Part:BBa_K4307014

nisABC

Nisin is a small peptide and it has been synthesized in bacteria before in literatures. In addition, some literatures have shown that the precursor of nisin, NisA, may have the function of inducing the two-component system after modified by NisB and NisC.

This time, We refer to the previous method of synthesizing nisin in E.coli, and construct the fusion protein of nisA and affibody Z_EGFR:2377 to recognize EGFR. According to the protein sequence information provided in UniProt, we commissioned a biotechnology company to optimize the codon, artificially synthesized NisA-affi containing 6His in C terminal, nisB and nisC sequences and inserted them into the pET28a vector with kanamycin resistance and chloramphenicol resistance respectively. Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 3418
  Illegal PstI site found at 1068
  Illegal PstI site found at 3234
  Illegal PstI site found at 3333
  Illegal PstI site found at 4170
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1068
  Illegal PstI site found at 3234
  Illegal PstI site found at 3333
  Illegal PstI site found at 4170
  Illegal NotI site found at 1774
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1076
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 3418
  Illegal PstI site found at 1068
  Illegal PstI site found at 3234
  Illegal PstI site found at 3333
  Illegal PstI site found at 4170
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 3418
  Illegal PstI site found at 1068
  Illegal PstI site found at 3234
  Illegal PstI site found at 3333
  Illegal PstI site found at 4170
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The following figure demonstrates our successful construction.

               Figure 1:  The construction results of NisA-affi-NisB+NisC.
               
           

Western blot was done to characterize the biobrick.

To ensure the expression of NisA-affi, we managed to transform the two plasmids into BL21(DE3). After it, we induced the production of nisA-affibody and used Ni column to purify the bacteria lysate. Western blots assay targeting 6xHis tag was conducted and nisA-affibody expression was detected(Figure 2).

               Figure 2:   Western blot results to detect NisA-affibody expression.
               

Flow cytometry was done to characterize the biobrick.

To measure the effectiveness of NisA-affi, we use it to induce expression of EGFP in pNisin2. If NisA-affi can function normally, the expression of downstream EGFP can be detected after addition of it.
We conducted flow cytometry for 4h induction bacteria(Figure 3) to detect the expression of EGFP under induced and control conditions. Flow cytometry result shows positive percentage of experimental group induced by 2μl NisA-affi is higher than control group, which means NisA-affi can act as the inducer of nisin TCS similar to nisin.

               Figure 3:   Flow cytometry results of pNisin2 induction using NisA-affibody.
               
          

Conclusion

We successfully expressed the NisA-affi in E.coli, and the results of the flow cytometry showed that NisA-affi could perform the same function of inducing Nisin TCS as Nisin. In addition, the fusion protein can play a role of detecting other proteins by replacing affibody ZEGFR:2377 into their ligands.