Difference between revisions of "Part:BBa K2332015:Design"
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===References=== | ===References=== | ||
| − | 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. | + | 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. |
| − | 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. | + | 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. |
| − | Mitra N. Incorporating Unnatural Amino Acids into Recombinant Proteins in Living Cells. Materials and Methods. 2013;3. | + | 3. Mitra N. Incorporating Unnatural Amino Acids into Recombinant Proteins in Living Cells. Materials and Methods. 2013;3. |
| − | Wang Y, Wu B, Wang Z, Huang Y, Wan W, Russell W et al. A genetically encoded photocaged Nε-methyl-l-lysine. Molecular BioSystems. 2010;6(9):1557. | + | 4. Wang Y, Wu B, Wang Z, Huang Y, Wan W, Russell W et al. A genetically encoded photocaged Nε-methyl-l-lysine. Molecular BioSystems. 2010;6(9):1557. |
Latest revision as of 22:41, 22 October 2017
Intimin'-mutSpyCatcher (Lys31X, for photocaging)
- 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 819
Illegal NgoMIV site found at 1560 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
This construct was designed to not contain any other amber stop codon but at position 31 (Lys31X) (Amberless E. coli must be used to see the effects of photocageing spyCatcher). 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.
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. Mitra N. Incorporating Unnatural Amino Acids into Recombinant Proteins in Living Cells. Materials and Methods. 2013;3.
4. Wang Y, Wu B, Wang Z, Huang Y, Wan W, Russell W et al. A genetically encoded photocaged Nε-methyl-l-lysine. Molecular BioSystems. 2010;6(9):1557.