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Recently there has been an increasing need for synthesizing valued chemicals through biorefineries. Lactams are an essential family of commodity chemicals widely used in the nylon industry with annual production of millions of tons. The bio-production of lactams can substantially benefit from high-throughput lactam sensing strategies for lactam producer screening. We present here a robust and living lactam biosensor that is directly compatible with high-throughput analytical means. The biosensor is a hydrogel microparticle encapsulating living microcolonies of engineered lactam-responsive Escherichia coli. The microparticles feature facile and ultra-high throughput manufacturing of up to 10,000,000 per hour through droplet microfluidics. We show that the biosensors can specifically detect major lactam species in a dose-dependent manner, which can be quantified using flow cytometry. The biosensor could potentially be used for high-throughput metabolic engineering of lactam biosynthesis. [ABSTRACT FROM AUTHOR]

Whole-cell biosensors are useful for monitoring heavy metal toxicity in public health and ecosystems, but their development has been hindered by intrinsic trade-offs between sensitivity and specificity. Here, we demonstrated an effective engineering solution by building a sensitive, specific, and high-response biosensor for carcinogenic cadmium ions. We genetically programmed the metal transport system of Escherichia coli to enrich intracellular cadmium ions and deprive interfering metal species. We then selected 16 cadmium-sensing transcription factors from the GenBank database and tested their reactivity to 14 metal ions in the engineered E. coli using the expression of the green fluorescent protein as the readout. The resulting cadmium biosensor was highly specific and showed a detection limit of 3 nM, a linear increase in fluorescent intensities from 0 to 200 nM, and a maximal 777-fold signal change. Using this whole-cell biosensor, a smartphone, and low-tech equipment, we developed a simple assay capable of measuring cadmium ions at the same concentration range in irrigation water and human urine. This method is user-friendly and cost-effective, making it affordable to screen large amounts of samples for cadmium toxicity in agriculture and medicine. Moreover, our work highlights natural gene repositories as a treasure chest for bioengineering.

Transcriptional regulatory protein (TRP)-based whole-cell biosensors are widely used nowadays. Here, they were demonstrated to have great potential application in screening cell efflux and influx pumps for small molecules. First, a vanillin whole-cell biosensor was developed by altering the specificity of a TRP, VanR, and strains with improved vanillin productions that were selected from a random genome mutagenesis library by using this biosensor as a high-throughput screening tool. A high intracellular vanillin concentration was found to accumulate due to the inactivation of the AcrA protein, indicating the involvement of this protein in vanillin efflux. Then, the application of this biosensor was extended to explore efflux and influx pumps, combined with directed genome evolution. Elevated intracellular vanillin levels resulting from efflux pump inactivation or influx pump overexpression could be rapidly detected by the whole-cell biosensor, markedly facilitating the identification of genome targets related to small-molecule transmembrane transport, which is of great importance in metabolic engineering.

Water contamination by pathogenic bacteria is a major public health concern globally. Monitoring bacterial contamination in water is critically important to protect human health, but this remains a critical challenge. Engineered whole-cell biosensors created through synthetic biology hold great promise for rapid and cost-effective detection of waterborne pathogens. In this study, we created a novel whole-cell biosensor to detect water contamination by Pseudomonas aeruginosa and Burkholderia pseudomallei , which are two critical bacterial pathogens and are recognized as common causative agents for waterborne diseases. The biosensor detects the target bacterial pathogens by responding to the relevant quorum sensing signal molecules. Particularly, this study constructed and characterized the biosensor on the basis of the QscR quorum sensing signal system for the first time. We first designed and constructed a QscR on the basis of the sensing module in the E. coli host cell and integrated the QscR sensing module with a reporting module that expressed an enhanced green fluorescent protein (EGFP). The results demonstrated that the biosensor had high sensitivity in response to the quorum sensing signals of the target bacterial pathogens. We further engineered a biosensor that expressed a red pigment lycopene in the reporting module to produce a visible signal readout for the pathogen detection. Additionally, we investigated the feasibility of a paper-based assay by immobilizing the lycopene-based whole-cell biosensor on paper with the aim to build a prototype for developing portable detection devices. The biosensor would provide a simple and inexpensive alternative for timely and point-of-care detection of water contamination and protect human health.

