Analysis of a phase variable restriction modification system of the human gut symbiont Bacteroides fragilis

The genomes of gut Bacteroidales contain numerous invertible regions, many of which contain promoters that dictate phase-variable synthesis of surface molecules such as polysaccharides, fimbriae, and outer surface proteins. Here, we characterize a different type of phase-variable system of Bacteroides fragilis, a Type I restriction modification system (R-M). We show that reversible DNA inversions within this R-M locus leads to the generation of eight specificity proteins with distinct recognition sites. In vitro grown bacteria have a different proportion of specificity gene combinations at the expression locus than bacteria isolated from the mammalian gut. By creating mutants, each able to produce only one specificity protein from this region, we identified the R-M recognition sites of four of these S-proteins using SMRT sequencing. Transcriptome analysis revealed that the locked specificity mutants, whether grown in vitro or isolated from the mammalian gut, have distinct transcriptional profiles, likely creating different phenotypes, one of which was confirmed. Genomic analyses of diverse strains of Bacteroidetes from both host-associated and environmental sources reveal the ubiquity of phase-variable R-M systems in this phylum

Automated Device for Multi-Stage Paper-Based Assays Enabled by an Electroosmotic Pumping Valve

This work presents the use of electroosmotic flow generation in porous media in combination with a hydrophobic air gap to create a controllable valve capable of operating in either finite dosing or continuous flow mode, enabling the implementation of multi-step biochemical assays on paper-based devices. A hierarchical superhydrophobic surface placed between two paper pads creates an air gap, keeping the valve nominally closed. To open the valve, a pair of electrodes are activated to generate electroosmotic pressure that overcomes the barrier. The study provides an experimentally validated model describing the governing parameters, and a detailed investigation of the closed valve stability. From these, a straightforward design for a compact and fully automated device is derived. The design is based on paper pads placed on printed circuit boards (PCB), equipped with heating and actuation electrodes and additional power and logic capabilities. The device is applied to the detection of SARS-CoV-2 sequences directly from raw saliva samples, using loop-mediated isothermal amplification (LAMP) requiring sample lysis followed by enzymatic deactivation and sample distribution to multiple amplification pads. Since PCB costs scale favorably with mass production, we believe that this approach could lead to low-cost diagnostic devices with the sensitivity of amplification methods