scholarly journals Comparative analysis of inducible promoters in cyanobacteria

2019 ◽  
Author(s):  
Anna Behle ◽  
Pia Saake ◽  
Ilka M. Axmann
Keyword(s):  
Escherichia Coli ◽  
Dynamic Range ◽  
Inducible Promoter ◽  
Model Organisms ◽  
Native Metal ◽  
Inducible Promoters ◽  
Advantages And Disadvantages ◽  
Great Progress ◽  
Dose Dependent ◽  
Synechocystis Sp

AbstractResearch in the field of synthetic biology highly depends on efficient, well-characterized promoters. While great progress has been made with other model organisms such as Escherichia coli, photosynthetic cyanobacteria still lag behind. Commonly used promoters that have been tested in cyanobacteria show weaker dynamic range or no regulation at all. Alternatives such as native metal-inducible promoters pose the problem of inducer toxicity.Here, we evaluate four different inducible promoters, both previously published and new, using the modular plasmid pSHDY, in the model cyanobacterium Synechocystis sp. PCC 6803 - namely the vanillate-inducible promoter PvanCC, the rhamnose-inducible Prha, and the aTc-inducible PL03, and the Co2+-inducible PcoaT. We estimate individual advantages and disadvantages, as well as dynamic range and strength of each promoter in comparison with well-established constitutive systems. We observed a delicate balance between transcription factor toxicity and sufficient expression to obtain a dose-dependent response to the inducer. In summary, we expand the current understanding and employability of inducible promoters in order to facilitate the construction of more complex regulatory synthetic networks, as well as more complicated biotechnological pathways for cyanobacteria.

scholarly journals An Estradiol-Inducible Promoter Enables Fast, Graduated Control of Gene Expression in Fission Yeast

2016 ◽  
Author(s):  
Makoto J. Ohira ◽  
David G. Hendrickson ◽  
R. Scott McIsaac ◽  
Nicholas Rhind
Keyword(s):  
Gene Expression ◽  
Fission Yeast ◽  
Dose Response ◽  
Dynamic Range ◽  
Inducible Promoter ◽  
Experimental Manipulation ◽  
Control Of Gene Expression ◽  
Inducible Promoters ◽  
Linear Dose ◽  
Promoter System

ABSTRACTThe fission yeast Schizosaccharomyces pombe lacks a diverse toolkit of inducible promoters for experimental manipulation. Available inducible promoters suffer from slow induction kinetics, limited control of expression levels and/or a requirement for defined growth medium. In particular, no S. pombe inducible promoter systems exhibit a linear dose response, which would allow expression to be tuned to specific levels. We have adapted a fast, orthogonal promoter system with a large dynamic range and a linear dose response, based on β-estradiol-regulated function of the human estrogen receptor, for use in S. pombe. We show that this promoter system, termed Z3EV, turns on quickly, can reach a maximal induction of 20 fold, and exhibits a linear dose response over its entire induction range, with few off target effects. We demonstrate the utility of this system by regulating the mitotic inhibitor Wee1 to create a strain in which cell size is regulated by β-estradiol concentration. This promoter system will be of great utility for experimentally regulating gene expression in fission yeast.

scholarly journals A rhamnose-inducible system for precise and temporal control of gene expression in cyanobacteria

2017 ◽  
Author(s):  
Ciarán L. Kelly ◽  
Andrew Hitchcock ◽  
Antonio Torres-Méndez ◽  
John T. Heap
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Linear Response ◽  
Inducible Promoter ◽  
External Control ◽  
Basal Transcription ◽  
Control Of Gene Expression ◽  
Biotechnological Potential ◽  
Genetic Tools ◽  
Inducible Promoters

