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 uses the in vivo mutagenesis capabilities of an error-prone polymerase I
BBa_K2082106.
This enzymes replace the normal polymerase I under certain conditions (see here) and introduces a high amount of mutations
inside our Evobody coding sequence. In that 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 directly in vivo.
Characterization
We characterized the error prone polymerase I per performing reversion assays, thus quantifiying the mutation rate.
Figure 1: Increase in mutation rate when using the error prone polymerase I (BBa_K2082106) in comparison the using wild type polymerase I (BBa_K2082107).
Furthermore we analyzed error-prone polymerase I mutation spectrum using Illumina sequencing.
Figure 2: Mutatagenic spectrum of error prone polymerase I (BBa_K2082106) in comparision to wild type polymerase I.
In combination we benchmarked the error-prone polymerase I by working out all necessary information to use this BioBrick in further directed evolution applications.
