Coding

Part:BBa_K4248003

Designed by: Kong Yangyang   Group: iGEM22_Shanghai_city   (2022-07-09)
Revision as of 08:01, 26 September 2022 by Sqh1028 (Talk | contribs)


Spheniscin-2

Spheniscin-2

Contribution

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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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