Difference between revisions of "Part:BBa K2332040:Design"

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===Design Notes===
 
===Design Notes===
−
Removal of ATG from GFP for a more efficient expression of this fusion protein
+
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===
 
===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
+
Intimin protein sequence (E. coli) obtained from UniProtKB: [http://www.uniprot.org/uniprot/P43261#sequences P43261], reverse translated and codon optimised for E. coli.
−
 
+
Intimin for the cell surface display of our tag was truncated according to Wentzel et al., 2001.
 +
SpyTag was obtained from: [https://parts.igem.org/Part:BBa_K1159201 BBa_K1159201].
 +
GFPmut3b was obtained from: [https://parts.igem.org/Part:BBa_E0040 BBa_E0040].
 +
The DNA sequence was synthesised by Integrated DNA Technologies (IDT)
  
 
===References===
 
===References===
 +
1. Jayaraman P, Devarajan K, Chua T, Zhang H, Gunawan E, Poh C. Blue light-mediated transcriptional activation and repression of gene expression in bacteria. Nucleic Acids Research. 2016;44(14):6994-7005.
 +
 +
2. Zakeri B, Fierer J, Celik E, Chittock E, Schwarz-Linek U, Moy V et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences. 2012;109(12):E690-E697.
 +
 +
3. Wentzel A, Christmann A, Adams T, Kolmar H. Display of Passenger Proteins on the Surface of Escherichia coli K-12 by the Enterohemorrhagic E. coli Intimin EaeA. Journal of Bacteriology. 2001;183(24):7273-7284.

Latest revision as of 23:29, 23 October 2017


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 (E. coli) obtained from UniProtKB: [http://www.uniprot.org/uniprot/P43261#sequences P43261], reverse translated and codon optimised for E. coli. Intimin for the cell surface display of our tag was truncated according to Wentzel et al., 2001. SpyTag was obtained from: BBa_K1159201. GFPmut3b was obtained from: BBa_E0040. The DNA sequence was synthesised by Integrated DNA Technologies (IDT)

References

1. Jayaraman P, Devarajan K, Chua T, Zhang H, Gunawan E, Poh C. Blue light-mediated transcriptional activation and repression of gene expression in bacteria. Nucleic Acids Research. 2016;44(14):6994-7005.

2. Zakeri B, Fierer J, Celik E, Chittock E, Schwarz-Linek U, Moy V et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences. 2012;109(12):E690-E697.

3. Wentzel A, Christmann A, Adams T, Kolmar H. Display of Passenger Proteins on the Surface of Escherichia coli K-12 by the Enterohemorrhagic E. coli Intimin EaeA. Journal of Bacteriology. 2001;183(24):7273-7284.