## 2 Results and Discussion
In the second round, 21 hits were isolated at 200 \(\upmu\)g/mL Carb and 10\({}^{-4}\) cell dilution. All hits were submitted for sequencing analysis. Surprisingly, 17 sequencing results displayed the exact same sequence, which was distinctly different from that of the C11 Nb and is a full-length Nb, while the other 4 sequencing results showed a stop codon within the Nbs. The sequence of the representative clone of these 17 Nbs, the K9 Nb, is illustrated in Figure S3. It had 2, 4, and 2 mutations in frameworks 1, 2, and 3, respectively. Surprisingly, the complementarity determining region 1 (CDR1) and CDR3 of the K9 Nb were very different from those of the 1.29 and C11 Nbs; the K9 Nb had an elongated CDR3 (19 amino acids) compared to that of the 1.29 and C11 Nbs (13 amino acids). We hypothesized that such differences between the template, the C11 Nb, and the isolated Nb (the K9 Nb) could be due to a phenomenon called \"replication slippage\" [17]. It should be noted that the diversity and mutation rate of the library produced by the second round of mutation were assessed by submitted random clones that appeared on the plate used for library size estimation and were not subjected to FLI-TRAP selection for sequencing. The sequencing results showed  
Figure 2: **Redirecting the 1.29 Nb to bind the RBD of BA.5 variant using FLI-TRAP. ** (**A**) Spot titers of serially diluted NEB10\(\upmu\) cells used for the determination of library selection conditions using FLI-TRAP. (**B**) Spot titers of serially diluted NEB10\(\upmu\) cells co-expressing TatABC with RBD BA.5-Bla and the 1.29 Nb or library-selected clones C1–C15 derived from the 1.29 Nb. (**C**) Spot titers of serially diluted NEB10\(\upmu\), MC4100, and DADE cells co-expressing TatABC with RBD BA.5-Bla and the 1.29 or K9 Nbs. that all Nbs had distinct Nucleotide sequences and that the K9 Nb should not have been a false positive from our FLI-TRAP selection. We also confirmed this by comparing the Carb resistance of cells containing RBD BA.5-Bla and K9 Nb fusion proteins in not only the _E. coli_ strain NEB10\(\upbeta\) but also in strains MC4100 and DADE using spot plating, as shown in Figure 2C. DADE is an isogenic AtatABCDAtatE derivative of MC4100; hence, it is not capable of performing Tat transport. The pDD18-Cm plasmid co-expressing RBD BA.5-Bla and ssTorA-K9-FLAG was co-transformed into MC4100 and DADE cells. Spot plating results showed a resistance of up to 50 \(\upmu\)g/mL Carb in MC4100 cells at the single colony level (10\({}^{-6}\)), up to 50 \(\upmu\)g/mL Carb while DADE cells showed only background growth at 10\({}^{-1}\). This result strongly confirmed that cell resistance was related to the Tat transport of the two binding proteins. At 100 \(\upmu\)g/mL Carb, NEB10\(\upbeta\) cells containing the K9 Nb grew at 10\({}^{-6}\), while cells containing the 1.29 Nb grew at only 10\({}^{-1}\), a 100,000-fold difference. Notably, the observable difference between the abilities of MC4100 and NEB10\(\upbeta\) to resist Carb could be due to many factors, for example, differences in abilities to produce recombinant protein and to resist toxicity of the arabinose used as an inducer. **Differences in Nb production of the two systems. ** For FLI-TRAP selection, Nbs were produced from the pDD18-Cm plasmid, which is derived from the pBAD18-Cm plasmid of _E. coli_ NEB10\(\upbeta\)[10]. For in vitro analysis, the pET vector system was employed, as high recombinant protein expression can be achieved via the T7 promoter system present in the pET vectors using _E. coli_ BL21(DE3) [18]. Western blot analysis (Figure 3) using Mouse Anti-DDDDK tag Monoclonal Antibody, HRP Conjugated, Clone M2 (1:3000; Abcam Cat# ab49763, RMID:AB_869428)to detect the FLAG epitope tag present in both systems revealed distinct bands corresponding to the estimated sizes of the 1.29 (17 kDa), C11 (17 kDa), and K9 (20 kDa) Nbs based on the identified amino acid sequences. Interestingly, differences in the solubility of the K9 Nb expressed in the two systems were quite apparent.