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

Abstract

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.

Detection of environmental pollutant cadmium in water using a visual bacterial biosensor

“…To characterize the overall response of the biosensor toward a wide concentration range of Cd(II), E. coli TOP10 harboring pPcad-vio was exposed to 0–800 μM Cd(II) using a double dilution method. Consistent with previous studies 21 , the cytotoxicity of Cd(II) was high upon exposure to above 200 μM Cd(II). Bacterial density decreased dramatically when the cytotoxicity of high concentrations of Cd(II) was obvious (Fig.…”

“…The violacein biosynthesis pathway has been heterogeneously reconstructed using the vioABCDE gene cluster derived from Chromobacterium violaceum in E. coli 20 . The metalloregulator CadR originally characterized in Pseudomonas putida 23 , has been successfully employed as the sensory element to develop Cd(II)-responsive biosensors with fluorescent proteins as the output signals in our previous studies 5 , 21 , 24 . In the present study, a Cd(II) biosensing construct was assembled by employing the CadR and its divergent promoter as the Cd(II) sensory module and the vioABCDE gene cluster as the visual reporter module.…”

“…The basal expression has been shown to be common in most identified heavy metal resistance operons and this property was believed to facilitate bacterial survival in the unpredictable and complex environment 25 , 26 . High-background biosensing platforms for cadmium have been reported due to the leaky expression of fluorescent reporters in these artificial cadmium resistance operons 5 , 7 , 21 . Pigment reporter, which is produced by biosynthetic enzymes catalysis, can be expected to constantly accumulate along with prolonging induction 15 .…”

“…The plasmid pET-vio with a violacein biosynthetic gene cluster ( vioABCDE ) inserted into the Nde I/ Sac I sites of pET-21a was previously constructed 20 . The plasmid pCadR 21 was digested with Bgl II and Xba I, and the resultant fragment containing the Cd(II) sensory module, the cadR gene and its divergent cad promoter, was inserted into the same sites of pET-vio to generate pPcad-vio. The resultant biosensing construct pPcad-vio was used for transformation into E. coli TOP10 and spread on LB agar plates containing 50 μg/mL ampicillin.…”

“…To investigate the performance of the biosensor toward increased concentrations of Cd(II), the early exponential-phase cultures of TOP10/pPcad-vio were exposed to 0, 0.0061, 0.012, 0.024, 0.049, 0.098, 0.195, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400, and 800 μM Cd(II) at 37 °C using a double dilution method as described previously 21 . After a 1.5-h induction, aliquots of 100 μL culture were read at 600 nm to determine the bacterial density, and aliquots of 100 μL butanol extract were read at 578 nm to determine the violacein-derived signal.…”