Team:Edinburgh/Bacterial/Blue light sensor

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Overview: The blue light sensor

The blue-light sensor, which is composed of the LovTAP hybrid protein designed by Strickland and made available by Prof. Sosnick, was biobricked by EPF-Lausanne in iGEM 2009. It is based on a α-helical domain linker between the LOV2 domain (the photoactive protein) and the E-coli trp repressor, which acts as a conduit for allosteric signals. The effective response of the sensor is at a wavelength of 470nm (as documented by the aforementioned Lausanne iGEM team).

The blue-light sensor （LovTAP） consists of five parts：

1. Photoreceptor1 （the shared helix between LOV domian and TrpR domain）
   • Dark blue when contacting the LOV domain (dark state).
   • Red when contacting the TrpR domain (light state).
2. Photoreceptor2 (falvin monoucleotide-FMN cofactor)
3. LOV domain-orange (photoactive protein)
4. TrpR domain-grey （DNA regulator)
5. Operator DNA

The figure above shows the whole process regarding how the light sensor works, from the dark state (A) to the light-activated state (B → C) and then returning to the stable state (D → A). In the dark state, the shared helix contacts the LOV2 domain, and the inactivated TrpR dissociates from the DNA; in the light state, the LOV2 domains absorb the blue light proton and form a covalent adduct between the FMN cofactor and a conserved cysteine residue, destroying the shared helix in the LOV domain and binding / populating an active formation of the TrpR domain. This in turn leads to LovTPR binding the DNA and repressing λcI. However, this binding is not stable, and thus it will eventually return to the initial state.

Problems

Transformants are often growing the wrong colour. LovTap controls the production of RFP in the cells. When LovTap is inactive (i.e. in the dark), the cells should be producing RFP and hence should be growing red. When LovTap is active (i.e. in white or blue light), the cells stop producing RFP and hence chould produce white colonies. At the moment some cells in the light are still growing red, although some are definitely growing white, and vice versa in the dark. One suggestion was that the plasmids are unstable and dividing randomly, altering the intensity of the colour in some cells. Attempts to stabilise the colours of the colonies are ongoing (see the video in the gallery which demonstrates one of the innovative methods of providing proper culture conditions).

Biobricks

BBa_K191006 (LovTAP)
BBa_K191009 (Mutated LovTAP (ILE427 -> PHE))
BBa_K191007 (Trp promoter)

References