Biology subjects

Evans, J. E.

Publications and source records attributed to Evans, J. E..

6 recordsLinked to original sources

bioRxiv2018

Considerations for imaging thick, low contrast, and beam sensitive samples with liquid cell transmission electron microscopy

Transmission electron microscopy of whole cells is hindered by the inherently large thickness and low atomic contrast intrinsic of cellular material. Liquid cell transmission electron microscopy allows samples to remain in their native hydrated state and may permit visualizing cellular dynamics in-situ. However, imaging biological cells with this approach remains challenging and identifying an optimal imaging regime using empirical data would help foster new advancements in the field. Recent questions about the role of the electron beam inducing morphological changes or damaging cellular structure and function necessitates further investigation of electron beam-cell interactions, but is complicated by variability in imaging techniques used across various studies currently present in literature. The necessity for using low electron fluxes for imaging biological samples requires finding an imaging strategy which produces the strongest contrast and signal to noise ratio for the electron flux used. Here, we experimentally measure and evaluate signal to noise ratios and damage mechanisms between liquid and cryogenic samples for cells using multiple electron imaging modalities all on the same instrument and with equivalent beam parameters to standardize the comparison. We also discuss considerations for optimal electron microscopy imaging conditions for future studies on whole cells within liquid environments.

cell biology↗

bioRxiv2018

Protein Structural Biology Using Cell-Free Platform from Wheat Germ

One of the biggest bottlenecks for structural analysis of proteins remains the creation of high yield and high purity samples of the target protein. Cell-free protein synthesis technologies are powerful and customizable platforms for obtaining functional proteins of interest in short timeframes while avoiding potential toxicity issues and permitting high-throughput screening. These methods have benefited many areas of genomic and proteomics research, therapeutics, vaccine development and protein chip constructions. In this work, we demonstrate a versatile and multistage eukaryotic wheat-germ cell-free protein expression pipeline to generate functional proteins of different sizes from multiple host organism and DNA source origins. We also developed a robust purification procedure, which can produce highly-pure (>98%) proteins with no specialized equipment required and minimal time invested. This pipeline successfully produced and analyzed proteins in all three major geometry formats used for structural biology including single particle analysis, and both two-dimensional and three-dimensional protein crystallography. The flexibility of the wheat germ system in combination with the multiscale pipeline described here provides a new workflow for rapid generation of samples for structural characterization that may not be amenable to other recombinant approaches.

molecular biology↗

bioRxiv2018

Cell-free production of full-length ice nucleating protein InaZ

Unlike inorganic or other synthetic alternatives, ice nucleating proteins (INPs) remain the most efficient ice nuclei today. Their potential applications in cryo-preservation, biomedicine, food industry and in the modulation of climate are widespread. Nevertheless, over several decades, cell-based recombinant methods have experienced multiple difficulties expressing these large proteins in full-length and in necessary yields while retaining functionality. As a result, our understanding of the structure and ice nucleation mechanism for this class of proteins is incomplete, and, most importantly, the full extent of possible applications unrealized. Using a wheat-germ cell-free expression pipeline, we successfully expressed and purified full-length ice nucleating protein InaZ from Pseudomonas syringae, known as a model INP. High protein yield and solubility has been achieved using this system. Ice nucleation experiments inside a dynamic environmental scanning electron microscope (ESEM) confirmed that the produced InaZ products remain functional. Preliminary structural assessments of these proteins using Transmission Electron Microscopy (TEM) showed experimental evidence for their structural organization as fibrils. We believe that the current platform will be suitable for expressing other INPs of interest and can be further employed as new engineering system either for industrial or scientific needs.

synthetic biology↗

bioRxiv2018

Integrated systems biology and imaging of the smallest free-living eukaryote Ostreococcus tauri

Ostreococcus tauri is an ancient phototrophic microalgae that possesses favorable genetic and cellular characteristics for reductionist studies probing biosystem design and dynamics. Here multimodal bioimaging and multi-omics techniques were combined to interrogate O. tauri cellular changes in response to variations in bioavailable nitrogen and carbon ratios. Confocal microscopy, stimulated Raman scattering, and cryo-soft x-ray tomography revealed whole cell ultrastructural dynamics and composition while proteomic and lipidomic profiling captured changes at the molecular and macromolecular scale.\n\nDespite several energy dense long-chain triacylglycerol lipids showing more than 40-fold higher abundance under N deprivation, only a few proteins directly associated with lipid biogenesis showed significant expression changes. However, the entire pathway for starch granule biosynthesis was highly upregulated suggesting much of the cellular energy is preferentially directed towards starch over lipid accumulation. Additionally, three of the five most downregulated and five of the ten most upregulated proteins during severe nitrogen depletion were unnamed protein products that warrant additional biochemical analysis and functional annotation to control carbon transformation dynamics in this smallest eukaryote.

systems biology↗

bioRxiv2018

Optimizing bioreactor growth of the smallest eukaryote

Photosynthetic organisms are adept at circumventing nutrient deprivation. Microalgae in particular present novel adaptations to nutrient and light starvation since they can scavenge external and internal nutrient pools to redistribute energy resources for survival. In this report, a turbidostatic photobioreactor was used to characterize environmental conditions and nutrient requirements for cultures of the smallest free-living eukaryote Ostreococcus tauri. Optimized growth conditions were identified that enable 4-times faster phototrophic growth-rates while increasing total biomass 10-fold. By achieving phototrophic doubling times shorter than 6 hours, these results highlight the potential of this smallest eukaryote for future industrial bioproduct applications.

cell biology↗

bioRxiv2018

Ostreococcus tauri is a high-lipid content green algae that extrudes clustered lipid droplets

Lipid droplet biogenesis, accumulation and secretion is an important field of research spanning biofuel feedstock production in algae and yeast to plant-microbe symbiosis or human metabolic disorders and other diseases. Here we evaluate the critical elements that influence lipid accumulation in the highly simplified and smallest known eukaryote Ostreococcus tauri and identify several conditions that satisfy its classification as an oleaginous green alga. In addition, these experiments revealed the release of excess lipids in pea-pod like structures where many dense lipid droplets are clustered in a linear fashion surrounded by an enveloping membrane which contrasts with known mechanisms from other eukaryotes. These results highlight the potential for Ostreococcus tauri to probe the evolution of lipid droplet dynamics as an emerging model organism with a compacted eukaryotic genome and also to impact lipid feedstock bioproduction applications either directly or using synthetic biology.\n\nOne Sentence SummaryThe smallest known eukaryote Ostreococcus tauri is oleaginous and sheds lipid droplets as pea-pod like membrane enclosed clusters.

cell biology↗