Plasmid Curing and Exchange Using a Novel Counter-Selectable Marker Based on Unnatural Amino Acid Incorporation at a...

A protocol was designed for plasmid curing using a novel counter-selectable marker, named pylS, in Escherichia coli. The pylS marker consists of the archaeal pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNA^pyl) with modification, and incorporates an unnatural amino acid (Uaa), N-benzyloxycarbonyl-l-lysine (ZK), at a sense codon in ribosomally synthesized proteins, resulting in bacterial growth inhibition or killing. Plasmid curing is performed by exerting toxicity on pylS located on the target plasmid, and selecting only proliferative bacteria. All tested bacteria obtained using this protocol had lost the target plasmid (64/64), suggesting that plasmid curing was successful. Next, we attempted to exchange plasmids with the identical replication origin and an antibiotic resistance gene without plasmid curing using a modified protocol, assuming substitution of plasmids complementing genomic essential genes. All randomly selected bacteria after screening had only the substitute plasmid and no target plasmid (25/25), suggesting that plasmid exchange was also accomplished. Counter-selectable markers based on PylRS-tRNA^pyl, such as pylS, may be scalable in application due to their independence from the host genotype, applicability to a wide range of species, and high tunability due to the freedom of choice of target codons and Uaa's to be incorporated.