Preprint Communication Version 1 Preserved in Portico This version is not peer-reviewed

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

Version 1 : Received: 27 September 2021 / Approved: 27 September 2021 / Online: 27 September 2021 (17:09:00 CEST)

A peer-reviewed article of this Preprint also exists.

Kato, Y. Plasmid Curing and Exchange Using a Novel Counter-Selectable Marker Based on Unnatural Amino Acid Incorporation at a Sense Codon. Int. J. Mol. Sci. 2021, 22, 11482. Kato, Y. Plasmid Curing and Exchange Using a Novel Counter-Selectable Marker Based on Unnatural Amino Acid Incorporation at a Sense Codon. Int. J. Mol. Sci. 2021, 22, 11482.

Journal reference: Int. J. Mol. Sci. 2021, 22, 11482
DOI: 10.3390/ijms222111482

Abstract

A protocol was designed for plasmid curing using a novel counter-selectable marker, named pylSZK-pylT, in Escherichia coli. The pylSZK-pylT marker consists of the archaeal pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNApyl) with modification, and incorporates an unnatural amino acid (Uaa), Nε-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 pylSZK-pylT 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-tRNApyl, such as pylSZK-pylT, 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.

Keywords

counter-selection; unnatural amino acids; pyrrolysyl tRNA synthetase; genetic code expansion; sence codon reassignment; plasmid curing

Subject

LIFE SCIENCES, Microbiology

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