A modular cell-based biosensor using engineered genetic logic circuits to detect and integrate multiple environmental signals

AbstractCells perceive a wide variety of cellular and environmental signals, which are often processed combinatorially to generate particular phenotypic responses. Here, we employ both single and mixed cell type populations, pre-programmed with engineered modular cell signalling and sensing circuits, as processing units to detect and integrate multiple environmental signals. Based on an engineered modular genetic AND logic gate, we report the construction of a set of scalable synthetic microbe-based biosensors comprising exchangeable sensory, signal processing and actuation modules. These cellular biosensors were engineered using distinct signalling sensory modules to precisely identify various chemical signals, and combinations thereof, with a quantitative fluorescent output. The genetic logic gate used can function as a biological filter and an amplifier to enhance the sensing selectivity and sensitivity of cell-based biosensors. In particular, an Escherichia coli consortium-based biosensor has been constructed that can detect and integrate three environmental signals (arsenic, mercury and copper ion levels) via either its native two-component signal transduction pathways or synthetic signalling sensors derived from other bacteria in combination with a cell-cell communication module. We demonstrate how a modular cell-based biosensor can be engineered predictably using exchangeable synthetic gene circuit modules to sense and integrate multiple-input signals. This study illustrates some of the key practical design principles required for the future application of these biosensors in broad environmental and healthcare areas

Synthetic biology tools for environmental protection

Synthetic biology transforms the way we perceive biological systems. Emerging technologies in this field affect many disciplines of science and engineering. Traditionally, synthetic biology approaches were commonly aimed at developing cost-effective microbial cell factories to produce chemicals from renewable sources. Based on this, the immediate beneficial impact of synthetic biology on the environment came from reducing our oil dependency. However, synthetic biology is starting to play a more direct role in environmental protection. Toxic chemicals released by industries and agriculture endanger the environment, disrupting ecosystem balance and biodiversity loss. This review highlights synthetic biology approaches that can help environmental protection by providing remediation systems capable of sensing and responding to specific pollutants. Remediation strategies based on genetically engineered microbes and plants are discussed. Further, an overview of computational approaches that facilitate the design and application of synthetic biology tools in environmental protection is presented

Biosensors for Environmental Monitoring

Real-time and reliable detection of molecular compounds and bacteria is essential in modern environmental monitoring. For rapid analyses, biosensing devices combining high selectivity of biomolecular recognition and sensitivity of modern signal-detection technologies offer a promising platform. Biosensors allow rapid on-site detection of pollutants and provide potential for better understanding of the environmental processes, including the fate and transport of contaminants.This book, including 12 chapters from 37 authors, introduces different biosensor-based technologies applied for environmental analyses

Optimization of Protein-Protein Interaction Measurements for Drug Discovery Using AFM Force Spectroscopy

Increasingly targeted in drug discovery, protein-protein interactions challenge current high throughput screening technologies in the pharmaceutical industry. Developing an effective and efficient method for screening small molecules or compounds is critical to accelerate the discovery of ligands for enzymes, receptors and other pharmaceutical targets. Here, we report developments of methods to increase the signal-to-noise ratio (SNR) for screening protein-protein interactions using atomic force microscopy (AFM) force spectroscopy. We have demonstrated the effectiveness of these developments on detecting the binding process between focal adhesion kinases (FAK) with protein kinase B (Akt1), which is a target for potential cancer drugs. These developments include optimized probe and substrate functionalization processes and redesigned probe-substrate contact regimes. Furthermore, a statistical-based data processing method was developed to enhance the contrast of the experimental data. Collectively, these results demonstrate the potential of the AFM force spectroscopy in automating drug screening with high throughput

Amperometric cytochrome c3-based biosensor for chromate determination

International audienceThe chromate reductase activity of cytochrome c3 (Cyt c3, Mr 13 000), isolated from the sulfate-reducing bacterium Desulfomicrobium norvegicum, was used to develop an amperometric biosensor to measure chromate (CrO42−) bioavailability. The performance of various biosensor configurations for qualitative and quantitative determination of Cr(VI) was studied. Biosensor properties depend on the technique used to immobilize the enzyme on the electrode (glassy carbon electrode). Immobilization of Cyt c3 by entrapment in poly 3,4-ethylenedioxythiophene films denatured the enzyme, while application of an adsorption technique did not affect enzyme activity but the detection range was limited. The best results were obtained with dialysis membranes, which allowed the determination of Cr(VI) from 0.20 to 6.84 mg l−1 (3.85–132 μM) with a sensitivity of 35 nA mg−1 l (1.82 nA μM−1). No interference was observed with As(V), As(III) and Fe(III). Only a small amount of Cyt c3 (372 ng of protein) was needed for this biosensor

Metalation and Structural Properties of apo-Metallothioneins

Metals are required by a quarter of all proteins to achieve their biological function, whether in an active site involved in catalytic chemistry or in a structural capacity. Metals are tightly regulated at the cellular level due to their propensity to cause unwanted side reactions and to be scavenged for use by pathogens. One of the proteins involved in this regulation of metal homeostasis is metallothionein (MT) which is a small, cysteine rich protein primarily involved in the regulation of zinc and copper homeostasis and heavy metal detoxification. MT is unique in its high cysteine content (~30% of the residues), its high capacity for metal binding and its fluxional structure in the absence of metal saturation. This fluxionality has made the structure of apo- and partially-metalated MTs difficult to study and as a result the binding pathway of MT for various metals remains unclear. This thesis describes the hard-to-characterize structure of apo- and partially-metalated MTs, their binding pathways and potential applications. Using primarily electrospray ionization mass spectrometry (ESI-MS) and covalent labeling, the structure of apo- and partially metalated MTs was probed. Modeling techniques that generate simulated ESI-MS data were used to recreate the covalent labeling spectra and aid in the interpretation of this complicated reaction. These experiments showed that apo-MT adopts a compact, globular conformation that is resistant to initial modification by alkylating reagents. Furthermore, this compact conformation is essential to the fast kinetics of cadmium binding and cluster formation. This cluster formation was found to be pH dependent and this insight was essential in the design of an MT-based biosensor for the detection of As(III) and Hg(II). Altogether, these results reconcile previous conflicting reports about the metal binding mechanisms of MTs, provide evidence of compact conformations of apo-MT and its role in binding kinetics and begin to demonstrate potential application of this fundamental knowledge in the design and testing of an electrochemical MT-based biosensor

Lincoln University Cooperative Extension and Research Annual Report 2012

This is LUCER-MC Report #04-12 Published by Lincoln University Cooperative Extension and Research (LUCER) Media Center; 900 Chestnut Street, 301 Allen Hall; Jefferson City, MO 65101.https://bluetigercommons.lincolnu.edu/lucer_reports/1001/thumbnail.jp