Journal: Methods in enzymology

Article Title: Recent Advances in the Genetic Manipulation of Methylosinus trichosporium OB3b

doi: 10.1016/bs.mie.2018.02.011

Figure Lengend Snippet: Bacterial Strains

Article Snippet: E. coli S17-1 , LB (Difco).

Techniques:

Journal: Journal of Industrial Microbiology & Biotechnology

Article Title: Engineering sequence and selectivity of late-stage C-H oxidation in the MycG iterative cytochrome P450

doi: 10.1093/jimb/kuab069

Figure Lengend Snippet: HPLC chromatograms of M-IV bioconversion with Escherichia coli expressing wild-type MycG and its mutants RM92, RM96, and RM98. The solid blue line represents the chromatograms at 220 nm. The dotted red lines indicate chromatograms at 280 nm. The upper panels show the UV spectrograms of M-II and M-V (1 mM). *The retention times of M-II and M-V are almost the same, and the peaks overlap. The absorption maximum for M-II occurs at 220 nm, while that of M-V is at 220 and 280 nm (upper panels). At same concentrations, M-II and M-V have equal peak areas at 220 nm. In addition, M-V has same peak areas at 220 and 280 nm. Therefore, M-V can be detected with a chromatogram at 280 nm, and M-II can be detected by subtracting the chromatogram at 280 nm from that at 220 nm.

Article Snippet: pMG522, pMG523, pMG524, pMG525, and pMG526 were introduced into the previously prepared mycG disruption mutant M. griseorubida TPMA0025 by intergeneric conjugation with E. coli S17-1 (Anzai et al., ).

Techniques: Expressing

Journal: Nature Communications

Article Title: Pancreatic glycoprotein 2 is a first line of defense for mucosal protection in intestinal inflammation

doi: 10.1038/s41467-021-21277-2

Figure Lengend Snippet: a Fecal total IgA concentration in WT and Gp2 –/– mice, as determined by ELISA ( n = 5). N.S. not significant (two-tailed unpaired t -test). Data are presented as mean values ± SEM. b 16S rRNA gene sequencing of fecal bacteria. Phylum levels of bacteria are shown. c qPCR analysis of mucosal bacteria from WT ( n = 9) and Gp2 –/– ( n = 8) mice were examined. * p < 0.05., N.S. indicates not significant (two-tailed unpaired t -test). Data are presented as mean values ± SEM. d Survival ratio and body weight change of WT and Gp2 –/– mice with acute colitis ( n = 12/group). * p < 0.05, ** p < 0.01 (two-tailed unpaired t -test). Data are presented as mean values ± SEM. e Colon length and representative pictures of colon are shown, intact; WT ( n = 3), KO ( n = 4), and colitis; WT, KO ( n = 12). *** p < 0.0001 (two-tailed unpaired t -test). Data are presented as mean values ± SEM. f Hematoxylin and eosin staining of colon at day 8 of DSS treatment are shown. Scale bars: 100 μm. Data are representative of three independent experiments. g Flow cytometry analysis of infiltrated neutrophils is shown. Representative data are shown. h Immunohistochemical analysis of colitis (DSS 2%, day 8) colon with luminal contents of WT and Gp2 –/– mice. Bacteria (red), MUC2 (green), DAPI (blue). Scale bars: upper panels, 100 μm; lower panels, 20 μm. Data are representative of three independent experiments. i Serum anti- E. coli IgM, intact ( n = 4), and colitis ( n = 5), IgA, intact WT ( n = 6), KO ( n = 10), and colitis WT ( n = 15), KO ( n = 11), IgG intact WT ( n = 12), KO ( n = 4), and colitis WT ( n = 11), KO ( n = 7) in intact and colitis WT and Gp2 –/– mice were analyzed by ELISA (Kruskal-Wallis test followed by Mann–Whitney U test). Data are presented as mean values ± SEM. j Body weight changes after the induction of acute TNBS-induced colitis in WT and Gp2 –/– mice. *** p < 0.01 (two-tailed unpaired t -test). Data are presented as mean values ± SEM. k Immunohistochemical analysis of colon and luminal contents during TNBS-induced colitis in Gp2 Panc and control mice. EUB338 bacteria (red), MUC2 (green), DAPI (blue). Scale bars: upper panels, 100 μm; lower panels, 20 μm. Data are representative of two independent experiments. Source data are provided as a Source Data file.

