IGEM:Imperial/2010/Output module: Difference between revisions
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(→GFP as output: relocating the link to EGFP) |
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This [http://www.springerlink.com/content/q34u8r7r7u76h045/fulltext.pdf review article] has some useful information on FRET. | This [http://www.springerlink.com/content/q34u8r7r7u76h045/fulltext.pdf review article] has some useful information on FRET. | ||
==Our autoinhibitory coiled-coil output constructs== | |||
Taken and adapted from the JACS article 'An Autoinhibited Coiled-Coil Design Strategy for Split-Protein | |||
Protease Sensors' [http://pubs.acs.org/doi/pdf/10.1021/ja9050857 ref] | |||
The construct design is of the order: | |||
A’-TEV-B-NFluc | |||
Cfluc-A-TEV-B‘2A | |||
Where in our case NFluc and CFluc will be NBlactamase CBlactamase//split eGFP and split TEV itself. The 2A refers to a mutated variation of the original coil sequence (AQLKKKLQANKKELAQLKWKLQALKKKLAQ)which produced a better coil activity. | |||
AQLEKELQALEKKLAQLEWENQALEKELAQ (A') | |||
AQAKKKAQANKKELAQLKWKLQALKKKLAQ (B'2A) | |||
Revision as of 02:40, 29 July 2010
Effector 1 (Protease) | Effector 2 (Dye,Enz) | Pigment biosynthetic pathways |
---|---|---|
transcr sigma 54 | 2 colourless | Bilins |
Activation (phosphorylation) | Enz-in-pathway
|
Anthocyanins
|
Target proteases to use
|
Protein scaffold | Fret pairs as back up for effectors |
2C DNA binding prot release |
This review article has some useful information on FRET.
Our autoinhibitory coiled-coil output constructs
Taken and adapted from the JACS article 'An Autoinhibited Coiled-Coil Design Strategy for Split-Protein Protease Sensors' ref
The construct design is of the order:
A’-TEV-B-NFluc Cfluc-A-TEV-B‘2A
Where in our case NFluc and CFluc will be NBlactamase CBlactamase//split eGFP and split TEV itself. The 2A refers to a mutated variation of the original coil sequence (AQLKKKLQANKKELAQLKWKLQALKKKLAQ)which produced a better coil activity.
AQLEKELQALEKKLAQLEWENQALEKELAQ (A')
AQAKKKAQANKKELAQLKWKLQALKKKLAQ (B'2A)
James- some suggested papers:
- Kerppola TK. Complementary methods for studies of protein interactions in living cells. Nat Methods. 2006 Dec;3(12):969-71. DOI:10.1038/nmeth1206-969 |
- Wehr MC, Laage R, Bolz U, Fischer TM, Grünewald S, Scheek S, Bach A, Nave KA, and Rossner MJ. Monitoring regulated protein-protein interactions using split TEV. Nat Methods. 2006 Dec;3(12):985-93. DOI:10.1038/nmeth967 |
GFP as output
- In vivo and in vitro protein solubility assays using split GFP
- Monitoring of conformational change in maltose binding protein using split green fluorescent protein
- Protein Splicing-Based Reconstitution of Split Green Fluorescent Protein for Monitoring Protein−Protein Interactions in Bacteria: Improved Sensitivity and Reduced Screening Time
- A Fluorescent Indicator for Detecting Protein−Protein Interactions in Vivo Based on Protein Splicing