Melbourne/Blue Photosensor
From 2007.igem.org
| Line 1: | Line 1: | ||
[[Melb:Background|<return to top of background>]] [[melbourne|<return to home page>]] [[Melb:And Gate |<next>]] | [[Melb:Background|<return to top of background>]] [[melbourne|<return to home page>]] [[Melb:And Gate |<next>]] | ||
| + | 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 srfA promoter (link required), as part of the AND gate. | ||
====[[Melbourne/Blue Photosensor Background|Blue Photosensor Background]]==== | ====[[Melbourne/Blue Photosensor Background|Blue Photosensor Background]]==== | ||
| Line 8: | Line 11: | ||
====Possible extensions:==== | ====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==== | ====References==== | ||
| Line 21: | Line 22: | ||
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. | 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 | + | It is proposed to replace Tsr with homolgouse ComP. |
| - | SRII-HtrII fusion to which | + | SRII-HtrII fusion to which ComP is fused |
| - | CopA when phosphorylated by | + | CopA when phosphorylated by ComP is an activator for PsfA promoter sequence from |
Dr Alan Grossman (M.I.T.) | Dr Alan Grossman (M.I.T.) | ||
Based on | Based on | ||
Revision as of 10:31, 6 August 2007
<return to top of background> <return to home page> <next>
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 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.. CheA,W,Y connected system.
It is proposed to replace Tsr with homolgouse ComP. SRII-HtrII fusion to which ComP is fused CopA 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.
