Toward a Boron-Doped Ultrananocrystalline Diamond Electrode-Based Dielectrophoretic Preconcentrator

This paper presents results on immunobeads-based isolation of rare bacteria and their capture at a boron-doped ultrananocrystalline diamond (BD-UNCD) electrode in a microfluidic dielectrophoretic preconcentrator. We systematically vary the bead surface chemistry and the BD-UNCD surface chemistry and apply dielectrophoresis to improve the specific and the nonspecific capture of bacteria or beads. Immunobeads were synthesized by conjugating antibodies to epoxy-/sulfate, aldehyde-/sulfate, or carboxylate-modified beads with or without poly­(ethylene glycol) (PEG) coimmobilization. The carboxylate-modified beads with PEG provided the highest capture efficiency (∼65%) and selectivity (∼95%) in isolating live Escherichia coli O157:H7 from cultures containing 1000 E. coli O157:H7 colony-forming units (cfu)/mL, or ∼500 E. coli O157:H7 and ∼500 E. coli K12 cfu/mL. Higher specificity was achieved with the addition of PEG to the antibody-functionalized bead surface, highest with epoxy-/sulfate beads (85−86%), followed by carboxylate-modified beads (76−78%) and aldehyde-/sulfate beads (74−76%). The bare BD-UNCD electrodes of the preconcentrator successfully withstood 240 kV/m for 100 min that was required for the microfluidic dielectrophoresis of 1 mL of sample. As expected, the application of dielectrophoresis increased the specific and the nonspecific capture of immunobeads at the BD-UNCD electrodes; however, the capture specificity remained unaltered. The addition of PEG to the antibody-functionalized BD-UNCD surface had little effect on the specificity in immunobeads capture. These results warrant the fabrication of electrical biosensors with BD-UNCD so that dielectrophoretic preconcentration can be performed directly at the biosensing electrodes

Genome-Wide Nucleosome Positioning Is Orchestrated by Genomic Regions Associated with DNase I Hypersensitivity in Rice

<div><p>Nucleosome positioning dictates the DNA accessibility for regulatory proteins, and thus is critical for gene expression and regulation. It has been well documented that only a subset of nucleosomes are reproducibly positioned in eukaryotic genomes. The most prominent example of phased nucleosomes is the context of genes, where phased nucleosomes flank the transcriptional starts sites (TSSs). It is unclear, however, what factors determine nucleosome positioning in regions that are not close to genes. We mapped both nucleosome positioning and DNase I hypersensitive site (DHS) datasets across the rice genome. We discovered that DHSs located in a variety of contexts, both genic and intergenic, were flanked by strongly phased nucleosome arrays. Phased nucleosomes were also found to flank DHSs in the human genome. Our results suggest the barrier model may represent a general feature of nucleosome organization in eukaryote genomes. Specifically, regions bound with regulatory proteins, including intergenic regions, can serve as barriers that organize phased nucleosome arrays on both sides. Our results also suggest that rice DHSs often span a single, phased nucleosome, similar to the H2A.Z-containing nucleosomes observed in DHSs in the human genome.</p></div

Association IPA1-binding sites with phased nucleosomes.

<p>An example of phased nucleosome arrays that flank an intergenic IPA1-binding site on rice chromosome 8. This binding site is overlapped with a DHS (red arrow). The distribution of MNase-seq data (dyad density calculated from paired-end reads by NucleR) and DNase-seq data (density calculated by F-seq) were used to present the nucleosome and DHS positions. Phased nucleosomes and DHS regions were also schematically marked.</p

Phased nucleosome arrays flanked TSSs of rice genes.

<p>(A) Nucleosome positioning profile associated with active genes. Phased nucleosome arrays are detectable after the TSSs. (B) Nucleosome positioning profile associated with non-expressed genes. Phased nucleosome arrays are detected on either side of the TSSs. (C) Distribution of DHS length for five different DHS categories. Note: the length of DHSs associated with proximal promoters (black line) are more variable than the lengths of other DHSs. (D) Heatmap of nucleosome positioning associated with active genes. Left panel: All expressed genes were sorted by the length of DHSs located in proximal promoters. The 5′ ends of the MNase-seq reads were mapped within 1 kb upstream and 1 kb downstream of the TSS of each gene to show the boundaries of nucleosomes core and linker. The red line on the left heatmap indicates the boundaries of DHSs. With the same order of the genes as in the left panel, the 5′ ends of DNase-seq reads (middle panel) and the fragments per kilobase of exon per million fragments mapped (FPKM) value log10 transformation (right panel) were mapped to show the DNase I sensitivity and the expression level of each gene, respectively.</p

Patterns of nucleosome positioning around DHSs in the rice genome.

