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Biosensors are powerful tools for monitoring specific metabolites or controlling metabolic flux towards the products in a single cell, which play important roles in microbial cell factory construction. Despite their potential role in metabolic flux monitoring, the development of biosensors for small molecules is still limited. Reported biosensors often exhibit bottlenecks of poor specificity and a narrow dynamic range. Moreover, fine-tuning the substrate binding affinity of a crucial enzyme can decrease its catalytic activity, which ultimately results in the repression of the corresponding essential metabolite biosynthesis and impairs cell growth. However, increasing intracellular substrate concentration can elevate the availability of the essential metabolite and may lead to restore cellular growth. Herein, a new strategy was proposed for constructing whole-cell biosensors based on enzyme encoded by essential gene that offer inherent specificity and universality. Specifically, S-adenosyl-methionine synthetase (MetK) in E. coli was chosen as the crucial enzyme, and a series of MetK variants were identified that were sensitive to L-methionine concentration. This occurrence enabled the engineered cell to sense L-methionine and exhibit L-methionine dose-dependent cell growth. To improve the biosensor's dynamic range, an S-adenosyl-methionine catabolic enzyme was overexpressed to reduce the intracellular availability of S-adenosyl-methionine. The resulting whole-cell biosensor effectively coupled the intracellular concentration of L-methionine with growth and was successfully applied to select strains with enhanced L-methionine biosynthesis from random mutagenesis libraries. Overall, our study presents a universal strategy for designing and constructing growth-coupled biosensors based on crucial enzyme, which can be applied to select strains overproducing high value-added metabolites in cellular metabolism. [ABSTRACT FROM AUTHOR]

The accurate and effective detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to preventing the spread of infectious diseases and ensuring human health. Herein, a nanobody-displayed whole-cell biosensor was developed for colorimetric detection of SARS-CoV-2 spike proteins. Serving as bioreceptors, yeast surfaces were genetically engineered to display SARS-CoV-2 binding of llama-derived single-domain antibodies (nanobodies) with high capture efficiency, facilitating the concentration and purification of SARS-CoV-2. Gold nanoparticles (AuNPs) employed as signal transductions were functionalized with horseradish peroxidase (HRP) and anti-SARS monoclonal antibodies to enhance the detection sensitivity. In the presence of SARS-CoV-2 spike proteins, the sandwiched binding will be formed by linking engineered yeast, SARS-CoV-2 spike proteins, and reporter AuNPs. The colorimetric signal was generated by the enzymatic reaction of HRP and its corresponding colorimetric substrate/chromogen system. At the optimal conditions, the developed whole-cell biosensor enables the sensitive detection of SARS-CoV-2 spike proteins in a linear range from 0.01 to 1 μg/mL with a limit of detection (LOD) of 0.037 μg/mL (about 4 × 108 virion particles/mL). Furthermore, the whole-cell biosensor was demonstrated to detect the spike protein of different SARS-CoV-2 variants in human serum, providing new possibilities for the detection of future SARS-CoV-2 variants.

Ten strains of the yeast Yarrowia lipolytica were constructed, the genomes of which contain the hrGFP gene under regulation of the formate dehydrogenase promoters. The resulting strains can act as whole-cell biosensors for the detection of formate ions in various media. By visual assessment of the biomass fluorescence, we selected the three most promising yeast strains. The main biosensor characteristics (threshold sensitivity, amplitude, and response time) of the selected strains were measured. As a result, in terms of characteristics, the B26 strain was recognized as the most suitable for the detection of formate ions. A carbon source for the nutrient medium that does not reduce the activation of the biosensor was selected. Furthermore, we showed that unlike formate and formaldehyde, methanol practically does not induce the biosensor fluorescence response. [ABSTRACT FROM AUTHOR]

Whole-cell biosensors could serve as eco-friendly and cost-effective alternatives for detecting potentially toxic bioavailable heavy metals in aquatic environments. However, they often fail to meet practical requirements due to an insufficient limit of detection (LOD) and high background noise. In this study, we designed a synthetic genetic circuit specifically tailored for detecting ionic mercury, which we applied to environmental samples collected from artisanal gold mining sites in Peru. We developed two distinct versions of the biosensor, each utilizing a different reporter protein: a fluorescent biosensor (Mer-RFP) and a colorimetric biosensor (Mer-Blue). Mer-RFP enabled real-time monitoring of the culture's response to mercury samples using a plate reader, whereas Mer-Blue was analysed for colour accumulation at the endpoint using a specially designed, low-cost camera setup for harvested cell pellets. Both biosensors exhibited negligible baseline expression of their respective reporter proteins and responded specifically to HgBr2 in pure water. Mer-RFP demonstrated a linear detection range from 1 nM to 1 μM, whereas Mer-Blue showed a linear range from 2 nM to 125 nM. Our biosensors successfully detected a high concentration of ionic mercury in the reaction bucket where artisanal miners produce a mercury–gold amalgam. However, they did not detect ionic mercury in the water from active mining ponds, indicating a concentration lower than 3.2 nM Hg2+—a result consistent with chemical analysis quantitation. Furthermore, we discuss the potential of Mer-Blue as a practical and affordable monitoring tool, highlighting its stability, reliance on simple visual colorimetry, and the possibility of sensitivity expansion to organic mercury. [ABSTRACT FROM AUTHOR]

The use of bacteriocins is a promising approach for addressing the immense threat of food-borne and drug-resistant pathogens. In recent years screening platforms for novel bacteriocins using whole-cell biosensors have been established. During screening cell-to-cell heterogeneity is currently neglected but might play a crucial role in signal development of the whole-cell biosensor after bacteriocin exposure. In this study, we explored the temporal dynamics of the signal heterogeneity of the biosensor Listeria innocua LMG2785/pNZpHin2Lm after nisin exposure using microfluidic single-cell analysis. The results provided novel and detailed insights into the dynamics of cell-to-cell heterogeneity in L. innocua LMG2785/pNZpHin2Lm at different nisin concentrations with a high spatio-temporal resolution. Furthermore, the formation of subpopulations during bacteriocin exposure was observed. In-depth single-cell tracking even revealed the regeneration of disrupted cells and recovery of pH homeostasis in rare instances. These findings are highly important for the future design and execution of bacteriocin assays and for the interpretation of fluorescence signal development at the population level after exposure to different concentrations of bacteriocins (here, nisin), as well as for obtaining deeper insights into single-cell persistence strategies to quantify the efficacy and efficiency of novel bacteriocins.

Abstract Cancer is one of the most life‐threatening diseases worldwide. Numerous diagnostic and therapeutic techniques, such as nanomaterials‐based cancer detection and imaging‐guided focal therapy, are successfully developed to achieve highly precise cancer theranostic strategies. However, due to unpredictive alterations occurring in cancer cell morphology, a lack of sensitivity and selectivity is a critical challenge among most biomarkers’ detection. To address this issue, instead of targeting proteins or biomarkers, the detection of cancer using whole cell‐based biosensors are ubiquitously discovered through a selective oligonucleotide, so‐called aptamer. Aptamer‐based whole‐cell detection is concurrently engaged with various nanomaterials, such as magnetic beads, gold (Au) nanoparticles, and graphene family to establish a better performance of the biosensor. In this research review, the recent strategies and prospects on whole‐cell cancer detection platform based on electrochemical aptasensors integrated with nanomaterials are thoroughly discussed and summarized. Finally, future challenges and prospects are also provided.

