scholarly journals Green Fluorescent Protein-Based Biosensor To Detect and Quantify Stress Responses Induced by DNA-Degrading Colicins

2011 ◽  
Vol 77 (18) ◽  
pp. 6691-6693 ◽  
Author(s):  
Sam Abraham ◽  
James Chin ◽  
Huub J. M. Brouwers ◽  
Bernadette Turner ◽  
Ren Zhang ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Stress Responses ◽  
Fluorescent Protein ◽  
Sos Response ◽  
Whole Cell ◽  
Whole Cell Biosensor ◽  
Green Fluorescent ◽  
Cell Biosensor

ABSTRACTHere we report the development of a whole-cell biosensor to detect and quantify the induction of the SOS response activated by DNA-degrading colicins. This biosensor utilizes the SOS-responsivecdapromoter to regulate the expression of green fluorescent protein. The biosensor assay revealed induction of stress for all DNA-degrading reference colicins (E2, E7, and E8).

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.

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 Bioengineering, a Cadmium Sensing Green Fluorescent Protein, Based Whole-Cell Biosensor From Pseudomonas aeruginosa

2020 ◽  
Vol 10 (2) ◽  
pp. 2032-2043
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Green Fluorescent Protein ◽  
Cadmium Chloride ◽  
Fluorescent Protein ◽  
Lead Nitrate ◽  
Gibson Assembly ◽  
Waste Production ◽  
Whole Cell Biosensor ◽  
Green Fluorescent ◽  
Promoter Interaction

Heavy metal toxicity is a significant issue due to an increase in industrial waste production, with cadmium being one of the major pollutants. An approach involving homologous cloning was made to bioengineer a microbial cadmium biosensor from a strain of Pseudomonas aeruginosa. The promoter pCadR from P. aeruginosa was cloned into a vector pEGFP-N2 using Gibson assembly. Escherichia coli DH5 alpha strain was used as the host cell, which on sensing cadmium, produced fluorescence using the reporter gene called green fluorescent protein (GFP). The clone, pEGFP-N2CadR, was subjected to increasing concentrations of cadmium chloride to determine the sensitivity. It was observed that pEGFP-N2CadR responded to micromolar concentrations of cadmium chloride; however, it was determined that the biosensor tested it with lead nitrate and copper sulfate solutions had non-specific interactions with other metals. The interaction of the promoter with the metals was weak compared to previous studies, which was attributed to several reasons mentioned in the paper. As the sensor's fluorescent intensities were dull and indistinguishable, it was not classified as a useful cadmium biosensor. Further studies for determining the promoter interaction and affinity towards various metals at varying concentrations are required to validate the results obtained.

scholarly journals Green Fluorescent Protein as a Noninvasive Stress Probe in Resting Escherichia coli Cells

1999 ◽  
Vol 65 (2) ◽  
pp. 409-414 ◽  
Author(s):  
Hyung Joon Cha ◽  
Ranjan Srivastava ◽  
Vikram N. Vakharia ◽  
Govind Rao ◽  
William E. Bentley
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Heat Shock ◽  
Stress Responses ◽  
Fluorescent Protein ◽  
Osmotic Shock ◽  
Cellular Stress ◽  
Stress Protein ◽  
Resting Cells ◽  
Green Fluorescent

ABSTRACT We constructed and characterized three stress probe plasmids which utilize a green fluorescent protein as a noninvasive reporter in order to elucidate Escherichia coli cellular stress responses in quiescent or resting cells. Cellular stress levels were easily detected by fusing three heat shock stress protein promoter elements, those of the heat shock transcription factor ς32, the protease subunit ClpB, and the chaperone DnaK, to the reporter genegfpuv . When perturbed by a chemical or physical stress (such as a heat shock, nutrient [amino acid] limitation, or addition of IPTG [isopropyl-β-d-thiogalactopyranoside], acetic acid, ethanol, phenol, antifoam, or salt [osmotic shock]), the E. coli cells produced GFPuv, which was easily detected within the cells as emitted green fluorescence. Temporal and amplitudinal mapping of the responses was performed, and the results revealed regions where quantitative delineation of cell stress was afforded.

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