The jellyfish Aequorea victoria produces a Green Fluorescent Protein (GFP, for short), which glows when exposed to light in the blue to ultraviolet range. Purified GFP solutions appear yellow under typical indoor lighting, but they glow with a bright green hue in the sunlight. The protein absorbs ultraviolet radiation and then emits it as lower-energy green light. Its usage in the world of genetic engineering and molecular cloning has garnered the attention of scientists and laymen alike, and its versatility is noteworthy. It is a biological marker that can monitor physiological processes, visualize protein localization, and detect transgenic expression in vivo (in live, isolated cells).
The protein is made up of 238 amino acids and three of them (Numbers 65 to 67) form a structure that emits visible green fluorescent light. The protein can attach to and mark other proteins with fluorescence, which enables scientists to mark the presence of another particular protein in organic structures. Gfp refers to the gene that produces the green fluorescent protein (GFP). Using DNA recombinant technology (usually the molecular cloning of foreign DNA into bacterial extrachromosomal DNA elements), scientists combine the Gfp gene to another gene that produces a protein that they want to study, and then they insert the complex into a cell (usually an Escherichia coli cell). If the cell fluoresces under blue light, scientists may infer that the cell expresses the other gene too. In essence, GFP can label specific organelles, cells, and tissues. Since the GFP gene is heritable, the descendants of labeled entities express it. This is helpful in DNA repair bioassays.
An example where GFP can be useful is in studying the effectiveness of RAD52, a gene that has a role in repairing damaged DNA. Since deficiencies in DNA repair mechanisms appear to be an underlying cause of cancer, RAD52's potential for use in cancer treatments is being heavily researched. But what’s the method for marking a gene in molecular cloning?
Firstly, restriction enzymes that can isolate the gene of interest need to be chosen. Restriction enzymes include EcoRI, EcoRII, BamHI, HindIII, etc. When they come into contact with a DNA sequence with a shape that matches a part of the enzyme, called the recognition site, they induce a break in both strands of the DNA molecule. Each restriction enzyme recognises a specific recognition site or DNA sequence. Recognition sites usually consist of 4-8 nucleotides.
After this, we must insert the gene of interest into a plasmid (a small, extrachromosomal DNA molecule within a cell, which is physically separated from chromosomal DNA) to form an expression vector. An expression vector is a plasmid that is designed to introduce a gene of interest into a target cell to produce the protein encoded by the gene of interest. E. coli is customarily utilised as the target cell for molecular cloning owing to its availability, reproduction rate, and convenience of utilisation. Adding the GFP gene to expression vectors makes it act as a reporter gene. Cells with the gene of interest and Gfp will glow under blue light. Basically, we can append the GFP gene to any substance we intend to monitor. For example, you can use it to mark a virus. As the virus spreads through a host, we can watch the spread by following the fluorescence.
The protein has numerous additional uses as well. As it does not interfere with biological processes in vivo, researchers use it to monitor how organisms develop (undergo an increase in complexity). For example, after 1994, Chalfie et al used GFP in the study of neuron development in C. elegans. This field of research has now broadened to include a multitude of organisms, inclusive of fruit flies, mice, and zebrafish among others. The world of art and commerce has also been introduced to the GFP. An artist, Eduardo Kac, has created a fluorescent green rabbit by genetically engineering GFP into its cells. Researchers are also employing GFP in the creation of fluorescent fishes and plants. GFP has also been added to countless other organisms. However, such practices are still controversial and are stimulating dialogue regarding the safety and morality of genetic engineering.
Surprise! GFP is not the only discovered fluorescent protein. Some corals contain a red fluorescent protein, DsRed. In fact, scientists have discovered over 150 distinct GFP-like proteins in many species. You can find a list of examples here. Now you have an amazing conversation starter if you’re stuck in a room full of biochemists.
“The Embryo Project Encyclopedia.” Green Fluorescent Protein | The Embryo Project Encyclopedia, embryo.asu.edu/pages/green-fluorescent-protein.
“Green Fluorescent Protein (Gfp).” Thermo Fisher Scientific - SG, www.thermofisher.com/sg/en/home/life-science/cell-analysis/fluorophores/green-fluorescent-protein.html.
“Home.” Center for Advanced Light Microscopy, nic.ucsf.edu/dokuwiki/doku.php?id=fluorescent_proteins.
“Online Courses & Credentials from Top Educators. Join for Free.” Coursera, www.coursera.org/learn/dna-decoded/home/welcome.
“Pdb101: Molecule of the Month: Green Fluorescent Protein (Gfp).” RCSB, pdb101.rcsb.org/motm/42.
“Recombinant Dna Technology.” Recombinant DNA Technology - an Overview | ScienceDirect Topics, www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/recombinant-dna-technology.
“Restriction Enzymes.” Science Learning Hub, www.sciencelearn.org.nz/resources/2035-restriction-enzymes.