Integral to our project is the directed evolution of our Evobodies. To continuously introduce new Evobody variants with potential binding capabilities into the population we used the in vivo mutagenesis capabilities of the error-prone polymerase I BBa_K2082106.
This enzyme replaces the normal polymerase I under certain conditions (further information) and introduces a high amount of mutations inside our Evobody coding sequence. In because the mutations are more frequently inside our Evobody sequence and rarely inside other genomic proteins this in vivo mutagenesis approach circumvents the main problems restraining in vivo mutagenesis: the unintentional mutagenesis of essential proteins of a bacteria.
By adding BBa_K2082106 to the iGEM parstreg and characterizing it we hope to give coming iGEM teams the possibility to easily optimize their proteins by means of directed evolution in vivo.

We characterized the error prone polymerase I by performing reversion assays, thus quantifiying the mutation rate.

Furthermore we analyzed error-prone polymerase I by high-throughput sequencing obtaining the mutation rate as well as the mutation spectrum.

In combination we benchmarked the error-prone polymerase I by working out all necessary information to use this BioBrick in further directed evolution applications.