e coli dh5α  (Thermo Fisher)


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    Structured Review

    Thermo Fisher e coli dh5α
    Influence of different chlorophyllin concentrations on the growth of ( A ) Escherichia coli <t>DH5α,</t> ( B ) Bacillus subtilis 168 and ( C ) Escherichia coli NR698 (initial cell number: ~10 8 cfu/mL). Bacteria were cultured for 24 h at 37 °C exposed to light (12 mW/cm 2 ; bright plots, upper row) or in darkness (grey plots, lower row). To determine the lower limit of efficacy, additional chlorophyllin concentrations were tested: ( D ) Escherichia coli DH5α, 20↑100 mg/L ( E ) Bacillus subtilis 168, 1.0↓0.01 mg/L and ( F ) Escherichia coli NR698, 1.0↓0.01 mg/L. Depicted are means (growth curves) together with corresponding 95% confidence limits (bar charts). Blue numbers indicate respective chlorophyllin concentrations. * p
    E Coli Dh5α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "What an Escherichia coli Mutant Can Teach Us About the Antibacterial Effect of Chlorophyllin"

    Article Title: What an Escherichia coli Mutant Can Teach Us About the Antibacterial Effect of Chlorophyllin

    Journal: Microorganisms

    doi: 10.3390/microorganisms7020059

    Influence of different chlorophyllin concentrations on the growth of ( A ) Escherichia coli DH5α, ( B ) Bacillus subtilis 168 and ( C ) Escherichia coli NR698 (initial cell number: ~10 8 cfu/mL). Bacteria were cultured for 24 h at 37 °C exposed to light (12 mW/cm 2 ; bright plots, upper row) or in darkness (grey plots, lower row). To determine the lower limit of efficacy, additional chlorophyllin concentrations were tested: ( D ) Escherichia coli DH5α, 20↑100 mg/L ( E ) Bacillus subtilis 168, 1.0↓0.01 mg/L and ( F ) Escherichia coli NR698, 1.0↓0.01 mg/L. Depicted are means (growth curves) together with corresponding 95% confidence limits (bar charts). Blue numbers indicate respective chlorophyllin concentrations. * p
    Figure Legend Snippet: Influence of different chlorophyllin concentrations on the growth of ( A ) Escherichia coli DH5α, ( B ) Bacillus subtilis 168 and ( C ) Escherichia coli NR698 (initial cell number: ~10 8 cfu/mL). Bacteria were cultured for 24 h at 37 °C exposed to light (12 mW/cm 2 ; bright plots, upper row) or in darkness (grey plots, lower row). To determine the lower limit of efficacy, additional chlorophyllin concentrations were tested: ( D ) Escherichia coli DH5α, 20↑100 mg/L ( E ) Bacillus subtilis 168, 1.0↓0.01 mg/L and ( F ) Escherichia coli NR698, 1.0↓0.01 mg/L. Depicted are means (growth curves) together with corresponding 95% confidence limits (bar charts). Blue numbers indicate respective chlorophyllin concentrations. * p

    Techniques Used: Cell Culture

    Effect of chlorophyllin on the early growth phase of ( A ) Escherichia coli DH5α, ( B ) Bacillus subtilis 168, and ( C ) Escherichia coli NR698. Liquid cultures of bacteria (initial cell number: 10 6 cfu/mL) were incubated in Erlenmeyer flasks in standard LB medium (red) and in presence of a chlorophyllin concentration of 22 mg/L (green). Cells grew either illuminated with 12 mW/cm 2 (bright plots, upper row) or in darkness (grey plots, lower row). Depicted are measured values (circles) and fitted curves (solid lines) with corresponding 95% confidence limits (red and green areas). Dashed lines describe growth of example cultures in LB medium + MeOH/KOH (solvent of chlorophyllin).
    Figure Legend Snippet: Effect of chlorophyllin on the early growth phase of ( A ) Escherichia coli DH5α, ( B ) Bacillus subtilis 168, and ( C ) Escherichia coli NR698. Liquid cultures of bacteria (initial cell number: 10 6 cfu/mL) were incubated in Erlenmeyer flasks in standard LB medium (red) and in presence of a chlorophyllin concentration of 22 mg/L (green). Cells grew either illuminated with 12 mW/cm 2 (bright plots, upper row) or in darkness (grey plots, lower row). Depicted are measured values (circles) and fitted curves (solid lines) with corresponding 95% confidence limits (red and green areas). Dashed lines describe growth of example cultures in LB medium + MeOH/KOH (solvent of chlorophyllin).

    Techniques Used: Incubation, Concentration Assay

    ( A ) Chemical structure of isolated chlorophyllin. ( B ) Alterations of chlorophyllin’s absorption spectrum in darkness and ( C ) exposed to light with 12 mW/cm 2 . ( D ) Reduction of chlorophyllin concentration exposed to different light intensities. ( E ) Required light exposure times to sterilize an E. coli DH5α suspension culture initial cell number: ~10 6 cfu/mL) with a light intensity of 12 mW/cm 2 using different chlorophyllin concentrations. ( F ) Required light exposure times to sterilize an E. coli DH5α suspension culture (initial cell number: ~10 6 cfu/mL) with a chlorophyllin concentration of 25 mg/L and different light intensities. For 12.5% light intensity no sterilization was observed within 300 min. Depicted are means ± standard deviation.
    Figure Legend Snippet: ( A ) Chemical structure of isolated chlorophyllin. ( B ) Alterations of chlorophyllin’s absorption spectrum in darkness and ( C ) exposed to light with 12 mW/cm 2 . ( D ) Reduction of chlorophyllin concentration exposed to different light intensities. ( E ) Required light exposure times to sterilize an E. coli DH5α suspension culture initial cell number: ~10 6 cfu/mL) with a light intensity of 12 mW/cm 2 using different chlorophyllin concentrations. ( F ) Required light exposure times to sterilize an E. coli DH5α suspension culture (initial cell number: ~10 6 cfu/mL) with a chlorophyllin concentration of 25 mg/L and different light intensities. For 12.5% light intensity no sterilization was observed within 300 min. Depicted are means ± standard deviation.

    Techniques Used: Isolation, Concentration Assay, Standard Deviation

    Schematic presentation of evaluation of CFU ability after incubation to chlorophyllin. Differently dense liquid cultures of ( A ) Escherichia coli DH5α, ( B ) Bacillus subtilis 168, and ( C ) Escherichia coli NR698 were supplemented with different chlorophyllin concentrations between 0.1 and 25 mg/L. Cells grew in 96-well matrix plates either illuminated with 12 mW/cm 2 ( A – C ) or protected from light ( D – F ). Samples (2.5 µL) were drawn at different time points and transferred onto LB agar plates. After overnight incubation at 37 °C in the dark, colony growth was analyzed. Dot size quantifies colony growth. Original pictures of the agar plates can be found in Figure S1 .
    Figure Legend Snippet: Schematic presentation of evaluation of CFU ability after incubation to chlorophyllin. Differently dense liquid cultures of ( A ) Escherichia coli DH5α, ( B ) Bacillus subtilis 168, and ( C ) Escherichia coli NR698 were supplemented with different chlorophyllin concentrations between 0.1 and 25 mg/L. Cells grew in 96-well matrix plates either illuminated with 12 mW/cm 2 ( A – C ) or protected from light ( D – F ). Samples (2.5 µL) were drawn at different time points and transferred onto LB agar plates. After overnight incubation at 37 °C in the dark, colony growth was analyzed. Dot size quantifies colony growth. Original pictures of the agar plates can be found in Figure S1 .

    Techniques Used: Incubation

    Fluorescence microscopic images of ( A ) Escherichia coli DH5α and ( B ) Escherichia coli NR698 after exposure to chlorophyllin (red fluorescence). Assumed modes of action of chlorophyllin against Gram-positive and Gram-negative bacteria. ( C ) Chlorophyllin (Chl) molecules cannot pass the intact outer membrane of Gram-negative bacteria. ( D ) Chlorophyllin is degraded in light. Degradation products can enter both Gram-negative and Gram-positive cells. The red crosses indicate cell death.
    Figure Legend Snippet: Fluorescence microscopic images of ( A ) Escherichia coli DH5α and ( B ) Escherichia coli NR698 after exposure to chlorophyllin (red fluorescence). Assumed modes of action of chlorophyllin against Gram-positive and Gram-negative bacteria. ( C ) Chlorophyllin (Chl) molecules cannot pass the intact outer membrane of Gram-negative bacteria. ( D ) Chlorophyllin is degraded in light. Degradation products can enter both Gram-negative and Gram-positive cells. The red crosses indicate cell death.

    Techniques Used: Fluorescence

    2) Product Images from "Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1"

    Article Title: Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1

    Journal: Journal of Biomedical Science

    doi: 10.1186/1423-0127-16-83

    Western blot analysis of the recombination proteins . Identification of the expression of the His-tagged recombinant proteins in E. Coli DH5α by Western blot. 1, DHFR; 2, rHMGB1 A box; 3, rHMGB1 B box; 4, rHMGB1; 5, tHMGB1; 6, mHMGB1 -211-215; 7, mHMGB1- 206-215; 8, mHMGB1 -201-215; 9, mHMGB1 -196-215; 10, mHMGB1 -191-215; 11, mHMGB1 -186-200; 12, mHMGB1 -196-210; 13, mHMGB1 -196-205; 14, mHMGB1 -198-207; 15, mHMGB1 -201-210; 16, mHMGB1 -201-205.
    Figure Legend Snippet: Western blot analysis of the recombination proteins . Identification of the expression of the His-tagged recombinant proteins in E. Coli DH5α by Western blot. 1, DHFR; 2, rHMGB1 A box; 3, rHMGB1 B box; 4, rHMGB1; 5, tHMGB1; 6, mHMGB1 -211-215; 7, mHMGB1- 206-215; 8, mHMGB1 -201-215; 9, mHMGB1 -196-215; 10, mHMGB1 -191-215; 11, mHMGB1 -186-200; 12, mHMGB1 -196-210; 13, mHMGB1 -196-205; 14, mHMGB1 -198-207; 15, mHMGB1 -201-210; 16, mHMGB1 -201-205.