Article Snippet: As previously described, E. coli strain S-17 were freeze-dried by Techno Suruga Laboratory.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Sequencing, Bacteria, Staining, Flow Cytometry, Immunohistochemical staining, MANN-WHITNEY, Control

Journal: Nature Communications

Article Title: Pancreatic glycoprotein 2 is a first line of defense for mucosal protection in intestinal inflammation

doi: 10.1038/s41467-021-21277-2

Figure Lengend Snippet: a Luminal contents of colon were stained with GP2 (red) and EUB338 (blue in left bottom and green in right bottom) were shown. Data are representative of three independent experiments. b Fecal unbound GP2 in WT and Gp2 –/– mice with or without acute colitis were measured by ELISA (WT, n = 8; Gp2 –/– , n = 4). N.S. indicates not significant (two-tailed unpaired t -test). Data are presented as mean values ± SEM. c Percentage of rGP2-bound fecal bacteria isolated from Gp2 –/– mice, PBS and rGP2 Intact n = 7, PBS colitis ( n = 7), rGP2 colitis ( n = 6). * p = 0.017 (two-tailed unpaired t -test). Data are presented as mean values ± SEM. d Representative flow cytometry analysis of fecal bacteria isolated from WT and Gp2 –/– mice with or without colitis were shown. e E. coli number of non-selected (rGP2 MACS −) and selected by MACS (rGP2 MACS +) fecal bacteria ( n = 9 /group). N.D.: not detected. Data are presented as mean values ± SEM. Source data are provided as a Source Data file.

Article Snippet: As previously described, E. coli strain S-17 were freeze-dried by Techno Suruga Laboratory.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Bacteria, Isolation, Flow Cytometry

Journal: Nature Communications

Article Title: Pancreatic glycoprotein 2 is a first line of defense for mucosal protection in intestinal inflammation

doi: 10.1038/s41467-021-21277-2

Figure Lengend Snippet: a Flow cytometry of E. coli strain S-17-GFP bound with or without rGP2 were performed. Representative data of three independent experiments were shown. *** p < 0.0001 (two-tailed unpaired t -test). Data are presented as mean values ± SEM. b CFU assay of S-17-GFP incubated with recombinant GP2 ( n = 4). N.S. indicates not significant (two-tailed unpaired t -test). c Approximately 1 × 10 6 CFU of WT S-17 cells and FimH-deficient S-17 cells (ΔFimH) were incubated with 1 μg of mouse or human GP2, and FACS analysis was conducted with anti-GP2 antibody. Representative data of three independent experiments are shown. d , e Immunohistochemical analysis of colon specimens isolated from ligated colon of S-17-GFP, pre-incubated with PBS or recombinant GP2 were shown. Scale bars, 50 μm (left panel) and 10 μm (right panels). f CFU assay of S-17-GFP injected looped colon (2% DSS day 6), n = 5 /group were shown. * p = 0.017 (two-tailed unpaired t -test). Data are presented as mean values ± SEM. g The amount of anti-GP2 IgG in the luminal contents of intact mice ( n = 5), DSS-treated mice ( n = 6), mice systemically administered GP2-bound heat-killed (70 °C, 30 min) S-17 cells ( n = 4), and GP2 knockout (GP2KO) mice immunized with rmGP2 and complete Freund’s adjuvant (CFA) as positive controls was determined by ELISA on day 27 after immunization ( n = 4). *** p < 0.01 (one-way ANOVA). Data are presented as mean values ± SEM. h Neutralizing assay for the binding of rGP2 to S-17 cells. Medium (Tris-HCL). Luminal contents from anti-GP2 IgG antibody–negative (intact) or -positive (DSS-treated) mice were pre-incubated with GP2 and added to S-17 cells. The GP2-bound SYTO9 + S-17 population is shown. Data are representative of two independent experiments. Source data are provided as a Source Data file.

Article Snippet: As previously described, E. coli strain S-17 were freeze-dried by Techno Suruga Laboratory.

Techniques: Flow Cytometry, Two Tailed Test, Colony-forming Unit Assay, Incubation, Recombinant, Immunohistochemical staining, Isolation, Injection, Knock-Out, Adjuvant, Enzyme-linked Immunosorbent Assay, Neutralizing Assay, Binding Assay

Journal: Frontiers in Microbiology

Article Title: Outer Membrane Vesicles and Soluble Factors Released by Probiotic Escherichia coli Nissle 1917 and Commensal ECOR63 Enhance Barrier Function by Regulating Expression of Tight Junction Proteins in Intestinal Epithelial Cells

doi: 10.3389/fmicb.2016.01981

Figure Lengend Snippet: Escherichia coli reference collection (ECOR) strains bearing tcpC gene increase TER in T-84 cell monolayers. The effect on TER of the indicated Escherichia coli strains from the ECOR collection was analyzed in T-84 monolayers in comparison with the probiotic EcN and the laboratory strain HB101. TER values were measured before and after 24 h incubation with cell-free supernatants (CF-SN) (2 mg protein/ml) collected from LB cultures ( n = 3 independent biological replicates). Data are presented as percentage of increase in TER from the initial value. a significance against untreated control cells ( p ≤ 0.001).

Article Snippet: The E. coli strains S17 (λpir) (Biomedal) and EB6193 (RP4-2 tet Mu-1 kan::Tn 7 integrant; leu-63 ::IS 10 recA1 creC510 hsdR17 endA1 zbf-5 uidA (MuI):: pir + thi Sp r /Sm r ) ( ) were used for cloning and propagation of the suicide plasmid pUT-miniTn5 Tc and derived recombinant constructs.

Techniques: Comparison, Incubation, Control