Part:BBa_K4307007
NisK
Originated from Lactococcus lactis subsp. Lactis, NisK is a member of the two-component regulatory system NisK/NisR involved in the regulation of the biosynthesis of lantibiotic nisin. NisK functions as a membrane-associated protein kinase that phosphorylates NisR in response to environmental nisin signals. We managed to express NisK in E.coli strain.
Functions together with NisR(BBa_K4307008), PnisA(BBa_K4307009), J23100 and EGFP, it forms composite part J23100-nisK-nisR+PnisA-EGFP(BBa_K4307013) to express fluorescence signal induced by nisin.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization
The following figure demonstrates our successful construction.
<img width="70%">
Figure 1: The construction results of NisK.
Fluorescence location assay was done to characterize the biobrick.
To determine whether the constitutively expressed NisK in our system could locate on cell membrane correctly, we design NisK-EGFP fusion protein, expressed the protein in MACH1-T1 strain and examined its cellular location under confocal laser scanning microscope. The imaging result showed stronger fluorescence signal located on bacterial membrane, and fluorescence intensity measured by computer also showed the same distribution(Figure 2), indicating that most NisK-EGFP fusion proteins were located correctly on the membrane.
Figure 2: NisK localization under confocal laser scanning microscope.
Conclusion
According to the location assay data, it can be found that NisK can locate on membrane correctly in E.coli, which indicates that it can act as a membrane receptor to sense nisin signal functionally in E.coli as in Lactococcus lactis. This ensures the effectiveness of nisin TCS in E.coli
| None |