    Techniques Used: Western Blot, Expressing, Recombinant

    Antibacterial activity assay of eleven different deleted mutants lacking different several amino acid residues in C-terminal acidic tail region of HMGB1 with the method of dilution in the test tubes . The antibacterial activities of eleven different deleted mutants lacking different several amino acid residues in C-terminal acidic tail region of HMGB1 were compared by the method of dilution in the test tubes. The bacteria used in this experiment included SA, JM109, ATCC 25922, DH5α and PA. Each experiment was performed in triplicate and repeated three times. DHFR protein purified by the same system was used as a negative control. Data showed as mean ± standard deviation and were analyzed with One-Way ANOVA. * P
    Figure Legend Snippet: Antibacterial activity assay of eleven different deleted mutants lacking different several amino acid residues in C-terminal acidic tail region of HMGB1 with the method of dilution in the test tubes . The antibacterial activities of eleven different deleted mutants lacking different several amino acid residues in C-terminal acidic tail region of HMGB1 were compared by the method of dilution in the test tubes. The bacteria used in this experiment included SA, JM109, ATCC 25922, DH5α and PA. Each experiment was performed in triplicate and repeated three times. DHFR protein purified by the same system was used as a negative control. Data showed as mean ± standard deviation and were analyzed with One-Way ANOVA. * P

    Techniques Used: Activity Assay, Purification, Negative Control, Standard Deviation

    Antibacterial activity assay of rHMGB1, tHMGB1, rHMGB1 A box, B box, and C peptide with the method of dilution in the test tubes . The antibacterial activities of rHMGB1, tHMGB1, rHMGB1 A box, B box, and C peptide were compared by the method of dilution in the test tubes as described in the Materials and Methods . The bacteria used in this experiment included SA, JM109, ATCC 25922, DH5α and PA. Each experiment was performed in triplicate and repeated three times. DHFR protein purified by the same system was used as a negative control. Data showed as mean ± standard deviation and were analyzed with One-Way ANOVA. * P
    Figure Legend Snippet: Antibacterial activity assay of rHMGB1, tHMGB1, rHMGB1 A box, B box, and C peptide with the method of dilution in the test tubes . The antibacterial activities of rHMGB1, tHMGB1, rHMGB1 A box, B box, and C peptide were compared by the method of dilution in the test tubes as described in the Materials and Methods . The bacteria used in this experiment included SA, JM109, ATCC 25922, DH5α and PA. Each experiment was performed in triplicate and repeated three times. DHFR protein purified by the same system was used as a negative control. Data showed as mean ± standard deviation and were analyzed with One-Way ANOVA. * P

    Techniques Used: Activity Assay, Purification, Negative Control, Standard Deviation

    3) Product Images from "Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74?sp chitinase inclusions, Cry crystals and spores for applied use"

    Article Title: Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74?sp chitinase inclusions, Cry crystals and spores for applied use

    Journal: Microbial Cell Factories

    doi: 10.1186/1475-2859-13-15

    Confirmation of the intracellular location of ChiA74∆s p in Escherichia coli and Bacillus thuringiensis 4Q7 using a chimeric construct with the green fluorescent protein (GFP) gene. (A) Schematic illustration that shows the fusion of chiA74 ∆s p with gfp . Panel 1, two constructs were developed, under regulation of the chiA74 wildtype promoter (wp) or under control of the cytA - p/ STAB-SD system. Lollipop indicates transcriptional terminator. Oligonucleotides used to make chimeric construct with gfp are shown in Table 1 . Panel 2, confirmation of chimeric constructs by PCR, primers used for amplification are showed in parenthesis and in Table 1 . L, 1 kb (kilobase) DNA Ladder (New England Biolabs); Lane 1, pEHchiA74∆ sp - gfp (primers: chiA74-1, chiA74-3); lane 2, pEHchiA74∆ sp - gfp (primers: chiA74-1, gfp-3); lane 3, pEHchiA74∆ sp - gfp (primers: gfp-1, chiA74-4); lane 4, pEB chi A74∆sp- gfp (primers: cytSTAB-1, chiA74-4); Lane 5, pEB chi A74∆sp- gfp (primers: cytSTAB-1, gfp-3); lane 6, pEB chi A74∆sp- gfp (primers: gfp-1, chiA74-4). (B) Phase contrast (left) and fluorescence micrographs (right) of recombinant strains of E. coli and B. thuringiensis strain 4Q7 expressing the chimeric protein ChiA74∆sp-GFP. Panel 1, E. coli- pEHchiA74∆ sp - gfp ; panel 2, 4Q7 - pEHchiA74∆ sp - gfp ; panel 3, E. coli- pEBchiA74∆ sp - gfp ; panel 4, 4Q7 - pEBchiA74∆ sp - gfp ; panel 5, E. coli DH5α; panel 6, 4Q7. Samples of recombinant strains of B. thuringiensis were collected at ~ 72 h.
    Figure Legend Snippet: Confirmation of the intracellular location of ChiA74∆s p in Escherichia coli and Bacillus thuringiensis 4Q7 using a chimeric construct with the green fluorescent protein (GFP) gene. (A) Schematic illustration that shows the fusion of chiA74 ∆s p with gfp . Panel 1, two constructs were developed, under regulation of the chiA74 wildtype promoter (wp) or under control of the cytA - p/ STAB-SD system. Lollipop indicates transcriptional terminator. Oligonucleotides used to make chimeric construct with gfp are shown in Table 1 . Panel 2, confirmation of chimeric constructs by PCR, primers used for amplification are showed in parenthesis and in Table 1 . L, 1 kb (kilobase) DNA Ladder (New England Biolabs); Lane 1, pEHchiA74∆ sp - gfp (primers: chiA74-1, chiA74-3); lane 2, pEHchiA74∆ sp - gfp (primers: chiA74-1, gfp-3); lane 3, pEHchiA74∆ sp - gfp (primers: gfp-1, chiA74-4); lane 4, pEB chi A74∆sp- gfp (primers: cytSTAB-1, chiA74-4); Lane 5, pEB chi A74∆sp- gfp (primers: cytSTAB-1, gfp-3); lane 6, pEB chi A74∆sp- gfp (primers: gfp-1, chiA74-4). (B) Phase contrast (left) and fluorescence micrographs (right) of recombinant strains of E. coli and B. thuringiensis strain 4Q7 expressing the chimeric protein ChiA74∆sp-GFP. Panel 1, E. coli- pEHchiA74∆ sp - gfp ; panel 2, 4Q7 - pEHchiA74∆ sp - gfp ; panel 3, E. coli- pEBchiA74∆ sp - gfp ; panel 4, 4Q7 - pEBchiA74∆ sp - gfp ; panel 5, E. coli DH5α; panel 6, 4Q7. Samples of recombinant strains of B. thuringiensis were collected at ~ 72 h.

    Techniques Used: Construct, Polymerase Chain Reaction, Amplification, Fluorescence, Recombinant, Expressing

    Expression of ChiA74Δsp in Escherichia coli DH5α and Bacillus thuringiensis 4Q7. (A) Schematic illustration of the strategy used to delete the secretion signal peptide-encoding sequence (shown as a rectangle inside the open reading frame) of chiA74 to generate chiA74∆ s p. Two constructs were developed, the first under regulation of wildtype promoter (wp) and the second under control of the strong cytA-p /STAB-SD promoter system. Lollipop indicates the putative transcriptional terminator site. Oligonucleotide sequences used to delete the signal peptide-coding sequence are shown in Table 1 . (B) Evaluation of endochitinase activity using solubilized intracellular proteins produced in E. coli. Panel 1: Zymogram using 4-MU-(GlcNAc) 3 for detection. Lane 1, E. coli- pEHchiA74∆s p ; lane 2, without sample; lane 3, E. coli- pEBchiA74∆s p; lane 4, E. coli DH5α . Black arrow indicates the position of ChiA74∆s p in recombinant E. coli strains . Panel 2: (a) E. coli- pEHchiA74∆s p , (b) E. coli- pEBchiA74∆s p. No endochitinase activity was observed with E. coli DH5α . (C) Phase contrast microscopy of recombinant strains of B. thuringiensis . Panel 1, 4Q7; panel 2, 4Q7 - pEHchiA74∆s p ; panel 3, 4Q7 - pEBchiA74∆s p . Black arrows indicate the positions of ChiA74∆s p inclusions. (D) Endochitinase activity determined using solubilized intracellular proteins of recombinant strains of B. thuringiensis. Panel 1: lane 1, 4Q7; lane 2, 4Q7 - pEHchiA74∆s p ; lane 3, 4Q7 - pEBchiA74∆s p. Black arrow indicates the position of ChiA74∆s p in recombinant 4Q7 strains . Panel 2: (a) 4Q7 - pEHchiA74∆s p , (b) 4Q7 - pEBchiA74∆s p . Activity of recombinant bacteria was normalized with the residual intracellular endochitinase activity of 4Q7.
    Figure Legend Snippet: Expression of ChiA74Δsp in Escherichia coli DH5α and Bacillus thuringiensis 4Q7. (A) Schematic illustration of the strategy used to delete the secretion signal peptide-encoding sequence (shown as a rectangle inside the open reading frame) of chiA74 to generate chiA74∆ s p. Two constructs were developed, the first under regulation of wildtype promoter (wp) and the second under control of the strong cytA-p /STAB-SD promoter system. Lollipop indicates the putative transcriptional terminator site. Oligonucleotide sequences used to delete the signal peptide-coding sequence are shown in Table 1 . (B) Evaluation of endochitinase activity using solubilized intracellular proteins produced in E. coli. Panel 1: Zymogram using 4-MU-(GlcNAc) 3 for detection. Lane 1, E. coli- pEHchiA74∆s p ; lane 2, without sample; lane 3, E. coli- pEBchiA74∆s p; lane 4, E. coli DH5α . Black arrow indicates the position of ChiA74∆s p in recombinant E. coli strains . Panel 2: (a) E. coli- pEHchiA74∆s p , (b) E. coli- pEBchiA74∆s p. No endochitinase activity was observed with E. coli DH5α . (C) Phase contrast microscopy of recombinant strains of B. thuringiensis . Panel 1, 4Q7; panel 2, 4Q7 - pEHchiA74∆s p ; panel 3, 4Q7 - pEBchiA74∆s p . Black arrows indicate the positions of ChiA74∆s p inclusions. (D) Endochitinase activity determined using solubilized intracellular proteins of recombinant strains of B. thuringiensis. Panel 1: lane 1, 4Q7; lane 2, 4Q7 - pEHchiA74∆s p ; lane 3, 4Q7 - pEBchiA74∆s p. Black arrow indicates the position of ChiA74∆s p in recombinant 4Q7 strains . Panel 2: (a) 4Q7 - pEHchiA74∆s p , (b) 4Q7 - pEBchiA74∆s p . Activity of recombinant bacteria was normalized with the residual intracellular endochitinase activity of 4Q7.

    Techniques Used: Expressing, Sequencing, Construct, Activity Assay, Produced, Recombinant, Microscopy

    4) Product Images from "Use of Chimeric Type IV Secretion Systems to Define Contributions of Outer Membrane Subassemblies for Contact-Dependent Translocation"

    Article Title: Use of Chimeric Type IV Secretion Systems to Define Contributions of Outer Membrane Subassemblies for Contact-Dependent Translocation

    Journal: Molecular microbiology

    doi: 10.1111/mmi.13700

    Chimeric T4SSs support conjugative DNA transfer and activate T6SS killing. A) Sequences encoding the outer membrane core complex (OMCC) subunits TraN, TraO, and the C-terminal half (residues 194–386) of TraF were replaced with corresponding genes or gene fragments from the A. tumefaciens VirB, E. coli R388 Trw, or B. pertussis Ptl systems on mini-pKM101 plasmid pCGR108. The chimeric T4SSs composed of the inner membrane complex (IMC) of pKM101 (yellow) joined to the OMCCs from the VirB, Trw, or Ptl systems (color-coded) are modeled on the R388 T4SS 3–10 ). B) E. coli donors carrying pCGR108 derivatives encoding the only the Tra pKM101 IMC or OMCC, or the IMC::OMCC chimeras transferred the mobilizable plasmid pJG42 at the frequencies shown in transconjugants per donor (Tc’s/D) in solid-surface (histogram, solid bars) or liquid (stippled bars) matings, and were resistant to IKe phage infection (S, sensitive; R, resistant). C) E. coli survival when cultivated in the absence or presence of P. aeruginosa PAO1. E. coli DH5α cells lacked or produced intact or variant forms of the Tra pKM101 T4SS depicted. Statistical significance is shown based on a Student’s t test corresponding to the values of plasmid-free DH5α or growth in the absence of P. aeruginosa (NS, not significant; * P
    Figure Legend Snippet: Chimeric T4SSs support conjugative DNA transfer and activate T6SS killing. A) Sequences encoding the outer membrane core complex (OMCC) subunits TraN, TraO, and the C-terminal half (residues 194–386) of TraF were replaced with corresponding genes or gene fragments from the A. tumefaciens VirB, E. coli R388 Trw, or B. pertussis Ptl systems on mini-pKM101 plasmid pCGR108. The chimeric T4SSs composed of the inner membrane complex (IMC) of pKM101 (yellow) joined to the OMCCs from the VirB, Trw, or Ptl systems (color-coded) are modeled on the R388 T4SS 3–10 ). B) E. coli donors carrying pCGR108 derivatives encoding the only the Tra pKM101 IMC or OMCC, or the IMC::OMCC chimeras transferred the mobilizable plasmid pJG42 at the frequencies shown in transconjugants per donor (Tc’s/D) in solid-surface (histogram, solid bars) or liquid (stippled bars) matings, and were resistant to IKe phage infection (S, sensitive; R, resistant). C) E. coli survival when cultivated in the absence or presence of P. aeruginosa PAO1. E. coli DH5α cells lacked or produced intact or variant forms of the Tra pKM101 T4SS depicted. Statistical significance is shown based on a Student’s t test corresponding to the values of plasmid-free DH5α or growth in the absence of P. aeruginosa (NS, not significant; * P

