A flow cytometry-optimized assay using an SOS–green fluorescent protein (SOS–GFP) whole-cell biosensor for the detection of genotoxins in complex environments

2006 ◽  
Vol 603 (2) ◽  
pp. 164-172 ◽  
Author(s):  
Anders Norman ◽  
Lars Hestbjerg Hansen ◽  
Søren J. Sørensen
Keyword(s):  
Flow Cytometry ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Complex Environments ◽  
Whole Cell ◽  
Whole Cell Biosensor ◽  
Green Fluorescent ◽  
Cell Biosensor

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.

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 Constitutive and Inducible Expression of Green Fluorescent Protein in Brucella suis

1999 ◽  
Vol 67 (12) ◽  
pp. 6695-6697 ◽  
Author(s):  
Stephan Köhler ◽  
Safia Ouahrani-Bettache ◽  
Marion Layssac ◽  
Jacques Teyssier ◽  
Jean-Pierre Liautard
Keyword(s):  
Flow Cytometry ◽  
Green Fluorescent Protein ◽  
Fluorescence Microscopy ◽  
Gene Fusion ◽  
Fluorescent Protein ◽  
Fusion System ◽  
Chromosomal Dna ◽  
Brucella Suis ◽  
Green Fluorescent ◽  
Inducible Genes

ABSTRACT A gene fusion system based on plasmid pBBR1MCS and the expression of green fluorescent protein was developed for Brucella suis, allowing isolation of constitutive and inducible genes. Bacteria containing promoter fusions of chromosomal DNA togfp were visualized by fluorescence microscopy and examined by flow cytometry. Twelve clones containing gene fragments induced inside J774 murine macrophages were isolated and further characterized.

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 High-Titer Retroviral Vectors Containing the Enhanced Green Fluorescent Protein Gene for Efficient Expression in Hematopoietic Cells

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3316-3321 ◽  
Author(s):  
Ana Limón ◽  
Javier Briones ◽  
Teresa Puig ◽  
Mercé Carmona ◽  
Oscar Fornas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Hematopoietic Cells ◽  
Retroviral Vectors ◽  
Green Fluorescent Protein Gene ◽  
Efficient System ◽  
Green Fluorescent

Abstract Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. We have developed a producer cell line that yields high titers of amphotropic retroviral vectors carrying the enhanced green fluorescent protein (EGFP) gene, a codon humanized, red-shifted variant of the green fluorescent protein (GFP) gene, which can be used as a selectable marker. We have used a hybrid vector that has been shown to efficiently drive gene expression in hematopoietic cells. Virtually all murine and human cell lines and primary human hematopoietic cells tested were transduced with varying efficiency after incubation with vector-containing supernatants. Human CD34+ cells obtained from cord blood or aphereses products were transduced using a protocol that involves daily addition of vector-containing supernatants for 6 consecutive days. At day 6, up to 16% of the cells expressed EGFP, as assessed by flow cytometry. Sorted EGFP-expressing cells were able to produce fluorescent hematopoietic colonies. EGFP's main advantages are its fast flow cytometry determination and the possibility of cell sorting and simultaneous evaluation of the transduction efficiency along with other phenotypic markers.

Differential CTX-M Expression from a Conserved Promoter: Role of Promoter-Associated Spacer Sequences Downstream of the blaCTX-M Regulon

2016 ◽  
Vol 26 (4) ◽  
pp. 284-290 ◽  
Author(s):  
Lin Liu ◽  
Xiangyan Zhang ◽  
Siyuan Yang ◽  
Yao Zhai ◽  
Weijia Liu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Green Fluorescent Protein ◽  
Promoter Activity ◽  
Expression Vector ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Qrt Pcr ◽  
Key Factor ◽  
Green Fluorescent

<b><i>Aims:</i></b> The aim of this project was to explore the different CTX-M expression levels occurring from a single conserved promoter with different spacer sequences, the variation of which is hypothesized to be a key factor in fluctuating levels of CTX-M. <b><i>Methods:</i></b> The <i>bla</i><sub>CTX-M</sub> promoter fragments with five different spacer sequences were amplified, sequenced and cloned into the pUA66 expression vector carrying the green fluorescent protein (GFP) gene. The expression of <i>bla</i><sub>CTX-M</sub> in the transconjugants was analyzed using fluorescence microscopy, flow cytometry and qRT-PCR. <b><i>Results:</i></b> The promoters of all the <i>bla</i><sub>CTX-M</sub> genes were provided by IS<i>Ecp1 </i>and were extremely conserved. The promoter-associated spacer sequences varied from 42 to 127 bp and variations in GFP expression in the five transconjugants were observed. A nucleic acid deletion and point mutation were detected in the spacer sequences by variations in which the expression of <i>bla</i><sub>CTX-M</sub> was influenced. <b><i>Conclusion:</i></b> The different spacer sequences have a significant impact on the activity of the conserved promoter. The shorter spacer sequence between the conserved promoter and the <i>bla</i><sub>CTX-M</sub> gene does not specifically enhance the expression of<i> bla</i><sub>CTX-M</sub>, contrary to previous reports. The expression of <i>bla</i><sub>CTX-M</sub> may be regulated by changes in promoter activity caused by diverse spacer sequences.

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