Composite

Part:BBa_K3187008

Designed by: iGEM TU_Darmstadt 2019   Group: iGEM19_TU_Darmstadt   (2019-10-12)
Revision as of 08:49, 16 October 2019 by Franziska (Talk | contribs)


GGGG-Tag for Sortase-mediated Ligation x mCherry Fluorescence Protein

Profile

Name mCherry-LPETGG
Base pairs 1028
Molecular weight 28.5 kDa
Origin synthetic, derived from Discosomasp.
Parts mCherry, T7-promoter, GGGG-sequence, lac-operator, GASPAG, Strep-Tag II, T7-terminator
Properties Red fluorescent, Ex λ: 587nm, Em λ: 610 nm

Usage and Biology

mCherry (BBa_K3187026)is a red fluorescent protein. Which is a synthetic protein derived from Discosoma sp. by directed evolution. The N-terminal GGGG-sequence (BBa_K3187018) can be fused to a protein with a C-terminal LPETGG-Sortase A link target="_blank">(BBa_K3187019) by Sortase A. We use mCherry as an easily imaged reporter for checking if the coupling worked.
The coding sequence was cloned in pDest vector, containing the sequence of mCherry, a GASPAG-Linker (BBa_K3187038), a Strep-Tag II (BBa_K3187025) for Purification, a T7 promoter with lac-operator and an RBS (BBa_K3187029), a T7TE terminator (BBa_K3187032), a GGGG-sequence, a Start-Codon (BBa_J70593) and a Stop-Codon (BBa_K2868029)(). The coding sequence consists of 851 bp which are translated to 260 amino acids.[1]

Methods

Purification

The GGGG-mCherry was heterologously expressed in E. coli BL21 and purified with GE Healthcare ÄKTA Pure machine which is a machine for FPLC. The used affinity tag was Strep-tagII.

SDS-Page and Western blot

To verify that the CP-LPETGG was produced, a SDS-Page SDS-Page followed by a Western blot was performed.

Results

Cloning and Expression

The successful cloning was proven with sanger sequencing and production with a Western blot.

Figure 1: Western blot of all produced and purified proteins.

Fig. 1 shows that the band of the GGGG-mCherry is approximatley found by the 28.5 kDa band. Consequently, the successful protein production was proven.CP-LPETGG is detected with Strep-Tactin-HRP.

References

  1. ↑ Nathan Shaner, Robert Campbell, Paul Steinbach, Ben Giepmans, Amy Palmer and Roger Tsien, Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein, Nature Biotechnology, 2004, 22: 1567-1572 [1]
Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 854
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 854
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 915
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 854
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 854
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None