Proof of Concept

Overview

The novel drug we proposed was to anchor nanobodies on the surface of probiotics, including yeast, Lactococcus, and E. coli, which were then delivered to the gut through a shellac-membraned capsule to combat pathogens like Shigella. Multiple objectives have been achieved to prove our product.

Construction of recombinant plasmids for yeast surface display

In order to enable yeast to express surface protein conjugated with target proteins, we cloned the nanobody gene sequence and the antigen gene sequences into the vectors. The vector pYD1 is a yeast surface display unit where the surface-sealed protein Aga2 was controlled by GAL1 promoter. Using XhoI and ApaI, the nanobody gene 20ipaD and the antigen gene IpaD was cloned into the corresponding restriction sites. The gene segment of 20ipaD was ~500bp, while that of IpaD was ~1000bp. The PCR results that amplify an originally ~400bp fragment of empty vector supported the success in plasmid construction (Figure 1, Figure 2). Sequencing data of the region further confirmed the construction of correct plasmids (Figure 3).

Construction of engineered yeasts that display nanobodies and are capable of being used

Using the plasmids constructed in the previous steps, the baker's yeast EBY100 was transformed. Considering the TRP1 maker in the plasmid, the yeast was selected on the SCDAA medium where tryptophan was absent. Colonies appeared after incubation, indicating the success of engineered probiotic construction (Figure 4). The yeasts that were transformed with recombinant plasmids were now able to display the nanobodies on their surface.

Then, the yeasts were induced by galactose, which can promote the transcriptional activity of GAL1 promoter. After 24h induction, a portion of the cells was shown to display the anticipated proteins on their surface by immunofluorescent microscopic imaging (Figure 5).

Summary and conclusion

As a drug of a novel strategy to target the pathogen, our engineered probiotics should combat the pathogenic Shigella by displaying nanobodies on surfaces to efficiently get over the antibiotic resistance problem. To prove the feasibility and efficiency of this proposed drug, evidence from different aspects should be taken into consideration. First, the most solid and important evidence should be the entity of the imagined product. In the wet lab, we have shown the successful construction of nanobody-displaying yeasts, proving the possibility and authenticity of producing such engineered probiotics. Second, whether the probiotic works to inhibit pathogens would be another important piece of evidence to prove the efficiency of this drug. We also showed the success of pathogen-mimicking yeasts construction and proposed the binding affinity assay that could prove the inhibition feature of the engineered probiotics. Third, evidence for optimization possibility is required to prove the improvement potential of the drug. For the collection of evidence from this aspect, multiple expression enhancement elements refitting and mutation-screening binding affinity enhancement were designed to get the optimized combination of expression abundance and binding affinity. Last but not least, solid storage and delivery method should be risen as evidence proving the practicability and productization ability. The shellac-based microencapsulation plus tablet/capsule wrapping theoretically achieved the room temperature storage and intestine-target release of engineered probiotics, thus proving the reliability of low-cost production and storage of the whole drug and the target specificity of the efficient component.