Part:BBa_K4307045

NisK-NisR-PnisA-EGFP

Consisting of inducer nisin, membrane receptor NisK, regulatory protein NisR and promoter PnisA activated by nisR, nisin TCS is an important signal transduction system in lactobacillus. After the extracellular nisin combining with NisK, nisK will phosphorylate the regulatory protein NisR, which activates the promoter PnisA and starts the expression of downstream genes.

Here, we constructed the J23100-nisK-nisR+PnisA-EGFP composite part with pFB20 backbone and transplanted it into E. coli to achieve our goal of detecting proteins. This composite part consists two basic parts, J23100-nisK-nisR and PnisA-EGFP. J23100-nisK-nisR play the role of constitutive expressing the membrane receptor NisK and the regulatory protein NisR, of which J23100 is a strong constitutive promoter. PnisA-EGFP plays the role of receiving activation signals from nisR and expressing the reporter gene EGFP.

It is worth mentioning that we are the first one to transform the NisK/R two-component system from lactobaccillus into E.coli and successfully characterize the function of it.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2934
  

Characterization

The following figure demonstrates our successful construction.

Figure 1: The construction results of J23100-nisK-nisR+PnisA-EGFP.

To measure the effectiveness of the nisin TCS, we added the gene of EGFP downstream of the promoter PnisA. If nisinTCS can function normally, the expression of downstream EGFP can be detected after addition of the inducer nisin.

Fluorescence spectrophotometry was done to characterize the biobrick.

We conducted 4-h induction assay at various nisin induction concentration, and results showed that 1-2 ng/ml nisin worked well in constitutive nisin induction system.

Figure 2: pNisin2 nisin 4-hour induction results at detailed induction concentration by fluorescence spectrophotometry.

Results from fluorescence microscope observation of 4-hour induction bacteria confirmed successful induction results as induction group showed stronger fluorescence signal.

Figure 3: pNisin2 nisin induction results under fluorescence microscope (100x).

Flow cytometry was done to characterize the biobrick.

We conducted flow cytometry for 4h induction bacteria(Figure 4) to detect the expression of EGFP under induced and control conditions. Flow cytometry result shows positive percentage of experimental group induced by 1ng/ml Nisin is higher than control group, which means successful fluorescence induction.

Figure 4: pNisin2 nisin induction results by flow cytometry.

Conclusion

Through the above verification, we proved that our modified nisin TCS can function normally in E.coli. It can sense extracellular protein signals, amplify them and transform them into expression of downstream gene. By changing the proteins fused with nisin, Our modified nisin TCS can also be used to detect different proteins, which makes it have potential to be used by other iGEM teams.