Difference between revisions of "Team:Bielefeld-CeBiTec"

We developed a novel and easy to use system for the generation of binding proteins in E. coli via in vivo directed evolution. Resulting proteins have the potential to be utilized for therapeutic or diagnostic purposes such as target-mediated drug delivery, immunodetection, or passive immunization. The system is particular useful for quick on-site generation of binders of evolving pathogens such as viruses. Moreover, many applications for basic research are within reach with limited hands on time and at low cost. As the starting point of our system, we designed and synthesized a library of sequences encoding binding proteins in E. coli based on Nanobodies as well as Monobodies. These libraries were cloned and are suggested as a new entity of BioBricks. The diversity of the library in E. coli is continuously increased by co-expressing a special DNA-Polymerase inducing plasmid restricted error-prone replication of the binding protein expressing plasmids. Finally, binding proteins with high affinity to the target protein are selected using a bacterial two-hybrid system conferring growth advantage to antibiotics in relation to protein-protein interaction. Ultimately, desired clones are enriched during fermentation either on plates or in continuous culture. Next to cloning the libraries, the selection and the evolution system, we validated the mutation system, defined the selection efficacy of our system and selected binding molecules.