Evolved Biosensor with High Sensitivity and Specificity for Measuring Cadmium in Actual Environmental Samples.

Bacterial biosensors have great potential in contaminant detection for sensitivity, specificity, cost-effectiveness, and easy operation. However, the existing cadmium-responsive bacterial biosensors cannot meet the real-world detection requirements due to lack of sensitivity, specificity, and anti-interference capability. This study aimed to develop a bacterial biosensor for detecting the total and extractable cadmium in actual environmental samples. We constructed the cadmium-responsive biosensor with the regulatory element (cadmium resistance transcriptional regulatory, CadR) and the reporting element (GFP) and improved its performance by directed evolution. The mutant libraries of biosensors were generated by error-prone PCR and screened by continuous five-round fluorescence-activated cell sorting (FACS), and a bacteria variant epCadR5 with higher performance was finally isolated. Biosensor fluorescence intensity was measured by a microplate reader, and results showed that the evolved cadmium-responsive bacterial biosensor was of high sensitivity and specificity in detecting trace cadmium, with a detection limit of 0.45 µg/L, which is 6.8 times more specific to cadmium than that of the wild-type. Furthermore, microscopic qualitative analysis results showed that the bacteria could produce fluorescence response in a cadmium-contaminated soil matrix, and quantitative analysis results showed that the values of cadmium from epCadR5 bacteria were close to that from inductively coupled plasma-mass spectrometry. These results suggest that the biosensor may have a broad application prospect in the detection of cadmium-contaminated soil and water.

Constructing of Bacillus subtilis-Based Lux-Biosensors with the Use of Stress-Inducible Promoters.

Here, we present a new lux-biosensor based on Bacillus subtilis for detecting of DNA-tropic and oxidative stress-causing agents. Hybrid plasmids pNK-DinC, pNK-AlkA, and pNK-MrgA have been constructed, in which the Photorhabdus luminescens reporter genes luxABCDE are transcribed from the stress-inducible promoters of B. subtilis: the SOS promoter PdinC, the methylation-specific response promoter PalkA, and the oxidative stress promoter PmrgA. The luminescence of B. subtilis-based biosensors specifically increases in response to the appearance in the environment of such common toxicants as mitomycin C, methyl methanesulfonate, and H2O2. Comparison with Escherichia coli-based lux-biosensors, where the promoters PdinI, PalkA, and Pdps were used, showed generally similar characteristics. However, for B. subtilis PdinC, a higher response amplitude was observed, and for B. subtilis PalkA, on the contrary, both the amplitude and the range of detectable toxicant concentrations were decreased. B. subtilis PdinC and B. subtilis PmrgA showed increased sensitivity to the genotoxic effects of the 2,2'-bis(bicyclo [2.2.1] heptane) compound, which is a promising propellant, compared to E. coli-based lux-biosensors. The obtained biosensors are applicable for detection of toxicants introduced into soil. Such bacillary biosensors can be used to study the differences in the mechanisms of toxicity against Gram-positive and Gram-negative bacteria.

Construction of a sensitive and specific lead biosensor using a genetically engineered bacterial system with a luciferase gene reporter controlled by pbr and cadA promoters.

BACKGROUND: A bacterial biosensor refers to genetically engineered bacteria that produce an assessable signal in the presence of a physical or chemical agent in the environment. METHODS: We have designed and evaluated a bacterial biosensor expressing a luciferase reporter gene controlled by pbr and cadA promoters in Cupriavidus metallidurans (previously termed Ralstonia metallidurans) containing the CH34 and pI258 plasmids of Staphylococcus aureus, respectively, and that can be used for the detection of heavy metals. In the present study, we have produced and evaluated biosensor plasmids designated pGL3-luc/pbr biosensor and pGL3-luc/cad biosensor, that were based on the expression of luc+ and under the control of the cad promoter and the cadC gene of S. aureus plasmid pI258 and pbr promoter and pbrR gene from plasmid pMOL30 of Cupriavidus metallidurans. RESULTS: We found that the pGL3-luc/pbr biosensor may be used to measure lead concentrations between 1-100 µM in the presence of other metals, including zinc, cadmium, tin and nickel. The latter metals did not result in any significant signal. The pGL3-luc/cad biosensor could detect lead concentrations between 10 nM to 10 µM. CONCLUSIONS: This biosensor was found to be specific for measuring lead ions in both environmental and biological samples.