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., 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 © 2021 Guo, Hui, Zhang, Liu, Li and Zheng.)

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.

Portable and inexpensive analytical tools are required to monitor pharmaceutical quality in technology limited settings including low- and middle-income countries (LMICs). Whole cell yeast biosensors have the potential to help meet this need. However, most of the readouts for yeast biosensors require expensive equipment or reagents. To overcome this challenge, we have designed a yeast biosensor that produces a unique scent as a readout. This inducible scent biosensor, or "scentsor", does not require the user to administer additional reagents for reporter development and utilizes only the user's nose to be "read". In this Letter, we describe a scentsor that is responsive to the hormone estradiol (E2). The best estimate threshold (BET) for E2 detection with a panel of human volunteers ( n = 49) is 39 nM E2 (15 nM when "non-smellers" are excluded). This concentration of E2 is sensitive enough to detect levels of E2 that would be found in dosage forms. This paper provides evidence that scent has the potential for use in portable yeast biosensors as a readout, particularly for use in LMICs.

The neurotransmitter phenylethylamine (PEA) is highly susceptible to oxidation to produce phenylacetic acid (PA). The fact that PEA and PA are both metabolites of phenylalanine (Phe) in humans makes them important indicators in the diagnosis of phenylketonuria. In this work, three-color whole-cell biosensors were developed to simultaneously detect these analytes (Phe, PEA, and PA). The tyrosine-responsive promoter was used to control the production of green fluorescent protein signals in response to Phe levels. The FeaR regulon was first used to indicate the presence of PEA, whereas the Paa regulon was used for the detection of PA. The combination of three sensor strains together made it possible to semiquantify the three analytes according to unique color outputs without cross-interference. We sought to optimize various modular components (ribosomal binding sites and fluorescent proteins) to ensure the rapid generation of fluorescent signals. Finally, the biosensors were implemented within a microchannel device to reduce sample consumption in point-of-care assays.

Enzyme engineering for gain of function requires navigating a large combinatorial sequence space efficiently. Typically, many mutations are needed to get significant improvements, while a single "bad" mutation can inactivate the enzyme. To establish high-throughput screening and achieve enhanced resolution between two variants, genetic libraries of the organophosphate hydrolase enzyme paraoxonase 1 (PON1) were rapidly screened via an engineered positive-feedback circuit: a p -nitrophenol (PNP)-specific transcription factor (TF) regulated expression of PON1, which catalyzed paraoxon breakdown and PNP production. Rare active mutant colonies, picked by simple visual fluorescence of a PON1-green fluorescent protein (GFP) fusion, were characterized. In a single screening round, high (library-scale) throughput enabled the discovery of enhanced paraoxon degradation activity in PON1, including structurally unexpected mutations.

Whole-cell biosensors have attracted considerable interests because they are robust, eco-friendly, and cost-effective. However, most of the biosensors harness the naturally occurring wild-type promoter, which often suffers from high background noise and low sensitivity. In this study, we demonstrate how to design the core elements (i.e., RNA polymerase binding site and transcription factor binding site) of the promoters to obtain a significant gain in the signal-to-noise output ratio of the whole-cell biosensor circuits. As a proof of concept, we modified the arsenite-regulated promoter from Escherichia coli K-12 genome, such that it has a lower background and higher expression. This was achieved by balancing the relationship between the number of ArsR binding sites (ABS) and the activity of the promoter and adjusting the location of the auxiliary ABS. A promoter variant ParsD-ABS-8 was obtained with an induction ratio of 179 (11-fold increase over the wild-type promoter) when induced with 1 μM arsenite. Importantly, the developed biosensor exhibited good dose-response in the range of 0.1 to 4 μM ( R 2 = 0.9928) of arsenite with a detection limit of ca. 10 nM. These results indicated that the engineered promoter modification approach could be used to improve the performance of whole-cell biosensors, thereby facilitating their practical application.

