Device

Part:BBa_K5477048:Design

Designed by: Kate Escobar   Group: iGEM24_UCopenhagen   (2024-09-30)


SUPERMOM: Dual-Function Biosensor for Detection and Detoxification


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1606
    Illegal XbaI site found at 4766
    Illegal PstI site found at 1790
    Illegal PstI site found at 1961
    Illegal PstI site found at 5712
    Illegal PstI site found at 5909
    Illegal PstI site found at 6293
    Illegal PstI site found at 6733
    Illegal PstI site found at 6793
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1606
    Illegal XbaI site found at 4766
    Illegal PstI site found at 1790
    Illegal PstI site found at 1961
    Illegal PstI site found at 5712
    Illegal PstI site found at 5909
    Illegal PstI site found at 6293
    Illegal PstI site found at 6733
    Illegal PstI site found at 6793
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1606
    Illegal XbaI site found at 4766
    Illegal PstI site found at 1790
    Illegal PstI site found at 1961
    Illegal PstI site found at 5712
    Illegal PstI site found at 5909
    Illegal PstI site found at 6293
    Illegal PstI site found at 6733
    Illegal PstI site found at 6793
    Illegal NgoMIV site found at 6115
    Illegal NgoMIV site found at 6234
    Illegal AgeI site found at 37
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Receptor Module

pRET2-LexA-mERα(LBD) | BBa_K5477029: This receptor module includes the pRET2 promoter, driving the expression of a LexA-mERα(LBD) fusion protein. The LexA DNA-binding domain (DBD) is fused to the ligand-binding domain (LBD) of a mutant Estrogen Receptor Alpha (mERα), which is engineered to exhibit high specificity towards BPA and related compounds. Upon ligand binding, the LexA-mERα(LBD) fusion protein undergoes a conformational shift, enabling it to bind to Lex6Op sequences in the reporter and detox modules, thus initiating the transcriptional response. Reporter Module

Lex6Op-pLEU2-NanoLuc | BBa_K5477031: The reporter module contains Lex6Op operator sequences, which serve as binding sites for the activated LexA-mERα(LBD) protein. Binding of LexA to these sequences activates the pLEU2 promoter, leading to the expression of NanoLuc luciferase. NanoLuc, a highly efficient luciferase, emits bioluminescence in the presence of its substrate. The intensity of this signal correlates directly with the concentration of the ligand (BPA or similar compounds) bound to the mERα receptor, enabling real-time detection. Detoxification Module

CYP3A4-MYC-pGAL1/10-POR | BBa_K5477039: The detoxification module incorporates the cytochrome P450 enzyme CYP3A4, which plays a key role in the oxidative metabolism of various EDCs, including BPA. CYP3A4 is regulated by the pGAL1/10 bidirectional promoter and is fused with a MYC tag for detection. The co-expression of NADPH-cytochrome P450 reductase (POR), driven by the same promoter, ensures efficient electron transfer to CYP3A4, facilitating its catalytic activity. This module enables the oxidation and detoxification of BPA, converting it into more hydrophilic and excretable metabolites, reducing its harmful effects.


Source

Homo sapiens and Saccharomyces cerevisiae

References