In situ microfluidic dialysis for biological small-angle X-ray scattering

Owing to the demand for low sample consumption and automated sample changing capabilities at synchrotron small-angle X-ray (solution) scattering (SAXS) beamlines, X-ray microfluidics is receiving continuously increasing attention. Here, a remote-controlled microfluidic device is presented for simultaneous SAXS and ultraviolet absorption measurements during protein dialysis, integrated directly on a SAXS beamline. Microfluidic dialysis can be used for monitoring structural changes in response to buffer exchange or, as demonstrated, protein concentration. By collecting X-ray data during the concentration procedure, the risk of inducing protein aggregation due to excessive concentration and storage is eliminated, resulting in reduced sample consumption and improved data quality. The proof of concept demonstrates the effect of halted or continuous flow in the microfluidic device. No sample aggregation was induced by the concentration process at the levels achieved in these experiments. Simulations of fluid dynamics and transport properties within the device strongly suggest that aggregates, and possibly even higher-order oligomers, are preferentially retained by the device, resulting in incidental sample purification. Hence, this versatile microfluidic device enables investigation of experimentally induced structural changes under dynamically controllable sample conditions

Finding the Needle in the Haystack-the Use of Microfluidic Droplet Technology to Identify Vitamin-Secreting Lactic Acid Bacteria

Efficient screening technologies aim to reduce both the time and the cost required for identifying rare mutants possessing a phenotype of interest in a mutagenized population. In this study, we combined a mild mutagenesis strategy with high-throughput screening based on microfluidic droplet technology to identify Lactococcus lactis variants secreting vitamin B2 (riboflavin). Initially, we used a roseoflavin-resistant mutant of L. lactis strain MG1363, JC017, which secreted low levels of riboflavin. By using fluorescence-activated droplet sorting, several mutants that secreted riboflavin more efficiently than JC017 were readily isolated from the mutagenesis library. The screening was highly efficient, and candidates with as few as 1.6 mutations per million base pairs (Mbp) were isolated. The genetic characterization revealed that riboflavin production was triggered by mutations inhibiting purine biosynthesis, which is surprising since the purine nucleotide GTP is a riboflavin precursor. Purine starvation in the mutants induced overexpression of the riboflavin biosynthesis cluster ribABGH. When the purine starvation was relieved by purine supplementation in the growth medium, the outcome was an immediate downregulation of the riboflavin biosynthesis cluster and a reduction in riboflavin production. Finally, by applying the new isolates in milk fermentation, the riboflavin content of milk (0.99 mg/liter) was improved to 2.81 mg/liter, compared with 0.66 mg/liter and 1.51 mg/liter by using the wild-type strain and the original roseoflavin-resistant mutant JC017, respectively. The results obtained demonstrate how powerful classical mutagenesis can be when combined with droplet-based microfluidic screening technology for obtaining microorganisms with useful attributes