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Addgene inc e coli k 12 strain mg1655
E Coli K 12 Strain Mg1655, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Addgene inc wt e coli strain mg1655
NT-seq simultaneously detects three types of methylation motifs in bacteria. a A to G frequency at known 6mA sites (G6mATC, A6mACN 6 GTGC, and GC6mACN 6 GTT) and unmethylated A sites in E. coli <t>MG1655</t> genome from native and PCR amplified DNA. b C to T frequency at known 4mC sites (T4mCTTC and 4mCCGG) and unmethylated C sites in H. pylori JP26 genome from native and PCR amplified DNA. c–e Negative log normalized A to G ratio of different 6mA motifs in E. coli WT strain MG1655 ( a ), hsdM deleted strain ( b ), and dam/dcm/hsdR mutated strain ( c ). 6mA position was underlined. f Negative log normalized C to T ratio of different 4mC motifs in H. pylori JP26. g Negative log normalized C to T ratio of 4mC sites identified by SMRT-seq in E. coli strain MG1655. h Negative log normalized C to T ratio of different 5mC motifs in H. pylori JP26. Only motifs with sequencing depth larger than 25× were considered for violin plots. Statistic test were performed by two-sided Mann-Whitney-Wilcoxon (MWW) test with Bonferroni correction (ns: P > 1.0e-3; ****: P ≤ 1.0e−6)
Wt E Coli Strain Mg1655, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "NT-seq: a chemical-based sequencing method for genomic methylome profiling"

Article Title: NT-seq: a chemical-based sequencing method for genomic methylome profiling

Journal: Genome Biology

doi: 10.1186/s13059-022-02689-9

NT-seq simultaneously detects three types of methylation motifs in bacteria. a A to G frequency at known 6mA sites (G6mATC, A6mACN 6 GTGC, and GC6mACN 6 GTT) and unmethylated A sites in E. coli MG1655 genome from native and PCR amplified DNA. b C to T frequency at known 4mC sites (T4mCTTC and 4mCCGG) and unmethylated C sites in H. pylori JP26 genome from native and PCR amplified DNA. c–e Negative log normalized A to G ratio of different 6mA motifs in E. coli WT strain MG1655 ( a ), hsdM deleted strain ( b ), and dam/dcm/hsdR mutated strain ( c ). 6mA position was underlined. f Negative log normalized C to T ratio of different 4mC motifs in H. pylori JP26. g Negative log normalized C to T ratio of 4mC sites identified by SMRT-seq in E. coli strain MG1655. h Negative log normalized C to T ratio of different 5mC motifs in H. pylori JP26. Only motifs with sequencing depth larger than 25× were considered for violin plots. Statistic test were performed by two-sided Mann-Whitney-Wilcoxon (MWW) test with Bonferroni correction (ns: P > 1.0e-3; ****: P ≤ 1.0e−6)
Figure Legend Snippet: NT-seq simultaneously detects three types of methylation motifs in bacteria. a A to G frequency at known 6mA sites (G6mATC, A6mACN 6 GTGC, and GC6mACN 6 GTT) and unmethylated A sites in E. coli MG1655 genome from native and PCR amplified DNA. b C to T frequency at known 4mC sites (T4mCTTC and 4mCCGG) and unmethylated C sites in H. pylori JP26 genome from native and PCR amplified DNA. c–e Negative log normalized A to G ratio of different 6mA motifs in E. coli WT strain MG1655 ( a ), hsdM deleted strain ( b ), and dam/dcm/hsdR mutated strain ( c ). 6mA position was underlined. f Negative log normalized C to T ratio of different 4mC motifs in H. pylori JP26. g Negative log normalized C to T ratio of 4mC sites identified by SMRT-seq in E. coli strain MG1655. h Negative log normalized C to T ratio of different 5mC motifs in H. pylori JP26. Only motifs with sequencing depth larger than 25× were considered for violin plots. Statistic test were performed by two-sided Mann-Whitney-Wilcoxon (MWW) test with Bonferroni correction (ns: P > 1.0e-3; ****: P ≤ 1.0e−6)

Techniques Used: Methylation, Amplification, Sequencing, MANN-WHITNEY


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Addgene inc e coli k 12 mg1655 rare

E Coli K 12 Mg1655 Rare, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Biosensor and chemo-enzymatic one-pot cascade applications to detect and transform PET-derived terephthalic acid in living cells"

Article Title: Biosensor and chemo-enzymatic one-pot cascade applications to detect and transform PET-derived terephthalic acid in living cells

Journal: iScience

doi: 10.1016/j.isci.2022.104326


Figure Legend Snippet:

Techniques Used: Recombinant, Purification, Variant Assay, Mutagenesis, Plasmid Preparation, Software


