Difference between revisions of "Part:BBa K4779001"

 
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This is a copper ion-responsive signaling pathway in Saccharomyces cerevisiae based on MAPK system. When the cells were induced by copper ions, the copper ion-inducible promoter cup1 expresses MFα2, generating pheromones. Through the amplification of pheromones signal pathway, the pprm1pro promoter was activated to express GFP, thereby reflecting the concentration of copper ions.
 
This is a copper ion-responsive signaling pathway in Saccharomyces cerevisiae based on MAPK system. When the cells were induced by copper ions, the copper ion-inducible promoter cup1 expresses MFα2, generating pheromones. Through the amplification of pheromones signal pathway, the pprm1pro promoter was activated to express GFP, thereby reflecting the concentration of copper ions.
  
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===Construction===
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===Usage and Biology===
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CMPG pro is a new composite part (BBa_K4779001). We obtained the gene sequences of the promoter of Cup1 (BBa_K945002), MFα2 (BBa_K110005), the promoter of prm1 pro (BBa_K3384313), green fluorescent protein (GFP) (BBa_K3112009), and the terminator CYC1 (BBa_K4278703) from IGEM, and sent them to GENEWIZ (Suzhou genewiz Biotechnology Co., Ltd.) for synthesis.
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<p> </p>
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<div>
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https://static.igem.wiki/teams/4779/wiki/nanjing-bioxstem-cmpg-pro.png
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</div>
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===Characterization===
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We subjected the engineered yeast strain BY4741-pRS415-CMPG Pro samples to a copper ion gradient treatment and subsequently conducted quantitative fluorescence measurements using flow cytometry, as illustrated in the accompanying figure. Within the range of 300 uM, we observed an increase in the signal output intensity of the sensor as the copper ion concentration rose, resulting in an expanded detection range compared to CMPG. Specifically, the signal output intensity of CMPG Pro, when exposed to copper ions at 300 uM, showed an 18.25% enhancement relative to the untreated state, which is 1.3 times greater than that of CMPG at 200 uM. This observation highlights an improved efficacy in terms of sensitivity.
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<p> </p>
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https://static.igem.wiki/teams/4779/wiki/nanjing-bioxstem-cmpg-pro2.png
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</div>
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Latest revision as of 19:05, 11 October 2023


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

This is a copper ion-responsive signaling pathway in Saccharomyces cerevisiae based on MAPK system. When the cells were induced by copper ions, the copper ion-inducible promoter cup1 expresses MFα2, generating pheromones. Through the amplification of pheromones signal pathway, the pprm1pro promoter was activated to express GFP, thereby reflecting the concentration of copper ions.

Construction

CMPG pro is a new composite part (BBa_K4779001). We obtained the gene sequences of the promoter of Cup1 (BBa_K945002), MFα2 (BBa_K110005), the promoter of prm1 pro (BBa_K3384313), green fluorescent protein (GFP) (BBa_K3112009), and the terminator CYC1 (BBa_K4278703) from IGEM, and sent them to GENEWIZ (Suzhou genewiz Biotechnology Co., Ltd.) for synthesis.


nanjing-bioxstem-cmpg-pro.png

Characterization

We subjected the engineered yeast strain BY4741-pRS415-CMPG Pro samples to a copper ion gradient treatment and subsequently conducted quantitative fluorescence measurements using flow cytometry, as illustrated in the accompanying figure. Within the range of 300 uM, we observed an increase in the signal output intensity of the sensor as the copper ion concentration rose, resulting in an expanded detection range compared to CMPG. Specifically, the signal output intensity of CMPG Pro, when exposed to copper ions at 300 uM, showed an 18.25% enhancement relative to the untreated state, which is 1.3 times greater than that of CMPG at 200 uM. This observation highlights an improved efficacy in terms of sensitivity.

nanjing-bioxstem-cmpg-pro2.png


Sequence and Features


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 2181
    Illegal SapI site found at 529