An Unnatural Amino Acid-Regulated Growth Controller Based on Informational Disturbance

We designed a novel growth controller regulated by feeding of an unnatural amino acid, N-benzyloxycarbonyl-l-lysine (ZK), using a specific incorporation system at a sense codon. This system is constructed by a pair of modified pyrrolisyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNA^pyl). Although ZK is non-toxic for normal organisms, the growth of Escherichia coli carrying the ZK incorporation system was inhibited in a ZK concentration-dependent manner without causing rapid bacterial death, presumably due to generation of non-functional or toxic proteins. The extent of growth inhibition strongly depended on the anticodon sequence of the tRNA^pyl gene. Taking advantage of the low selectivity of PylRS for tRNA^pyl anticodons, we experimentally determined the most effective anticodon sequence among all 64 nucleotide sequences in the anticodon region of tRNA^pyl gene. The results suggest that the ZK-regulated growth controller is a simple, target-specific, environmental noise-resistant and titratable system. This technique may be applicable to a wide variety of organisms because the growth inhibitory effects are caused by "informational disturbance", in which the highly conserved system for transmission of information from DNA to proteins is perturbed.