Bacterial Biosensors Research Articles

Polymer Encapsulation of Bacterial Biosensors Enables Coculture with Mammalian Cells.

Coexistence of different populations of cells and isolation of tasks can provide enhanced robustness and adaptability or impart new functionalities to a culture. However, generating stable cocultures involving cells with vastly different growth rates can be challenging. To address this, we developed living analytics in a multilayer polymer shell (LAMPS), an encapsulation method that facilitates the coculture of mammalian and bacterial cells. We leverage LAMPS to preprogram a separation of tasks within the coculture: growth and therapeutic protein production by the mammalian cells and l-lactate biosensing by Escherichia coli encapsulated within LAMPS. LAMPS enable the formation of a synthetic bacterial–mammalian cell interaction that enables a living biosensor to be integrated into a biomanufacturing process. Our work serves as a proof-of-concept for further applications in bioprocessing since LAMPS combine the simplicity and flexibility of a bacterial biosensor with a viable method to prevent runaway growth that would disturb mammalian cell physiology.

Rapid printing of a Bacterial array for a Solid-Phase Assay (BacSPA) of heavy metal ions

Bacterial whole-cell biosensors have been used to detect numerous pollutants for decades. In this work, a rapid sensing method using patterned bacterial arrays on solidified agar media was reported. A printing system, which quickly patterns 384-spot bacterial arrays via inertial impact, was developed in this study. The droplet formation was observed by a high-speed camera and simulated in COMSOL software to investigate the printing conditions. We found that the droplet volume and speed were influenced by the accelerations of the impact. Following adjustment, the printer could eject discrete droplets and pattern arrays with high accuracy. The printed bacteria were incubated, and images acquired by a commercial digital camera were analyzed by the Python program. The areas of 384 spots in a single array were similar, with a high roundness and low deviations. We found that the printer plate quality influenced the printing results more than the printing conditions. Finally, the printing system was applied to determine heavy metals concentrations in solid growth media by bioluminescent sensor bacteria. The luminescence intensity patterns varied between heavy metals. We propose the printing system as a useful tool for rapid patterning of bacterial whole cell sensor arrays on solid agar.

Topologies of synthetic gene circuit for optimal fold change activation in bacterial biosensors

Biological regulatory networks in nature comprise feedforward and feedback loops allowing cells to perform sophisticated tasks such as DNA repair, cell division, and apoptosis. During the past decades, network motifs have been introduced into living cells to scale–up the computational complexity of synthetic gene circuits. For example, mutual inhibition was the first network that has been implemented in Escherichia coli cells to create a bistable switch. Recently, an incoherent feedforward loop and integral feedback controller have been applied to achieve robust adaptation in Escherichia coli cells.

Low-cost bacterial nanocellulose-based interdigitated biosensor to detect the p53 cancer biomarker

Low-cost sensors to detect cancer biomarkers with high sensitivity and selectivity are essential for early diagnosis. Herein, an immunosensor was developed to detect the cancer biomarker p53 antigen in MCF7 lysates using electrical impedance spectroscopy. Interdigitated electrodes were screen printed on bacterial nanocellulose substrates, then coated with a matrix of layer-by-layer films of chitosan and chondroitin sulfate onto which a layer of anti-p53 antibodies was adsorbed. The immunosensing performance was optimized with a 3-bilayer matrix, with detection of p53 in MCF7 cell lysates at concentrations between 0.01 and 1000 Ucell. mL−1, and detection limit of 0.16 Ucell mL−1. The effective buildup of the immunosensor on bacterial nanocellulose was confirmed with polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) and surface energy analysis. In spite of the high sensitivity, full selectivity with distinction of the p53-containing cell lysates and possible interferents required treating the data with a supervised machine learning approach based on decision trees. This allowed the creation of a multidimensional calibration space with 11 dimensions (frequencies used to generate decision tree rules), with which the classification of the p53-containing samples can be explained.

