Bioactivity test. Upon the success of ligation between 1f and the peptide FYTKPT to give insulin analog 2, the activity of ligated 2 was characterized through a cell-based assay by using pAKT levels as measurements of IR downstream signaling (Figure ). Noted, insulin analog 2, which has a single Glu to Ala mutation at B21 position, was previously reported to have similar binding affinity as hIns to IR. We showed that the ligated 2 had a comparable EC50 with recombinant hIns from a commercial source, as well as the same analog 2 from the completely synthetic full B chain by SPPS (Scheme S4). The hydrolyzed byproduct 6 from 1f during the ligation reaction had a nearly 100-fold decreased activity compared to hIns and 2, which is consistent with the previous reports that PheB25 is critical for receptor engagement. Omniligase-1-mediated selective M13 phage labeling. Tremendous progress was made in expanding the phage display library with synthetic functional groups by chemical or chem-enzymatic reactions. There are two main M13 phage surface proteins that can be used for peptide modifications, pIII and pVIII. pIII-library is the preferred version for high affinity ligands due to its lower copy number (5 vs up to 2700 for pVIII). Unlike previous examples such as phage libraries generated from sortase ligations where a specific epitope is required for the ligase, omniligase-1 has a broader substrate scope, which will further enhance the library diversity. However, this also creates a new selectivity challenge because both pIII and pVIII can potentially be modified. To avoid pVIII modifications (Figure ), we first introduced a Glu to Pro mutation in the second residue of pVIII with FYTPKT displayed at the N-terminus of pIII. We hypothesize that this can prevent any pVIII ligations because Pro at P2' is a disallowed substrate for omniligase-1. This pVIII-mutated phage was incubated with different concentrations of omniligase-1 and a biotin-labeled substrate (biotin-EYNAAPF-Gc-R-NH2), which allow us to monitor the ligation using streptavidin-HRP. After 20 hours at 37 o C (Figure ), the biotin-containing band was only detected at 55 kDa, which is the same molecular weight as pIII. We did not observe any pVIII modifications. We also performed the same ligation reaction using the pVIIIwild type (WT) phage clone. To our surprise, there was no difference between the two phage clones, and no streptavidin signal was detected at pVIII size, even for the phage with wild type pVIII. The result indicated that the omniligase-1mediated ligation was selective toward pIII. To our best knowledge, this is the first study using omniligase-1 for phage display. Although pVIII has a much higher copy number, omniligase-1 still preferentially reacted with pIII likely due to steric hindrance of the environment surrounding pVIII.