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Molecular biology is one of the most significant fields of biological science. The main aim of molecular biology  is to explore and understand biological functions including all living beings. The knowledge gained could be extended to practical use in cell biology, pharmacy or medicine. Cellular biology is a key to visualise a blueprint of life; development of drugs, the use of biomarkers and the study of gene expression rely on molecular biology methodology.1-4

Back in the 20th century, limitations in experiments were common due to the complexity of cellular structures. Real-time monitoring of cellular processes was very difficult in the past with the tools available at the time.5-9 The scientific community was waiting for a better and well-defined monitoring tool for further cellular exploration.1

A revolutionary technique was developed in the early 21st century which made a vast contribution to biological sciences.2 The discovery of Green Fluorescent Protein (GFP) and its application enabled real-time monitoring of cellular functions in living organisms. This novel tool was a ground-breaking discovery. In the present day, many forms of cellular expression are described and clarified using GFP-based methodology.1,3,4,10-16

It had been observed for decades that some living organisms such as jelly fish or other sea creatures, lit up when photographed with a flash light. At first it was described in the 1950s that a green fluorescent substance within a jellyfish was responsible for a light emission effect. In the 1960s, after a dedicated hard working mission to extract more than 10,000 jellyfish to find a fluorescent substance, Osamu Shimomura finally isolated an associated protein from the crystal jelly fish Aequorea Victoria and visually described its bioluminescent effect.5 Basically, GFP absorbs light between the blue to ultraviolet band and emits a bright green fluorescence after binding with Ca2+. The term ‘Green Fluorescent Protein’ or GFP was coined later in the 1970s by Morin and Hastings.3 During the time from the 1970-1990s, GFP structures were broadly studied  to gain a better understanding of its scientific merit.1,3,6-13 Douglas Prasher realised its potential illumination effect and proposed that GFP could be inserted into cellular structures to observe their function. In the 1990s, he successfully cloned and mapped its genetic sequence. Unfortunately, his funding ran out and he was unable to carry out further experiments to merge the GFP gene into a living organism. The samples and data were sent to several institutions to carry on the study.6,11

In 1994, Martin Chalfie successfully incorporated the GFP gene, a native GFP extracted from Aequorea Victoria (also called wild-type GFP), into E.coli and C.elegans and induced bacteria to exhibit a green fluorescence after exposure to blue light.12 Previous studies expanded the use of GFP by developing GFP derivatives and produced various colors with quicker illumination. This development extended GFP applications to diverse laboratory techniques for studying biological science.2,17,18

A wild-type GFP (wtGFP) contains 238 amino acids. The GFP crystal structures were fully described in 1996. Its shape looks like a soda can, a typical barrel structure composed of one β-sheet with alpha helixes. It contains a chromophore within the centre of the barrel shape.8 The chromophore is responsible for the bioluminescence.5,10,11 Lukyanov described GFP’s natural function as a light activated electron donor similar to the process that chlorophyll donates electrons in photosynthesis. The peak excitation spectrum is in a wavelength at 395nm and a smaller maximum at about 470nm. The fluorescent emission spectrum is at 505nm which is approximately   a green colour band of visible light spectrum.19

GFP is non-toxic and provides low host specificity, which is why it can be used in various species of organisms from single cell organisms to multi-cellular living beings. When merging GFP into the organism’s gene, it will mutually express a bioluminescent effect along with its normal cellular function without compro- mising the general physiology. Cellular activities can be observed in real-time with instant visualisation. The most common use of GFP is to monitor protein activities within a cell; the location, movement and protein cycle, or it is tagged to display gene expressions.2,11,13,15 The flow of dynamicity can be imaged as a successful temporal study. GFP can be used as a biosensor to monitor intracellular parameters such as pH or metabolic activity. In medicine, GFP is widely used for functional tissue imaging for example when studying neuron activity. GFP is also valuable for studying cancer from its basic molecular properties to developing new treatments.2

Given its huge impact on biological sciences, including subsequent benefits from GFP and the dedicated work   of high academic merit, the GFP and  its  applications was awarded the Noble Prize in 2008 for chemistry. Osamu Shimomura, Marty Chalfie and Roger Tsien were acknowledged “for the discovery and development of the green fluorescent protein, GFP.” Also, Douglas Prasher was highly praised as a co-contributor of GFP development.