ABSTRACTCyanobacteria are important for fundamental studies of photosynthesis and have great biotechnological potential. In order to better study and fully exploit these organisms, the limited repertoire of genetic tools and parts must be expanded. A small number of inducible promoters have been used in cyanobacteria, allowing dynamic external control of gene expression through the addition of specific inducer molecules. However, the inducible promoters used to date suffer from various drawbacks including toxicity of inducers, leaky expression in the absence of inducer and inducer photolability, the latter being particularly relevant to cyanobacteria which, as photoautotrophs, are grown under light. Here we introduce the rhamnose-induciblerhaBADpromoter ofEscherichia coliinto the model freshwater cyanobacteriumSynechocystissp. PCC 6803 and demonstrate it has superior properties to previously reported cyanobacterial inducible promoter systems, such as a non-toxic, photostable, non-metabolizable inducer, a linear response to inducer concentration and crucially no basal transcription in the absence of inducer.

scholarly journals Accumulation of Inorganic Polyphosphate in phoU Mutants of Escherichia coli and Synechocystis sp. Strain PCC6803

2002 ◽  
Vol 68 (8) ◽  
pp. 4107-4110 ◽  
Author(s):  
Tomohiro Morohoshi ◽  
Tatsuya Maruo ◽  
Yoko Shirai ◽  
Junichi Kato ◽  
Tsukasa Ikeda ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Biological Process ◽  
Negative Regulator ◽  
Wild Type ◽  
Inorganic Polyphosphate ◽  
Insertional Inactivation ◽  
Synechocystis Sp ◽  
Polyp Accumulation

ABSTRACT The biological process for phosphate (Pi) removal is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyP). We obtained Escherichia coli mutants which accumulate a large amount of polyP. The polyP accumulation in these mutants was ascribed to a mutation of the phoU gene that encodes a negative regulator of the Pi regulon. Insertional inactivation of the phoU gene also elevated the intracellular level of polyP in Synechocystis sp. strain PCC6803. The mutant could remove fourfold more Pi from the medium than the wild-type strain removed.

scholarly journals Collecting eco-evolutionary data in the dark: Impediments to subterranean research and how to overcome them

2020 ◽  
Author(s):  
Stefano Mammola ◽  
Enrico Lunghi ◽  
Helena Bilandžija ◽  
Pedro Cardoso ◽  
Volker Grimm ◽  
...  
Keyword(s):  
Model Systems ◽  
Ex Situ ◽  
Model Organisms ◽  
Physiological Tolerance ◽  
Advantages And Disadvantages ◽  
General Scientific ◽  
Experimental Model Systems ◽  
Scientific Questions ◽  
Subterranean Species

(1) Caves and other subterranean habitats fulfill the requirements of experimental model systems to address general questions in ecology and evolution. Yet, the harsh working conditions of these environments and the uniqueness of the subterranean organisms have challenged most attempts to pursuit standardized research(2) Two main obstacles have synergistically hampered previous attempts. First, there is a habitat impediment related to the objective difficulties of exploring subterranean habitats and our inability to access the network of fissures that represent the elective habitat for the so-called “cave species.” Second, there is a biological impediment illustrated by the rarity of most subterranean species and their low physiological tolerance, often limiting sample size and complicating lab experiments.(3) We explore the advantages and disadvantages of four general experimental setups (in-situ, quasi in-situ, ex-situ, and in-silico) in the light of habitat and biological impediments. We also discuss the potential of indirect approaches to research. Furthermore, using bibliometric data, we provide a quantitative overview of the model organisms that scientists have exploited in the study of subterranean life.(4) Our over-arching goal is to promote caves as model systems where one can perform standardised scientific research. This is important not only to achieve an in-depth understanding of the functioning of subterranean ecosystems but also to fully exploit their long-discussed potential in addressing general scientific questions with implications beyond the boundaries of this discipline.

scholarly journals Receiver Designs for Electronic Toll Collection Systems : A Survey

2020 ◽  
pp. 576-581
Author(s):  
Rarika Ravi ◽  
Anu Assis
Keyword(s):  
Dynamic Range ◽  
Supply Voltage ◽  
Automatic Gain Control ◽  
Power Level ◽  
Cmos Technology ◽  
Electronic Toll Collection ◽  
Chip Area ◽  
Advantages And Disadvantages ◽  
Digitally Controlled ◽  
Toll Collection