We present an integral smartphone-based whole-cell biosensor, LumiCellSense (LCS), which incorporates a 16-well biochip with an oxygen permeable coating, harboring bioluminescent Escherichia coli bioreporter cells, a macro lens, a lens barrel, a metal heater tray, and a temperature controller, enclosed in a light-impermeable case. The luminescence emitted by the bioreporter cells in response to the presence of the target chemicals is imaged by the phone's camera, and a dedicated phone-embedded application, LCS_Logger, is employed to calculate photon emission intensity and plot it in real time on the device's screen. An alert is automatically given when light intensity increases above the baseline, indicating the presence of the target. We demonstrate the efficacy of this system by the detection of residues of an antibiotic, ciprofloxacin (CIP), in whole milk, with a detection threshold of 7.2 ng/mL. This value is below the allowed maximum as defined by European Union regulations. [ABSTRACT FROM AUTHOR]

Whole-cell biosensors can form the basis of affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing, but to date the detection of analytes such as proteins that cannot easily diffuse across the cell membrane has been challenging. Here we developed a novel biosensing platform based on cell agglutination using an E. coli whole-cell biosensor surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design to detect a model analyte at nanomolar concentrations. Moreover, we show that the design architecture is flexible by building assays optimized to detect a range of model analyte concentrations using straightforward design rules and a mathematical model. Finally, we re-engineer our whole-cell biosensor for the detection of a medically relevant biomarker by the display of two different nanobodies against human fibrinogen and demonstrate a detection limit as low as 10 pM in diluted human plasma. Overall, we demonstrate that our agglutination technology fulfills the requirement of POC testing by combining low-cost nanobody production, customizable detection range and low detection limits. This technology has the potential to produce affordable diagnostics for field-testing in the developing world, emergency or disaster relief sites, as well as routine medical testing and personalized medicine.

This review reflects back over almost 40 years of the author's basic research conducted at Graduate School of Pharmaceutical Sciences, Osaka University, Japan. After performing postdoctoral research in USA, the author became a research associate at Prof. Yoshiharu Miura's lab and started research on Biochemical Engineering in 1984. At that time, the main research purpose was to solve global environmental issues for maintaining human health. The author's achievements included novel useful material production system under inorganic conditions and genetically engineered whole-cell bacterial sensors detecting arsenite by naked eye without a detecting device. Another theme in the lab was to construct bioartificial liver support system. Various scaffolds for hepatocytes were newly prepared for constructing the compact reactor. Besides the bioreactor study, the author conducted cell transplantation research for the treatment of chronic liver diseases. It was shown that mesenchymal stem cells derived from third molars (wisdom teeth) could differentiate into hepatocytes and exhibit therapeutic effects in liver-damaged animals. After 2006, the lab started research on drug delivery systems, including noninvasive delivery of drugs such as peptides and nucleic acids by regulating epithelial tight junctions. Many substances enabling drug delivery through "paracellular" route were newly prepared. The author started basic research on Biochemical Engineering in the 1970s. Although these studies eventually shifted into the pharmaceutical field, the underlying concept was based on "engineering" throughout a 40-year research period. The author cordially thanks all colleagues for supporting engineering research in our lab.

To improve the performance of a whole-cell biosensor for lead detection, we designed six gene circuits by re-configuring the regulatory elements and incorporating positive feedback loops to the circuits. The lead resistance operon pbr encodes six genes with pbrRT on one side of the promoter and pbrABCD on the other side. PbrR, the divergent promoter it regulates, and GFP were used to design the lead biosensors. One has pbrR and gfp on opposite sides of the promoter mimicking the native operon. We re-configured it by placing pbrR and gfp on the same side or under two separate promoters. The one with pbrR and gfp on the same side demonstrated lead sensitivity 10 times higher than the others. Positive feedback loop was introduced to these circuits. The strength of the output signal from the designs with positive feedback loop was 1.5–2 times stronger than those without positive feedback. This study demonstrates the importance of configuration and positive feedback as effective strategies to improve the performance of lead biosensors and they can be extended to the design of other whole-cell biosensors. [ABSTRACT FROM AUTHOR]

