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 pylSZK-pylT, in Escherichia coli. The pylSZK-pylT marker consists of the archaeal pyrrolysyl-tRNA synthetas...

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Autor principal:	Yusuke Kato
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	counter-selection unnatural amino acids pyrrolysyl tRNA synthetase genetic code expansion sence codon reassignment plasmid curing Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/d59f0af6f8df45939509f832370e82ea
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spelling	oai:doaj.org-article:d59f0af6f8df45939509f832370e82ea2021-11-11T16:56:40ZPlasmid Curing and Exchange Using a Novel Counter-Selectable Marker Based on Unnatural Amino Acid Incorporation at a Sense Codon10.3390/ijms2221114821422-00671661-6596https://doaj.org/article/d59f0af6f8df45939509f832370e82ea2021-10-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/21/11482https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067A 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.Yusuke KatoMDPI AGarticlecounter-selectionunnatural amino acidspyrrolysyl tRNA synthetasegenetic code expansionsence codon reassignmentplasmid curingBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 11482, p 11482 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	counter-selection unnatural amino acids pyrrolysyl tRNA synthetase genetic code expansion sence codon reassignment plasmid curing Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	counter-selection unnatural amino acids pyrrolysyl tRNA synthetase genetic code expansion sence codon reassignment plasmid curing Biology (General) QH301-705.5 Chemistry QD1-999 Yusuke Kato Plasmid Curing and Exchange Using a Novel Counter-Selectable Marker Based on Unnatural Amino Acid Incorporation at a Sense Codon
description	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.
format	article
author	Yusuke Kato
author_facet	Yusuke Kato
author_sort	Yusuke Kato
title	Plasmid Curing and Exchange Using a Novel Counter-Selectable Marker Based on Unnatural Amino Acid Incorporation at a Sense Codon
title_short	Plasmid Curing and Exchange Using a Novel Counter-Selectable Marker Based on Unnatural Amino Acid Incorporation at a Sense Codon
title_full	Plasmid Curing and Exchange Using a Novel Counter-Selectable Marker Based on Unnatural Amino Acid Incorporation at a Sense Codon
title_fullStr	Plasmid Curing and Exchange Using a Novel Counter-Selectable Marker Based on Unnatural Amino Acid Incorporation at a Sense Codon
title_full_unstemmed	Plasmid Curing and Exchange Using a Novel Counter-Selectable Marker Based on Unnatural Amino Acid Incorporation at a Sense Codon
title_sort	plasmid curing and exchange using a novel counter-selectable marker based on unnatural amino acid incorporation at a sense codon
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/d59f0af6f8df45939509f832370e82ea
work_keys_str_mv	AT yusukekato plasmidcuringandexchangeusinganovelcounterselectablemarkerbasedonunnaturalaminoacidincorporationatasensecodon
_version_	1718432206260535296

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

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