Composite

Part:BBa_K3009005

Designed by: Carolin Ruckes   Group: iGEM19_Freiburg   (2019-10-15)


sfGFP with Amber outside the chrom.

Group: Freiburg
Author: Alisa
Summary: Superfolder GFP is an improved variant of the original Green Fluorescent Protein and is used as a fluorescent reporter Protein. By mutating position 66 to an amber stop codon, the incorporation of a noncanonical amino acid instead of Tyrosine in the chromophore is made possible. .
Documentation:

Usage

sfGFP is a widely used fluorescent reporter protein [1]. It is used to study protein expression and dynamics in living cells. Superfolder GFP is a faster-folding variant of the original Green Fluorescent Protein. Fluorescence can be observed when exciting the chromophore of the protein with a laser and detecting the emitted fluorescence. By mutating a Phenylalanine at a permissive site to an amber stop codon, the incorporation of noncanonical amino acids can ne studied. The Biobrick is especially useful as a control for noncanonical incorporation into the chromophore. [2]

Biology

GFP was first extracted from the Aequorea victoria, where it is responsible for Bioluminescence. [3]


Characterization

Without an orthogonal translational machienery able to incorporate noncanonical amino acids, only truncated, unfunctional proteins should be translated, and no fluorescence should be visible.


References

[1]Pédelacq et. al . (2006): Engineering and characterization of a superfolder green fluorescent protein. In: Nature biotechnology 24 (1), S. 79–88
[2] Wang et. al (2003): Unnatural amino acid mutagenesis of green fluorescent protein. In: The Journal of organic chemistry 68 (1), S. 174–176.
[3] Shimomura et. al (1962): Extraction, Purification and Properties of Aequorin, a Bioluminescent Protein from the Luminous Hydromedusan,Aequorea. In: J. Cell. Comp. Physiol. 59 (3), S. 223–239



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1107
    Illegal EcoRI site found at 4055
    Illegal XbaI site found at 3608
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1107
    Illegal EcoRI site found at 4055
    Illegal NheI site found at 1689
    Illegal NheI site found at 3572
    Illegal NheI site found at 3595
    Illegal NheI site found at 4049
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1107
    Illegal EcoRI site found at 4055
    Illegal BamHI site found at 3988
    Illegal XhoI site found at 4509
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1107
    Illegal EcoRI site found at 4055
    Illegal XbaI site found at 3608
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1107
    Illegal EcoRI site found at 4055
    Illegal XbaI site found at 3608
    Illegal NgoMIV site found at 2622
    Illegal NgoMIV site found at 5917
    Illegal AgeI site found at 503
    Illegal AgeI site found at 1775
    Illegal AgeI site found at 2099
    Illegal AgeI site found at 3120
    Illegal AgeI site found at 3823
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4981
    Illegal BsaI site found at 5605
    Illegal SapI site found at 3805



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1107
    Illegal EcoRI site found at 4055
    Illegal XbaI site found at 3608
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1107
    Illegal EcoRI site found at 4055
    Illegal NheI site found at 1689
    Illegal NheI site found at 3572
    Illegal NheI site found at 3595
    Illegal NheI site found at 4049
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1107
    Illegal EcoRI site found at 4055
    Illegal BamHI site found at 3988
    Illegal XhoI site found at 4509
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1107
    Illegal EcoRI site found at 4055
    Illegal XbaI site found at 3608
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1107
    Illegal EcoRI site found at 4055
    Illegal XbaI site found at 3608
    Illegal NgoMIV site found at 2622
    Illegal NgoMIV site found at 5917
    Illegal AgeI site found at 503
    Illegal AgeI site found at 1775
    Illegal AgeI site found at 2099
    Illegal AgeI site found at 3120
    Illegal AgeI site found at 3823
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4981
    Illegal BsaI site found at 5605
    Illegal SapI site found at 3805


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