We developed a novel system for generating binding proteins in E. coli via directed evolution. Our system can be subdivided into a library, mutagenesis and selection. The library was designed and created by randomizing binding protein sequences in E. coli. Our library reached the size of over one hundred thousand of each, Monobodies and Nanobodies.

The mutagenesis system uses a error prone polymerase to introduce mutations in the coding regions for our binding protein. Therefore creating an even greater variety. We verified the functionality of the system by various reversion experiments to show that it is functional. Additionally the mutation rate was measured by Miseq sequencing.

Our selection system uses 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 this we run several experiments (expression control, binding control, interaction control, in vivo test ) and verified its functionality.

For our future prospect we designed a business plan where we considered the necessary points to introduce our project as the system to generate Evobodys itself as well as a company that produces binding proteins. To further develop that thought the necessary process operations were determined and are shown in flow diagrams to showcase the necessary steps to be taken from a custom binder order to the finished lyophilized product for shipping. Because we were only working in the dimensions of our lab we reached out to experts and asked for their opinion on up scaling our idea. They provided a lot of parameters to be considered and asked questions we up to this point did not thought about. We integrated their knowledge on the topic into the development of the up scaling and business plan as well as the process development. The ultimate aim for our system would be that it can generate binders to every target. Therefore we ran a Phagemid display in order to evaluate the capabilities of our system. We already tested a protein domain, a transcription factor and an enzyme successfully. We look forward to further improve our system in the future!