Team:Valencia UPV/Project/modules/methodology

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<div align="center"><img class="img-title" alt="Methodology" src="https://static.igem.org/mediawiki/2014/1/10/VUPVMethodology-title.png"></img></div><br/>
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<p><h3 class="hook" align="left"><a>Project</a> > <a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules">Modules</a> > <a>Methodology</a> </h3></p><br/><br/>
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        <li class="active"><a href="#tab1">Clonning</a></li>
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        <li><a href="#tab2">Expression analysis</a></li>
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        <li><a href="#tab3">Pheromone analysis</a></li>
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<div align="center"><span class="coda"><roja>M</roja>ethodology</span> </div><br/><br/>
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            <br/><p class="subpart">SAMPLE PREPARATION- HEADSPACE-SOLID-PHASE MICROEXTRACTION</p><br/><br/>
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<p>Headspace solid-phase microextraction is one of the most convenient techniques for analysing Volatile Organic Compounds (VOCs). It is a very sensitive, inexpensive, robust and easy-to-use technique since it does not require the use of solvents and is able to detect metabolites up to ppt (parts per trillion) with a low sample quantity.</p><br/><br/>
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        <a name="cloning"><h3>Cloning</h3></a>
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<p>In our case, we chose this technique coupled to a Gas Chromatography-Mass Spectrometry detection (see GC-MS) to analyse the presence of pheromones ((Z)-11 hexadec-1-ol, (Z)-11 hexadecenal, (Z)-11 hexadecenyl acetate)[see biosynthesis] in our samples, Nicotiana benthamiana leaves [see agroinfiltration].</p><br/><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/parts_construction" class="normal-link-page">from GB to BB procedure.</a> </p>
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<h3>Expression</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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<p>This technique is based on the detection of volatiles present in the headspace of a vial. The volatiles diffuse from the sample (Solid phase) to the headspace (Gas phase) and are captured by an absorbent, a polymer-coated fiber with high affinity for them. After desorption from the fiber, volatiles are analysed by GC-MS.</p><br/><br/>
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        <h3>Measurements</h3><br/>
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<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" class="normal-link-page">Gas Chromatography-Mass Spectrometry.</a></p>
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<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" class="normal-link-page"> Dynamic Headspace sampling technique.</a></p>
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<div align="center"><img width="600px" src="https://static.igem.org/mediawiki/2014/8/88/VUPVSample_preparation_1.jpg" alt="solid_phase_extraction" title="Solid Phase Microextraction"></img></div><br/>
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<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" class="normal-link-page">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" class="normal-link-page">Wind tunnel assay</a> to observe male moths behaviour under stimulation with our pheromones.</p>
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<div align="center"><p style="font-size: 0.8em; width: 70%;">Source: Metabolomics: Methods and Protocols. Humana press, Totowa, New Jersey.</p></div><br/>
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<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/luciferase" class="normal-link-page">Luciferase expression assay</a>. </p>
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<p>In order to perform an accurate analysis of the sample, this technique must follow 3 simple steps:</p><br/><br/>
 
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<li><a class="black-bold">Sample preparation</a>: it is vital for a proper analysis that samples are unaltered during the whole process.
 
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    <li>Therefore the biological material, leaves in this case, must be immediately kept on liquid Nitrogen after the removal from the plant.</li>
 
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    <li>Then, leaves are ground to a fine powder with mortar and pestle and introduced in a screw cap headspace vial.</li>
 
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    <li>Afterwards, EDTA and saturated solution of CaCl2 are added to inhibit enzymatic activity and stabilize the sample.</li>
 
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    <li>Finally, they are sonicated to induce the release of volatiles from the solid phase to the gas phase in the headspace of the vial.</li>
 
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<li><a class="black-bold">Trapping of volatiles</a>: volatiles present in the gas phase must be extracted for further analysis. Therefore a fiber coated with an adsorbent polymer is introduced in the headspace, and ‘traps’ the volatiles from the sample.</li>
 
