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| - | ==Overview==
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| - | ===Uvr8===
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| - | UVR8 is a plant protein first identified in Arabidopsos thaliana. UVR8 is a photoreceptor and a crucial part in the plant stress response to UV-B (280-315 nm).
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| - | In absence of UV-B the protein occurs as a dimer. At the dimeric interface it contains tryptophane residues which absorb the UV-B light and thereby changing conformation of the protein which results in the dissociation of the dimer. The monomeric UVR8 is then able to initiate downstream reactions that trigger the transcription of proteins needed for the stress response.
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| - | We want to make use of this photoreceptor by incorporating this protein as an transcription factor in bacteria. Since UVR8 is not able to interact with DNA by itself we are fusing it with a TetR monomer, a known repressor. We make use of the fact that the Tet repressor is only active when it binds as a dimer to the DNA. The fusion of a UVR8 unit with the DNA binding domain of TetR should result in a repression when UVR8 is a dimer and a activation when UVR8 and therefore also TetR is a monomer. With this we would be able to activate the pTet promoter upon UV exposure, which causes the dissociation of the UVR8 dimer.
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| - | [[File:UVR8.png|frameless|400px|center]]
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| - | ===LovTap===
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| - | LovTap is a protein fusion of the LOV2 domain from Avena sativa phototropin1 and trp repressor from E.coli.
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| - | Phototropin is a plant blue-light receptor, with a flavin mononucleotide (FMN) cofactor and conserved cysteine residues responsible for the conformation change upon blue-light exposure. The LovTap fusion makes use of this conformational change. In the dark state the LovTap is not able to bind to DNA. After light activation the deformation of the TetR domain is reversed and TetR is able to bind to the TetR operon.
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| - | We would like to use LovTap as a blue light sensor for our multiplexer.
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| - | [[File:LovTap_1.png|frameless|500px|center]]
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| - | Fig: The figure shows in blue the Lov2 domain, in yellow the TrpR and in dark blue the linker helix.
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| - | ===Cph8===
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| - | Cph8 is a chimeric light receptor engineered by Levskaya et al. in 2005. It contains the photoreceptor Cph1 and the EnvZ histidine kinase. The light sensing unit is connected to transcription via the EnvZ-OmpR signaling pathway, phosphorylated OmpR acting as a transcription factor.
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| - | Cph1 is only active when it binds the chromophore phycocyanobiline (PCB). Since PCB is not naturally produced in E.coli we also need to express the PCB generators: ho1 and pcyA.
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| - | In this construct far red light activates the EnvZ kinase which results in the phosphorylation of OmpR and its activation as a transcription factor. Red light on the other hand deactivates the kinase and therefore the gene can not be expressed.
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| - | [[File:Cph8.png|frameless|400px|center]]
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| - | Fig: This figure shows the fusion of the Cph1 (green) with the PCB chromophore linked with the EnvZ Kinase and OmpR (organe). Phosphorylated OmpR binds to the ompC promoter and activates transcription.
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