Optimizing the production of dsRNA biocontrols in microbial systems using multiple transcriptional terminators.

Crop pests and pathogens annually cause over $220 billion in global crop damage, with insects consuming 5%-20% of major grain crops. Current crop pest and disease control strategies rely on insecticidal and fungicidal sprays, plant genetic resistance, transgenes, and agricultural practices. Double-stranded RNA (dsRNA) is emerging as a novel sustainable method of plant protection as an alternative to traditional chemical pesticides. Successful commercialization of dsRNA-based biocontrols requires the economical production of large quantities of dsRNA combined with suitable delivery methods to ensure RNAi efficacy against the target pest. In this study, we have optimized the design of plasmid DNA constructs to produce dsRNA biocontrols in Escherichia coli, by employing a wide range of alternative synthetic transcriptional terminators before measurement of dsRNA yield. We demonstrate that a 7.8-fold increase of dsRNA was achieved using triple synthetic transcriptional terminators within a dual T7 dsRNA production system compared to the absence of transcriptional terminators. Moreover, our data demonstrate that batch fermentation production dsRNA using multiple transcriptional terminators is scalable and generates significantly higher yields of dsRNA generated in the absence of transcriptional terminators at both small-scale batch culture and large-scale fermentation. In addition, we show that application of these dsRNA biocontrols expressed in E. coli cells results in increased insect mortality. Finally, novel mass spectrometry analysis was performed to determine the precise sites of transcriptional termination at the different transcriptional terminators providing important further mechanistic insight.

Lithium conductivity in glasses of the Li2O-Al2O3-SiO2 system.

To improve the understanding of Li-dynamics in oxide glasses, i.e. the effect of [AlO4](-) tetrahedra and non-bridging oxygens on the potential landscape, electrical conductivity of seven fully polymerized and partly depolymerized lithium aluminosilicate glasses was investigated using impedance spectroscopy (IS). Lithium is the only mobile particle in these materials. Data derived from IS, i.e. activation energies, pre-exponential factors and diffusivities for lithium, are interpreted in light of Raman spectroscopic analyses of local structures in order to identify building units, which are crucial for lithium dynamics and migration. In polymerized glasses (compositional join LiAlSiO4-LiAlSi4O10) the direct current (DC) electrical conductivity continuously increases with increasing lithium content while lithium diffusivity is not affected by the Al/Si ratio in the glasses. Hence, the increase in electrical conductivity can be solely assigned to lithium concentration in the glasses. An excess of Li with respect to Al, i.e. the introduction of non-bridging oxygen into the network, causes a decrease in lithium mobility in the glasses. Activation energies in polymerized glasses (66 to 70 kJ mol(-1)) are significantly lower than those in depolymerized networks (76 to 78 kJ mol(-1)) while pre-exponential factors are nearly constant across all compositions. Comparison of the data with results for lithium silicates from the literature indicates a minimum in lithium diffusivity for glasses containing both aluminium tetrahedra and non-bridging oxygens. The findings allow a prediction of DC conductivity for a large variety of lithium aluminosilicate glass compositions.