Part:BBa_K4846019
pRSFduet-DarL-NisL-His-DarA-NisA-DarE
pRSFduet-DarL-NisL-His-DarA-NisA-DarE
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Composite Part: BBa_K4846019
pRSFduet-DarL-NisL-His-DarA-NisA-DarE
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.
Figure1.Expression frame of the plasmid pRSFduet-DarL-NisL-His-DarA-NisA-DarE
Experimental Approach
Through enzymatic digestion and ligation, we ligated the vector backbones pRSFduet and pETduet with the fragments DarL-NisL-His-DarA-NisA-DarE to construct plasmid pRSFduet-DarL-NisL-His-DarA-NisA-DarE.
Figure2.The generation of plasmid pRSFduet-DarL-NisL-His-DarA-NisA-DarE constructed.
After constructing the recombinant plasmid pRSFduet-DarL-NisL-His-DarA-NisA-DarE, we needed to co-transform them into the same bacterial strain. pRSFduet-DarL-NisL-His-DarA-NisA-DarE was first transformed, and positive transformants were selected to prepare competent cells containing pRSFduet-DarL-NisL-His-DarA-NisA-DarE, and 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 plasmid pRSFduet-DarL-NisL-His-DarA-NisA-DarE and pETduet-NisB-NisC.
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
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 of DarA-NisA fusion antimicrobial peptide on Bacillus subtilis
Discussion and Future plan
Co-expressing the precursors and PTM enzymes on the same plasmid enabled the modification of DarA and NisA to confer antimicrobial activity. At the molecular level, we fused DarA and NisA to generate a peptide with anti-Gram-positive effect. Perhaps it is because after expressing the fusion peptide, in vitro cleavage experiments are required, and the fusion peptide is not completely cleaved, resulting in weak activity and little 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 149
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 305
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 324
Illegal AgeI site found at 566
Illegal AgeI site found at 5297 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 639
Illegal SapI site found at 1659
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