Team:NEU-China/PartOR1D2


Part OR1D2 Document

Part:BBa_K2288001

Designed by: Jiawen Yang Group: iGEM17_NEU-China (2017-10-10)

Rho-OR1D2(HEK293)

Encoding the fragments of the olfactory receptor OR1D2 with Rho-tag in its N-terminal in HEK293 cells. Once the specific odor, bourgeonal, binds to OR1D2 on the cell membrane, it leads to a series of signaling rally and consequently generates a flux of cAMP which could be detected by a cAMP-activated reporter gene system. The Rho tag was added to its N-terminal, which could improve the olfactory receptor to express on the surface of HEK293.
The whole OR1D2 signaling exists primarily in the olfactory epithelia cells which are not easily cultured and amplified to generate the ex vivo biosensor, nor does a simplified prokaryote can reconstitute the whole OR1D2 pathway. Therefore we chose an easily cultured eukaryotic HEK293FT cells instead to reconstitute the olfactory signaling cascade for the biosensor with a CRE-luciferase construct as the reporter.

Function Receptor
RFC standard RFC 10
Backbone pSB1C3
Organism Human
Source Human HEK293FT
Submitted by NEU-China 2017

Usage and Biology

Protein data table for BioBrick BBa_K2288001 automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 10: (underlined part encodes the protein)
  ATGACCGAG ... AGGCTGACATAA
  ORF from nucleotide position 1 to 969 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
1 
101 
201 
301 		 MTETSQVAPAGGDGGNQSEGSEFLLLGMSESPEQQQILFWMFLSMYLVTVVGNVLIILAISSDSRLHTPVYFFLANLSFTDLFFVTNTIPKMLVNLQSHN
KAISYAGCLTQLYFLVSLVALDNLILAVMAYDRYVAICCPLHYTTAMSPKLCILLLSLCWVLSVLYGLIHTLLMTRVTFCGSRKIHYIFCEMYVLLRMAC
SNIQINHTVLIATGCFIFLIPFGFVIISYVLIIRAILRIPSVSKKYKAFSTCASHLGAVSLFYGTLCMVYLKPLHTYSVKDSVATVMYAVVTPMMNPFIY
SLRNKDMHGALGRLLDKHFKRLT*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
None of the supported features appeared in the sequence
Amino acid composition:
Ala (A) 20 (6.2%)
Arg (R) 10 (3.1%)
Asn (N) 11 (3.4%)
Asp (D) 8 (2.5%)
Cys (C) 11 (3.4%)
Gln (Q) 8 (2.5%)
Glu (E) 6 (1.9%)
Gly (G) 16 (5.0%)
His (H) 10 (3.1%)
Ile (I) 24 (7.4%)
Leu (L) 48 (14.9%)
Lys (K) 12 (3.7%)
Met (M) 15 (4.6%)
Phe (F) 19 (5.9%)
Pro (P) 11 (3.4%)
Ser (S) 27 (8.4%)
Thr (T) 21 (6.5%)
Trp (W) 2 (0.6%)
Tyr (Y) 17 (5.3%)
Val (V) 27 (8.4%)
Amino acid counting
Total number: 323
Positively charged (Arg+Lys): 22 (6.8%)
Negatively charged (Asp+Glu): 14 (4.3%)
Aromatic (Phe+His+Try+Tyr): 48 (14.9%)
Biochemical parameters
Atomic composition: C 1671H 2624N 406O 436S 26
Molecular mass [Da]: 36211.2
Theoretical pI: 8.90
Extinction coefficient at 280 nm [M -1 cm -1]: 36330 / 37018 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges 
Codon usage
Organism: E. coli B. subtilis S. cerevisiae A. thaliana P. patens Mammals
Codon quality ( CAI): good (0.68) good (0.69) acceptable (0.57) good (0.67) excellent (0.81) excellent (0.80)
Alignments (obtained from PredictProtein.org)
   There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours.
Predictions (obtained from PredictProtein.org)
   There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours.
The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation.
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How to use

If you want to use it, you should digest it with the enzyme PstI and XbaI to get the fragments. Then you need to add the restriction sites on both sides of the sequence by PCR. A pair of reference primers below is provided.
Forward: cgtaagcttatgaccgagacatctcaggtggcccctgccggcggcgatggaggcaac
Reverse: tatctcgagttatgtcagcctcttaaagtgtttatctagg
Most importantly, you need to clone the parts into the commonly used eukaryotic expression vector pcDNA3.1+. As shown, you also need to digest the vector with HindIII and XhoI endonuclease in advance.

Sequencing results of OR1D2

Proof of expression
Sensitivity Testing

Test the sensitivity and specificity of this basic odor biosensors with their cognate odors bourgeonal by qPCR.