    Techniques Used: Plasmid Preparation, Infection, Produced, Variant Assay

    Requirements for activation of T6SS killing by P. aeruginosa PAO1. E. coli DH5α lacking or producing the Tra pKM101 T4SS composed of the His 6 -TraF variants shown; in each case, the traF allele was substituted for wild-type traF by incorporation into the pKM101 tra locus on plasmid pCGR108. Statistical significance is shown based on a Student’s t test corresponding to the values of plasmid-free DH5α or growth in the absence of P. aeruginosa (NS, not significant; * P
    Figure Legend Snippet: Requirements for activation of T6SS killing by P. aeruginosa PAO1. E. coli DH5α lacking or producing the Tra pKM101 T4SS composed of the His 6 -TraF variants shown; in each case, the traF allele was substituted for wild-type traF by incorporation into the pKM101 tra locus on plasmid pCGR108. Statistical significance is shown based on a Student’s t test corresponding to the values of plasmid-free DH5α or growth in the absence of P. aeruginosa (NS, not significant; * P

    Techniques Used: Activation Assay, Plasmid Preparation

    5) Product Images from "Improving Acetate Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein (CRP)"

    Article Title: Improving Acetate Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein (CRP)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0077422

    Growth profile. Mutant strain A2, the control (DH5α Δ crp harbouring plasmid pKCP containing native crp operon) and DH5α Δ crp strain harbouring blank plasmid pKC in M9 medium at 37 °C ( A ) 0 g/L sodium acetate ( B ) 10 g/L sodium acetate ( C ) 15 g/L sodium acetate. Average values and standard deviations were calculated from triplicate experiments.
    Figure Legend Snippet: Growth profile. Mutant strain A2, the control (DH5α Δ crp harbouring plasmid pKCP containing native crp operon) and DH5α Δ crp strain harbouring blank plasmid pKC in M9 medium at 37 °C ( A ) 0 g/L sodium acetate ( B ) 10 g/L sodium acetate ( C ) 15 g/L sodium acetate. Average values and standard deviations were calculated from triplicate experiments.

    Techniques Used: Mutagenesis, Plasmid Preparation

    Cell growth of E. coli DH5α overexpressing uxaB in M9 medium, with E. coli DH5α harbouring pCA24N as control. ( A ) 0 g/L sodium acetate ( B ) 5 g/L sodium acetate. Average values and standard deviations were calculated from triplicate experiments.
    Figure Legend Snippet: Cell growth of E. coli DH5α overexpressing uxaB in M9 medium, with E. coli DH5α harbouring pCA24N as control. ( A ) 0 g/L sodium acetate ( B ) 5 g/L sodium acetate. Average values and standard deviations were calculated from triplicate experiments.

    Techniques Used:

    6) Product Images from "Natural microbial communities supporting the transfer of the IncP-1β plasmid pB10 exhibit a higher initial content of plasmids from the same incompatibility group"

    Article Title: Natural microbial communities supporting the transfer of the IncP-1β plasmid pB10 exhibit a higher initial content of plasmids from the same incompatibility group

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2014.00637

    Stability of plasmid pB10 and Escherichia coli DH5α in three river sediment microcosms (A–C) . Numbers indicate rate of disappearance (expressed in log per day) calculated from an exponential curve fit of the data (dotted lines). Each data point is an average of three values obtained from triplicate qPCR reactions, error bars represent standard deviation. Part of the data were presented previously in Bonot and Merlin (2010) .
    Figure Legend Snippet: Stability of plasmid pB10 and Escherichia coli DH5α in three river sediment microcosms (A–C) . Numbers indicate rate of disappearance (expressed in log per day) calculated from an exponential curve fit of the data (dotted lines). Each data point is an average of three values obtained from triplicate qPCR reactions, error bars represent standard deviation. Part of the data were presented previously in Bonot and Merlin (2010) .

    Techniques Used: Plasmid Preparation, Real-time Polymerase Chain Reaction, Standard Deviation

    Dissemination of plasmid pB10 (A) and relative abundance of IncP-1α/β plasmids (B) in various environmental communities maintained in microcosms. The ability of each environmental matrix to support the dissemination of plasmid pB10 was deduced from the comparison of pB10 and E. coli DH5α DNA relative stabilities in microcosms overtime (rate of disappearance), as exemplified in Figure 1 for sediments A–C. Transfer of pB10 was considered as effective (+) when pB10 stability appeared significantly greater than the stability of its initial donor host. Close rate of disappearance for both pB10 and E. coli DH5α reflects an absence of transfer (-). Values between brackets represent the number of the corresponding WWTPs. effluents [#1] were sampled twice in May 2011(#1A) and February 2012 (#1B). Each data point is an average of 3 values obtained from triplicate qPCR, where error bars represent standard deviation.
    Figure Legend Snippet: Dissemination of plasmid pB10 (A) and relative abundance of IncP-1α/β plasmids (B) in various environmental communities maintained in microcosms. The ability of each environmental matrix to support the dissemination of plasmid pB10 was deduced from the comparison of pB10 and E. coli DH5α DNA relative stabilities in microcosms overtime (rate of disappearance), as exemplified in Figure 1 for sediments A–C. Transfer of pB10 was considered as effective (+) when pB10 stability appeared significantly greater than the stability of its initial donor host. Close rate of disappearance for both pB10 and E. coli DH5α reflects an absence of transfer (-). Values between brackets represent the number of the corresponding WWTPs. effluents [#1] were sampled twice in May 2011(#1A) and February 2012 (#1B). Each data point is an average of 3 values obtained from triplicate qPCR, where error bars represent standard deviation.

    Techniques Used: Plasmid Preparation, Real-time Polymerase Chain Reaction, Standard Deviation

    7) Product Images from "BioVector, a flexible system for gene specific-expression in plants"

    Article Title: BioVector, a flexible system for gene specific-expression in plants

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-13-198

    The general map of GECs. MCS I and MCS II, the multiple cloning sites; att L1 and att L2, recombination sites; ccd B, a negative selection marker for DH5α; CmR, Chloramphenicol resistant in E. coli ; Entr-F and sp6, sequencing primers; the numbers in parenthesis, the position of corresponding restrict enzymes.
    Figure Legend Snippet: The general map of GECs. MCS I and MCS II, the multiple cloning sites; att L1 and att L2, recombination sites; ccd B, a negative selection marker for DH5α; CmR, Chloramphenicol resistant in E. coli ; Entr-F and sp6, sequencing primers; the numbers in parenthesis, the position of corresponding restrict enzymes.

    Techniques Used: Clone Assay, Selection, Marker, Sequencing

    The rationale of BioVector. The LR reaction among GEC, PEC and BDV shows the basic principle of BioVector, in which a gene and a promoter in individual entry clones are positioningly and directionally joined together in a destination vector. att L1/2/3/4, att R1/2/3/4, and att P1/2/3/4, recombination sites; ccd B, a negative selection marker for DH5α; LR Clonase II®, recombination enzyme from Invitrogen.
    Figure Legend Snippet: The rationale of BioVector. The LR reaction among GEC, PEC and BDV shows the basic principle of BioVector, in which a gene and a promoter in individual entry clones are positioningly and directionally joined together in a destination vector. att L1/2/3/4, att R1/2/3/4, and att P1/2/3/4, recombination sites; ccd B, a negative selection marker for DH5α; LR Clonase II®, recombination enzyme from Invitrogen.

    Techniques Used: Clone Assay, Plasmid Preparation, Selection, Marker

    8) Product Images from "Chronic Prosthetic Hip Infection Caused by a Small-Colony Variant of Escherichia coli"

    Article Title: Chronic Prosthetic Hip Infection Caused by a Small-Colony Variant of Escherichia coli

    Journal: Journal of Clinical Microbiology

    doi:

    Immunoblotting. Cell lysates of E. coli DH5α and the two colony types of Z-2376 grown under aerobic or anaerobic conditions on TSA-blood agar plates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets, and tested against antisera. (A) Antiserum from the patient diluted 1:500; (B) pooled antisera from 10 healthy volunteers diluted 1:100. Lanes 1, DH5α; lanes 2, large-colony type of Z-2376 grown aerobically; lanes 3, small-colony type of Z-2376 grown aerobically; lanes 4, large-colony type of Z-2376 grown anaerobically; lanes 5, small-colony type of Z-2376 grown anaerobically; lanes 6, prestained low-molecular-weight marker.
    Figure Legend Snippet: Immunoblotting. Cell lysates of E. coli DH5α and the two colony types of Z-2376 grown under aerobic or anaerobic conditions on TSA-blood agar plates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets, and tested against antisera. (A) Antiserum from the patient diluted 1:500; (B) pooled antisera from 10 healthy volunteers diluted 1:100. Lanes 1, DH5α; lanes 2, large-colony type of Z-2376 grown aerobically; lanes 3, small-colony type of Z-2376 grown aerobically; lanes 4, large-colony type of Z-2376 grown anaerobically; lanes 5, small-colony type of Z-2376 grown anaerobically; lanes 6, prestained low-molecular-weight marker.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Molecular Weight, Marker

    Southern hybridization. Two identical nylon membranes carrying immobilized, Eco RI-digested, genomic DNA were hybridized with the hemB probe (A) and the hemD probe (B). Lanes 1, DH5α; lanes 2: small-colony type of Z-2376; lanes 3, large-colony type of Z-2376; lanes M, marker ( Hin dIII-digested λ DNA). The localization of the different fragments at 23.7, 9.46, 6.66, 4.2, 2.25, and 1.96 kb (top to bottom) are indicated by lines.
    Figure Legend Snippet: Southern hybridization. Two identical nylon membranes carrying immobilized, Eco RI-digested, genomic DNA were hybridized with the hemB probe (A) and the hemD probe (B). Lanes 1, DH5α; lanes 2: small-colony type of Z-2376; lanes 3, large-colony type of Z-2376; lanes M, marker ( Hin dIII-digested λ DNA). The localization of the different fragments at 23.7, 9.46, 6.66, 4.2, 2.25, and 1.96 kb (top to bottom) are indicated by lines.

