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Team:Valencia UPV/prueba pag
From 2014.igem.org
Project > Modules > Methodology
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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.
Remember that we introduced an Inducible genetic switch 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.
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 Renilla 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.
Figure 1. Bioluminescent firefly Source: Emmanuelm at en.wikipedia
Figure 2. Sea pansy, Renilla reniformis Source: http://www.jaxshells.org/
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.
Genes naturally encoded in the T-DNA can be replaced with the gene or genes of interest so Agrobacterium 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 Agrobacterium.
LB medium with the appropriate antibiotics is inoculated with a single Agrobacterium 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.
After incubation of the cultures for 2 hours, N. benthamiana 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.
- On syringe-agroinfiltration, the Agrobacterium 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.
- 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.
Once inside the leaf the Agrobacterium 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.
