This project develops a product containing the natural antimicrobial peptide (AMP) Hydramacin-1, which is discovered while investigating the epithelial defense of the ancient metazoan Hydra. It is active both against Gram-positive and Gram-negative bacteria including multi-resistant human pathogenic strains. And it has many advantages such as low side effects and not easy development of drug resistance. Our experimental data indicated its significant effect on cleaning the water in the fish tank. Therefore, our product will provide a water quality improvement plan for fish farming.

Because the T7 promoter and T7 RNA polymerase have strong ability in translation and usually be used as protein expression, we choose pET28a-vector and E.coli BL21(DE3), with T7 promoter and T7 RNA polymerase respectively, to express our target protein Hydramacin-1. To achieve this, we designed the DNA sequences of Hydramacin-1 to be inserted into the XbaI and XhoI sites of the pET-28a(+) vector (Figure 1.), transferred the recombinant plasmid into E.coli BL21(DE3) for protein expression.

In order to obtain the AMP protein, expanded the culture pET-28a(+)- Hydramacin-1/BL21(DE3) 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 Hydramacin-1 protein was measured as 0.212mg/mL. At this point, we got the Hydramacin-1 protein solutions we wanted.

To confirm the ability of our purified Hydramacin-1 protein to inhibit bacterial growth, we used E.coli DH5α as bacteria for bacteriostatic test experiments. 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 protein coded according to the empty pET-28a(+) plasmid. As shown in the graph, the less diluted the Hydramacin-1 protein is, the more impact it has on bacterial growth. Just like the experiments we ran on LL-37, Spheniscin-28, Sparamosin_26-54, and Fusion. Although the average OD₆₀₀ absorbance for the solution that contains 100μl of bacteria and 100μl of the Hydramacin-1 AMP 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 AMP plays an insignificant role in bacterial inhibition. However, there is a decrease in OD₆₀₀ absorbance after applying the AMP that has been diluted 5 times. The drop in OD₆₀₀ absorbance is further emphasized when we directly applied the AMP 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 AMP as a way to inhibit bacterial growth. While there is a drastic decrease in the OD₆₀₀ absorbance where the Hydramacin–1 AMP 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 AMP may be a better bacterial inhibitor compared to Kanamycin or other antibodies.

After confirming the feasibility of using AMP against E. coli DH5α which is cultured in the laboratory, we used the same experimental set-up to test their effectiveness in inhibiting the growth of real mixed bacteria acquired from Haichang Ocean Park. While the positive and negative control group are all composed of the same solutions, we conducted this experiment with 11 different concentrations, each increasing with a ratio of 2. Through doing this, we hope to find the best concentration at which our antimicrobial peptide works. Similar to the trends presented in our results section, the less diluted the peptide, the more effective it is. Just like the OD₆₀₀ absorbance results when we tested the Hydramacin-1 AMP against DH5α, the OD₆₀₀ absorbance is only significantly lower than that of the negative control group when the peptide solution was diluted 8 times or less. Specifically, we see a drastic decrease in the OD₆₀₀ absorbance when the peptide was only diluted 4 times. Stopping at an OD₆₀₀ absorbance of 0.1296 when the peptide was undiluted at all, the OD₆₀₀ absorbance of the undiluted AMP is slightly higher than that of the positive control group. Although the graph shows that Kanamycin, the antibody used in the positive control group, was able to inhibit bacterial growth better, the Hydramacin-1 can plausibly be a better bacterial inhibitor if we concentrate the protein more. With the current concentration being 0.212mg/mL, increasing it to 0.4mg/mL or 0.5mg/mL may make it a better bacterial growth inhibitor when compared to existing antibodies. In conclusion, our Hydramacin-1 products still have great potential in inhibiting real mixed bacteria growth.

Based on the test results using the double dilution method to dilute the antimicrobial peptide and test on DH5α and Oceanarium bacteria, we conducted another round of dilution and testing for more accurate data and effect analysis. In this test, we uniformly diluted the antimicrobial peptide to micromolar concentrations and constructed at least nine gradients for the peptide. Similar to the trend of our first experimental results, the lower the dilution of the antimicrobial peptide, the more effective it was. When the concentration of Hydramacin-1 protein reached 8μM, it could sterilize and inhibit bacteria to OD₆₀₀ absorbance value 0.5 and below. When the concentration of Hydramacin-1 protein reaches 15μM, it can sterilize and inhibit bacteria growth to the OD₆₀₀ absorbance value of 0.25 or below, and its antimicrobial effect is even better than that of kanamycin. This confirms our inference above. After the concentration of Hydramacin-1 protein reaches a certain level (equal to or greater than 15μM), our peptide products can achieve the effect of sterilization beyond antibiotics. This result is really encouraging.