Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells.
Yuichi Taniguchi,Paul J. Choi,Gene-Wei Li,Huiyi Chen,Mohan Babu,Jeremy Hearn,Andrew Emili,X. Sunney Xie +7 more
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TLDR
System-wide analyses of protein and mRNA expression in individual cells with single-molecule sensitivity using a newly constructed yellow fluorescent protein fusion library for Escherichia coli found that almost all protein number distributions can be described by the gamma distribution with two fitting parameters which, at low expression levels, have clear physical interpretations as the transcription rate and protein burst size.Abstract:
Protein and messenger RNA (mRNA) copy numbers vary from cell to cell in isogenic bacterial populations. However, these molecules often exist in low copy numbers and are difficult to detect in single cells. We carried out quantitative system-wide analyses of protein and mRNA expression in individual cells with single-molecule sensitivity using a newly constructed yellow fluorescent protein fusion library for Escherichia coli. We found that almost all protein number distributions can be described by the gamma distribution with two fitting parameters which, at low expression levels, have clear physical interpretations as the transcription rate and protein burst size. At high expression levels, the distributions are dominated by extrinsic noise. We found that a single cell's protein and mRNA copy numbers for any given gene are uncorrelated.read more
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tCRISPRi: tunable and reversible, one-step control of gene expression.
Xintian Li,Yonggun Jun,Michael Erickstad,Steven D. Brown,Adam R. Parks,Donald L. Court,Suckjoon Jun +6 more
TL;DR: The results show that tCRISPRi, in combination with recombineering, provides a simple and easy-to-implement tool for gene expression control, and is ideally suited for construction of both individual strains and high-throughput tunable knockdown libraries.
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Levels of Germination Proteins in Dormant and Superdormant Spores of Bacillus subtilis
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New aspects of RNA processing in prokaryotes
TL;DR: High-resolution analyses of whole prokaryotic transcriptomes allow comprehensive mapping of processed transcripts, detection of essentially all expressed regulatory RNAs, and monitoring of the global impact of ribonucleases and other processing factors, and opens new perspectives for the understanding of the molecular mechanisms responsible for mRNA decay in proKaryotes.
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ODE constrained mixture modelling: a method for unraveling subpopulation structures and dynamics.
TL;DR: This work uses ODE constrained mixture modelling to study NGF-induced Erk1/2 phosphorylation in primary sensory neurones, a process relevant in inflammatory and neuropathic pain and proposes a mechanistic pathway model for this process and reconstructed static and dynamical subpopulation characteristics across experimental conditions.
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Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells
Burak Okumus,Dirk Landgraf,Ghee Chuan Lai,Somenath Bakhsi,Juan Carlos Arias-Castro,Juan Carlos Arias-Castro,Sadik Yildiz,Dann Huh,Raul Fernandez-Lopez,Celeste Peterson,Erdal Toprak,Meriem El Karoui,Meriem El Karoui,Johan Paulsson +13 more
TL;DR: A microfluidics-based platform that enables single-molecule counting of low-abundance proteins by mechanically slowing-down their diffusion within the cytoplasm of live Escherichia coli cells is developed.
References
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Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.
Tomoya Baba,Takeshi Ara,Miki Hasegawa,Yuki Takai,Yoshiko Okumura,Miki Baba,Kirill A. Datsenko,Masaru Tomita,Barry L. Wanner,Hirotada Mori,Hirotada Mori +10 more
TL;DR: These mutants—the ‘Keio collection’—provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome‐wide testing of mutational effects in a common strain background, E. coli K‐12 BW25113.
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Stochastic Gene Expression in a Single Cell
TL;DR: This work constructed strains of Escherichia coli that enable detection of noise and discrimination between the two mechanisms by which it is generated and reveals how low intracellular copy numbers of molecules can fundamentally limit the precision of gene regulation.
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Global analysis of protein localization in budding yeast
Won-Ki Huh,James V. Falvo,Luke C. Gerke,Adam S. Carroll,Russell W. Howson,Jonathan S. Weissman,Erin K. O'Shea +6 more
TL;DR: The construction and analysis of a collection of yeast strains expressing full-length, chromosomally tagged green fluorescent protein fusion proteins helps reveal the logic of transcriptional co-regulation, and provides a comprehensive view of interactions within and between organelles in eukaryotic cells.
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Fabrication of microfluidic systems in poly(dimethylsiloxane)
J.C. McDonald,David C. Duffy,Janelle R. Anderson,Daniel T. Chiu,Hongkai Wu,Olivier Schueller,George M. Whitesides +6 more
TL;DR: Fabrication of microfluidic devices in poly(dimethylsiloxane) (PDMS) by soft lithography provides faster, less expensive routes to devices that handle aqueous solutions.