Reporter

Part:BBa_K3945010:Design

Designed by: Anika Zaman   Group: iGEM21_Calgary   (2021-10-20)


BRET: A fluorescence-based measurement system for rare earth elements


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 811
    Illegal NgoMIV site found at 1150
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Previous experiments using a LanM-based FRET system were done in the past, where the authors established a certain R0,(Forster distance) at which the system worked [3]. Forster distance refers to the distance at which BRET undergoes 50% of resonance energy transfer [2]. As that paper demonstrated a successful signal, we decided to utilize a BRET system with a similar R0. The mCherry and NanoLuc combination was shown to have the closest R0 out of a number of BRET systems modelled by Weihs et al, which is why it became our chosen system [3].

As physical distance plays such a large role in the success of this system, linkers between LanM and the BRET proteins were modelled. This was done to ensure that the proteins wouldn’t interact when LanM was in an unbound state, but would fall within the minimum distance required when LanM was bound and folded. Furthermore, we needed to ensure that the presence of the BRET proteins would not interfere with the folding and unfolding mechanisms of LanM. This was determined through molecular docking simulations. The linkers used for the BRET system provided the optimized values for reaching the R0, as well as provided the best physical distance between BRET components and LanM.

According to previous studies, the signal output of the BRET system changes depending on the protein-terminus the luminophore and fluorophore are fused to [2]. As such, in order to increase our signal output, the mCherry was fused to the N-terminus of LanM and NanoLuc to the C-terminus.

Source

The sequence was obtained from the genome of Methylobacterium extorquens and the NanoBIT sequence was obtained from Promega. The mCherry sequence was obtain from genome of Discosoma