When one takes a look into a cell he quickly finds the mechanism of DNA replication. The polymerases are responsible for the replication of the entire DNA. One of this polymerases is the DNA polymerase I, which plays a role in lagging-strand replication of chromosomal DNA. Moreover, the DNA polymerase I also has the task to do proofreading and plays a role in replicating ColE1 plasmids [Camps, 2003]. ColE1 plasmids are characterized by the ColE1 origin of replication (ori). During the lagging-strand synthesis the DNA polymerase processes RNA primer (˜20 nt) and fills gaps during DNA repair reactions [Allen, 2011]. Sometimes the polymerase does mistakes while synthesizing a DNA strand. These mistakes can have big effects. It can lead to diseases or death of the organism, when a protein is damaged that is essential for survival. To reduce the frequency of mistakes the DNA polymerase I checks his work through proofreading.

To produce more diversity in our library we use an in vivo mutagenesis system, which is based on an error prone DNA polymerase I (EP-Pol I) [Troll, 2011]. Thus, the mutagenesis system generates mutations during replication. It is designed as a two plasmid system and is beneficial to our approach due to the selectivity towards a specific plasmid. Only the plasmid encoding the target protein sequence is mutated.

Prof. Dr. Manel Camps from the University of Santa Cruz investigated a DNA polymerase I, called EP-Pol I, which lacks in correct synthesis and in proof-reading [Camps, 2003]. In contrast to the normal DNA polymerase I the EP-Pol I has three point mutations, I709N, A759R and D424A [Camps, 2003]. The I709N mutation is located in motif A. This is a conserved sequence in the palm domain of the polymerase active site. The mutations leads to an enlargement of the substrate-binding pocket, which is a possible explanation for the increased error rate [Camps, 2003]. The D424A mutation in the exonuclease domain leads to deactivation of the proofreading activity of the EP-Pol I. The amino acid replacements A759R is located in the O-helix, which is a conserved sequence (motif B) that lies close to the polymerase active site on dNTP binding. This may stabilized the enzyme with similar conformation, which leads to miss integration [Camps, 2003].

According to Jennifer M. Allen, the EP-Pol I produces evenly distributed mutations and generates base pair substitutions (transitions) and transversions [Allen, 2011]. Another advantage of the EP-Pol I is that it only replicates and thus mutates plasmids that have a ColE1 ori and that the EP-Pol I processes Okazaki fragments [Allen, 2011]. Therefore, no significant increase in the mutation rate in the chromosomal DNA was observed [Camps, 2003]. During replication the EP-Pol I is replaced by DNA-Polymerase III. This leads to the conclusion that with increasing distance from the switch the frequency of EP-Pol I mutations decrease [Allen, 2011]. In this paper they also wrote that the EP-Pol I synthesizes 400 nt to 500 nt after the ori, but during a skype conversation with Manel Camps, he said that this is not the case. It seems that the mutations are randomly distributed over the plasmid. According to [Alexander, 2014] the EP-Pol I does more than 1 mutations per kb. The substitution of the several bases has different probabilities (figure 1).

For efficient use of the EP-Pol I it is recommended to use the E. coli JS00 strain that carries a knockout of the native Pol I as well as a temperature sensitive DNA polymerase I, which can compensate the knockout at certain temperatures [Camps, 2003]. With the use of JS200 it is possible to switch between EP-Pol I (mutation phase) and DNA polymerase I (no mutation phase). At 30 °C the DNA polymerase I is active and synthesizes the plasmids, because it is more efficient and faster than the low fidelity EP-Pol I [Camps, 2003]. When changing to 37 °C the DNA polymerase I is no longer active and the EP-Pol I comes to work and begins with mutation [Alexander, 2014].

To effectively occupy the EP-Pol I we use a two-plasmid system Manel Camps. One plasmid has a pSC101 ori (Pol I-independent) and the EP-Pol I sequence and the other plasmid has the ColE1 ori and our Evobody sequence (Fig. XXXXX). Therefore, only the plasmid with the Evobody sequence will be mutated. This is a form of directed evolution, because it doesn't mutate everywhere [Alexander, 2014]. Also with the use of JS200 strain we are able to switch between mutation and no mutation phase.

• [Alexander, 2014] Alexander DL, 2014. Random mutagenesis by error-prone pol plasmid replication in Escherichia coli. Methods In Molecular Biology (Clifton, N.J.), 2014, 1179, 31.
• [Allen, 2011] Allen JM, 2011. Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication. Nucleic Acids Research, 2011, 39, 16, 7020.
• [Badran, 2015] Badran AH, 2015. Development of potent in vivo mutagenesis plasmids with broad mutational spectra. Nature Communications, 2015, 6, 8425.
• [Camps, 2003] Camps, Manel, 2003. Targeted Gene Evolution in Escherichia coli Using a Highly Error-Prone DNA Polymerase I. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100, 17, 9727.
• [Troll 2011] Troll, C, 2011. Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli. JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, 2011, 49.