Confocal fluorescence microscopy allows us to observe a specific protein in cells by fluorescent markers for proteins. The most frequently used is the green fluorescent protein, which fluoresces in the green spectrum. New markers are being discovered, and one of these is a modified haloalkane dehalogenase from bacteria Rhodococcus rhodochorus. Modified haloalkane dehalogenase has one amino acid residue replaced in the active site, allowing the fluorescent ligand to bind to the active site. To observe the MLKL protein in cells, we constructed a fusion of the modified haloalkane dehalogenase with the MLKL. We used MLKL because it is localised in the cell membrane when active, which allows us to verify the applicability of the modified dehalogenase for confocal fluorescence microscopy. Our work was focused on the preparation of the plasmid, containing MLKL and the modified dehalogenase domain DNA, with the new IVA cloning method. The results were verified by agarose gel electrophoresis and sequencing and confirmed that we made a plasmid that included DNA for both proteins, linked and ready for the cell expression and fluorescence microscopy studies.
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