Future Directions

What comes next?

Future Directions and Outlook

Our team sought to develop a novel synthetic sensor capable of detecting Fusarium graminearum in cereal crops. Due to time constraints, we were unable to complete everything we had envisioned and did not have the opportunity to fully construct the sensor with its nanobody and “pseudo antigen.” However, we developed a foundation through our research and laid out steps to be undertaken. Any future iterations of the UAlberta team that take on this project can follow our framework and start from an accelerated position.

Cameleon-Q-Sensor

We successfully transformed the IDT synthesized Cameleon-Q-Sensor (BBa_K4755013) into E. coli Rosetta-Gami B(DE3). Despite the successful transformation, IPTG-induced expression of the sensor was poor, meaning no significant fluorescent data could be obtained. However, when plated without IPTG, protein expression occurred at basal levels and produced a prominent pale purple colour. This probably indicates that our sensor is properly folded and functioning as intended. Due to time constraints we were unable to fully assemble the Biobrick compatible Cameleon-Q-Sensor (BBa_K4755022). Overall, no significant data pertaining to our sensors proposed FRET-quenching functionality could be obtained. Future iGEM teams can easily work with and improve upon the design of this sensor by using the Biobrick parts we designed this year.

Pseudo-Antigen Screening Assay Using Alfa-Tag

Even the short sequences of our antigen fragments represent a massive sequence space. Meaning the identification of pseudo-antigens could prove quite challenging. As a proof of concept we designed a screening assay that should effectively and efficiently identify weak nanobody-antigen complexes, Figure 1. This assay requires three things: 1. Ni-NTA Agarose columns, 2. His-Tagged NBs, and 3. Antigen-Fragment-Tagged sfGFP. NBs are left to bind to the Ni-NTA Agarose columns. Mutant versions of the Antigen-Fragment, fused to sfGFP are passed through the column. Resulting green fluorescence in the flow-through and elution could provide broad estimates of binder dynamics. Future teams can use this design for fast and “low-resolution” binder dynamics analysis. This design could also be improved upon to produce any number of colour-based screening assays.

Pseudo-Antigen Screening Assay.
Figure 1. Pseudo-Antigen Screening Assay. A. The Alfa-Tag NB is bound inside the Ni-NTA Agarose columns. Add WT, single mutant, double mutant, and triple mutant Alfa-tags, fused to sfGFP to their respective columns. We expect the WT tag to bind tightly with the NB and reside in the column until elution with Imidazole. It is assumed that each additional mutation to the Alfa-Tag will reduce its affinity for the NB. Therefore, as we move to the right along image A we anticipate an increased concentration of sfGFP in the flow through and less in the elution, indicating a weaker bind. B. By adding time intervals, rough estimates of binder dissociation rates can be determined. The plate in image B provides the example of three consecutive wash steps followed by an elution, with each of the four steps having six replicates.

Nanobodies & The Antigen Fragments

After screening the Nanobody (NB) library, we successfully isolated a binder with affinity for our antigen fragment. We also identified strategies to improve scaffold stability and simplicity for future fragments. All of which validates our initial assumption regarding rapid target diversification. Not only can we quickly and easily identify novel binders to small helical sequences, but we can probably do so repeatedly for a range of fungal species. It is important to note that while these Nanobodies might bind our scaffolds, they may not retain this behaviour on the full-sized fungal antigen. Regardless, we are optimistic about the broader applicability of our system. In addition, the pseudo-antigen screening assay, using mutated versions of Alfa-Tag may prove to be a valuable system for identifying Nanobody-Antigen complexes that provide desired sensor dynamics (1). Future teams can use this information as a stepping stone on their way towards developing novel small binders.

REFERENCES

  1. Götzke, H., Kilisch, M., Martínez-Carranza, M., Sograte-Idrissi, S., Rajavel, A., Schlichthaerle, T., Engels, N., Jungmann, R., Stenmark, P., Opazo, F., & Frey, S. (2019). The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications. Nature Communications, 10(1), 4403. https://doi.org/10.1038/s41467-019-12301-7