Composite

Part:BBa_K3009035

Designed by: Tahira Aslan   Group: iGEM19_Freiburg   (2019-12-13)


sfGFP (Amber inside chrom.) with MS2 loops

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 fusing three ms2 loop structures to the untranslated 3’ end of the transcribed mRNA, MCP fusion proteins can be recruited to the mRNA. An amber stop codon inside the chromophore enables the incorporation of unnatural amino acids into the chromophore of sfGFP.
Documentation:

Usage

By fusing ms2 loops to the untranslated 3’ end of the protein, MCP can be recruited to the transcribed mRNA. Fusion proteins of MCP are brought into close proximity to the mRNA. [1 ]By mutating the fluorescence-inducing tyrosine to an amber stop codon (TAG), the incorporation of various noncanonical amino acids into the chromophore is made possible. Aromatic noncanonical amino acids are known to shift the spectral properties of the protein. [2]


Biology

GFP was first extracted from the Aequorea victoria, where it is responsible for Bioluminescence. [3] stem-loops are part of the regulatory system of the MS2 phage which is active in early infection stages, and block replicase activity by binding the MS2 coat protein. [4]


Characterization

We expressed sfGFP-ms2 constructs with an amber stop codon with and without an orthogonal aminoacyl tRNA Synthetase pair which is able to incorporate D-Phenylalanine into the chromophore of sfGFP.

(PLACEHOLDER)

Figure 4: fluorescence intensity quantification of sfGFP with amber outside the chromophore with (green) and without (grey) orthogonal aminoacyl tRNA Synthetase in the cytosol. Untransformed C321.deltaA cells serve as negative control. Signal intensities were acquired by Fluorescence Microscopy and quantified by ImageJ.Graph shows values normalized to the sfGFP control

References

[1] Peabody, D. S. (1993): The RNA binding site of bacteriophage MS2 coat protein. In: The EMBO Journal 12 (2), S. 595–600.
[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. 223–23
[4] Peabody, D. S. (1993): The RNA binding site of bacteriophage MS2 coat protein. In: The EMBO Journal 12 (2), S. 595–600.



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
    Illegal XbaI site found at 6147
    Illegal XbaI site found at 6256
    Illegal PstI site found at 6159
    Illegal PstI site found at 6229
    Illegal PstI site found at 6268
  • 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
    Illegal PstI site found at 6159
    Illegal PstI site found at 6229
    Illegal PstI site found at 6268
  • 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
    Illegal XbaI site found at 6147
    Illegal XbaI site found at 6256
    Illegal PstI site found at 6159
    Illegal PstI site found at 6229
    Illegal PstI site found at 6268
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1107
    Illegal EcoRI site found at 4055
    Illegal XbaI site found at 3608
    Illegal XbaI site found at 6147
    Illegal XbaI site found at 6256
    Illegal PstI site found at 6159
    Illegal PstI site found at 6229
    Illegal PstI site found at 6268
    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


[edit]
Categories
Parameters
None