A novel synthetic sRNA promoting protein overexpression in cell-free systems.

Bacterial small RNAs (sRNAs) that regulate gene expression have been engineered for uses in synthetic biology and metabolic engineering. Here, we designed a novel non-Hfq-dependent sRNA scaffold that uses a modifiable 20 nucleotide antisense binding region to target mRNAs selectively and influence protein expression. The system was developed for regulation of a fluorescent reporter in vivo using Escherichia coli, but the system was found to be more responsive and produced statistically significant results when applied to protein synthesis using in vitro cell-free systems (CFS). Antisense binding sequences were designed to target not only translation initiation regions but various secondary structures in the reporter mRNA. Targeting a high-energy stem loop structure and the 3' end of mRNA yielded protein expression knock-downs that approached 70%. Notably, targeting a low-energy stem structure near a potential RNase E binding site led to a statistically significant 65% increase in protein expression (p < 0.05). These results were not obtainable in vivo, and the underlying mechanism was translated from the reporter system to achieve better than 75% increase in recombinant diaphorase expression in a CFS. It is possible the designs developed here can be applied to improve/regulate expression of other proteins in a CFS.

Resolving complex phenotypes with Raman spectroscopy and chemometrics.

Raman spectroscopy and chemometric analyses are used to characterize phenotypes of biological samples. The approach is relevant in biotechnology to identify and monitor productive cell cultures. It can also detect the presence of pathogens in food products and screen for disease in clinical applications. Raman-based phenotyping is of interest because it is inexpensive, rapid, label-free, and is not obscured by water molecules. Here, recent applications in microbial species and tissue identification, isogenic cell/tissue phenotype changes, and characterizing biological fluids were surveyed along with the myriad spectral processing and chemometric analysis approaches. Suggestions are also given to help standardize and solidify Raman-based phenotyping as an -omics analysis method. These include offering repositories for raw spectral data and molecular assignment libraries.

Mitigating nutrient accumulation with microalgal growth towards enhanced nutrient removal and biomass production in an osmotic photobioreactor.

Forward osmosis (FO) has great potential for low energy consumption wastewater reuse provided there is no requirement for draw solutes (DS) regeneration. Reverse solute flux (RSF) can lead to DS build-up in the feed solution. This remains a key challenge because it can cause significant water flux reduction and lead to additional water quality problems. Herein, an osmotic photobioreactor (OsPBR) system was developed to employ fast-growing microalgae to consume the RSF nutrients. Diammonium phosphate (DAP) was used as a fertilizer DS, and algal biomass was a byproduct. The addition of microalgae into the OsPBR proved to maintain water flux while reducing the concentrations of NH4+-N, PO43--P and chemical oxygen demand (COD) in the OsPBR feed solution by 44.4%, 85.6%, and 77.5%, respectively. Due to the forward cation flux and precipitation, intermittent supplements of K+, Mg2+, Ca2+, and SO42- salts further stimulated algal growth and culture densities by 58.7%. With an optimal hydraulic retention time (HRT) of 3.33 d, the OsPBR overcame NH4+-N overloading and stabilized key nutrients NH4+-N at âˆ¼ 2.0 mg L-1, PO43--P < 0.6 mg L-1, and COD < 30 mg L-1. A moderate nitrogen reduction stress resulted in a high carbohydrate content (51.3 ± 0.1%) among microalgal cells. A solids retention time (SRT) of 17.82 d was found to increase high-density microalgae by 3-fold with a high yield of both lipids (9.07 g m-3 d-1) and carbohydrates (16.66 g m-3 d-1). This study encourages further exploration of the OsPBR technology for simultaneous recovery of high-quality water and production of algal biomass for value-added products.