Team:Edinburgh/Bacterial/Green light sensor

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Overview: The green light sensor

If we are to make a complete repressilator, we will need a green light-sensing BioBrick to go with the red and blue. To our knowledge, there has been no green light sensor successfully used in E. coli. Tokyo-Nokogen 2009 attempted to make a green light sensor but couldn't get it to work, due to problems with the BioBricks stored in the Registry.

Tokyo-Nokogen's design adapted the red light sensor designed by UT Austin 2004 to sense green light, by replacing the Cph1 domain of the protein (which reacts to red light) with the light sensing domain of CcaS (which senses green light). However, the second part of the protein is the inner membrane region of EnvZ, which duplicates with the red sensor.

With this system, OmpR will be phosphorylated by both green light and red light sensors (since we use Austin 2005's sensor as well), and the cell would be colour blind. While CcaS would be a good protein to use for the purpose of sensing green light, we therefore need to link it to a different two-component receptor system if we want to make a separate green light sensor.

Strategy

At first, we hoped to use the α-helical domain of an allosteric protein to couple the light receptor and repressor, a strategy that has been successfully utilised previously in the blue light sensor fusion.

However, this method would have likely required the usage of Statistical Coupling Analysis to measure how much the amino acid distribution at some position i changes upon a perturbation of the amino acid distribution at another position j. It was thought that such analysis would be beyond the time constraints imposed upon the project, and thus this strategy was abandoned in favour of the strategy described below.

Our next design involved the fusion of PhoR and CcaS to create a green light sensor, by replacing the downstream portion of the CcaS with the downstream portion of PhoR based on the fact that both contain the same domains HisKA and HATPase-C. PhoR phosphorylates the response regulator PhoB, which then will binds to conserved DNA sequences called phoboxes and interacts with the σ70 subunit of RNA polymerase to control transcription. Thus, we can add the reporter gene here as a response to the detection of green light.

The 3D structure of CcaS and PhoR has not been fully documented, although portions of their domains have. Thus, we are not able to accurately predict the fusion from their structure.

CcaS (light receptor)

• GAF: The phytochrome domain, which is PCB here, is covalently anchored at a conserved cysteine residue in the GAF domain.

• PCB (phycobilin, which is introduced into the E-coli): Light activates the PCB to trigger a Z to E isomerization as well as subsequent conformational changes of the chromophore and the apoprotein, leading to the downstream processes.

• HK: Domain B of the HK phosphorylates and then transfers the phosphorylation to domain A, which then phosphorylates PhoB and leads to the expression of the reporter gene.

The cut will be positioned between amino acids 475 and 525, which is between the PAS and HisKA domains.

PhoR (response regulator)

The cut will be positioned between amino acids 150 and 195.

Problems

We are having to wait on DNA from our main collaborators, Mexico UNAM-Genomics, before we can continue with this particular part of the project. It would be a good one to complete since no other team besides our two are working on this particular part.

BioBricks

The overall construct, once built, will be a new BioBrick, with credit for submission going to Mexico UNAM-Genomics.

Characterisation

***

References

Hirose, Y., Shimada, T., Narikawa, R., Katayama, M. & Ikeuchi, M. (2008). Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. Proceedings of the National Academy of Sciences 105, 9528-9533.

Tokyo-Nokogen 2009 team wiki, https://2009.igem.org/Team:Tokyo-Nokogen.