Ion Selective Amperometric Biosensors for Environmental Analysis of Nitrate, Nitrite and Sulfate.

Inorganic ions that can be redox-transformed by living cells can be sensed by biosensors, where the redox transformation gives rise to a current in a measuring circuit. Such biosensors may be based on enzymes, or they may be based on application of whole cells. In this review focus will be on biosensors for the environmentally important ions NO3-, NO2-, and SO42-, and for comparison alternative sensor-based detection will also be mentioned. The developed biosensors are generally characterized by a high degree of specificity, but unfortunately also by relatively short lifetimes. There are several investigations where biosensor measurement of NO3- and NO2- have given new insight into the functioning of nitrogen transformations in man-made and natural environments such as sediments and biofilms, but the biosensors have not become routine tools. Future modifications resulting in better long-term stability may enable such general use.

Borosilicate Glass Fiber-Optic Biosensor for the Detection of Escherichia coli.

Polyclonal antibodies against Escherichia coli and fluorescent, secondary, antibodies were immobilized on borosilicate glass fibers pre-treated with 3-glycidyloxypropyl trimethoxysilane (GPS). Light with an average wavelength of 627 nm, emitted by a diode placed at one end of the glass fiber, was detected by an ultrasensitive photodiode with peak sensitivity at 640 nm. Changes in fluorescence, caused by binding of E. coli to the antibodies, changed the net refractive index of the glass fiber and thus the internal reflection of light. These evanescent changes in photon energy were recorded by an ultrasensitive photodiode. Signals were amplified and changes in voltage recorded with a digital multimeter. A linear increase in voltage readings was recorded over 2 h when 3.0 × 107 CFU/ml and 2.77 × 109 CFU/ml E. coli were adhered to the antibodies. Voltage readings were recorded with E. coli cell numbers from 2 × 103 CFU/ml to 2 × 106 CFU/ml, but readings remained unchanged for 2 h, indicating that the limit of detection is 3.0 × 107 CFU/ml. This simple technology may be used to develop a low-cost, portable, fiber-optic biosensor to detect E. coli in infections and may have applications in the medical field. Research is in progress to optimize the sensitivity of the fiber-optic biosensor and determine its specificity.

Integration of a Gold-Specific Whole E. coli Cell Sensing and Adsorption Based on BioBrick.

Detection and recovery of heavy metals from environmental sources is a major task in environmental protection and governance. Based on previous research into cell-based visual detection and biological adsorption, we have developed a novel system combining these two functions by the BioBrick technique. The gold-specific sensory gol regulon was assembled on the gold-chaperone GolB (Gold-specific binding protein), which is responsible for selectively absorbing gold ions, and this led to an integration system with increased probe tolerance for gold. After being incorporated into E. coli, this system featured high-selective detection and recycling of gold ions among multi-metal ions from the environment. It serves as an efficient method for biological detection and recovery of various heavy metals. We have developed modular methods for cell-based detection and adsorption of heavy metals, and these offer a quick and convenient tool for development in this area.

Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production.

Many high-value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimize the productivity of these cultures it is important to monitor cellular metabolism, for example the utilization of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole-cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity, and robustness. Importantly, our whole-cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole-cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalize on the wealth of genetic operons for metabolite sensing available in nature for the development of other whole-cell biosensors. Biotechnol. Bioeng. 2017;114: 1290-1300. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Numerical modeling of the dynamic response of a bioluminescent bacterial biosensor.