Phenolic acids including hydroxybenzoic and hydroxycinnamic acids are secondary plant and fungal metabolites involved in many physiological processes offering health and dietary benefits. They are often utilised as precursors for production of value-added compounds. The limited availability of synthetic biology tools, such as whole-cell biosensors suitable for monitoring the dynamics of phenolic acids intracellularly and extracellularly, hinders the capabilities to develop high-throughput screens to study their metabolism and forward engineering. Here, by applying a multi-genome approach, we have identified phenolic acid-inducible gene expression systems composed of transcription factor-inducible promoter pairs responding to eleven different phenolic acids. Subsequently, they were used for the development of whole-cell biosensors based on model bacterial hosts, such as Escherichia coli , Cupriavidus necator and Pseudomonas putida. The dynamics and range of the biosensors were evaluated by establishing their response and sensitivity landscapes. The specificity and previously uncharacterised interactions between transcription factor and its effector(s) were identified by a screen of twenty major phenolic acids. To exemplify applicability, we utilise a protocatechuic acid-biosensor to identify enzymes with enhanced activity for conversion of p -hydroxybenzoate to protocatechuate. Transcription factor-based biosensors developed in this study will advance the analytics of phenolic acids and expedite research into their metabolism. • A multi-genome approach for identifying phenolic acid-inducible gene expression systems. • Developed biosensors for detection of eleven different phenolic acids. • Full parameterisation of biosensors response, sensitivity, and specificity. • The application of protocatechuic acid-biosensor for identification of enzymes with enhanced activity. • The toolbox for application in synthetic biology and studying the metabolism of phenolic acids. [ABSTRACT FROM AUTHOR]

Synthetic microbial community (SynCom) biosensors allow for the use of genetically edited or natural microbial communities in a biosensing system to capture and convert biosignals into digital outputs. The workflow of SynCom biosensor construction contains three basic modules, including selection of microbial target candidates, construction and validation of SynComs, and detection of biosignals. SynCom biosensors have the potential to revolutionize biosensing technology by improving sensitivity, specificity, cost effectiveness, and real-time monitoring capabilities. Synthetic microbial community (SynCom) biosensors are a promising technology for detecting and responding to environmental cues and target molecules. SynCom biosensors use engineered microorganisms to create a more complex and diverse sensing system, enabling them to respond to stimuli with enhanced sensitivity and accuracy. Here, we give a definition of SynCom biosensors, outline their construction workflow, and discuss current biosensing technology. We also highlight the challenges and future for developing and optimizing SynCom biosensors and the potential applications in agriculture and food management, biotherapeutic development, home sensing, urban and environmental monitoring, and the One Health foundation. We believe SynCom biosensors could be used in a real-time and remote-controlled manner to sense the chaos of constantly dynamic environments. [ABSTRACT FROM AUTHOR]

Whole-cell biosensors could serve as eco-friendly and cost-effective alternatives for detecting potentially toxic bioavailable heavy metals in aquatic environments. However, they often fail to meet practical requirements due to an insufficient limit of detection (LOD) and high background noise. In this study, we designed a synthetic genetic circuit specifically tailored for detecting ionic mercury, which we applied to environmental samples collected from artisanal gold mining sites in Peru. We developed two distinct versions of the biosensor, each utilizing a different reporter protein: a fluorescent biosensor (Mer-RFP) and a colorimetric biosensor (Mer-Blue). Mer-RFP enabled real-time monitoring of the culture’s response to mercury samples using a plate reader, whereas Mer-Blue was analysed for colour accumulation at the endpoint using a specially designed, low-cost camera setup for harvested cell pellets. Both biosensors exhibited negligible baseline expression of their respective reporter proteins and responded specifically to HgBr2 in pure water. Mer-RFP demonstrated a linear detection range from 1 nM to 1 μM, whereas Mer-Blue showed a linear range from 2 nM to 125 nM. Our biosensors successfully detected a high concentration of ionic mercury in the reaction bucket where artisanal miners produce a mercury–gold amalgam. However, they did not detect ionic mercury in the water from active mining ponds, indicating a concentration lower than 3.2 nM Hg2+—a result consistent with chemical analysis quantitation. Furthermore, we discuss the potential of Mer-Blue as a practical and affordable monitoring tool, highlighting its stability, reliance on simple visual colorimetry, and the possibility of sensitivity expansion to organic mercury.

In this study, we designed a Cd2+ whole-cell biosensor with both positive and negative feedback cascade amplifiers in Pseudomonas putida KT2440 (LTCM) based on our previous design with only a negative feedback amplifier (TCM). The results showed that the newly developed biosensor LTCM was greatly improved compared to TCM. Firstly, the linear response range of LTCM was expanded while the maximum linear response range was raised from 0.05 to 0.1 μM. Meanwhile, adding a positive feedback amplifier further increased the fluorescence output signal of LTCM 1.11–2.64 times under the same culture conditions. Moreover, the response time of LTCM for detection of practical samples was reduced from 6 to 4 h. At the same time, LTCM still retained very high sensitivity and specificity, while its lowest detection limit was 0.1 nM Cd2+ and the specificity was 23.29 (compared to 0.1 nM and 17.55 in TCM, respectively). In summary, the positive and negative feedback cascade amplifiers effectively improved the performance of the biosensor LTCM, resulting in a greater linear response range, higher output signal intensity, and shorter response time than TCM while retaining comparable sensitivity and specificity, indicating better potential for practical applications.

ABSTRACT A green fluorescent protein (GFP)-based whole-cell biosensor (WCB-GFP) for monitoring arsenic (As) was developed in Bacillus subtilis. To this end, we designed a reporter gene fusion carrying the gfpmut3a gene under the control of the promoter/operator region of the arsenic operon (Pars::gfpmut3a) in the extrachromosomal plasmid pAD123. This construct was transformed into B. subtilis 168, and the resultant strain was used as a whole-cell biosensor (BsWCB-GFP) for the detection of As. The BsWCB-GFP was specifically activated by inorganic As(III) and As(V), but not by dimethylarsinic acid [DMA(V)], and exhibited high tolerance to the noxious effects of arsenic. Accordingly, after 12 h exposure, B. subtilis cells carrying the Pars::gfpmut3a fusion exhibited 50 and 90% lethal doses (LD50 and LD90) to As(III) of 0.89 mM and As 1.71 mM, respectively. Notably, dormant spores from the BsWCB-GFP were able to report the presence of As(III) in a concentration range from 0.1 to 1,000 μM 4 h after the onset of germination. In summary, the specificity and high sensitivity for As, as well as its ability to proliferate under concentrations of the metal that are considered toxic in water and soil, makes the B. subtilis biosensor developed here a potentially important tool for monitoring environmental samples contaminated with this pollutant. IMPORTANCE Arsenic (As) contamination of groundwater is associated with serious worldwide health risks. Detection of this pollutant at concentrations that are established as permissible for water consumption by WHO is a matter of significant interest. Here, we report the generation of a whole-cell biosensor for As detection in the Gram-positive spore former B. subtilis. This biosensor reports the presence of inorganic As, activating the expression of the green fluorescent protein (GFP) under the control of the promoter/operator of the ars operon. The biosensor can proliferate under concentrations of As(III) that are considered toxic in water and soil and detect this ion at concentrations as low as 0.1 μM. Of note, spores of the Pars-GFP biosensor exhibited the ability to detect As(III) following germination and outgrowth. Therefore, this novel tool has the potential to be directly applied to monitor As contamination in environmental samples.

