Composite

Part:BBa_K4385014

Designed by: Min An Zhao   Group: iGEM22_Jilin_China   (2022-10-01)
Revision as of 03:13, 12 October 2022 by Minan (Talk | contribs) (→‎Characterization)


Lead constitutive surface display

Lead constitutive surface display consists of the constitutive promoter J23100, the lead-inducible regulatory protein PbrR and the anchoring protein Lpp-OmpA.


Usage and Biology

J23100 continuously expresses PbrR for lead ion adsorption, and Lpp-OmpA is used as an anchoring protein to anchor PbrR on the surface of E. coli to construct a whole-cell biosorption device for lead.

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 (in Fig.1)

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. 2B, 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 SpeI site found at 938
    Illegal PstI site found at 880
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 1120
    Illegal NheI site found at 1143
    Illegal SpeI site found at 938
    Illegal PstI site found at 880
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1255
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 938
    Illegal PstI site found at 880
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 938
    Illegal PstI site found at 880
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 671


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