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 called Evobodies have the potential to bind specifically to target proteins enabling various medical and analytical applications. Great advantages of our low-cost system are the short hands on time and the short generation time.
As the starting point of our system, we designed and synthesized genetic libraries encoding binding proteins based on Nanobodies as well as Monobodies. The diversity of the respective library in E. coli is continuously increased by co-expressing a special DNA-Polymerase conferring 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 providing growth advantage to antibiotics in relation to protein-protein interaction strength. Ultimately, desired clones are enriched during fermentation in batch or in continuous culture.
Binding capability of our libraries, efficiency of our selection system and potential of our mutagenesis system were demonstrated. Moreover, library diversity and mutation system characteristics were analyzed in detail by high-throughput sequencing.
As the starting point of our system, we designed and synthesized genetic libraries encoding binding proteins based on Nanobodies as well as Monobodies. The diversity of the respective library in E. coli is continuously increased by co-expressing a special DNA-Polymerase conferring 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 providing growth advantage to antibiotics in relation to protein-protein interaction strength. Ultimately, desired clones are enriched during fermentation in batch or in continuous culture.
Binding capability of our libraries, efficiency of our selection system and potential of our mutagenesis system were demonstrated. Moreover, library diversity and mutation system characteristics were analyzed in detail by high-throughput sequencing.
Achievements
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Design and construction of an Evobody library and invention of a new BioBrick class |
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Over 100,000 clones per library generated |
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High diversity of the plasmid library confirmed by high-throughput sequencing |
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Functionality of the library was demonstrated by binding to various targets |
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Construction and detailed characterization via high-throughput sequencing of a plasmid-specific mutagenesis system |
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Construction and characterization of several parts for reversion assays |
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Plasmid sequence improvement by re-sequencing and de novo assembly |
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Construction and characterization of a bacterial two-hybrid selection system |
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Prediction of the outcome of our system |
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Evolution-based human practice projects were perfectly integrated |
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Concept development for industrial upscaling |
