Generator

Part:BBa_K3161003

Designed by: Shan Dong   Group: iGEM19_XHD-WS-Wuhan-B   (2019-06-18)
Revision as of 13:47, 21 October 2019 by Zhai 0391 (Talk | contribs)


Pc-RBS-PbrR-W

PbrR protein expressed under the control of weak Anderson promoter J23117


Usage and Biology

Aim of experiment

PbrR is a lead-binding protein in the form of dimer. In the presence of lead ions, Pb2+ binds to the PbrR protein dimer, resulting in a conformational change that causes lead sensitive promoters to begin transcription. In order to design biosensor that can detect different concentration of lead threshold, we designed a series of parts expressing PbrR protein under the control of different strength constitutive promoter, including strong promoter J23100, medium promoter J23106 and weak promoter J23117.

Results

PbrR-W (K3161003) was expressed under the control of weak Anderson promoter J23117. We measured the protein expression of three kinds of PbrR, and the results were as follows As can be seen from figure 6, PbrR protein expressed under different strengths of constitutive promoters J23100(S), J23106 (M) and J23117 (W). The order of expression intensity is S>M>W. This also explains why biosensors constructed in S, M and W have different tolerance to lead. As shown in figure 2, biosensors containing PbrR-S were more resistant to Pb2+, while biosensors composed of PbrR-M (K3161002) or PbrR-W(K3161003) were less resistant to high concentrations of lead (5ppm and 10ppm), indicating that PbrR expression played a role in the adsorption of lead by cells.

T--XHD-WS-Wuhan-B--W.jpeg

Fig1. PbrR protein expression under different strength of Anderson promoters in SDS-PAGE. S, strong Anderson promoter J23100, M, medium promoter, J23106, W, weak promoter J23117.

T--XHD-WS-Wuhan-B--growth.png

Fig2. Effect of PbrR protein expression under the control of different strength of promoters on bacteria resistance to lead. S, strong Anderson promoter J23100, M, medium promoter, J23106, W, weak promoter J23117.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None