1. History. Discovery of fluorescent proteins, their natural sources and biological functions. Principles of bioluminescence. 2. Physical, chemical and molecular properties of fluorescent proteins. Three-dimensional structure and variations in structure as the basis of functional variability. 3. Green fluorescent protein from Aequorea victoria (GFP) and its mutated allelic forms. Changes in spectral properties, photostability and phototoxicity. Types and properties of naturally occurring fluorescent proteins from other species. 4. Spectral properties of fluorescent proteins and their use in cell biology. Maturation of fluorochromes and expression of excitation and emission properties. The problem of dimerization and oligomerization, targeted development of monomeric forms. 5. Molecular biology of recombinant GFP technologies. Cloning, vectors, construction of plasmids for gene transfer. Creation of fusion products of fluorescent proteins. Methods of propagation, isolation and transformation. Sequences of chimeric genes. 6. Passive applications of GFP technologies. Functional fusion of GFP and recombinant expression. Cell markers, reference genes, fusion proteins. 7. Spectrum of use of GFP technologies in various species of organisms. Applications in functional cell studies. 8. Specific tagging of fluorescent proteins for dynamic study of cell organelles. Monitoring the dynamics of organelles and GFP-fusion proteins, subcellular localization, multicolor imaging and colocalization. 9. Use of fluorescent protein technology in plants: preparation of constructs, transformation, expression, detection. Variants of fluorescent proteins for multispectral detection in living cells. Expression of non-toxic markers by non-invasive method in real time. 10. Practical applications of real-time study of living cells: Quantitative analysis of proteins, their interactions and dynamic redistribution in cells by FRET, FLIM, FRAP, FLIP methods. Visualization of molecular interactions in living cells by bimolecular fluorescence complementation. Principle, possibilities of use and application. Fluorescence correlation spectroscopy. 11. Optical variants of fluorescent proteins and their applications. Photoactivation, photoconversion, photoinduction, time-spectral activation. 12. Genetically encoded fluorescent molecular biosensors: Active application of GFP technologies for sensitive detection of physiological and metabolic cellular changes.
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