    Techniques Used: Hybridization, Marker

    9) Product Images from "An engineered autotransporter-based surface expression vector enables efficient display of Affibody molecules on OmpT-negative E. coli as well as protease-mediated secretion in OmpT-positive strains"

    Article Title: An engineered autotransporter-based surface expression vector enables efficient display of Affibody molecules on OmpT-negative E. coli as well as protease-mediated secretion in OmpT-positive strains

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-014-0179-z

    Evaluation of OmpT-mediated release and small-scale purification of displayed Affibody molecule. A . Flow-cytometric analysis for comparison of surface expression of recombinant proteins between p AraBAD -Z IgG -EC in OmpT-negative E. coli BL21 and p AraBAD -Z IgG -EC in OmpT-positive E. coli DH5α. B . SDS-PAGE showing IMAC-purified supernatants from p AraBAD -Z IgG -EC in OmpT-negative E. coli BL21 and p AraBAD -Z IgG -EC in OmpT-positive E. coli DH5α. Supernatants from non-induced samples, indicated with (−), were included as controls. C . Mass spectrum from ESI-MS analysis of IMAC-purified supernatants from p AraBAD -Z IgG -EC in OmpT-positive E. coli DH5α. Theoretical molecular weight of the OmpT-cleaved Z IgG -His 6 is 9712 Da. D . SPR-based biosensor analysis on the IMAC-purified Z IgG -His 6 . Response units (RU) on the y-axis and time on the x-axis. Representative sensorgrams from injection of Z IgG -His 6 at three different concentrations over human IgG immobilized on the chip surface. Injections were performed in duplicates.
    Figure Legend Snippet: Evaluation of OmpT-mediated release and small-scale purification of displayed Affibody molecule. A . Flow-cytometric analysis for comparison of surface expression of recombinant proteins between p AraBAD -Z IgG -EC in OmpT-negative E. coli BL21 and p AraBAD -Z IgG -EC in OmpT-positive E. coli DH5α. B . SDS-PAGE showing IMAC-purified supernatants from p AraBAD -Z IgG -EC in OmpT-negative E. coli BL21 and p AraBAD -Z IgG -EC in OmpT-positive E. coli DH5α. Supernatants from non-induced samples, indicated with (−), were included as controls. C . Mass spectrum from ESI-MS analysis of IMAC-purified supernatants from p AraBAD -Z IgG -EC in OmpT-positive E. coli DH5α. Theoretical molecular weight of the OmpT-cleaved Z IgG -His 6 is 9712 Da. D . SPR-based biosensor analysis on the IMAC-purified Z IgG -His 6 . Response units (RU) on the y-axis and time on the x-axis. Representative sensorgrams from injection of Z IgG -His 6 at three different concentrations over human IgG immobilized on the chip surface. Injections were performed in duplicates.

    Techniques Used: Purification, Flow Cytometry, Expressing, Recombinant, SDS Page, Mass Spectrometry, Molecular Weight, SPR Assay, Injection, Chromatin Immunoprecipitation

    10) Product Images from "Murine immunization with CS21 pili or LngA major subunit of enterotoxigenic Escherichia coli (ETEC) elicits systemic and mucosal immune responses and inhibits ETEC gut colonization"

    Article Title: Murine immunization with CS21 pili or LngA major subunit of enterotoxigenic Escherichia coli (ETEC) elicits systemic and mucosal immune responses and inhibits ETEC gut colonization

    Journal: Veterinary microbiology

    doi: 10.1016/j.vetmic.2016.02.001

    LngA-recombinant and CS21 pili antigens recognized by monoclonal anti-LngA and polyclonal murine sera Panel A. LngA-his tag recombinant and CS21purified native pili preparation were separated in an SDS-PAGE gel. The Coomassie blue stained gel is shown on the left and the Western blot labeled with anti-LngA monoclonal antibody is shown on the right. CS21 pili preparations were derived from E9034A ETEC strain; LngA-his tag protein was obtained from Genscript; BL21 E. coli was used as negative control; and E9034AΔ lngA pili preparation was used as LngA negative control. Panel B . Western blot of CS21 and CS8 pili from ETEC strains from different geographic locations using murine anti-LngA polyclonal sera. Purified CS21 and CS8 pili from ETEC strains originated in different countries were evaluated by Western blot using two different antisera, one derived from a mouse immunized IP with LngA-his tag antigen plus IFA and the second obtained from a mouse immunized IP with CS21 pili antigen plus IFA. Pili preparations from E. coli DH5α E. coli BL21 and CS21 + ETEC E9034A strains were used as negative and positive controls and LngA-His recombinant protein was used as LngA protein control. The size of LngA is 22 kDa.
    Figure Legend Snippet: LngA-recombinant and CS21 pili antigens recognized by monoclonal anti-LngA and polyclonal murine sera Panel A. LngA-his tag recombinant and CS21purified native pili preparation were separated in an SDS-PAGE gel. The Coomassie blue stained gel is shown on the left and the Western blot labeled with anti-LngA monoclonal antibody is shown on the right. CS21 pili preparations were derived from E9034A ETEC strain; LngA-his tag protein was obtained from Genscript; BL21 E. coli was used as negative control; and E9034AΔ lngA pili preparation was used as LngA negative control. Panel B . Western blot of CS21 and CS8 pili from ETEC strains from different geographic locations using murine anti-LngA polyclonal sera. Purified CS21 and CS8 pili from ETEC strains originated in different countries were evaluated by Western blot using two different antisera, one derived from a mouse immunized IP with LngA-his tag antigen plus IFA and the second obtained from a mouse immunized IP with CS21 pili antigen plus IFA. Pili preparations from E. coli DH5α E. coli BL21 and CS21 + ETEC E9034A strains were used as negative and positive controls and LngA-His recombinant protein was used as LngA protein control. The size of LngA is 22 kDa.

    Techniques Used: Recombinant, SDS Page, Staining, Western Blot, Labeling, Derivative Assay, Negative Control, Purification, Immunofluorescence

    11) Product Images from "Maturation of molybdoenzymes and its influence on the pathogenesis of non-typeable Haemophilus influenzae"

    Article Title: Maturation of molybdoenzymes and its influence on the pathogenesis of non-typeable Haemophilus influenzae

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2015.01219

    Neutrophil-dependent killing of HI2019 strains and E. coli Dh5α . Neutrophils (2 * 10 ∧6 cells/mL) were incubated with 1:10 HI2019 WT , HI2019 Δ mobA , and HI2019 Δ mobA _comp strains and after 2 h of incubation, viable bacteria were measured by CFUs counting as described in Materials and Methods. Values shown are % viable bacteria at 2 h compared to the initial inoculum. E. coli Dh5α cells were used as a control.
    Figure Legend Snippet: Neutrophil-dependent killing of HI2019 strains and E. coli Dh5α . Neutrophils (2 * 10 ∧6 cells/mL) were incubated with 1:10 HI2019 WT , HI2019 Δ mobA , and HI2019 Δ mobA _comp strains and after 2 h of incubation, viable bacteria were measured by CFUs counting as described in Materials and Methods. Values shown are % viable bacteria at 2 h compared to the initial inoculum. E. coli Dh5α cells were used as a control.

    Techniques Used: Incubation

    12) Product Images from "Characterization of a minimal pKW2124 replicon from Weissella cibaria KLC140 and its application for the construction of the Weissella expression vector pKUCm1"

    Article Title: Characterization of a minimal pKW2124 replicon from Weissella cibaria KLC140 and its application for the construction of the Weissella expression vector pKUCm1

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2015.00035

    Evaluation of pKUCm series and pMR1 vector re-transformation efficiencies from W. confusa ATCC 10881 into the same strain (black scale bar), compared with the vectors prepared from Escherichia coli DH5α into W. confusa ATCC 10881 (gray scale bar). Re-transformation of the vectors from Weissella improved the transformation efficiency.
    Figure Legend Snippet: Evaluation of pKUCm series and pMR1 vector re-transformation efficiencies from W. confusa ATCC 10881 into the same strain (black scale bar), compared with the vectors prepared from Escherichia coli DH5α into W. confusa ATCC 10881 (gray scale bar). Re-transformation of the vectors from Weissella improved the transformation efficiency.

    Techniques Used: Plasmid Preparation, Transformation Assay

    13) Product Images from "Transfer of Herb-Resistance Plasmid From Escherichia coli to Staphylococcus aureus Residing in the Human Urinary Tract"

    Article Title: Transfer of Herb-Resistance Plasmid From Escherichia coli to Staphylococcus aureus Residing in the Human Urinary Tract

    Journal: Jundishapur Journal of Microbiology

    doi: 10.5812/jjm.15056

    Resistance Plasmid Pattern Lane M, molecular size marker; lane D, plasmid fragment of E. coli CP9 as a donor; lane R1 and R2, trans conjugants of E. coli DH5α obtained using E. coli CP9 as a donor.
    Figure Legend Snippet: Resistance Plasmid Pattern Lane M, molecular size marker; lane D, plasmid fragment of E. coli CP9 as a donor; lane R1 and R2, trans conjugants of E. coli DH5α obtained using E. coli CP9 as a donor.

    Techniques Used: Plasmid Preparation, Marker

    14) Product Images from "Decontamination of 16S rRNA gene amplicon sequence datasets based on bacterial load assessment by qPCR"

    Article Title: Decontamination of 16S rRNA gene amplicon sequence datasets based on bacterial load assessment by qPCR

    Journal: BMC Microbiology

    doi: 10.1186/s12866-016-0689-4

    Bacterial load assessed by qPCR. The universal bacterial primers used in qPCR target the V3 segment of the 16S rRNA gene. Bacterial loads were determined using the standard curves obtained with S. aureus MW2 ( a ) or E. coli DH5α ( b ) genomic DNA. The S. aureus and E. coli genomes weigh approximately 2.9 and 4.8 fg and contain six and seven 16S rRNA gene copies, respectively. Each symbol (Exp1, Exp2 and Exp3) corresponds to the series of aliquots processed at a given point and represents the mean of duplicate measurements with relative deviations from the mean
    Figure Legend Snippet: Bacterial load assessed by qPCR. The universal bacterial primers used in qPCR target the V3 segment of the 16S rRNA gene. Bacterial loads were determined using the standard curves obtained with S. aureus MW2 ( a ) or E. coli DH5α ( b ) genomic DNA. The S. aureus and E. coli genomes weigh approximately 2.9 and 4.8 fg and contain six and seven 16S rRNA gene copies, respectively. Each symbol (Exp1, Exp2 and Exp3) corresponds to the series of aliquots processed at a given point and represents the mean of duplicate measurements with relative deviations from the mean

    Techniques Used: Real-time Polymerase Chain Reaction

    15) Product Images from "A Novel Epimerase That Converts GlcNAc-P-P-undecaprenol to GalNAc-P-P-undecaprenol in Escherichia coli O157 *"

    Article Title: A Novel Epimerase That Converts GlcNAc-P-P-undecaprenol to GalNAc-P-P-undecaprenol in Escherichia coli O157 *

    Journal:

    doi: 10.1074/jbc.M109.061630

    Z3206 does not complement glycosylation of AcrA in a Gne-dependent glycosylation system. Periplasmic extracts prepared from E. coli DH5α cells carrying the AcrA expression plasmid and the pgl operon Δ gne complemented with pMLBAD:Z3206
    Figure Legend Snippet: Z3206 does not complement glycosylation of AcrA in a Gne-dependent glycosylation system. Periplasmic extracts prepared from E. coli DH5α cells carrying the AcrA expression plasmid and the pgl operon Δ gne complemented with pMLBAD:Z3206

    Techniques Used: Expressing, Plasmid Preparation

    16) Product Images from "Kinetic Properties of Four Plasmid-Mediated AmpC ?-Lactamases"

    Article Title: Kinetic Properties of Four Plasmid-Mediated AmpC ?-Lactamases

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.49.10.4240-4246.2005

    MICs of E. coli DH5α strains and overproduction of the plasmid-encoded class C β-lactamases.
    Figure Legend Snippet: MICs of E. coli DH5α strains and overproduction of the plasmid-encoded class C β-lactamases.

