Composite

Part:BBa_K4385014

Designed by: Min An Zhao   Group: iGEM22_Jilin_China   (2022-10-01)
Revision as of 03:27, 12 October 2022 by Minan (Talk | contribs)

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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

Lpp-OmpA-PbrR can be anchored on the surface of E. coli to improve the adsorption ability of lead. Firstly, to determine the adsorption capacity of PbrR for lead ions, PJ23100 promoter was used (in Fig.1)

Figure 1.J23100-Lpp-OmpA-PbrR-J23100-SUMO-MTs.


To make sure the construction of our plasmid was successful, EcoR I and Spe I were used to digest the plasmid, we obtained two fragments of 2047 bp and 2110 bp by gel electrophoresis. The results confirmed that our construction of PJ23100-Lpp-OmpA-PbrR was correct (Fig. 2A).

We set up a control group and an experimental group to determine the adsorption capacity of engineered bacteria. The content of lead ions in the culture medium was determined by Amplite Immunofluorescence Kit.

The results showed that the lead ion content in the culture medium of all groups was lower than the initial concentration, while the lead ion content in the culture medium of the control group was higher than that of the experimental group，which indicates that the engineered bacteria can better absorb lead ions (Fig. 2B).

Figure 2.PbrR absorption of Pb2+. (A)Digestion and electrophoresis of PJ23100-Lpp-OmpA-PbrR-ST. (B)The results of the absorption. The residual lead ion concentration in the solution is calculated by a four-parameter logic curve calculator. (C)Standard curve obtained by four parameter logic curve calculators. *，P<0.05 from control group. △，P<0.05 from respective control.


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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