Bacterial luminescent biosensors in the system for assessing the mechanisms of antibacterial activity of carbon-based nanomaterials

The study presents the results of an analysis of possible mechanisms of antibacterial activity of nanocarbon compounds. It was found that the contact of carbon-based nanomaterials with bacterial cells is not accompanied by a violation of their structural integrity and the development of various types of stress (oxidative stress, reaction of the protein-chaperone system, SOS response). The result of this interaction is a change in the magnitude of the surface zeta potential of cells and the permeability of their membrane, which negatively affects the energy processes occurring in target cells. The direct manifestation of this condition is a decrease in the level of luminescence of luminescent strains and a loss of viability.

Bacterial Biosensor Supported in Montmorillonite Clay Intercalated with Ionic Liquids for General Toxicity Assessment

Bacterial Biosensor Supported in Montmorillonite Clay Intercalated with Ionic Liquids for General Toxicity Assessment

Increased sensitivity of heavy metal bioreporters in transporter deficient Synechocystis PCC6803 mutants

The detection and identification of heavy metal contaminants are becoming increasingly important as environmental pollution causes an ever-increasing health hazard in the last decades. Bacterial heavy metal reporters, which constitute an environmentally friendly and cheap approach, offer great help in this process. Although their application has great potential in the detection of heavy metal contamination, their sensitivity still needs to be improved. In this study, we describe a simple molecular biology approach to improve the sensitivity of bacterial heavy metal biosensors. The constructs are luxAB marker genes regulated by the promoters of heavy metal exporter genes. We constructed a mutant strain lacking the cluster of genes responsible for heavy metal transport and hence achieved increased intracellular heavy metal content of the Synechocystis PCC6803 cyanobacterium. Taking advantage of this increased intracellular heavy metal concentration the Ni2+; Co2+ and Zn2+ detection limits of the constructs were three to tenfold decreased compared to the sensitivity of the same constructs in the wild-type cyanobacterium.

Bacterial Lighthouses-Real-Time Detection of Yersinia enterocolitica by Quorum Sensing.

Foodborne zoonotic pathogens have a severe impact on food safety. The demand for animal-based food products (meat, milk, and eggs) is increasing, and therefore faster methods are necessary to detect infected animals or contaminated food before products enter the market. However, conventional detection is based on time-consuming microbial cultivation methods. Here, the establishment of a quorum sensing-based method for detection of foodborne pathogens as Yersinia enterocolitica in a co-cultivation approach using a bacterial biosensor carrying a special sensor plasmid is described. We combined selective enrichment with the simultaneous detection of pathogens by recording autoinducer-1-induced bioluminescent response of the biosensor. This new approach enables real-time detection with a calculated sensitivity of one initial cell in a sample after 15.3 h of co-cultivation, while higher levels of initial contamination can be detected within less than half of the time. Our new method is substantially faster than conventional microbial cultivation and should be transferrable to other zoonotic foodborne pathogens. As we could demonstrate, quorum sensing is a promising platform for the development of sensitive assays in the area of food quality, safety, and hygiene.

Recent advances in bacterial biosensing and bioremediation of cadmium pollution: a mini-review.

Cadmium (Cd) pollution has become a global environmental issue because Cd gets easily accumulated and translocated in the food chain, threatening human health. Considering the detrimental effects and non-biodegradability of environmental Cd, this is an urgent issue that needs to be addressed through the development of robust, cost-effective, and eco-friendly green routes for monitoring and remediating toxic levels of Cd. This article attempts to review various bacterial approaches toward biosensing and bioremediation of Cd in the environment. This review focuses on the recent development of bacterial cell-based biosensors for the detection of bioavailable Cd and the bioremediation of toxic Cd by natural or genetically-engineered bacteria. The present limitations and future perspectives of these available bacterial approaches are outlined. New trends for integrating synthetic biology and metabolic engineering into the design of bacterial biosensors and bioadsorbers are additionally highlighted.

