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| - | <p>Then the reagent containing the necessary substrates to start the Firefly luciferase reaction (Dual-Glo® LuciferaseAssay Reagent is added and the luminescence values are detected in a GloMax 96 Microplate Luminometer (Promega) and recorded.</p></br/>
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| - | <p>Afterwards, another reagent (Dual-Glo® Stop & Glo® Reagent) is added which can quench the Firefly luciferase luminescence and activate the <i>Renilla</i> luciferase reaction. Then, <i>Renilla</i> luminescence is also detected in the Luminometer and the values are recorded.</p></br/>
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| - | <p>Finally, once both values are recorded for each well, the ratio Firefly/<i>Renilla</i> luminescence is calculated. Since Renilla luminescence is supposed to be constant between samples, this ratio, as mentioned before, allows knowing whether the changes in firefly luminescence are due to changes in expression or external factors. In addition, samples must be normalized with a negative control.</p></br/>
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| - | <div align="center"><img width="800px" src="https://static.igem.org/mediawiki/2014/f/fb/VUPVLuciferasa.png" alt="luciferase" title="Luciferase"></img></div><br/>
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| - | <div align="center"><p style="text-align: justify; font-style: italic; font-size: 0.8em; width: 700px;"><span class="black-bold">Figure 3</span>. Dual-Glo ® Luciferase Assay system procedure
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| - | Source: Dual-Glo ® Luciferase Assay system Technical Manual (Promega)
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| - | .</p></div><br/>
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| - | <p>We also wanted to compare the expression of this chimeric promoter and terminator (<a class="normal-link-page" href="#">see Constructs: Switch</a>) with other well-known promoters and terminators. Therefore, using the following equation, the relative expression ratio or experimental transcriptional activity (ETA) compared to the known expression values of the PNos promoter and the TNos terminator can be calculated. The ETA of PNos and TNos is arbitrarily set as 1.</p><br/>
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| - | <p><span class="red-bold">FALTA METER UNA ECUACION AQUI!!</span></p><br/>
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| - | <p>With all this setup, we are ready to know if our Copper inducible switch is really working and quantify its induction of the Luciferase reporter gene expression(<a class="normal-link-page" href="https://2014.igem.org/Team:Valencia_UPV/Project/results">See Results: Luciferase assay</a>).</p><br/>
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| - | <p align="center" class="black-bold">REFERENCES</p><br/>
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| - | <div style="position: relative; left: 3%; width: 96%;"><ol>
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| - | <li>Dual-Glo ® Luciferase Assay system Technical Manual (Promega).</li>
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| - | <li>Sarrion-Perdigones A, Vazquez-Vilar M, Palací J, Castelijns B, Forment J, Ziarsolo P, Blanca J, Granell A, Orzaez D. (2013) GoldenBraid2.0: A comprehensive DNA assembly framework for Plant Synthetic Biology. Plant Physiol Epub ahead of print, doi: 10. 1104/ pp. 113. 217661.</li>
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| - | <a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/sample_preparation">SAMPLE PREPARATION- HEADSPACE-SOLID-PHASE MICROEXTRACTION</a><br/><br/>
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| - | <a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/sample_analysis">SAMPLE ANALYSIS - GAS CHROMATOGRAPHY-MASS SPECTROMETRY</a><br/><br/>
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| - | <a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/dynamic_headspace">DYNAMIC HEADSPACE SAMPLING TECHNIQUE</a><br/><br/>
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