IGEM:Imperial/2010/Output module

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(Beta-Lactamase construct)
(Our autoinhibitory coiled-coil output constructs)
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ggcggcggcggcgaaaacctgtattttcagggcggcaaactgggcggcggcggc  LINKER
ggcggcggcggcgaaaacctgtattttcagggcggcaaactgggcggcggcggc  LINKER
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===Overall sequence for Coiled-coil construct===
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C-TERMINAL sGFP/sB-Lac -AQLEKELQALEKKLAQLEWENQALEKELAQGGGGENLYFQGGKLGGGGAQAKKKAQANKKELAQLKWKLQALKKKLAQ
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gcgcagctggaaaaagaactgcaggcgctggaaaaaaaactggcgcagctggaatgggaa
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aaccaggcgctggaaaaagaactggcgcagggcggcggcggcgaaaacctgtattttcag
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ggcggcaaactgggcggcggcggcgcgcaggcgaaaaaaaaagcgcaggcgaacaaaaaa
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gaactggcgcagctgaaatggaaactgcaggcgctgaaaaaaaaactggcgcag
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AQLEKELQALEKKLAQLEWENQALEKELAQGGGGENLYFQGGKLGGGGAQLKKKLQANKKELAQLKWKLQALKKKLAQ -N-TERMINAL sGFP/sB-Lac
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gcgcagctggaaaaagaactgcaggcgctggaaaaaaaactggcgcagctggaatgggaa
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aaccaggcgctggaaaaagaactggcgcagggcggcggcggcgaaaacctgtattttcag
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ggcggcaaactgggcggcggcggcgcgcagctgaaaaaaaaactgcaggcgaacaaaaaa
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gaactggcgcagctgaaatggaaactgcaggcgctgaaaaaaaaactggcgcag
===Beta-Lactamase construct===  
===Beta-Lactamase construct===  

Revision as of 06:23, 29 July 2010

Effector 1 (Protease) Effector 2 (Dye,Enz) Pigment biosynthetic pathways
transcr sigma 54 2 colourless Bilins
Activation (phosphorylation) Enz-in-pathway
  • short pathways
  • ensure - colourless -> colour
Anthocyanins
  • short pathways
  • ensure - colourless -> colour
Target proteases to use
  • not many AA
  • non-toxic
  • can work in E.Coli
  • quantize speed, efficiency
Protein scaffold Fret pairs as back up for effectors
2C DNA binding prot release

This review article has some useful information on FRET.

Contents

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

Overall sequence for Coiled-coil construct

C-TERMINAL sGFP/sB-Lac -AQLEKELQALEKKLAQLEWENQALEKELAQGGGGENLYFQGGKLGGGGAQAKKKAQANKKELAQLKWKLQALKKKLAQ

gcgcagctggaaaaagaactgcaggcgctggaaaaaaaactggcgcagctggaatgggaa aaccaggcgctggaaaaagaactggcgcagggcggcggcggcgaaaacctgtattttcag ggcggcaaactgggcggcggcggcgcgcaggcgaaaaaaaaagcgcaggcgaacaaaaaa gaactggcgcagctgaaatggaaactgcaggcgctgaaaaaaaaactggcgcag


AQLEKELQALEKKLAQLEWENQALEKELAQGGGGENLYFQGGKLGGGGAQLKKKLQANKKELAQLKWKLQALKKKLAQ -N-TERMINAL sGFP/sB-Lac

gcgcagctggaaaaagaactgcaggcgctggaaaaaaaactggcgcagctggaatgggaa aaccaggcgctggaaaaagaactggcgcagggcggcggcggcgaaaacctgtattttcag ggcggcaaactgggcggcggcggcgcgcagctgaaaaaaaaactgcaggcgaacaaaaaa gaactggcgcagctgaaatggaaactgcaggcgctgaaaaaaaaactggcgcag

Beta-Lactamase construct

NβLac-A-TEV-B’4A βLactamase (26-196) TEV B-CβLac βLactamase (198-290)


fluorocilin green (Invitrogen) was used as the fluorescing substrate.

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:

  1. Kerppola TK. . pmid:17117150. PubMed HubMed [1]
  2. Wehr MC, Laage R, Bolz U, Fischer TM, Grünewald S, Scheek S, Bach A, Nave KA, and Rossner MJ. . pmid:17072307. PubMed HubMed [2]
All Medline abstracts: PubMed HubMed

GFP as output

EGFP


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