    Techniques Used: Plasmid Preparation

    17) Product Images from "Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) of Escherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants"

    Article Title: Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) of Escherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants

    Journal:

    doi: 10.1128/AEM.71.11.7224-7228.2005

    Growth of E. coli DH5α strains expressing tyrA fbr genes in the presence of m -fluoro- d , l -tyrosine. Eight strains of E. coli DH5α harboring different plasmids were monitored: — , pZE21:: tyrA WT ; + , pZE21:: tyrA fbr-5 ; ▪,
    Figure Legend Snippet: Growth of E. coli DH5α strains expressing tyrA fbr genes in the presence of m -fluoro- d , l -tyrosine. Eight strains of E. coli DH5α harboring different plasmids were monitored: — , pZE21:: tyrA WT ; + , pZE21:: tyrA fbr-5 ; ▪,

    Techniques Used: Expressing

    18) Product Images from "Development of a Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Detecting and Quantifying CMY-2 and SHV ?-Lactamases"

    Article Title: Development of a Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Detecting and Quantifying CMY-2 and SHV ?-Lactamases

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.40.6.1947-1957.2002

    Immunoblotting. (a) Immunoblot of various amounts of purified CMY-2 β-lactamase probed with 1 μg of anti-CMY-2 antibody/ml. (b) Immunoblot of various amounts of purified SHV-1 β-lactamase probed with 1 μg of anti-SHV-1 antibody/ml. (c) Immunoblot of various β-lactamase-producing strains probed with 1 μg of anti-CMY-2 antibody/ml. Strains, listed from left to right, included E coli DH10B carrying plasmid pBC SK(−) with the SHV-1 β-lactamase, strains producing K-1 and ACT-1 β-lactamases, strain DH5α/pUC18 producing the TEM-1 β-lactamase, a cefepime-resistant E. aerogenes strain producing a β-lactamase (EA), and a strain expressing the P99 Amp C β-lactamase; in addition, E. coli J53-2-derived strains 194-61 and 194 and E. coli strain 20 (EC20) are clinical and laboratory strains producing CMY-2 β-lactamase. (d) Identical immunoblots of strains E. coli DH10B/pUC18 producing TEM-1 β-lactamase, E. coli DH10B/pBC SK(−) producing SHV-1 β-lactamase, and E. coli J53-2-derived 194-61 producing CMY-2 β-lactamase probed with anti-TEM antibody (1:100 dilution) or 1 μg of anti-SHV antibody/ml.
    Figure Legend Snippet: Immunoblotting. (a) Immunoblot of various amounts of purified CMY-2 β-lactamase probed with 1 μg of anti-CMY-2 antibody/ml. (b) Immunoblot of various amounts of purified SHV-1 β-lactamase probed with 1 μg of anti-SHV-1 antibody/ml. (c) Immunoblot of various β-lactamase-producing strains probed with 1 μg of anti-CMY-2 antibody/ml. Strains, listed from left to right, included E coli DH10B carrying plasmid pBC SK(−) with the SHV-1 β-lactamase, strains producing K-1 and ACT-1 β-lactamases, strain DH5α/pUC18 producing the TEM-1 β-lactamase, a cefepime-resistant E. aerogenes strain producing a β-lactamase (EA), and a strain expressing the P99 Amp C β-lactamase; in addition, E. coli J53-2-derived strains 194-61 and 194 and E. coli strain 20 (EC20) are clinical and laboratory strains producing CMY-2 β-lactamase. (d) Identical immunoblots of strains E. coli DH10B/pUC18 producing TEM-1 β-lactamase, E. coli DH10B/pBC SK(−) producing SHV-1 β-lactamase, and E. coli J53-2-derived 194-61 producing CMY-2 β-lactamase probed with anti-TEM antibody (1:100 dilution) or 1 μg of anti-SHV antibody/ml.

    Techniques Used: Purification, Plasmid Preparation, Activated Clotting Time Assay, Transmission Electron Microscopy, Expressing, Derivative Assay, Western Blot

    19) Product Images from "Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) of Escherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants"

    Article Title: Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) of Escherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants

    Journal:

    doi: 10.1128/AEM.71.11.7224-7228.2005

    Growth of E. coli DH5α strains expressing tyrA fbr genes in the presence of m -fluoro- d , l -tyrosine. Eight strains of E. coli DH5α harboring different plasmids were monitored: — , pZE21:: tyrA WT ; + , pZE21:: tyrA fbr-5 ; ▪,
    Figure Legend Snippet: Growth of E. coli DH5α strains expressing tyrA fbr genes in the presence of m -fluoro- d , l -tyrosine. Eight strains of E. coli DH5α harboring different plasmids were monitored: — , pZE21:: tyrA WT ; + , pZE21:: tyrA fbr-5 ; ▪,

    Techniques Used: Expressing

    Related Articles

    Clone Assay:

    Article Title: BioVector, a flexible system for gene specific-expression in plants
    Article Snippet: Therefore, genes, tags, and any regulatory elements of interest from PCR amplification can be directly cloned into GECs after digestion. .. A ccd B gene in REL sites (Figure ) served as a negative selection marker for E. coli DH5α as all GATEWAY entry vectors employ (Invitrogen) [ , , ].

    Article Title: Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1
    Article Snippet: Other fragments were cloned into the Kpn I/Hin dIII sites of pQE-80L vector. .. Briefly, the lysates of E. coli DH5α expressing the corresponding proteins were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Pierce).

    Centrifugation:

    Article Title: Enhanced performance of the microalga Chlorella sorokiniana remotely induced by the plant growth-promoting bacteria Azospirillum brasilense and Bacillus pumilus
    Article Snippet: In experiments that measured the potential effect of CO2 , Escherichia coli DH5α (Invitrogen, Carlsbad, CA) served as the negative control for microalgal growth and promoting metabolites because it does not have any plant growth-promoting effects; it also served as a positive control in the CO2 experiment because it produces CO2 , as any E. coli . .. Bacteria were then harvested by centrifugation at 3720 × g for 7 min, rinsed twice in 0.85% saline solution, and subsequently transferred to minimal mineral Brunner’s medium (DSMZ medium 457) composed of (in g L−1 ): Na2 HPO4 (2.44), KH2 PO4 (1.52), (NH4 )2 SO4 (0.50), MgSO4 •7H2 O (0.20), CaCl2 •2H2 O (0.05), EDTA (0.50), FeSO4 •7H2 O (0.20), and (in μg·L−1 ): ZnSO4 •7H2 O (0.10), MnCl2 •4H2 O (0.03), H3 BO3 (0.30), CoCl2 •6H2 O (0.20), CuCl2 •2H2 O (0.01), NiCl2 •6H2 O (0.02), Na2 MoO4 •2H2 O (0.03).

    Amplification:

    Article Title: BioVector, a flexible system for gene specific-expression in plants
    Article Snippet: Therefore, genes, tags, and any regulatory elements of interest from PCR amplification can be directly cloned into GECs after digestion. .. A ccd B gene in REL sites (Figure ) served as a negative selection marker for E. coli DH5α as all GATEWAY entry vectors employ (Invitrogen) [ , , ].

    Article Title: Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1
    Article Snippet: The DNA sequences encoding mHMGB1 -186-200, mHMGB1 -196-210, mHMGB1 -196-205, mHMGB1 -198-207, mHMGB1 -201-210 and mHMGB1 -201-205 (rHMGB1 lacking amino acid residues 186-200, 196-210, 196-205, 198-207, 201-210 and 201-205 respectively) were amplified by one-step opposite-direction PCR using MutanBEST Kit (TaKaRa) according to the manufacturer's instructions (taking pUC19-rHMGB1 as template). .. Briefly, the lysates of E. coli DH5α expressing the corresponding proteins were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Pierce).

    Positive Control:

    Article Title: Maturation of molybdoenzymes and its influence on the pathogenesis of non-typeable Haemophilus influenzae
    Article Snippet: .. E. coli Dh5α (Life Technologies) was used as a positive control. ..

    Article Title: Enhanced performance of the microalga Chlorella sorokiniana remotely induced by the plant growth-promoting bacteria Azospirillum brasilense and Bacillus pumilus
    Article Snippet: .. In experiments that measured the potential effect of CO2 , Escherichia coli DH5α (Invitrogen, Carlsbad, CA) served as the negative control for microalgal growth and promoting metabolites because it does not have any plant growth-promoting effects; it also served as a positive control in the CO2 experiment because it produces CO2 , as any E. coli . .. For initial culturing of the microalga, 10 mL axenic C. sorokiniana culture, cultivated in sterile mineral medium (C30), was added to a sterile flask containing 90 mL sterile C30 medium, composed of (in g·L−1 ): KNO3 (25), MgSO4 •7H2 O (10), KH2 PO4 (4), K2 HPO4 (1), FeSO4 •7H2 O (1), and (in μg·L−1 ): H3 BO3 (2.86), MnCl2 •4H2 O (1.81), ZnSO4 •7H2 O (0.11), CuSO4 •5H2 O (0.09), NaMoO4 (0.021), pH 5.25 and incubated at 27 ± 2 °C on a rotary shaker at 120 rpm under 60 μmol photon·m−2 ·s−1 continuous light intensity for 6 days .

    Construct:

    Article Title: Non-contact positions impose site selectivity on Cre recombinase
    Article Snippet: .. Plasmids were constructed and propagated using Escherichia coli DH5α (Invitrogen). ..

    Article Title: Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74?sp chitinase inclusions, Cry crystals and spores for applied use
    Article Snippet: .. All constructs (Figure A) were propagated in E. coli DH5α [supE44, DlacU169 (F80lacZDM15 ) hsdR17 recA1 end A1 gyrA96 thi-1 relA1 ] (Invitrogen, Carlsbad CA, USA) and then used for transforming the acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7, (hereafter 4Q7), and B. thuringiensis subsp. kurstaki HD1 (hereafter HD1) (Bacillus Genetic Stock Center, Columbus, OH). .. Plasmid pGLO is a vector that harbors the green fluorescent protein (gfp ) gene under the control arabinose (araC ) promoter and contains an ampicillin (bla ) resistance gene marker (Bio-Rad, Hercules CA, USA).

    Article Title: BioVector, a flexible system for gene specific-expression in plants
    Article Snippet: We constructed a set of alternative GECs for labeling a desired protein with different tags at the N- or C-terminus (Table ). .. A ccd B gene in REL sites (Figure ) served as a negative selection marker for E. coli DH5α as all GATEWAY entry vectors employ (Invitrogen) [ , , ].

    Article Title: Improving Acetate Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein (CRP)
    Article Snippet: .. Materials The host strain E. coli DH5α ∆crp was constructed by knocking out crp from E. coli DH5α (Invitrogen, San Diego, US) according to previously established protocol [ ]. .. Overnight culture was prepared in Luria-Bertani (LB) medium containing 1% tryptone (Oxoid, Hampshire, UK), 0.5% yeast extract (Merck, Damstadt, Germany), and 1% sodium chloride (Merck, Damstadt, Germany).

