Figure 2.

Generation of iPhage vector and library cloning strategy. The parental f88-4 phage vector contains two capsid genes encoding a wild-type protein VIII (pVIII, depicted in grey) and a recombinant protein VIII (rpVIII, depicted in green). The recombinant gene VIII contains a foreign DNA insert with a HindIII and a PstI cloning site, which allows the cloning of annealed oligonucleotides encoding the pen peptide in frame with the recombinant gene VIII. For generation of the iPhage vector, f88-4 displaying the pen peptide motif (RQIKIWFQNRRMKWKK) and fUSE5 phage plasmids are digested with BamHI and XbaI restriction enzymes, purified, and fused by standard ligation protocol. Next, an annealed random oligonucleotide library (e.g. X₄YX₄) is digested with the BglI restriction enzyme and cloned within the SfiI restriction site of the pIII coat protein gene (pIII, depicted in light blue) on the iPhage vector. MC1061 E. coli electro competent cells are transformed with the library cloned iPhage vector to produce a random peptide iPhage library. TetR – tetracycline resistance gene.