Simultaneous Visualization of Multiple Gene Expression in Single Cells Using an Engineered Multicolor Reporter Toolbox and Approach of Spectral Crosstalk Correction

2019 ◽  
Vol 8 (11) ◽  
pp. 2536-2546 ◽  
Author(s):  
Jundong Han ◽  
Aiguo Xia ◽  
Yajia Huang ◽  
Lei Ni ◽  
Wenhui Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cells ◽  
Multiple Gene ◽  
Spectral Crosstalk ◽  
Simultaneous Visualization ◽  
Crosstalk Correction

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.

scholarly journals Simultaneous Visualization of Both Signaling Cascade Activity and End-Point Gene Expression in Single Cells

PLoS ONE ◽  
2011 ◽  
Vol 6 (5) ◽  
pp. e20148 ◽  
Author(s):  
Irene Weibrecht ◽  
Ida Grundberg ◽  
Mats Nilsson ◽  
Ola Söderberg
Keyword(s):  
Gene Expression ◽  
Single Cells ◽  
Signaling Cascade ◽  
End Point ◽  
Simultaneous Visualization

scholarly journals Artificial complementary chromatic acclimation gene expression system in Escherichia coli

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Dwi Ariyanti ◽  
Kazunori Ikebukuro ◽  
Koji Sode
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Light Exposure ◽  
Expression System ◽  
Red Light ◽  
Green Light ◽  
Light Illumination ◽  
Multiple Gene ◽  
Gene Expression System ◽  
Chromatic Acclimation

Abstract Background The development of multiple gene expression systems, especially those based on the physical signals, such as multiple color light irradiations, is challenging. Complementary chromatic acclimation (CCA), a photoreversible process that facilitates the control of cellular expression using light of different wavelengths in cyanobacteria, is one example. In this study, an artificial CCA systems, inspired by type III CCA light-regulated gene expression, was designed by employing a single photosensor system, the CcaS/CcaR green light gene expression system derived from Synechocystis sp. PCC6803, combined with G-box (the regulator recognized by activated CcaR), the cognate cpcG2 promoter, and the constitutively transcribed promoter, the PtrcΔLacO promoter. Results One G-box was inserted upstream of the cpcG2 promoter and a reporter gene, the rfp gene (green light-induced gene expression), and the other G-box was inserted between the PtrcΔLacO promoter and a reporter gene, the bfp gene (red light-induced gene expression). The Escherichia coli transformants with plasmid-encoded genes were evaluated at the transcriptional and translational levels under red or green light illumination. Under green light illumination, the transcription and translation of the rfp gene were observed, whereas the expression of the bfp gene was repressed. Under red light illumination, the transcription and translation of the bfp gene were observed, whereas the expression of the rfp gene was repressed. During the red and green light exposure cycles at every 6 h, BFP expression increased under red light exposure while RFP expression was repressed, and RFP expression increased under green light exposure while BFP expression was repressed. Conclusion An artificial CCA system was developed to realize a multiple gene expression system, which was regulated by two colors, red and green lights, using a single photosensor system, the CcaS/CcaR system derived from Synechocystis sp. PCC6803, in E. coli. The artificial CCA system functioned repeatedly during red and green light exposure cycles. These results demonstrate the potential application of this CCA gene expression system for the production of multiple metabolites in a variety of microorganisms, such as cyanobacteria.

scholarly journals Massively parallel gene expression variation measurement of a synonymous codon library

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Alexander Schmitz ◽  
Fuzhong Zhang
Keyword(s):  
Gene Expression ◽  
Codon Usage ◽  
Single Cells ◽  
Massively Parallel ◽  
Protein Abundance ◽  
Translation Efficiency ◽  
Gene Expression Variation ◽  
Expression Variation ◽  
Change In Mean ◽  
Adaptation Index

Abstract Background Cell-to-cell variation in gene expression strongly affects population behavior and is key to multiple biological processes. While codon usage is known to affect ensemble gene expression, how codon usage influences variation in gene expression between single cells is not well understood. Results Here, we used a Sort-seq based massively parallel strategy to quantify gene expression variation from a green fluorescent protein (GFP) library containing synonymous codons in Escherichia coli. We found that sequences containing codons with higher tRNA Adaptation Index (TAI) scores, and higher codon adaptation index (CAI) scores, have higher GFP variance. This trend is not observed for codons with high Normalized Translation Efficiency Index (nTE) scores nor from the free energy of folding of the mRNA secondary structure. GFP noise, or squared coefficient of variance (CV2), scales with mean protein abundance for low-abundant proteins but does not change at high mean protein abundance. Conclusions Our results suggest that the main source of noise for high-abundance proteins is likely not originating at translation elongation. Additionally, the drastic change in mean protein abundance with small changes in protein noise seen from our library implies that codon optimization can be performed without concerning gene expression noise for biotechnology applications.

scholarly journals Using single-cell transcriptomics to understand functional states and interactions in microbial eukaryotes

2019 ◽  
Vol 374 (1786) ◽  
pp. 20190098 ◽  
Author(s):  
Chuan Ku ◽  
Arnau Sebé-Pedrós
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Interspecific Interactions ◽  
Single Cells ◽  
Life Cycles ◽  
Modern Biology ◽  
Microbial Eukaryotes ◽  
Eukaryotic Microorganisms

Understanding the diversity and evolution of eukaryotic microorganisms remains one of the major challenges of modern biology. In recent years, we have advanced in the discovery and phylogenetic placement of new eukaryotic species and lineages, which in turn completely transformed our view on the eukaryotic tree of life. But we remain ignorant of the life cycles, physiology and cellular states of most of these microbial eukaryotes, as well as of their interactions with other organisms. Here, we discuss how high-throughput genome-wide gene expression analysis of eukaryotic single cells can shed light on protist biology. First, we review different single-cell transcriptomics methodologies with particular focus on microbial eukaryote applications. Then, we discuss single-cell gene expression analysis of protists in culture and what can be learnt from these approaches. Finally, we envision the application of single-cell transcriptomics to protist communities to interrogate not only community components, but also the gene expression signatures of distinct cellular and physiological states, as well as the transcriptional dynamics of interspecific interactions. Overall, we argue that single-cell transcriptomics can significantly contribute to our understanding of the biology of microbial eukaryotes. This article is part of a discussion meeting issue ‘Single cell ecology’.

Antioxidants increase number of progenitor endothelial cells through multiple gene expression pathways

2008 ◽  
Vol 42 (8) ◽  
pp. 754-762 ◽  
Author(s):  
Carmela Fiorito ◽  
Monica Rienzo ◽  
Ettore Crimi ◽  
Raffaele Rossiello ◽  
Maria Luisa Balestrieri ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Multiple Gene ◽  
Expression Pathways
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