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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.

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.

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.

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.

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.

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 development of genetic engineering, especially synthetic biology, greatly contributes to the development of novel metal biosensors. The cad operon encoding cadmium resistance was previously characterized from Pseudomonas putida. In this study, single-, dual-, and triple-signal output Cd(II) biosensors were successfully developed using artificial translationally coupled cad operons. Sensitivity, selectivity, and response toward Cd(II) and Hg(II), of three biosensors were all determined. Reporter signals of three biosensors all increased within the range 0.1–3.125 μM Cd(II). Three biosensors responded strongly to Cd(II), and weakly to Hg(II). However, the detection ranges of Cd(II) and Hg(II) do not overlap in all three biosensors. Next, novel Cd(II) biosensing coupled with bioadsorptive artificial cad operons were assembled for the first time. Cd(II)-induced fluorescence emission, enzymatic indication, and Cd(II) binding protein surface display can be achieved simultaneously. This study provides an example of one way to realize multiple signal outputs and bioadsorption based on the redesigned heavy metal resistance operons, which may be a potential strategy for biodetection and removal of toxic metal in the environment, facilitating the study of the mechanism and dynamics of bioremediation.

Rapid progress in the genetic circuit design enabled whole-cell biosensors (WCBs) to become prominent in detecting an extensive range of analytes with promise in many fields, from medical diagnostics to environmental toxicity assessment. However, several drawbacks, such as high background signal or low precision, limit WCBs to transfer from proof-of-concept studies to real-world applications, particularly for heavy metal toxicity monitoring. For an alternative WCB module design, we utilized Bxb1 recombinase that provides tight control as a switch to increase dose-response behavior concerning leakiness. The modularity of Bxb1 recombinase recognition elements allowed us to combine an engineered semi-specific heat shock response (HSR) promoter, sensitive to stress conditions including toxic ions such as cadmium, with cadmium resistance regulatory elements; a cadmium-responsive transcription factor and its cognitive promoter. We optimized the conditions for the recombinase-based cadmium biosensor to obtain increased fold change and shorter response time. This system can be expanded for various heavy metals to make an all-in-one type of WCB, even using semi-specific parts of a sensing system.

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

Aliivibrio fischeri LuxR and Aliivibrio logei LuxR1 and LuxR2 regulatory proteins are quorum sensing transcriptional (QS) activators, inducing promoters of luxICDABEG genes in the presence of an autoinducer (3-oxo-hexanoyl-l-homoserine lactone). In the Aliivibrio cells, luxR genes are regulated by HNS, CRP, LitR, etc. Here we investigated the role of the luxR expression level in LuxI/R QS system functionality and improved the whole-cell biosensor for autoinducer detection. Escherichia coli-based bacterial lux-biosensors were used, in which Photorhabdus luminescensluxCDABE genes were controlled by LuxR-dependent promoters and luxR, luxR1, or luxR2 regulatory genes. We varied either the dosage of the regulatory gene in the cells using additional plasmids, or the level of the regulatory gene expression using the lactose operon promoter. It was shown that an increase in expression level, as well as dosage of the regulatory gene in biosensor cells, leads to an increase in sensitivity (the threshold concentration of AI is reduced by one order of magnitude) and to a two to threefold reduction in response time. The best parameters were obtained for a biosensor with an increased dosage of luxRA. fischeri (sensitivity to 3-oxo-hexanoyl-l-homoserine lactone reached 30–100 pM).

High levels of blood glucose are always associated with numerous complications including cholesterol abnormalities. Therefore, it is important to simultaneously monitor blood glucose and cholesterol levels in patients with diabetes during the management of chronic diseases. In this study, a glucose dehydrogenase from Aspergillus oryzae TI and a cholesterol oxidase from Chromobacterium sp. DS-1 were displayed on the surface of Saccharomyces cerevisiae, respectively, using the yeast surface display system at a high copy number. In addition, two whole-cell biosensors were constructed through the immobilization of the above yeast cells on electrodes, for electrochemical detection of glucose and cholesterol. The assay time was 8.5 s for the glucose biosensors and 30 s for the cholesterol biosensors. Under optimal conditions, the cholesterol biosensor exhibited a linear range from 2 to 6 mmol·L−1. The glucose biosensor responded efficiently to the presence of glucose at a concentration range of 20–600 mg·dL−1 (1.4–33.3 mmol·L−1) and showed excellent anti-xylose interference properties. Both biosensors exhibited good performance at room temperature and remained stable over a three-week storage period.

Biosensors have major advantages over chemical or physical analyses with regard to specificity, sensitivity, and portability. Recently, many types of whole-cell bacterial biosensors have been developed using recombinant DNA technology. The bacteria are genetically engineered to respond to the presence of chemicals or physiological stresses by synthesizing a reporter protein, such as luciferase, β-galactosidase, or green fluorescent protein. In addition to an overview of conventional biosensors, this minireview discusses a novel type of biosensor using a photosynthetic bacterium as the sensor strain and the crtA gene, which is responsible for carotenoid synthesis, as the reporter. Since bacteria possess a wide variety of stress-response mechanisms, including antioxidation, heat-shock responses, nutrient-starvation, and membrane-damage responses, DNA response elements for several stress-response proteins can be fused with various reporter genes to construct a versatile set of bacterial biosensors for a variety of analytes. Portable biosensors for on-site monitoring have been developed using a freeze-dried biosensing strain, and cell array biosensors have been designed for high-throughput analysis. Moreover, in the future, the use of single-cell biosensors will permit detailed analyses of samples. Signals from such sensors could be detected with digital imaging, epifluorescence microscopy, and/or flow cytometry. [ABSTRACT FROM AUTHOR]