scholarly journals A split ribozyme that links detection of a native RNA to orthogonal protein outputs

2022 ◽  
Author(s):  
Lauren Gambill ◽  
August Staubus ◽  
Andrea Ameruoso ◽  
James Chappell
Keyword(s):  
Antibiotic Resistance ◽  
Synthetic Biology ◽  
Protein Production ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Tetrahymena Thermophila ◽  
Laboratory Evolution ◽  
Input Signals ◽  
Different Types

Individual RNA remains a challenging signal to synthetically transduce into different types of cellular information. Here, we describe Ribozyme-ENabled Detection of RNA (RENDR), a plug-and-play strategy that uses cellular transcripts to template the assembly of split ribozymes, triggering splicing reactions that generate orthogonal protein outputs. To identify split ribozymes that require templating for splicing, we used laboratory evolution to evaluate the activities of different split variants of the Tetrahymena thermophila ribozyme. The best design delivered a 93-fold dynamic range of splicing with RENDR controlling fluorescent protein production in response to an RNA input. We resolved a thermodynamic model to guide RENDR design, showed how input signals can be transduced into diverse visual, chemical, and regulatory outputs, and used RENDR to detect an antibiotic resistance phenotype in bacteria. This work shows how transcriptional signals can be monitored in situ using RNA synthetic biology and converted into different types of biochemical information.

Development of a Fluorescent Multiwell Assay for Evaluating the Capacity of the Ciliated Protozoan Tetrahymena for Bacterivory in Water Samples

2006 ◽  
Vol 41 (3) ◽  
pp. 307-315 ◽  
Author(s):  
Mary E. Power ◽  
Dana Sotornik ◽  
Marcel D.O. Pinheiro ◽  
Vivian R. Dayeh ◽  
Barbara J. Butler ◽  
...  
Keyword(s):  
Water Samples ◽  
Ecosystem Health ◽  
Cytochalasin B ◽  
Microbial Population ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Microtitre Plate ◽  
Population Composition ◽  
Different Types ◽  
Water Ecosystems

Abstract Bacterivory by ciliates in various water ecosystems, both natural and artificial, plays a significant role on the microbial population composition and consequently affects water quality. A convenient, rapid and inexpensive methodology to evaluate the capacity of the ciliate protozoan Tetrahymena thermophila for bacterivory was developed utilizing fluorescent protein expressing bacteria (FPEB) in a microtitre plate fluorimeter. Bacterivory was correlated with a loss in fluorescence measured in the fluorimeter and confirmed by fluorescence microscopy showing that the FPEB were engulfed during the assay and subsequently lost their fluorescence, whereas Cytochalasin B, a known inhibitor of phagocytosis, prevented a decrease in relative fluorescence units (RFUs). The ciliate bacterivory (CB) assay has a great dynamic range allowing the assay to be performed with a variety of predator:prey concentrations. A model toxicant, CuCl2, known to have a toxicological impact on protozoa and often present in different types of wastewater, resulted in measurable decreases in bacterivory. As well, starvation of Tetrahymena for 24 h resulted in reduced bacterivory. In the future, the CB assay could be developed for water monitoring purposes to rapidly assess water samples for the capacity to support bacterivory as an indicator of ecosystem health.

Pb Electrodeposition in the Presence of Chlorine Ions on Cu(100): An in Situ Optical Reflectivity Difference Study

2003 ◽  
Vol 781 ◽  
Author(s):  
J. Gray ◽  
W. Schwarzacher ◽  
X.D. Zhu
Keyword(s):  
Oblique Incidence ◽  
Opposite Sign ◽  
Bulk Phase ◽  
Phase Layer ◽  
Optical Reflectivity ◽  
Scan Rate ◽  
Different Types ◽  
Chlorine Ions ◽  
High Overpotentials

AbstractWe studied the initial stages of the electrodeposition of Pb in the presence of chlorine ions on Cu(100), using an oblique-incidence optical reflectivity difference (OIRD) technique. The OI-RD results reveal that immediately following the underpotential deposition (UPD) of the first Pb monolayer, two different types of bulk-phase films grow depending upon the magnitude of overpotential and cyclic voltammetry (CV) scan rate. At low overpotentials and/or slow scan rates, we propose that a bulk-phase Pb film grows on top of the UPD monolayer. At high overpotentials and/or fast scan rates, either a PbO, PbCl2, or a rough Pb bulk-phase layer grows on top of the UPD layer such that the reflectivity difference signal from such a film has an opposite sign.

scholarly journals Selected In Situ Hybridization Methods: Principles and Application

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3874
Author(s):  
Dominika Veselinyová ◽  
Jana Mašlanková ◽  
Katarina Kalinová ◽  
Helena Mičková ◽  
Mária Mareková ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Imaging Techniques ◽  
Cell Communication ◽  
Probe Design ◽  
Rapid Progress ◽  
In Situ Techniques ◽  
Different Types ◽  
Laboratory Procedures ◽  
The Individual

