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)

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