A whole-cell biosensor based on synthetic biology provides a promising new method for the on-site detection of food contaminants. The basic components of whole-cell biosensors include the sensing elements, such as transcription factors and riboswitches, and reporting elements, such as fluorescence, gas, etc. The sensing and reporting elements are coupled through gene expression regulation to form a simple gene circuit for the detection of target substances. Additionally, a more complex gene circuit can involve other functional elements or modules such as signal amplification, multiple detection, and delay reporting. With the help of synthetic biology, whole-cell biosensors are becoming more versatile and integrated, that is, integrating pre-detection sample processing, detection processes, and post-detection signal calculation and storage processes into cells. Due to the relative stability of the intracellular environment, whole-cell biosensors are highly resistant to interference without the need of complex sample preprocessing. Due to the reproduction of chassis cells, whole-cell biosensors replicate all elements automatically without the need for purification processing. Therefore, whole-cell biosensors are easy to operate and simple to produce. Based on the above advantages, whole-cell biosensors are more suitable for on-site detection than other rapid detection methods. Whole-cell biosensors have been applied in various forms such as test strips and kits, with the latest reported forms being wearable devices such as masks, hand rings, and clothing. This paper examines the composition, construction methods, and types of the fundamental components of synthetic biological whole-cell biosensors. We also introduce the prospect and development trend of whole-cell biosensors in commercial applications. [ABSTRACT FROM AUTHOR]

Aims Whole-cell biosensors are increasingly utilized in various applications. These platforms integrate cells with a signal measurement device. One of the main challenges in the development of such platforms is the immobilization matrix that is used to keep the cells stable, which also affects the portability of the device. In this study, a portable and simple immobilization of bioluminescent bacterial cells in calcium alginate hydrogel was examined. Methods and results The effects of several physical parameters were investigated (e.g. calcium alginate solution volume, drying, incubation time, mixing procedure, bacterial concentration, and tablet location within the cylinder). An alginate solution volume of 3 ml was preferred as well as the addition of 400 μl solution after the 15 min of compressing step and before the polymerization step. Also, a stirring mixing mode is favored over vortexing due to the creation of better homogenized tablets, as well as a bacterial concentration of 0.15 OD600nm that produced a high light response while maintaining a lower variance. Lastly, the findings showed a significantly higher response [induction factor (IF)] in the tablets using the optimized immobilization protocol (IF = 8.814) than the old one (IF = 1.979). Conclusions To conclude, bacterial cells immobilization in calcium alginate tablets provides improved sensitivity and storability. [ABSTRACT FROM AUTHOR]

Sulforaphane (SFN), a defense secondary metabolite, can be used to predict the health status of plants and also has pharmacological effects, including anticancer, antioxidant, and anti‐inflammatory properties. The detection of SFN is therefore of great significance for the prevention and treatment of diseases. In this study, a "turn off" whole‐cell biosensor that can rapidly and robustly respond to the presence of SFN was constructed based on the orthogonal genetic components (hrpR, hrpS, and PhrpL) of Pseudomonas syringae (PS). The final optimized biosensor, p114(30R‐30S), was able to inhibit 91.7% of the fluorescence intensity in the presence of 100‐μM SFN. Subsequently, a HrpRS‐regulated OFF–ON genetic switch was designed by reconstituting a reverse σ70 promoter on the σ54‐PhrpL promoter sequence; this was coupled with dual‐color reporter genes to construct a "turn off–on" whole‐cell SFN biosensor. The PhrpLB variant increased the expression of green fluorescence a factor of 11.9 and reduced the expression of red fluorescence by 85.8% compared with the system in the absence of SFN. Thus, a robust switching of signal output from "turn off" to "turn on" was realized. In addition, the biosensor showed good linearity in the SFN concentration ranges of 0.1–10 μM (R2 = 0.99429) and 10–100 μM (R2 = 0.99465) and a detection limit of ~0.1 μM. [ABSTRACT FROM AUTHOR]

Synthetic biology-based whole-cell sensing technology provides an alternative approach for rapid and on-site detection of heavy metals. Because the intracellular environment is relatively stable, whole-cell biosensor presents strong anti-interference ability; the biosensor cells can proliferate with cell self-replication, so the whole-cell biosensor has the characteristics of simple, cheap and fast in production. Therefore, whole-cell biosensor demonstrates a promising application prospect in the rapid detection of food pollutants. This article, the composition, construction methods, and types of core elements for whole-cell biosensor are summarized. Then synthetic biology-based gene circuits for multi-function detection technologies are introduced; further the commercialized applications of whole-cell biosensor in rapid food safety testing are enumerated; and finally the challenges and development prospects of cell biosensors in the rapid detection of food safety are illustrated. [ABSTRACT FROM AUTHOR]

We designed and constructed a whole-cell biosensor capable of detecting the presence and quantity of carbon monoxide (CO) using the CO regulatory transcription factor. This biosensor utilizes CooA, a CO-sensing transcription regulator that activates the expression of carbon monoxide dehydrogenase (CODH), to detect the presence of CO and respond by triggering the expression of a GUS reporter protein (β-glucuronidase). The GUS reporter protein is expressed from a CO-induced CooA-binding promoter (PcooF) by CooA and enables the effective colorimetric detection of CO. An Escherichia coli strain used to validate the biosensor showed growth and GUS activity under anaerobic conditions; this study used the inert gas (Ar) to create anaerobic conditions. The pBRCO biosensor could successfully detect the presence of CO in the headspace. Moreover, the GUS-specific activity of pBRCO according to the CO strength as partial pressure followed Michaelis-Menten kinetics (R2 = 0.98). It was confirmed that the GUS-specific activity of pBRCO increased linearly up to 30.39 kPa (R2 = 0.98), and thus, a quantitative analysis of CO concentration (i.e., partial pressure) was possible.

This review aims to systematize data on the construction and applications of bacterial lux-biosensors in various fields ranging from investigation of gene regulation and regulatory networks to the new probiotics' search and ecotoxicological research. The typical technical solutions and devices required for diverse tasks applying lux-biosensors are reviewed. Aspects of the application of lux-biosensors in fundamental researches, such as the study of oxidative stress, heat shock, DNA-damaging, pro- and antioxidant activities, are also considered. This technology allows rapid screening of the biological activities of newly synthesized compounds, which could be applied as components for fuels, household chemicals, and drugs. Works related to the ecological state assessment on water resources are also described. The use of lux-biosensor complexes based on different organisms, including both gram-positive and gram-negative bacteria, makes toxicological investigations more comprehensive. Bacterial lux-biosensors based on Escherichia coli can be used as a model for evaluation of the effect of certain substances on the transmembrane potential in mitochondria, albeit with extrapolation to a certain extent. Another aspect that draws our interest is that biosensors are able to help predict some systemic properties of probiotics. In the future, it's quite promising to see more applications of lux-biosensors for environmental control, microbial-microbial interaction assessment, antioxidant action mechanism studies and toxicological studies in the development of new drugs.

