What is measured
Summary
In our project, we have used the Green Fluorescent Protein (GFP) as the expression gene in order to see the efficiency of our promoters.
GFP
The GFP has got a huge importance in synthetic biology. Its foremost benefit over traditional fluorescent dyes is the fact that it is non-poisonous and may be expressed in living cells, allowing the examination of dynamic, physiological processes.
The GFP has fluorescence in its own structure so all the organisms capable of transcribing that protein would be capable of expressing that characteristic green color. It can perdure in those organisms without the need for other agents of chromophores.
GFP is a protein that can be fused to other proteins, providing them with additional fluorescent domains as optical tags for monitoring their activity in vivo, selecting and isolating those cells that produce proteins fused to GFP, or the amount of protein produced at a given time.
We have used GFP aware of those details for our plants. We wanted to see the fluorescent activity inside the leaf and for that, the sample needs to be alive.
Our experiment design
All our plasmids contained the different selected promoters and the GFP as an expression gene. The plasmids were infiltrated into the plants. We place transformed Nicotiana benthamiana in a chamber with ambient CO2 concentration and in a chamber with high CO2 concentration.
How is measured
Quantification
We used a confocal microscope to analyze our samples. First, we took different pictures of different parts of the leaves to compare the expression levels. After that, we used the program ImageJ to quantify the intensity of the GFP signal in the position where the line was drawn. The units of the graph are intensity over pixels. This is indeed a quantification method for intensity.
