A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit

Experiments in synthetic biology and microbiology can benefit from protein expression systems with low cell-to-cell variability (noise) and expression levels precisely tunable across a useful dynamic range. Despite advances in understanding the molecular biology of microbial gene regulation, many experiments employ protein-expression systems exhibiting high noise and nearly all-or-none responses to induction. I present an expression system that incorporates elements known to reduce gene expression noise: negative autoregulation and bicistronic transcription. I show by stochastic simulation that while negative autoregulation can produce a more gradual response to induction, bicistronic expression of a repressor and gene of interest can be necessary to reduce noise below the extrinsic limit. I synthesized a plasmid-based system incorporating these principles and studied its properties in Escherichia coli cells, using flow cytometry and fluorescence microscopy to characterize induction dose-response, induction/repression kinetics and gene expression noise. By varying ribosome binding site strengths, expression levels from 55–10,740 molecules/cell were achieved with noise below the extrinsic limit. Individual strains are inducible across a dynamic range greater than 20-fold. Experimental comparison of different regulatory networks confirmed that bicistronic autoregulation reduces noise, and revealed unexpectedly high noise for a conventional expression system with a constitutively expressed transcriptional repressor. I suggest a hybrid, low-noise expression system to increase the dynamic range.publishe

Entropy-driven formation of a chiral liquid-crystalline phase of helical filaments

Author Posting. © The Authors, 2006. This article is posted here by permission of American Physical Society for personal use, not for redistribution. The definitive version was published in Physical Review Letters 96 (2006): 018305, doi:10.1103/PhysRevLett.96.018305.We study the liquid-crystalline phase behavior of a concentrated suspension of helical flagella isolated from Salmonella typhimurium. Flagella are prepared with different polymorphic states, some of which have a pronounced helical character while others assume a rodlike shape. We show that the static phase behavior and dynamics of chiral helices are very different when compared to simpler achiral hard rods. With increasing concentration, helical flagella undergo an entropy-driven first order phase transition to a liquid-crystalline state having a novel chiral symmetry.M. S. and R. O. are supported by NIH Grant No. EB002583

On the value of preprints: an early career researcher perspective

Peer-reviewed journal publication is the main means for academic researchers in the life sciences to create a permanent, public record of their work. These publications are also the de facto currency for career progress, with a strong link between journal brand recognition and perceived value. The current peer-review process can lead to long delays between submission and publication, with cycles of rejection, revision and resubmission causing redundant peer review. This situation creates unique challenges for early career researchers (ECRs), who rely heavily on timely publication of their work to gain recognition for their efforts. ECRs face changes in the academic landscape including the increased interdisciplinarity of life sciences research, expansion of the researcher population and consequent shifts in employer and funding demands. The publication of preprints, publicly available scientific manuscripts posted on dedicated preprint servers prior to journal managed peer-review, can play a key role in addressing these ECR challenges. Preprinting benefits include rapid dissemination of academic work, open access, establishing priority or concurrence, receiving feedback and facilitating collaborations. While there is a growing appreciation for and adoption of preprints, a minority of all articles in life sciences and medicine are preprinted. The current low rate of preprint submissions in life sciences and ECR concerns regarding preprinting needs to be addressed. We provide a perspective from an interdisciplinary group of early career researchers on the value of preprints and advocate the wide adoption of preprints to advance knowledge and facilitate career development

Energy injustice and Nordic electric mobility: inequality, elitism, and externalities in the electrification of vehicle-to-grid (V2G) transport

Much research on electric mobility transitions has been descriptive or positive, rather than normative or critical, assessing the deeper ethical, justice, or moral issues that arise. To address this gap, this study qualitatively assesses the ongoing transition to Nordic electric vehicles (EVs) and vehicle-to-grid (V2G) systems. It does so through the various lenses of distributive justice, procedural justice, cosmopolitan justice, and recognition justice. It asks: what are the types of injustices associated with electric mobility and V2G? In what ways do emerging patterns of electric mobility worsen socio-environmental risks or vulnerabilities? Based on original primary data collected from 257 experts across Denmark, Finland, Iceland, Norway, and Sweden, the study finds that electric mobility can erode elements of distributive justice for being accessible only to the rich, and for raising risks related to privacy, hacking, and cyberterrorism. Electric mobility may contravene aspects of procedural justice by reinforcing exclusion and elitism in national planning. It can erode cosmopolitan justice by producing negative environmental externalities, and exacerbating rural (and global) vulnerability. It may threaten recognition justice through unemployment, disruption to traditional businesses, and the entrenchment of patriarchy. Thankfully, the study also proposes a suite of policy mechanisms to address many of these concerns

In Vivo Structure of the E. coli FtsZ-ring Revealed by Photoactivated Localization Microscopy (PALM)

