Melbourne/Blue Photosensor

From 2007.igem.org

(Difference between revisions)
Line 7: Line 7:
====[[Melbourne/Plan/Blue Photosensor|Method]] ====
====[[Melbourne/Plan/Blue Photosensor|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.
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.

Revision as of 10:03, 6 August 2007

<return to top of background> <return to home page> <next>


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.