Coding

Part:BBa_K2332311

Designed by: Camillo Moschner   Group: iGEM17_UCL   (2017-09-27)
Revision as of 14:44, 8 October 2017 by CamilloMoschner (Talk | contribs) (→‎Functional Parameters)


PhoCl, a mammalian photocleavable protein

PhoCl, a Mammalian Photocleavable Protein
Function Photocleavable Linker
Use in Mammalian cells
Abstraction Hierarchy Part
RFC standard RFC10, RFC23 & RFC25 compatible
Backbone pSB1C3
Submitted by [http://2017.igem.org/Team:UCL UCL iGEM 2017]

As part of the UCL 2017's project "Light-induced Technologies" we investigated light sensitive proteins and their possible applications in synthetic genetic circuits. PhoCl is a novel (April 2017) photocleavable protein engineered from a green-to-red photoconvertible fluorescent protein (FP).


Usage and Biology

As stated in the original paper: "The photoconversion reaction is a violet light (~400 nm)-induced β-elimination reaction that extends the conjugated system of the chromophore with concomitant cleavage of the polypeptide backbone to form an ~66-residue N-terminal fragment and an ~166-residue C-terminal fragment that remain associated."

The original protein has been engineered by Zhang et al. 2017 (Robert E. Campbell lab). The Campbell lab has sent the original plasmid to the UCL iGEM 2017 team as part of a collaboration and the team has made a BioBrick out of the engineered protein.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 37
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 93
    Illegal XhoI site found at 102
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 607



Functional Parameters

color-NA-

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)
 ATGGGGGGT ... GGAGGTACCTAA
 ORF from nucleotide position 1 to 849 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
1 
101 
201 		MGGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPSSSVIPDYFKQSFPEGYSWERSMTYEDGGICIATNDITMEGDSFINKIHFKGTNFPPNGPVMQKRTV
GWEASTEKMYERDGVLKGDVKMKLLLKGGGHYRCDYRTTYKVKQKPVKLPDYHFVDHRIEILSHDKDYNKVKLYEHAVARNSTDSMDELYKGGSGGMVSK
GEETITSVIKPDMKNKLRMEGNVNGHAFVIEGEGSGKPFEGIQTIDLEVKEGAPLPFAYDILTTAFHYGNRVFTKYPRGGGGT*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
His6-tag: 5 to 10
Enterokinase cleavage site: 27 to 31
Amino acid composition:
Ala (A)9 (3.2%)
Arg (R)11 (3.9%)
Asn (N)10 (3.5%)
Asp (D)22 (7.8%)
Cys (C)2 (0.7%)
Gln (Q)6 (2.1%)
Glu (E)18 (6.4%)
Gly (G)35 (12.4%)
His (H)14 (4.9%)
Ile (I)14 (4.9%)
Leu (L)13 (4.6%)
Lys (K)25 (8.8%)
Met (M)13 (4.6%)
Phe (F)11 (3.9%)
Pro (P)13 (4.6%)
Ser (S)17 (6.0%)
Thr (T)17 (6.0%)
Trp (W)2 (0.7%)
Tyr (Y)15 (5.3%)
Val (V)16 (5.7%)
Amino acid counting
Total number:283
Positively charged (Arg+Lys):36 (12.7%)
Negatively charged (Asp+Glu):40 (14.1%)
Aromatic (Phe+His+Try+Tyr):42 (14.8%)
Biochemical parameters
Atomic composition:C1398H2143N387O429S15
Molecular mass [Da]:31716.6
Theoretical pI:6.39
Extinction coefficient at 280 nm [M-1 cm-1]:33350 / 33475 (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 (0.70)good (0.65)acceptable (0.53)good (0.63)excellent (0.90)excellent (0.91)
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.
If you have any questions, comments or suggestions, please leave us a comment.

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

[edit]
Categories
Parameters
color