CRH/GFPT Recombinant GFP Technologies and their Use

Lecturers: Miroslav Ovečka, Pavol Vadovič
Lecture: 2 hours/week + seminar 1 hour/week
Credits: 2
Summer semester
Form of course completion: exam

• History. The discovery of fluorescent proteins, their natural sources and biological functions. Principles of bioluminescence.
• Physical, chemical and molecular properties of fluorescent proteins. The three-dimensional structure and variations in the structure as a basis for functional variability.
• The green fluorescent protein from Aequorea victoria (GFP) and its mutant allelic forms. Changes in the spectral properties, phototoxicity and photostability. Types and properties of naturally occurring fluorescent proteins from other species.
• Spectral properties of fluorescent proteins and their use in cell biology. Fluorochrome maturation and expression of the excitation and emission properties. Dimerization and oligomerization, targeted development of the monomeric forms.
• Molecular biology of recombinant GFP technology. Cloning vectors, the construction of plasmids for gene transfer. Formation of fluorescent protein fusion products. Methods of propagation, isolation and transformation. The sequences of the chimeric genes.
• Passive application of GFP technology. Functional GFP fusion and recombinant expression. Cell markers, reporter genes, fusion proteins.
• Spectrum of GFP technology utilization in different species of organisms. Applications in functional cell studies.
• Specific fluorescent tagging of proteins to study dynamic cellular organelles. Monitoring of dynamic organelles and GFP-fusion proteins, the subcellular localization, multicolour imaging and colocalization.
• Utilization of fluorescent proteins in plants: the preparation of constructs, transformation, expression, detection. Variants of fluorescent proteins for multispectral detection in living cells. Expression of nontoxic markers by non-invasive method in real time.
• Practical applications of the study of living cells in real time: Quantitative analysis of proteins, their interactions and dynamic redistribution in cells by FRET, FLIM, FRAP, FLIP. Visualization of molecular interactions in living cells by bimolecular fluorescence complementation. Principle, possible uses and applications. Fluorescence correlation spectroscopy.
• Optical variants of fluorescent proteins and their applications. Photoactivation, photoconversion, photoinduction, time-spectral activation.
• Genetically encoded fluorescent molecular biosensors: Active applications of GFP technologies for sensitive detection of physiological and metabolic cellular changes.