Melbourne/Blue Photosensor

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[[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>]]
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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).
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The kinase domain of HtrII will be replaced with the kinase domain of ComP. ComP forms part of a two-component system from Bacillus subtilis - and this will not affect any endogenous networks in e.coli.
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The two component system involves: ComP, a two-component sensor histidine kinase and ComA, a two-component response regulator. Phosphorylated comA will upregulate transcription at the psfA/srfA promoter [[Melbourne/Lab_BL_Notebook/PsrfA_sequence|psfA/srfA]], as part of the AND gate.
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'''In summary''':
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[[Image:Melbourne SrII-HtrII.JPG|200px|SrII-HtrII]] + [[Image:Melbourne ComP.JPG|200px|ComP]] >>>>>> [[Image:Melbourne HtrII-ComPchimera.jpg|200px|HtrII-ComP fusion]]
====[[Melbourne/Blue Photosensor Background|Blue Photosensor Background]]====
====[[Melbourne/Blue Photosensor Background|Blue Photosensor Background]]====
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====Possible extensions:====
====Possible extensions:====
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*Determine proteins in vesicles and hence relevant genes:
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*Determination of optimal wavelength:
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**Perform 2DGE on expressed vesicle proteins.
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**use of different substrates (different retinals)
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**Seperate vesicles by floatation and do an SDS PAGE.
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*Separate variants all submitted as BioBricks.
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**Apply MALDI TOF MS to determine Amino acid sequences and hence degenerate code and genes.
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*Submitted synthesized ComP and ComA as BioBricks
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*Seperate coding regions into individual biobricks.
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*Model the pathway to determine rate-limiting step
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*Recombine in different ways and observe effect on gas vessicle structure.
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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====
====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.
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.
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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.  
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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.  
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It is proposed to replace Tsr with homolgouse CopP.
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It is proposed to replace Tsr fusion with homolgouse ComP.
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SRII-HtrII fusion to which CopP is fused
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SRII-HtrII fusion to which ComP is fused
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CopA when phosphorylated by CopP is an activator for PsfA promoter sequence from
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ComA 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  
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SRII is from Natronomonas pharaonis.
SRII is from Natronomonas pharaonis.
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Tsr fusion was made by Jung et al J Bacteriol 183 6365-6371 (2001) they propose a mechanism.
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Tsr fusion was made by Jung et al J Bacteriol 183 6365-6371 (2001) they propose a mechanism.
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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.
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Also what are the normal functions of ComP etc.
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Latest revision as of 13:44, 26 October 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).

The kinase domain of HtrII will be replaced with the kinase domain of ComP. ComP forms part of a two-component system from Bacillus subtilis - and this will not affect any endogenous networks in e.coli. The two component system involves: ComP, a two-component sensor histidine kinase and ComA, a two-component response regulator. Phosphorylated comA will upregulate transcription at the psfA/srfA promoter psfA/srfA, as part of the AND gate.

In summary:

SrII-HtrII + ComP >>>>>> HtrII-ComP fusion

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.

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