Site-specific properties of Tn7 transposition into the E. coli Chromosome

Abstract Self-cleaving ribozymes are catalytic RNAs and can be found in all domains of life. They catalyze a site-specific cleavage that results in a 5′ fragment with a 2′,3′ cyclic phosphate (2′,3′ cP) and a 3′ fragment with a 5′ hydroxyl (5′ OH) end. Recently, several strategies to enrich self-cleaving ribozymes by targeted biochemical methods have been introduced by us and others. Here, we develop an alternative strategy in which 5ʹ OH RNAs are specifically ligated by RtcB ligase, which first guanylates the 3′ phosphate of the adapter and then ligates it directly to RNAs with 5′ OH ends. Our results demonstrate that adapter ligation to highly structured ribozyme fragments is much more efficient using the thermostable RtcB ligase from Pyrococcus horikoshii than the broadly applied Escherichia coli enzyme. Moreover, we investigated DNA, RNA and modified RNA adapters for their suitability in RtcB ligation reactions. We used the optimized RtcB-mediated ligation to produce RNA-seq libraries and captured a spiked 3ʹ twister ribozyme fragment from E. coli total RNA. This RNA-seq-based method is applicable to detect ribozyme fragments as well as other cellular RNAs with 5ʹ OH termini from total RNA.

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Two related recombinases are required for site-specific recombination at dif and cer in E. coli K12

Cell ◽

10.1016/0092-8674(93)80076-q ◽

1993 ◽

Vol 75 (2) ◽

pp. 351-361 ◽

Cited By ~ 214

Author(s):

Garry Blakely ◽

Gerhard May ◽

Richard McCulloch ◽

Lidia K. Arciszewska ◽

Mary Burke ◽

...

Keyword(s):

Site Specific ◽

Site Specific Recombination ◽

E Coli

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Waterborne illness and injury: a feasibility study of a site-specific predictive model for beach water quality at Beachway Park in the City of Burlington

10.32920/ryerson.14668362 ◽

2021 ◽

Author(s):

Ramien Sereshk

Keyword(s):

Water Quality ◽

Predictive Model ◽

Real Time ◽

Site Specific ◽

E Coli ◽

Time Basis ◽

A Site ◽

Persistence Model ◽

Beach Water Quality ◽

Beach Water

It is commonly assumed that the persistence model, using day-old monitoring results, will provide accurate estimates of real-time bacteriological concentrations in beach water. However, the persistence model frequently provides incorrect results. This study: 1. develops a site-specific predictive model, based on factors significantly influencing water quality at Beachway Park; 2. determines the feasibility of the site-specific predictive model for use in accurately predicting near real-time E. coli levels. A site-specific predictive model, developed for Beachway Park, was evaluated and the results were compared to the persistence model. This critical performance evaluation helped to identify the inherent inaccuracy of the persistence model for Beachway Park, which renders it an unacceptable approach for safeguarding public health from recreational water-borne illnesses. The persistence model, supplemented with a site-specific predictive model, is recommended as a feasible method to accurately predict bacterial levels in water on a near real-time basis.

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Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

Biomolecules ◽

10.3390/biom9070255 ◽

2019 ◽

Vol 9 (7) ◽

pp. 255 ◽

Cited By ~ 16

Author(s):

Sviatlana Smolskaya ◽

Yaroslav Andreev

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Nonsense Suppression ◽

Expression Vectors ◽

Site Specific ◽

Suppressor Trna ◽

E Coli ◽

Orthogonal Pair

More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.

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Decoupling Protein Production from Cell Growth Enhances the Site-Specific Incorporation of Noncanonical Amino Acids in E. coli

ACS Synthetic Biology ◽

10.1021/acssynbio.0c00298 ◽

2020 ◽

Vol 9 (11) ◽

pp. 3052-3066

Author(s):

Meritxell Galindo Casas ◽

Patrick Stargardt ◽

Juergen Mairhofer ◽

Birgit Wiltschi

Keyword(s):

Amino Acids ◽

Cell Growth ◽

Protein Production ◽

Noncanonical Amino Acids ◽

Site Specific ◽

E Coli

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Functional Analysis of the Acinetobacter baumannii XerC and XerD Site-Specific Recombinases: Potential Role in Dissemination of Resistance Genes

Antibiotics ◽

10.3390/antibiotics9070405 ◽

2020 ◽

Vol 9 (7) ◽

pp. 405

Author(s):

David L. Lin ◽

German M. Traglia ◽

Rachel Baker ◽

David J. Sherratt ◽

Maria Soledad Ramirez ◽

...

Keyword(s):

Escherichia Coli ◽

Functional Analysis ◽

Acinetobacter Baumannii ◽

Potential Role ◽

Covalent Attachment ◽

Recombination Reaction ◽

Site Specific ◽

Site Specific Recombination ◽

E Coli ◽

Resistant Genes

Modules composed of a resistance gene flanked by Xer site-specific recombination sites, the vast majority of which were found in Acinetobacter baumannii, are thought to behave as elements that facilitate horizontal dissemination. The A. baumannii xerC and xerD genes were cloned, and the recombinant clones used to complement the cognate Escherichia coli mutants. The complemented strains supported the resolution of plasmid dimers, and, as is the case with E. coli and Klebsiella pneumoniae plasmids, the activity was enhanced when the cells were grown in a low osmolarity growth medium. Binding experiments showed that the partially purified A. baumannii XerC and XerD proteins (XerCAb and XerDAb) bound synthetic Xer site-specific recombination sites, some of them with a nucleotide sequence deduced from existing A. baumannii plasmids. Incubation with suicide substrates resulted in the covalent attachment of DNA to a recombinase, probably XerCAb, indicating that the first step in the recombination reaction took place. The results described show that XerCAb and XerDAb are functional proteins and support the hypothesis that they participate in horizontal dissemination of resistant genes among bacteria.

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The MaoP/maoS Site-Specific System Organizes the Ori Region of the E. coli Chromosome into a Macrodomain

PLoS Genetics ◽

10.1371/journal.pgen.1006309 ◽

2016 ◽

Vol 12 (9) ◽

pp. e1006309 ◽

Cited By ~ 18

Author(s):

Michèle Valens ◽

Axel Thiel ◽

Frédéric Boccard

Keyword(s):

Specific System ◽

Site Specific ◽

E Coli

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