Team:ETH Zurich/Construct2/Theory

From 2012.igem.org

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LovTap

LovTap is a protein fusion of the LOV2 domain from Avena sativa phototropin1 and trp repressor from E.coli.

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 TrpR domain is reversed and TrpR is able to bind to the trp operon.  

We would like to use LovTap as a blue light sensor for our multiplexer. 


LovTap 1.png

Fig: The figure shows in blue the Lov2 domain, in yellow the TrpR and in dark blue the linker helix.

Cph8

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.

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.

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. 

Cph8.png

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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