The FtsZ protein, a tubulin-like GTPase, plays a pivotal role in prokaryotic cell division. In vivo it localizes to the midcell and assembles into a ring-like structure-the Z-ring. The Z-ring serves as an essential scaffold to recruit all other division proteins and generates contractile force for cytokinesis, but its supramolecular structure remains unknown. Electron microscopy (EM) has been unsuccessful in detecting the Z-ring due to the dense cytoplasm of bacterial cells, and conventional fluorescence light microscopy (FLM) has only provided images with limited spatial resolution (200–300 nm) due to the diffraction of light. Hence, given the small sizes of bacteria cells, identifying the in vivo structure of the Z-ring presents a substantial challenge. Here, we used photoactivated localization microscopy (PALM), a single molecule-based super-resolution imaging technique, to characterize the in vivo structure of the Z-ring in E. coli. We achieved a spatial resolution of ∼35 nm and discovered that in addition to the expected ring-like conformation, the Z-ring of E. coli adopts a novel compressed helical conformation with variable helical length and pitch. We measured the thickness of the Z-ring to be ∼110 nm and the packing density of FtsZ molecules inside the Z-ring to be greater than what is expected for a single-layered flat ribbon configuration. Our results strongly suggest that the Z-ring is composed of a loose bundle of FtsZ protofilaments that randomly overlap with each other in both longitudinal and radial directions of the cell. Our results provide significant insight into the spatial organization of the Z-ring and open the door for further investigations of structure-function relationships and cell cycle-dependent regulation of the Z-ring

Induction dose-response and noise characterization for bicistronic autoregulation circuits with a range of expression levels.

<p>(<b>A</b>) Simulated gene expression mean and noise for the bicistronic, autoregulatory construct with different translation rates in the absence (dashed lines) and presence (solid lines) of extrinsic noise. (<b>B</b>) GFP expression means for strains with mutated GFP ribosome binding sites at various 2, 8, and 32 nM ATc. Strains are colored by predicted ribosome binding site efficiencies. (<b>C</b>) GFP expression in unrelated cells for the highest-expression strain pZH509 at 0, 0.5, 8 and 32 nM ATc. Maximum fluorescence intensities normalized by the mean number of GFP molecules per cell (<b>Table B in</b> <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187259#pone.0187259.s001" target="_blank">S1 Text</a>). Scale bar 2 μm. (<b>D</b>) Fluorescence of the non-GFP-expressing plasmid pZH501 is compared to the lowest-expression-level plasmid pZH511 without induction. Intensity scaling identical for both images. Scale bar 5 μm. (<b>E</b>) GFP expression mean (molecules/cell normalized by cell area) and noise for plasmids pZH509, pZH511, and pZH512 at 0, 0.5, 8 and 32 nM ATc. Dashed line indicates the approximate global extrinsic noise limit [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187259#pone.0187259.ref001" target="_blank">1</a>].</p

Comparison of regulatory circuits and increased dynamic range with a hybrid circuit.

<p>(<b>A</b>) GFP induction is measured by flow cytometry and fit using the Hill equation for pZH509 (blue, <i>n</i>_<i>h</i> = 0.60 +/- 0.16), pZH517 (green, <i>n</i>_<i>h</i> = 0.65 +/- 0.14) and pZH520 (red, <i>n</i>_<i>h</i> = 2.24 +/- 0.22). Data and fit curves are normalized to the fit value at 256 nM ATc. Data not shown for 0 nM ATc (<b>Figure F in</b> <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187259#pone.0187259.s001" target="_blank">S1 Text</a>). (<b>B</b>) Noise dependence on mean expression level; coloring identical to <b>Fig 4A</b>. Black dot, pZH514 at 32 nM ATc. Noise cannot be calculated for pZH509 at 0 nM ATc because of low expression (<b>Figure F in</b> <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187259#pone.0187259.s001" target="_blank">S1 Text</a>). (<b>C</b>) A hybrid scheme is proposed (inset) in which repressor (white box) expression occurs both from autoregulated (black arrow) and relatively weak (gray arrow) promoters that share a transcription terminator (black box). This achieves an inducer dose-response in the gene of interested (orange) that is less steep than in the absence of autoregulation (red) while increasing the dynamic range relative to bicistronic autoregulatory circuit (blue). (<b>D</b>) The hybrid system reduces noise relative to the system with constitutively expressed repressor, with noise at or below the extrinsic limit (black). All simulations include extrinsic noise.</p

Data files 2: A plasmid-based E. coli gene expression system with cell-to-cell variation below the extrinsic noise limit

<p>This zip archive contains flow cytometry data used to generate the figures in the submitted manuscript "A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit" (PONE-D-17-11810). Text files within the archive describe how to open files and which data corresponds to manuscript figures.</p

List of chemical reactions in stochastic simulations and reaction rates.

<p>The network schemes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187259#pone.0187259.g001" target="_blank">Fig 1A</a> can be simulated by including the reactions with black squares for (i) constitutive expression, (ii) constitutive repressor, (iii) autoregulated repressor and (iv) bicistronic autoregulation. Rates fixed in all simulations (when not set to zero) are listed below along with names of variable reaction rates. The number of free inducer molecules was kept constant.</p