We are experiencing rapid progress in all types of imaging techniques used in the detection of various numbers and types of mutation. In situ hybridization (ISH) is the primary technique for the discovery of mutation agents, which are presented in a variety of cells. The ability of DNA to complementary bind is one of the main principles in every method used in ISH. From the first use of in situ techniques, scientists paid attention to the improvement of the probe design and detection, to enhance the fluorescent signal intensity and inhibition of cross-hybrid presence. This article discusses the individual types and modifications, and is focused on explaining the principles and limitations of ISH division on different types of probes. The article describes a design of probes for individual types of in situ hybridization (ISH), as well as the gradual combination of several laboratory procedures to achieve the highest possible sensitivity and to prevent undesirable events accompanying hybridization. The article also informs about applications of the methodology, in practice and in research, to detect cell to cell communication and principles of gene silencing, process of oncogenesis, and many other unknown processes taking place in organisms at the DNA/RNA level.

scholarly journals The Sorting of Mitochondrial DNA and Mitochondrial Proteins in Zygotes: Preferential Transmission of Mitochondrial DNA to the Medial Bud

1998 ◽  
Vol 142 (3) ◽  
pp. 613-623 ◽  
Author(s):  
Koji Okamoto ◽  
Philip S. Perlman ◽  
Ronald A. Butow
Keyword(s):  
Mitochondrial Dna ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Mitochondrial Matrix ◽  
Outer Membranes ◽  
Marker Proteins ◽  
The Matrix ◽  
Green Fluorescent ◽  
Mitochondrial Reticulum

Green fluorescent protein (GFP) was used to tag proteins of the mitochondrial matrix, inner, and outer membranes to examine their sorting patterns relative to mtDNA in zygotes of synchronously mated yeast cells in ρ+ × ρ0 crosses. When transiently expressed in one of the haploid parents, each of the marker proteins distributes throughout the fused mitochondrial reticulum of the zygote before equilibration of mtDNA, although the membrane markers equilibrate slower than the matrix marker. A GFP-tagged form of Abf2p, a mtDNA binding protein required for faithful transmission of ρ+ mtDNA in vegetatively growing cells, colocalizes with mtDNA in situ. In zygotes of a ρ+ × ρ+ cross, in which there is little mixing of parental mtDNAs, Abf2p–GFP prelabeled in one parent rapidly equilibrates to most or all of the mtDNA, showing that the mtDNA compartment is accessible to exchange of proteins. In ρ+ × ρ0 crosses, mtDNA is preferentially transmitted to the medial diploid bud, whereas mitochondrial GFP marker proteins distribute throughout the zygote and the bud. In zygotes lacking Abf2p, mtDNA sorting is delayed and preferential sorting is reduced. These findings argue for the existence of a segregation apparatus that directs mtDNA to the emerging bud.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

scholarly journals Maximizing protein translation rate in the non-homogeneous ribosome flow model: a convex optimization approach

2014 ◽  
Vol 11 (100) ◽  
pp. 20140713 ◽  
Author(s):  
Gilad Poker ◽  
Yoram Zarai ◽  
Michael Margaliot ◽  
Tamir Tuller
Keyword(s):  
Synthetic Biology ◽  
Convex Optimization ◽  
Production Rate ◽  
Protein Production ◽  
Flow Model ◽  
Protein Translation ◽  
Optimization Approach ◽  
Initiation Rate ◽  
Translation Rate ◽  
Protein Production Rate

Translation is an important stage in gene expression. During this stage, macro-molecules called ribosomes travel along the mRNA strand linking amino acids together in a specific order to create a functioning protein. An important question, related to many biomedical disciplines, is how to maximize protein production. Indeed, translation is known to be one of the most energy-consuming processes in the cell, and it is natural to assume that evolution shaped this process so that it maximizes the protein production rate. If this is indeed so then one can estimate various parameters of the translation machinery by solving an appropriate mathematical optimization problem. The same problem also arises in the context of synthetic biology, namely, re-engineer heterologous genes in order to maximize their translation rate in a host organism. We consider the problem of maximizing the protein production rate using a computational model for translation–elongation called the ribosome flow model (RFM). This model describes the flow of the ribosomes along an mRNA chain of length n using a set of n first-order nonlinear ordinary differential equations. It also includes n + 1 positive parameters: the ribosomal initiation rate into the mRNA chain, and n elongation rates along the chain sites. We show that the steady-state translation rate in the RFM is a strictly concave function of its parameters. This means that the problem of maximizing the translation rate under a suitable constraint always admits a unique solution, and that this solution can be determined using highly efficient algorithms for solving convex optimization problems even for large values of n . Furthermore, our analysis shows that the optimal translation rate can be computed based only on the optimal initiation rate and the elongation rate of the codons near the beginning of the ORF. We discuss some applications of the theoretical results to synthetic biology, molecular evolution, and functional genomics.