<p>This paper discusses about different receiver designs adopted so far for various electronic toll collection systems. A comparative analysis based on the discussions is also provided. It shows that each design has it's own advantages and disadvantages compared to others. The main aim of this paper is to identify the most suitable design. The researches shows that the receiver design described in the 5.8GHz digitally controlled DSRC receiver for Chinese electronic toll collection system is the most suitable one. Here all RF, IF blocks and digital baseband for on-chip automatic gain control, are integrated on an RF-SoC. The proposed digitally controlled LNA and mixer circuits are elaborated. The technology used is 0.13μm CMOS technology. The RF block occupies a chip area of 0.75mm2. It consumes 22mA under a 1.5V supply voltage. The bit error rate maintains better than 10-6, the input power level varies from -75dBm to -8dBm. This design provides a receiver sensitivity improvement of at least 25%, and a dynamic range enhancement of at least 12%.</p>

Measurement of Hypozincaemia in Mice: A Sensitive Test for Detection of Pyrogens

1981 ◽  
Vol 61 (4) ◽  
pp. 445-449 ◽  
Author(s):  
Susan Boobis ◽  
Rose E. Hartley
Keyword(s):  
Escherichia Coli ◽  
Zinc Concentration ◽  
Shigella Dysenteriae ◽  
Metabolic Inhibitor ◽  
Repeated Treatment ◽  
Plasma Zinc ◽  
Sensitive Test ◽  
Zinc Concentrations ◽  
Zinc Levels ◽  
Dose Dependent

1. The effect of bacterial pyrogens on plasma zinc concentration in mice was studied as a method of bioassay for these substances. 2. A dose-related depression of plasma zinc concentrations was observed 4 h after intravenous injection of doses of 0.05-500 ng of purified endotoxins of Salmonella abortus equi, Shigella dysenteriae and Escherichia coli. Responses were dose-dependent and reproducible for each endotoxin in five strains of mice. 3. Tolerance to endotoxin could be induced in mice by injection of doses of 500 ng but was not seen with doses of <5 ng, even after repeated treatment. 4. Dinitrophenol, a metabolic inhibitor, and amphetamine, a metabolic enhancer, did not affect plasma zinc levels, indicating that changes in basal metabolic rate do not affect the outcome of this assay. The effects of pyrogenic materials other than endotoxin on plasma zinc levels were also tested and the results suggest that measurement of hypozincaemia in mice provides the basis for a simple, practical and inexpensive test for endotoxins and other pyrogens.

Use of the Escherichia coli gene for asparagine synthetase as a selective marker in a shuttle vector capable of dominant transfection and amplification in animal cells

1987 ◽  
Vol 7 (5) ◽  
pp. 1623-1628
Author(s):  
M Cartier ◽  
M W Chang ◽  
C P Stanners
Keyword(s):  
Escherichia Coli ◽  
Cell Lines ◽  
Animal Cell ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Asparagine Synthetase ◽  
Translational Initiation ◽  
Shuttle Vectors ◽  
Advantages And Disadvantages

A new dominant amplifiable selective system for use in bacterium-animal cell shuttle vectors was developed by the insertion of a 2-kilobase genomic fragment containing the cloned Escherichia coli gene for asparagine synthetase (AS) into the pBR322-simian virus 40 recombinant vector pSV2 so as to place the translational initiator codon for the bacterial AS about 1,000 base pairs downstream from the simian virus 40 early promoter. This new construct, pSV2-AS, retains bacterial sequences for transcriptional and translational initiation and so can express AS in bacteria. The construct can also complement AS- mutants of mammalian cells, giving AS+ transfectants capable of growth in medium lacking asparagine, with relatively high efficiency (about 300 colonies per microgram of DNA per 10(6) cells exposed). The vector can be amplified up to 100-fold in such AS+ transfectants by selection in asparagine-free medium containing increasing concentrations of the AS inhibitor beta-aspartyl hydroxamate. AS+ transfectants were found to be much more resistant to a second AS inhibitor, Albizziin, than were normal AS+ animal cell lines. This difference, which may indicate a strong resistance of the bacterial AS enzyme to Albizziin, was exploited to develop an effective selection for bacterial AS transfectants of a number of wild-type AS+ cell lines of rat, Chinese hamster, mouse, and human origin. LR-73 cells, a Chinese hamster AS+ cell line, were transfected with pSV2-AS with an efficiency of about 1,000 colonies per 0.5 microgram of DNA per 10(6) cells. The integrated construct in these cells was amplified by incubation of the transfectants in increasing concentrations of beta-aspartyl hydroxamate. Advantages and disadvantages of this new dominant, selectable, and amplifiable marker over markers commonly used in shuttle vectors are discussed.