An Intracellular Sensing and Signal Transduction System That Regulates the Metabolism of Polycyclic Aromatic Hydrocarbons in Bacteria.

ABSTRACTMany bacteria utilize polycyclic aromatic hydrocarbon (PAH) as carbon and energy sources for growth. These bacteria play an important role in the amelioration of PAH pollution in various environments. However, it is unclear how bacteria sense PAHs and how PAH degradation pathways are regulated via signal transduction. Here, we investigated these mechanisms in Cycloclasticus, a ubiquitous PAH-degrading bacterium in marine environments. We identified the key genes involved in intracellular PAH sensing, signal transduction, and the differential regulation of degradation pathways for each PAH examined. Our results showed that PAHs bind specifically to a diguanylate cyclase PdgC, leading to the generation of cyclic dimeric GMP (c-di-GMP), which subsequently binds to two CRP/FNR family regulators, DPR-1 and DPR-2. c-di-GMP activates the transcription of DPR-1 and DPR-2 to positively regulate degradation pathways specific to pyrene and phenanthrene/naphthalene, respectively. This is the first report of an intracellular signal transduction pathway associated with PAH degradation in bacteria. Our results improve our understanding of the intracellular responses to PAHs. The existence of the identified genes in other bacteria indicates that the strategy described here is widely used by other PAH-degrading bacteria.IMPORTANCE Polycyclic aromatic hydrocarbons (PAHs) are widely distributed and have been found indoors, in the atmosphere, in terrestrial soils, in marine waters and sediments, and even in outer space. Bacteria degrade PAHs via degradation pathways. PAH signal sensing and transduction, as well as the regulation of PAH degradation pathways, are crucial for bacterial PAH biodegradation. However, prior to this study, these processes were poorly known. This study employed multiple molecular approaches to better understand the regulatory networks controlling PAH metabolism in bacteria. This report illustrates, for the first time, PAH-specific intracellular sensing, signal transduction, and metabolic regulatory pathways. Our results will help to increase our understanding of the hydrocarbon-metabolism regulatory network as well as the regulatory intricacies that control microbial biodegradation of organic matter. These key data should be considered to improve the rational design and efficiency of recombinant biodegradable, bacterial biosensors, and biocatalysts in modern green chemistry.

Programmable receptors enable bacterial biosensors to detect pathological biomarkers in clinical samples

Bacterial biosensors, or bactosensors, are promising agents for medical and environmental diagnostics. However, the lack of scalable frameworks to systematically program ligand detection limits their applications. Here we show how novel, clinically relevant sensing modalities can be introduced into bactosensors in a modular fashion. To do so, we have leveraged a synthetic receptor platform, termed EMeRALD (Engineered Modularized Receptors Activated via Ligand-induced Dimerization) which supports the modular assembly of sensing modules onto a high-performance, generic signaling scaffold controlling gene expression in E. coli. We apply EMeRALD to detect bile salts, a biomarker of liver dysfunction, by repurposing sensing modules from enteropathogenic Vibrio species. We improve the sensitivity and lower the limit-of-detection of the sensing module by directed evolution. We then engineer a colorimetric bactosensor detecting pathological bile salt levels in serum from patients having undergone liver transplant, providing an output detectable by the naked-eye. The EMeRALD technology enables functional exploration of natural sensing modules and rapid engineering of synthetic receptors for diagnostics, environmental monitoring, and control of therapeutic microbes.

Development of bacterial biosensor for sensitive and selective detection of acetaldehyde

