Green Fluorescent Protein based Whole Cell Bacterial Biosensor for the detection of bioavailable heavy metals in soil environment

2021 ◽  
pp. 101785
Author(s):  
I.V.N. Rathnayake ◽  
Mallavarapu Megharaj ◽  
Ravi Naidu
Keyword(s):  
Heavy Metals ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Soil Environment ◽  
Bacterial Biosensor ◽  
Whole Cell ◽  
Green Fluorescent

Electrophysiological Analysis of Dorsal Root Ganglion Neurons Pre- and Post-Coexpression of Green Fluorescent Protein and Functional 5-HT3Receptor

1997 ◽  
Vol 77 (6) ◽  
pp. 3115-3121 ◽  
Author(s):  
George M. Smith ◽  
Richard L. Berry ◽  
Jay Yang ◽  
Darrell Tanelian
Keyword(s):  
Green Fluorescent Protein ◽  
Dorsal Root Ganglion ◽  
Dorsal Root ◽  
Fluorescent Protein ◽  
Dorsal Root Ganglion Neurons ◽  
Drg Neurons ◽  
Whole Cell ◽  
Electrophysiological Analysis ◽  
Green Fluorescent ◽  
Ganglion Neurons

Smith, George M., Richard L. Berry, Jay Yang, and Darrell Tanelian. Electrophysiological analysis of dorsal root ganglion neurons pre- and post-coexpression of green fluorescent protein and functional 5-HT3receptor. J. Neurophysiol. 77: 3115–3121, 1997. Aequorea green fluorescent protein (GFP) is an excellent marker to examine genetically altered live cells in whole animals or culture. Its potential use in identifying genetically modified neurons, however, has not been investigated extensively. To examine the usefulness, toxicity, and potential electrophyiological effects of GFP expression in neurons, we generated adenovirus containing the mGFP4 cDNA. One week after virus transfection of dorsal root ganglion neurons (DRG), 10% of postnatal DRG neurons appeared brightly fluorescent, labelling the soma and neurites. Temporal examination of these neurons demonstrated no toxicity to DRG neurons even after several weeks in culture with repeated daily epifluorescent exposure. Electrophysiological analysis and comparison of control and viral exposed (GFP− and GFP+) DRG neurons did not demonstrate any differences in whole cell resistance, resting potential, action potential (AP) threshold, AP duration, AP amplitude, or whole cell capacitance. To investigate the usefulness of GFP as a marker for identifying neurons genetically altered to express a novel neurotransmitter receptor, a second adenovirus construct was generated containing both GFP and serotonin type 3 (5-HT3) receptor cDNAs. Transfection of DRG neurons with this virus produced an inward current in the presence of serotonin only in DRG neurons that were GFP-positive. It is concluded that adenoviral transfection of neurons with GFP, for cellular labeling, and coexpression of GFP-neurotransmitter constructs are safe, nontoxic, methods for electrophysiologically investigating neurons over several weeks. The uniqueness of the vector used in these experiments is that it was constructed to express GFP in a second cassette so that it would label the transduced cells, but have no potential for interfering with the function of the foreign 5-HT3receptor.

scholarly journals Construction of a ColD cda Promoter-Based SOS-Green Fluorescent Protein Whole-Cell Biosensor with Higher Sensitivity toward Genotoxic Compounds than Constructs Based on recA, umuDC, or sulA Promoters

2005 ◽  
Vol 71 (5) ◽  
pp. 2338-2346 ◽  
Author(s):  
Anders Norman ◽  
Lars Hestbjerg Hansen ◽  
Søren J. Sørensen
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Reporter System ◽  
Whole Cell ◽  
In The Wild ◽  
Whole Cell Biosensor ◽  
Green Fluorescent ◽  
Higher Sensitivity ◽  
Cell Biosensor

ABSTRACT Four different green fluorescent protein (GFP)-based whole-cell biosensors were created based on the DNA damage inducible SOS response of Escherichia coli in order to evaluate the sensitivity of individual SOS promoters toward genotoxic substances. Treatment with the known carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) revealed that the promoter for the ColD plasmid-borne cda gene had responses 12, 5, and 3 times greater than the recA, sulA, and umuDC promoters, respectively, and also considerably higher sensitivity. Furthermore, we showed that when the SOS-GFP construct was introduced into an E. coli host deficient in the tolC gene, the minimal detection limits toward mitomycin C, MNNG, nalidixic acid, and formaldehyde were lowered to 9.1 nM, 0.16 μM, 1.1 μM, and 141 μM, respectively, which were two to six times lower than those in the wild-type strain. This study thus presents a new SOS-GFP whole-cell biosensor which is not only able to detect minute levels of genotoxins but, due to its use of the green fluorescent protein, also a reporter system which should be applicable in high-throughput screening assays as well as a wide variety of in situ detection studies.

Monitoring Oxidative Stress and DNA Damage Induced by Heavy Metals in Yeast Expressing a Redox-Sensitive Green Fluorescent Protein

2009 ◽  
Vol 58 (5) ◽  
pp. 504-510 ◽  
Author(s):  
Shanshan Yu ◽  
Wei Qin ◽  
Guoqiang Zhuang ◽  
Xianen Zhang ◽  
Guanjun Chen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heavy Metals ◽  
Dna Damage ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals Development and Characterization of a Green Fluorescent Protein-Based Bacterial Biosensor for Bioavailable Toluene and Related Compounds

2002 ◽  
Vol 68 (4) ◽  
pp. 1962-1971 ◽  
Author(s):  
Lawrence Stiner ◽  
Larry J. Halverson
Keyword(s):  
Flow Cytometry ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fluorescence Emission ◽  
Broad Host Range ◽  
Bacterial Biosensor ◽  
Minimum Concentration ◽  
Fluorescence Response ◽  
Green Fluorescent ◽  
Related Compounds

ABSTRACT A green fluorescent protein-based Pseudomonas fluorescens strain A506 biosensor was constructed and characterized for its potential to measure benzene, toluene, ethylbenzene, and related compounds in aqueous solutions. The biosensor is based on a plasmid carrying the toluene-benzene utilization (tbu) pathway transcriptional activator TbuT from Ralstonia pickettii PKO1 and a transcriptional fusion of its promoter PtbuA1 with a promoterless gfp gene on a broad-host-range promoter probe vector. TbuT was not limiting, since it was constitutively expressed by being fused to the neomycin phosphotransferase (nptII) promoter. The biosensor cells were readily induced, and fluorescence emission after induction periods of 3 h correlated well with toluene, benzene, ethylbenzene, and trichloroethylene concentrations. Our experiments using flow cytometry show that intermediate levels of gfp expression in response to toluene reflect uniform induction of cells. As the toluene concentration increases, the level of gfp expression per cell increases until saturation kinetics of the TbuT-PtbuA1 system are observed. Each inducer had a unique minimum concentration that was necessary for induction, with K app values that ranged from 3.3 ± 1.8 μM for toluene to 35.6 ± 16.6 μM for trichloroethylene (means ± standard errors of the means), and maximal fluorescence response. The fluorescence response was specific for alkyl-substituted benzene derivatives and branched alkenes (di- and trichloroethylene, 2-methyl-2-butene). The biosensor responded in an additive fashion to the presence of multiple inducers and was unaffected by the presence of compounds that were not inducers, such as those present in gasoline. Flow cytometry revealed that, in response to toxic concentrations of gasoline, there was a small uninduced population and another larger fully induced population whose levels of fluorescence corresponded to the amount of effectors present in the sample. These results demonstrate the potential for green fluorescent protein-based bacterial biosensors to measure environmental contaminants.

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