It is utmost importance to our project to separate bacteria with high affinity binding proteins from bacteria with low or moderate affinity binding proteins. Therefore, we need an efficient selection system. One approach is based on the concept of the very common yeast two-hybrid system, but optimized for bacterial selection directly in E. coli (Badran et al. 2016).
An overview of the system in action is given in Figure(1).

The bacterial two-hybrid system is a possibility to separate cells from each other through differences in protein-protein interaction strength(Hu et al. 2000). The whole system works in vivo and gives bacteria with strong interaction of two chosen proteins a high advantage in their growth ability. This growth difference distinguishes such bacteria from bacteria with very weak protein interactions. This method can be applied to our Evobody generation system by constructing a hybrid transcriptional activator system. This system comprises two fusion proteins. One protein contains a DNA binding domain fused with our target protein (1). The second protein consists of our Evobody fused to a subunit of the RNA polymerase(2). The DNA binding domain of the first fusion protein binds to a specific DNA pattern upstream of the promoter sequence of a reporter gene (3). If there is an interaction between the target protein and the Evobody (4), the interaction leads to a recruitment of the fused subunit of the RNA polymerase to the promoter (5). Therefore, the RNA polymerase can only express the reporter gene, for example a beta-lactamase, if the interaction of the target and the Evobody is strong enough (6). Otherwise, a weak or unavailable interaction (7) leads to no reporter gene transcription at all (8). Therefore, it is possible to raise the antibiotic concentration to increase the selection pressure on cells. The results are optimized Evobodies with high affinity towards the target (9).

Our system is based on two fusion proteins. The first protein consists of our target protein fused with a DNA binding domain. In very early hybrid transcriptional activation system the frequently binding domains were different zinc finger proteins (Joung et al. 2000). Each of these proteins has a so called zinc finger domain, which is able to bind at specific DNA sequences (Klug & Rhodes 1987). The zinc finger domain of the murein transcription factor Zif268 was most frequently used in two-hybrid systems. Three individual zinc finger motifs collectively bind a nine base-pair long sequence (Pavletich & Pabo 1991). Due to the development of more complicated experiments more zinc finger proteins per experiment were needed. This results in a big prone for faults, because the specificities of individiual zinc finer proteins can overlap and can depend on the context of surrounding zinc fingers and DNA (Ramirez et al. 2008). Off-target effects were observed instead of a high affinity to the intended target. The revision of the zinc fingers make them on the one hand way more effective but also on the other hand way more complicated to use (Maeder et al. 2008; Sander et al. 2011).
Therefore, DNA binding domains, which are a lot easier to use for bacterial two-hybrid systems are necessary. Examples are, the repressor proteins cI of the phages lambda (Dove & Hochschild 2004) and 434 (Hays et al. 2000), respectively. The protein family cI are repressor proteins that compete with the phage Cro proteins for DNA binding. The main function of them is the binding at the binding sites OR1 and OR2 on the DNA. If they bind at these sites they prevent that Cro can bind at the binding sites OR2 and OR3 and thereby inhibit the expression of the cro gene (Brooks & Clark 1967). Further researches revealed that next to the binding sequences OR1 and OR2, also OL1 and OL2 downstream of the OR1 and OR2 sites are necessary for complete repression of the cro gene. Therefore, cI binds at the DNA as an octamere (Dodd et al. 2001).
The sites OR1 and OR2 are important, because the binding domain is only used to anchor the fusion protein close to the promoter. Afterwards, gene expression is increased by attracting the activation domain which is fused to the binding protein (Joung et al. 2000). All cI protein have specific binding sequences namely OR1 and OR2. These cI binding sequences are species specific and therefore different between diverse varying phages isolates. However, the design of a hybrid transcriptional activation system with cI as binding domain is easier than the use of a zinc finger protein. This is the due to the binding sequences for a specific cI, which does not require optimization like zinc fingers do.
Comparison of cI proteins to zinc finger proteins revealed a much higher expression rate of a reporter in a designed bacterial two hybrid system, if cI proteins is used as the DNA binding domain (Badran et al. 2016). Due to the easier design and higher expected profit, we decided to use the cI protein of the phage 434, as the DNA binding domain for our system.

