Composite

Part:BBa_K1769002

Designed by: Ho-Tsang Tsai   Group: iGEM15_NYMU-Taipei   (2015-09-14)

Salicylic acid sensing Oscillator

Inducible promoter,J61051,act as salicylic acid sensor, when the promoter is activated, it will produce luxI which in turn triggers the oscillator. GFP is the reporter of this circuit.

Usage and Biology

This sensor system consists of a negative feedback and positive feedback loop. The positive feedback loop is initiated when the inducible promoter, J61051, is induced by salicylic acid and produced protein LuxR. LuxR then forms a complex with AHL, a protein produced by LuxI. This complex upregulates the production of LuxI, Aiia, and GFP. However, once Aiia is matured, it will degrade AHL much faster then LuxI can produce and downregulate the production of GFP LuxI and Aiia itself. The production of Aiia will stop which allows the expression of Luxi to produce of AHL faster than Aiia can degrade.Moreover, AHL is a quorum sensing molecule which allows cells to synchronize thier production of GFP reporter.

Result

By adding different amount of salicylic acid, we want to test whether the oscillator will work. The GFP response is mearured by microplate reader every 15 minutes. After a overnight incubation, the bacteria were induced by different concentrations of salicylic acid(0, 10-6, 10-5, 10-4).


The oscillation of GFP exists even when there is no salicylic acid, this is maybe due to the leakiness of the J61051 inducible promoter. However, the amplitude and duration of the oscillation is much higher when the bacteria is iduced with 10-6 of salicylic acid. ALthough the production of GFP keeps building up, which makes the oscillation not so perfect, the oscillation is stable even after 500 minutes after induction.


In our project, we want to detect the salicylic acid produced by potatoes. From paper research, we learned that the salicylic acid produced by potatoes is about 10-6 M ~ 10-5 M. The oscillation with that concentration of salicylic acid is show on the picture above. The period of the oscillation is about 80 minutes

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 786
    Illegal BglII site found at 2938
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 77
    Illegal NgoMIV site found at 618
    Illegal AgeI site found at 3249
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2218
    Illegal BsaI.rc site found at 3087
    Illegal BsaI.rc site found at 4107
    Illegal BsaI.rc site found at 4837


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