Engineering Success

Building on the 2023 ULethbridge Collegiate iGEM Team’s Club² project, where the target protein PbEL04 was identified and designed to bind with a chimeric GFP (COAPE-GFP) for detecting the Plasmodiophora brassicae pathogen, we wanted to focus on demonstrating the feasibility of a protein-based detection kit. We aims to develop a chimeric protein by transferring the GFP binding site into a nanobody or fragment antigen-binding (FAB) with PbEL04. This addresses the key challenge of optimizing binding specificity for improved detection.

This year we wanted to be more specific with our detection, so we utilized traditional and computational methods to develop engineered antibody fragments against PbEL04.

Upon the completion of successful transformation of the constructs (nanobody and FAB) in DH5a and Rosetta (DE3), we began to outline a plan for expression screening.

With low levels of nanobody expression as well as the calendar moving quickly, we opted to focus solely on optimizing our expression screening of our nanobody after consulting with our PI and advisors. Furthermore, a literature review revealed the difficulty of purifying the FAB compared to the nanobody leading to this shift in our approach. Our discussions with advisors and our PI outlined a plan for the nanobody expression screening involving 6 conditions with 37°C and LB likely to be selected.

After encountering challenges with the purification of PbEL04 due to dimer formation, the team shifted focus to refining purification techniques. Advisors suggested using size exclusion chromatography (SEC) to separate monomers from dimers, while adding beta-mercaptoethanol (BME) to buffers was recommended to prevent dimerization.

In order to understand how Pbel04, FAB and/or the Nanobody interact or clash we need to run a contact analysis. We decided to use Chimera to do this because of its simplicity compared to alternative softwares such as Pymol. In this way, newer Drylab members can contribute to both contact analysis and various other analyses with a relatively small learning curve to overcome.

In order for sufficient contacts to be demonstrated in silico, alteration of our constructs needed to be done, this meant the addition of a lysine region to Fab and the Nanobody.

In order to determine the name and function of the pbel04 and FAB/nanobody (as well as discover any alternative proteins) we need to run a protein BLAST. BLAST was run using Plasmodiophora Brassicae as our reference organism with all other settings set to default.In order to run BLAST we used a FASTA sequence found in Uniprot. Ahead of the BLAST results, we will then run ALPHA FOLD with each protein to determine its structure, how the structure relates to its function and how we can utilize its structure in future iterations of our project. We ran both softwares for proteins encoded by the ascension number BBF98784, R4I7S9 A0A0G4IKW4 and A0A0G4IRM5.

Gramm docking is used to determine how effectively our RECEPTOR and LIGAND will bind. By performing this test in-silico, we can further iterate on and understand wet lab results. In order to setup gramm docking, our docking and methodology was set to free docking, our number of results was limited to the top 5 and the number of scan matches was set to 150000. Our receptor was PBEL04 and our ligand was either FAB or the FAB Nanobody.

After the addition of the lysine region binding occurred precisely where other analysis determined it to be.

To predict the behavior of PbEL04 at specific temperatures, molecular simulations were conducted. This approach aims to assess the effectiveness of our lateral flow in situ. Initially, GROMACS was employed on a gaming PC due to the infeasibility of accessing a supercomputer or server cluster. Two preliminary simulations were performed to evaluate the viability of using a gaming PC for molecular dynamics studies.

Following recommendations, simulations were run at 25, 30, 35, 40, 45, and 70°C. Due to the shorter lengths of our simulations compared to various studies, varying temperatures were used to identify significant changes in output data more quickly. GPU acceleration was not implemented due to insufficient understanding of the computer systems. Future analyses of RMSD, RMSF, SASA, hydrogen bonds, and radius of gyration will provide insights into how protein structure varies with temperature.