Overview

Modelling

With regards to any experiment or project, there must be a high degree of preparatory work done before entering the lab to run experiments. This helps mitigate any safety concerns, prevent loss of resources, and helps greatly with the overall success of the experiment. A large amount of this preparatory work is encompassed by modelling the components of the experiment and how they interact with each other and with the surrounding environment. Many modelling software allows us to predict the folding patterns, interactions, and more with great accuracy which we can then use to design our protocols and order our lab reagents. Since our team is expressing chimeric proteins as well as proteins not found on the iGEM registry, our team dedicated a lot of time and energy to modelling all the components of our biosensor. This will not only aid in proving our concept but also ensuring our lab work would be successful!

Since our biosensor system is comprised of many working parts, we divided each aspect of the model between various teams. The wet lab team worked on modelling the protein aspect of the biosensor system, which was comprised of the target protein and the bioreceptor. The dry lab team modelled the physical device as well as the testing strip apparatus that accompanies the device and functions as the active testing site.

Wet Lab

Before integrating the bioreceptor and the proteins into a functional model, the wet lab team broke up the biosensor into discrete 3D structures. The team used these structures to predict the folding patterns of the protein and its interactions with targets to test the proof of concepts. The first and largest aspect of the model was the creation of the antibody fragment (ScFv). The antibody fragment is the expressed protein designed to bind to the target protein. To prove the concept, the team modelled a green fluorescent protein (GFP) attachment to the ScFv and for the biosensor system, the team created an alkaline phosphatase linked to the ScFv model.

Figure 1 - scFv with alkaline phosphate attached graphic, made with BioRender.com

Demonstrates the principle of a chromogenic enzyme which is intended to catalyze the conversion of large amounts of clear substrate to a coloured substrate. This allows for the identification of minute quantities of target protein to be detected. Attached to the enzyme is our single chain variable fragment (scFv) which binds to our target protein.

In earlier renditions of the project, the team also created a modular chimeric protein with a ScFv, an alkaline phosphatase, a GFP with a TEV protease cut site before the GFP, and a His-tag on the C-terminal end of the protein to create one large, multifunctional protein. However, this design was too large, therefore the team designed the two individual bioreceptors as described above. The team also modelled our target protein, ospA .

Dry Lab

The dry lab team modelled the hardware components by creating a device housing and a testing strip. The device housing consists of numerous components including the strip storage, the testing bed, and the tick puncher which were all carefully modelled and assembled inmodelling software such as SolidEdge before being 3D printed. The testing strip focuses on modelling fluid flow, capillary flow, antibody adhesion, solvent and buffer delivery, and much more. This aspect of the model deals with complex chemistry-based principles.