magistrsko delo
Alenka Kastelic (Author), Mojca Benčina (Mentor), Maja Meško (Co-mentor)

Abstract

Nabor dobro okarakteriziranih in ortogonalnih transkripcijskih dejavnikov je v naravi omejen, zato smo z orodji sintezne biologije pripravili od kalcija odvisne umetne transkripcijske dejavnike s sintetičnimi domenami na osnovi obvitih vijačnic. Večdelni transkripcijski dejavnik smo sestavili iz treh delov: regulatorne domene proteina NFAT, DNA-vezavne domene efektorja TAL in aktivacijske domene VP64. Za medsebojno povezavo vseh treh delov smo uporabili pare obvitih vijačnic N7-N8, P3-P4 in P5A-P6A. Posamezen peptid, ki tvori obvito vijačnico, smo vezali na posamezno komponento sistema. Povezovanje prek parov obvitih vijačnic je omogočilo sestavljanje transkripcijskega dejavnika. Učinkovitost transkripcijskega dejavnika smo pokazali v sesalskih celicah HEK293T, v katere smo s transfekcijo vnesli pripravljene plazmidne konstrukte z deli transkripcijskega dejavnika in poročevalski plazmid z zapisom za kresničkino luciferazo. Transficirane celice smo stimulirali s kalcijevim ionoforom in aktivacijo našega sistema spremljali z merjenjem luminiscence. Ugotovili smo, da lahko konstitutivno aktivnost celic v odsotnosti stimulacije s kalcijevim ionoforom uspešno zmanjšamo z uporabo sidrnega peptida KRφ.

Keywords

biotehnologija;transkripcijski dejavniki;obvite vijačnice;NFAT;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [A. Kastelic]
UDC: 601.4:577.21(043.2)
COBISS: 125645059 Link will open in a new window
Views: 40
Downloads: 13
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Other data

Secondary language: English
Secondary title: Engineering calcium-dependent transcription factors with synthetic dimerization domains based on coiled coils
Secondary abstract: Since well characterized and orthogonal transcription factors are rare in nature, we employed the tools of synthetic biology to engineer artificial calcium-dependent transcription factors with synthetic domains based on coiled coils. The split transcription factor consists of three parts, the regulatory domain of the NFAT protein, the DNA-binding TAL effector domain and the activation domain VP64. Pairs of coiled coils N7-N8, P3-P4, and P5A-P6A were used to combine the three constituent parts. Each part of the split transcription factor was tethered to a coiled coil forming peptide. The linking via pairs of coiled coils enabled the reconstitution of the split transcription factor. Its effectiveness was demonstrated in mammalian HEK293T cells. Via transfection we then introduced prepared plasmid constructs with parts of the transcription factor and a reporting plasmid coding for firefly luciferase. The transfected cells were stimulated with calcium ionophore and then the activation of the system was monitored by luminescence measuring. It was determined that the constituent cell activity in the absence of calcium ionophore stimulation can be successfully reduced using the KRφ anchor peptide.
Secondary keywords: biotechnology;transcription factors;NFAT;coiled coils;
Type (COBISS): Master's thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Študij biotehnologije
Pages: 1 spletni vir (1 datoteka PDF (XI, 42 f., [7] f. pril.))
ID: 16469468