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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            <p class="subpart">SAMPLE PREPARATION- HEADSPACE-SOLID-PHASE MICROEXTRACTION</p>
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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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<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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<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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        <a name="cloning"><h3>Cloning</h3></a>
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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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        <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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<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>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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<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>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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<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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<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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<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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<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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<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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<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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<p>For further information check the detailed protocol (<a class="blue-bold">see protocol</a>)</p><br/><br/>
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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 align="center"><strong>References</strong></p><br/>
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<li>Wolfram Weckwerth., 2007. <a class="black-bold">Metabolomics: Methods and Protocols</a>. Humana press, Totowa, New Jersey.</li>
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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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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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