Fig. 1: Engineering and evolution of an UAA-dependent P. aeruginosa vaccine.

a X-ray structure overlay of the homodimeric interface of P. aeruginosa (gold; PDB: 6AMS) and E. coli (blue; PDB: 1MMI) DnaN, with side chains involved in dimer interaction shown as sticks. The dotted line indicates the boundary of the homodimeric interface, with residues labeled according to the P. aeruginosa structure. Residues for wild-type (WT) and mutant (M3, M5, M8) amino acid sequences of E. coli DnaN evolved to form a BzF-dependent dimer are provided in the table, as well as the structure-based consensus sites in P. aeruginosa wild-type DnaN. b Outline of P. aeruginosa engineering campaign. An origin-less knock-in/knock-out plasmid (KI/KO) integrates up or downstream of genomic dnaN. A wild-type copy of dnaN is supplied on a plasmid containing a temperature sensitive origin (TS ori), allowing the KI/KO cassette to be excised and genomic dnaN deleted to afford a TS strain. This strain is transformed with a vector encoding a BzF aminoacyl-tRNA synthetase (BzFRS/tRNA) and the evolved BzF-dependent DnaN, and the TS plasmid encoding wild-type DnaN is cured at elevated temperature resulting in a BzF-dependent Vaccine strain. c Design of the directed evolution platform for P. aeruginosa dnaN in E. coli using a cAMP generating bacterial two-hybrid system. Four independent sequence clusters were obtained and are shown in the table. A western blot of DnaN variant containing Pa M2 and Ec M3 mutations formed a photocrosslink-trapped dimer.