Coding

Part:BBa_K3809010:Design

Designed by: Francisco Javier Castaeda Villagran   Group: iGEM21_TecCEM   (2021-10-07)
Revision as of 14:47, 16 October 2021 by FCASTANEDA (Talk | contribs) (→‎Design Notes)


ESR1 with Linker, His Tag and Signal Peptide


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 48
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1264


Design Notes

The idea of the desing of this part was that if we could express this protein receptor, purify it and immobilize it, we could potentially use it to capture Endocrine Disruptive Compounds in a sample of water. In order to check if EDCs could bind to the receptor, we used molecular docking with the estrogen receptor obtained from PDB (code: 2IOG) and different chemicals that include estradiol, BPA, phenol, benzene and different pthalates. The result we obtained was adequate, since the binding constants of all the EDCs were similar or better to the binding constant of the natural ligand of the receptor (estradiol). We figured that we needed to design a way to purify and immobilize the protein after its expression. We therefore added a Signal Peptide Sequence to guarantee its secretion from E. coli; a His Tag to purify it using a Ni Affinity Column; and a linker sequence so that there would not be any problems in the folding of both domains. The linker sequence has an Cystein that could help us immobilize our protein in a functionilized surface. To verify that the receptor could fold with all the changes we made, we used a model prediction software and checked if there werer any structural changes compared to the original protein. Therefore, we made a structure alignment between our model and protein 2IOG from PDB which was the original sequence we used in the first place.

In order to express the protein in a bacterial system, we optimized the codons for E. coli using Benchling.

Source

The Signal Peptide sequence NPS4 was obtained from BBa_K3606042, the linker was a modified version of BBa_K1486004, with the addition of a cistein; ESR1 sequence was obtained from Homo sapiens estrogen receptor 1 (ESR1), transcript variant 1, mRNA, NCBI Reference Sequence: NM_000125.4.

References