IGEM:Imperial/2010/Output module
| 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')
gcgcagctggaaaaagaactgcaggcgctggaaaaaaaactggcgcagctggaatgggaa aaccaggcgctggaaaaagaactggcgcag
AQAKKKAQANKKELAQLKWKLQALKKKLAQ (B'2A)
gcgcaggcgaaaaaaaaagcgcaggcgaacaaaaaagaactggcgcagctgaaatggaaa ctgcaggcgctgaaaaaaaaactggcgcag
Tobacco etch virus (TEV) protease-cleavable linker (GGGGENLYFQGGKLGGGG)was used.
ggcggcggcggcgaaaacctgtattttcagggcggcaaactgggcggcggcggc LINKER
Beta-Lactamase construct
NβLac-A-TEV-B’4A βLactamase (26-196) TEV B-CβLac βLactamase (198-290)
Superfolder split GFP
NOTE readily self assembles as does split Beta-galactosidase
'One fragment of the split GFP contains the first 214 residues of the exceptionally stable, fast-folding ‘‘superfolder’’ GFP protein (Pedelacq et al., 2006), further evolved to be stable as a protein fragment. This fragment includes ten of the eleven strands of the beta-barrel structure of GFP and will be called spGFP1-10. The second split-GFP fragment consists of just 16 residues, 215– 230, which make up the 11th strand of the GFP b-barrel. This second fragment, spGFP11, acts as a small protein tag that can be inserted into many different proteins without affecting their solubility' ref
Amino acid and sequence of the GFP 11 cassette:
SSLKRRKIPMGSSHHHHHHSSGLVPRGSHM*(frame shift +1)
- LIGSDGGSGGGSTSRDHMVLHEYVNAAGIT*GT*LEHHHHHH*DPAANKARKEAELAAATAEQ*LA*PLEA
DNA sequence of the GFP 11 cassette 0421007 TAGAGATACTGAGCACATCAGCAGGACGCACTGACCGAGTTCATTAAAGAGGAGAAAGATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGTAATTAATTAATTGGATCCGATGGAGGGTCTGGTGGCGGATCAACAAGTCGTGACCACATGGTCCTTCATGAGTACGTAAATGCTGCTGGGATTACATAAGGTACCTAACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCTCTAGAGGCCTC 02129811
Amino acid sequence of the GFP 1-10 cassette DRDLDPAKLIRLTIHMGGTSMSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK*GTLEHHHHHH*DPAANKARKEAELAAATAEQ*LA*PLEA
DNA sequence of the GFP 1-10 cassette
01079936
AGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATACATATGGGTGGCACTAGTATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGAGGAGAGGGTGAAGGTGATGCTACAATCGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAAAGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAAATACAAGACGCGTGCTGTAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTACTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCACAGTTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAAACTGTCCTTTCGAAAGATCCCAACGAAAAGTAAGGTACCCTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCTCTAGAGGCCTC
02129811
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
