The core of our system is the combination of an initial library with in vivo mutation and selection, to optimize Evobodies by directed evolution. To get in this evolutional process, some binding proteins must show at least a marginal affinity to the chosen target protein. Low affinity binders than get mutated and selected in vivo during the culture growth under a rising selective pressure.
To make sure the designed binding proteins can attach several target proteins and the Monobody and Nanobody libraries can deliver initial Evobodies, which can be optimized by mutation and selection, we performed a phagemid display, using phagemid pAK100 and M13 derived helper phages.(Link to protocol)

After assembling the pAK100 backbone with the CDS of our designed bindng proteins via Gibson cloning, the phagemid was transformed into electrocompetent E. coli ER2738 and plated on LB agar, containing chloramphenicol and tetracycline. Thus, we generated a comparative small library of about 3000 clones for each, Monobody and Nanobody coding phagemids, as test library for use in phagemid display.
Infecting the eluted and incubated culture with M13 derived helper phages and the following purification of the composed phages leads to pelleted phages, diluted in PBS. The UV-VIS absorbance of an 1:50 aliquot at 269 nm and 320 nm was measured, to calculate the phage concentration, based on the following formula:

We gained a concentration of 2.99 x 10¹³ of Monobody carrying phages per mL and 1.599 x 10¹³ of Nanobody carrying phages per mL in an elution volume of 1.5 mL each.
These phages were used to accomplish one stage of phage panning on immuno-tubes, coated with several different proteins, from transcription factors to enzymes and protein domains. We explicit relinquished further panning rounds, to simulate the conditions of our system. Thereby just an initial binder is required to get optimized by in vivo mutation and selection. After an exhaustive washing procedure and the following elution of phages with affinity imparting binding proteins an exponential growing culture of ER2738 was infected and plated on LB agar, containing chloramphenicol. After incubation at 37°C, several colonies were detectable on agar plates (Fig. 1).

As an example, we isolated and sequenced one of the Monobody CDSs coding for a formylglycine-generating enzyme (FGE) binder (Fig. 2).

Summarized, the phagemid display was a good opportunity, to screen our Monobody and Nanobody libraries at initial binding proteins for our directed evolution system. We could show, that in a small extract of 3.000 variable Monobodies, as well as Nanobodies, out of a theoretical library size of about 1 billion CDS contains some potential candidates for getting an optimal Evobody by directed evolution!