Fig. 2: A schematic representation of a method for constructing an amber-obligate phage display library by superinfection-immunity-based selection.
From: An amber obligate active site-directed ligand evolution technique for phage display
a, b Following cloning, the naive phagemid library is used to transform non-amber-suppressing E. coli bearing an F sex pilus. The expression of pIII is induced with IPTG, and shortly after, cells are superinfected with the CM13 helper phage (a). The expression of pIII in cells harboring a copy of the library that contains only sense codons renders these cells immune to superinfection (b). c Changing the media to one containing kanamycin allows for the selective growth of cells harboring a copy of the library that contains in-frame amber codons. Cells harboring a copy of the library with deleterious mutations also pass this selection. d, e The phagemid library is purified (d) and used to transform DH5α, an amber-suppressing strain of E. coli (e). Complementation of the phagemid with a pIII-knockout helper phage in E. coli DH5α allows for the production of phagemid particles only from cells harboring a copy of the peptide-pIII library containing in-frame amber codons.