    Article Title: Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1
    Article Snippet: After being sequenced, the fragments encoding rHMGB1 A box and B box were separately ligated into the Kpn I/Hin dIII cloning sites in the pQE-80L/DHFR prokaryotic expression vector (pQE-80L/DHFR vector constructed by us from pQE-80L). .. Briefly, the lysates of E. coli DH5α expressing the corresponding proteins were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Pierce).

    Real-time Polymerase Chain Reaction:

    Article Title: Natural microbial communities supporting the transfer of the IncP-1β plasmid pB10 exhibit a higher initial content of plasmids from the same incompatibility group
    Article Snippet: .. Basically, sets of qPCR primers were designed to prime on both sides of a unique junction between building blocks of the element targeted for quantification: a unique assembly between two truncated transposons for plasmid pB10 (target coordinates 45968–46102; GenBank NC_004840), and both sides of a unique 97 kb-deletion in the chromosome of E. coli DH5α (target coordinates on K12 274654–372933; GenBank U00096; ; ). qPCRs were performed in triplicate using a “Step One Plus Real-Time PCR System” (Applied Biosystems, driver: StepOne Software v2.2) with thermocycling conditions set as follows: 2 min at 50°C, then 10 min at 95°C followed by 45 cycles of 15 s at 95°C and 1 min at 60°C. .. Quantifications were carried out from 25 ng of community DNA using a “TaqMan® Universal PCR Master Mix, NoAmpErase® UNG” (Applied Biosystems) as recommended by the manufacturer, with 800 nM of each primer, and 300 nM of TaqMan probe, in a 25 μL reaction volume.

    Incubation:

    Article Title: Maturation of molybdoenzymes and its influence on the pathogenesis of non-typeable Haemophilus influenzae
    Article Snippet: Plates were centrifuged at 500 × g for 10 min then incubated at 37°C with 5% CO2 for 2 h. After incubation, neutrophils were lysed with water, the content of each well serially diluted in BHI and plated on sBHI-agar for overnight incubation and enumeration of CFU. .. E. coli Dh5α (Life Technologies) was used as a positive control.

    Article Title: Enhanced performance of the microalga Chlorella sorokiniana remotely induced by the plant growth-promoting bacteria Azospirillum brasilense and Bacillus pumilus
    Article Snippet: In experiments that measured the potential effect of CO2 , Escherichia coli DH5α (Invitrogen, Carlsbad, CA) served as the negative control for microalgal growth and promoting metabolites because it does not have any plant growth-promoting effects; it also served as a positive control in the CO2 experiment because it produces CO2 , as any E. coli . .. For initial culturing of the microalga, 10 mL axenic C. sorokiniana culture, cultivated in sterile mineral medium (C30), was added to a sterile flask containing 90 mL sterile C30 medium, composed of (in g·L−1 ): KNO3 (25), MgSO4 •7H2 O (10), KH2 PO4 (4), K2 HPO4 (1), FeSO4 •7H2 O (1), and (in μg·L−1 ): H3 BO3 (2.86), MnCl2 •4H2 O (1.81), ZnSO4 •7H2 O (0.11), CuSO4 •5H2 O (0.09), NaMoO4 (0.021), pH 5.25 and incubated at 27 ± 2 °C on a rotary shaker at 120 rpm under 60 μmol photon·m−2 ·s−1 continuous light intensity for 6 days .

    Article Title: Characterization of a minimal pKW2124 replicon from Weissella cibaria KLC140 and its application for the construction of the Weissella expression vector pKUCm1
    Article Snippet: .. All Weissella species were incubated anaerobically at 37°C in de Man-Rogosa-Sharpe (MRS) medium (Difco, Detroit, MI, USA) and E. coli DH5α (Invitrogen, Carlsbad, CA, USA) was grown with shaking in Luria-Bertani (LB) medium (Difco) at 37°C. .. Ampicillin sulfate (Sigma, St. Louis, MO, USA) was added to the E. coli growth medium for selection at 50 μg/ml.

    Article Title: Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1
    Article Snippet: Briefly, the lysates of E. coli DH5α expressing the corresponding proteins were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Pierce). .. After blocked with 5% non-fat milk in phosphate buffered saline containing 0.1% Tween 20 (PBS-T), the membranes were incubated with mouse anti-His monoclonal antibody (Qiagen).

    Luciferase:

    Article Title: Murine immunization with CS21 pili or LngA major subunit of enterotoxigenic Escherichia coli (ETEC) elicits systemic and mucosal immune responses and inhibits ETEC gut colonization
    Article Snippet: E. coli DH5α (Life Technologies, Grand Island, NY), and CS21+ or CS8+ ETEC strains were cultured on Luria broth (LB), Terrific broth (TB) or TB agar plates at 37 °C overnight ( ). .. CS21+ ETEC E0934A strain was electroporated with plasmid pCM17 containing luciferase genes ( , ).

    Cell Culture:

    Article Title: What an Escherichia coli Mutant Can Teach Us About the Antibacterial Effect of Chlorophyllin
    Article Snippet: .. Bacteria Strains and Cell Culture Experiments were performed with E. coli DH5α (Invitrogen, Carlsbad, CA, USA), E. coli NR698 (MC4100 lptD4213 ; kindly provided by M. Grabowicz, Princeton University, NJ, USA) [ ] and B. subtilis 168 (trpC2 ; laboratory stock). ..

    Article Title: Chronic Prosthetic Hip Infection Caused by a Small-Colony Variant of Escherichia coli
    Article Snippet: E. coli DH5α was purchased from Gibco-BRL (Eggenstein, Germany). .. Unless otherwise stated, bacteria were cultured on Trypticase soy agar (TSA; Oxoid, Unipath Ltd., Basingstoke, England) supplemented with 7% defibrinated sheep blood (Oxoid, Unipath Ltd.) for 48 h at 37°C under aerobic conditions.

    Article Title: Improving Acetate Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein (CRP)
    Article Snippet: Materials The host strain E. coli DH5α ∆crp was constructed by knocking out crp from E. coli DH5α (Invitrogen, San Diego, US) according to previously established protocol [ ]. .. M9 minimal medium was used for cells cultured under acetate stress, which is composed of the following chemicals (per liter): 6.78 g Na2 HPO4 , 3 g KH2 PO4 , 0.5 g NaCl, 1 g NH4 Cl, 0.49 g MgSO4 .7H2 O, 0.011 g CaCl2 , 2 g glucose and 1 ml of trace metal stock solution.

    Article Title: Murine immunization with CS21 pili or LngA major subunit of enterotoxigenic Escherichia coli (ETEC) elicits systemic and mucosal immune responses and inhibits ETEC gut colonization
    Article Snippet: .. E. coli DH5α (Life Technologies, Grand Island, NY), and CS21+ or CS8+ ETEC strains were cultured on Luria broth (LB), Terrific broth (TB) or TB agar plates at 37 °C overnight ( ). .. CS21+ ETEC E0934A strain was electroporated with plasmid pCM17 containing luciferase genes ( , ).

    Expressing:

    Article Title: Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1
    Article Snippet: .. Briefly, the lysates of E. coli DH5α expressing the corresponding proteins were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Pierce). .. After blocked with 5% non-fat milk in phosphate buffered saline containing 0.1% Tween 20 (PBS-T), the membranes were incubated with mouse anti-His monoclonal antibody (Qiagen).

    Western Blot:

    Article Title: Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1
    Article Snippet: Then, all of these prokaryotic expression vectors were separately transformed into E. coli DH5α and induced by IPTG to express the corresponding His-tagged proteins, which were determined by Western blot. .. Briefly, the lysates of E. coli DH5α expressing the corresponding proteins were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Pierce).

    Transformation Assay:

    Article Title: Non-contact positions impose site selectivity on Cre recombinase
    Article Snippet: Plasmids were constructed and propagated using Escherichia coli DH5α (Invitrogen). .. Transformation of DH5α and BS583 with the loxK2 2 plasmid pBS584 yielded the indicator/selector strains BS1491 and BS1494, respectively.

    Article Title: Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1
    Article Snippet: Then, all of these prokaryotic expression vectors were separately transformed into E. coli DH5α and induced by IPTG to express the corresponding His-tagged proteins, which were determined by Western blot. .. Briefly, the lysates of E. coli DH5α expressing the corresponding proteins were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Pierce).

    Electroporation:

    Article Title: Characterization of a minimal pKW2124 replicon from Weissella cibaria KLC140 and its application for the construction of the Weissella expression vector pKUCm1
    Article Snippet: All Weissella species were incubated anaerobically at 37°C in de Man-Rogosa-Sharpe (MRS) medium (Difco, Detroit, MI, USA) and E. coli DH5α (Invitrogen, Carlsbad, CA, USA) was grown with shaking in Luria-Bertani (LB) medium (Difco) at 37°C. .. Weissella and other electroporation host bacteria were selected using chloramphenicol (USB Corporation, Santa Clara, CA, USA) with the following appropriate concentrations, 6.0 μg/ml for Weissella, Lactobacillus and Bifidobacterium, 5.0 μg/ml for Lactococcus and Leuconostoc , and 3.0 μg/ml for Streptococcus .

    Chromatography:

    Article Title: Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1
    Article Snippet: Briefly, the lysates of E. coli DH5α expressing the corresponding proteins were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Pierce). .. The expressed proteins were purified by Ni2+ -NTA chromatography kit (Qiagen) under the natural condition following the instructions of the manufacturer.

    Infection:

    Article Title: Chronic Prosthetic Hip Infection Caused by a Small-Colony Variant of Escherichia coli
    Article Snippet: E. coli Z-2376 was obtained from two different specimens of the scar area of a patient with a prosthetic hip infection. .. E. coli DH5α was purchased from Gibco-BRL (Eggenstein, Germany).

    Article Title: Use of Chimeric Type IV Secretion Systems to Define Contributions of Outer Membrane Subassemblies for Contact-Dependent Translocation
    Article Snippet: E. coli DH5α (GIBCO-BRL) was used for plasmid constructions and the type VI secretion system (T6SS) killing assay. .. E. coli MG1655 ( E. coli Genetic Stock Center) served as donors in the conjugation assays and for the phage infection assays.

    Generated:

    Article Title: Non-contact positions impose site selectivity on Cre recombinase
    Article Snippet: Plasmids were constructed and propagated using Escherichia coli DH5α (Invitrogen). .. Lysogenization of DH5Δ lac U169 ( ) with the resulting phage generated the loxP 2 lacZ indicator strain BS583.

    Article Title: Natural microbial communities supporting the transfer of the IncP-1β plasmid pB10 exhibit a higher initial content of plasmids from the same incompatibility group
    Article Snippet: Basically, sets of qPCR primers were designed to prime on both sides of a unique junction between building blocks of the element targeted for quantification: a unique assembly between two truncated transposons for plasmid pB10 (target coordinates 45968–46102; GenBank NC_004840), and both sides of a unique 97 kb-deletion in the chromosome of E. coli DH5α (target coordinates on K12 274654–372933; GenBank U00096; ; ). qPCRs were performed in triplicate using a “Step One Plus Real-Time PCR System” (Applied Biosystems, driver: StepOne Software v2.2) with thermocycling conditions set as follows: 2 min at 50°C, then 10 min at 95°C followed by 45 cycles of 15 s at 95°C and 1 min at 60°C. .. Except for manure microcosm experiments, standard curves correlating the detection threshold of the fluorescence signal (Cq) to known concentrations of target DNA were generated by qPCR on pure template DNA obtained as follows: pB10 was extracted from E. coli DH5α(pB10) using a Wizard® Plus SV Minipreps kit (Promega, Madison, WI, USA), DNA was then linarized by digestion with Bam HI (Promega), and repurified using a QIAquick® PCR purification kit (Qiagen).

