Coding

Part:BBa_K5466017:Design

Designed by: Adrián Gómez Lara, Daniel Bulnes Roldán   Group: iGEM24_UMA-MALAGA   (2024-09-23)


Nb28-S102D Aga2P


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
    COMPATIBLE WITH RFC[1000]


Design Notes

Fusions of the N-terminus of scFv and sdAb may interfere with antigen binding so we decided to fuse the sdAb C-terminal with the Aga2p N-terminal, with a flexible linker.

AG was added to the C-terminus based on findings from Wang et al. (2005). After cloning the scFv insert directly between the Aga2p leader sequence and the Aga2P protein, the fusion protein was not properly cleaved, resulting in no detectable signal. It was later discovered that, without the scFv insert, the EF dipeptide—derived from the EcoRI recognition site used to fuse the desired displayed protein with Aga2P—was positioned at the terminus of the Aga2 signal peptide, which interfered with proper cleavage. Previous studies indicated that the +1 and +2 amino acids at the N-terminus of the fusion protein are critical for signal peptidase processing. Several sequences (EF, EA, EAEA, and AG) were tested, all of which successfully induced display. AG, being neutral and minimally bulky, was chosen as the default peptide spacer.


S. cerevisiae codon optimized.

Source

Source in each individual part.