Team:Tec-Chihuahua/Design

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Design Overview

Considerations

1. In order to obtain the antimicrobial peptides of interest (apidaecin, abaecin, defensin 1 and defensin 2) and to be able to use them against both pathogens (Paenibacillus larvae and Melissococcus plutonius) an appropriate design is required; specialized in expressing heterologous proteins that are meant to be purified.


2. Given that the peptides will be exposed to the hive's environment, it is essential to provide them protection which can increase their half-life.

Genetic Design

The need for an efficient peptide production led to the use of a strong promoter. The answer was the T7 promoter which possesses a high specificity to the T7 RNA polymerase; only DNA cloned downstream from T7 promoter can serve as a template for T7 RNA Polymerase-directed RNA synthesis¹. The choice of this promoter was the first step to create the genetic design. The iGEM registry already had a T7 promoter, which also included an RBS (BBa_K525998) and this part was elected for the experimentation.

E. coli BL21(DE3), a chassis specialized in high-level expression of recombinant proteins, harbors a prophage DE3 derived from a bacteriophage λ, which carries the T7 RNA polymerase gene under the control of the lacUV5 promoter.² Given that expression control was sought out, and that the peptide expression depended on the presence of T7 RNA polymerase, this strain was chosen. When IPTG is added, RNA polymerase is produced.

Looking for better expression of proteins, further evaluation of reported parts was carried; the pelB leader sequence found in the iGEM registry (BBa_J32015) was selected. It directs the protein to the bacterial periplasmic membrane and reduces or even eliminates inclusion body formation³.

In order to be able to purify the peptides of interest, a 6X his-tag downstream the antimicrobial peptide sequence was considered, in addition an efficient terminator of transcription from the iGEM registry was selected (BBa_B0010).

Finally the complete composite is obtained: (T7 promoter + RBS) + (PelB) + (antimicrobial peptide) + (6x His-Tag) + (T1 terminator)

Experimental plan to test the genetic design

1. Synthesis of the complete composite, flanked by the iGEM prefix and suffix.
2. Digestion with EcoRI and PstI of both the synthesized composite and the pSB1C3 linearized vector.
3. Ligation of the pSB1C3 backbone to the composite.
4. Transformation of chemically competent BL21 (DE3) cells.
5. Plasmidic extraction of transformed BL21(DE3) cells.
6. Electrophoresis gel.
7. Induction with IPTG of BL21 (DE3) transformed cells.
8. Extraction of soluble and insoluble proteins.
9. Affinity chromatography purification.
10. SDS-PAGE and antibiograms.

Final application

The final application implements the proteins produced (after performing the antibiograms with different combinations of AMP's and selecting the best option) in a product that is suitable for beekeepers to apply to their hives.

We knew that the peptides required a cover to extend their half-life, so methods for peptide protection were investigated, and thanks to the characteristics PLGA conferred to the covering, the usage of this copolymer was chosen. Poly Lactic-co-Glycolic Acid (PLGA) is biocompatible and a biodegradable FDA approved polymer that has been extensively studied as a delivery vehicle for drugs, proteins, and various other macromolecules⁴. PLGA nanoparticles are reported to control drug release, to protect the compounds from inactivation before reaching their site of action⁵, and they also exhibit a wide range of pH-dependent erosion times⁴.

The choice of the final method of application was possible thanks to our integrated human practices. (You can see the exact trajectory on how the application method was changing in base to our human practices.)

After considering applying the nanocapsules in the bee bread, the idea of using powder or sprinkler to apply the product in the hive was contemplated, however studying the best option, it was finally concluded that the application method would consist in adding the nanoencapsulated peptides to the liquid food beekeepers give to their hives.

References

1. Promega. (2018). T7 RNA Polymerase. Retrieved from https://worldwide.promega.com/products/cloning-and-dna-markers/molecular-biology-enzymes-and-reagents/t7-rna-polymerase/?catNum=P2075
2. Promega. (2018). T7 RNA Polymerase. Retrieved from https://worldwide.promega.com/products/cloning-and-dna-markers/molecular-biology-enzymes-and-reagents/t7-rna-polymerase/?catNum=P2075
3. Kovalskaya, N., & Hammond, R. W. (2009). Expression and functional characterization of the plant antimicrobial snakin-1 and defensin recombinant proteins. Protein Expression and Purification, 63(1), 12–17. doi:10.1016/j.pep.2008.08.013
4. Makadia, H. & Siegel, S. (2011). Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3347861/
5. Teixeira, M., Alonso, M., Pinto, M. & Barbosa, C. (2004). Development and characterization of PLGA nanospheres and nanocapsules containing xanthone and 3-methoxyxanthone. Retrieved from https://pdfs.semanticscholar.org