Development Of Whole-cell Biosensors Research Articles

Involvement of organic acids and amino acids in ameliorating Ni(II) toxicity induced cell cycle dysregulation in Caulobacter crescentus: a metabolomics analysis

Nickel (Ni(II)) toxicity is addressed by many different bacteria, but bacterial responses to nickel stress are still unclear. Therefore, we studied the effect of Ni(II) toxicity on cell proliferation of α-proteobacterium Caulobacter crescentus. Next, we showed the mechanism that allows C. crescentus to survive in Ni(II) stress condition. Our results revealed that the growth of C. crescentus is severely affected when the bacterium was exposed to different Ni(II) concentrations, 0.003mM slightly affected the growth, 0.008mM reduced the growth by 50%, and growth was completely inhibited at 0.015mM. It was further shown that Ni(II) toxicity induced mislocalization of major regulatory proteins such as MipZ, FtsZ, ParB, and MreB, resulting in dysregulation of the cell cycle. GC-MS metabolomics analysis of Ni(II) stressed C. crescentus showed an increased level of nine important metabolites including TCA cycle intermediates and amino acids. This indicates that changes in central carbon metabolism and nitrogen metabolism are linked with the disruption of cell division process. Addition of malic acid, citric acid, alanine, proline, and glutamine to 0.015mM Ni(II)-treated C. crescentus restored its growth. Thus, the present work shows a protective effect of these organic acids and amino acids on Ni(II) toxicity. Metabolic stimulation through the PutA/GlnA pathway, accelerated degradation of CtrA, and Ni-chelation by organic acids or amino acids are some of the possible mechanisms suggested to be involved in enhancing C. crescentus's tolerance. Our results shed light on the mechanism of increased Ni(II) tolerance in C. crescentus which may be useful in bioremediation strategies and synthetic biology applications such as the development of whole cell biosensor.

Synthetic biology for microbial heavy metal biosensors.

Using recombinant DNA technology, various whole-cell biosensors have been developed for detection of environmental pollutants, including heavy metal ions. Whole-cell biosensors have several advantages: easy and inexpensive cultivation, multiple assays, and no requirement of any special techniques for analysis. In the era of synthetic biology, cutting-edge DNA sequencing and gene synthesis technologies have accelerated the development of cell-based biosensors. Here, we summarize current technological advances in whole-cell heavy metal biosensors, including the synthetic biological components (bioparts), sensing and reporter modules, genetic circuits, and chassis cells. We discuss several opportunities for improvement of synthetic cell-based biosensors. First, new functional modules must be discovered in genome databases, and this knowledge must be used to upgrade specific bioparts through molecular engineering. Second, modules must be assembled into functional biosystems in chassis cells. Third, heterogeneity of individual cells in the microbial population must be eliminated. In the perspectives, the development of whole-cell biosensors is also discussed in the aspects of cultivation methods and synthetic cells.

Development of whole cell biosensors mediated by bacteria chemoreceptors (614.5)

Whole cell biosensors are part of an emerging strategy to build new tools for diagnostic and therapeutic applications. The goal is to re‐engineer bacterial cells to respond to environmental cues or chemicals. Whole cell biosensors typically express a sensor protein targeting the ligand of interest that is coupled with a genetic regulatory system to produce a measureable signal by the host cell. We have adapted an AraC‐based transcriptional reporter assay to create a new whole cell biosensor that may be used for the creation of new sensor proteins. In our bio‐reporter system, Trg is translationally‐fused to AraC such that dimerization of the Trg‐AraC results in expression of the reporter gene. The presence of analyte will be detected in the periplasm of the cell by inclusion of an engineered periplasmic binding protein (PBP) specific for this molecule. The bound protein then interacts with Trg causing a conformational change. This dimerization of the Trg‐AraC fusion in the cytoplasm will then switch on gene expression of the reporter gene, green fluorescent protein (GFP). This mechanism reports dose‐response fluorescence detection in the presence of the analyte. Ultimately this bio‐reporter system will allow us to design new PBPs with selectivity for specific chemical entities and for construction of a genetic switch inside bacterial cells that precisely reports the presence of the analyte using a fluorescence reporter system.

