scholarly journals Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level

2000 ◽  
Vol 97 (13) ◽  
pp. 7260-7265 ◽  
Author(s):  
V. Baubet ◽  
H. Le Mouellic ◽  
A. K. Campbell ◽  
E. Lucas-Meunier ◽  
P. Fossier ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Single Cell ◽  
Fluorescent Protein ◽  
Single Cell Level ◽  
Cell Level ◽  
Green Fluorescent

scholarly journals Bacterial Activity in the Rhizosphere Analyzed at the Single-Cell Level by Monitoring Ribosome Contents and Synthesis Rates

2000 ◽  
Vol 66 (2) ◽  
pp. 801-809 ◽  
Author(s):  
Cayo Ramos ◽  
Lars Mølbak ◽  
Søren Molin
Keyword(s):  
Growth Rate ◽  
Single Cell ◽  
Monitoring System ◽  
Fluorescent Protein ◽  
Transient Gene Expression ◽  
Single Cell Level ◽  
Cell Level ◽  
Gene Encoding ◽  
Rate Dependent ◽  
Green Fluorescent

ABSTRACT The growth activity of Pseudomonas putida cells colonizing the rhizosphere of barley seedlings was estimated at the single-cell level by monitoring ribosomal contents and synthesis rates. Ribosomal synthesis was monitored by using a system comprising a fusion of the ribosomal Escherichia coli rrnBP1 promoter to a gene encoding an unstable variant of the green fluorescent protein (Gfp). Gfp expression in a P. putida strain carrying this system inserted into the chromosome was strongly dependent on the growth phase and growth rate of the strain, and cells growing exponentially at rates of ≥0.17 h−1 emitted growth rate-dependent green fluorescence detectable at the single-cell level. The single-cell ribosomal contents were very heterogeneous, as determined by quantitative hybridization with fluorescently labeled rRNA probes in P. putida cells extracted from the rhizosphere of 1-day-old barley seedlings grown under sterile conditions. After this, cells extracted from the root system had ribosomal contents similar to those found in starved cells. There was a significant decrease in the ribosomal content of P. putida cells when bacteria were introduced into nonsterile bulk or rhizosphere soil, and the Gfp monitoring system was not induced in cells extracted from either of the two soil systems. The monitoring system used permitted nondestructive in situ detection of fast-growing bacterial microcolonies on the sloughing root sheath cells of 1- and 2-day-old barley seedlings grown under sterile conditions, which demonstrated that it may be possible to use the unstable Gfp marker for studies of transient gene expression in plant-microbe systems.

scholarly journals Generation of genetic tools for gauging multiple gene expression at single cell level

2021 ◽  
Author(s):  
Marta Mellini ◽  
Massimiliano Lucidi ◽  
Francesco Imperi ◽  
Paolo Visca ◽  
Livia Leoni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Image Processing ◽  
Single Cell ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Phenotypic Heterogeneity ◽  
Single Cell Level ◽  
Cell Level ◽  
Multiple Gene ◽  
Genetic Tools

Key microbial processes in many bacterial species are heterogeneously expressed in single cells of bacterial populations. However, the paucity of adequate molecular tools for live, real-time monitoring of multiple gene expression at the single cell level has limited the understanding of phenotypic heterogeneity. In order to investigate phenotypic heterogeneity in the ubiquitous opportunistic pathogen Pseudomonas aeruginosa, a genetic tool that allows gauging multiple gene expression at the single cell level has been generated. This tool, named pRGC, consists in a promoter-probe vector for transcriptional fusions that carries three reporter genes coding for the fluorescent proteins mCherry, green fluorescent protein (GFP) and cyan fluorescent protein (CFP). The pRGC vector has been characterized and validated via single cell gene expression analysis of both constitutive and iron-regulated promoters, showing clear discrimination of the three fluorescence signals in single cells of a P. aeruginosa population, without the need of image-processing for spectral crosstalk correction. In addition, two pRGC variants have been generated for either i) integration of the reporter gene cassette into a single neutral site of P. aeruginosa chromosome, that is suitable for long-term experiments in the absence of antibiotic selection, or ii) replication in bacterial genera other than Pseudomonas. The easy-to-use genetic tools generated in this study will allow rapid and cost-effective investigation of multiple gene expression in populations of environmental and pathogenic bacteria, hopefully advancing the understanding of microbial phenotypic heterogeneity. IMPORTANCE Within a bacterial population single cells can differently express some genes, even though they are genetically identical and experience the same chemical and physical stimuli. This phenomenon, known as phenotypic heterogeneity, is mainly driven by gene expression noise and results in the emergence of bacterial sub-populations with distinct phenotypes. The analysis of gene expression at the single cell level has shown that phenotypic heterogeneity is associated with key bacterial processes, including competence, sporulation and persistence. In this study, new genetic tools have been generated that allow easy cloning of up to three promoters upstream of distinct fluorescent genes, making it possible to gauge multiple gene expression at the single cell level by fluorescent microscopy, without the need of advanced image-processing procedures. A proof of concept has been provided by investigating iron-uptake and iron-storage gene expression in response to iron availability in P. aeruginosa.

