Introduction to the reporters
eYGFPuv: It comes from the crustacean Chiridius poppei, a marine copepod. The singularity of this fluorescent protein is that there is no need to observe the gene expression with a confocal microscope, because it is observed by naked eyes under ultraviolet light.
RUBY: This is a cassette reporter that produces betalain and turns the plant to a reddish color when it is expressed. The advantage of this gene is that the expression of the protein is noticeable to the naked eye without any kind of excitation.
GFP: The GFP has a huge importance in synthetic biology. It allows us to see in a visible way how fluorescent dyes express in living cells, allowing the examination of dynamic, physiological processes. The GFP has fluorescence in its 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.
It 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.
What have we measured?
Throughout the whole research, we have conducted various experiments that included the gathering of data of the biological systems created.
Concentrations of plasmids with NanoDrop:
The microvolume spectrophotometer NanoDrop is a specialized instrument designed to measure the concentration and purity of nucleic acids (DNA, RNA) and proteins in small sample volumes.
Here's how a Nanodrop works:
- Sample Application: A small droplet (usually 1-2 μL) of the sample is placed onto a specialized surface, typically a quartz or disposable plastic pedestal.
- Absorbance Measurement: The Nanodrop uses ultraviolet-visible (UV-Vis) spectroscopy to measure how much light is absorbed by the sample at specific wavelengths.
- Calculation of Concentration: Based on the absorbance measurements at the appropriate wavelengths, the Nanodrop software calculates the concentration of nucleic acids in the sample. For nucleic acids, the concentration is usually expressed in units of ng/μL.
It is very important to clean the area of the sensors with ultrapure water before starting to measure, to calibrate it as accurately as possible. After measuring the desired samples, it is important to end the experiment, and again clean the sensor with ultrapure water three more times.
Fluorescence of GFP
As aforementioned, the GFP, or Green Fluorescent Protein, is a biomolecule that gets excited to a certain wavelength, specifically at 475 nm and at 395 nm.
A way of checking if the plants were responding properly to the transformation and checking which promoters were better for the task, was using this method.
For that task, the measurement equipment used is the Fluorometer. A fluorometer is a scientific instrument designed to measure the fluorescence of a substance. It functions by emitting a specific excitation wavelength of light onto the sample (like the ones explained before in case of the GFP), causing the sample to emit fluorescence at a longer wavelength. By detecting and quantifying the intensity and spectrum of this emitted fluorescence, fluorometers provide valuable insights into the composition, concentration, and characteristics of fluorescent materials.
Protein measurement
This procedure is equivalent to the one mentioned before, and we can arrive at the same conclusions regarding GFP expression, but an exact measurement requires multiple screenings to have as much information as possible about our modified biological systems.
To make the quantification of protein, we have used the Bradford Method.
Protein concentration signifies the total quantity of protein within a given sample. The primary aim is to determine protein levels in each sample using a colorimetric technique, specifically the Bradford method, renowned for its protein quantification capabilities. The reagent utilized for this assay is commonly known as the "Bradford Reagent," with Coomassie Brilliant Blue G-250 serving as the solvent. This method relies on alterations in the coloration of the solution, which vary in response to varying protein concentrations.