Improve

New Improved Part: BBa_K4846017  (pETduet-NisB-NisC)

Old Existing Parts: BBa_K4307011 (NisB) ; BBa_K4307012 (NisC)

 

Summary

We have made improvements on the original components BBa_K4307011 and BBa_K4307012. We connected these two parts in to the same plasmid pETduet (BBa_K4846011) , then obtained the new funtional 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-transform ed 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 in to 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 funtional 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.