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

International Journal of Molecular Sciences ◽

10.3390/ijms222111482 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11482

Author(s):

Yusuke Kato

Keyword(s):

Amino Acid ◽

Selectable Marker ◽

Antibiotic Resistance Gene ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Plasmid Curing ◽

Host Genotype ◽

Wide Range ◽

Sense Codon ◽

Target Plasmid

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.

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

Life ◽

10.3390/life11090920 ◽

2021 ◽

Vol 11 (9) ◽

pp. 920

Author(s):

Yusuke Kato

Keyword(s):

Amino Acid ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Dependent Manner ◽

Inhibitory Effects ◽

Concentration Dependent Manner ◽

Growth Inhibitory ◽

Transmission Of Information ◽

Sense Codon ◽

Cognate Trna

We designed a novel growth controller regulated by feeding of an unnatural amino acid, Nε-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 (tRNApyl). 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 tRNApyl gene. Taking advantage of the low selectivity of PylRS for tRNApyl anticodons, we experimentally determined the most effective anticodon sequence among all 64 nucleotide sequences in the anticodon region of tRNApyl 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.

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

10.20944/preprints202107.0210.v1 ◽

2021 ◽

Author(s):

Yusuke Kato

Keyword(s):

Amino Acid ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Dependent Manner ◽

Inhibitory Effects ◽

Concentration Dependent Manner ◽

Growth Inhibitory ◽

Transmission Of Information ◽

Sense Codon ◽

Cognate Trna

We designed a novel growth controller regulated by feeding of an unnatural amino acid, Nε-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 (tRNApyl). 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 tRNApyl gene. Taking advantage of the low selectivity of PylRS for tRNApyl anticodons, we experimentally determined the most effective anticodon sequence among all 64 nucleotide sequences in the anticodon region of tRNApyl 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.

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Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽

10.1007/s00726-020-02927-z ◽

2020 ◽

Author(s):

Thomas L. Williams ◽

Debra J. Iskandar ◽

Alexander R. Nödling ◽

Yurong Tan ◽

Louis Y. P. Luk ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Code ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation ◽

Genetic Code Expansion ◽

Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

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Improving orthogonal tRNA-synthetase recognition for efficient unnatural amino acid incorporation and application in mammalian cells

Molecular BioSystems ◽

10.1039/b904228h ◽

2009 ◽

Vol 5 (9) ◽

pp. 931 ◽

Cited By ~ 46

Author(s):

Jeffrey K. Takimoto ◽

Katrina L. Adams ◽

Zheng Xiang ◽

Lei Wang

Keyword(s):

Amino Acid ◽

Mammalian Cells ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation

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An archaebacteria-derived glutamyl-tRNA synthetase and tRNA pair for unnatural amino acid mutagenesis of proteins in Escherichia coli

Nucleic Acids Research ◽

10.1093/nar/gkg903 ◽

2003 ◽

Vol 31 (23) ◽

pp. 6700-6709 ◽

Cited By ~ 48

Author(s):

S. W. Santoro

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Unnatural Amino Acid Mutagenesis ◽

Amino Acid Mutagenesis

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Efficient Multisite Unnatural Amino Acid Incorporation in Mammalian Cells via Optimized Pyrrolysyl tRNA Synthetase/tRNA Expression and Engineered eRF1

Journal of the American Chemical Society ◽

10.1021/ja5069728 ◽

2014 ◽

Vol 136 (44) ◽

pp. 15577-15583 ◽

Cited By ~ 123

Author(s):

Wolfgang H. Schmied ◽

Simon J. Elsässer ◽

Chayasith Uttamapinant ◽

Jason W. Chin

Keyword(s):

Amino Acid ◽

Mammalian Cells ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation

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High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

Applied and Environmental Microbiology ◽

10.1128/aem.03417-13 ◽

2013 ◽

Vol 80 (5) ◽

pp. 1718-1725 ◽

Cited By ~ 16

Author(s):

Masaomi Minaba ◽

Yusuke Kato

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Unnatural Amino Acids ◽

Mammalian Cells ◽

Expression System ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Translational Efficiency ◽

Site Specific ◽

Content Type

ABSTRACTSynthetic biologists construct complex biological circuits by combinations of various genetic parts. Many genetic parts that are orthogonal to one another and are independent of existing cellular processes would be ideal for use in synthetic biology. However, our toolbox is still limited with respect to the bacteriumEscherichia coli, which is important for both research and industrial use. The site-specific incorporation of unnatural amino acids is a technique that incorporates unnatural amino acids into proteins using a modified exogenous aminoacyl-tRNA synthetase/tRNA pair that is orthogonal to any native pairs in a host and is independent from other cellular functions. Focusing on the orthogonality and independency that are suitable for the genetic parts, we designed novel AND gate and translational switches using the unnatural amino acid 3-iodo-l-tyrosine incorporation system inE. coli. A translational switch was turned on after addition of 3-iodo-l-tyrosine in the culture medium within minutes and allowed tuning of switchability and translational efficiency. As an application, we also constructed a gene expression system that produced large amounts of proteins under induction conditions and exhibited zero-leakage expression under repression conditions. Similar translational switches are expected to be applicable also for eukaryotes such as yeasts, nematodes, insects, mammalian cells, and plants.

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