<p>The nucleosome positioning profiles were shown around the DHSs located in (A) proximal promoters (within 200 bp upstream of a TSS); (B) distal promoters (200–1000 bp upstream of a TSS); (C) within genes; (D) downstream regions of genes (within 200 bp downstream of gene transcription); (E) intergenic regions and (F) 10,000 randomly selected genomic regions. Y-axes show normalized reads (read number in per bp genome in per million reads) within 1 kb upstream and downstream around the DHSs. Ellipses indicate the nucleosomes within (grey) and outside (black) of DHSs. Arrows in (A–D) indicate the direction of gene transcription. Single-end MNase-seq reads were used in mapping nucleosome positioning.</p

Boxplots of estimated lengths of linkers (A) and spacing (B) between the phased nucleosomes mapped close to DHSs.

<p>"***","**","*" indicated <i>p</i><0.001, <i>p</i><0.01, <i>p</i><0.05, respectively, for the comparison of linker length/spacing between intergenic region and either regions within genes (“gene”) or in proximal promoters (“200 bp”).</p

Patterns of nucleosome positioning around DHSs in the human genome.

<p>DHSs (data from CD4+ T cell line) were also divided into five different categories based on their genomic locations: <b>(A)</b> proximal promoters (within 200 bp upstream of a TSS); <b>(B)</b> within genes; and <b>(C)</b> intergenic regions. Y-axes show normalized MNase-seq reads (read number in per bp genome in per million reads). Zero on the x-axes indicates the most sensitive site of the aligned DHSs. Ellipses indicate phased nucleosomes with H2A.Z. Arrows in (A, B) indicate the direction of gene transcription.</p

Nucleosome positioning profiles associated with DHSs with different lengths in proximal promoters.

<p>(A) DHSs in 320–480 bp. (B) DHSs in 200–320 bp. (C) DHSs in 140–200 bp. (D) DHSs in 80–140 bp. (E) DHSs in 20–80 bp. Y-axes show normalized reads of DNase-seq and MNase-seq. Zero on the X-axis indicates the boundary of DHSs toward short arm of the chromosomes. Black ellipses indicate the inferred nucleosomes. Grey ellipses indicate -1 nucleosomes within DHSs. Black vertical lines in (d, e) indicate the left and right boundaries of the DHSs inferred by DNase-seq reads.</p

Efficient Production of a Functional Human Milk Oligosaccharide 3′-Sialyllactose in Genetically Engineered Escherichia coli

3′-Sialyllactose (3′-SL) is one of the most important and simplest sialylated human milk oligosaccharides. In this study, a plasmid-based pathway optimization along with chromosomal integration strategies was applied for 3′-SL production. Specifically, the precursor CMP-Neu5Ac synthesis pathway genes and α2,3-sialyltransferase-encoding gene were introduced into Escherichia coli BL21­(DE3)­ΔlacZ to realize 3′-SL synthesis. Genes nanA and nanK involved in Neu5Ac catabolism were further deleted to reduce the metabolic flux of competitive pathway. Several α2,3-sialyltransferases from different species were selected to evaluate the sialylation effect. The precursor pools were balanced and improved by optimizing key enzyme expression involved in the UDP-GlcNAc and CMP-Neu5Ac synthesis pathway. Finally, an additional α2,3-sialyltransferase expression cassette was integrated into chromosome to maximize 3′-SL synthesis, and 4.5 g/L extracellular 3′-SL was produced at a shake-flask level. The extracellular 3′-SL concentration was raised to 23.1 g/L in a 5 L bioreactor fermentation, which represents the highest extracellular value ever reported

Phase-Separated Synthetic Organelles Based on Intrinsically Disordered Protein Domain for Metabolic Pathway Assembly in <i>Escherichia coli</i>

Extensive research efforts have been focused on spatial organization of biocatalytic cascades or catalytic networks in confined cellular environments. Inspired by the natural metabolic systems that spatially regulate pathways via sequestration into subcellular compartments, formation of artificial membraneless organelles through expressing intrinsically disordered proteins in host strains has been proven to be a feasible strategy. Here we report the engineering of a synthetic membraneless organelle platform, which can be used to extend compartmentalization and spatially organize pathway sequential enzymes. We show that heterologous overexpression of the RGG domain derived from the disordered P granule protein LAF-1 in an Escherichia coli strain can form intracellular protein condensates via liquid–liquid phase separation. We further demonstrate that different clients can be recruited to the synthetic compartments via directly fusing with the RGG domain or cooperating with different protein interaction motifs. Using the 2′-fucosyllactose de novo biosynthesis pathway as a model system, we show that clustering sequential enzymes into synthetic compartments can effectively increase the titer and yield of the target product compared to strains with free-floating pathway enzymes. The synthetic membraneless organelle system constructed here gives a promising approach in the development of microbial cell factories, wherein it could be used for the compartmentalization of pathway enzymes to streamline metabolic flux