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.
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