Water quality and water management are worldwide issues. The analysis of pollutants and in particular, heavy metals, is generally conducted by sensitive but expensive physicochemical methods. Other alternative methods of analysis, such as microbial biosensors, have been developed for their potential simplicity and expected moderate cost. Using a biosensor for a long time generates many changes in the growth of the immobilized bacteria and consequently alters the robustness of the detection. This work simulated the operation of a biosensor for the long-term detection of cadmium and improved our understanding of the bioluminescence reaction dynamics of bioreporter bacteria inside an agarose matrix. The choice of the numerical tools is justified by the difficulty to measure experimentally in every condition the biosensor functioning during a long time (several days). The numerical simulation of a biomass profile is made by coupling the diffusion equation and the consumption/reaction of the nutrients by the bacteria. The numerical results show very good agreement with the experimental profiles. The growth model verified that the bacterial growth is conditioned by both the diffusion and the consumption of the nutrients. Thus, there is a high bacterial density in the first millimeter of the immobilization matrix. The growth model has been very useful for the development of the bioluminescence model inside the gel and shows that a concentration of oxygen greater than or equal to 22 % of saturation is required to maintain a significant level of bioluminescence. A continuous feeding of nutrients during the process of detection of cadmium leads to a biofilm which reduces the diffusion of nutrients and restricts the presence of oxygen from the first layer of the agarose (1 mm) and affects the intensity of the bioluminescent reaction. The main advantage of this work is to link experimental works with numerical models of growth and bioluminescence in order to provide a general purpose model to understand, anticipate, or predict the dysfunction of a biosensor using immobilized bioluminescent bioreporter in a matrix.

Construction and evaluation of a genetic construct for specific detection and measurement of propionate by whole-cell bacteria.

Anaerobic digestion is a microbiological technology that converts biomass wastes into biogas, achieving both waste treatment and bioenergy production. Accumulation of volatile fatty acids (VFA) during acidogenesis, particularly propionate, often causes upset or failure of digesters. Early detection and monitoring of propionate concentration in digesters allow for just-in-time interventions to prevent irreversible costly process breakdown. In an attempt to develop a rapid method of measuring propionate concentration and bioavailability, we constructed a genetic construct for specific detection of bioavailable propionate. The genetic construct was constructed by transcriptional fusion of the regulatory gene (prpR) and the promoter of the prp operon (PprpB ) of Escherichia coli W3110 with the reporter gene cassette luxCDABE. When the genetic construct was carried on a plasmid and transformed into E. coli (referred to as plasmid-based biosensor), it resulted in stronger emission of luminescence than when it was inserted into the chromosome of E. coli (referred to as chromosome-based biosensor). The biosensor responded specifically to propionate. The luminescence signal increased linearly with increasing concentration of propionate from 1 to 10 mM. The utility of the biosensor was evaluated using samples collected from anaerobic digesters. Once instrumented in future studies, the whole-cell bacterial biosensor developed in this study may provide an alternative technology for real-time detection and measurement of propionate in digesters.

Visual arsenic detection in environmental waters: Innovating with a naked-eye biosensor for universal application.

Arsenic contamination in environmental water sources poses a significant threat to human health, necessitating the development of sensitive and accessible detection methods. This study presents a multidimensional optimization of a bacterial biosensor for the susceptible and deoxyviolacein (DV)-based visual detection of arsenic. The research involved screening six different arsenic resistance (ars) operons and optimizing the genetic circuit to minimize background noise. Introducing an arsenic-specific transport channel enhanced the sensor's sensitivity to 1 nM with a quantitative range from 0.036 to 1.171 µM. The pigment-based biosensor offers a simple colorimetric approach for arsenic detection without complex instrumentation. The preferred biosensor demonstrated characteristics of anti-chelating agent interference, consistently quantified As(III) concentrations ranging from 0.036 to 1.171 µM covering the World Health Organization (WHO) drinking water limit. Innovatively, it effectively detects arsenic in seawater within a linear regression range of 0.071 to 1.125 µM. The biosensor's selectivity for arsenic was confirmed, with minimal cross-response to group 15 metals. Our naked-eye biosensor offers a novel approach for the rapid, on-site detection of arsenic in various water sources. Its simplicity, cost-effectiveness, and versatility make it a valuable tool for environmental monitoring and public health initiatives.

A novel antimony-selective ArsR transcriptional repressor and its specific detection of antimony trioxide in environmental samples via bacterial biosensor.

