We design five plasmids: The DNA sequences of the four antimicrobial peptides Hydramacin-1, Spheniscin-2, LL-37, and Sparamosin26-54 were inserted into the XbaI and XhoI sites of the pET-28a(+) vector, respectively. The fifth one was a fusion antimicrobial peptide that connects LL-37 and Sparamosin26-54 through a linker, which was also inserted into the pET-28a(+) vector for protein expression, as shown in Figure1.

Then, we amplify Hydramacin-1, LL-37, and Sparamosin_26-54 by PCR (Figure2.), double-enzyme digestion, and ligase to pET28a(+) carrier, respectively, to obtain the other three plasmids Hydramacin-1- pET28a(+), LL-37- pET28a(+), and Sparamosin_26-54- pET28a(+).

We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure3-7.), and the plasmid was successfully constructed. So far, we have successfully obtained five recombinant plasmids, which were respectively on the pET28a(+) vector, which can be used to express antimicrobial peptide proteins.

In order to obtain the five antimicrobial peptide proteins, we transferred the recombinant plasmids into E.coli BL21(DE3), expanded the culture in the LB medium, and added IPTG to induce protein expression when the OD₆₀₀ reached 0.4. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used nickel column purification to purify the antibacterial peptide protein we wanted. The concentration of each protein was measured as:
0.212mg/L Hydramacin-1, 0.208mg/mL Sparamosin_26-54, 0.42mg/mL LL-37, 0.74mg/mL Spheniscin-2, and 0.431mg/mL Fusion.
At this point, we got the five antimicrobial peptide protein solutions we wanted.

To confirm the ability of the our purified antimicrobial peptide to inhibit bacterial growth, we used E.coli DH5-alpha as bacteria, and antibiotics as a positive control for bacteriostatic test experiments.
To better show the relationship between the concentration of antimicrobial peptides and the inhibition of bacterial growth, we added 100 μL of DH5α and 100 μL of different concentrations of the antimicrobial peptide to each of the five test tubes. Our five test tubes were filled with the antimicrobial peptide stock solution and diluted 1, 5, 25, 125, and 625 times solution, and repeated three times for each concentration to form the average data graph with error bars.
The results showed that the five antimicrobial peptide proteins had significant antibacterial effects at double dilution concentrations. A single antimicrobial peptide protein can be seen to inhibit the growth of about 60% of bacteria in the range of 5 to 25 times dilution. The antibacterial effect of the Fusion antimicrobial peptide is the best, with about 60% when diluted 625 times. It shows that our antimicrobial peptide products do have a significant antibacterial function, and the antibacterial function of the fusion antimicrobial peptide is the best. A detailed analysis of the antibacterial effect of each antimicrobial peptide is given below.

We confirmed the ability to use the Hydramacin-1 antimicrobial peptide to inhibit bacterial growth by first testing it against DH5α. As shown in the graph, the less diluted the antimicrobial peptide is, the more impact it has on bacterial growth. Just like the experiments we ran on Fusion, LL-37, Spheniscin-28, and Sparamosin-26, the positive control group is composed of 100μl of DH5α along with 100μl of liquid LB broth that contains Kanamycin and the negative control group contains 100μl of DH5α and 100μl of the peptide coded according to the empty pET-28a(+) plasmid. Although the average OD₆₀₀ absorbance for the solution that contains 100μl of bacteria and 100μl of the Hydramacin-1 antimicrobial peptide that has been diluted 625, 125, and 25 times are relatively lower than the OD₆₀₀ absorbance of the negative control group, the error bars of these variations overlap with each other. The overlap in their error bars hints that their OD₆₀₀ absorbance is not significantly different from each other, meaning that when diluted 625, 125, and 25 times, the antimicrobial peptide plays an insignificant role in bacterial inhibition. However, there is a decrease in OD₆₀₀ absorbance after applying the antimicrobial peptide that has been diluted 5 times. The drop in OD₆₀₀ absorbance is further emphasized when we directly applied the antimicrobial peptide that has not been diluted to the DH5α bacteria. With the error bars of both of these variations not overlapping with those of the negative control group, the data shows the feasibility of using Hydramacin-1 antimicrobial peptide as a way to inhibit bacterial growth. While there is a drastic decrease in the OD₆₀₀ absorbance where the Hydramacin–1 antimicrobial peptide that has not been diluted was applied, the graph shows that Kanamycin, the antibody that was used in the positive control group, is still a better bacterial inhibitor. Nevertheless, we can continue to concentrate the peptide solution, making its concentration higher than 0.212mg/ml. With a higher concentration, the Hydramacin-1 antimicrobial peptide may be a better bacterial inhibitor compared to Kanamycin or other antibodies.

The graph above indicates that the less diluted the protein Spheniscin-2 solution is, the more bactericidal it is. Compared to other bars in the chart, the one with 1:1 dilution has the most significant effect on sterilization, which can eliminate almost 77.79% of bacteria. However, the histograms of Spheniscin-2 antimicrobial peptides diluted by 625, 125, and 25-fold LB medium were virtually identical to the graph of the negative control group, indicating that they had a little sterilizing effect. And the antimicrobial peptide diluted by five times had some antibacterial ability, but it was not as significant as the sterilization effect of Spheniscin-2 antimicrobial peptide stock solution.

We also used the same operation to dilute an antimicrobial peptide called LL37. In this histogram, the heights of antimicrobial peptides that have been diluted 625x and 125x with LB medium are almost the same as that of the negative control group, indicating that the sterilization ability of these two samples is practically absent. In contrast, the sample diluted 25 times LB had a significant sterilizing effect but was not as good as the one with five times. The best sterilization effect was the LL37 stock solution, which could sterilize up to 75.74%.

We performed dilution of the antimicrobial peptide Sparamosin26-54 using the same procedure and operation. By comparing the height of the histograms and the margin of error, we learned that the antimicrobial peptide sterilization after 625-fold LB solution dilution was not competent because its height was higher than the image height of the negative control. The samples diluted by 125x and 5x were similar in sterilization ability, but the 25x was a bit worse than both. Among all models, the Sparamosin26-54 stock solution had the best antimicrobial effect, which could reach 76.20%.

As the graph shows, the less the Fusion antimicrobial peptide was diluted, the stronger its ability to inhibit bacterial growth. However, even considering the error bars, the 625 times dilution had a better ability to inhibit bacterial growth than the 125-fold dilution. Therefore, we compared the experimental results of the Fusion antimicrobial peptides with the crude extracts in the preliminary experiment and found that these antimicrobial peptides have a slightly better ability to inhibit bacterial growth at lower concentrations than at concentrations around the median. The OD₆₀₀ absorbance was not significantly different from that of the negative control group when it was located at the median concentration, i.e., 125-fold and 25-fold diluted solutions, which shows that the antimicrobial peptides did not work efficiently at this concentration. However, with the decrease in dilution and the increase of antimicrobial peptide concentration, the OD₆₀₀ absorbance decreased significantly and reached an average of 0.1602 at double dilution. Although this value is still somewhat high compared to the positive control with kanamycin, it can be inferred from the above table that at higher concentrations of the antimicrobial peptide, Fusion antimicrobial peptide is likely to replace kanamycin as a more effective bacterial inhibitor.