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Team:Valencia UPV/Project/modules/methodology
From 2014.igem.org
Project > Modules > Methodology
Clonning
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 analysis
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.
Pheromone analysis
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 byGas 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.
