Melbourne/Blue Photosensor
From 2007.igem.org
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As part of the overall system design. A blue light sensitive pathway is required in addition to the red light sensitive pathway. Described below is the blue photosensor. This involves the design of a chimeric trans-membrane protein. A blue light sensitive (~500nm) integral photo receptor SopII that dimerizes with a histidine kinase; HtrII (As described in 2001 paper).
This will be fused to a two-component system from Bacillus subtilis - so as to not affect any endogenous networks. The two component system involves: comP - two-component sensor histidine kinase, comA - two-component response regulator. Phosphorylated comA will upregulated transcription via psfA/srfA promoter (link required), as part of the AND gate.
Contents |
Blue Photosensor Background
Method
Possible extensions:
- Determination of optimal wavelength:
- use of different substrates (different retinals)
- Separate variants all submitted as BioBricks.
- Submitted synthesized ComP and ComA as BioBricks
- Model the pathway to determine rate-limiting step
References
This part is based on “Photostimulation of a Sensory Rhodopsin II/HtrII/Tsr Fusion Chimera Activates CheA-Autophosphorylation and CheY-Phosphotransfer in Vitro” by Vishwa D. Trivedi and John L. Spudich, Biochemistry 2003, 42, 13887-13892. Acording to this article the peak sensitivity is to 500+/-5nm, and results in a 3 fold activation of the Tsr (wild type). CheA,W,Y connected system.
It is proposed to replace Tsr fusion with homolgouse ComP. SRII-HtrII fusion to which ComP is fused ComA when phosphorylated by ComP is an activator for PsfA promoter sequence from Dr Alan Grossman (M.I.T.) Based on
- SRII-HtrII-Tsr fusion from Prof J.L. Spudich (university of Texas)melb:spudich N sequence
- BBa_J51000 (ComP) kinase
- BBa_J51001 (ComA) activator
PARTS:
- SrfA promoter
- ComA protein generator
- SRII-ComP photosensor
- Any phyco construction genes?
SRII is from Natronomonas pharaonis.
Tsr fusion was made by Jung et al J Bacteriol 183 6365-6371 (2001) they propose a mechanism. I don’t see why anyone thinks this will work!!! Currently a conformational change induced by light increases affinity in TSR for Che family which leads to cross phosphorylation. To replace TSR with a kinase would require the kinase activity to be modulated – hence matching using homology as was done for tsr is not likely to work. Also what are the normal functions of ComP etc.