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<li><a class="black-bold">Desorption</a>: release of the volatiles from the fiber by thermal desorption which takes place in the insertion port of the Gas chromatograph.</li>
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<a class="button-content" id="goto-left" align="center" href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/switch"><strong>&larr; Go to Switch</strong></a>
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<a class="button-content" id="goto-middle" align="center" href="https://2014.igem.org/Team:Valencia_UPV/Project/modules"><strong>Go to Modules</strong></a>
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<a class="button-content" id="goto-right" align="center" href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/biosafety"><strong>Go to Biosafety &rarr;</strong></a></div><br/><br/><br/><br/>
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<p>Now the sample is ready to be analysed by GC-MS. (<a class="blue-bold">see GC-MS</a>)</p><br/><br/>
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<p>For further information check the detailed protocol (<a class="blue-bold">see protocol</a>)</p><br/><br/>
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<p align="center"><strong>References</strong></p><br/>
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<div style="position: relative; left: 3%; width: 96%;"><ol>
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<li>Wolfram Weckwerth., 2007. <a class="black-bold">Metabolomics: Methods and Protocols</a>. Humana press, Totowa, New Jersey.</li><br/>
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<li>Zhouyao Zhang , Janusz Pawliszyn (1993). <a class="black-bold">Headspace solid-phase microextraction</a>. Anal. Chem., 1993, 65 (14), pp 1843–1852.</li>
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<br/><p class="subpart">SAMPLE ANALYSIS - GAS CHROMATOGRAPHY-MASS SPECTROMETRY</p><br/><br/>
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<p>When it comes to analysing volatile compounds Gas Chromatography (GC) coupled to Mass spectrometry (MS) is unequivocally the first choice. The combination of both techniques allows the separation and identification of each single volatile molecule present in the sample.</p><br/><br/>
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<p>First of all, the sample must be prepared and volatilized (in case the sample is not in gas state). This is not our situation, since we are analysing volatiles extracted by HS-SPME (<a class="blue-bold">see Sample preparation - HS-SPME</a>). Once the sample is in gas state it can be introduced in the gas Chromatograph.</p><br/><br/>
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<p class="subpart">GAS CROMATOGRAPHY</p><br/><br/>
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<p>As every chromatography technique, it is based on the separation of the components of a mixture, which is called the mobile phase or eluent, when it is flowing through a stationary phase.</p><br/><br/>
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<p>The molecules of the mixture are separated by selective retention, it is to say, if they have more affinity for the mobile phase, they will flow faster while if they have higher affinity for the stationary phase, they will be more retained by it. Therefore molecules will last different times flowing through the stationary phase, it is called retention time.</p><br/><br/>
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<div align="center"><img width="600px" src="https://static.igem.org/mediawiki/2014/4/47/VUPVGas_chromatography_1.jpg" alt="analytes"></img></div><br/>
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<p>In the case of Gas chromatography, the mobile phase is composed of pure gases that act as carriers, N2, He or H. The stationary phase is a capillary column with a inner hollow where the mobile phase flows through. The inner surface of the column is coated with the stationary material. The column is inside an oven where the temperature is raised in order to increase the volatility of the analytes, decreasing the analysis time without losing resolution.</p><br/><br/>
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<p>The retention characteristics of the column depend on its length, material and the temperature of the oven.</p><br/><br/>
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<p>As the analytes flow through the column and become separated, they arrive at the detector where they will be identified.</p><br/><br/>
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<div align="center"><img width="600px" src="https://static.igem.org/mediawiki/2014/2/22/VUPVGas_chromatography_2.jpg" alt="sample_injector"></img></div><br/>
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<div align="center"><p style="font-size: 0.8em; width: 70%;">K. Murray/ Wikimedia Commons / CC-BY-SA-3.0.</p></div><br/>
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<p>Example of different molecules separated by GC:</p><br/><br/>
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<br/><p class="subpart">MASS SPECTROMETRY</p><br/><br/>
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<p>Once the gas molecules arrive at the detector, they can be identified by mass spectrometry. This technique works by ionizing the gas molecules coming from the GC and measuring the relative abundance of the formed ions.</p><br/><br/>
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<p>The first step is to ionize the gas molecules. There are many methods but the one used was Electron Ionization (EI). This basically works by bombing the molecules with high-energy electrons until they break into fragments of different charge and mass.</p><br/><br/>
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<p>Then, the resulting fragments must be analysed according to their mass/charge (m/z) ratio. There are many different mass analysers; the quadrupole analyser is the chosen one in this case. It consists of four cylindrical rods, two of them having positive electric potential while the other two are negatively charged. A radio frequency voltage is applied between the rod pairs creating an oscillating electric field. Only the ions with a given m/z will maintain its trajectory and cross the quadrupole to be detected, the rest will be deflected.
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<p>Finally, a detector can define the amount of ions with a given m/r. There are also many types of detectors. In this case, an electron multiplier was used. These detectors can amplify the signal of a given ion into an electronic current, like a cascade. The more quantity of ions that arrive, the greater the electron current. Therefore, the system is capable of quantifying the arriving ions by measuring the produced electric signal.</p><br/><br/>
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<p>After all the analysis project, the pheromone analysis results were obtained (<a class="blue-bold">see Results: pheromone analysis</a>)</p><br/><br/>
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<p>To see more details about GC-MS conditions <a class="blue-bold">see Protocol</a>.</p><br/><br/>
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Latest revision as of 02:17, 18 October 2014

Project > Modules > Methodology



Methodology


Cloning

In order to assemble the necessary BioBricks (BB) to create the Sexy Plant, we employed a modular DNA cloning method called GoldenBraid (GB). The GB constructs were assembled following this procedure Flowchart. To convert GoldenBraid assemblies to the BioBricks standards, we followed the conversion from GB to BB procedure.



Expression


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 Transient gene expression in the host plant.



Measurements


Such a complex project as the Sexy Plant, requires many different measurement techniques.

In order to analyse the pheromone production in the plant, we collected transformed Nicotiana benthamiana leaf samples and performed a Headspace SPME, a technique that traps the volatile organic compounds produced in the sample. Then, the volatiles were analysed and identified by Gas Chromatography-Mass Spectrometry.

Willing to test if the plants efficiently released the pheromone, we also performed a Dynamic Headspace sampling technique.

We also wanted to study moth’s response to pheromones produced by our genetically engineered plants. Therefore we performed an Electroantennography to test the antennae detection and signal transmission upon stimulation with our plant samples. In addition, we performed a Wind tunnel assay to observe male moths behaviour under stimulation with our pheromones.

Finally, to test the induction of gene expression triggered by our cupper-activated switch, we performed a Luciferase expression assay.











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