We developed a novel in vivo system for generating binding proteins in E. coli via directed evolution. The concept of our system subdivides into a library for initial diversity, a system for mutagenesis to further increase the diversity and a selection system to identify high affinity binding proteins.

At first, we designed and created two libraries of partly random binding protein sequences in E. coli based on Monobodies and Nanobodies, respectively. Each library reached a size of over one hundred thousand clones posing a solid start point of our system. High diversity of the library was confirmed by stade-of-the-art high-throughput sequencing. Applicability of our library was confirmed by finding multiple potential binders against diverse targets. This library poses a valuable resource for our following directed evolution. Moreover, we provide an unprecedented part collection to the whole iGEM community enabling the application of libraries in future projects.

Furthermore, we used an error prone polymerase I for targeted mutagenesis of the binding protein encoding plasmid. This system is well suited for the diversification of initial binding proteins derived from our library. This mutagenesis system was functionally characterized by various reversion experiments. Precise mutation rate and mutation types were analyzed via high-throuput sequencing. In contrast to genome-wide mutation system, our system is especially useful to future iGEM projects to apply mutagenesis of BioBricks within the standard plasmid.

Finally, we used a bacterial two hybrid system to give cells with fitting binding proteins to the target protein an advantage in growth by developing an antibiotic resistance. Functionality of this system was demonstrated by several experiments, ranging expression controls over binding controls to interaction controls and in vivo tests. In summary, we provide a functional bacterial two hybrid system for other iGEM teams to work with.

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In a nutshell, we provide evidence that all devices of our project work as expected. Moreover, we made it iteratively even better by modeling our system, integrating experts and public reviews. Industry scale applicability was indicated by continuous fermentation experiments and a business plan.
In particular, we reached all of our milestones: (1) We designed and created two functional libraries with high diversities, (2) we assembled a working system for plasmid-targeted mutagenesis and (3) we implemented a functional bacterial two hybrid selection system.