Part:BBa_K2332014:Design
Blue light inducible expression of Intimin'-SpyCatcher (Pblind Intimin'-SpyCatcher)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 898
Illegal NgoMIV site found at 1639 - 1000COMPATIBLE WITH RFC[1000]
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 (Wentzel et al., 2001). We also included a linker between Intimin and SpyCatcher to facilitate mobility of SpyCatcher on the cell surface and a HisTag for protein purification. 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. SpyCatcher was obtained from: BBa_K1159200. 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.