scholarly journals Multiplex Real-Time Polymerase Chain Reaction for Simultaneous Quantification ofSalmonellaspp.,Escherichia coli, andStaphylococcus aureusin Different Food Matrices: Advantages and Disadvantages

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Amanda Teixeira Sampaio Lopes ◽  
George Rêgo Albuquerque ◽  
Bianca Mendes Maciel
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Limit Of Detection ◽  
Microbiological Quality ◽  
Rapid Screening ◽  
Multiplex Qpcr ◽  
Advantages And Disadvantages ◽  
Simultaneous Quantification ◽  
Polymerase Chain ◽  
Food Matrices

Quantitative real-time polymerase chain reactions (qPCRs) of the most prevalent bacteria causing foodborne diseases worldwide, such asSalmonellaspp.,Escherichia coli, andStaphylococcus aureus,can be an important tool for quantitative microbial risk assessment, which requires numerical data to determine the level of contamination at a specific stage of food production. However, most of qPCR assays described in the literature for these pathogens are qualitative; their objective is pathogen detection and not pathogen quantification. Thus, the aim of our work was to develop a qPCR for the simultaneous quantification ofSalmonellaspp.,E. coli, andS. aureusand to propose its use in the analysis of foods, as a tool for microbiological quality monitoring. For this, a multiplex qPCR was standardized for the simultaneous quantification of specific fragments of target genes (ssf,phoA, andnuc) corresponding to each one of the mentioned bacteria. The limit of detection of the technique was 13, 10, and 12 gene copies forssf,phoA, andnuc, respectively; standard curves showed R2> 0.99, with efficiencies ranging from 99 to 110%, and inter- and intraexperiment reproducibility presented a low coefficient of variation in all trials. This methodology was applied in different food matrices (milk, ground beef, and oyster meat), and the results were compared with official microbiological culture methodology and with ready-to-use test. Advantages and disadvantages of each methodology used in this study are pointed out. We suggest that this multiplex qPCR can be used as a rapid screening technique for the analysis of food microbiological quality.

scholarly journals Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus

2006 ◽  
Vol 72 (1) ◽  
pp. 102-111 ◽  
Author(s):  
S.-V. Albers ◽  
M. Jonuscheit ◽  
S. Dinkelaker ◽  
T. Urich ◽  
A. Kletzin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Shuttle Vector ◽  
Sulfolobus Solfataricus ◽  
Inducible Promoter ◽  
Single Step ◽  
Vector System ◽  
Hyperthermophilic Archaea ◽  
Model Organisms ◽  
Hyperthermophilic Archaeon ◽  
High Level

ABSTRACT Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for archaea, and no system for high-level gene expression existed for hyperthermophilic organisms. Recently, a virus-based shuttle vector with a reporter gene was developed for the crenarchaeote Sulfolobus solfataricus, a model organism of hyperthermophilic archaea that grows optimally at 80°C (M. Jonuscheit, E. Martusewitsch, K. M. Stedman, and C. Schleper, Mol. Microbiol. 48:1241-1252, 2003). Here we have refined this system for high-level gene expression in S. solfataricus with the help of two different promoters, the heat-inducible promoter of the major chaperonin, thermophilic factor 55, and the arabinose-inducible promoter of the arabinose-binding protein AraS. Functional expression of heterologous and homologous genes was demonstrated, including production of the cytoplasmic sulfur oxygenase reductase from Acidianus ambivalens, an Fe-S protein of the ABC class from S. solfataricus, and two membrane-associated ATPases potentially involved in the secretion of proteins. Single-step purification of the proteins was obtained via fused His or Strep tags. To our knowledge, these are the first examples of the application of an expression vector system to produce large amounts of recombinant and also tagged proteins in a hyperthermophilic archaeon.

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