Description and theory behind Green fluorescent protein

Aequorea Victoria is a type of jellyfish found off the cost of North America and can grow up to 5cm to 10cm. Tiny Aequorea Victoria are asexually budded of their hydroid colonies and feed on soft bodied prey which includes other types of jelly fish as well.

The interesting fact about this jelly fish is its ability to bioluminescent in the sea. They actually produce blue luminescent due to the protein aeqourin; however it appears as green to us; due to the fact that it is emitted through the molecule green fluorescent protein (GFP).

GFP was first discovered by the scientist Osamu Shimomura in the jellyfish Aequorea Victoria, and is a type of green fluorescent substance. The function of GFP is to transduce the blue fluorescent emitted by the jellyfish to green, through energy transfer. Since its discovery, GFP has been used in a wide range of application, for example the measurement of protein to protein interaction. Structure of GFP below:

The fluorophore of the GFP which is responsible for the fluorescent, originates from the internal Ser-Tyr-Gly sequence which has been modified to a 4-(p-hydroxybenzylidene) - imidazolidin-5-one structure. It is form by a series of events in an auto-catalytic process and no cofactors or enzymatic components are needed.

Due to the GFP being able to tolerate N- and C-terminal fusion to wide range proteins, it has been expressed in other animals such as the zebra fish and E.Coli. Furthermore, as GFP is non toxic and non invasive, it is being used as a marker for gene expression.

Transformation of E coli using pGLO gene

The pGLO vector contains several important bioreporter and biomarker sites, those of which include the Green Fluroscent Protein (GFP) site, ampicillin resistant site(Amp R) and arabinose site (araC)which codes for a protein that 'turns on' the GFP gene and allows it to fluoresce, hence the use of arabinose as feed in the experiment.

The GFP site allows the cell to fluoresce with help from the protein that is produced from the araC site.

The Amp R site codes for beta-lactamase. This allows the bacteria to break down ampicillin, hence confering ampicillin resistance to the bacteria.

A plasmid map of pGLO showing the three important sites- araC, GFP and Ampr

The GFP gene is first isolated and cut from jellyfish DNA with a restriction enzyme, and the gene is inserted into the plasmid ( depending on what kind of restriction enzyme is used) using ligase to stick the sticky ends to the plasmid.

The plasmid is then inserted inside the E coli cells, which hav

e had their cell membranes modified in order to take up the plasmids.

E coli cells are then grown on LB-Amp plates. The bacteria which do not acquire the plasmid die off as they get killed by the ampicillin present in the agar medium. Those bacteria that may have picked up the plasmid are able to glow in the dark as they possess the GFP gene.

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