We are the Pepsick iGEM team dedicated to developing antimicrobial peptide products for cleaning fish tanks. In order to carry forward the spirit of iGEM, and inherit and spread the value of iGEM, we specially searched the iGEM Biological Parts library for related antimicrobial peptides and picked BBa_K1162002, Spheniscin-2 antimicrobial peptide from the king penguin (Aptenodytes patagonicus). This is a biological part submitted by iGEM13_Utah_State in 2013, with only DNA sequence information and simple text description information. Our team carried out a comprehensive characterization of this part in the laboratory, adding data from antibacterial testing to dedicate its function and properties in inhibiting bacterial growth. This information can be a good reference for future iGEM teams working on antimicrobial peptides.
In addition, through literature research, we found three other new types of antimicrobial peptides, Hydramacin-1, LL-37, and Sparamosinsub>26-54. We upload their DNA sequence information and basic introduction information in the registry of standard biological parts to provide more choices of antimicrobial peptides for future iGEM teams.
What's more, we have added a new fusion antimicrobial peptide, which is formed by fusing LL-37 and Sparamosin_26-54 through a protein linker, which can effectively combine the antibacterial functions of LL-37 and Sparamosin26-54, not only inhibiting bacterial growth but also inhibiting fungal growth, that is an original and new form of fusion antimicrobial peptides, which also provides more ideas for future iGEM teams to optimize antimicrobial peptide products.

Male penguins can keep undigested food in their stomachs for weeks without spoilage, precisely because of the presence of antimicrobial peptides Spheniscin-2, which have antibacterial and antifungal activity against Gram-positive and Gram-negative bacteria.

We let the synthetic company synthesize the DNA fragments of Spheniscin-2, and inserted into the XbaI and XhoI sites of the pET-28a(+) vector. The certificate of recombinant plasmid sequencing results is as Figure1.

In order to obtain the antimicrobial peptide protein, 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 to purify the antibacterial peptide protein we wanted. The concentration of Spheniscin-2 protein was measured as 0.74mg/mL.

To confirm Spheniscin-2’s function and properties in inhibiting bacterial growth, we firstly 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 Spheniscin-2 protein to each of the five test tubes. Our five test tubes were filled with the protein 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.

In order to test the antibacterial effect of the Spheniscin-2 in the real fish tank environment, we specially retrieved some water samples from the aquarium from Haichang Ocean Park. After culturing the mixed bacteria in the water samples from the aquarium, we used the same experimental set-up to test their effectiveness in inhibiting the growth of mixed bacteria. 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 which Spheniscin-2 works at. Similar to the trends presented showed in the results, the less diluted the peptide, the more effective it is.

This graph presents a positive relationship between the concentration of the Spheniscin-2 antimicrobial peptide and the OD₆₀₀ absorbance. In other words, the higher the concentration of the antimicrobial peptide, or the less diluted it is, the lower the OD₆₀₀ absorbance, indicate the stronger inhibition. It suggested a successful inhibition of mixed bacteria growth. The OD₆₀₀ absorbance continues to decrease as the antimicrobial peptide concentration increases. When we directly applied our antimicrobial peptide with a concentration of 0.74mg/ml to the bacteria, the average of the OD₆₀₀ absorbance is 0.1523, which is still slightly higher than the OD₆₀₀ absorbance of the positive control group where we used the Kanamycin antibody. However, this does not mean that Spheniscin-2 is a weaker bacterial growth inhibitor than Kanamycin. With the trend showing that an increase in concentration will lead to a better inhibition of mixed bacterial growth, we can concentrate Spheniscin-2 protein so that non-diluted antimicrobial peptide would have a higher concentration, thus leading to a higher inhibition of mixed bacterial growth.

Based on the test results using the double dilution method to dilute the antimicrobial peptide and test on DH5-alpha and Oceanarium bacteria, we conducted another round of dilution and testing for more accurate data and effect analysis. The graph shows the histogram of OD₆₀₀ absorbance values of different concentrations of Spheniscin-2 antimicrobial peptide. The graph shows that the higher the concentration of Spheniscin-2 antimicrobial peptide, the smaller the OD₆₀₀ value, which means the better the antimicrobial effect. When the antimicrobial peptide concentration reaches 25μM, its OD₆₀₀ absorbance value drops to about 0.5, which means that from this concentration, the use of Spheniscin-2 antimicrobial peptide will have a more significant effect. When its concentration reaches 50 μM, the antimicrobial effect of Spheniscin-2 antimicrobial peptide is even similar to the antimicrobial effect of the positive control kanamycin. Then, according to the trend, the antibacterial effect of Spheniscin-2 antimicrobial peptide will be better than that of kanamycin when it is further concentrated to a higher concentration.

Hydramacin-1 is a novel antimicrobial protein recently discovered during investigations of the epithelial defense of the ancient metazoan Hydra.. The amino acid sequence of hydramacin-1 shows no sequence homology to any known antimicrobial proteins. Determination of the solution structure revealed that hydramacin-1 possesses a disulfide bridge-stabilized alphabeta motif. The bacteria hydramacin-1 used in our experiment has good antibacterial effect.

Antimicrobial peptide LL-37 is from the cathelicidin family of peptides and it’s the only one who from this family of peptides. This antimicrobial worked with the immune system. It same as the antibiotics but it can directly distract on bacteria, virus and fungi. Also LL-37 can cooperate with the natural immune system to get the sex immune and other else. This antimicrobial can also worked on the endothelial cells and epithelial cells that live in the blood vessels to stimulate them for vessels reborn. One investigation about ICM, they used SWT to released LL-37, then LL-37 stimulate the cardiac muscle to reborn.

Sparamosin_26-54 is the new antimicrobial peptide that was found at Scylla’s gonad. By continued research about this new peptide, it was found that the sparamosin_1-25 and Sparamosin_26-54 were good antibacterial to the fungi cryptococcosis that is a disease that fatality rate can reached 40%. This kind of antimicrobial peptide might be an important medicine in the future.

In the fusion antibacterial peptides (AMPs), the functional gene for LL-37 and Sparamosin_26-54 are combined, intended to express a new AMP with both proficiencies of the two AMPs in bacterial and fungal inhibition. We specifically add a linker in the middle of the two AMPs. The linker is an indispensable component of recombinant fusion proteins, linkers have shown increasing importance in the construction of stable, bioactive fusion proteins. Besides the basic role in linking the functional domains together (as in flexible and rigid linkers) or releasing the free functional domain in vivo (as in in vivo cleavable linkers), linkers may offer many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles.
This new composite part is a technical innovation of the project as a new attempt.

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2. Michalek, Matthias, et al. "Hydramacin-1 in action: scrutinizing the barnacle model." Antimicrobial agents and chemotherapy 57.7 (2013): 2955-2966.
3. Hassan, M., et al. "Natural antimicrobial peptides from bacteria: characteristics and potential applications to fight against antibiotic resistance." Journal of applied microbiology 113.4 (2012): 723-736.
4. Jung, Sascha, et al. "Hydramacin-1, structure and antibacterial activity of a protein from the basal metazoan Hydra." Journal of Biological Chemistry 284.3 (2009): 1896-1905.