Semiconducting polymer dots (Pdots) are rapidly becoming one of the most studied nanoparticles in fluorescence bioimaging and sensing. Their small size, high brightness, and resistance to photobleaching make them one of the most attractive fluorophores for fluorescence imaging and sensing applications. This paper highlights our recent advances in fluorescence bioimaging and sensing with nanoscale luminescent Pdots, specifically the use of organic dyes as dopant molecules to modify the optical properties of Pdots to enable deep red and near infrared fluorescence bioimaging applications and to impart sensitivity of dye doped Pdots towards selected analytes. Building on our earlier work, we report the formation of secondary antibody-conjugated Pdots and provide Cryo-TEM evidence for their formation. We demonstrate the selective targeting of the antibody-conjugated Pdots to FLAG-tagged FLS2 membrane receptors in genetically engineered plant leaf cells. We also report the formation of a new class of luminescent Pdots with emission wavelengths of around 1000 nm. Finally, we demonstrate the formation and utility of oxygen sensing Pdots in aqueous media.
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Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump–probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals.more » « less
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Abstract The conversion of lignocellulose‐rich biomass to bio‐based chemicals and higher order fuels remains a grand challenge, as single‐microbe approaches often cannot drive both deconstruction and chemical production steps. In contrast, consortia based bioprocessing leverages the strengths of different microbes to distribute metabolic loads and achieve process synergy, product diversity, and bolster yields. Here, we describe a biphasic fermentation scheme that combines the lignocellulolytic action of anaerobic fungi isolated from large herbivores with domesticated microbes for bioproduction. When grown in batch culture, anaerobic fungi release excess sugars from both cellulose and crude biomass due to a wealth of highly expressed carbohydrate active enzymes (CAZymes), converting as much as 49% of cellulose to free glucose. This sugar‐rich hydrolysate readily supports growth of
Saccharomyces cerevisiae , which can be engineered to produce a range of value‐added chemicals. Further, construction of metabolic pathways from transcriptomic data reveals that anaerobic fungi do not catabolize all sugars that their enzymes hydrolyze from biomass, leaving other carbohydrates such as galactose, arabinose, and mannose available as nutritional links to other microbes in their consortium. Although basal expression of CAZymes in anaerobic fungi is high, it is drastically amplified by cellobiose breakout products encountered during biomass hydrolysis. Overall, these results suggest that anaerobic fungi provide a nutritional benefit to the rumen microbiome, which can be harnessed to design synthetic microbial communities that compartmentalize biomass degradation and bioproduct formation.
