Composite

Part:BBa_K4779003

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


CMPS Pro:A yeast copper-induced reporter pathway with Positive feedback loop with prm1pro promoter

It is a copper ion reaction reporting pathway in Saccharomyces cerevisiae. We utilized the pprm1 Pro for optimizing GFP expression, and employed the prm1 promoter to express Ste5ΔN-CTM for positive feedback regulation in the yeast copper-induced reporting pathway.

Construction

CMPS Pro is a new composite part (BBa_K4779003). We obtained the gene sequences of the promoter of Cup1 (BBa_K945002), MFα2 (BBa_K110005), the promoter of prm1(BBa_K1346004), the promoter of prm1 pro(BBa_K3384313)，green fluorescent protein (GFP) (BBa_K3112009), Ste5ΔN-CTM(BBa_K3384315) and the terminator CYC1(BBa_K4278703) from IGEM, and sent them to GENEWIZ (Suzhou Genewiz Biotechnology Co., Ltd.) for synthesis.

nanjing-bioxstem-cmps-pro.png

Characterization

We subjected the engineered yeast strain BY4741-pRS415-CMPS Pro samples to a copper ion gradient treatment and quantitatively measured their fluorescence intensity at different copper ion concentrations, as depicted in the graph. Within 300 uM, the signal output intensity of the CMPS Pro sensor increased with an ascending copper ion concentration and nearly reached its peak at 50 uM. In comparison to CMPG, which reached its signal output peak at 200 uM, CMPS Pro achieved comparable signal output intensity at lower copper ion concentrations, indicating an improved sensitivity. Unexpectedly, replacing pprm1 with pprm1 Pro did not further enhance the sensor's sensitivity.


nanjing-bioxstem-cmps-pro2.png


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3138
    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 SpeI site found at 4393
    Illegal SpeI site found at 4939
    Illegal PstI site found at 49
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3138
    Illegal SpeI site found at 343
    Illegal SpeI site found at 523
    Illegal SpeI site found at 4393
    Illegal SpeI site found at 4939
    Illegal PstI site found at 49
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3138
    Illegal BglII site found at 55
    Illegal BglII site found at 3586
    Illegal BglII site found at 4894
    Illegal BamHI site found at 510
    Illegal XhoI site found at 8
    Illegal XhoI site found at 4831
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3138
    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 SpeI site found at 4393
    Illegal SpeI site found at 4939
    Illegal PstI site found at 49
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3138
    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 SpeI site found at 4393
    Illegal SpeI site found at 4939
    Illegal PstI site found at 49
    Illegal NgoMIV site found at 36
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2694
    Illegal SapI site found at 529


[edit]
Categories
Parameters
None