Composite

Part:BBa_K4779000:Design

Designed by: Ziqi Mi   Group: iGEM23_Nanjing-BioXstem   (2023-09-26)


CMPG:A yeast copper-induced reporter pathway with prm1 promoter


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 279
    Illegal XbaI site found at 517
    Illegal SpeI site found at 343
    Illegal SpeI site found at 523
    Illegal PstI site found at 49
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 343
    Illegal SpeI site found at 523
    Illegal PstI site found at 49
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 55
    Illegal BamHI site found at 510
    Illegal XhoI site found at 8
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 279
    Illegal XbaI site found at 517
    Illegal SpeI site found at 343
    Illegal SpeI site found at 523
    Illegal PstI site found at 49
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 279
    Illegal XbaI site found at 517
    Illegal SpeI site found at 343
    Illegal SpeI site found at 523
    Illegal PstI site found at 49
    Illegal NgoMIV site found at 36
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2213
    Illegal SapI site found at 529


Design Notes

When designing the sequence in detail, one of the key considerations was how to convert copper ion signals into information molecule signals. We achieved this signal conversion through the pCup1 and MFα2 elements, and amplified the signals through a cascade amplification system based on the MAPK pathway. We leveraged the high sensitivity of the MAPK pathway to pheromone to enhance the system's sensitivity to copper ion concentration signals. Here, we placed the pheromone-responsive promoter (in this case, pprm1) in-frame with a yeast enhanced green fluorescent protein, thereby generating a measurable fluorescence signal and facilitating the quantification of copper ion concentrations.



Source

We obtained the gene sequences of the basic parts from IGEM, such as the promoter of Cup1 (BBa_K945002), MFα2 (BBa_K110005), the promoter of prm1 (BBa_K1346004), green fluorescent protein (GFP) (BBa_K3112009), and the terminator CYC1 (BBa_K4278703), and sent them to GENEWIZ (Suzhou Genewiz Biotechnology Co., Ltd.) for synthesis. The new composite part (CMPG) (BBa_K4779000) is also synthesized.

References