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Multiple antibiotic resistance regulators (MarRs) control the transcription of genes in the mar operon of Escherichia coli in the presence of salicylic acid (SA). The interaction with SA induces conformational changes in the MarR released from the promoter of the mar operon, turning on transcription. We constructed an SA-specific E. coli cell-based biosensor by fusing the promoter of the mar operon (PmarO) and the gene that encodes an enhanced green fluorescent protein (egfp). Because SA and aspirin are structurally similar, a biosensor for monitoring aspirin can be obtained by genetically engineering MarR to be aspirin (ASP)-responsive. To shift the selectivity of MarR toward ASP, we changed the residues around the ligand-binding sites by site-directed mutagenesis. We examined the effects of genetic engineering on MarR by introducing MarRs with PmarO-egfp into E. coli. Among the tested mutants, MarR T72A improved the ASP responses by approximately 3 times compared to the wild-type MarR, while still showing an SA response. Although the MarR T72A biosensor exhibited mutual interference between SA and ASP, it accurately determined the ASP concentration in spiked water and medicine samples with over 90% accuracy. While the ASP biosensors still require improvement, our results provide valuable insights for developing E. coli cell-based biosensors for ASP and transcription factor-based biosensors in general. [ABSTRACT FROM AUTHOR]

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]

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.

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]

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.

Multiple antibiotic resistance regulators (MarRs) control the transcription of genes in the mar operon of Escherichia coli in the presence of salicylic acid (SA). The interaction with SA induces conformational changes in the MarR released from the promoter of the mar operon, turning on transcription. We constructed an SA-specific E. coli cell-based biosensor by fusing the promoter of the mar operon (PmarO) and the gene that encodes an enhanced green fluorescent protein (egfp). Because SA and aspirin are structurally similar, a biosensor for monitoring aspirin can be obtained by genetically engineering MarR to be aspirin (ASP)-responsive. To shift the selectivity of MarR toward ASP, we changed the residues around the ligand-binding sites by site-directed mutagenesis. We examined the effects of genetic engineering on MarR by introducing MarRs with PmarO-egfp into E. coli. Among the tested mutants, MarR T72A improved the ASP responses by approximately 3 times compared to the wild-type MarR, while still showing an SA response. Although the MarR T72A biosensor exhibited mutual interference between SA and ASP, it accurately determined the ASP concentration in spiked water and medicine samples with over 90% accuracy. While the ASP biosensors still require improvement, our results provide valuable insights for developing E. coli cell-based biosensors for ASP and transcription factor-based biosensors in general.

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]

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.

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.

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]

This study was undertaken to develop a high-throughput screening strategy using a whole-cell biosensor to enhance methyl-group transfer, a rate-limiting step influenced by intracellular methyl donor availability and methyltransferase efficiency. An l -homocysteine biosensor was designed based on regulatory protein MetR from Escherichia coli , which rapidly reported intracellular l -homocysteine accumulation resulted from S-adenosyl- l -homocysteine (SAH) formation after methyl-group transfer. Using S-adenosyl- l -methionine (SAM) as a methyl donor, this biosensor was applied to caffeic acid 3- O -methyltransferase derived from Arabidopsis thaliana (AtComT). After several rounds of directed evolution, the modified enzyme achieved a 13.8-fold improvement when converting caffeic acid to ferulic acid. The best mutant exhibited a 5.4-fold improvement in catalytic efficiency. Characterization of beneficial mutants showed that improved O -methyltransferase dimerization greatly contributed to enzyme activity. This finding was verified when we switched and compared the N-termini involved in dimerization across different sources. Finally, with tyrosine as a substrate, the evolved AtComT mutant greatly improved ferulic acid biosynthesis, yielding 3448 mg L−1 with a conversion rate of 88.8%. These results have important implications for high-efficiency O -methyltransferase design, which will greatly benefit the biosynthesis of a wide range of natural products. In addition, the l -homocysteine biosensor has the potential for widespread applications in evaluating the efficiency of SAM-based methyl transfer. [ABSTRACT FROM AUTHOR]

5-Aminolevulinic acid (5-ALA) is a non-protein amino acid widely used in agriculture, animal husbandry and medicine. Currently, microbial cell factories are a promising production pathway, but the lack of high-throughput fermentation strain screening tools often hinders the exploration of engineering strategies to increase cell factory yields. Here, mutant AC103-3H was screened from libraries of saturating mutants after response-specific engineering of the transcription factor AsnC of L-asparagine (Asn). Based on mutant AC103-3H, a whole-cell biosensor EAC103-3H with a specific response to 5-ALA was constructed, which has a linear dynamic detection range of 1–12 mM and a detection limit of 0.094 mM, and can be used for in situ screening of potential high-producing 5-ALA strains. With its support, overexpression of the C5 pathway genes using promoter engineering assistance resulted in a 4.78-fold enhancement of 5-ALA production in the engineered E. coli. This study provides an efficient strain screening tool for exploring approaches to improve the 5-ALA productivity of engineered strains. [ABSTRACT FROM AUTHOR]

