Difference between revisions of "Part:BBa K2332025:Design"

 
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===Design Notes===
 
===Design Notes===
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Amberless E. coli must be used to see the effects of photocageing spyCatcher
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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.
  
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===Source===
  
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Intimin protein sequence obtained from UniProtKB: [http://www.uniprot.org/uniprot/P43261#sequences P43261], reverse translated and codon optimised for E. coli.
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Intimin for the cell surface display of our tag was truncated according to Wentzel et al., 2001.
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SpyCatcher was obtained from: [https://parts.igem.org/Part:BBa_K1159200 BBa_K1159200]. The DNA sequence was synthesised by Integrated DNA Technologies (IDT)
  
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===References===
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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.
  
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===Source===
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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.
  
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SpyCatcher: BBa_K1159200
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3. Mitra N. Incorporating Unnatural Amino Acids into Recombinant Proteins in Living Cells. Materials and Methods. 2013;3.
  
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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.
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===References===
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Latest revision as of 00:10, 23 October 2017


Intimin'-mutSpyCatcher (Lys31X, for photocaging)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 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

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.