The Stochastic Nature of Gene Expression Revealed at the Single-Molecule Level

Chen, Xin; Cremer, Paul S.

doi:10.1021/cb600129z

Cited by 2 publications

(2 citation statements)

References 13 publications

(21 reference statements)

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“…40 Hence, droplets containing identical genes show quite wide phenotypic variance (variance in fluorescence): the coefficient of variation (CV) of green fluorescence of lacZ genes expressed in vitro in double emulsions and analyzed by flow cytometry was 104%. 40 The variance in fluorescence is at least in part due to the polydispersity, but biological variance in the expression of single genes may also contribute: high levels of variance have been reported for expression of single genes in vivo 56 and in vitro: a CV of 44% was reported for the expression of single GFP genes in picolitre droplets in a microfluidic system. 20 Second, it is difficult to add new reagents to preformed droplets, which limits the range of assays that can be performed.…”

Section: Discussionmentioning

confidence: 99%

A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution

Fallah-Araghi¹,

Baret²,

Ryckelynck³

et al. 2012

Lab Chip

225

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In vitro screening systems based on the coupled transcription and translation of genes using cell-free systems have a number of attractive features for protein engineering and directed evolution. We present a completely in vitro ultrahigh-throughput screening platform using droplet-based microfluidics. Single genes are compartmentalized in aqueous droplets, dispersed in inert carrier oil, and amplified using the polymerase chain reaction (PCR). After amplification, the droplets, now containing 30,000 copies of each gene, are fused one-to-one with droplets containing a cell-free coupled transcription-translation (IVTT) system and the reagents for a fluorogenic assay. Fluorescence-activated electrocoalescence with an aqueous stream is then used to selectively recover genes from droplets containing the desired activity. We demonstrate, by selecting mixtures of lacZ genes encoding the enzyme β-galactosidase and lacZmut genes encoding an inactive variant, that this system can sort at 2000 droplets s(-1): lacZ genes were enriched 502-fold from a 1 : 100 molar ratio of lacZ : lacZmut genes. Indeed, the false positive and false negative error rates were both <0.004 and the results indicate that enrichment is not limited by the sorting efficiency, but by the co-encapsulation of multiple genes in droplets, which is described by the Poisson distribution. Compared to screening using microtiter plate-based systems, the volume and cost of PCR and IVTT reagents are reduced by almost 10(5)-fold, allowing the screening of 10(6) genes using only 150 μL of reagents.

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Section: Discussionmentioning

confidence: 99%

A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution

Fallah-Araghi¹,

Baret²,

Ryckelynck³

et al. 2012

Lab Chip

225

View full text Add to dashboard Cite

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“…Such stochastic events that trigger changes in the phenotype have been recently described at a single-molecule/single-cell level for E. coli (30,36,53,75,245). It has been shown that even a single mRNA copy within a single cell can lead to bursts in protein expression and that therefore not only transcription but also posttranscriptional effects are responsible for stochastic protein expression profiles (34). It has been demonstrated that the random dissociation of a single protein molecule (repressor) from the DNA can result in large bursts of protein expression in E. coli, ultimately determining the cell's phenotype (36).…”

Section: Listening Carefully: Bacterial Communicationsmentioning

confidence: 99%

Central Role of the Cell in Microbial Ecology

Zengler

2009

Microbiol Mol Biol Rev

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SUMMARY Over the last few decades, advances in cultivation-independent methods have significantly contributed to our understanding of microbial diversity and community composition in the environment. At the same time, cultivation-dependent methods have thrived, and the growing number of organisms obtained thereby have allowed for detailed studies of their physiology and genetics. Still, most microorganisms are recalcitrant to cultivation. This review not only conveys current knowledge about different isolation and cultivation strategies but also discusses what implications can be drawn from pure culture work for studies in microbial ecology. Specifically, in the light of single-cell individuality and genome heterogeneity, it becomes important to evaluate population-wide measurements carefully. An overview of various approaches in microbial ecology is given, and the cell as a central unit for understanding processes on a community level is discussed.

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The Stochastic Nature of Gene Expression Revealed at the Single-Molecule Level

Cited by 2 publications

References 13 publications

A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution

A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution

Central Role of the Cell in Microbial Ecology

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