## 4 Materials and Methods
### Plasmid Construction  
The protein sequence of the 1.29 Nb was obtained from [PERSON] et al. [9]. The protein sequence of the SARS-CoV-2 spike RBD (amino acids R319-F541 of the S protein) from the Omicron subvariant BA.5 is based on wild-type SARS-CoV-2 (YP_009724390.1) with G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, and Y505H mutations. The DNA sequences of both proteins were synthesized with codon optimization for _E. coli_ expression by GenScript, Inc. (Piscataway, NJ, USA). Both genes were then cloned into the pDD18-Cm bicistronic plasmid to create pDD18-Cm RBD-Bla::ssTorA-1.29 Nb-FLAG. The sequences of all constructs were verified using Sanger method (U2 Bio Co., Ltd., Seoul, South Korea).  
Error-prone mutations were introduced to the 1.29 or C11 Nb genes by PCR amplification using the GeneMorph II Random Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) with the 1.29 Nb XbaI forward primer (5\({}^{\prime}\)-GCGATGCTAGACAGGTGCAACTGGTTG-3') and the 1.29 Nb SaII reverse primer (5'-GCGATGGTCGACGCCTGTAACGGT CACTTG-3'). For the library for the first round of mutation, PCRs were performed for 35 cycles with 5 ng pDD18-Cm RBD-Blaz::ssTorA-1.29 Nb-FLAG (containing the 1.29 Nb template) in each reaction. The resulting PCR products were cloned into the Xbal and SaII cut sites of pDD18-Cm RBD-Blaz::ssTorA-1.29 Nb-FLAG. Ligation mixtures were transformed into NEB10\(\updelta\) cells using electroporation. Cells were retrieved from an electroporation cuvette by resuspending in 1 mL LB medium. Cells from 10 cuvettes were pooled together into a 250-mL shake flask and allowed to recover at 37 \({}^{\circ}\)C with 200 RPM agitation for 1 h. To determine the library size, spread plate method was used. Briefly, 100 \(\upmu\)L of 10\({}^{-2}\)-10\({}^{-4}\) serially diluted cells were spread onto LB agar plates supplemented with 25 \(\upmu\)g/mL Cm and incubated at 37 \({}^{\circ}\)C for 24 h. After incubation, the numbers of colonies appeared on the plates containing colonies between 20-300 CFU/mL were counted and used for library size calculation. To evaluate the library quality, 30 colonies were randomly picked, miniprepped and submitted to sequencing analysis by Sanger method (U2 Bio Co., Ltd.) with sequencing primer, ssTorA for (5'-AACAATAACGATCTTTCAGGC-3'), which binds to the Tat signal peptide of TorA at the C-terminus of Nbs. The error rate determination was reported by counting the number of DNA point mutations observed in each sequenced Nb gene obtained from the library prior to subjected to FLI-TRAP isolation. A similar strategy was used to construct the library for the second round of mutation; however, to increase the mutation rate, 35 cycles of PCR were performed with 1 ng pDD18-Cm RBD-Blaz::ssTorA-C11 Nb-FLAG instead.  
For cytoplasmic expression of Nbs without the ssTorA signal peptide, the 1.29, C11, and K9 Nb genes were PCR amplified from pDD18-Cm and cloned into the pET-28a anti-GCN4-FLAG-His [46] plasmid between the Ncol and SaII cut sites using the 1.29 Nb Ncol forward primer (5'-GCGATGCCATGGCCAGGTGCAACTGGTTGAG-3') and the 1.29 Nb Salt reverse primer (5'-GCGATGGTCGACGCCTGTAACGGTCACTTG-3'), resulting in pET-28a Nb-FLAG-His.