We developed a novel system for generating binding proteins in E. coli via directed evolution. The concept of our system subdivides into a library, a system for mutagenesis and a selection system:

At first, we designed and created a library of random binding protein sequences in E. coli to form the starting point of our project. We reached a library size of over one hundred thousand of each, Monobodies and Nanobodies. We verified our library by finding multiple potential binders against diverse targets and further by sequencing the library with different techniques. By that, we created a great foundation for our following directed evolution and an unprecedented part collection for all other iGEM teams, who want to build their own library.

Furthermore, we used an error prone polymerase I by which we introduced mutations in the coding regions for our binding proteins. By this, we created an even greater variety of different Mono- or Nanobodies. We verified the functionality by various reversion experiments to show that our error prone polymerase is working correctly. Moreover, we examined the precise mutation rate and positions by Miseq sequencing. This working mutation system can be used by future iGEM teams for various applications.

Last but not least, 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. To proof that our selection system is working, we executed several experiments, ranging from expression controls over binding controls to interaction controls and in vivo tests. With this, we provide a functional bacterial two hybrid system for other iGEM teams to work with.

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In a nutshell, we have proven that all parts of our project work as expected by various control experiments and made it iteratively even better by modeling our system, integrating experts and public reviews and also fermentation.
In particular, we have reached all of our following three milestones: We designed and created two functional libraries with high diversities, we assembled a working mutation system and we implemented a functional bacterial two hybrid selection system.