Part:BBa_K3009034
sfGFP 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.</b>
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 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. [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
[2] Peabody, D. S. (1993): The RNA binding site of bacteriophage MS2 coat protein. In: The EMBO Journal 12 (2), S. 595–600.
[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
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1107
Illegal EcoRI site found at 4055
Illegal XbaI site found at 3608 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1107
Illegal EcoRI site found at 4055
Illegal XbaI site found at 3608 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 4981
Illegal BsaI site found at 5605
Illegal SapI site found at 3805
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 3
Illegal XbaI site found at 112
Illegal PstI site found at 15
Illegal PstI site found at 85
Illegal PstI site found at 124 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 137
Illegal PstI site found at 15
Illegal PstI site found at 85
Illegal PstI site found at 124 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 3
Illegal XbaI site found at 112
Illegal PstI site found at 15
Illegal PstI site found at 85
Illegal PstI site found at 124 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 3
Illegal XbaI site found at 112
Illegal PstI site found at 15
Illegal PstI site found at 85
Illegal PstI site found at 124 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 441
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