The second fusion protein of the hybrid transcriptional activation system is our Evobody fused to an activation domain. This domain is the impeller of the system. If the activation domain is recruited to the promoter through the interaction of the binding protein (the Evobody) and the DNA anchored target protein the gene expression of the reporter gene is strongly increased(Dove & Hochschild 2004). The most commonly used activation domain is the alpha subunit of the DNA-dependent RNA polymerase A or in short RpoA. Several early publications about bacterial one- or two-hybrid systems are based on this subunit as activator (Joung et al. 2000; Dove & Hochschild 2004, Fu 2004, Durai et al. 2006). RpoA is a subunit of the core complex of the DNA-dependent RNA polymerase. The interaction with the promoter as well as the connection to the regulatory proteins is established via RpoA. Therefore, it is essential for the initiation of the subunit assembly of the RNA polymerase (Ishihama 1992).
RpoZ, the much smaller omega subunit of the RNA polymerase, which could also be used as transcriptional activator. It is mainly important for the facilitation and stabilization of the assembly of the RNA polymerase (Mathew & Chatterji 2006). RpoZ outperformed RpoA and a special phage polymerase subunit in a gene activation comparison (Badran et al. 2016). Therefore, we decided to use the RpoZ as activator domain.

Every system should be validate by analyzing a positive control. The two hybrid transcriptional activation system converts the binding affinity of two proteins in corresponding expression intensity. A well-documented protein-protein interaction is the binding of the regulator protein Gal4 of Saccharomyces cerevisiae with a modified version of the Gal11 regulatory protein Gal11P (Jeong et al. 2001). The transformation from Gal11 to Gal11P results in a mutation in only one amino acid (Himmelfarb et al. 1990), which allows the binding of Gal11P in the dimerization domain of Gal4 (Hidalgo et al. 2001).

In addition to the Gal4-Gal11P interaction a second positiv control should be tested. Having our library in mind we looked for a control that was very similar to our Evobodies. One protein with good structural similarities is the antibody mimic or monobody HA4. (Strukturelle Ähnlichkeit zeigen) This monobody was designed by a working group in Chicago searching for an antibody mimic, that can bind the SH2 domain of the tyrosine kinase Abelson with low nanomolar affinity (Wojcik et al. 2010). The results of this work show an equally well interaction in vitro and in vivo, respectively. To validate the potential of the hybrid transcriptional activation system HA4 mutants are also necessary. Single amino acid substitutions change in most cases the structure of the protein and lower or increase the interaction affinity with other proteins (Brender & Zhang 2015). In single mutation R38A in HA4 causes a reduction of transcriptional activation of the reporter gene of 35 to 40%. The mutation Y87A is even more detrimental leading to only 5 to 10% of the native binding affinity (Badran et al. 2016).
These controls allowed a detail characterization of our system. It is possible to show that different binding affinities result in differences in the reporter gene expression.

A selection system should be characterized based on different proteins with known mutual affinity values. A strong correlation between the binding affinity of the interacting proteins and the reporter gene activity is required. The selection of hogh affiniy Evobodies necessitates a reporter that confers growth advantage to the cells of interest. A good choice for such a reporter is an antibiotic resistance gene like bla against ampicillin (Livermore 1995) or tetA againsttetracycline (Roberts 1996), respectively. Increase of the antibiotic concentration would lead to a decrease of a bacterial population. Finally, the best Evobodies should allow the survival of the cells. The native lacZ pomoter was modified to achieve a ten times greater expression rate when bound by a cI-RpoZ fusion protein (Badran et al 2016). Moreover, the distance of the 434 cI binding site to the transcription start site was optimized by the working group (Badran et al 2016). The optimal distance of the binding site is exactly 61 basepairs upstream of the transcription start site (Badran et al. 2016).
The final design of our promoter is given in figure (X).

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