Access to affordable and field deployable diagnostics are key barriers to the control and eradication of many endemic and emerging infectious diseases. While cost, accuracy, and usability have all improved in recent years, there remains a pressing need for even less expensive and more scalable technologies. To that end, we explored new methods to inexpensively produce and couple protein-based biosensing molecules (affinity reagents) with scalable electrochemical sensors. Previous whole-cell constructs resulted in confounding measurements in clinical testing due to significant cross-reactivity when probing for host-immune (antibody) response to infection. To address this, we developed two complimentary strategies based on either the release of surface displayed or secretion of fusion proteins. These dual affinity biosensing elements couple antibody recognition (using antigen) and sensor surface adhesion (using gold-binding peptide-GBP) to allow single-step reagent production, purification, and biosensor assembly. As a proof-of-concept, we developed Hepatitis C virus (HCV)-core antigen-GBP fusion proteins. These constructs were first tested and optimized for consistent surface adhesion then the assembled immunosensors were tested for cross-reactivity and evaluated for performance in vitro. We observed loss of function of the released reagents while secreted constructs performed well in in vitro testing with 2 orders of dynamic range, and a limit of detection of 32 nM. Finally, we validated the secreted platform with clinical isolates (n = 3) with statistically significant differentiation of positive vs. non-infected serum (p < 0.0001) demonstrating the ability to clearly distinguish HCV positive and negative clinical samples., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A novel toxicity-warning sensor for water quality monitoring in recirculating aquaculture systems (RAS) is presented. The design of the sensor system mainly comprises a whole-cell biosensor. Aliivibrio fischeri , a luminescent bacterium widely used in toxicity analysis, was tested for a mixture of known fish-health stressors, namely nitrite, un-ionized ammonia, copper, aluminum and zinc. Two toxicity predictive models were constructed. Correlation, root mean squared error, relative error and toxic behavior were analyzed. The linear concentration addition (LCA) model was found suitable to ally with a machine learning algorithm for prediction of toxic events, thanks to additive behavior near the limit concentrations for these stressors, with a root-mean-squared error (RMSE) of 0.0623, and a mean absolute error of 4%. The model was proved to have a smaller relative deviation than other methods described in the literature. Moreover, the design of a novel microfluidic chip for toxicity testing is also proposed, which is to be integrated in a fluidic system that functions as a bypass of the RAS tank to enable near-real time monitoring. This chip was tested with simulated samples of RAS water spiked with zinc, with an EC50 of 6,46E-7 M. Future work will be extended to the analysis of other stressors with the novel chip.

A novel whole-cell biosensor was developed to noninvasively and simultaneously monitor the in situ genetic activities of the four quorum sensing (QS) networks in Pseudomonas aeruginosa PAO1, including the las, rhl, pqs, and iqs systems. P. aeruginosa PAO1 is a model bacterium for studies of biofilm and pathogenesis while both processes are closely controlled by the QS systems. This biosensor worked well by selectively monitoring the expression of one representative gene from each network. In the biosensor, the promoter regions of lasI, rhlI, pqsA, and ambB (QS genes) controlled the fluorescent reporter genes of Turbo YFP, mTag BFP2, mNEON Green, and E2-Orange, respectively. The biosensor was successful in monitoring the impact of an important environmental factor, salt stress, on the genetic regulation of QS networks. High salt concentrations (≥ 20 g·L -1 ) significantly downregulated rhlI, pqsA, and ambB after the biosensor was incubated for 17 h to 18 h at 37 °C, resulting in slow bacterial growth.

