IGEM:Imperial/2010/Output module

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

Overall sequence for Coiled-coil construct
C-TERMINAL sGFP/sB-Lac -GGGSGGGSGGGS-AQLEKELQALEKKLAQLEWENQALEKELAQGGGGENLYFQGGKLGGGGAQAKKKAQANKKELAQLKWKLQALKKKLAQ

ggcggcggcagcggcggcggcagcggcggcggcagcgcgcagctggaaaaagaactgcaggcgctggaaaaaaaactggcgcagctggaatgggaa aaccaggcgctggaaaaagaactggcgcagggcggcggcggcgaaaacctgtattttcag ggcggcaaactgggcggcggcggcgcgcaggcgaaaaaaaaagcgcaggcgaacaaaaaa gaactggcgcagctgaaatggaaactgcaggcgctgaaaaaaaaactggcgcag

AQLEKELQALEKKLAQLEWENQALEKELAQGGGGENLYFQGGKLGGGGAQLKKKLQANKKELAQLKWKLQALKKKLAQ-GGGSGGGSGGGS-N-TERMINAL sGFP/sB-Lac

gcgcagctggaaaaagaactgcaggcgctggaaaaaaaactggcgcagctggaatgggaa aaccaggcgctggaaaaagaactggcgcagggcggcggcggcgaaaacctgtattttcag ggcggcaaactgggcggcggcggcgcgcagctgaaaaaaaaactgcaggcgaacaaaaaa gaactggcgcagctgaaatggaaactgcaggcgctgaaaaaaaaactggcgcagggcggcggcagcggcggcggcagcggcggcggcagc

(GGGS)3 is the flexible linker between the split protease/enzyme and the coil!!

Beta-Lactamase construct
NβLac-A-TEV-B’4A βLactamase (26-196)(Glu) TEV B-CβLac βLactamase (Leu)(198-290)

LacB from pQE32 plasmid []

Showing sequences for the B-Lactamase, the sequences are split into the two halves with the gap in the middle.

Protein 286aa MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMAttlrklltg

ellt

LASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW

Nucleotide atgagcattcagcattttcgcgtggcgctgattccgttttttgcggcgttttgcctgccggtgtttgcgcatccggaaaccctggtgaaagtgaaagatgcggaagatcagctgggcgcgcgcgtgggctatattgaactggatctgaacagcggcaaaattctggaaagctttcgcccggaagaacgctttccgatgatgagcacctttaaagtgctgctgtgcggcgcggtgctgagccgcattgatgcgggccaggaacagctgggccgccgcattcattatagccagaacgatctggtggaatatagcccggtgaccgaaaaacatctgaccgatggcatgaccgtgcgcgaactgtgcagcgcggcgattaccatgagcgataacaccgcggcgaacctgctgctgaccaccattggcggcccgaaagaactgaccgcgtttctgcataacatgggcgatcatgtgacccgcctggatcgctgggaaccggaactgaacgaagcgattccgaacgatgaacgcgataccaccatgccggtggcgatggcgaccaccctgcgcaaactgctgaccggc

gaactgctgacc

ctggcgagccgccagcagctgattgattggatggaagcggataaagtggcgggcccgctgctgcgcagcgcgctgccggcgggctggtttattgcggataaaagcggcgcgggcgaacgcggcagccgcggcattattgcggcgctgggcccggatggcaaaccgagccgcattgtggtgatttataccaccggcagccaggcgaccatggatgaacgcaaccgccagattgcggaaattggcgcgagcctgattaaacattgg

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

Split luciferase
entire seq:

Luciferase [Photinus pyralis] GenBank: AAA29795.1

can be obtained from Promega as a reporter plasmid pGL3

medaknikkgpapfypledgtageqlhkamkryalvpgtiaftdahievnityaeyfemsvrlaeamkryglntnhrivvcsenslqffmpvlgalfigvavapandiynerellnsmnisqptvvfvskkglqkilnvqkklpiiqkiiimdsktdyqgfqsmytfvtshlppgfneydfvpesfdrdktialimnssgstglpkgvalphrtacvrfshardpifgnqiipdtailsvvpfhhgfgmfttlgylicgfrvvlmyrfeeelflrslqdykiqsallvptlfsffakstlidkydlsnlheiasggaplskevgeavakrfhlpgirqgygltettsailitpegddkpgavgkvvpffeakvvdldtgktlgvnqrgelcvrgpmimsgyvnnpeatnalidkdgwlhsgdiaywdedehffivdrlkslikykgyqvapaelesillqhpnifdagvaglpdddagelpaavvvlehgktmtekeivdyvasqvttakklrggvvfvdevpkgltgkldarkireilikakkggkskl

Firefly Luciferase(2-416) Firefly Luciferase( 398-550)

N-FLUC protein daknikkgpapfypledgtageqlhkamkryalvpgtiaftdahievnityaeyfemsvrlaeamkryglntnhrivvcsenslqffmpvlgalfigvavapandiynerellnsmnisqptvvfvskkglqkilnvqkklpiiqkiiimdsktdyqgfqsmytfvtshlppgfneydfvpesfdrdktialimnssgstglpkgvalphrtacvrfshardpifgnqiipdtailsvvpfhhgfgmfttlgylicgfrvvlmyrfeeelflrslqdykiqsallvptlfsffakstlidkydlsnlheiasggaplskevgeavakrfhlpgirqgygltettsailitpegddkpgavgkvvpffeakvvdldtgktlgvnqrgelcvrgpmimsgyvnnpeatnalidkdg

Nucleotide gatgcgaaaaacattaaaaaaggcccggcgccgttttatccgctggaagatggcaccgcgggcgaacagctgcataaagcgatgaaacgctatgcgctggtgccgggcaccattgcgtttaccgatgcgcatattgaagtgaacattacctatgcggaatattttgaaatgagcgtgcgcctggcggaagcgatgaaacgctatggcctgaacaccaaccatcgcattgtggtgtgcagcgaaaacagcctgcagttttttatgccggtgctgggcgcgctgtttattggcgtggcggtggcgccggcgaacgatatttataacgaacgcgaactgctgaacagcatgaacattagccagccgaccgtggtgtttgtgagcaaaaaaggcctgcagaaaattctgaacgtgcagaaaaaactgccgattattcagaaaattattattatggatagcaaaaccgattatcagggctttcagagcatgtatacctttgtgaccagccatctgccgccgggctttaacgaatatgattttgtgccggaaagctttgatcgcgataaaaccattgcgctgattatgaacagcagcggcagcaccggcctgccgaaaggcgtggcgctgccgcatcgcaccgcgtgcgtgcgctttagccatgcgcgcgatccgatttttggcaaccagattattccggataccgcgattctgagcgtggtgccgtttcatcatggctttggcatgtttaccaccctgggctatctgatttgcggctttcgcgtggtgctgatgtatcgctttgaagaagaactgtttctgcgcagcctgcaggattataaaattcagagcgcgctgctggtgccgaccctgtttagcttttttgcgaaaagcaccctgattgataaatatgatctgagcaacctgcatgaaattgcgagcggcggcgcgccgctgagcaaagaagtgggcgaagcggtggcgaaacgctttcatctgccgggcattcgccagggctatggcctgaccgaaaccaccagcgcgattctgattaccccggaaggcgatgataaaccgggcgcggtgggcaaagtggtgccgttttttgaagcgaaagtggtggatctggataccggcaaaaccctgggcgtgaaccagcgcggcgaactgtgcgtgcgcggcccgatgattatgagcggctatgtgaacaacccggaagcgaccaacgcgctgattgataaagatggc

C-FLUC Protein msgyvnnpeatnalidkdgwlhsgdiaywdedehffivdrlkslikykgyqvapaelesillqhpnifdagvaglpdddagelpaavvvlehgktmtekeivdyvasqvttakklrggvvfvdevpkgltgkldarkireilikakkggkskl