Extensively industrial applications and ever-accelerated anthropogenic activities have resulted in the dramatic accumulation of Sb2O3 contaminant in the environment, leading to adverse health effects on humans and ecosystems. Although arsenite has been subjected to numerous studies and ArsR-based whole-cell biosensors have been successfully applied in field testing of arsenite, there is limited information on the biological recognition element of Sb2O3 and its actual application in biosensor construction and environmental monitoring. In this study, we identified a specific recognition element of Sb2O3, SxArsR, in Sphingobium xenophagum C1 by the induced bioluminescent signal analysis of gene expression in response to Sb2O3 exposure. Compared to the other four groups of characterized ArsRs, the novel SxArsR lacks the third cysteine residue for binding of arsenite and has a conserved histidine-cysteine "HCXC" binding site that directly and specifically binds for Sb2O3. Sb2O3 can remove SxArsR from the core operator/promoter binding sequence in the -79 region upstream of the start codon of sxarsR. Based on the specificity of SxArsR protein and the sensitivity of SxArsR-binding DNA sequence, SxArsR-based whole-cell biosensor was constructed and showed a linear relationship (R2 = 0.99) from 0.01 to 6.0 µM of Sb2O3 with a detection limit of 0.01 µM. The novel bacterial biosensor also exhibited a good performance in the detection of Sb2O3 in environmental water and sediment samples. Overall, SxArsR-based biosensor represents a promising strategy for Sb2O3 detection and may have a profound impact on further practical application of ArsR biosensor in the dual-signal simultaneous detection of arsenite and Sb2O3.

Anthocyanin biosynthetic pathway switched by metalloregulator PbrR to enable a biosensor for the detection of lead toxicity.

Environmental lead pollution mainly caused by previous anthropogenic activities continuously threatens human health. The determination of bioavailable lead is of great significance to predict its ecological risk. Bacterial biosensors using visual pigments as output signals have been demonstrated to have great potential in developing minimal-equipment biosensors for environmental pollutant detection. In this study, the biosynthesis pathway of anthocyanin was heterogeneously reconstructed under the control of the PbrR-based Pb(II) sensory element in Escherichia coli. The resultant metabolic engineered biosensor with colored anthocyanin derivatives as the visual signal selectively responded to concentrations as low as 0.012 µM Pb(II), which is lower than the detection limit of traditional fluorescent protein-based biosensors. A good linear dose-response pattern in a wide Pb(II) concentration range (0.012-3.125 µM) was observed. The color deepening of culture was recognized to the naked eye in Pb(II) concentrations ranging from 0 to 200 µM. Importantly, the response of metabolic engineered biosensors toward Pb(II) was not significantly interfered with by organic and inorganic ingredients in environmental water samples. Our findings show that the metabolic engineering of natural colorants has great potential in developing visual, sensitive, and low-cost bacterial biosensors for the detection and determination of pollutant heavy metals.

Metabolic engineering of the violacein biosynthetic pathway toward a low-cost, minimal-equipment lead biosensor.

Metabolic engineered bacteria have been successfully employed to produce various natural colorants, which are expected to be used as the visually recognizable signals to develop mini-equipment biological devices for monitoring toxic heavy metals. The violacein biosynthetic pathway has been reconstructed in Escherichia coli (E. coli). Here the successful production of four violacein derivatives was achieved by integrating metabolic engineering and synthetic biology. Lead binding to the metalloregulator enables whole-cell colorimetric biosensors capable of assessing bioavailable lead. Deoxyviolacein-derived signal showed the most satisfied biosensing properties among prodeoxyviolacein (green), proviolacein (blue), deoxyviolacein (purple), and violacein (navy). The limit of detection (LOD) of pigment-based biosensors was 2.93 nM Pb(II), which is lower than that of graphite furnace atomic absorption spectrometry. Importantly, a good linear dose-response model in a wide dose range (2.93-6000 nM) was obtained in a non-cytotoxic deoxyviolacein-based biosensor, which was significantly better than cytotoxic violacein-based biosensor (2.93-750 nM). Among ten metal ions, only Cd(II) and Hg(II) exerted a slight influence on the response of the deoxyviolacein-based biosensor toward Pb(II). The deoxyviolacein-based biosensor was validated in detecting bioaccessible Pb(II) in environmental samples. Factors such as low cost and minimal-equipment requirement make this biosensor a suitable biological device for monitoring toxic lead in the environment.

