Plasmid Curing and Exchange Using a Novel Counter-Selectable Marker Based on Unnatural Amino Acid Incorporation at a Sense Codon
A protocol was designed for plasmid curing using a novel counter-selectable marker, named <i>pylS<sup>ZK</sup>-pylT</i>, in <i>Escherichia coli</i>. The <i>pylS<sup>ZK</sup>-pylT</i> marker consists of the archaeal pyrrolysyl-tRNA synthetas...
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| Formato: | article |
| Lenguaje: | EN |
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MDPI AG
2021
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| Materias: | |
| Acceso en línea: | https://doaj.org/article/d59f0af6f8df45939509f832370e82ea |
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| Sumario: | A protocol was designed for plasmid curing using a novel counter-selectable marker, named <i>pylS<sup>ZK</sup>-pylT</i>, in <i>Escherichia coli</i>. The <i>pylS<sup>ZK</sup>-pylT</i> marker consists of the archaeal pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNA<sup>pyl</sup>) with modification, and incorporates an unnatural amino acid (Uaa), <i>N<sup>ε</sup></i>-benzyloxycarbonyl-<span style="font-variant: small-caps;">l</span>-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 <i>pylS<sup>ZK</sup>-pylT</i> 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<sup>pyl</sup>, such as <i>pylS<sup>ZK</sup>-pylT</i>, 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. |
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