Toxic heavy metal accumulation is one of anthropogenic environmental pollutions, which poses risks to human health and ecological systems. Conventional heavy metal remediation approaches rely on expensive chemical and physical processes leading to the formation and release of other toxic waste products. Instead, microbial bioremediation has gained interest as a promising and cost-effective alternative to conventional methods, but the genetic complexity of microorganisms and the lack of appropriate genetic engineering technologies have impeded the development of bioremediating microorganisms. Recently, the emerging synthetic biology opened a new avenue for microbial bioremediation research and development by addressing the challenges and providing novel tools for constructing bacteria with enhanced capabilities: rapid detection and degradation of heavy metals while enhanced tolerance to toxic heavy metals. Moreover, synthetic biology also offers new technologies to meet biosafety regulations since genetically modified microorganisms may disrupt natural ecosystems. In this review, we introduce the use of microorganisms developed based on synthetic biology technologies for the detection and detoxification of heavy metals. Additionally, this review explores the technical strategies developed to overcome the biosafety requirements associated with the use of genetically modified microorganisms.

Due to the toxicity of mercury and its harmful effects on human health, it is essential to establish a low-cost, highly sensitive and highly specific monitoring method with a wide detection range, ideally with a simple visual readout. In this study, a whole-cell biosensor with adjustable detection limits was developed for the detection of mercury ions in water samples, allowing controllable threshold detection with an expanded detection range. Gene circuits were constructed by combining the toehold switch system with lactose operon, mercury-ion-specific operon, and inducible red fluorescent protein gene. Using MATLAB for design and selection, a total of eleven dual-input single-output sensing logic circuits were obtained based on the basic logic of gene circuit construction. Then, biosensor DTS-3 was selected based on its fluorescence response at different isopropyl β-D-Thiogalactoside (IPTG) concentrations, exhibiting the controllable detection threshold. At 5–20 μM IPTG, DTS-3 can achieve variable threshold detection in the range of 0.005–0.0075, 0.06–0.08, 1–2, and 4–6 μM mercury ion concentrations, respectively. Specificity experiments demonstrated that DTS-3 exhibits good specificity, not showing fluorescence response changes compared with other metal ions. Furthermore spiked sample experiments demonstrated its good resistance to interference, allowing it to distinguish mercury ion concentrations as low as 7.5 nM by the naked eye and 5 nM using a microplate reader. This study confirms the feasibility and performance of biosensor with controllable detection threshold, providing a new detection method and new ideas for expanding the detection range of biosensors while ensuring rapid and convenient measurements without compromising sensitivity. [ABSTRACT FROM AUTHOR]

As a heavy metal pollutant, Cd2+ often enters the human body through the food chain causing great harm to human health. Whole-cell biosensor is an emerging technology for rapid on-site detection of heavy metals with the advantages of inexpensive, fast to mass-produce, and strong in anti-interference resistance, but suffering from insatisfactory specificity. In this study, a strategy of Adjacent Site Saturation Mutation (ASSM) was designed to improve the specificity of transcription factor CadR, which acted as the recognition element and determined the specificity of whole cell Cd2+ biosensors. A specific saturated library was constructed using the strategy of adjacent mutation. After two rounds of high-throughput visual screening, a whole-cell biosensor with good response to Cd2+, and with significant weakened Hg2+ interference was obtained. The optimized whole-cell biosensor showed a linear dynamic concentration range from 500 nM to 100 μM, a detection limit of 0.079 μM, and has satisfactory specificity and anti-interference. The ASSM strategy proposed in this study can provide a new method for the application of synthetic biology in food safety detection, indicating the importance of whole-cell biosensors for the detection of heavy metals. [ABSTRACT FROM AUTHOR]

There is an urgent need to develop novel antibiotics since antibiotic resistance is an increasingly serious threat to global public health. Whole-cell biosensors are one of the promising strategies for new antibiotic discovery. The peptidoglycan (PG) of the bacterial cell wall is one of the most important targets for antibiotics. However, the biosensors for the detection of PG-targeting antibiotics in Gram-negative bacteria have not been developed, mainly because of the lack of the regulatory systems that sense and respond to PG stress. Recently, we identified a novel two-component signal transduction system (PghKR) that is responsible for sensing and responding to PG damage in the Gram-negative bacterium Shewanella oneidensis. Based on this system, we developed biosensors for the detection of PG-targeting antibiotics. Using ampicillin as an inducer for PG stress and the bacterial luciferase LuxCDABE as the reporter, we found that the PghKR biosensors are specific to antibiotics targeting PG synthesis, including b-lactams, vancomycin, and D-cycloserine. Deletion of genes encoding PG permease AmpG and β-lactamase BlaA improves the sensitivity of the biosensors substantially. The PghKR biosensor in the background of DblaA is also functional on agar plates, providing a simple method for screening bacteria that produce PG-targeting antibiotics. [ABSTRACT FROM AUTHOR]

The rise of methicillin‐resistant Staphylococcus aureus (MRSA) infections has gained concern throughout the world over the past decades. Alternative therapeutic agents to antibiotics are rapidly growing to impede the proliferation of MRSA‐caused infections. Lately, synthetic biology techniques have developed whole‐cell biosensors by designing gene circuitry capable of sensing quorum‐sensing (QS) molecules of pathogens and triggering expression of an antimicrobial moiety that kills MRSA and therefore prevents its further proliferation. Here, an E. coli was engineered in silico to act as a whole‐cell biosensor that senses QS molecules from MRSA and triggers the expression of a bacteriocin that kills MRSA. To achieve this functionality, biosensor and bacteriocin modules were constructed and assembled into a vector. Both modules were codon‐optimized to increase the yield production of the recombinant proteins. We then demonstrate in silico that the construction of a dual biosensor‐killer plasmid, which holds two genetical modules known as biosensor and bacteriocin modules, enables the recombinant host to sense QS molecules from MRSA. Our designed whole‐cell biosensor demonstrates in silico its ability to produce and secrete the bacteriocin as a function of the external concentration of autoinducer peptide from MRSA. These in silico results unravel the possibility of designing antimicrobial smarter therapeutics against resistant pathogens. [ABSTRACT FROM AUTHOR]

Timely surveillance of airborne pathogens is essential to preventing the spread of infectious diseases and safeguard human health. Methods for sensitive, efficient, and cost-effective detection of airborne viruses are needed. With advances in synthetic biology, whole-cell biosensors have emerged as promising platforms for environmental monitoring and medical diagnostics. However, the current design paradigm of whole-cell biosensors is mostly based on intracellular detection of analytes that can transport across the cell membrane, which presents a critical challenge for viral pathogens and large biomolecules. To address this challenge, we developed a new type of whole-cell biosensor by expressing and displaying VHH-based quenchbody (Q-body) on the surface of the yeast Saccharomyces cerevisiae for simple one-step detection of influenza A (H1N1) virus. Seventeen VHH antibody fragments targeting the hemagglutinin protein H1N1-HA were displayed on the yeast cells and screened for the H1N1-HA binding affinity. The functionally displayed VHHs were selected to create surface-displayed Q-body biosensors. The surface-displayed Q-body exhibiting the highest quenching and dequenching efficiency was identified. The biosensor quantitatively detected H1N1-HA in a range from 0.5 to 16 μg/mL, with a half-maximal concentration of 2.60 μg/mL. The biosensor exhibited high specificity for H1N1-HA over other hemagglutinin proteins from various influenza A virus subtypes. Moreover, the biosensor succeeded in detecting the H1N1 virus at concentrations from 2.4 × 10 4 to 1.5 × 10 7 PFU/mL. The results from this study demonstrated a new whole-cell biosensor design that circumvents the need for transport of analytes into biosensor cells, enabling efficient detection of the target virus particles.

