Our computational methods, including the pipeline to generate candidate mutations that might improve binding affinity between a protein and a target ligand, are freely available for any future team to use, and instructions on how to use it are included. Moreover, our educational resources, documented in our Education Section, are available for other teams to use and reference.
The goal of our project was to create a robust computational pipeline that can speed up protein engineering in all kinds of research. Thus, our pipeline is fully accessible for researchers to use, including future iGEM teams. This computational tool can assist iGEM teams in their protein development, and marks our primary contribution to future teams.
For teams to use:
Step 1: Determine the sequences of receptors that are most evolutionary related to the Ste2 receptor.
Step 2: Use the ColabFold AlphaFold2 using MMSeqs2 (found here) to predict protein fold of the reference and evolutionary-related receptors complexed with Alpha-Factor and Cystatin-C.
Step 3: Determine the binding residues involved for each receptor when bound to Alpha-Factor and Cystatin C through the use of Prodigy, and find the common binding residues between these groups.
Step 4: Determine the pairwise alignment between the reference Ste2 and the evolutionarily related receptors.
Step 5: Create a list of candidate residues to mutate (using the criteria of “binding” and high Shannon entropy described in Project Modeling).
Step 6: Computationally mutate to the evolutionarily related amino acids and calculate the binding energies of the mutated receptor with Cystatin-C
All relevant scripts for each step can be found on our Github page, available to all teams for future use! Code may be used as long as it is attributed to Harvard iGEM.
Through our collaboration with McGill, we have seen how other teams can benefit from using this pipeline, needing only to make project specific modifications to the code and changing the inputs accordingly. The feedback we received from McGill helped us validate and hone our project’s usefulness to other teams. You can read more about our collaboration with them here. We hope that our project is a useful contribution not only to future teams, but to researchers from many institutions as well.
In addition, our team created an educational lecture series on synthetic biology, which you can read more about on our Education and Communication page. These materials are available to all iGEM teams, and to everyone who wants to learn more about synthetic biology!