Composite

Part:BBa_K4385015

Designed by: Min An Zhao   Group: iGEM22_Jilin_China   (2022-10-04)
Revision as of 03:43, 12 October 2022 by Minan (Talk | contribs)

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Copper constitutive surface display

This part was mainly composed of constitutive promoter J23100, the anchoring protein OmpC-CBP and the heavy metal detoxification protein. Promoter J23100 continuously expressed downstream copper ion surface display proteins to verify the function of outer membrane protein C and lipoprotein of copper ion surface display system. The 6*His Tags were added to this sequence to purify the recombinant protein.


Usage and Biology

Metallothioneins (MTs) are a family of heavy metal detoxification protein. To counter the detrimental effect of heavy metal on our engineered bacteria, MTs will be continuously expressed in our engineered bacteria to increase their tolerance to heavy metals.Promoter J23100 continuously expressed downstream copper ion surface display proteins to verify the function of outer membrane protein C and lipoprotein of copper ion surface display system. The 6*His Tags were added to this sequence to purify the recombinant protein.

Characterization

To verify the effect of soluble MTs have on the cells’ metal tolerance abilities, two plasmids, PJ23100-OmpC-CBP and PJ23100-OmpC-CBP-PJ23100-ST，which was shorted for PJ23100-OmpC-CBP-ST, were constructed. E. coli harboring PJ23100-OmpC-CBP-ST and PJ23100-OmpC-CBP were subjected to medium containing different concentrations of copper ions. OD600 measurements were conducted to analyze the effects of ST on bacterial growth. As shown in Fig. 1C, ST enhanced the tolerance of bacteria in heavy metal solutions. Therefore, we added ST in all other composite parts of surface display system.

Figure 1.Validation of ST(A) The gene circuit. (B) Digestion and gel electrophoresis of two plasmids. (C) The growth curve of E. coli BL21(DE3). Cu2+ with gradient concentration is added to the culture medium, and OD600 value is detected at indicated time.


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 1289
    Illegal NheI site found at 1312
    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 1424
  • 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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