Gut metabolites are pivotal mediators of host-microbiome interactions and provide an important window on human physiology and disease. However, current methods to monitor gut metabolites rely on heavy and expensive technologies such as liquid chromatography-mass spectrometry (LC-MS). In that context, robust, fast, field-deployable, and cost-effective strategies for monitoring fecal metabolites would support large-scale functional studies and routine monitoring of metabolites biomarkers associated with pathological conditions. Living cells are an attractive option to engineer biosensors due to their ability to detect and process many environmental signals and their self-replicating nature. Here we optimized a workflow for feces processing that supports metabolite detection using bacterial biosensors. We show that simple centrifugation and filtration steps remove host microbes and support reproducible preparation of a physiological-derived media retaining important characteristics of human feces, such as matrix effects and endogenous metabolites. We measure the performance of bacterial biosensors for benzoate, lactate, anhydrotetracycline, and bile acids, and find that they are highly sensitive to fecal matrices. However, encapsulating the bacteria in hydrogel helps reduce this inhibitory effect. Sensitivity to matrix effects is biosensor-dependent but also varies between individuals, highlighting the need for case-by-case optimization for biosensors’ operation in feces. Finally, by detecting endogenous bile acids, we demonstrate that bacterial biosensors could be used for future metabolite monitoring in feces. This work lays the foundation for the optimization and use of bacterial biosensors for fecal metabolites monitoring. In the future, our method could also allow rapid pre-prototyping of engineered bacteria designed to operate in the gut, with applications to in situ diagnostics and therapeutics.

A whole-cell biosensor based on synthetic biology provides a promising new method for the on-site detection of food contaminants. The basic components of whole-cell biosensors include the sensing elements, such as transcription factors and riboswitches, and reporting elements, such as fluorescence, gas, etc. The sensing and reporting elements are coupled through gene expression regulation to form a simple gene circuit for the detection of target substances. Additionally, a more complex gene circuit can involve other functional elements or modules such as signal amplification, multiple detection, and delay reporting. With the help of synthetic biology, whole-cell biosensors are becoming more versatile and integrated, that is, integrating pre-detection sample processing, detection processes, and post-detection signal calculation and storage processes into cells. Due to the relative stability of the intracellular environment, whole-cell biosensors are highly resistant to interference without the need of complex sample preprocessing. Due to the reproduction of chassis cells, whole-cell biosensors replicate all elements automatically without the need for purification processing. Therefore, whole-cell biosensors are easy to operate and simple to produce. Based on the above advantages, whole-cell biosensors are more suitable for on-site detection than other rapid detection methods. Whole-cell biosensors have been applied in various forms such as test strips and kits, with the latest reported forms being wearable devices such as masks, hand rings, and clothing. This paper examines the composition, construction methods, and types of the fundamental components of synthetic biological whole-cell biosensors. We also introduce the prospect and development trend of whole-cell biosensors in commercial applications.

Indole is a biologically active compound naturally occurring in plants and some bacteria. It is an important specialty chemical that is used as a precursor by the pharmaceutical and chemical industries, as well as in agriculture. Recently, indole has been identified as an important signaling molecule for bacteria in the mammalian gut. The regulation of indole biosynthesis has been studied in several bacterial species. However, this has been limited by the lack of in vivo tools suitable for indole-producing species identification and monitoring. The genetically encoded biosensors have been shown to be useful for real-time quantitative metabolite analysis. This paper describes the identification and characterization of the indole-inducible system PpTrpI/PPP_RS00425 from Pseudomonas putida KT2440. Indole whole-cell biosensors based on Escherichia coli and Cupriavidus necator strains are developed and validated. The specificity and dynamics of biosensors in response to indole and its structurally similar derivatives are investigated. The gene expression system PpTrpI/PPP_RS00425 is shown to be specifically induced up to 639.6-fold by indole, exhibiting a linear response in the concentration range from approximately 0.4 to 5 mM. The results of this study form the basis for the use of whole-cell biosensors in indole metabolism-relevant bacterial species screening and characterization. [ABSTRACT FROM AUTHOR]

Genetically programmed biosensors have been widely used to monitor bioavailable heavy metal pollutions in terms of their toxicity to living organisms. Most bacterial biosensors were initially designed to detect specific heavy metals such as mercury and cadmium. However, most available biosensors failed to distinguish cadmium from various heavy metals, especially mercury. Integrating diverse sensing elements into a single genetic construct or a single host strain has been demonstrated to quantify several heavy metals simultaneously. In this study, a dual-sensing construct was assembled by employing mercury-responsive regulator (MerR) and cadmium-responsive regulator (CadR) as the separate sensory elements and enhanced fluorescent protein (eGFP) and mCherry red fluorescent protein (mCherry) as the separate reporters. Compared with two corresponding single-sensing bacterial sensors, the dual-sensing bacterial sensor emitted differential double-color fluorescence upon exposure to 0–40 μM toxic Hg(II) and red fluorescence upon exposure to toxic Cd(II) below 200 μM. Bioavailable Hg(II) could be quantitatively determined using double-color fluorescence within a narrow concentration range (0–5 μM). But bioavailable Cd(II) could be quantitatively measured using red fluorescence over a wide concentration range (0–200 μM). The dual-sensing biosensor was applied to detect bioavailable Hg(II) and Cd(II) simultaneously. Significant higher red fluorescence reflected the predominant pollution of Cd(II), and significant higher green fluorescence suggested the predominant pollution of Hg(II). Our findings show that the synergistic application of various sensory modules contributes to an efficient biological device that responds to concurrent heavy metal pollutants in the environment. [ABSTRACT FROM AUTHOR]

In recent years, whole-cell biosensors (WCBs) have emerged as a potent approach for environmental monitoring and on-site analyte detection. These biosensors harness the biological apparatus of microorganisms to identify specific analytes, offering advantages in sensitivity, specificity, and real-time monitoring capabilities. A critical hurdle in biosensor development lies in ensuring the robust attachment of cells to surfaces, a crucial step for practical utility. In this study, we present a comprehensive approach to tackle this challenge via engineering Escherichia coli cells for immobilization on paper through the Curli biofilm pathway. Furthermore, incorporating a cellulose-binding peptide domain to the CsgA biofilm protein enhances cell adhesion to paper surfaces, consequently boosting biosensor efficacy. To demonstrate the versatility of this platform, we developed a WCB for copper, optimized to exhibit a discernible response, even with the naked eye. To confirm its suitability for practical field use, we characterized our copper sensor under various environmental conditions—temperature, salinity, and pH—to mimic real-world scenarios. The biosensor-equipped paper discs can be freeze-dried for deployment in on-site applications, providing a practical method for long-term storage without loss of sensitivity paper discs demonstrate sustained functionality and viability even after months of storage with 5 μM limit of detection for copper with visible-to-naked-eye signal levels. Biofilm-mediated surface attachment and analyte sensing can be independently engineered, allowing for flexible utilization of this platform as required. With the implementation of copper sensing as a proof-of-concept study, we underscore the potential of WCBs as a promising avenue for the on-site detection of a multitude of analytes. The steps for the procedures involved in cellular immobilization on paper using biofilm, sensor optimization, and analyte detection through whole-cell biosensors. 1) Bacterial cells have been modified to adhere to paper surfaces through the expression of extracellular biofilms. 2) The copper-specific biosensor unit has been engineered to generate a response within the naked-eye detectable range. 3) The biofilm-based immobilization and sensory circuits are combined in a single cell. Paper discs were freeze-dried and stored for future experiments. 4) Paper discs were incubated with a water source to determine if it is contaminated with the target analyte of interest, copper (Cu). [Display omitted] [ABSTRACT FROM AUTHOR]

