Oxidative Stress Detection With Escherichia Coli Harboring A katG\u27::lux Fusion

A plasmid containing a transcriptional fusion of the Escherichia coli katG promoter to a truncated Vibrio fischeri lux operon (luxCDABE) was constructed. An E. coli strain bearing this plasmid (strain DPD2511) exhibited low basal levels of luminescence, which increased up to 1,000-fold in the presence of hydrogen peroxide, organic peroxides, redox-cycling agents (methyl viologen and menadione), a hydrogen peroxide-producing enzyme system (xanthine and xanthine oxidase), and cigarette smoke. An oxyR deletion abolished hydrogen peroxide-dependent induction, confirming that oxyR controlled katG\u27::lux luminescence. Light emission was also induced by ethanol by an unexplained mechanism. A marked synergistic response was observed when cells were exposed to both ethanol and hydrogen peroxide; the level of luminescence measured in the presence of both inducers was much higher than the sum of the level of luminescence observed with ethanol and the level of luminescence observed with hydrogen peroxide. It is suggested that this construction or similar constructions may be used as a tool for assaying oxidant and antioxidant properties of chemicals, as a biosensor for environmental monitoring and as a tool for studying cellular responses to oxidative hazards

Detection Of DNA Damage By Use Of Escherichia Coli Carrying recA\u27::lux, uvrA\u27::lux, And alkA\u27::lux Reporter Plasmids

Plasmids were constructed in which DNA damage-inducible promoters recA, uvrA, and alkA from Escherichia coli were fused to the Vibrio fischeri luxCDABE operon. Introduction of these plasmids into E. coli allowed the detection of a dose-dependent response to DNA-damaging agents, such as mitomycin and UV irradiation. Bioluminescence was measured in real time over extended periods. The fusion of the recA promoter to luxCDABE showed the most dramatic and sensitive responses. lexA dependence of the bioluminescent SOS response was demonstrated, confirming that this biosensor\u27s reports were transmitted by the expected regulatory circuitry. Comparisons were made between luxCDABE and lacZ fusions to each promoter. It is suggested that the lux biosensors may have use in monitoring chemical, physical, and genotoxic agents as well as in further characterizing the mechanisms of DNA repair

Bioluminescent Escherichia Coli Strains For The Quantitative Detection Of Phosphate And Ammonia In Coastal And Suburban Watersheds

Accumulation of phosphate and ammonia in estuarine systems and subsequent dinoflagellate and algal blooms has been implicated in fish kills and in health risks for fishermen. Analytic chemistry kits are used to measure phosphate and ammonia levels in water samples, but their sensitivity is limited due to specificity for inorganic forms of these moieties. An Escherichia coli bioluminescent reporter system measured the bioavailability of inorganic nutrients through fusion of E. coli promoters (phoA or glnAp2) to the luxCDABE operon of Vibrio fischeri carried either on the chromosome or on a multicopy plasmid vector, resulting in emission of light in response to phosphate or ammonia starvation. Responses were shown to be under the control of expected physiological regulators, phoB and glnFG, respectively. Standard curves were used to determine the phosphate and ammonia levels in water samples from diverse watersheds located in the northeastern United States. Bioluminescence produced in response to nutrient starvation correlated with concentrations of phosphate (1-24 ppm) and ammonia (0.1-1.6 ppm). While the ammonia biosensor measured nutrient concentrations in tested water samples that were comparable to the amounts reported by a commercial kit, the phosphate biosensor reported higher levels of phosphate in Chesapeake water samples than did the kit

Responses To Toxicants Of An Escherichia Coli Strain Carrying A uspA\u27::lux Genetic Fusion And An E. Coli Strain Carrying A grpE\u27::lux Fusion Are Similar

A transcriptional fusion of the Escherichia coli uspA promoter to luxCDABE was characterized and compared with a heat shock-responsive grpE\u27::lux fusion. Similarities in range and rank order of inducing conditions were observed; however, the magnitude of induction was typically greater for the grpE\u27::lux fusion strain

Hypothesis Testing and Power Calculations for Taxonomic-Based Human Microbiome Data

This paper presents new biostatistical methods for the analysis of microbiome data based on a fully parametric approach using all the data. The Dirichlet-multinomial distribution allows the analyst to calculate power and sample sizes for experimental design, perform tests of hypotheses (e.g., compare microbiomes across groups), and to estimate parameters describing microbiome properties. The use of a fully parametric model for these data has the benefit over alternative non-parametric approaches such as bootstrapping and permutation testing, in that this model is able to retain more information contained in the data. This paper details the statistical approaches for several tests of hypothesis and power/sample size calculations, and applies them for illustration to taxonomic abundance distribution and rank abundance distribution data using HMP Jumpstart data on 24 subjects for saliva, subgingival, and supragingival samples. Software for running these analyses is available

How to do a grounded theory study: a worked example of a study of dental practices

<p>Abstract</p> <p>Background</p> <p>Qualitative methodologies are increasingly popular in medical research. Grounded theory is the methodology most-often cited by authors of qualitative studies in medicine, but it has been suggested that many 'grounded theory' studies are not concordant with the methodology. In this paper we provide a worked example of a grounded theory project. Our aim is to provide a model for practice, to connect medical researchers with a useful methodology, and to increase the quality of 'grounded theory' research published in the medical literature.</p> <p>Methods</p> <p>We documented a worked example of using grounded theory methodology in practice.</p> <p>Results</p> <p>We describe our sampling, data collection, data analysis and interpretation. We explain how these steps were consistent with grounded theory methodology, and show how they related to one another. Grounded theory methodology assisted us to develop a detailed model of the process of adapting preventive protocols into dental practice, and to analyse variation in this process in different dental practices.</p> <p>Conclusions</p> <p>By employing grounded theory methodology rigorously, medical researchers can better design and justify their methods, and produce high-quality findings that will be more useful to patients, professionals and the research community.</p