Details about the construction of the library can be found in the Appendix. In short, a synthetic design was ordered based on a statistic analysis of the diversity found in natural VHH and aiming at reducing hydrophobicity at some position. The size of the CDR1 and CR2 was fixed at 7 amino acids while 4 sizes of CDR3 were chosen (9, 12, 15 and 18 amino acids). Large -scale PCR was then carried out ensuring that at least 1010 DNA molecules were used as a matrix. Fragments were then cut and inserted into the pHEN2-3myc plasmid. The ligated DNA material was used to transform electrocompetent E. coli TG1 cells (Lucigen Corp., Middleton, United States). Serial dilution was used to count the total number of bacteria transformed. A potential diversity of 3 × 109 was calculated. Transformed bacteria plated on 430 2xYT-ampicillin agar dishes (140 mm), grown overnight at 37°C, scrapped and stored in 30% of glycerol at −80°C.

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