Acetaldehyde is a human carcinogen and widely existed in alcoholic beverages and polluted air. In this study, a simple, fast, convenient and sensitive acetaldehyde biosensor was developed based on an acetaldehyde dehydrogenase (AldDH) bacteria surface display system. The whole-cell catalyst facilitated the dehydrogenation of acetaldehyde, while coenzyme NAD+ was reduced and the resultant NADH can be detected spectrometrically at 340 nm. The correct location of AldDH on the bacteria surface was confirmed by the subcellular fraction and immunofluorescence analysis. By comparing the fusion protein expression level and whole-cell activity, the proper display system for anchoring AldDH on the cell surface was obtained. The results of kinetics analysis towards both surface-displayed AldDH and intracellular expressed AldDH demonstrated that the mass-transport resistance was dramatically alleviated by cell-surface display strategy. Under optimal conditions, AldDH-surface display strain with the highest whole-cell activity (3.41 ± 0.3 mU/OD600) was applied to spectrophotometry acetaldehyde detection system. An excellent linear relationship between the increases of absorbance at 340 nm and acetaldehyde concentration over the range from 1 μM to 300 μM was reached. The proposed approach offered adequate sensitivity for the detection of acetaldehyde at 0.33 μM. Most importantly, the developed biosensor showed the narrowest substrate specificity towards acetaldehyde, which has been employed for quick determination of acetaldehyde in real samples with good accuracy. The total detection time was within 20 min. The method reported here provided a simple, rapid, and low-cost strategy for the sensitive and selective measurement of acetaldehyde. Therefore, genetically engineered cells may find broad application in biosensors and biocatalysts.

Green fluorescent protein based whole cell bacterial biosensor for the detection of bioavailable heavy metals in soil environment

A Green fluorescent protein (GFP) based whole cell bacterial biosensor was prepared using a bacterial strain sensitive to several heavy metals in order to detect bioavailable heavy metals in soils. The transformant, named as Bacillus megaterium VR1 was immobilized in silica matrix using sol–gel technology, and optimized for its effective pH range, cell density, exposure time, and storage stability. The lowest detection limit (LOD) for each metal was also determined. The pH range for the bacterial strain was found to be between pH 5–8.5. The optimum exposure time for the transformed bacterial strain to respond to the lowest tested concentration of heavy metal at 25% of inhibition compared to the control was determined as 4 h, 4 h, and 7 h, for Cd, Cu and Zn, respectively. SiNa/LUDOX 1/1 was selected as the optimum immobilization matrix. Storage up to 2 weeks did not show any reduction in the fluorescence in all the matrices. The linear range of the whole cell bacterial biosensor was determined as 0-10; 0–20 and 0–100 mg/L for Cd, Cu and Zn respectively. The lowest detection limit was determined as 1.42 × 10 −4 , 3.16 × 10 −4 , and 2.42 × 10 −4 mg/L for Cd, Cu and Zn, respectively. • Biosensor, Bacillus megaterium VR1 showed sensitivity to Cadmium, Copper, and Zinc. • The optimum pH for the biosensor was found to be pH 5–8.5. • Storage of biosensor up to 2 weeks did not show any decrease in fluorescence. • This biosensor has a great potential for monitoring heavy metal toxicity.

Modeling and characterization of an engineered microbial biosensor for high-throughput screening of arsenic in rural water

Current arsenic analysis methods in groundwater samples rely on expensive apparatus, complicated procedures, and dangerous chemical reagents. Also, delays in detecting arsenic harm to public health, the environment, agriculture and food sectors. Therefore, in this study, a bioluminescent biosensor has been optimized and used to detect and measure arsenic concentration in groundwater. Optimum conditions for the appropriate performance of E. coli DH5α (pJAMA-arsR) were determined and the luminescent calibration curve was drawn. The optimization results showed that maximum luminescent light output could occur at the end of the logarithmic phase or the beginning of the stationary phase, the temperature of 37 °C, and pH between 5.5 and 7 upon adding 10 μl n-decanal (18 mM). Increasing the duration of bacterial induction by arsenic leads to elevation of biosensor luminescent light yield. Functional stability of the biosensor with 20 % glycerol (V/V) at −20 °C was verified for at least six months. Luminescence reaction of the bacterial biosensor cells to arsenic concentration in the range of 0–90 ppb was promising (R2 = 0.948 by linear regression), but higher arsenic concentration had poisonous effect on biosensor cells. The modified Gompertz model derived here could successfully predict the bacterial biosensor growth under the optimum condition compared with experimental data. In this study critical challenges, such as technical and appropriate performance, are defined to interpret the bacterial biosensor’s true perspective to promote its broad adoption and usage. The work is concluded with closing remarks and potential perspectives to emphasize the importance of the bacterial biosensor, which could detect arsenic from a wide scope in real-time, quickly, and environmentally friendly signaling tool with high sensitivity and selectivity.

