Results

Overview

In this project, we genetically fused nisin (or bsjA) with darobactin via molecular cloning and heterologously expressed the fusion peptides in E. coli. Two strategies were also used to modify nisin, bsjA, and darobactin, one was the separate expression of the precursor peptide and the post-translational modifying (PTM) enzyme, and the other was the co-expression of the precursor peptide and the PTM enzyme. After obtaining the fusion proteins, we performed in vitro cleavage assays to obtain the core peptides and verified their activity.

Our experimental results are mainly composed of parts, which are (1) plasmid construction and transformation, (2) expression and purification of the fusion peptide, and (3) validation of the structure and activity of the fusion peptide.

 

Plasmid construction and transformation

For the construction of the following four plasmids, gene sequences: nisB, nisC, bsjM, bsjA1-L, darL, darA-core, and bsjA1-core were handed over to the company for synthesis and then cloned directly into the pUC57 cloning vector. The fragments were ligated to the vector and by enzymatic ligation, resulting in four recombinant plasmids:

Plasmid A： pRSFduet-DarL-BsjL-His-DarA-BsjA

Plasmid B： pRSFduet-DarL-NisL-His-DarA-NisA-DarE

Plasmid C： pETduet-DarE-BsjM

Plasmid D：pETduet-NisB-NisC

Plasmid A and plasmid C will be transferred into the same strain, while plasmid B and plasmid D will be transferred into the same strain

 

Figure 1 T he  plasmids constructed in the project

 

We first extracted four plasmids containing the target genes synthesized by the company and amplified the target fragments using PCR. As shown in Figure 2, DNA electrophoresis results proved that we successfully amplified four fragments. After obtaining the target fragments, we processed the target fragment and the vector (pRSFduet and pETduet) using double enzyme digestion and formed the recombinant plasmids by enzymatic ligation.

 

Figure 2 DNA electrophoresis results of PCR amplified target fragments

 

Subsequently, the plasmids A and B were transferred into the BL21(DE3) competent cell, respectively. As shown in Figure 3, the transformants were successfully grown after overnight culture. The results of colony PCR showed that both transformants amplified the target bands, indicating that both plasmids A and B were successfully transformed into the BL21(DE3).

 

 

Figure 3 PCR results of colonies of transformants

 

On the premise that both A and B plasmids are successfully transformed, these two plasmids were transformed into competent cells, and then C or D plasmid was transformed into the competent cells containing A or B plasmid, respectively. As shown in Figure 4, for the transformants containing the A and C plasmids, all transformants could amplify two target bands, indicating that the A and C plasmids were successfully transferred into BL21(DE3). For the transformants containing the B and D plasmids, transformants 3, 4, 5, 6, and 7 were able to amplify two target bands, indicating that the strain was successfully constructed.

 

 

Figure 4 Colony PCR and sequencing results of transformants containing dual plasmids

 

3. Expression and purification of proteins

We inoculated the positive transformants and when the OD₆₀₀ value of the bacterial solution was about 0.8, IPTG was added to induce expression. After centrifugation to obtain the bacterium, the bacterium was resuspended with a buffer containing 8 M urea and sonicated. As shown in Figure 5, we did not observe target bands on SDS-PAGE, likely due to the small sizes of the final products (His-DarA-BsjA: 13.4 kDa, His-DarA-NisA: 8 kDa). And the nickel column purification was not effective. This may be because we did not elute the impurities sufficiently, which resulted in the low purities of the final target proteins.

 

Figure 5 SDS-PAGE result of the protein expression and purification

 

4.  Agar diffusion growth inhibition assay

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.

 

 

Figure 6. Antibacterial effect of DarA-BsjA fusion peptide antimicrobial peptides on Escherichia coli

 

 

Figure 7. Antibacterial effect of two antimicrobial peptides on Bacillus subtilis

We used Gram negative bacteria Escherichia coli and Gram positive bacteria Bacillus subtilis as indicator bacteria to verify the antibacterial effect of antimicrobial peptides. From the Figure 6 and 7, it can be seen that antimicrobial peptides have little inhibitory effect on Escherichia coli, while they have antibacterial effect on Bacillus subtilis, but the antibacterial effect is not very significant. This result is not very ideal, possibly because after expressing the fusion peptide, in vitro cleavage experiments are required, and the cleavage results need to be detected by mass spectrometry. The fusion peptide was not completely cleaved, resulting in weak activity and no antibacterial effect. 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 in the future.