    Article Title: Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1
    Article Snippet: The DNA sequences encoding rHMGB1 A box, B box, tHMGB1 and mHMGB1 -211-215, mHMGB1 - 206-215, mHMGB1 -201-215, mHMGB1 -196-215, mHMGB1 -191-215 (rHMGB1 lacking amino acid residues 211-215, 206-215, 201-215, 196-215 and 191-215 respectively) were generated by PCR taking pUC19-rHMGB1 as template. .. Briefly, the lysates of E. coli DH5α expressing the corresponding proteins were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Pierce).

    Polymerase Chain Reaction:

    Article Title: Natural microbial communities supporting the transfer of the IncP-1β plasmid pB10 exhibit a higher initial content of plasmids from the same incompatibility group
    Article Snippet: Basically, sets of qPCR primers were designed to prime on both sides of a unique junction between building blocks of the element targeted for quantification: a unique assembly between two truncated transposons for plasmid pB10 (target coordinates 45968–46102; GenBank NC_004840), and both sides of a unique 97 kb-deletion in the chromosome of E. coli DH5α (target coordinates on K12 274654–372933; GenBank U00096; ; ). qPCRs were performed in triplicate using a “Step One Plus Real-Time PCR System” (Applied Biosystems, driver: StepOne Software v2.2) with thermocycling conditions set as follows: 2 min at 50°C, then 10 min at 95°C followed by 45 cycles of 15 s at 95°C and 1 min at 60°C. .. Quantifications were carried out from 25 ng of community DNA using a “TaqMan® Universal PCR Master Mix, NoAmpErase® UNG” (Applied Biosystems) as recommended by the manufacturer, with 800 nM of each primer, and 300 nM of TaqMan probe, in a 25 μL reaction volume.

    Article Title: Characterization of a minimal pKW2124 replicon from Weissella cibaria KLC140 and its application for the construction of the Weissella expression vector pKUCm1
    Article Snippet: Paragraph title: BACTERIAL STRAINS, PLASMIDS, PCR PRIMERS, AND GROWTH CONDITIONS ... All Weissella species were incubated anaerobically at 37°C in de Man-Rogosa-Sharpe (MRS) medium (Difco, Detroit, MI, USA) and E. coli DH5α (Invitrogen, Carlsbad, CA, USA) was grown with shaking in Luria-Bertani (LB) medium (Difco) at 37°C.

    Article Title: Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1
    Article Snippet: The DNA sequences encoding mHMGB1 -186-200, mHMGB1 -196-210, mHMGB1 -196-205, mHMGB1 -198-207, mHMGB1 -201-210 and mHMGB1 -201-205 (rHMGB1 lacking amino acid residues 186-200, 196-210, 196-205, 198-207, 201-210 and 201-205 respectively) were amplified by one-step opposite-direction PCR using MutanBEST Kit (TaKaRa) according to the manufacturer's instructions (taking pUC19-rHMGB1 as template). .. Briefly, the lysates of E. coli DH5α expressing the corresponding proteins were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Pierce).

    Recombinant:

    Article Title: Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1
    Article Snippet: Paragraph title: Preparation of recombinant proteins and C peptide ... Briefly, the lysates of E. coli DH5α expressing the corresponding proteins were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Pierce).

    Article Title: An engineered autotransporter-based surface expression vector enables efficient display of Affibody molecules on OmpT-negative E. coli as well as protease-mediated secretion in OmpT-positive strains
    Article Snippet: .. E. coli DH5α (Invitrogen) was used for OmpT-mediated release of recombinant protein into the medium. .. Construction of expression vectors DNA fragments encoding ZIgG , His6 , OmpT cleavage site and ABP were subcloned into the vector pMK90 [ ], containing parts of the aidA gene under control of the aidA promoter [ ].

    In Vivo:

    Article Title: Murine immunization with CS21 pili or LngA major subunit of enterotoxigenic Escherichia coli (ETEC) elicits systemic and mucosal immune responses and inhibits ETEC gut colonization
    Article Snippet: E. coli DH5α (Life Technologies, Grand Island, NY), and CS21+ or CS8+ ETEC strains were cultured on Luria broth (LB), Terrific broth (TB) or TB agar plates at 37 °C overnight ( ). .. E9034A/pCM17, used for in vivo challenge mice experiments, was cultured in LB broth supplemented with Kanamycin at 37 °C overnight and then sub-cultured in DMEM/F12 (1:1) medium (25 mM glucose, 15 mM HEPES and 0.5% mannose) at ratio of 1:10 for 3 hours at 37 °C.

    Fluorescence:

    Article Title: Natural microbial communities supporting the transfer of the IncP-1β plasmid pB10 exhibit a higher initial content of plasmids from the same incompatibility group
    Article Snippet: Basically, sets of qPCR primers were designed to prime on both sides of a unique junction between building blocks of the element targeted for quantification: a unique assembly between two truncated transposons for plasmid pB10 (target coordinates 45968–46102; GenBank NC_004840), and both sides of a unique 97 kb-deletion in the chromosome of E. coli DH5α (target coordinates on K12 274654–372933; GenBank U00096; ; ). qPCRs were performed in triplicate using a “Step One Plus Real-Time PCR System” (Applied Biosystems, driver: StepOne Software v2.2) with thermocycling conditions set as follows: 2 min at 50°C, then 10 min at 95°C followed by 45 cycles of 15 s at 95°C and 1 min at 60°C. .. Except for manure microcosm experiments, standard curves correlating the detection threshold of the fluorescence signal (Cq) to known concentrations of target DNA were generated by qPCR on pure template DNA obtained as follows: pB10 was extracted from E. coli DH5α(pB10) using a Wizard® Plus SV Minipreps kit (Promega, Madison, WI, USA), DNA was then linarized by digestion with Bam HI (Promega), and repurified using a QIAquick® PCR purification kit (Qiagen).

    Conjugation Assay:

    Article Title: Use of Chimeric Type IV Secretion Systems to Define Contributions of Outer Membrane Subassemblies for Contact-Dependent Translocation
    Article Snippet: E. coli DH5α (GIBCO-BRL) was used for plasmid constructions and the type VI secretion system (T6SS) killing assay. .. E. coli MG1655 ( E. coli Genetic Stock Center) served as donors in the conjugation assays and for the phage infection assays.

    Isolation:

    Article Title: Maturation of molybdoenzymes and its influence on the pathogenesis of non-typeable Haemophilus influenzae
    Article Snippet: Neutrophil killing assays Human neutrophils were isolated and purified from venous blood using the PolyMorphPrep kit (Axis-Shield) as per the manufacturer's instructions and seeded into 96-well plates at 2* 105 cells/well. .. E. coli Dh5α (Life Technologies) was used as a positive control.

    Subcloning:

    Article Title: An engineered autotransporter-based surface expression vector enables efficient display of Affibody molecules on OmpT-negative E. coli as well as protease-mediated secretion in OmpT-positive strains
    Article Snippet: Bacterial strains Escherichia coli strain RR1∆M15 [ ] and E. coli strain DH5α (Invitrogen, Carlsbad, CA) were used for subcloning work. .. E. coli DH5α (Invitrogen) was used for OmpT-mediated release of recombinant protein into the medium.

    Microscopy:

    Article Title: Chronic Prosthetic Hip Infection Caused by a Small-Colony Variant of Escherichia coli
    Article Snippet: E. coli DH5α was purchased from Gibco-BRL (Eggenstein, Germany). .. The sizes of the colonies were determined by plate microscopy.

    Purification:

    Article Title: Maturation of molybdoenzymes and its influence on the pathogenesis of non-typeable Haemophilus influenzae
    Article Snippet: Neutrophil killing assays Human neutrophils were isolated and purified from venous blood using the PolyMorphPrep kit (Axis-Shield) as per the manufacturer's instructions and seeded into 96-well plates at 2* 105 cells/well. .. E. coli Dh5α (Life Technologies) was used as a positive control.

    Article Title: Natural microbial communities supporting the transfer of the IncP-1β plasmid pB10 exhibit a higher initial content of plasmids from the same incompatibility group
    Article Snippet: Basically, sets of qPCR primers were designed to prime on both sides of a unique junction between building blocks of the element targeted for quantification: a unique assembly between two truncated transposons for plasmid pB10 (target coordinates 45968–46102; GenBank NC_004840), and both sides of a unique 97 kb-deletion in the chromosome of E. coli DH5α (target coordinates on K12 274654–372933; GenBank U00096; ; ). qPCRs were performed in triplicate using a “Step One Plus Real-Time PCR System” (Applied Biosystems, driver: StepOne Software v2.2) with thermocycling conditions set as follows: 2 min at 50°C, then 10 min at 95°C followed by 45 cycles of 15 s at 95°C and 1 min at 60°C. .. Except for manure microcosm experiments, standard curves correlating the detection threshold of the fluorescence signal (Cq) to known concentrations of target DNA were generated by qPCR on pure template DNA obtained as follows: pB10 was extracted from E. coli DH5α(pB10) using a Wizard® Plus SV Minipreps kit (Promega, Madison, WI, USA), DNA was then linarized by digestion with Bam HI (Promega), and repurified using a QIAquick® PCR purification kit (Qiagen).

    Article Title: Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1
    Article Snippet: Briefly, the lysates of E. coli DH5α expressing the corresponding proteins were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Pierce). .. The expressed proteins were purified by Ni2+ -NTA chromatography kit (Qiagen) under the natural condition following the instructions of the manufacturer.

    Sequencing:

    Article Title: Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74?sp chitinase inclusions, Cry crystals and spores for applied use
    Article Snippet: These plasmids (pEHchiA74 and pEBchiA74) were used for deleting the signal peptide-encoding sequence of ChiA74 to obtain ChiA74Δsp (see below). .. All constructs (Figure A) were propagated in E. coli DH5α [supE44, DlacU169 (F80lacZDM15 ) hsdR17 recA1 end A1 gyrA96 thi-1 relA1 ] (Invitrogen, Carlsbad CA, USA) and then used for transforming the acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7, (hereafter 4Q7), and B. thuringiensis subsp. kurstaki HD1 (hereafter HD1) (Bacillus Genetic Stock Center, Columbus, OH).

    Article Title: BioVector, a flexible system for gene specific-expression in plants
    Article Snippet: Therefore, GECs saved both time and cost for cloning and sequencing of genes. .. A ccd B gene in REL sites (Figure ) served as a negative selection marker for E. coli DH5α as all GATEWAY entry vectors employ (Invitrogen) [ , , ].

    Labeling:

    Article Title: BioVector, a flexible system for gene specific-expression in plants
    Article Snippet: We constructed a set of alternative GECs for labeling a desired protein with different tags at the N- or C-terminus (Table ). .. A ccd B gene in REL sites (Figure ) served as a negative selection marker for E. coli DH5α as all GATEWAY entry vectors employ (Invitrogen) [ , , ].