Selective point-of-care detection of pathogenic bacteria using sialic acid functionalized gold nanoparticles

In resource-limited settings, fast and simple point-of-need tests should facilitate healthcare providers the identification of pathogens avoiding empirical suboptimal treatments with broad-spectrum antibiotics. A rapid optical whole cell bacterial biosensor has been here developed using sialic acid functionalized gold nanoparticles allowing the selective screening of Gram-positive Staphylococcus aureus ATCC 25923 and Methicillin Resistant Staphylococcus aureus (MRSA) USA300 and Gram-negative bacteria (Pseudomonas aeruginosa ATCC 15442) by selecting the appropriate dispersing media. Those bacteria were selected due to their common presence in wound bed tissue of chronic infected topical wounds. The discrimination of bacterial pathogens has been attempted in different media including water, two independent buffers, bacterial broth, human serum and human urine. The identification of Gram + bacterial pathogens was also assessed under simultaneous co-culture of S. Aureus and Pseudomonas aeruginosa. High bacterial loads were required to provide with a statistically significant optical pathogen identification in human serum whereas it was not possible to detect the presence of bacteria at clinically relevant levels in urine.

The Integration of Whole-Cell Biosensors for the Field-Ready Electrochemical Detection of Arsenic

Rapid on-site measurements of arsenic (As) are essential for the timely remediation of As-contaminated groundwater for both municipal and emergency response applications. Current field tests suffer from either complicated end-user instructions or a lack of accuracy and specificity. The system presented here combines a whole-cell bacterial biosensor with an electrochemical measurement that provides enhanced accuracy and signal intensity compared to traditional bacterial-detection approaches. When integrated within a customized hardware system, this whole-cell sensor demonstrated excellent specificity and sensitivity. This fast, sensitive, and easy-to-use approach is a viable alternative for on-site arsenic testing.

A Whole-Cell Bacterial Biosensor for Blood Markers Detection in Urine

The early detection of blood in urine (hematuria) can play a crucial role in the treatment of serious diseases (e.g., infections, kidney disease, schistosomiasis, and cancer). Therefore, the development of low-cost portable biosensors for blood detection in urine has become necessary. Here, we designed an ultrasensitive whole-cell bacterial biosensor interfaced with an optoelectronic measurement module for heme detection in urine. Heme is a red blood cells (RBCs) component that is liberated from lysed cells. The bacterial biosensor includes Escherichia coli cells carrying a heme-sensitive synthetic promoter integrated with a luciferase reporter (luxCDABE) from Photorhabdus luminescens. To improve the bacterial biosensor performance, we re-engineered the genetic structure of luxCDABE operon by splitting it into two parts (luxCDE and luxAB). The luxCDE genes were regulated by the heme-sensitive promoter, and the luxAB genes were regulated by either constitutive or inducible promoters. We examined the genetic circuit's performance in synthetic urine diluent supplied with heme and in human urine supplied with lysed blood. Finally, we interfaced the bacterial biosensor with a light detection setup based on a commercial optical measurement single-photon avalanche photodiode (SPAD). The whole-cell biosensor was tested in human urine with lysed blood, demonstrating a low-cost, portable, and easy-to-use hematuria detection with an ON-to-OFF ratio of 6.5-fold for blood levels from 5 × 104 to 5 × 105 RBC per mL of human urine.