Fluorescent protein-based FRET sensor for intracellular monitoring of redox status in bacteria at single cell level

2014 ◽  
Vol 406 (28) ◽  
pp. 7195-7204 ◽  
Author(s):  
Bobin George Abraham ◽  
Ville Santala ◽  
Nikolai V. Tkachenko ◽  
Matti Karp
Keyword(s):  
Single Cell ◽  
Fluorescent Protein ◽  
Redox Status ◽  
Single Cell Level ◽  
Cell Level

scholarly journals An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level

F1000Research ◽  
2014 ◽  
Vol 3 ◽  
pp. 60 ◽  
Author(s):  
Shin-ichiro Kojima ◽  
Gary G. Borisy
Keyword(s):  
Single Cell ◽  
Fluorescent Protein ◽  
Selection Marker ◽  
Target Sequence ◽  
Fluorescence Intensity Ratio ◽  
Single Cell Level ◽  
Reporter Assay ◽  
Red Fluorescent Protein ◽  
Dual Fluorescence ◽  
Cell Level

RNA interference (RNAi) is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA) in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA) together with an enhanced green fluorescent protein (EGFP); the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry). Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls) is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3) and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker (ccdB) method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification.

In Situ Detection of High Levels of Horizontal Plasmid Transfer in Marine Bacterial Communities

1998 ◽  
Vol 64 (7) ◽  
pp. 2670-2675 ◽  
Author(s):  
Cecilia Dahlberg ◽  
Maria Bergström ◽  
Malte Hermansson
Keyword(s):  
Gene Transfer ◽  
Pseudomonas Putida ◽  
Single Cell ◽  
Fluorescent Protein ◽  
Detection System ◽  
Plasmid Transfer ◽  
Conjugative Plasmid ◽  
Epifluorescence Microscopy ◽  
Single Cell Level ◽  
Cell Level

ABSTRACT Gene transfer of the conjugative plasmid pBF1 fromPseudomonas putida to indigenous bacteria in seawater was investigated with a detection system for gene transfer based on the green fluorescent protein (GFP) (C. Dahlberg et al., Mol. Biol. Evol. 15:385–390, 1998). pBF1 was tagged with the gfp gene controlled by a lac promoter which is down regulated in the donor cell by a chromosomal repressor (lacI q). The plasmid donor cells (Pseudomonas putida KT2442) subsequently do not express gfp. Transfer to recipient strains lacking the repressor results in expression of gfp. The transconjugant can subsequently be detected by epifluorescence microscopy on a single-cell level. By using this method, transfer of pBF1::gfp and expression of the gfpgene were first shown to occur during nutrient-limiting conditions to several defined recipient bacteria in artificial seawater. Second, we measured transfer of pBF1 from P. putida to the marine bacterial community directly in seawater samples, on a single-cell level, without limiting the detection of gene transfer to the culturable fraction of bacteria. Plasmid transfer was detected on surfaces and in bulk seawater. Seawater bacteria with different morphologies were shown to receive the plasmid. Gene transfer frequencies of 2.3 × 10−6 to 2.2 × 10−4 transconjugants per recipient were recorded after 3 days of incubation.

scholarly journals An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level

F1000Research ◽  
2014 ◽  
Vol 3 ◽  
pp. 60 ◽  
Author(s):  
Shin-ichiro Kojima ◽  
Gary G. Borisy
Keyword(s):  
Single Cell ◽  
Fluorescent Protein ◽  
Selection Marker ◽  
Target Sequence ◽  
Fluorescence Intensity Ratio ◽  
Single Cell Level ◽  
Reporter Assay ◽  
Red Fluorescent Protein ◽  
Dual Fluorescence ◽  
Cell Level

RNA interference (RNAi) is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA) in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA) together with an enhanced green fluorescent protein (EGFP); the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry). Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls) is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3) and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi.To further facilitate testing of RNAi efficacy, we developed a negative selection marker (ccdB) method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification.

Detection of HIV-1 ribonuclease H activity in single-cell by using RNA mimics green fluorescent protein based biosensor

2019 ◽  
Vol 281 ◽  
pp. 439-444 ◽  
Author(s):  
Kai Zhang ◽  
Qianlu Yang ◽  
Wanting Huang ◽  
Ke Wang ◽  
Xue Zhu ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Single Cell ◽  
Fluorescent Protein ◽  
Ribonuclease H ◽  
Green Fluorescent ◽  
Hiv 1
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