The green fluorescent protein (GFP) is derived from the Aequorea victoria. It was first discovered by Shimomura in 1960 during the purification of aequorin. He reported a protein that gives slight green fluorescence in sunlight, yellow fluorescence only under tungsten light, and very bright green fluorescence when exposed to ultraviolet of a minerality. Then he proteolyzed the GFP and could successfully analyse the peptide that is responsible for absorbance, and correctly reported that the structure of the chromophore is 4-(p-hydroxybenzylidene)imidazolidin-5-one linked to a peptide backbone 1- and 2-positions of the ring. After the discovery of the GFP, it was cloned by parsher and the gene was expressed by Chalfie and Inouye & Tsuji in another living organism to create fluorescence. This was the beginning of the crucial breakthrough, and the green fluorescent protein has become the centre of attraction for many scientists due to marvellous interest as the first and only cloned protein that generates visible fluorescence upon expression with no external cofactors. The fluorescence property of the green fluorescent protein is due to a covalent rearrangement between three amino acids. These three amino acids are serine, tyrosine, and glycine, that are in positions 65, 66, and 67 respectively. The mechanism of chromophore formation is: the GFP folds first into almost a native conformation, then the imidazolinone is formed when the Gly67 amide undergoes nucleophilic attack on the carbonyl residue 65, then dehydration takes place. The last step is, the conjugation of the aromatic group of the molecular oxygen by dehydrogenating the α-β bond of residue 66. The chromophore obtains its visible fluorescence only at this stage. The structure of the green fluorescent protein shows that the chromophore is buried in the centre of the β-can, which is 11 stranded β-barrel that is attached to an α-helix to form a cylinder that protects the chromophore from quenching occurring due to water dipoles, paramagnetic oxygen or cis-trans isomerisation. Disadvantage of wild type GFP and how to overcome it The wild-type GFP has many disadvantages that decreased the green fluorescent protein efficiency in cell imaging. The folding ability declines dramatically at the 37C (the physiological temperature). And as a result, the fluorescent signal dropped. The rate of its maturation is low and it drifts strongly to aggregation. Moreover, there are two separate peaks occur because of the presence of neutral (lex,395 nm) and phenolate (lex,490 nm) chromophore forms. To overcome the drawbacks of the GFP, some modifications were necessary to be done to enhance its efficiency. The site-directed mutagenesis using the plasmid vector to introduce the mutated DNA, a process which provides accurate and precise changes and manageable segments of the genome. The mutation and alteration of GFP excitation and emission spectra are beneficial for these most critical three reasons: first, to produce detectable marker in monitoring the multiple cellular events concurrently; second, to act as donors and acceptors for the fluorescence resonance energy transfer or FRET; third, to highlight the relationship between the structure and function of the protein.