Coding

Part:BBa_K2332315

Designed by: Camillo Moschner   Group: iGEM17_UCL   (2017-10-22)
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Plasma membrane delivery system (PMDS, mammalian)

Plasma Membrane Delivery System (PMDS)
Function Delivery of protein (e.g. mCherry) to the PM
Use in Mammalian cells
Abstraction Hierarchy Part
RFC standard RFC10, RFC12, RFC23 & RFC25 compatible
Backbone pSB1C3
Submitted by [http://2017.igem.org/Team:UCL UCL iGEM 2017]


This coding region translates a transmembrane domain which is destined to go to the plasma membrane via the secretory pathway. While doing so it delivers any 'cytosolic domain insert' that has been cloned into this BioBrick in a plug-and-play fashion (via the KpnI-BamHI restriction sites) to the plasma membrane. The delivered protein is localised to the plasma membrane but activation of the photocleavable linker PhoCl with 400 nm, violet light allows irreversible breakage of the connection with the PM and releases the 'cytosolic domain insert' into the cytoplasm. For characterisation purposes mCherry has been chosen 'cytosolic domain insert' in this BioBrick.



Usage and Biology

As stated in the original paper:


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 2485
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 704
    Illegal BsaI.rc site found at 1471


Functional Parameters

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 10: (underlined part encodes the protein)
 ACCATGCGGGCC ... GTCGGATCCTAG
 ORF from nucleotide position 4 to 2490 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
1 
101 
201 
301 
401 
501 
601 
701 
801 		MRAWIFFLLCLAGRALAATGRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQH
DFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADH
YQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKDYKDDDDKTGGGGGSGGGGSPSIATGMVGALLLLLVVALGIGL
FMRRRHIVRKRGGGGSGGGGSGSVIPDYFKQSFPEGYSWERSMTYEDGGICIATNDITMEGDSFINKIHFKGTNFPPNGPVMQKRTVGWEASTEKMYERD
GVLKGDVKMKLLLKGGGHYRCDYRTTYKVKQKPVKLPDYHFVDHRIEILSHDKDYNKVKLYEHAVARNSTDSMDELYKGGSGGMVSKGEETITSVIKPDM
KNKLRMEGNVNGHAFVIEGEGSGKPFEGIQTIDLEVKEGAPLPFAYDILTTAFHYGNRVFTKYPRGGGGTGGGGSGGGGSVSKGEEDNMAIIKEFMRFKV
HMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDG
EFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYER
AEGRHSTGGMDELYKGGGGSPKKKRKVGS*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
RFC25 scar (shown in bold): 19 to 20, 266 to 267, 807 to 808
Flag-tag: 258 to 265
Enterokinase cleavage site: 261 to 265
SV40 nuclear localization sequence: 821 to 827
Amino acid composition:
Ala (A)36 (4.3%)
Arg (R)33 (4.0%)
Asn (N)30 (3.6%)
Asp (D)53 (6.4%)
Cys (C)5 (0.6%)
Gln (Q)20 (2.4%)
Glu (E)58 (7.0%)
Gly (G)114 (13.8%)
His (H)25 (3.0%)
Ile (I)40 (4.8%)
Leu (L)55 (6.6%)
Lys (K)75 (9.0%)
Met (M)26 (3.1%)
Phe (F)37 (4.5%)
Pro (P)36 (4.3%)
Ser (S)42 (5.1%)
Thr (T)46 (5.5%)
Trp (W)7 (0.8%)
Tyr (Y)38 (4.6%)
Val (V)53 (6.4%)
Amino acid counting
Total number:829
Positively charged (Arg+Lys):108 (13.0%)
Negatively charged (Asp+Glu):111 (13.4%)
Aromatic (Phe+His+Try+Tyr):107 (12.9%)
Biochemical parameters
Atomic composition:C4078H6316N1110O1228S31
Molecular mass [Da]:91535.6
Theoretical pI:6.82
Extinction coefficient at 280 nm [M-1 cm-1]:95120 / 95433 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges 
Codon usage
Organism:E. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
Codon quality (CAI):good (0.69)good (0.68)acceptable (0.57)good (0.67)excellent (0.85)excellent (0.85)
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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Reference

Zhang W, Lohman AW, Zhuravlova Y, Lu X, Wiens MD, Hoi H, Yaganoglu S, Mohr MA, Kitova EN, Klassen JS, Pantazis P, Thompson RJ, Campbell RE. Optogenetic control with a photocleavable protein, PhoCl. Nat Methods. 14(4):391-394 (2017) NCBI

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Categories
Parameters
None