Nucleotide atgagcggctatgtgaacaacccggaagcgaccaacgcgctgattgataaagatggctggctgcatagcggcgatattgcgtattgggatgaagatgaacatttttttattgtggatcgcctgaaaagcctgattaaatataaaggctatcaggtggcgccggcggaactggaaagcattctgctgcagcatccgaacatttttgatgcgggcgtggcgggcctgccggatgatgatgcgggcgaactgccggcggcggtggtggtgctggaacatggcaaaaccatgaccgaaaaagaaattgtggattatgtggcgagccaggtgaccaccgcgaaaaaactgcgcggcggcgtggtgtttgtggatgaagtgccgaaaggcctgaccggcaaactggatgcgcgcaaaattcgcgaaattctgattaaagcgaaaaaaggcggcaaaagcaaactg

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

Re-confirmed sequence for S219D TEV construct
Protein: GRSLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASnftntnnyftSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMDKPEEPFQPVKEATQLMN

Nucleotide: ggccgcagcctgtttaaaggcccgcgcgattataacccgattagcagcaccatttgccatctgaccaacgaaagcgatggccataccaccagcctgtatggcattggctttggcccgtttattattaccaacaaacatctgtttcgccgcaacaacggcaccctgctggtgcagagcctgcatggcgtgtttaaagtgaaaaacaccaccaccctgcagcagcatctgattgatggccgcgatatgattattattcgcatgccgaaagattttccgccgtttccgcagaaactgaaatttcgcgaaccgcagcgcgaagaacgcatttgcctggtgaccaccaactttcagaccaaaagcatgagcagcatggtgagcgataccagctgcacctttccgagcagcgatggcattttttggaaacattggattcagaccaaagatggccagtgcggcagcccgctggtgagcacccgcgatggctttattgtgggcattcatagcgcgagcaactttaccaacaccaacaactattttaccagcgtgccgaaaaactttatggaactgctgaccaaccaggaagcgcagcagtgggtgagcggctggcgcctgaacgcggatagcgtgctgtggggcggccataaagtgtttatggataaaccggaagaaccgtttcagccggtgaaagaagcgacccagctgatgaac

C23O
Catechol 2,3-dioxygenase (also known as C23O or MPC) catalyses the conversion of a colourless substrate (catechol or substituted catechols) into a bright yellow product (2-hydroxymuconic semialdehyde) within seconds of substrate addition.

C23O is a homotetramer and its crystal structure has been solved. See Kita et al or use the accession code 1mpy.

C23O has also been expressed successfully in B. subtilis by Zukowski et al.

We would make a fusion protein by adding, for example, GST to either the C- or N- terminus. This would hopefully prevent oligomerisation until the TEV protease is activated. TEV would specifically cleave the linker between GST and C23O, resulting in C23O monomers that are capable of oligomerising. Homotetramers would then be active and could catalyse the chromogenic reaction.

The N-terminus could be favoured because it seems to be more accessible to the protease as it is on the surface of the monomer. However, it is further away from the oligomerisation interfaces and so might not actually prevent oligomerisation.

The C-terminus, on the other hand, is less accessible, but fusing GST there could prevent entry of the substrate into the funnel that runs through the enzyme. It is also closer to the oligomerisation domain and so fusing GST here is more likely to prevent oligomerisation.

However, when we looked at C23O in Pymol, we noticed that the N-termini were probably closer to the oligomerisation interfaces (see below). Also, we were also worried that we might still get dimerisation if we used the N-terminus for our fusion protein. However, Kita et al show that the projecting loop is needed for dimerisation, and this is very near to the N-terminus.



The C23O gene is already part of a BioBrick (Part:BBa_K118021).

James- some suggested papers:

 * 1) 1 pmid=17117150
 * 2) 2 pmid=17072307

GFP as output
EGFP


 * 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