Part:BBa_K4307012

NisC

Improvement by 2023 iGEM Team FSHS-GD 2023

Summary

We have made improvements on the original components BBa_K4307011 and BBa_K4307012. We connected these two parts into the same plasmid pETduet (BBa_K4846011), then obtained the new functional part pETduet-NisB-NisC (BBa_K4846017). Throughout the design, we used T7 promoters (BBa_K3521000) to induce protein efficient expression using IPTG induced.

Figure 1 The plasmid map of NisB and NisC

Usage and Biology

Nisin is synthesized by a putative membrane-associated lantibiotic synthetase complex composed of the dehydratase NisB, the cyclase NisC, and the ABC transporter NisT in Lactococcus lactis. Earlier work has demonstrated that NisB and NisT are linked via NisC to form such a complex[1-3].

Construction Design

To achieve co-expression of the precursors and PTM enzymes, we designed two expression frames. One was T7-DarL-NisL-His-DarA-NisA-T7-DarE, where the first MCS contained DarL-NisL-His-DarA-NisA, and the second MCS contained the darobactin PTM enzyme DarE. DarL and NisL are leaders of DarA and NisA for recognition and binding by the PTM enzymes. The second expression frame was NisB-NisC, the PTM enzyme of NisA.

Through enzymatic digestion and ligation, we ligated the vector backbones pRSFduet and pETduet with the fragmentsNisB-NisC to construct pETduet-NisB-NisC.

Figure 2 The generation of the plasmids constructed.

Experimental Approach

After constructing the recombinant plasmid pRSFduet-DarL-NisL-His-DarA-NisA-DarE and pETduet-NisB-NisC, we co-transformed them into the same bacterial strain. plasmid pRSFduet-DarL-NisL-His-DarA-NisA-DarE was first transformed, and positive transformants were selected to prepare competent cells containing it, and plasmid pETduet-NisB-NisC was then transformed into it. We performed gel electrophoresis and sequencing to verify the successful transformation of two plasmids into E. coli.

Figure 3 Colony PCR and sequencing results of transformants containing both.

Characterization/Measurement

We inoculated the positive transformant and induced protein expression with IPTG. The bacterial cells were then lysed by sonication, and nickel purification was performed to obtain the fusion peptides at higher purity (Figure 4).

Figure 4 SDS-PAGE result of the protein expression and purification.

Compared to parts BBa_K4307011 and BBa_K4307012, we not only successfully amplified these NisB and NisC two gene fragments, but also linked them into the same vector to express the fusion protein. By combining with another plasmid pRSFduet-DarL-NisL-His-DarA-NisA-DarE, a fusion peptide of nisin and darobactin was formed.

Improved functional test

After obtaining the purified proteins, we performed in vitro cleavage experiments using lysyl endopeptidase to obtain the core peptide for subsequent testing in inhibition experiments. Finally, agar diffusion assays showed the cleaved DarA-NisA fusion peptide inhibited Bacillus subtilis (Gram-positive), but the antibacterial effect is not very significant (Figure 5).

Figure 5. Antibacterial effect on Bacillus subtilis

Discussion and Future plan

Perhaps it is because after expressing the fusion peptide, in vitro cleavage experiments are required, and the fusion peptide is not completely cleaved. In the future, we will use mass spectrometry to detect the cutting effect and ensure the formation of fusion peptides for antibacterial purposes. In addition, we will also conduct antibacterial tests on other common Gram negative and Gram positive bacteria.

References:

1. Chen, J., & Kuipers, O. P. (2021). Isolation and Analysis of the Nisin Biosynthesis Complex NisBTC: further Insights into Their Cooperative Action. mBio, 12(5), e0258521.
2. Imai, Y., Meyer, K. J., Iinishi, A., Favre-Godal, Q., Green, R., Manuse, S., Caboni, M., Mori, M., Niles, S., Ghiglieri, M., Honrao, C., Ma, X., Guo, J. J., Makriyannis, A., Linares-Otoya, L., Böhringer, N., Wuisan, Z. G., Kaur, H., Wu, R., Mateus, A., & Lewis, K. (2019). A new antibiotic selectively kills Gram-negative pathogens. Nature, 576(7787), 459–464.
3. Chen, J., & Kuipers, O. P. (2021). Isolation and Analysis of the Nisin Biosynthesis Complex NisBTC: further Insights into Their Cooperative Action. mBio, 12(5), e0258521.

Originated from Lactococcus lactis subsp. Lactis, NisC is involved in the post-translational modification of the lantibiotic nisin. We managed to express NisC in E.coli for post-translational modification of NisA-affi fusion protein.
Functioned together with NisA-affi(BBa_K4307010) and NisB(BBa_K4307011), it forms composite part NisA-affi-NisB+NisC (BBa_K4307046) to produce functional NisA-affibody peptide.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 541
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 541
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 541
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 541
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The following figure demonstrates our successful construction.

Figure 1: The construction results of NisC.