Phenol and p -cresol are two common toxic small molecules related to various diseases. Existing reports confirmed that high L -tyrosine in the daily diet can increase the concentration of phenolic compounds in blood and urine. L-tyrosine is a common component of protein-rich foods. Some anaerobic bacteria in the gut can convert non-toxic l-tyrosine into these two toxic phenolic compounds, phenol and p -cresol. Existing methods have been constructed for measuring the concentration of phenolic compound in feces. However, there is still a lack of direct visual evidence to measure the phenolic compounds in the intestine. In this study, we aimed to construct a whole-cell biosensor for phenolic compounds detection based on the dmpR , the regulator from the phenol metabolism cluster. The commensal bacterium Citrobacter amalonaticus PS01 was selected and used as the chassis. Compared with the biosensor based on ECN1917, the biosensor PS01[ dmpR ] could better implant into the mouse gut through gavage and showed a higher sensitive to phenolic compound. And the concentration of phenolic compounds in the intestines could be observed with the help of in vivo imaging system using PS01[ dmpR ]. This paper demonstrated endogenous phenol synthesis in the gut and the strategy of using commensal bacteria to construct whole-cell biosensors for detecting small molecule compounds in the intestines. [Display omitted] • A plasmid for detecting phenol and p -cresol was constructed. • The biosensor was constructed with a native bacterium chassis from the host. • The limit of quantitation of the biosensor was 948.4 nmol/L for phenol and 9.52 µmol/L for p -cresol. • The whole biosensor could be used in vivo to detect the level of toxic phenolic compounds in the host. [ABSTRACT FROM AUTHOR]

Genetically programmed biosensors have been widely used to monitor bioavailable heavy metal pollutions in terms of their toxicity to living organisms. Most bacterial biosensors were initially designed to detect specific heavy metals such as mercury and cadmium. However, most available biosensors failed to distinguish cadmium from various heavy metals, especially mercury. Integrating diverse sensing elements into a single genetic construct or a single host strain has been demonstrated to quantify several heavy metals simultaneously. In this study, a dual-sensing construct was assembled by employing mercury-responsive regulator (MerR) and cadmium-responsive regulator (CadR) as the separate sensory elements and enhanced fluorescent protein (eGFP) and mCherry red fluorescent protein (mCherry) as the separate reporters. Compared with two corresponding single-sensing bacterial sensors, the dual-sensing bacterial sensor emitted differential double-color fluorescence upon exposure to 0–40 μM toxic Hg(II) and red fluorescence upon exposure to toxic Cd(II) below 200 μM. Bioavailable Hg(II) could be quantitatively determined using double-color fluorescence within a narrow concentration range (0–5 μM). But bioavailable Cd(II) could be quantitatively measured using red fluorescence over a wide concentration range (0–200 μM). The dual-sensing biosensor was applied to detect bioavailable Hg(II) and Cd(II) simultaneously. Significant higher red fluorescence reflected the predominant pollution of Cd(II), and significant higher green fluorescence suggested the predominant pollution of Hg(II). Our findings show that the synergistic application of various sensory modules contributes to an efficient biological device that responds to concurrent heavy metal pollutants in the environment.

The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick), is a major pest of citrus fruits and other plants and is defined as a quarantine pest in several countries. Supervision and visual monitoring are crucial for the detection of eggs and larvae. There is a growing need to implement alternative pest control strategies and early detection methodologies in the packing house before shipping. Biosensors may provide a better-suited solution, as they are portable, do not require expensive equipment, and may be used with simple methodologies. In this study, a whole-cell bacterial biosensor was developed to identify the presence of Thaumatotibia leucotreta larvae in citrus fruits. The volatile organic compounds (VOCs) released by the larvae were identified (n = 11), which were not previously tested. The change in the VOCs composition in infected and uninfected mandarin (Citrus reticulata) was examined using gas chromatography-mass spectrometry (GC-MS) and the bioluminescent bacterial panel. During this work, two fixation methods (alginate beads and tablets) were tested for the bioluminescent bacteria cells, and tablets were preferred due to their higher uniformity and consistency of the light signal from the cells. Furthermore, the response of the bacteria to the changes in the concentration of VOCs as a result of a change in the amount of the larvae was examined (larvae n = 3, 6, or 9). In addition, in order to demonstrate the ability of the whole-cell biosensor to detect the presence of larva within the fruit, infected and uninfected mandarins were tested on the 14th day of larval hatching. The results showed that the bioluminescent bacteria could differentiate between mandarins with and without larva. The differences in the light signals (Induction Factor (IF)) between the mandarin fruits can be seen in the results with the larva (cytotoxicity IF = 3.046, genotoxicity IF = 2.643, and quorum sensing IF = 1.954) and without (cytotoxicity IF = 1.589, genotoxicity IF = 1.149 and quorum sensing IF = 1.205). The findings of this study make it possible to further develop a prototype using bioluminescent bacteria in order to detect pests of citrus fruits. The early detection of infected fruits will allow their filtering from the crop and therefore reduce the damage to additional fruits. Even one larva can invalidate the whole stock and the plot for future export. • The false codling moth (FCM), Thaumatotibia leucotreta , is a major pest. • Volatile organic compounds (VOCs) profile between infected and non-infected. • Whole-cell bacterial biosensor to identify Thaumatotibia leucotreta in citrus fruits. • Gas chromatography-mass spectrometry (GC-MS) for volatiles from the larvae. • The bioluminescent bacteria can identify mandarins with larvae. [ABSTRACT FROM AUTHOR]

IMP(Inosinic acid) is a raw material with extremely important uses in food manufacturing and agriculture. In the selection process of IMP producing strains, there has been no tool available to assist in high-throughput screening, which greatly affects the speed of strain development. To address this issue, we have developed a whole cell IMP biosensor based on the characteristics of GlxR , a member of the CRP/FNR transcription factor family in Corynebacterium stationis. By modifying and altering the specificity of GlxR , we transformed the effector molecule from cAMP(Cyclic adenosine monophosphate) to IMP. Our strategies involved optimizing the reporter gene, engineering the GlxR transcription factor via Fluorescence-Activated Cell Sorting (FACS), and manipulating the Pcg3195 promoter. We constructed a synthetic biosensor called pSenIMP-MUT1-Pmmm, which utilized GlxR-MUT1, Pcg3195 engineered Pmmm promoters, and EGFP reporter genes. This biosensor facilitated the establishment of a high-throughput screening strategy using FACS. Among the screened cells, 53.33% were identified as the purATTG guaBTTG strain, exhibiting the highest IMP production, while 16.67% were selected as the purATTG strain with the second highest IMP production. Overall, our study presents a novel method for high-throughput screening of IMP producing strains using a synthetic biosensor based on GlxR characteristics. This approach significantly accelerates the strain development process and holds great potential for various industrial applications. [Display omitted] • High-throughput tool has been made for the screening of IMP producing strains. • The effector from cAMP to IMP was transformed by modifying the specificity of GlxR. • Optimising biosensors with multiple approaches. [ABSTRACT FROM AUTHOR]

