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.
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