Coding

Part:BBa_K2332032:Design

Designed by: Paola Handal   Group: iGEM17_UCL   (2017-10-19)


GFP-mutSpyCatcher (Lys31X, for photocaging)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 662


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 removed the stop codon in GFP and placed the SpyCatcher sequence after a linker sequence and included a HisTag for protein purification.

Source

SpyCatcher sequence was obtained from BBa_K1159200 and GFP from: BBa_E0040. 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. Mitra N. Incorporating Unnatural Amino Acids into Recombinant Proteins in Living Cells. Materials and Methods. 2013;3.

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.