diplomsko delo
Luka Fratina (Author), Gregor Gunčar (Mentor)

Abstract

Konfokalna fluorescenčna mikroskopija omogoča opazovanje določenega proteina v celicah z uporabo fluorescenčnih označevalcev za proteine. Najpogosteje se uporablja zeleni fluorescenčni protein, ki fluorescira v zelenem spektru. A odkriva se še več novih označevalcev in eden od teh je modificirana haloalkan dehalogenaza iz bakterije Rhodococcus rhodochrous. V aktivnem mestu modificrane haloalkan dehalogenaze je zamenjan en aminokislinski ostanek, kar omogoča vezavo fluorescenčnega liganda v aktivno mesto. Za opazovanje proteina MLKL v celicah, smo zapis modificirane domene haloalkan dehalogenaze sklopili z zapisom za protein MLKL. MLKL smo uporabili, ker je v aktivni obliki v celični membrani, kar omogoča preverjanje uporabnosti modificirane dehalogenaze za konfokalno fluorescenčno mikroskopijo. Osredotočili smo se na pripravo plazmida, ki vsebuje zapis za MLKL in modificirano domeno dehalogenaze. Za to smo uporabili novo metodo kloniranja IVA. Rezultate smo preverili z agarozno gelsko elektroforezo in z določitvijo nukleotidnega zaporedja ter s tem potrdili, da smo ustvarili plazmid, ki vključuje zapis za oba proteina, ki sta sklopljena in pripravljena za izražanje v celicah in fluorescenčno mikroskopiranje.

Keywords

proteini;psevdokinaza MLKL;modificirana dehalogenaza;kloniranje IVA;konfokalna fluorescenčna mikroskopija;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [L. Fratina]
UDC: 577.112(043.2)
COBISS: 32379651 Link will open in a new window
Views: 412
Downloads: 118
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Other data

Secondary language: English
Secondary title: Use of haloalkane dehalogenase domain for labeling of MLKL protein
Secondary abstract: 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.
Secondary keywords: confocal fluorescence microscopy;modified dehalogenase;IVA cloning;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages: 27 str.
ID: 12033162