Part:BBa_K3009004

sfGFP with amber inside 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 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 tested whether the stem loops structures impede the translation of the GFP sequence. GFP-ms2 constructs exhibit fluorescence at 475 nm under fluorescence microscopy, the loops strucures therefore do not impede the translation.

References

[1]Pédelacq et. al . (2006): Engineering and characterization of a superfolder green fluorescent protein. In: Nature biotechnology 24 (1), S. 79–88</b> [2] Wang et. al (2003): Unnatural amino acid mutagenesis of green fluorescent protein. In: The Journal of organic chemistry 68 (1), S. 174–176.</b> [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