It is an urgent need to develop novel antibiotics to treat infections caused by multi-drug-resistant bacteria. One promising strategy could be the use of whole-cell biosensors, which have been extensively studied to monitor environmental pollutants and intracellular metabolites. Here, we used the σM-mediated regulatory system of Bacillus subtilis to construct a whole-cell biosensor for the detection of cell envelope-acting antibiotics. Using polymyxin B as the inducer for bacterial cell envelope stress and enhanced green fluorescent protein (EGFP) as the reporter, we found that the promoter of ypuA (PypuA) had the lowest background noise and the most significant changes in the fluorescence output. The whole-cell biosensor displayed dose-dependent and time-dependent responses in fluorescence signals. The detection range of this biosensor for polymyxin B was between 0.125 and 12 μg/mL. The response of the biosensor is specific to antibiotics that target the cell envelope. Besides determination in liquid cultures, the output signal of the biosensor can be easily determined on agar surfaces. Using this biosensor, we successfully detected polymyxins secreted by its producing strain and bacteria that produce cell envelope-acting antibiotics. Key points: • A whole-cell biosensor was constructed based on the σM-mediated regulatory system. • The response of the biosensor is specific to cell envelope-acting antibiotics. • The biosensor can be used to screen novel cell envelope-acting antibiotics. [ABSTRACT FROM AUTHOR]

Bioluminescence occurs through a chemical reaction in organisms that spontaneously produce light. Luminescent bacteria are unique among bioluminescent organisms. Their bioluminescence intensity is an indicator of their metabolic activity, which can directly reflect the influence of environmental factors on cell viability. Moreover, the whole bioluminescence process is totally gene encoded without the addition of extra substrates. As a result, bacterial bioluminescence has been a powerful tool for whole-cell biosensors and bio-reporters in bioanalysis and bioimaging. This review aims to cover the applications of wild-type and recombinant luminescent bacteria to detect the toxicity of environmental pollutants and biological molecules. The bacterial bioluminescence analytical assay has characteristics such as high sensitivity, short-term detection, and easy operation. Meanwhile, due to the development of gene engineering and optical technology, bacterial luciferase as a reporter protein has been successfully expressed in prokaryotic and eukaryotic cells, tissues, and organs of animals. The major applications for bacterial luciferase-based bioluminescence imaging, such as infectious diseases, cancer therapy, and stem cell tracing, are discussed in this review. [ABSTRACT FROM AUTHOR]

Although many whole-cell biosensors (WCBs) for the detection of Cd2+ have been developed over the years, most lack sensitivity and specificity. In this paper, we developed a Cd2+ WCB with a negative feedback amplifier in P. putida KT2440. Based on the slope of the linear detection curve as a measure of sensitivity, WCB with negative feedback amplifier greatly increased the output signal of the reporter mCherry, resulting in 33% greater sensitivity than in an equivalent WCB without the negative feedback circuit. Moreover, WCB with negative feedback amplifier exhibited increased Cd2+ tolerance and a lower detection limit of 0.1 nM, a remarkable 400-fold improvement compared to the WCB without the negative feedback circuit, which is significantly below the World Health Organization standard of 27 nM (0.003 mg/L) for cadmium in drinking water. Due to the superior amplification of the output signal, WCB with negative feedback amplifier can provide a detectable signal in a much shorter time, and a fast response is highly preferable for real field applications. In addition, the WCB with negative feedback amplifier showed an unusually high specificity for Cd2+ compared to other metal ions, giving signals with other metals that were between 17.6 and 41.4 times weaker than with Cd2+. In summary, the negative feedback amplifier WCB designed in this work meets the requirements of Cd2+ detection with very high sensitivity and specificity, which also demonstrates that genetic negative feedback amplifiers are excellent tools for improving the performance of WCBs.

The use of bacteriocins is a promising approach for addressing the immense threat of food-borne and drug-resistant pathogens. In recent years screening platforms for novel bacteriocins using whole-cell biosensors have been established. During screening cell-to-cell heterogeneity is currently neglected but might play a crucial role in signal development of the whole-cell biosensor after bacteriocin exposure. In this study, we explored the temporal dynamics of the signal heterogeneity of the biosensor Listeria innocua LMG2785/pNZpHin2 Lm after nisin exposure using microfluidic single-cell analysis. The results provided novel and detailed insights into the dynamics of cell-to-cell heterogeneity in L. innocua LMG2785/pNZpHin2 Lm at different nisin concentrations with a high spatio-temporal resolution. Furthermore, the formation of subpopulations during bacteriocin exposure was observed. In-depth single-cell tracking even revealed the regeneration of disrupted cells and recovery of pH homeostasis in rare instances. These findings are highly important for the future design and execution of bacteriocin assays and for the interpretation of fluorescence signal development at the population level after exposure to different concentrations of bacteriocins (here, nisin), as well as for obtaining deeper insights into single-cell persistence strategies to quantify the efficacy and efficiency of novel bacteriocins., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Fante, Desiderato, Riedel and Grünberger.)

Cadmium (Cd) is carcinogenic to humans and can accumulate in the liver, kidneys, and bones. There is widespread presence of cadmium in the environment as a consequence of anthropogenic activities. It is important to detect cadmium in the environment to prevent further exposure to humans. Previous whole-cell biosensor designs were focused on single-sensing constructs but have had difficulty in distinguishing cadmium from other metal ions such as lead (Pb) and mercury (Hg). We developed a dual-sensing bacterial bioreporter system to detect bioavailable cadmium by employing CadC and CadR as separate metal sensory elements and eGFP and mCherry as fluorescent reporters in one genetic construct. The capability of this dual-sensing biosensor was proved to simultaneously detect bioavailable cadmium and its toxic effects using two sets of sensing systems while still maintaining similar specificity and sensitivity of respective signal-sensing biosensors. The productions of double-color fluorescence were directly proportional to the exposure concentration of cadmium, thereby serving as an effective quantitative biosensor to detect bioavailable cadmium. This novel dual-sensing biosensor was then validated to respond to Cd(II) spiked in environmental water samples. This is the first report of the development of a novel dual-sensing, whole-cell biosensor for simultaneous detection of bioavailable cadmium. The application of two biosensing modules provides versatile biosensing signals and improved performance that can make a significant impact on monitoring high concentration of bioavailable Cd(II) in environmental water to reduce human exposure to the harmful effects of cadmium. [ABSTRACT FROM AUTHOR]

