Team:Edinburgh/Bacterial/Red light sensor


Revision as of 14:50, 25 October 2010 by JRWK (Talk | contribs)

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 (Figure 1).

Figure 1: The activity of the chimaeric light receptor Cph8, described in further detail below..

  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.




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.


The red light sensor has seen frequent use throughout the history of iGEM, beginning with the original coliroid parts by UT Austin 2004 to their adaptation by Harvard 2008. We have adapted their parts to the pSB1C3 chassis along with a number of different reporter systems for characterisation.

BBa_K322122: phycocyanobilin synthesis operon (Harvard 2008's BBa_K098010 in pSB1C3).

BBa_K322123: phycocyanobilin synthesis operon without terminator.

BBa_K322124: Cph8 light sensing protein (UT Austin 2004's BBa_I15010 in pSB1C3).

BBa_K322125: Cph8 with lacZ reporter system.

BBa_K322126: Cph8 with EYFP reporter system.

BBa_K322127: phycocyanobilin synthesis genes with cph8.

BBa_K322128: phycocyanobilin synthesis genes with cph8 and EYFP reporter system.


Strains made by transformation with required DNA. Overnight cultures with cells of the required strains in 2.5ml LB + 40mg/ml Cml were grown in the dark at 37C with shaking. ONs were as follows:

  • JM109 – RLS.lacZ.YFP
  • envZ – RLS.lacZ.YFP
  • JM109 – YFP Control
  • envZ – YFP Control

100μl of ONs were used to inoculate two sterile LB + 40mg/ml Cml making up a total volume of 4ml in 5ml flasks which were then incubated in both light and dark, at 37C with shaking, as follows:

  • JM109 – RLS.lacZ.YFP (Light / Dark)
  • envZ – RLS.lacZ.YFP (Light / Dark)
  • JM109 – YFP Control (Light / Dark)
  • envZ – YFP Control (Light / Dark)

At 50 minute time intervals 100μl of each sample was taken and mixed with 800μl of sterile water in plastic cuvettes. The optical density of each sample was taken after the spectrophotometer was set with a sample of 100μl of sterile LB and 800μl water as a control, as was the luminescence, with readings for the background luminescence being taken for the sample of LB and water.

Two readings for each sample at each time interval were taken and then an average was calculated for each time interval for both optical density and luminescence.

For each sample at each time interval the average luminescence (normalised by the background luminescence) was divided by the optical density and plotted on a graph, shown below as Figure 2.

Figure 2: Characterisation data for the red light sensor.




Throughout this wiki there are words in bold that indicate a relevance to human aspects. It will become obvious that human aspects are a part of almost everything in iGEM.