This paper presents a whole-cell biosensor that operates in conjunction with a smartphone-based fluorescence diagnostic system on a paper device to monitor the concentration of gold ions in human urine. The heavy metal-tolerant bacteria Cupriavidus metallidurans was genetically engineered for use as a chassis in a red fluorescent protein (RFP)-based microbial sensor. The biosensor is highly sensitive to gold ions, with a detection limit of 110 nM. The proposed smartphone-based analysis system provides a user-friendly approach to design tools of personal health monitoring for reporting the presence of gold ions in human urine.

The quantitative monitoring of intracellular metabolites with in vivo biosensors provides an efficient means of identifying high-yield strains and observing product accumulation in real time. In this study, a shikimic acid (SA) biosensor was constructed from a LysR-type transcriptional regulator (ShiR) of Corynebacterium glutamicum. The SA biosensor specifically responded to the increase of intracellular SA concentration over a linear range of 19.5 ± 3.6 to 120.9 ± 1.2 fmole at the single-cell level. This new SA biosensor was successfully used to (1) monitor the SA production of different C. glutamicum strains; (2) develop a novel result-oriented high-throughput ribosome binding site screening and sorting strategy that was used for engineering high-yield shikimate-producing strains; and (3) engineer a whole-cell biosensor through the coexpression of the SA sensor and a shikimate transporter shiA gene in C. glutamicum RES167. This work demonstrated that a new intracellular SA biosensor is a valuable tool facilitating the fast development of microbial SA producer.

Improved easy-to-use diagnostic tools for infections are in strong demand worldwide. Yet, despite dramatic advances in diagnostic technologies, the gold-standard remains culturing. Here we offer an alternative tool demonstrating that a bacterial biosensor can efficiently detect Pseudomonas aeruginosa infections in patients suffering from otitis externa. Detection was based on specific binding between the biosensor and 2-aminoacetophenone (2-AA), a volatile produced by P. aeruginosa in high amounts. We collected pus samples from ears of 26 subjects exhibiting symptoms of otitis externa. Detection of P. aeruginosa using the biosensor was compared to detection using gold-standard culturing assay and to gas-chromatograph-mass-spectrometry (GC-MS) analyses of 2-AA. The biosensor strain test matched the culture assay in 24 samples (92%) and the GC-MS analyses in 25 samples (96%). With this result in hand, we designed a device containing a whole-cell luminescent biosensor combined with a photo-multiplier tube. This device allowed detection of 2-AA at levels as low as 2 nmol, on par with detection level of GC-MS. The results of the described study demonstrate that the volatile 2-AA serves as an effective biomarker for P. aeruginosa in ear infections, and that activation of the biosensor strain by 2-AA provides a unique opportunity to design an easy-to-use device that can specifically detect P. aeruginosa infections., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Deficiencies in vitamins and minerals (micronutrients) are a critical global health concern, in part due to logistical difficulties in assessing population micronutrient status. Whole-cell biosensors offer a unique opportunity to address this issue, with the potential to move sample analysis from centralized, resource-intensive clinics to minimal-resource, on-site measurement. Here, we present a proof-of-concept whole-cell biosensor in Escherichia coli for detecting zinc, a micronutrient for which deficiencies are a significant public health burden. Importantly, the whole-cell biosensor produces readouts (pigments) that are visible to the naked eye, mitigating the need for measurement equipment and thus increasing feasibility for sensor field-friendliness and affordability at a global scale. Two zinc-responsive promoter/transcription factor systems are used to differentially control production of three distinctly colored pigments in response to zinc levels in culture. We demonstrate strategies for tuning each zinc-responsive system to turn production of the different pigments on and off at different zinc levels, and we demonstrate production of three distinct color regimes over a concentration range relevant to human health. We also demonstrate the ability of the sensor cells to grow and produce pigment when cultured in human serum, the ultimate target matrix for assessing zinc nutritional status. Specifically, we present approaches to overcome innate immune responses that would otherwise hinder bacterial sensor survival, and we demonstrate production of multiple pigment regimes in human serum with different zinc levels. This work provides proof of principle for the development of low-cost, minimal-equipment, field-deployable biosensors for nutritional epidemiology applications.

