Composite

Part:BBa_K2332040:Design

Designed by: Paola Handal   Group: iGEM17_UCL   (2017-10-20)
Revision as of 23:27, 23 October 2017 by Paola handal (Talk | contribs) (→‎Design Notes)


Blue light inducible expression of Intimin'-GFP-SpyTag (Pblind Intimin'-GFP-SpyTag)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 2844
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 2844
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 2844
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 2844
    Illegal NgoMIV site found at 880
    Illegal NgoMIV site found at 1621
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2715


Design Notes

We decided to use Intimin' as this has been previously proven to function effectively for cell surface display of up to 30kDa passenger proteins (Wentzel et al., 2001). We included a linker between Intimin' and GFP and between GFP and SpyTag to facilitate mobility of GFP and SpyTag on the cell surface. The stop codon in GFP was removed for the generation of this fusion protein. Pblind promoter was designed by Jayaraman P. et al. (2016)

Source

Intimin protein sequence obtained from: http://www.uniprot.org/uniprot/P43261#sequences And reverse translated using: http://www.bioinformatics.org/sms2/rev_trans.html Intimin for the cell surface display of our tag was truncated according to Wentzel et al., 2001 SpyTag: BBa_K1159201 GFPmut 3b: Part:BBa_E0040


References