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Priprava in karakterizacija od kalcija odvisnega transkripcijskega dejavnika na osnovi kalmodulina in od kalcija in kalmodulina odvisne protein kinaze II
ID Fuhrmann, Sara Kimm (Author), ID Benčina, Mojca (Mentor) More about this mentor... This link opens in a new window, ID Dolinar, Marko (Co-mentor)

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Abstract
Transkripcijski dejavniki so nepogrešljivi elementi sintezne biologije. Ker je število dobro okarakteriziranih in ortogonalnih naravnih transkripcijskih dejavnikov omejeno, želimo s pripravo umetnih transkripcijskih dejavnikov omogočiti načrtovanje obširnejših sinteznih genetskih vezij. V okviru magistrske naloge smo želeli pripraviti sistem za uravnavanje izražanja genov na osnovi interakcij med kalmodulinom in njegovim tarčnim peptidom iz od kalcija in kalmodulina odvisne protein kinaze II, katerega aktivnost bi lahko nadzorovali z uravnavanjem znotrajcelične koncentracije prostih kalcijevih ionov. Pripravili smo transkripcijski dejavnik iz dveh delov. Prvi del smo pripravili s fuzijo tarčnega peptida MKII in DNA-vezavne domene efektorja TAL, drugi del pa s fuzijo kalmodulina in aktivacijske domene VPR. Pripravili smo več konstruktov z različnim zaporedjem domen. Učinkovitost aktivacije transkripcije poročevalskega gena smo spremljali z dvojnim luciferaznim testom v celicah HEK293T. Pričakovali smo, da se bo načrtovani transkripcijski dejavnik ustrezno sestavil in sprožil izražanje poročevalskega gena ob stimulaciji celic s kalcijevim ionoforom A23187 in posledičnem dvigu znotrajcelične koncentracije kalcija. Testirali smo konstrukte z različnim zaporedjem domen kalmodulin/VPR in MKII/efektor TAL in spreminjali količine in razmerja posameznih vektorjev z zapisom za del transkripcijskega dejavnika. Učinkovite aktivacije transkripcije nismo dosegli v nobenem primeru. Neaktivnost pripravljenega sistema bi lahko bila posledica več dejavnikov, kot so nezadostno izražanje, neenakomerna transfekcija celic z obema deloma transkripcijskega dejavnika, neustrezna znotrajcelična lokalizacija posameznih konstruktov, nezmožnost sestave delov transkripcijskega dejavnika v aktivno celoto ter interakcije z endogenimi proteini.

Language:Slovenian
Keywords:sintezna biologija, umetni transkripcijski dejavniki, kalmodulin, efektorji TAL
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Year:2019
PID:20.500.12556/RUL-112680 This link opens in a new window
COBISS.SI-ID:1538495683 This link opens in a new window
Publication date in RUL:04.11.2019
Views:1196
Downloads:296
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Secondary language

Language:English
Title:Construction and characterization of a calcium-dependent transcription factor based on calmodulin and calcium/calmodulin-dependent protein kinase II
Abstract:
Transcription factors are essential elements in synthetic biology. The number of well-characterized and orthogonal natural transcription factors is limited, which poses a limitation on the size of synthetic networks that can be constructed. To overcome this problem, artificial transcription factors have been created. The aim of this master's thesis was the preparation of a system for gene expression regulation, based on interactions between calmodulin and its target peptide, originating from calcium/calmodulin-dependent protein kinase II, the activity of which could be controlled by regulating the intracellular concentration of free calcium ions. We prepared a two-part transcription factor. We fused calmodulin and its target peptide MKII to a transcriptional activation domain (VPR) and a DNA-binding domain (TAL effector), respectively. We prepared several gene constructs with a different domain order. The efficacy of transcription activaton was monitored by a luciferase reporter assay in HEK293T cells. We expected that the stimulation of cells by the calcium ionophore A23187 and the consequent increase of the intracellular calcium concentration would cause the assembly of transcription factor which could then promote expressing of effector. We tested constructs with a different calmodulin/VPR and MKII/TAL effector domain order and varied the amount and ratios of vectors, but we did not detect effective transcription activation. The inactivity of the prepared system could be attributed to several factors, such as insufficient expression level, uneven cell transfection with both parts of the transcription factor, inadequate intracellular localization of individual parts, inability to assemble into intact transcription factor and interactions with endogenous proteins.

Keywords:synthetic biology, artificial transcription factors, calmodulin, TAL effectors

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