Fig. 1: In vivo assembly and detection of eukaryotic nucleosome core in E. coli.
From: Creating a bacterium that forms eukaryotic nucleosome core particles
A schematic of the polycistronic construct for expression of Xenopus histones in E. coli. In (a, b, e, and f), “tag-H2A” is a histone H2A construct with an N-terminal S-tag and a hexahistidine (6 × His) tag, while “tag-H4” is a histone H4 construct with a C-terminal 6 × His tag. b Expression of Xenopus histones in E. coli Rosetta(DE3) grown at 37 °C, induced with (+) or without (−) 400 μM IPTG. T, total lysate; S, supernatant; P, pellet; M, protein standards. tag-H2A, 18.15 kDa; H2B, 13.64 kDa; H3, 15.07 kDa; tag-H4, 13.31 kDa. The figure is representative of three independent experiments. c A schematic of in situ micrococcal nuclease digestion assay for E. coli cells (ecMNase assay). d DNA fragmentation profiles of two E. coli strains grown at 37 °C, induced with 400 μM IPTG, using ecMNase assay. Arrows mark mono-, di- and tri-nucleosome bands. M, DNA standards. In (b) and (d), Ec-r-pET29a represents the control strain with pET29a, while Ec-r-pXen represents the nucleosome-forming strain. The figure is representative of three independent experiments. e Elution diagram of the nucleosome complexes from the size exclusion chromatography monitored by UV (280 nM) absorption (top), and SDS-PAGE analysis of fractions 27–46 (bottom). The 8–15 mL elution peak corresponds to these fractions. The figure is representative of three independent experiments. f A schematic of two polycistronic constructs for expression of Xenopus histones with H3 either deleted (pET-Xen-H3del) or truncated between amino acid residues 101–132 (pET-Xen-H3Δ). g Expression of Xenopus histones in E. coli strains Ec-r-pXen-H3del and Ec-r-pXen-H3Δ containing pET-Xen-H3del and pET-Xen-H3Δ, grown at 37 °C, induced with (+) or without (−) 400 μM IPTG, respectively. H3Δ, 11.55 kDa. The figure is representative of three independent experiments. h DNA fragmentation profiles of strains Ec-r-pXen-H3del and Ec-r-pXen-H3Δ using ecMNase assay. i AFM images of nucleosome complexes (bead-like structure) in Xenopus (left) and Ec-r-pXen (right), but not in Ec-r-pET29a (middle). The height profiles were provided in Supplementary Fig. 1e. Arrows indicate the nucleosome core. Bar length = 100 nm. The figure is representative of two independent experiments. Source data are provided as a Source Data file.