    Mouse Assay:

    Article Title: Murine immunization with CS21 pili or LngA major subunit of enterotoxigenic Escherichia coli (ETEC) elicits systemic and mucosal immune responses and inhibits ETEC gut colonization
    Article Snippet: E. coli DH5α (Life Technologies, Grand Island, NY), and CS21+ or CS8+ ETEC strains were cultured on Luria broth (LB), Terrific broth (TB) or TB agar plates at 37 °C overnight ( ). .. E9034A/pCM17, used for in vivo challenge mice experiments, was cultured in LB broth supplemented with Kanamycin at 37 °C overnight and then sub-cultured in DMEM/F12 (1:1) medium (25 mM glucose, 15 mM HEPES and 0.5% mannose) at ratio of 1:10 for 3 hours at 37 °C.

    SDS Page:

    Article Title: Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1
    Article Snippet: .. Briefly, the lysates of E. coli DH5α expressing the corresponding proteins were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Pierce). .. After blocked with 5% non-fat milk in phosphate buffered saline containing 0.1% Tween 20 (PBS-T), the membranes were incubated with mouse anti-His monoclonal antibody (Qiagen).

    Plasmid Preparation:

    Article Title: Non-contact positions impose site selectivity on Cre recombinase
    Article Snippet: Plasmids were constructed and propagated using Escherichia coli DH5α (Invitrogen). .. Transformation of DH5α and BS583 with the loxK2 2 plasmid pBS584 yielded the indicator/selector strains BS1491 and BS1494, respectively.

    Article Title: Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74?sp chitinase inclusions, Cry crystals and spores for applied use
    Article Snippet: All constructs (Figure A) were propagated in E. coli DH5α [supE44, DlacU169 (F80lacZDM15 ) hsdR17 recA1 end A1 gyrA96 thi-1 relA1 ] (Invitrogen, Carlsbad CA, USA) and then used for transforming the acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7, (hereafter 4Q7), and B. thuringiensis subsp. kurstaki HD1 (hereafter HD1) (Bacillus Genetic Stock Center, Columbus, OH). .. Plasmid pGLO is a vector that harbors the green fluorescent protein (gfp ) gene under the control arabinose (araC ) promoter and contains an ampicillin (bla ) resistance gene marker (Bio-Rad, Hercules CA, USA).

    Article Title: Natural microbial communities supporting the transfer of the IncP-1β plasmid pB10 exhibit a higher initial content of plasmids from the same incompatibility group
    Article Snippet: .. Basically, sets of qPCR primers were designed to prime on both sides of a unique junction between building blocks of the element targeted for quantification: a unique assembly between two truncated transposons for plasmid pB10 (target coordinates 45968–46102; GenBank NC_004840), and both sides of a unique 97 kb-deletion in the chromosome of E. coli DH5α (target coordinates on K12 274654–372933; GenBank U00096; ; ). qPCRs were performed in triplicate using a “Step One Plus Real-Time PCR System” (Applied Biosystems, driver: StepOne Software v2.2) with thermocycling conditions set as follows: 2 min at 50°C, then 10 min at 95°C followed by 45 cycles of 15 s at 95°C and 1 min at 60°C. .. Quantifications were carried out from 25 ng of community DNA using a “TaqMan® Universal PCR Master Mix, NoAmpErase® UNG” (Applied Biosystems) as recommended by the manufacturer, with 800 nM of each primer, and 300 nM of TaqMan probe, in a 25 μL reaction volume.

    Article Title: BioVector, a flexible system for gene specific-expression in plants
    Article Snippet: A ccd B gene in REL sites (Figure ) served as a negative selection marker for E. coli DH5α as all GATEWAY entry vectors employ (Invitrogen) [ , , ]. .. Plenty of REL sites in GECs provide a flexible platform for ad arbitrium modifying the vector by researcher themselves for their own individual study.

    Article Title: Transfer of Herb-Resistance Plasmid From Escherichia coli to Staphylococcus aureus Residing in the Human Urinary Tract
    Article Snippet: .. Plasmid Characterization Total plasmid DNA was prepared (Plasmid Extraction Kit, Beijing TIANGEN) and transferred into E. coli DH5α (Invitrogen, Carlsbad, CA, USA). ..

    Article Title: Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1
    Article Snippet: Other fragments were cloned into the Kpn I/Hin dIII sites of pQE-80L vector. .. Briefly, the lysates of E. coli DH5α expressing the corresponding proteins were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Pierce).

    Article Title: Murine immunization with CS21 pili or LngA major subunit of enterotoxigenic Escherichia coli (ETEC) elicits systemic and mucosal immune responses and inhibits ETEC gut colonization
    Article Snippet: E. coli DH5α (Life Technologies, Grand Island, NY), and CS21+ or CS8+ ETEC strains were cultured on Luria broth (LB), Terrific broth (TB) or TB agar plates at 37 °C overnight ( ). .. CS21+ ETEC E0934A strain was electroporated with plasmid pCM17 containing luciferase genes ( , ).

    Article Title: Use of Chimeric Type IV Secretion Systems to Define Contributions of Outer Membrane Subassemblies for Contact-Dependent Translocation
    Article Snippet: .. E. coli DH5α (GIBCO-BRL) was used for plasmid constructions and the type VI secretion system (T6SS) killing assay. .. E. coli MG1655 ( E. coli Genetic Stock Center) served as donors in the conjugation assays and for the phage infection assays.

    Software:

    Article Title: Natural microbial communities supporting the transfer of the IncP-1β plasmid pB10 exhibit a higher initial content of plasmids from the same incompatibility group
    Article Snippet: .. Basically, sets of qPCR primers were designed to prime on both sides of a unique junction between building blocks of the element targeted for quantification: a unique assembly between two truncated transposons for plasmid pB10 (target coordinates 45968–46102; GenBank NC_004840), and both sides of a unique 97 kb-deletion in the chromosome of E. coli DH5α (target coordinates on K12 274654–372933; GenBank U00096; ; ). qPCRs were performed in triplicate using a “Step One Plus Real-Time PCR System” (Applied Biosystems, driver: StepOne Software v2.2) with thermocycling conditions set as follows: 2 min at 50°C, then 10 min at 95°C followed by 45 cycles of 15 s at 95°C and 1 min at 60°C. .. Quantifications were carried out from 25 ng of community DNA using a “TaqMan® Universal PCR Master Mix, NoAmpErase® UNG” (Applied Biosystems) as recommended by the manufacturer, with 800 nM of each primer, and 300 nM of TaqMan probe, in a 25 μL reaction volume.

    Negative Control:

    Article Title: Enhanced performance of the microalga Chlorella sorokiniana remotely induced by the plant growth-promoting bacteria Azospirillum brasilense and Bacillus pumilus
    Article Snippet: .. In experiments that measured the potential effect of CO2 , Escherichia coli DH5α (Invitrogen, Carlsbad, CA) served as the negative control for microalgal growth and promoting metabolites because it does not have any plant growth-promoting effects; it also served as a positive control in the CO2 experiment because it produces CO2 , as any E. coli . .. For initial culturing of the microalga, 10 mL axenic C. sorokiniana culture, cultivated in sterile mineral medium (C30), was added to a sterile flask containing 90 mL sterile C30 medium, composed of (in g·L−1 ): KNO3 (25), MgSO4 •7H2 O (10), KH2 PO4 (4), K2 HPO4 (1), FeSO4 •7H2 O (1), and (in μg·L−1 ): H3 BO3 (2.86), MnCl2 •4H2 O (1.81), ZnSO4 •7H2 O (0.11), CuSO4 •5H2 O (0.09), NaMoO4 (0.021), pH 5.25 and incubated at 27 ± 2 °C on a rotary shaker at 120 rpm under 60 μmol photon·m−2 ·s−1 continuous light intensity for 6 days .

    Selection:

    Article Title: BioVector, a flexible system for gene specific-expression in plants
    Article Snippet: .. A ccd B gene in REL sites (Figure ) served as a negative selection marker for E. coli DH5α as all GATEWAY entry vectors employ (Invitrogen) [ , , ]. .. The chloramphenicol (Cm) was employed as a selection marker in E. coli , which may be compatible to most of binary vectors available.

    Article Title: Characterization of a minimal pKW2124 replicon from Weissella cibaria KLC140 and its application for the construction of the Weissella expression vector pKUCm1
    Article Snippet: All Weissella species were incubated anaerobically at 37°C in de Man-Rogosa-Sharpe (MRS) medium (Difco, Detroit, MI, USA) and E. coli DH5α (Invitrogen, Carlsbad, CA, USA) was grown with shaking in Luria-Bertani (LB) medium (Difco) at 37°C. .. Ampicillin sulfate (Sigma, St. Louis, MO, USA) was added to the E. coli growth medium for selection at 50 μg/ml.

    Agarose Gel Electrophoresis:

    Article Title: Transfer of Herb-Resistance Plasmid From Escherichia coli to Staphylococcus aureus Residing in the Human Urinary Tract
    Article Snippet: Plasmid Characterization Total plasmid DNA was prepared (Plasmid Extraction Kit, Beijing TIANGEN) and transferred into E. coli DH5α (Invitrogen, Carlsbad, CA, USA). .. Plasmid DNA was separated on a 0.7% agarose gel, stained with ethidium bromide, and visualized under UV-light.

    Concentration Assay:

    Article Title: What an Escherichia coli Mutant Can Teach Us About the Antibacterial Effect of Chlorophyllin
    Article Snippet: Bacteria Strains and Cell Culture Experiments were performed with E. coli DH5α (Invitrogen, Carlsbad, CA, USA), E. coli NR698 (MC4100 lptD4213 ; kindly provided by M. Grabowicz, Princeton University, NJ, USA) [ ] and B. subtilis 168 (trpC2 ; laboratory stock). .. Prior to the experiments, cell concentration was determined optically at 590 nm and set to OD590 = 0.1 before cells were diluted in LB as required.

    Marker:

    Article Title: Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74?sp chitinase inclusions, Cry crystals and spores for applied use
    Article Snippet: All constructs (Figure A) were propagated in E. coli DH5α [supE44, DlacU169 (F80lacZDM15 ) hsdR17 recA1 end A1 gyrA96 thi-1 relA1 ] (Invitrogen, Carlsbad CA, USA) and then used for transforming the acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7, (hereafter 4Q7), and B. thuringiensis subsp. kurstaki HD1 (hereafter HD1) (Bacillus Genetic Stock Center, Columbus, OH). .. Plasmid pGLO is a vector that harbors the green fluorescent protein (gfp ) gene under the control arabinose (araC ) promoter and contains an ampicillin (bla ) resistance gene marker (Bio-Rad, Hercules CA, USA).

    Article Title: BioVector, a flexible system for gene specific-expression in plants
    Article Snippet: .. A ccd B gene in REL sites (Figure ) served as a negative selection marker for E. coli DH5α as all GATEWAY entry vectors employ (Invitrogen) [ , , ]. .. The chloramphenicol (Cm) was employed as a selection marker in E. coli , which may be compatible to most of binary vectors available.

    Staining:

    Article Title: Transfer of Herb-Resistance Plasmid From Escherichia coli to Staphylococcus aureus Residing in the Human Urinary Tract
    Article Snippet: Plasmid Characterization Total plasmid DNA was prepared (Plasmid Extraction Kit, Beijing TIANGEN) and transferred into E. coli DH5α (Invitrogen, Carlsbad, CA, USA). .. Plasmid DNA was separated on a 0.7% agarose gel, stained with ethidium bromide, and visualized under UV-light.

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    Thermo Fisher max efficiency dh5α competent cells
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