Part:BBa_K4385019

Copper-inducible surface display

This part is composed of CusSR two-component signal transport system, PcusC and OmpC-CBP.

Usage and Biology

Promoter PCusC can express downstream genes under the induction of Cu2+. When the zinc ion concentration in the environment exceeds a certain concentration, the PcusC promotes the transcription of downstream OmpC-CBP, and realizes the adsorption of zinc ions in the environment. The 6*His Tag was added to this sequence to purify the recombinant protein.

Characterization

Therefore, PCusC-OmpC-CBP-ST was constructed so that the OmpC-CBP would be expressed only the presence of copper ions in the environment.

Figure 1.PCusC-OmpC-CBP-ST.

To verify the construction of the PCusC-OmpC-CBP-ST, the enzyme digestion and agarose gel electrophoresis were performed (Fig. 2A). After 4 hrs of induction with 0.5, 1.0, 2.0 mM CuSO4 respectively, the concentration of Cu2+ in the supernatant was measured. The result also shows that our engineered bacteria have better capacity of copper absorption (Fig. 2B)

Figure 2. Validation of the absorption of copper ions with PCusC.(A) Digestion and gel electrophoresis of PCusC-OmpC-CBP-ST. (B) The measurement of the concentration of Cu2+ in the supernatant. The Cu2+ 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 535
  Illegal XbaI site found at 28
  Illegal SpeI site found at 694
  Illegal PstI site found at 1042
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 535
  Illegal NheI site found at 1410
  Illegal NheI site found at 1433
  Illegal SpeI site found at 694
  Illegal PstI site found at 1042
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 535
  Illegal BglII site found at 243
  Illegal BglII site found at 1545
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 535
  Illegal XbaI site found at 28
  Illegal SpeI site found at 694
  Illegal PstI site found at 1042
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 535
  Illegal XbaI site found at 28
  Illegal SpeI site found at 694
  Illegal PstI site found at 1042
  Illegal AgeI site found at 361
  
• 1000
  COMPATIBLE WITH RFC[1000]