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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Understanding bacterial resistance to antimicrobial peptides: From the surface to deep inside.
Simone Maria-Neto,Keyla C. de Almeida,Maria Lígia Rodrigues Macedo,Octavio L. Franco,Octavio L. Franco +4 more
TL;DR: Multiple strategies by which bacteria could develop enhanced antimicrobial peptide resistance are discussed, focusing on sub-cellular regions from the surface to deep inside, evaluating bacterial membranes, cell walls and cytoplasmic metabolism.
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Counting protein molecules using quantitative fluorescence microscopy
Valerie C. Coffman,Jian-Qiu Wu +1 more
TL;DR: Two popular methods for quantification of absolute protein numbers in cellular structures using fluorescence microscopy are reviewed: stepwise photobleaching to count discrete changes in intensity from a small number of fluorescent fusion proteins, and comparing the fluorescence intensity of a protein to a known in vivo or in vitro standard.
Journal ArticleDOI
Multiplexed, targeted profiling of single-cell proteomes and transcriptomes in a single reaction
Alex S. Genshaft,Shuqiang Li,Caroline J. Gallant,Spyros Darmanis,Spyros Darmanis,Sanjay M. Prakadan,Carly G. K. Ziegler,Martin Lundberg,Simon Fredriksson,Joyce Hong,Aviv Regev,Aviv Regev,Aviv Regev,Kenneth J. Livak,Ulf Landegren,Alex K. Shalek +15 more
TL;DR: This work profiles the transcriptomic and proteomic response of a human breast adenocarcinoma cell line to a chemical perturbation using the Fluidigm C1™ system and highlights the potential for molecular metadata to inform highly-multiplexed single-cell analyses.
Journal ArticleDOI
Bacterial transcriptomics: what is beyond the RNA horiz-ome?
TL;DR: Transcription in bacteria resembles that in eukaryotes in terms of complexity more closely than was previously thought.
Journal ArticleDOI
Tools for the Microbiome: Nano and Beyond.
Julie S. Biteen,Paul C. Blainey,Zoe G. Cardon,Miyoung Chun,George M. Church,Pieter C. Dorrestein,Scott E. Fraser,Jack A. Gilbert,Jack A. Gilbert,Janet K. Jansson,Rob Knight,Jeff F. Miller,Aydogan Ozcan,Kimberly A. Prather,Stephen R. Quake,Edward G. Ruby,Pamela A. Silver,Sharif A. Taha,Ger van den Engh,Paul S. Weiss,Gerard C. L. Wong,Aaron T. Wright,Thomas D. Young +22 more
TL;DR: In this paper, the authors describe the opportunities, technical needs, and potential approaches to address these challenges, based on recent and upcoming advances in measurement and control at the nanoscale and beyond, and provide the basis for advancing the largely descriptive studies of the microbiome to the theoretical and mechanistic understandings that will underpin the discipline of microbiome engineering.
References
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Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.
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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.