Team:Valencia UPV/prueba pag

Project > Modules > Methodology

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/

Therefore, we collected leaf samples from transformed N. benthamiana with our construct, which was comprised of the switch, the Firefly luciferase reporter, and the Renilla luciferase internal standard. We cut small discs from the leaves and introduced them in a multiwall plate, containing CuSO4 dilutions at different concentrations.

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.

Afterwards, another reagent (Dual-Glo® Stop & Glo® Reagent) is added which can quench the Firefly luciferase luminescence and activate the Renilla luciferase reaction. Then, Renilla luminescence is also detected in the Luminometer and the values are recorded.

Finally, once both values are recorded for each well, the ratio Firefly/Renilla 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.

We also wanted to compare the expression of this chimeric promoter and terminator (see Constructs: Switch) 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.

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(See Results: Luciferase assay).

1. 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.


2. 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.