Team:Queens Canada/Engineering
Engineering Success
Engineering Success
Throughout the project development and testing process, the team followed the engineering cycle suggested by iGEM: Design --> Build --> Test --> Learn --> Design. This cycle has not only helped our team create successful parts and components for our prototype but has helped improve and optimize various aspects of our project. The cycle has helped us create a device that has real-world applications and has a greater functionality than the initial designs.
Examples of Engineering Design Cycle
Development of anti-ospA ScFv
During the initial phases of the project, our team focused on sifting through as much literature as possible to find ScFv candidates to use in our biosensor. Eventually finding our winner with the 3-24 cell line ScFv fragments published by Ghosh et al. Which we adapted to our binding partner in the detection process.
Design (1)
Our first iteration of the ScFv was comprised of the sequence deposted on GenBank by Ghosh et al. Which we codon-optimized for E.Coli expression.
Build (1)
To test for proper folding, we chose to build a 3D model of our ScFv using the software ABodyBuilder before testing its binding to ospA using the ClusPro molecular docking software. In this first rendition, we were unable to create a successful 3D build as the sequence appeared to be missing two critical complementarities determining region (CDR) anchor domains.
Design (2)
Our second iteration of the ScFv used the CDR anchor domains from a previous year's QGEM project to try to produce a successful render of our 3D structure as all other sequences we created were failing.
Build (2)
Using our new ScFv sequence (which we codon-optimized for E.Coli again), we managed to build a successful antibody fragment structure using ABodyBuilder software which we saved as a PDB file.
Test (2)
To test the successful creation of our ScFv, we uploaded our structure to the ClusPro molecular docking software and ran it against the ospA protein structure (PDB ID: 1fj1). A successful test would show the amino acids in the CDR regions interacting with ospA in the same locations as predicted by the literature. Unfortunately, all of the docking orientations did not match the literature and many did not bind using the CDR regions of the ScFv, suggesting we needed to redesign our antibody fragment structure.
Learn (2)
From our test results, we made the deduction that the problem likely does not lie in the heavy chain and the light chain main sequences as they have been shown to work in the literature but is likely in the regions we created ourselves. Thus, redesigning the CDR anchor regions and the linker regions will likely solve our problems or at least take our team closer to our functioning ScFv.
Design (3)
Using ABodyBuilder once more, we created a new ScFv 3D structure which appeared to have folded correctly.
Test (3)
Using ClusPro once more, our new ScFv structure was uploaded along with its binding partner ospA (PDB ID 1FJ1). After processing on the supercomputer, we discovered that the lowest binding conformation was the conformation that matched our literature sources, suggesting we had created a build that would successfully bind ospA. Our lowest energy score also had the most members included in the binding interaction by 37 residues, providing further justification for the success of our build.
| Cluster | Members | Representative | Weighted Score |
|---|---|---|---|
| 0 | 118 | Center | -323.9 |
| Lowest Energy | -330.9 | ||
| 1 | 81 | Center | -306.9 |
| Lowest Energy | -320.1 | ||
| 2 | 40 | Center | -275.4 |
| Lowest Energy | -306.0 | ||
| 3 | 40 | Center | -273.2 |
| Lowest Energy | -306.0 | ||
| 4 | 37 | Center | -337.2 |
| Lowest Energy | -337.2 | ||
| 5 | 28 | Center | -268.3 |
| Lowest Energy | -298.1 |
Learn (3)
Although we observed positive test results from our simulations, we cannot be sure the structure we created is functional until we perform our proof-of-concept test. We will continue to learn as we progress with the development of our biosensor.
Other Uses of the Engineering Design Cycles
This iterative process was also utilized when designing our prototype. The iterations for the design of the casing and device are detailed in the CAD Modeling Iterations Page.