Our project initiated by the using of black soldier fly larvae for the treatment of food waste at school. There is no sting smell from the food waste mixture with the larvae for whole week of digestion treatment. We started reviewing the literature to figure out what chemicals or enzymes may be produced by the larvae to stop the bacterial growth and thus eliminate the bad smell. DLP4, an antimicrobial peptide (AMP), was found to be the possible candidate for killing the bacteria [1]. By phylogenetic analysis of AMP in black soldier fly through Molecular Evolutionary Genetics Analysis, we selected the DLP family and CLP family peptide as our target to produce AMPs for further testing and characterization.
Before the plasmid design, we have an interview with Professor Shi Zheng Ren, an entomologist and a expert in bioindustry communication and development. Prof. Shi has two concerns on our project. First is how we purify the AMP and the second is will the AMP kill the E. coli during the expression.
After this interview, we know that there is a problem to express AMPs in E.coli expression system directly as the peptide may inhibit the growth of E.coli. Therefore, it is important to generate a system to minimize this effect. After considering this challenges that have arisen from our discussion, we concluded that the project was still feasible for using an inhibitor system to secure the expression of AMPs in the E.coli. Moreover, we have to add some tag system to facilitate the protein purification and extraction.
After some research in the literature and databases, we have selected intein as a fusion protein to inhibit the action of AMPs inside E.coli before the purification and cleavage process. Intein-AMPs are inactivated antimicrobial peptide so that it will not kill E.coli during expression to ensure continuous expression of AMPs. These aggregates are easily and cheaply be purified by Ni-NTA resin (QIAGEN, Hilden, Germany) with the presence of His tag as well. Autocleavage excises itself when the recombinant protein is in pH 10 buffers. [2] in the meantime, SUMO tag is also used to increase the solubility of AMPs and make it easier to extract.
To validate the selected AMPs and SUMO-intein-AMP expression system, we constructed plasmids with SUMO and 6 His-tag as the backbone and linked to intein and different AMPs, which were registered in the iGEM’s Part's Registry page (BBa_ K4794000 to BBa_ K4794006). The part was designed with T7 promoters, followed by the 6 His tag, SUMO tag, intein, AMP and ending with the terminator as depicted in Figure 2. For the first step of cloning, SalI and KpnI are used for the assembly of intein-AMPs to pET-SUMO plasmid, while for the change of other AMP sequence, SacI and KpnI were used. After the AMP expression, SDS-PAGE was used to determine the expression and solubility of AMP. Then, protein purification and antimicrobial tests would be followed.
However, the results of SDS-PAGE showed that most of expressed proteins for DLP1-3, were found in the pellet instead of supernatant, which greatly reduced the yield of AMPs collected for the purification and thus the antimicrobial test. Only DLP4 and CLP1 were enough to proceed to the next stage. Also, the result show that intein self-cleavage system works but the efficiency can be increased with further fine tuning of the cleavage environment or even with alternate design of cleavage system. Maybe another expression system was required to increase the yield of AMPs.
Two different expression systems were proposed to ease this situation.
For the first approach, SUMO-tag can be simply deleted and obtain a new composite part, T7-His-intein-AMP [BBa_K4794009], to check whether the solubility can be increased by reducing the size of protein synthesized. If this new system works, high concentration of AMPs should be collected after purification. However, this system cannot solve the problem of low efficiency of intein self-cleavage, so we proposed another approach.
For the second approach, SUMO tag and His tag can be replaced by ELK-16 system, T7-AMP-intein-PT linker-ELK16 [BBa_K4794010] to change the purification mechanism by changing the solubility of AMP from insoluble to soluble form[4]. The ELK-16 system includes ELK16, Mxe GyrA intein, and PT linker. The protein expressed first is insoluble, but by adding DTT, the AMPs can be removed from intern and become soluble. This is a easy way to purify target protein with intein system. This system would be applied for future experiments (2nd engineering cycle). If this new system works, we can collect higher concentration of AMPs for antimicrobial test to achieve our final goals – finding an alternative of antibiotics and make an antimicrobial black soldier fly ecoplaster.
During the expression, purification and testing of antimicrobial properties of the AMPs, we visited a black soldier fly farm and they suggested different parts and stages of the black soldier fly have different usages. From the shell of black soldier fly larvae obtained from the farm, we further have some research on its properties and found that it is possible to produce a hydrogel [3] and apply the purified AMPs to form an antiseptic plaster.
Production of hydrogel plaster was studied and carried out in our project. Demineralization and deproteinization of BSF shells in 2M HNO3 was done and followed by deacetylation with excess 16.7M NaOH to the BSF chitin samples. The hydrogel was formed using excess acetic acid. [5] With high temperature treatment, a water-proof layer can be produced to fulfill the property of plaster for daily use. Finally, we can broaden application the use of AMPs expressed in our system and engineered a marketable product, which production process can meet the Sustainable Development Goals 12, Responsible Consumption. This contributes to changing unsustainable consumption and production patterns, including through the easing the food waste problem, setting up a circular economy model, and innovative design of antimicrobial black soldier fly plaster to move towards more sustainable patterns of consumption and production.
We have an interview with Mr. Gilbert, Finance Bussiness Partner from British Standards Institution. We have learnt that in order to commercialize the product, we have to increase the variety of tape types and sizes, consider ISO certifications (e.g. ISO 13485, 10933, and 9001) and estimate the cost. High variety of tape types and sizes can position our product as a consumer favorite. Getting ISO certifications can give customers’ confidence in our plaster. To estimate the cost can make sure it has a attractive price.
In the future, we should try give some colours and have some designs to the plaster to make it more attractive and fit to consumer favorite. Also, more tests should be done to apply the ISO certifications. For the cost, we can emphaize on how to convert food waste into valuable things. Black soldier fly feeds on the food waste to solve the pollution. When it grow up, its shell can be used to make plaster and other body parts can be the fish feed. If all these application works, the cost of plaster should be much lower.
1. Xia J, Ge C, Yao H. Antimicrobial Peptides from Black Soldier Fly (Hermetia illucens) as Potential Antimicrobial Factors Representing an Alternative to Antibiotics in Livestock Farming. Animals (Basel). 2021;11(7):1937. Published 2021 Jun 29. doi:10.3390/ani11071937
2. Wu C-L, Chih Y-H, Hsieh H-Y, Peng K-L, Lee Y-Z, Yip B-S, Sue S-C, Cheng J-W. High Level Expression and Purification of Cecropin-like Antimicrobial Peptides in Escherichia coli. Biomedicines. 2022; 10(6):1351. https://doi.org/10.3390/biomedicines10061351
3. Gafri, Hasan. F., Fathiah Mohamed Zuki, Mohamed Kheireddine Taeib Aroua and Nur Awanis Hashim. “Mechanism of bacterial adhesion on ultrafiltration membrane modified by natural antimicrobial polymers (chitosan) and combination with activated carbon (PAC).” Reviews in Chemical Engineering 35 (2018): 421 - 443.
4. Wang, M., Zheng, K., Lin, J., Huang, M., Ma, Y., Li, S., Luo, X., and Wang, J. (2018). Rapid and efficient production of cecropin A antibacterial peptide in Escherichia coli by fusion with a self-aggregating protein. BMC Biotechnol. 18, 62
5. Pires, Cléo T. G. V. M. T. et al. “The Effect of Chitin Alkaline Deacetylation at Different Condition on Particle Properties.” Procedia Chemistry 9 (2014): 220-225.