We implement a basic diversity of initial binding proteins by creating a high-quality library consisting of over one hundred thousand Monobodies and over onehundredsixty thousand Nanobodies. By this and our part collection we set up the foundation to build our system on and set a cornerstone for utilizing and generating libraries in iGEM. We verified our binding protein libraries with Phagemid display and high-throughput sequencing techniques.
Figure 1: Nanobody randomized CDR3 of 24 colonies. Top to bottom: Ordered sequence, chromatogram and sequencing result. Randomized regions show the occurence of the expected bases.
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 for in vivo mutagenesis, demonstrated and quantified mutagenesis by reversion assays (figure 2) and determined the mutation spectrum by next-generation sequencing.
Furthermore, we determined the mutation rate of our error prone polymerase I as well as of our genome wide mutator
BioBrick BBa_K2082117 by high-throughput sequencing.
The main selection system applied for the whole Evobody generator was a bacterial two-hybrid system. This method is able to select whole bacterial cultures based on their binding strength of the Evobody against a desired target. A lot of parameters of the system were checked by showing the interaction possibility of the positive controls, the binding of the cI DNA binding domain and the expression rate of the fusion proteins itself. However, the most important achievement was the demonstration of a whole working bacterial two-hybrid system.
With our mutagenesis system, we want to find the best adapted E. coli culture, and we want to find this culture as quickly as possible. Therefore, we looked for a tool that can predict us, if the desired culture can be found at all, and, if yes, which culture will be the winner. Additionally, the prediction should provide us optimal parameters for the laboratory, the best cultivation time and inoculation number. The program computes a minimal cultivation period of 8 hours and at least 11 inoculations, when an initial binder exists (figure 4).
One of the fundamental aspects when establishing a directed evolution approach is to grant an advantage to the individuals with favorable properties. In our setup we coupled correct binding properties of our Evobody proteins to a growth advantage in the form of a gradual ampicillin resistance. To determine the applicability of this resistance and how selective pressure can be applied, we did several fermentations to characterize the used strain.h