An autonomous bioluminescent bacterial biosensor module for outdoor sensor networks, and its application for the detection of buried explosives

We describe a miniaturized field-deployable biosensor module, designed to function as an element in a sensor network for standoff monitoring and mapping of environmental hazards. The module harbors live bacterial sensor cells, genetically engineered to emit a bioluminescent signal in the presence of preselected target materials, which act as its core sensing elements. The module, which detects and processes the biological signal, composes a digital record that describes its findings, and can be transmitted to a remote receiver. The module is an autonomous self-contained unit that can function either as a standalone sensor, or as a node in a sensor network. The biosensor module can potentially be used for detecting any target material to which the sensor cells were engineered to respond. The module described herein was constructed to detect the presence of buried landmines underneath its footprint. The demonstrated detection sensitivity was 0.25 mg 2,4-dinitrotoluene per Kg soil.

Analysis of bioavailable toluene by using recombinant luminescent bacterial biosensors with different promoters

In this study, we constructed recombinant luminescent Escherichia coli with T7, T3, and SP6 promoters inserted between tol and lux genes as toluene biosensors and evaluated their sensitivity, selectivity, and specificity for measuring bioavailable toluene in groundwater and river water. The luminescence intensity of each biosensor depended on temperature, incubation time, ionic strength, and concentrations of toluene and coexisting organic compounds. Toluene induced the highest luminescence intensity in recombinant lux-expressing E. coli with the T7 promoter [T7-lux-E. coli, limit of detection (LOD) = 0.05 μM], followed by that in E. coli with the T3 promoter (T3-lux-E. coli, LOD = 0.2 μM) and SP6 promoter (SP6-lux-E. coli, LOD = 0.5 μM). Luminescence may have been synergistically or antagonistically affected by coexisting organic compounds other than toluene; nevertheless, low concentrations of benzoate and toluene analogs had no such effect. In reproducibility experiments, the biosensors had low relative standard deviation (4.3–5.8%). SP6-lux-E. coli demonstrated high adaptability to environmental interference. T7-lux-E. coli biosensor—with low LOD, wide measurement range (0.05–500 μM), and acceptable deviation (− 14.3 to 9.1%)—is an efficient toluene biosensor. This is the first study evaluating recombinant lux E. coli with different promoters for their potential application in toluene measurement in actual water bodies.

Bacterial-Derived Plant Protection Metabolite 2,4-Diacetylphloroglucinol: Effects on Bacterial Cells at Inhibitory and Subinhibitory Concentrations.

2,4-Diacetylphloroglucinol (2,4-DAPG) is a well-known bacterial secondary metabolite, however, its mechanism of inhibitory and subinhibitory action on bacterial cells is still poorly understood. The mechanism of 2,4-DAPG action on model bacterial strains was investigated using fluorescent spectroscopy and the action of the antibiotic was found to involve a rapid increase in membrane permeability that was accompanied by a reduction in its viability in nutrient-poor medium. At the same time, antibacterial action in nutrient-rich medium developed for several hours. Atomic force microscopy demonstrated time-dependent disturbances in the outer membrane of Escherichia coli when exposed to 2,4-DAPG, while Staphylococcus aureus cells have been visualized with signs of intracellular leakage. In addition, 2,4-DAPG inhibited the metabolic activity of S. aureus and E. coli bacterial cells in mature biofilms. Observed differences in the antibiofilm activity were dependent upon antibiotic concentration. The intracellular targets of the action of 2,4-DAPG were assessed using bacterial biosensors with inducible bioluminescence corresponding to DNA and protein damage. It was unable to register any positive response from either sensor. As a result, the bactericidal action of 2,4-DAPG is believed to be associated with the destruction of the bacterial barrier structures. The subinhibitory effect of 2,4-diacetylphloroglucinol was tested on quorum-sensing mediated processes in Pectobacterium carotovorum. Subinhibitory concentrations of 2,4-DAPG were found to lower the biosynthesis of acyl-homoserine lactones in P. carotovorum in a dose-dependent manner. Further investigation elucidated that 2,4-DAPG inhibits the metabolic activity of bacteria without affecting their viability.