Our composite part BBa_K4248005 was improved based on the existing part BBa_K1162006.
There is a mini-review of the development of LL-37 protein-related parts. In 2009, group iGEM09_Slovenia firstly designed a basic part BBa_K245114, LL37. In 2012, group iGEM12_Trieste designed the part LL37 (BBa_K875009) using Assembly Standard #10 for both the prefix and suffix and optimizing the coding sequence for E.coli. In 2013, group iGEM13_Utah_State further improved the part LL37 (BBa_K1162006) using Assembly Standard #23 and by removing the stop codon at the end of the coding region. In 2019, group Jilin_China 2019 characterized this part to kill C. Albicans. In 2021, group HZAU-China 2021 characterized this part to kill Salmonella Typhimurium SL1344. Today, our team further improved the LL-37 by adding fusing a new peptide Sparamosin26-54 with a Linker between the two APMs. Furthermore, the codon has been optimized for E. coli to develop our new composite part BBa_K4248005.

In order to prove the function of our new fusion part LL37-Linker- Sparamosin_26-54 , we expressed and purified the Fusion AMP, LL-37 AMP, and Sparamosin26-54 AMP, then detected the inhibition in mixed bacteria acquired from Haichang Ocean Park. As the result shown above, our Fusion AMP has achieved nearly twice bacteriostatic ability as efficiency as that of LL-37 peptide. Besides, our project aimed to clean fish tanks. And Fusion AMP could well inhibit mixed bacterial growth in fish tanks, which is in accord with our initial expectation.

We fashioned the target gene Sparamosin_26-54 with a famous human anti-microbial peptide LL-37. LL-37 is a famous antibacterial peptide (AMP), derived from mammals, mainly to the bacteria has solid antibacterial ability, in this case, we will imagine if the antifungal activity of antimicrobial peptides with LL-37 fuses together, form an artificial modification of bifunctional peptide, so will give the fusion peptide, has the function of antibacterial and antifungal, Therefore, Sparamosin_26-54 , which has anti-fungal activity, was fused with LL-37, and the fusion peptide was expressed and purified through the Escherichia coli expression system, which will have broader application scenarios and application scope.

We design three plasmids: The DNA sequences of the AMPs LL-37 and Sparamosin_26-54 were inserted into the XbaI and XhoI sites of the pET-28a(+) vector, respectively. The third one was a fusion AMP that connects LL-37 and Sparamosin_26-54 through a linker, which was also inserted into the pET-28a(+) vector for protein expression, as shown in Figure 2.

To build the plasmid, we let the synthetic company synthesize the DNA fragment of Fusion and integrate it into the pET-28a(+) vector. Then, we amplify LL-37 and Sparamosin_26-54 by PCR (Figure3.), double-enzyme digestion, and ligase to pET28a(+) carrier, respectively, to obtain the other two plasmids 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 (Figure 4.). Thus, the three expression plasmids were successfully constructed. And the last step was extracting the recombinant plasmids from E.coli DH5α and transferring them into E.coli BL21(DE3) competent, so that can be used to express AMP proteins.

In order to obtain the three AMPs’ proteins, we 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: 0.208mg/mL Sparamosin_26-54 , 0.42mg/mL LL-37, and 0.431mg/mL Fusion. At this point, we got the three AMPs’ protein solutions we wanted.

To confirm the function of the AMPs to inhibit bacterial growth, we used the real mixed bacteria acquired from Haichang Ocean Park 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 mixed bacteria and 100 μL of different concentrations of the AMPs to each tube and repeated them three times for each concentration to form the average data graph with error bars.
As we have seen in Figure 5, the Fusion peptide’s performance (Figure 6C) at each concentration was almost always the best of the three. Furthermore, Fusion widens the gap when the concentration reaches 15 μm. The Fusion peptide did nearly twice as efficient as the rest of the two peptides (Figure 6A, 6B) in the concentration of 15μm, 20μm, and 25μm, proving the success of using the Fusion peptide to enhance our performance of the AMPs.