Knowledge of intracellular metabolite levels is important for the understanding of metabolic flux distributions. Whole-cell biosensors of key metabolites are ideal for the monitoring of carbon flow in important metabolic pathways, thus guiding metabolic engineering for microbial improvement. However, lack of biosensors for metabolites of interests has limited their applications. In this study, a genetically encoded whole-cell biosensor specifically responding to shikimic acid has been developed by screening a site-saturation mutagenesis library of the binding pocket of a uric acid-responsive regulatory protein. This biosensor has been successfully applied in analyzing and engineering metabolic flux in the shikimic acid pathway, through genome-wide screening of gene targets critical for the pathway flux, and by improving the specific activity of pathway key enzyme, AroG. This work demonstrates the feasibility of monitoring metabolic flux with the aid of whole-cell biosensors designed for key metabolites., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Whole cell biosensors have been seldom used in the pharmaceutical and cosmetics industries for preservative efficacy testing (PET). According to several pharmacopoeias, preservatives should be tested for microbial activity using traditional viable count techniques; the use of whole cell microbial biosensors potentially provides an alternative, fast, and efficient method. The aim of the study was to assess the applicability of Pseudomonas aeruginosa ATCC9027 and its validated bioluminescent strains for preservative efficacy tests using benzalkonium chloride (BKC). Applicability of five constitutively-expressed bioluminescent strains was evaluated for preservative efficacy tests (PET) using bacterial replication, bioluminescence and fluorescence in a three-way study. PET using BKC showed no significant difference between bioluminescence and enumeration. Good correlations between bioluminescence, colony-forming units (CFU) count and fluorescence were obtained for BKC concentrations (R>0.9) between 0.0003-0.0025% against strains containing the constructs lys-pMElux, lpp-pMElux and tat-pMElux. Furthermore, two-way ANOVA analysis showed that the bioluminescent method and traditional plate counting method were equivalent for concentrations of BKC (0.0003-0.01%) during preservative efficacy tests. PET testing with BKC showed that tat-pMElux (R>0.9) had consistently high correlation coefficients between CFU and relative bioluminescence; P. aeruginosa ATCC9027 tatH5-pMElux is the best construct for testing various antimicrobial agents., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Malonyl-coenzyme A (CoA) is an important biosynthetic precursor in vivo. Although Escherichia coli is a useful organism for biosynthetic applications, its malonyl-CoA level is too low., Results: To identify strains with the best potential for enhanced malonyl-CoA production, we developed a whole-cell biosensor for rapidly reporting intracellular malonyl-CoA concentrations. The biosensor was successfully applied as a high-throughput screening tool for identifying targets at a genome-wide scale that could be critical for improving the malonyl-CoA biosynthesis in vivo. The mutant strains selected synthesized significantly higher titers of the type III polyketide triacetic acid lactone (TAL), phloroglucinol, and free fatty acids compared to the wild-type strain, using malonyl-CoA as a precursor., Conclusion: These results validated this novel whole-cell biosensor as a rapid and sensitive malonyl-CoA high-throughput screening tool. Further analysis of the mutant strains showed that the iron ion concentration is closely related to the intracellular malonyl-CoA biosynthesis.

The cell envelope is an essential bacterial structure that consists of the cytoplasmic membrane, the cell wall, and-in Gram-negative bacteria-the outer membrane. Because of its crucial functions, it represents a prime antibiotic target. Monitoring and maintaining its integrity are therefore keys to survival, especially in competitive environments where antibiotics represent one means of suppressing the growth of competitors. Resistance against external antibiotic threat, as well as auto-immunity against self-produced antibiotics, is often mediated by two-component systems (2CSs). They respond to antibiotic threat by inducing gene expression that results in the production of specific resistance determinants. The underlying transcriptional control is exhibited at the level of specific target promoters, which usually share a number of relevant features: They are tightly controlled and only induced in the presence of specific (sets of) antibiotics. This induction is dose dependent and often very sensitive, that is, it occurs well below inhibitory antibiotic concentrations. Because of these characteristics, a number of well-characterized cell envelope stress-inducible promoters have been developed for two different applied purposes: first, as whole-cell biosensors for antibiotic detection and mechanism-of-action studies, and second, as antibiotic-inducible expression systems for biotechnological purposes. The current state of research in both fields will be discussed in this review, focusing on 2CS-regulated promoters from Firmicutes bacteria that are induced to mediate resistance against antimicrobial peptides (AMPs) targeting the cell envelope.