During the last few decades, whole-cell biosensors have attracted increasing attention for their enormous potential in monitoring bioavailable heavy metal contaminations in the ecosystem. Visual and measurable output signals by employing natural pigments have been demonstrated to offer another potential choice to indicate the existence of bioavailable heavy metals in recent years. The biosynthesis of the blue pigment indigoidine has been achieved in E. coli following heterologous expression of both BpsA (a single-module non-ribosomal peptide synthetase) and PcpS (a PPTase to activate apo-BpsA). Moreover, we demonstrated herein the development of the indigoidine-based whole-cell biosensors to detect bioavailable Hg(II) and Pb(II) in water samples by employing metal-responsive transcriptional regulator MerR and PbrR as the sensory elements, and the indigoidine biosynthesis gene cluster as a reporter element. The resulting indigoidine-based biosensors presented a good selectivity and high sensitivity to target metal ions. High concentration of target metal exposure could be clearly recognized by the naked eye due to the color change by the secretion of indigoidine, and quantified by measuring the absorbance of the culture supernatants at 600 nm. Dose–response relationships existed between the exposure concentrations of target heavy metals and the production of indigoidine. Although fairly good linear relationships were obtained in a relatively limited concentration range of the concentrations of heavy metal ions, these findings suggest that genetically controlled indigoidine biosynthesis triggered by the MerR family transcriptional regulator can enable a sensitive, visual, and qualitative whole-cell biosensor for bioindicating the presence of bioaccessible heavy metal in environmental water samples. Key points: • Biosynthesis pathway of indigoidine reconstructed in a high copy number plasmid in E. coli. • Visual and colorimetric detection of Hg(II) and Pb(II) by manipulation of indigoidine biosynthesis through MerR family metalloregulator. •Enhanced detection sensitivity toward Hg(II) and Pb(II) achieved using novel pigment-based whole-cell biosensors. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is carcinogenic to humans and can accumulate in the liver, kidneys, and bones. There is widespread presence of cadmium in the environment as a consequence of anthropogenic activities. It is important to detect cadmium in the environment to prevent further exposure to humans. Previous whole-cell biosensor designs were focused on single-sensing constructs but have had difficulty in distinguishing cadmium from other metal ions such as lead (Pb) and mercury (Hg). We developed a dual-sensing bacterial bioreporter system to detect bioavailable cadmium by employing CadC and CadR as separate metal sensory elements and eGFP and mCherry as fluorescent reporters in one genetic construct. The capability of this dual-sensing biosensor was proved to simultaneously detect bioavailable cadmium and its toxic effects using two sets of sensing systems while still maintaining similar specificity and sensitivity of respective signal-sensing biosensors. The productions of double-color fluorescence were directly proportional to the exposure concentration of cadmium, thereby serving as an effective quantitative biosensor to detect bioavailable cadmium. This novel dual-sensing biosensor was then validated to respond to Cd(II) spiked in environmental water samples. This is the first report of the development of a novel dual-sensing, whole-cell biosensor for simultaneous detection of bioavailable cadmium. The application of two biosensing modules provides versatile biosensing signals and improved performance that can make a significant impact on monitoring high concentration of bioavailable Cd(II) in environmental water to reduce human exposure to the harmful effects of cadmium.

In this study, an Escherichia coli (E. coli) whole-cell biosensor for the specific detection of bioavailable arsenic was developed by placing a green fluorescent protein (GFP) reporter gene under the control of the ArsR1 (GSU2952) regulatory circuit from Geobacter sulfurreducens. E. coli cells only emitted green fluorescence in the presence of arsenite and were more sensitive to arsenite when they were grown in M9 supplemented medium compared to LB medium. Under optimal test conditions, the Geobacter arsR1 promoter had a detection limit of 0.01 μM arsenite and the GFP expression was linear within a range of 0.03–0.1 μM (2.25–7.5 μg/l). These values were well below World Health Organization’s drinking water quality standard, which is 10 μg/l. The feasibility of using this whole-cell biosensor to detect arsenic in water samples, such as arsenic polluted tap water and landfill leachate was verified. The biosensor was determined to be just as sensitive as atomic fluorescence spectrometry. This study examines the potential applications of biosensors constructed with Geobacter ArsR-Pars regulatory circuits and provides a rapid and cost-effective tool that can be used for arsenic detection in water samples.

Whole-cell biosensors could be helpful for in situ disease diagnosis. However, their use in analyzing biological samples has been hindered by unstable responses, low signal enhancement, and growth inhibition in complex media. Here, we offered a solution by building a visual whole-cell biosensor for urinary mercury determination. With deoxyviolacein as the preferred signal for the mercury biosensor for the first time, it enabled the quantitative detection of urinary mercury with a favorable linear range from 1.57 to 100 nM. The biosensor can accurately diagnose urine mercury levels exceeding the biological exposure index with 95.8% accuracy. Thus, our study provided a biosensing platform with great potential to serve as a stable, user-friendly, and high-throughput alternative for the daily monitoring or estimating of urinary mercury. [Display omitted] [ABSTRACT FROM AUTHOR]

Methylmercury is primarily responsible for most food mercury pollution cases. However, most biosensors developed for mercury pollution analysis can only detect mercury ions. Although oxidative strong-acid digestion or microwave-assisted digestion can convert methylmercury into mercury ions, it is unsuitable for on-site detection. This study designed a bio-digestion gene circuit and integrated it into a mercury ion whole-cell biosensor，creating a novel on-site methylmercury detection method. Five alkyl mercury lyases from different bacterial genomes were screened via bioinformatics analysis, of which goMerB from Gordonia otitis showed the highest catalytic biological digestion efficiency. The goMerB site-specific saturation and random mutation libraries were constructed. After two rounds of high-throughput visualization screening, the catalytic activity of the mutant increased 2.5-fold. The distance between the three crucial amino acid sites and methylmercury changed in the mutant, which likely contributed to the enhanced catalytic efficiency. The optimized whole-cell biosensor showed a linear dynamic concentration range of 100 nM to 100 μM (R2 =0.991), satisfactory specificity, and interference resistance. The detection limit of the goMerBt6-MerR-RFP biosensor was 0.015 μM, while the limit of quantitation was 0.049 μM. This study demonstrated the application of synthetic biology for food safety detection and highlighted the future potential of "Lab in a Cell" for hazard analysis. [Display omitted] • Based on synthetic biology, this article used genetic circuits instead of mechanical instruments, and provides a case study. • In this paper, the visual high-throughput directed evolution of alkyl mercury lyase is realized. • This study solved the problem that most biosensors could only detect mercury ions but not methylmercury. [ABSTRACT FROM AUTHOR]

Redox-active small molecules containing quinone functional groups play important roles as pharmaceuticals, but can be toxic if overdosed. Despite the need for a fast and quantitative method to detect quinone and its derivatives, current sensing strategies are often slow and struggle to differentiate between structural analogs. Leveraging the discovery that microorganisms use certain quinones to perform extracellular electron transfer (EET), we investigated the use of Lactiplantibacillus plantarum as a whole-cell bioelectronic sensor to selectively sense quinone analogs. By tailoring the native EET pathway in L. plantarum , we enabled quantitative quinone sensing of 1,4-dihydroxy-2-naphthoic acid (DHNA) - a gut bifidogenic growth stimulator. We found that L. plantarum could respond to environmental DHNA within seconds, producing concentration-dependent electrical signals. This sensing capacity was robust in different assay media and allowed for continuous monitoring of DHNA concentrations. In a simulated gut environment containing a mixed pool of quinone derivatives, this tailored EET pathway can selectively sense pharmacologically relevant quinone analogs, such as DHNA and menadione, amongst other structurally similar quinone derivatives. We also developed a multivariate model to describe the mechanism behind this selectivity and found a predictable correlation between quinone physiochemical properties and the corresponding electrical signals. Our work presents a new concept to selectively sense quinone using whole-cell bioelectronic sensors and opens the possibility of using probiotic L. plantarum for bioelectronic applications in human health. [ABSTRACT FROM AUTHOR]

Abstract Background Whole cell biosensors provide a simple method for the detection of heavy metals. However, previous designs of them rely primarily on simulation of heavy metal resistance systems of bacteria. Results This study proposes a strategy for the rational design of metal detection circuits based on sensor proteins of the MerR family. Our results indicate the expression level of sensor protein can be used as a “rheostat” for tuning detection sensitivity with parabola curves to represent the relationships between the detection slopes and the sensor protein levels. This circuits design strategy (named as “Parabola Principle”), is used as a guide for the discovery of optimum metal detection circuits, and the design of biosensors with specific metal detection characteristics. For example, visible qualitative Hg (II) biosensors with a threshold of 0.05 mg/L are successfully constructed. Conclusions These results indicate the feasibility of developing a sensor that is much more tunable than what is presented. Graphical abstract