Coding

Part:BBa_K3809010

Designed by: Francisco Javier Castañeda Villagran   Group: iGEM21_TecCEM   (2021-10-07)
Revision as of 14:54, 16 October 2021 by FCASTANEDA (Talk | contribs)


ESR1 with Linker, His Tag and Signal Peptide

This sequence codes for the Human Estrogen Receptor Alpha. It was modified in order to express the ligand binding domain and the DNA binding domain. The sequence also includes a 6X Histidine Tag (for its purification), a linker sequence of 4 glycines, 1 serine and 1 cistein (for its immobilization) and a signal peptide sequence (NPS4). This protein is a natural ocurring receptor found in human cells that acts as an enhacer protein which activates after its binding with its ligand. ESR1's natural ligand is Estradiol and after the molecule binds in its domain, it will undergo structural changes that activate the receptor by the formation of a dimer. It has been established that the most important aminoacids for the correct binding of estradiol and ESR1 are Gly521, His524, Leu525 and Met528.

For the purification of ESR1, we added a 6X His Tag and for the immobilization of the protein in a piezoelectric sensor, we added a linker which includes a sequence of 4 glycines, 1 serine and 1 cistein. The addition of the cistein is very important since the immobilization method we chose is based on the formation of a disulfide bond. Furthermore, we chose to mantain the DNA binding domain because it has a region rich in cisteins. Finally, we added a signal peptide sequence NPS4 (which comes from BBa_K3606042), in order to guarantee the secretion of our protein and make it easier for us to purify.

To verify that ESR1 would fold correctly and there would be no steric obstacle, we simulated the protein with its corresponding additions using the tool Robetta (https://robetta.bakerlab.org/). When we recieved the model, we made an structural alignment using the natural protein as reference with a PDB code 2IOG (Protein Data Bank, rcsb.org). Therefore, we concluded that the addition of the Linker, His Tag and Signal Peptide were not interfering with the correct folding of ESR1. Furthermore, we proved that the His Tag and the linker were exposed, so we could use them for the purposed we established previously.


Sequence and Features


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


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