Strategies for Improving Small-Molecule Biosensors in Bacteria.

In recent years, small-molecule biosensors have become increasingly important in synthetic biology and biochemistry, with numerous new applications continuing to be developed throughout the field. For many biosensors, however, their utility is hindered by poor functionality. Here, we review the known types of mechanisms of biosensors within bacterial cells, and the types of approaches for optimizing different biosensor functional parameters. Discussed approaches for improving biosensor functionality include methods of directly engineering biosensor genes, considerations for choosing genetic reporters, approaches for tuning gene expression, and strategies for incorporating additional genetic modules.

A method for detecting perfluorooctanoic acid and perfluorooctane sulfonate in water samples using genetically engineered bacterial biosensor.

A simple, reagent and pre-treatment (i.e. dilution, sample purification and pH adjustment) free approach based genetically engineered bacterial biosensor is developed and demonstrated for the detection of perfluorinated compounds in water samples. The bacterial biosensor was developed by integrating two genes called regulatory (defluorinase gene) and reporter gene (green fluorescence gene) through genetic engineering techniques. The as-developed bacterial biosensor was employed to detect perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) in water samples upon induction of regulatory gene and expression of green fluorescence protein. The induced fluorescence emission by the biosensor was visualized using fluorescence microscopic images. The specificity of biosensor was evaluated with different types of organic pollutants such as chlorinated compounds, polyaromatic hydrocarbons and pesticides etc., in both presence and absence of PFOA and PFOS. The biosensor was employed to detect the perfluorinated compounds at nano gram level in both standard solutions and natural water samples like river water, wastewater and drinking water with an analysis time of 24 h. The detection of PFOA and PFOS by the developed-bacterial sensor is validated by liquid chromatography coupled with mass spectrometer. The developed biosensor has demonstrated a rapid and sensitive detection of PFOA and PFOS in various water samples.

Transcription factor based whole-cell biosensor for specific and sensitive detection of sodium dodecyl sulfate.

Extensive use of Sodium Dodecyl Sulfate (SDS) in households, agricultural operations, and industries is leading to its subsequent disposal in waterways. There is an apprehension of the adverse effect of such detergents on various living organisms. Thus, an efficient, specific, and simple detection method to monitor SDS reliably in the environment is needed. We used sdsB1 activator protein and SDS-responsive promoter of sdsA1 gene along with Green Fluorescent Protein (GFP) to construct a novel SDS biosensor in Pseudomonas aeruginosa chassis. The GFP intensity of the biosensor showed a linear relationship (R2 = 0.99) from 0.4 to 62.5 ppm of SDS with a detection limit of 0.1 ppm. This biosensor is highly specific for SDS and has minimal interference from other detergents, metals, and inorganic ions. The biosensor showed a satisfactory and reproducible recovery rate for the detection of SDS in real samples. Overall, this is a low cost, easy-to-use, selective, and reliable biosensor for monitoring SDS in the environment.

Rapid BOD assessment with a microbial array coupled to a neural machine learning system.

The domestic usage of water generates approximately 310 km3 of wastewater worldwide (2015, AQUASTAT, Food and Agriculture Organization of United Nations). This sewage contains an important organic load due to the use of this water; this organic load is characterized using a standard method, namely, the biological oxygen demand measurement (BOD5). The BOD5 provides information about the biodegradable organic load (standard ISO 5815). However, this measurement protocol is very time-consuming (5 days) and may produce variability in approximately 20% of results mainly due to variation in the environmental inocula. To remedy these limitations, this work proposes an innovative concept relying on the implementation of a set of rigorously selected bacterial strains. This publication depicts the different steps used in this study, from bio-indicator selection to validation with real wastewater samples. The results obtained in the final step show a strong correlation between the developed approach and the reference method (ISO 5815) with a correlation rate of approximately 0.9. In addition, the optimization of the experimental conditions and the use of controlled strains (8 selected strains) allow significant reduction in the duration of the BOD5 analysis, with only 3 h required for the proposed method versus 5 days for the reference method. This technological breakthrough should simplify the monitoring of wastewater treatment plants and provide quicker, easier and more coherent control in terms of the treatment time.

