Composite

Part:BBa_K4385017

Designed by: Min An Zhao   Group: iGEM22_Jilin_China   (2022-10-06)


Zinc constitutive surface display

This part consists of the constitutive promoters J23100, OmpC (BBa_K4385003) and ZBP (BBa_K4385004), which is used to verify the function of the zinc ion surface display parts. The 6*His tag was added to this sequence to purify the recombinant protein.


Usage and Biology

OmpC-ZBP can be anchored on the surface of E. coli cell membrane and enhance its absorption capacity of zinc.

Characterization

The constitutive promoter PJ23100 was placed upstream of OmpC-ZBP to verify its function (Fig. 1A). To verify the construction of PJ23100-OmpC-ZBP-ST, the enzyme digestion and gel electrophoresis were performed (Fig. 1B). The concentration of zinc was measured by Micro Serum Zinc Concentration Assay Kit. As shown in Fig. 1C, the concentration of zinc ions in the supernatant is lower than that of the control. And the absorption capacity of cells is higher than that in the control, which indicate that our engineered bacteria can better absorb zinc ions.

Figure 1.Validation of the absorption of Zn2+.(A) Digestion and gel electrophoresis of PZraP-OmpC-ZBP-ST (B) The measurement of the concentration of Zn2+ in the supernatant. The Zn2+ was added when the OD600 value of bacteria reached 0.5. The concentration in supernatant was measured after 4 hrs of induction and normalized by the same OD600 value. *，P<0.05 from control group. △，P<0.05 from respective control.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 414
    Illegal SpeI site found at 573
    Illegal PstI site found at 921
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 414
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 1304
    Illegal NheI site found at 1327
    Illegal SpeI site found at 573
    Illegal PstI site found at 921
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 414
    Illegal BglII site found at 122
    Illegal BglII site found at 1439
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 414
    Illegal SpeI site found at 573
    Illegal PstI site found at 921
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 414
    Illegal SpeI site found at 573
    Illegal PstI site found at 921
    Illegal AgeI site found at 240
  • 1000
    COMPATIBLE WITH RFC[1000]


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