Difference between revisions of "Part:BBa K1162002"

Latest revision as of 15:13, 12 October 2022

Spheniscin-2 antimicrobial peptide from the king penguin (Aptenodytes patagonicus)

Spheniscin-2 is a potent antimicrobial peptide that is produced by the king penguin. In nature, the king penguin produces Spheniscin-2 in its stomach to preserve food for long periods of time by preventing bacterial decomposition of the food. Studies by ( ) have shown that Spheniscin-2 has a wide range of activity against many gram positive, gram negative, and even fungal organisms, allowing the king penguin to store food for long periods of time.

Design

This part was codon optimized for E. coli using the Life Technologies GeneArt ® software program. By design, this part is Assembly Standard #23 (Silver Fusion) and contains a Methionine(atg) residue at the start of the coding region.

References

Contribution by 2022 iGEM Team Shanghai_city

Spheniscin-2 is a potent antimicrobial peptide that is produced by the king penguin. In nature, the king penguin produces Spheniscin-2 in its stomach to preserve food for long periods of time by preventing bacterial decomposition of the food. Studies by Group BBa_K1162002 have shown that Spheniscin-2 has a wide range of activity against many gram positive, gram negative, and even fungal organisms, allowing the king penguin to store food for long periods of time.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, which was a 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.

Engineering Success

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.

a) Construction of Spheniscin-2 expression plasmids

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.

Figure 1. The sequencing blast results of the recombinant plasmid Spheniscin-2-pET28a(+).

b) Spheniscin-2 protein expression and purification

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.

Figure 2. The process of purification of Spheniscin-2 protein by nickel column.

c) Functional test of Spheniscin-2

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.

Figure 3. Test results of protein Spheniscin-2 inhibiting a single species of bacterial growth.

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 (Figure 3). In conclusion, the data show that the Spheniscin-2 protein concentration greater than 0.37mg/ml has more than 77.79% ability to inhibit the growth of a single species of bacteria E.coli DH5-alpha. 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.

Figure 4. Test results of protein Spheniscin-2 inhibiting real mixed bacterial growth.

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.

Figure 5. Test results of protein Spheniscin-2 inhibiting real mixed bacterial growth with more specific concentrations.

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.