In the Evobody generating process we want to obtain a high affinity binding protein by directed evolution.
To further evolve binding proteins with an innate ability to interact with our target we want to mutagenize
them and thereby enable an ongoing adaptation to the target.
We successfully cloned two different approaches to in vivo mutagenesis, demonstrated and quantified
mutagenesis by reversion assays and determined the mutation spectrum by next-generation sequencing.
Cloning
We added several in vivo mutagensis parts to the iGEM parts registry
From our mutators we quantified the mutation by reversion assays using a stop beta lactamase.
Figure 1: Mutagensis as reversion rate for the error prone polymerase I in comparison to wild type polymerase I. Frequency of ampicillin resistance colonies per complete cell count was determined.
Figure 2: Mutagenesis rate of our genome wide mutators as reversion rate for the error prone polymerase I in comparison to wild type polymerase I. Frequency of ampicillin resistance colonies per complete cell count was determined.
Furthermore we determined the mutation rate of our error prone polymerase I as well as from our genome wide mutator
BioBrick BBa_K2082117 by high-throughput sequencing.
Figure 3: Mutagenesis rate of our genome wide mutators BBa_K2082116 and BBa_K2082117.
Figure 4: Mutagenic spectrum of our error prone polymerase I and our genome wide mutators BBa_K2082117 and BBa_K2082116
