Composite

Part:BBa_K3187010

Designed by: iGEM TU_Darmstadt 2019   Group: iGEM19_TU_Darmstadt   (2019-10-12)
Revision as of 19:48, 16 October 2019 by Franziska (Talk | contribs)


TEV Cleavage Site x GGGG-Tag for Sortase-mediated Ligation X mCherry Fluorescence Protein

Profile

Name TVMV-GGGG-mCherry
Base pairs 1052
Molecular weight 29.4 kDa
Origin synthetic, derived from Discosomasp.
Parts mCherry, GGGG-Sequence, TVMV site, T7 Promoter, lac Operator, RBS, araBAD promoter + RBS, GASPAG Linker, Strep-Tag II, Double Terminator for pDEStara2
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. In order to remove the first methionine in front of the GGGG-Sequence a TEVMV-restriction site is cloned in the sequence. By removing the first methionine the linkage of LPETGG and GGGG-Sequences should work better. We use mCherry as an easily imaged reporter for checking if the coupling worked.
The coding sequence was cloned in pDEStara2 vector, containing the sequence of mCherry, a GGGG-sequence, a TVMV restriction site (BBa_K3187020), 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 Start-Codon (BBa_J70593) and a Stop-Codon (BBa_K2868029). Since pDEStara2 is a vector for dual expression it also contains an araBAD promoter with an RBS (BBa_K3187041) and a Double Terminator (BBa_K3187042). The coding sequence consists of 851 bp which are translated to 260 amino acids.[1]

Methods

Purification

The GGGG-mCherry with TEV Restrictionsite 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

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 XbaI site found at 324
    Illegal PstI site found at 1133
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1197
    Illegal PstI site found at 1133
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 239
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 324
    Illegal PstI site found at 1133
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 324
    Illegal PstI site found at 1133
    Illegal AgeI site found at 74
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 56


[edit]
Categories
Parameters
None