The contamination of mycotoxins is a serious problem around the world. It has detrimental effects on human beings and leads to tremendous economic loss. It is essential to develop a rapid and non-destructive method for contamination recognition particularly for early alarm. In this study, the whole-cell biosensor array was constructed and employed for rapid recognition of wheat contamination by combining with machine learning algorithms. Seven key VOCs were explored through univariate coupling to multivariate analysis of orthogonal partial least squares-discrimination analysis (OPLS-DA) models. The promoters of dnaK , katG , oxyR , soxS obtained from the stress-responsive of key VOCs were fused to the bacterial operon and fabricated on the whole-cell biosensor. The constructed whole-cell biosensor array was consisted with four kinds of sensors and 18 sensor unit. The bioluminescent intensity combined with linear machine learning algorithm of partial least squares discriminant analysis (PLS-DA) and non-linear algorithms of back propagating artificial neural network (BP-ANN) and least square support vector machine (LS-SVM) were employed to establish discrimination models for mold contamination especially for early warning. The Monte-Carlo strategy was performed to generate thirty subsets for modeling to give more reliable results. As a result, the whole-cell biosensor combined with non-linear algorithm of LS-SVM was practicable for detecting mold identification for wheat early-warning with the accuracy of 97.24%. Additionally, this study provides practical and effective methods not only for wheat quality guarantee and supervision but also for other foodstuffs. [Display omitted] [ABSTRACT FROM AUTHOR]

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.

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]

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]

As a threat to human health and public health, cadmium (Cd) pollution has received widespread social concern. Our previously constructed CadR-based bacterial whole cell biosensor (WCB) epCadR5 showed high sensitivity and specificity in cadmium detection. However, the application of the sensor is still hindered by the need for laboratory equipment to read the fluorescence signal output. In this study, we aimed to optimizing the sensor to make it available for visual detection of environmental cadmium and simplify the detection process to advance practical application of the sensor. By replacing the constitutive promoter with J110, the fluorescence signal output of the sensor was significantly increased and the fluorescence leakage was decreased. In addition, the fluorescence signal output of green fluorescence protein (GFP) was enhanced by the addition of a 5′ untranslated region (5′-UTR) mlcR10. The fluorescence signal output of the WCB is sufficiently robust to be visible and distinguishable to the naked eye, which is of paramount importance for visual detection. The sensor readout can be conveniently recorded by mobile phone camera and quantified. For ease of on-site application, the WCB's visual detection procedures and conditions were further optimized and simplified. The WCB demonstrated good linearity and detection limit (1.81 μg/L) for visual detection of Cd2+ without the assistance of bulky laboratory equipment. For the detection of real environmental samples, the WCB visual detection results were close to those of WCB-flow cytometry (FACS) and graphite furnace atomic absorption spectroscopy (GFAAS). In this work, we developed an easy-to-use, on-site and visual detection biosensor for monitoring environmental Cd2+. It will advance the utilization of cadmium WCBs in practical settings. The optimization and simplification strategy in the study also provide new insights into the visualization of other bacterial biosensors, and will advance the practical application of WCBs. [Display omitted] • Improvement of CadR-based cadmium bacteria whole cell biosensor by optimizing of promoter and adding of 5′-UTR sequence. • Visualization of CadR-based whole cell biosensor. • The visualized cadmium WCB provided an easy-to-use, on-site and visual detection tool for monitoring Cd2+. [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]

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]

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.

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]

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.

Phosphonates (PHTs), organic compounds with a stable C-P bond, are widely distributed in nature. Glyphosate (GP), a synthetic PHT, is extensively used in agriculture and has been linked to various human health issues and environmental damage. Given the prevalence of GP, developing cost-effective, on-site methods for GP detection is key for assessing pollution and reducing exposure risks. We adopted Agrobacterium tumefaciens CHLDO, a natural GP degrader, as a host and the source of genetic parts for constructing PHT biosensors. In this bacterial species, the phn gene cluster, encoding the C-P lyase pathway, is regulated by the PhnF transcriptional repressor. We selected the phnG promoter, which displays a dose-dependent response to GP, to build a set of whole-cell biosensors. Through stepwise genetic optimization of the transcriptional cascade, we created a whole-cell biosensor capable of detecting GP in the 0.25-50 μM range in various samples, including soil and water.

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.

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.

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.