Development of Colorimetric Whole-Cell Biosensor for Detection of Heavy Metals in Environment for Public Health

Heavy metals cause various fetal diseases in humans. Heavy metals from factory wastewater can contaminate drinking water, fish, and crops. Inductively coupled plasma-mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS) are commonly used to analyze heavy metal contents; however, these methods require pre-treatment processes and are expensive and complex. To overcome these limitations, three metal-sensing materials using a whole-cell biosensor in Escherichia coli (E. coli) were developed. Strains were engineered to harbor three kinds of plasmids containing the copA, zntA, and mer promoters for sensing copper, cadmium, and mercury, respectively. The luciferase (lux) gene was inserted as a reporter into the plasmid, which was later replaced with a fused protein sequence containing OmpA (1–159) and mCherry for optical detection. The constructed strains could detect mercury, cadmium, and copper at 0.1–0.75 ppm, 0.2–0.75 ppm, and 2–7.5 ppm, respectively, with linearity values of 0.99030, 0.99676, and 0.95933, respectively. The immobilization linearity value was 0.99765. Notably, these three heavy metals could be detected by visual analysis of the strains. Overall, these findings establish this novel sensor as a potential approach for heavy metal detection in biological samples and foods.

Illuminate the hidden: in vivo mapping of microscale pH in the mycosphere using a novel whole-cell biosensor

The pH of an environment is both a driver and the result of diversity and functioning of microbial habitats such as the area affected by fungal hyphae (mycosphere). Here we used a novel pH-sensitive bioreporter, Synechocystis sp. PCC6803_peripHlu, and ratiometric fluorescence microscopy, to spatially and temporally resolve the mycosphere pH at the micrometre scale. Hyphae of the basidiomycete Coprionopsis cinerea were allowed to overgrow immobilised and homogeneously embedded pH bioreporters in an agarose microcosm. Signals of >700 individual cells in an area of 0.4 × 0.8 mm were observed over time and used to create highly resolved (3 × 3 µm) pH maps using geostatistical approaches. C. cinerea changed the pH of the agarose from 6.9 to ca. 5.0 after 48 h with hyphal tips modifying pH in their vicinity up to 1.8 mm. pH mapping revealed distinct microscale spatial variability and temporally stable gradients between pH 4.4 and 5.8 over distances of ≈20 µm. This is the first in vivo mapping of a mycosphere pH landscape at the microscale. It underpins the previously hypothesised establishment of pH gradients serving to create spatially distinct mycosphere reaction zones.

Detection of Bioavailable Cadmium by Double-Color Fluorescence Based on a Dual-Sensing Bioreporter System

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.

Computer-Aided Rational Engineering of Signal Sensitivity of Quorum Sensing Protein LuxR in a Whole-Cell Biosensor

LuxR, a bacterial quorum sensing-related transcription factor that responds to the signaling molecule 3-oxo-hexanoyl-homoserine lactone (3OC6-HSL). In this study, we employed molecular dynamics simulation and the Molecular Mechanics Generalized Born Surface Area (MM-GB/SA) method to rationally identify residues in Vibrio fischeri LuxR that are important for its interaction with 3OC6-HSL. Isoleucine-46 was selected for engineering as the key residue for interaction with 3OC6-HSL-LuxR-I46F would have the strongest binding energy to 3OC6-HSL and LuxR-I46R the weakest binding energy. Stable wild-type (WT) LuxR, I46F and I46R variants were produced in Escherichia coli (E. coli) in the absence of 3OC6-HSL by fusion with maltose-binding protein (MBP). Dissociation constants for 3OC6-HSL from MBP-fusions of WT-, I46F- and I46R-LuxR determined by surface plasmon resonance confirmed the binding affinity. We designed and constructed a novel whole-cell biosensor on the basis of LuxR-I46F in E. coli host cells with a reporting module that expressed green fluorescent protein. The biosensor had high sensitivity in response to the signaling molecule 3OC6-HSL produced by the target bacterial pathogen Yersinia pestis. Our work demonstrates a practical, generalizable framework for the rational design and adjustment of LuxR-family proteins for use in bioengineering and bioelectronics applications.

Influence of the luxR Regulatory Gene Dosage and Expression Level on the Sensitivity of the Whole-Cell Biosensor to Acyl-Homoserine Lactone

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).