Without mutation our world would look very different to how we see it today. The evolutionary process, as we know it, is mainly based on the variability produced by mutagenesis and recombination. While recombination redistributes already existing features in nature, mutagenesis may vary existing ones and create completely new ones. In nature the process of mutagenesis is random which means quite a lot of unsuccessful variants regarding survival. A successful mutation, on the other hand, may be the first step to a new and better adapted species. But an advantageous mutation in one species may bring chaos to another. A predator for example may profit from a mutation but its prey could quickly become endangered. In this race more mutations in a shorter period of time could make the difference in survival or extinction. In a way this, so called mutation rate, could be seen as the speed of evolution. One of the highest mutation rates known today is present in the family of Flaviviridae. This group of viruses contains popular members like Dengue and Zika virus. Less than 150 years ago, in the blink of an evolutionary eye, Dengue was only able to infect apes. Mutations enabled it to extend the host range to infect humans as well. But not only the host range but also the mortality of a virus could be changed by only a few mutations.
That is why fight fire with fire. By applying a special mutagenesis system, we are able to strongly enhance mutation rate compared to the numbers present in nature. To create an effective evolutionary system we do not only need an efficient mutagenesis system, but also need to use it during growth of the mutating organism. This is why we use in vivo mutagenesis. It may seem less efficient than conventional laboratory mutagenesis but it provides an advantage which can not be bestowed by in vitro techniques: Evolution.

One of the elementary requirements of every evolutionary process is variability, which can be achieved by mutagenesis. The alteration of existing genotypes is the only way to discover new features and abilities of proteins and organisms. The aim of our mutagenesis system is to create a diversity of binding proteins during the course of fermentation to gain as much different Evobodies as possible. Building on the library approach and leading to the selection system, the mutagenesis represents the central part of our project. After transformation of the library and an initial selection round the mutagenesis has to take over. That is why we are using a system that is capable of in vivo DNA alteration. Two different approaches were applied to reach this aim.

The mutagenesis plasmid is rather unspecific and leads to a general raise of mutations in the DNA of host organisms. This system was used to get a manifold increase of base pair exchanges and a strong repression of bacterial DNA repair mechanisms. Six different proteins build this mutation system.

A more specific approach is the two plasmid mutagenesis system (Camps et al., 2003). It is based on a modified DNA polymerase I, which replicates the first part of plasmids belonging to the ColE1-family, like pSB1C3.

In the following, we will elaborate on why and how we implemented these two systems in detail in our project.