Composite
PcusC-GFP

Part:BBa_K4385011

Designed by: Min An Zhao   Group: iGEM22_Jilin_China   (2022-10-01)


Lead sensor

The lead sensor is composed of lead-inducible promoter and GFP.


Usage and Biology

When there is no lead ion in the environment, PbrR protein is expressed in the presence of Pbr constitutive promoter and binds to its downstream inducible promoter Pbc to repress GFP expression; when lead ion reaches a certain concentration in the environment, PbrR protein chelates lead ion so that it cannot bind to the inducible promoter Pbc and downstream GFP can be expressed and the engineered bacteria can see green fluorescence.

Characterization

To verify the sensitivity of Pb2+-inducible promoter, a GFP was connected to report the expression intensity. Pbr-PbrR-Pbc-GFP was constructed to pSB1C3.

Figure 1.Pbr-PbrR-Pbc-GFP.


Different concentrations of Pb2+ were added to the reaction system, and the response of the promoter were reflected by detecting the change of fluorescence intensity. As shown in Fig. 3B, the expression of Pb2+-inducible promoter was optimal under 100 μM Pb2+ after being induced 12 hrs. Moreover, the Pb2+ inducible promoter continues to be effective after 16hours of induction, which shows our system’s stability.

Figure 2.Duration of Pb2+ induction. (A) Digestion and gel electrophoresis of Pbr-PbrR-Pbc-GFP. (B) Fluorescence intensity of GFP under different concentration of Pb2+. The design construct was transformed into E. coli BL21(DE3). The Pb2+ was added when the OD600 value was reached 0.5. The fluorescence intensity was detected at indicated time and normalized by the OD600 value. *，P<0.05 compared to control using Student’s t test.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 462
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 462
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 462
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 462
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 253
    Illegal BsaI.rc site found at 1394


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Categories
Parameters
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