Melbourne/Blue Photosensor
From 2007.igem.org
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Contents |
Blue Photosensor Background
Method
Possible extensions:
- Determine proteins in vesicles and hence relevant genes:
- Perform 2DGE on expressed vesicle proteins.
- Seperate vesicles by floatation and do an SDS PAGE.
- Apply MALDI TOF MS to determine Amino acid sequences and hence degenerate code and genes.
- Seperate coding regions into individual biobricks.
- Recombine in different ways and observe effect on gas vessicle structure.
Knock out/modify some of the genes to observe the effect on vesicles and buoyancy, aiming to improve lifting speed and capability of cells.
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.. CheA,W,Y connected system.
It is proposed to replace Tsr with homolgouse CopP. SRII-HtrII fusion to which CopP is fused CopA when phosphorylated by CopP 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.