Contribution of nonconsensus base pairs within ArsR binding sequences toward ArsR-DNA binding and arsenic-mediated transcriptional induction.

BACKGROUND: A transcriptional reporter is the key component in bacterial biosensors which are employed to monitor the induction or repression of a reporter gene corresponding to environmental change. Interaction of a transcription factor with its consensus sequence generated by using a position weight matrix (PWM) model is crucial for its sensitivity of the reporter. However, recent studies suggest that PWM model based on independent contribution of individual consensus base pairs to protein interaction is often insufficient to explain complex regulation, such as the effect of nonconsensus sequences on the protein-DNA binding affinity. In the present study, we employed a simpler prokaryotic arsenic repressor (ArsR) regulation system to access the protein-DNA recognition. Contribution of nonconsensus base pairs within ArsR binding sequences toward ArsR-DNA binding and arsenic-mediated transcriptional induction was studied. RESULTS: We constructed a series of arsenic responsive reporters, each comprising two copies of the ArsR binding sequences from different resources. We found that high arsenic-mediated induction specifically requires the binding sequence from Escherichia coli to be placed at the first binding sequence; however, no such preference was observed for the second binding sequence, which could be from Acidithiobacillus ferrooxidans, plasmid R773, Synechococcus, or a core binding sequence of arsR. By creating a series of reporters differed at the nonconsensus base pairs of the second binding sequence, we observed that some constructs bound weakly while others strongly to ArsR. Most interestingly, although a number of these reporters showed similar binding affinity to ArsR, their arsenic-dependent induction differed significantly. CONCLUSIONS: The results indicated that nonconsensus base pairs could have profound influence on protein binding and may also modulate post-binding function. These findings provide new insights into the complex regulation of gene expression and facilitate the development of transcriptional reporter-based biosensors.

Ligand-Doped Copper Oxo-hydroxide Nanoparticles are Effective Antimicrobials.

Bacterial resistance to antimicrobial therapies is an increasing clinical problem. This is as true for topical applications as it is for systemic therapy. Topically, copper ions may be effective and cheap antimicrobials that act through multiple pathways thereby limiting opportunities to bacteria for resistance. However, the chemistry of copper does not lend itself to facile formulations that will readily release copper ions at biologically compatible pHs. Here, we have developed nanoparticulate copper hydroxide adipate tartrate (CHAT) as a cheap, safe, and readily synthesised material that should enable antimicrobial copper ion release in an infected wound environment.First, we synthesised CHAT and showed that this had disperse aquated particle sizes of 2-5 nm and a mean zeta potential of - 40 mV. Next, when diluted into bacterial medium, CHAT demonstrated similar efficacy to copper chloride against Escherichia coli and Staphylococcus aureus, with dose-dependent activity occurring mostly around 12.5-50 mg/L of copper. Indeed, at these levels, CHAT very rapidly dissolved and, as confirmed by a bacterial copper biosensor, showed identical intracellular loading to copper ions derived from copper chloride. However, when formulated at 250 mg/L in a topically applied matrix, namely hydroxyethyl cellulose, the benefit of CHAT over copper chloride was apparent. The former yielded rapid sustained release of copper within the bactericidal range, but the copper chloride, which formed insoluble precipitates at such concentration and pH, achieved a maximum release of 10 ± 7 mg/L copper by 24 h.We provide a practical formulation for topical copper-based antimicrobial therapy. Further studies, especially in vivo, are merited.

Method with high-throughput screening potential for antioxidative substances using Escherichia coli biosensor katG'::lux.

A new method is described for the rapid real-time screening of antioxidative properties using a recombinant Escherichia coli DPD2511 biosensor. This microplate technique, without time-consuming pre-incubations and handling, has potential for a high-throughput search of bioactive compounds. Special emphasis was given to obtaining highly reliable and repeatable results.