One big step of our system is the fact, that we need something to separate the bacteria with the good binding proteins from the bacteria with bad binders. Therefore, we need a selection system. This selection system is based on the concept of the very common yeast two-hybrid system, but optimized for bacterial selection directly in E. coli.
An overview how the system should work is given in the illustration below.

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. One approach to use this method in our Evobody generation system is the possibility to build a hybrid transcriptional activator system. This system comprises two fusion proteins. One protein contains a DNA binding domain fused with our target protein. The second protein consists of our Evobody fused to a subunit of the RNA polymerase. DNA binding domain of the first fusion protein binds to a specific DNA pattern near the promoter side of our reporter gene. If there is an interaction between our target protein and our Evobody these interaction leads to a recruitment of the fused subunit of the RNA polymerase to the promoter. Therefore, the RNA polymerase can only express the reporter gene, in example a β-lactamase, when the interaction of the target and the Evobody is strong enough. For the selection it is possible to use a raising antibiotic pressure to filter out our best Evobodies with the strongest affinity to our target.

Our system is based on two fusion proteins. The first protein is parted in our target protein fused with a DNA binding domain. In very early uses of a hybrid transcriptional activation system the main binding domains in use 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). In two-hybrid systems the zinc finger based on the zinc finger domain of the murine transcription factor Zif268 is the most popular one. Three individual zinc finger motifs collectively bind a 9 base_pair sequence (Pavletich & Pabo 1991). With time and more complicated experiments the zinc finger assays were more and more prone for faults. They started to fail to bind their intended target (Ramirez et al. 2008). The revision of the zinc fingers make them on the one hand way more effective but also on the othr 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, like the repressor proteins cI of the phages lambda (Dove & Hochschild 2004) and 434 (Hays et al. 2000). The protein family cI are repressor proteins which 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 inhibit the expression of the cro gene (Brooks & Clark 1967). Further researches has shown, 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. In the next step 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 OR1 and OR2. These sequences are different for diverse cI proteins of varying phages. But the design of a hybrid transcriptional activation system with cI as binding domain is easier than the use of a zinc finger protein, because the binding sequences for a specific cI are always the same and you do not have to optimize it for every new experiment like for the zinc finger proteins.
The direct comparison of an zinc finger protein and the cI proteins of 434 and lambda shows, that the expression rate of an reporter in a designed bacterial two hybrid system is much higher, if one of the 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 a cI protein, namely cI of the phage 434, as the binding domain for our system.

The second fusion protein of the hybrid transcriptional activation system is our Evobody fused with a activation domain. This domain is the impeller of the system. If it is recruited 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. A lot of early publications about bacterial two-hybrid systems are based on this subunit as activator (Joung et al. 2000; Dove & Hochschild 2004). RpoA is a subunit of the core complex of the DNA-dependent RNA polymerase and is on the one hand necessary for the interaction with the promoter DNA and with the other regulatory elements and on the other hand necessary for the initiation of the subunit assembly of the RNA polymerase (Ishihama 1992).
Another possible activator is next to the alpha subunit, the much smaller omega subunit of the RNA polymerase called RpoZ. RpoZ is the smallest part of the RNA polymerase and is mainly important for the facilitation and stabilization of the assembly of the RNA polymerase (Mathew & Chatterji 2006). Both subunits and a special phage polymerase subunit would be compared with each other to test which one has the biggest expression activation. The comparison show very clearly that the omega subunit leads to the highest expression rate of the reporter protein (Badran et al. 2016). Based on this publication we decided, that the use of the RpoZ as activator domain is the most promising choice for our system.

Every system should be compared to a positive control to test the functionality. 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).

Next 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 with the goal to find an antibody mimic, that bound the SH2 domain of the tyrosine kinase Abelson with low nanomolar affinity (Wojcik et al. 2010). In the publication of Wojcik et al is shown that the interaction worked in vitro and in vivo equally well. To validate the potential of the hybrid transcriptional activation system HA4 mutants are also necessary. Single mutations with an exchange of one amino acid changed in most cases the structure of the protein and lowered or increased the interaction affinity with other proteins (Brender & Zhang 2015). In case of HA4 a single mutation R38A reduces the transcriptional activation of the report gene about 35-40%. Another mutation Y87A reduces the transcriptional activation even more to only 5-10% of the native binding affinity (Badran et al. 2016).
With all of these controls it is possible to show that the system itself is working and that different interaction levels results in differences in the activity of the reportergene expression.

A suitable selection system requires the select of samples based on tested parameters. In the case of the hybrid transcriptional activation system, the higher binding affinity has to lead to an selection advantage, to distinguish the high affinity Evobodies from the weakly binding Evobodies. A reporter is necessary which gives our bacteria an advantage if the reporter has a high expression rate. A good choice for such a reporter is an antibiotic resistence cassette like beta-lactamase (Livermore 1995) or tetracycline (Roberts 1996). Every increase of the antibiotic concentration would lead to more bacteria who cannot survive. However, at last the best Evobodies should allow the survival of the cells. The construction of the pomoter of the reporter gene is based on data of the publication of Badran et al from 2016. They tested a lot of different mutated promotor sequences of the native lacZ promoter and found a promoter sequence, that exhibits a ten times greater expression rate with cI of lambda 434 as binding domain and RpoZ as activation domain. Also the distance of the binding site of the 434 cI away from the transcription start site would be optimized by the working group. The optimal distance of the binding site is exactly 61 basepairs upstream of the transcription start site (Badran et al. 2016).

Coming soon