Difference between revisions of "Part:BBa K2332011:Design"

 
(→‎Design Notes)
 
(3 intermediate revisions by the same user not shown)
Line 7: Line 7:
  
 
===Design Notes===
 
===Design Notes===
−
We decided to fuse SpyCatcher to the N-terminal of a truncated version of Intimin as this has been previously proven to function effectively for cell surface display of up to 30kDa passenger proteins
+
We decided to fuse SpyTag to the N-terminal of a truncated version of 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 also included a linker between Intimin and SpyTag to facilitate mobility of SpyTag on the cell surface.
−
 
+
Blue light inducible promoter was designed by Jayaraman P. et al. (2016)
−
 
+
  
 
===Source===
 
===Source===
  
−
All parts were obtained as synthetic gBlocks
+
Intimin protein sequence obtained from UniProtKB: [http://www.uniprot.org/uniprot/P43261#sequences P43261], reverse translated and codon optimised for E. coli.
−
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
+
Intimin for the cell surface display of our tag was truncated according to Wentzel et al., 2001.
−
Pblind:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5001607/
+
SpyTag was obtained from: [https://parts.igem.org/Part:BBa_K1159201 BBa_K1159201]. The DNA sequence was synthesised by Integrated DNA Technologies (IDT)
  
 
===References===
 
===References===
 +
1. 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.
 +
 +
2. 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.
 +
 +
3. 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.

Latest revision as of 23:59, 22 October 2017


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 880
    Illegal NgoMIV site found at 1621
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We decided to fuse SpyTag to the N-terminal of a truncated version of 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 also included a linker between Intimin and SpyTag to facilitate mobility of SpyTag on the cell surface. Blue light inducible promoter was designed by Jayaraman P. et al. (2016)

Source

Intimin protein sequence 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. The DNA sequence was synthesised by Integrated DNA Technologies (IDT)

References

1. 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.

2. 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.

3. 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.