Data S1 - A Novel Helper Phage Enabling Construction of Genome-Scale ORF-Enriched Phage Display Libraries

Figure S1. Schematic representation of the vector pVCEPI23964. Only the relevant genes and restriction sites are shown. The map is not to scale. lacPO, lac promoter-operator; RBS, ribosome-binding site; PelB, pectate lyase signal sequence; Stuffer, 1.8 Kbp nucleotide sequence flanked by BsaI sites (a) and (c); Tryp, trypsin protease cleavage site; S, spacer; gIIIP, segment encoding amino acid residues 2–405 of the gene III of filamentous phage; fori, origin of replication of filamentous phage; Ampr, β-lactamase gene; Ori, ColE1 origin of replication; tHP, transcriptional terminator. Details of regions marked a, b and c by double-headed arrows are shown in A, B and C, respectively. ‘D’ shows the sequence flanking the ORF after cloning into the phagemid vector harboring attB1 and attB2 site-specific recombination sites (in bold) based on Gateway Technology. Amino acids are shown in single-letter code below the nucleotide sequence. Restriction enzyme sites are shown above the nucleotide. Figure S2. Western blot analysis. Trypsin-untreated and -treated VCSM13 and AGM13 phages (1.5×1010) were separated by 10% SDS-PAGE under reducing conditions, transferred onto 0.45 µ PVDF membranes and probed with anti-gIIIP MAb 30421, which targets an epitope located in the N2 domain of gIIIP from the bacteriophage M13. M, Prestained marker; Lanes 1–5, 1.5×1010 VCSM13 phages treated with 0, 0.1, 1, 10 and 100 µg/ml trypsin. Table S1. Analysis of random clones after treatment of primary phages (P01) with various concentrations of trypsin. Table S2. Size distribution of randomly sequenced clones at various stages of library construction.
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