Detection and quantification of small peptides, such as yeast pheromones, are often challenging. We developed a highly sensitive and robust affinity-assay for the quantification of the α-factor pheromone of Saccharomyces cerevisiae based on recombinant hydrophobins. These small, amphipathic proteins self-assemble into highly stable monolayers at hydrophilic-hydrophobic interfaces. Upon functionalization of solid supports with a combination of hydrophobins either lacking or exposing the α-factor, pheromone-specific antibodies were bound to the surface. Increasing concentrations of the pheromone competitively detached the antibodies, thus allowing for quantification of the pheromone. By adjusting the percentage of pheromone-exposing hydrophobins, the sensitivity of the assay could be precisely predefined. The assay proved to be highly robust against changes in sample matrix composition. Due to the high stability of hydrophobin layers, the functionalized surfaces could be repeatedly used without affecting the sensitivity. Furthermore, by using an inverse setup, the sensitivity was increased by three orders of magnitude, yielding a novel kind of biosensor for the yeast pheromone with the lowest limit of detection reported so far. This assay was applied to study the pheromone secretion of diverse yeast strains including a whole-cell biosensor strain of Schizosaccharomyces pombe modulating α-factor secretion in response to an environmental signal.

: Bioluminescence is light production by living organisms, which can be observed in numerous marine creatures and some terrestrial invertebrates. More specifically, bacterial bioluminescence is the "cold light" produced and emitted by bacterial cells, including both wild-type luminescent and genetically engineered bacteria. Because of the lively interplay of synthetic biology, microbiology, toxicology, and biophysics, different configurations of whole-cell biosensors based on bacterial bioluminescence have been designed and are widely used in different fields, such as ecotoxicology, food toxicity, and environmental pollution. This chapter first discusses the background of the bioluminescence phenomenon in terms of optical spectrum. Platforms for bacterial bioluminescence detection using various techniques are then introduced, such as a photomultiplier tube, charge-coupled device (CCD) camera, micro-electro-mechanical systems (MEMS), and complementary metal-oxide-semiconductor (CMOS) based integrated circuit. Furthermore, some typical biochemical methods to optimize the analytical performances of bacterial bioluminescent biosensors/assays are reviewed, followed by a presentation of author's recent work concerning the improved sensitivity of a bioluminescent assay for pesticides. Finally, bacterial bioluminescence as implemented in eukaryotic cells, bioluminescent imaging, and cancer cell therapies is discussed.

The increasing molecular understanding of many diseases today permits the development of new diagnostic methods. However, few easy-to-handle and inexpensive tools exist for common diseases such as food disorders. Here we present a living material based analytical sensor (LiMBAS) containing genetically modified bacteria (Escherichia coli) immobilized and protected in a thin layer between a nanoporous and support polymer membrane for a facile quantification of disease-relevant oligosaccharides. The bacteria were engineered to fluoresce in response to the analyte to reveal its diffusion behavior when using a blue-light source and optical filter. We demonstrated that the diffusion zone diameter was related semi-logarithmically to the analyte concentration. LiMBAS could accurately quantify lactose or galactose in undiluted food samples and was able to measure food intolerance relevant concentrations in the range of 1-1000 mM requiring a sample volume of 1-10 μL. LiMBAS was storable for at least seven days without losing functionality at 4 °C. A wide range of genetic tools for E. coli are readily available thus allowing the reprogramming of the material to serve as biosensor for other molecules. In combination with smartphones, an automated diagnostic analysis becomes feasible which would also allow untrained people to use LiMBAS., (Copyright © 2015 Elsevier Ltd. All rights reserved.)