Contribution
We designed cell surface display platforms to display nanobodies on probiotics including yeast, E.coli Nissle 1917, and L.lactis. We were able to verify the possibility of expressing nanobodies using the Aga2 surface display system. We also designed a novel way to detect protein surface expression by using fluorescence-activated cell sorting (FACS). Another contribution is the protein modeling protocol. We designed an integrated protocol involving the usage of PyMol, RobeTTA, and Alphafold2. Through literature review, we were able to determine multiple parameters that measure the accuracy of the protein model and evaluated our protein design. We also contributed to previous parts by designing our parts with improvements based on past iGEM teams. Another contribution is the PCR troubleshooting guide we made during the collaboration with Duke. It contains actual wet lab gel pictures and troubleshooting advice for future iGEM teams to reference. Last but not least, we created a model for our iGEM team members to collaborate using GitLab as an open source platform.
Cell surface display platforms
Cell surface display platforms can be utilized to cater to many protein engineering and therapeutic drug design purposes. In our case, the therapeutic effect and the nanobody localization on the cellular membrane of yeast yield future possibilities for iGEM teams to utilize this surface display platform based on the yeast Aga2 protein, specifically for therapeutic designs, but possible to be utilized to express other proteins as well.
Integrated protein modeling protocol
Online programs help visualize and analyze the structure of different proteins and their docking scenarios. We use Robetta and Alphafold2 to model the structures of our protein, and used HADDOCK to visualize the docking structure. The evaluation focuses on different parameters: Ramachandran plot, Overall G-factors, Atomic Z-score RMS, Percentage of the amino acids having scored >= 0.2 in the 3D/1D profile, Overall quality factor, and Z-score. These parameters determine whether a certain structure is stable. For more details, please see our Model Page.
Improvement of parts
PCR troubleshooting guide for future iGEM teams
In our collaboration with Duke iGEM, we put together a PCR troubleshooting guide, summarizing all the different problems we encountered during PCR in wet lab.
See our trouble shooting guide here.
Collaborations using GitLab
To ease the writing of the Wiki and manage resources efficiently, we conducted all collaborations within our team using GitLab. We created a monolithic repository for all our documentation way before iGEM announced that GitLab will be their new Wiki platform. And, we use it to track every event and task using GitLab Issues, including our brainstorming, lab design, and Wiki design process.
GitLab is very helpful for our collaborations. All issues, responses to issues, and document changes are well recorded on the Activity tab, and due dates and assignments for individual are used to help us finish our tasks in a timely manner. GitLab CI allows us to build our Wiki and preview it after every push without manual intervention. The power of Git also enables us to track down mistakes made to our Wiki.
We believe that our model collaborating using GitLab will be a good reference for future iGEM teams so we decided to open source our repository to help them scale their documentation project well.