Coding

Part:BBa_K5477014

Designed by: Kate Malana Escobar   Group: iGEM24_UCopenhagen   (2024-09-26)
Revision as of 16:12, 26 September 2024 by Kateesc1700 (Talk | contribs)


LexA-mERα Chimeric activator with LexA DNA binding domain fused with the mutant ERa

LexA-mERα Chimeric activator with LexA DNA binding domain fused with the mutated ligand-binding domain of Estrogen receptor alpha Full Description: The LexA-mERα (LexA-mutant Estrogen Receptor Alpha) encoding for the chimeric activator where the ligand-binding domain (LBD) of the Estrogen Receptor alpha (ERα) has been mutated. Like the wild-type version, it combines the DNA-binding domain (DBD) of the LexA with the mutated LBD of ERα, designed to bind specific ligands—in this case, modified to potentially alter ligand specificity or binding efficiency. The LexA DBD remains responsible for binding to LexA operator sequences (Lex6Op) in the promoter region, while the mutant LBD (mERα) is responsible for recognizing and responding to specific estrogen-like molecules or analogs, such as bisphenol A (BPA), with altered dynamics compared to the wild-type ERα.

table-bpa-estrogen.png

The figure shows the limits of detection (LOD) and half-maximal effective concentrations (EC50) for both wild-type (WT) and various mutant versions of the ERα receptor in response to bisphenol A (BPA) and 17β-estradiol. The mutant P4E C8 demonstrates high sensitivity to BPA, with a low EC50 (1.1 x 10-6 M) and LOD (2.7 x 10-7 M), making it highly responsive to BPA while showing minimal sensitivity to 17β-estradiol (EC50 > 900 nM). This mutant is ideal for specific BPA detection.

era-mutants-alignment.png

From the figure above which is from the paper of Rajasärkkä et al. 2011, the mutant P4E C8 mutant version of ERα demonstrates a high sensitivity to bisphenol A (BPA) with a low EC50 value of 1.1 x 10-6 M, indicating it is more responsive to BPA compared to other variants. Additionally, it shows low sensitivity to 17β-estradiol, with an EC50 > 900 nM, demonstrating that this mutant is effective for detecting BPA while minimizing interaction with its native ligand, estradiol.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 900
    Illegal PstI site found at 1084
    Illegal PstI site found at 1255
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1084
    Illegal PstI site found at 1255
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1034
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 900
    Illegal PstI site found at 1084
    Illegal PstI site found at 1255
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 900
    Illegal PstI site found at 1084
    Illegal PstI site found at 1255
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 934


References

Rajasärkkä, J., Hakkila, K. and Virta, M. (2011), Developing a compound-specific receptor for bisphenol a by directed evolution of human estrogen receptor ᆇ. Biotechnol. Bioeng., 108: 2526-2534. https://doi.org/10.1002/bit.23214 Zhou, T., Liang, Z. & Marchisio, M.A. Engineering a two-gene system to operate as a highly sensitive biosensor or a sharp switch upon induction with β-estradiol. Sci Rep 12, 21791 (2022). https://doi.org/10.1038/s41598-022-26195-x

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