Mixing and Comparative Properties of NR Compounds Filled with Different Types of Reinforcing Fillers

2017 ◽  
Vol 266 ◽  
pp. 172-176
Author(s):  
Pattarawadee Maijan ◽  
Nitinart Saetung ◽  
Wisut Kaewsakul
Keyword(s):  
Carbon Black ◽  
Silica Surface ◽  
High Viscosity ◽  
Cure Rate ◽  
Modified Silica ◽  
Reinforcing Fillers ◽  
Silane Coupling ◽  
Different Types ◽  
Cure Time

Mixing behaviors of the compounds filled with different reinforcing fillers were studied in correlation with compound and vulcanizate properties. Four filler systems were used including: 1) silica plus small amount of silane coupling agent; 2) carbon black; 3) pre-modified silica; and 4) silica+silane-carbon black mixed one. The results have shown that silica provides longer optimum cure time and shorter cure rate than carbon black due to accelerator adsorption on silica surface. In addition, owing to highly polar nature on silica surface the silica-based compounds show rather high viscosity, attributed to stronger filler-filler interaction as can be confirmed by Payne effect and reinforcement index. However, the commercial surface treatment or pre-modified form of silica shows superior properties than in-situ modification of silica by silane during mixing, while it gives comparable properties to carbon black-based compound. Tensile properties of vulcanizates show a good correlation with the basic properties of their compounds.

scholarly journals Fate of Salmonella Typhimurium in laboratory-scale drinking water biofilms

2013 ◽  
Vol 11 (4) ◽  
pp. 629-635 ◽  
Author(s):  
L. M. Schaefer ◽  
V. S. Brözel ◽  
S. N. Venter
Keyword(s):  
Drinking Water ◽  
Green Fluorescent Protein ◽  
Health Risk ◽  
Salmonella Typhimurium ◽  
Fluorescent Protein ◽  
Laboratory Scale ◽  
Green Fluorescent ◽  
In Situ Detection

Investigations were carried out to evaluate and quantify colonization of laboratory-scale drinking water biofilms by a chromosomally green fluorescent protein (gfp)-tagged strain of Salmonella Typhimurium. Gfp encodes the green fluorescent protein and thus allows in situ detection of undisturbed cells and is ideally suited for monitoring Salmonella in biofilms. The fate and persistence of non-typhoidal Salmonella in simulated drinking water biofilms was investigated. The ability of Salmonella to form biofilms in monoculture and the fate and persistence of Salmonella in a mixed aquatic biofilm was examined. In monoculture S. Typhimurium formed loosely structured biofilms. Salmonella colonized established multi-species drinking water biofilms within 24 hours, forming micro-colonies within the biofilm. S. Typhimurium was also released at high levels from the drinking water-associated biofilm into the water passing through the system. This indicated that Salmonella could enter into, survive and grow within, and be released from a drinking water biofilm. The ability of Salmonella to survive and persist in a drinking water biofilm, and be released at high levels into the flow for recolonization elsewhere, indicates the potential for a persistent health risk to consumers once a network becomes contaminated with this bacterium.

The mineralogy and firing behaviour of pottery clays of the Lake Van region, eastern Turkey

Clay Minerals ◽  
2017 ◽  
Vol 52 (4) ◽  
pp. 453-468 ◽  
Author(s):  
A. Aras ◽  
S. Kiliç
Keyword(s):  
East Coast ◽  
Chemical Characterization ◽  
Mineralogical Composition ◽  
Lake Van ◽  
X Ray Diffraction ◽  
Pottery Production ◽  
X Ray ◽  
Different Types ◽  
Firing Shrinkage

AbstractThe present study focused on the mineralogical and chemical characterization and firing behaviour of clays from the Lake Van region and compared them with the same characteristics established for two ancient pot sherds. Four pottery clays collected from Kutki and Kuşluk in the Kesan Valley to the south, from Kavakbaşı to the southwest and from Bardakçı village on the east coast of Lake Van were analysed by X-ray diffraction to identify mineralogical composition (bulk clays and <2 μm fractions after heating at 300–500°C and ethylene glycol solvation). Further analyses were conducted to determine the size distribution, chemical composition and physical properties of test bodies derived from these clays. The in situ weathered schist forming the primary micaceous red clays which are suitable for local pottery production are characterized by large muscovite-sericite-illite and small calcite contents. In contrast, the Bardakçı clays are dominated by large smectite contents and are only used sparingly in mixtures of local pottery production because they undergo firing shrinkage and present drying and firing flaws in the fired bodies. Firing ranges of ~800–900°C were inferred from the mineralogy and colours of the two ancient sherds from Kutki. As a result of mineralogical analysis of fired and unfired test bodies of these pottery clays and pot sherds, two different types of pastes were determined for pottery production in the Lake Van region: metamorphic and volcanic paste, the former characterized by a calcite-poor and mica-sericite-rich matrix and the latter by large smectite and small calcite contents.

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