Part:BBa_K2332040:Design
Blue light inducible expression of Intimin'-GFP-SpyTag (Pblind Intimin'-GFP-SpyTag)
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 2844
- 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 2844
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 2844
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 2844
Illegal NgoMIV site found at 880
Illegal NgoMIV site found at 1621 - 1000INCOMPATIBLE 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.