Team:Edinburgh/Bacterial/Red light sensor

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Overview: The red light sensor

The red light sensor was first engineered by UT Austin in iGEM 2004. In this project, we will try to use it as one of the photo-activated sensors in the repressilator network. The maximum response of the sensor is 660nm.

The red-light sensor (Cph8) contains　three parts:

1. PCB - Phycocyanobilin, coded for by HO and pcyA, required for light sensing
2. Cph8, a combination of cph1 and envZ which, together with PCB, form a transmembrane, histidine kinase-based light sensing system
3. OmpR, a natural E. coli protein which couples with EnvZ and, when phosphorylated, activates genes downstream of the ompC promoter

In the absence of red light, phosphorylated OmpR activates Envz, which in turn promotes transcription from the OmpC promoter and represses transcription from the OmpF promoter, thus leading to the expression of LacZ. This catalyses the formation of a black precipitate from S-gal(3,4-cyclohexenoesculetin-β-D-galactopyranoside).

When exposed to red light, an isomerization in the Cph1 and a structure change in the phycocyanobilin (PCB) part of the photoreceptor inactivates the histidine kinase acitity of EnvZ.

1. The chimaeric light receptor Cph8 contains the photoreceptor from Cph1 (green) and the histidine kinase and response regulator from EnvZ–OmpR (orange); inset, conversion of haem to phycocyanobilin (PCB), which forms part of the photoreceptor. Red light drives the sensor to a state in which autophosphorylation is inhibited (right), turning off gene expression.
2. Miller assay showing that Cph8 is active in the dark (black bars) in the presence of PCB and inactive in the light(white bars). There is no light dependent activity in the absence of Cph8 (-) and there is constitutive activity when only the histidine kinase domain of EnvZ is expressed (+), or when the PCB metabolic pathway is not included (-PCB).
3. When an image is projected on to a bacterial lawn, the LacZ reporter is expressed only in the dark regions.
4. Transfer function of the circuit. As the intensity of the light is increased by using a light gradient projected from a 35mm slide, the circuit output gives a graded response.

Strategy

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Problems

The whole construct would not transform and the transformations of the individual BioBricks which make up the whole construct have also failed. We attempted to amplify products out of the BioBrick and see if they were actually there. The only thing we recovered was the sensing component, cph8. We have retrieved transformants from the kanamycin-resistant version of K098010, the HO-pcyA fusion.

Biobricks

BBa_I15008 (Heme oxygenase (ho1) from Synechocystis (not working))
BBa_I15009　(Ferredoxin oxidoreductase (PcyA) from synechocystis (not working))
BBa_I15010　(Photosensing Chimaera (Cph8) (not working, but amplified out of the whole construct))
BBa_M30109 (Whole construct (the sequence in the registry does not match the database))
BBa_R0082 (OmpC promoter)
BBa_R0084 (OmpF promoter)
BBa_J25500 (light repressed mCherry that uses the coliroid photosensor)
BBa_K098010 (fusion of HO and pcyA, available in chloramphenicol resistance (not working) and kanamycin resistance (working but not miniprepping))

References

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