A Direct Technique for Magnetic Functionalization of Living Human Cells

Functionalized living cells are regarded as effective tools in directed cell delivery and tissue engineering. Here we report the facile functionalization of viable isolated HeLa cells with superparamagnetic cationic nanoparticles via a single-step biocompatible process. Nanoparticles are localized on the cellular membranes and do not penetrate into the cytoplasm. The magnetically responsive cells are viable and able to colonize and grow on substrates. Magnetically facilitated microorganization of functionalized cells into viable living clusters is demonstrated. We believe that the technique described here may find a number of potential applications in cell-based therapies and in development of whole-cell biosensors.

Nonlinear dielectric spectroscopy as an indirect probe of metabolic activity in thylakoid membrane.

Nonlinear dielectric spectroscopy (NDS) is a non-invasive probe of cellular metabolic activity with potential application in the development of whole-cell biosensors. However, the mechanism of NDS interaction with metabolic membrane proteins is poorly understood, partly due to the inherent complexity of single cell organisms. Here we use the light-activated electron transport chain of spinach thylakoid membrane as a model system to study how NDS interacts with metabolic activity. We find protein modification, as opposed to membrane pump activity, to be the dominant source of NDS signal change in this system. Potential mechanisms for such protein modifications include reactive oxygen species generation and light-activated phosphorylation.

Development and applications of whole cell biosensors for ecotoxicity testing

Whole cell biosensors are the focus of considerable and increasing interest worldwide as methods for detecting and quantifying environmental toxicity, including biochemical oxygen demand (BOD), heavy metals, antibiotics, pesticides and herbicides. This review follows the development of whole cell biosensors from attempts to utilise changes in cellular metabolism to determine BOD and general toxicity, through the exploitation of unique metabolic pathways to detect specific toxicants, to the increasingly widespread use of genetic engineering to build new, and modify existing, sensing pathways.

Bioluminescent-bioreporter integrated circuits form novel whole-cell biosensors

The bioluminescent-bioreporter integrated circuit represents a new advance in the development of whole-cell biosensors, consisting of a genetically engineered bioreporter organism interfaced with an integrated circuit. The bioreporter is engineered to luminesce when a targeted substance is encountered, with the circuit being designed to detect this luminescence, process the signal and communicate the results. The bioreporters are thus available to the circuit designer as another component, analogous to a transistor or a resistor, albeit a living entity. The concept has been demonstrated using Pseudomonas putida TVA8 to sense the environmental pollutant toluene.

The development of whole cell biosensors for on‐line screening of herbicide pollution of surface waters

AbstractThe use of mediator assisted amperometric whole‐cell biosensors, monitoring microbial photosynthetic electron transfer, to screen for herbicides in intake protection by the water industry is described. Cells harvested from axenic cultures are loaded onto bacteriological filters and held against a working carbon electrode polarised against the silver chloride reference. Redox mediators are reduced by the microorganisms and then reoxidised at the working electrode resulting in a flow of current. For on‐line applications the sensors are controlled by a programmable electrochemical analyser (Artek) and housed in specially designed perspex flow cells which incorporate light emitting diodes (LEDs) for photosynthetic stimulation. A range of cyanobacteria and algae have been tested, but biosensors incorporating unicellular cyanobacteria have proven the most successful. Results obtained using the cyanobacterium Synechococcus show detection levels of less than 200 ppb with response times of less than ten minutes for selected herbicides. Sensor stability in the absence of pollutant poisoning has been found to be greatly influenced by the choice of mediator. Membrane penetrating mediators such as ‐benzoquinone reduce sensor life to about 24 hours, while a sensor life of up to six days has been achieved with ferricyanide.