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Addgene inc e coli k 12 mg1655 δcrp genome
E Coli K 12 Mg1655 δcrp Genome, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Addgene inc e coli mg1655
Lon overexpression rescues colony formation in OFX-treated Δ lon cells. (A and B) WT <t>E.</t> <t>coli</t> <t>MG1655</t> (A) and Δ lon E. coli MG1655 (B) containing the sulA-gfp reporter plasmid (pMSs201-P sulA -gfp ) were grown to stationary phase and diluted 100-fold in fresh LB medium. Diluted cells were then treated with 5 μg/mL OFX for 6 h. After treatment, phase-contrast and fluorescence (GFP) micrographs of cells were obtained. Images at 0 ( t = 0) and 6 h ( t = 6 h) from a representative replicate are shown, but similar data were obtained from four biological replicates ( n = 4). Scale bar, 25 μm. (C and D) E. coli cells (WT and Δ lon ) containing an inducible lon overexpression plasmid (pUA66- lon ) or empty vector control (pUA66-EV) were treated with OFX (5 μg/mL) for 6 h and transferred to LB agar plates supplemented with isopropyl β- d -1-thiogalactopyranoside (IPTG) at the indicated concentrations. CFU were measured for each strain before and after treatment. n = 4. (E) WT and Δ lon E. coli containing pUA66- lon were cultured with 1 mM IPTG before and/or during OFX treatment. After treatment, cells were transferred to agar plates with or without 1 mM IPTG. CFU were measured for each strain before and after treatment. n = 4. Statistical significance for pairwise comparisons was assessed using one-way analysis of variance (ANOVA) with Dunnett’s post hoc test. * * , P < 0.01; ** * , P < 0.001; *** * , P < 0.0001; ns, nonsignificant.
E Coli Mg1655, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "lon Deletion Impairs Persister Cell Resuscitation in Escherichia coli"

Article Title: lon Deletion Impairs Persister Cell Resuscitation in Escherichia coli

Journal: mBio

doi: 10.1128/mbio.02187-21

Lon overexpression rescues colony formation in OFX-treated Δ lon cells. (A and B) WT E. coli MG1655 (A) and Δ lon E. coli MG1655 (B) containing the sulA-gfp reporter plasmid (pMSs201-P sulA -gfp ) were grown to stationary phase and diluted 100-fold in fresh LB medium. Diluted cells were then treated with 5 μg/mL OFX for 6 h. After treatment, phase-contrast and fluorescence (GFP) micrographs of cells were obtained. Images at 0 ( t = 0) and 6 h ( t = 6 h) from a representative replicate are shown, but similar data were obtained from four biological replicates ( n = 4). Scale bar, 25 μm. (C and D) E. coli cells (WT and Δ lon ) containing an inducible lon overexpression plasmid (pUA66- lon ) or empty vector control (pUA66-EV) were treated with OFX (5 μg/mL) for 6 h and transferred to LB agar plates supplemented with isopropyl β- d -1-thiogalactopyranoside (IPTG) at the indicated concentrations. CFU were measured for each strain before and after treatment. n = 4. (E) WT and Δ lon E. coli containing pUA66- lon were cultured with 1 mM IPTG before and/or during OFX treatment. After treatment, cells were transferred to agar plates with or without 1 mM IPTG. CFU were measured for each strain before and after treatment. n = 4. Statistical significance for pairwise comparisons was assessed using one-way analysis of variance (ANOVA) with Dunnett’s post hoc test. * * , P < 0.01; ** * , P < 0.001; *** * , P < 0.0001; ns, nonsignificant.
Figure Legend Snippet: Lon overexpression rescues colony formation in OFX-treated Δ lon cells. (A and B) WT E. coli MG1655 (A) and Δ lon E. coli MG1655 (B) containing the sulA-gfp reporter plasmid (pMSs201-P sulA -gfp ) were grown to stationary phase and diluted 100-fold in fresh LB medium. Diluted cells were then treated with 5 μg/mL OFX for 6 h. After treatment, phase-contrast and fluorescence (GFP) micrographs of cells were obtained. Images at 0 ( t = 0) and 6 h ( t = 6 h) from a representative replicate are shown, but similar data were obtained from four biological replicates ( n = 4). Scale bar, 25 μm. (C and D) E. coli cells (WT and Δ lon ) containing an inducible lon overexpression plasmid (pUA66- lon ) or empty vector control (pUA66-EV) were treated with OFX (5 μg/mL) for 6 h and transferred to LB agar plates supplemented with isopropyl β- d -1-thiogalactopyranoside (IPTG) at the indicated concentrations. CFU were measured for each strain before and after treatment. n = 4. (E) WT and Δ lon E. coli containing pUA66- lon were cultured with 1 mM IPTG before and/or during OFX treatment. After treatment, cells were transferred to agar plates with or without 1 mM IPTG. CFU were measured for each strain before and after treatment. n = 4. Statistical significance for pairwise comparisons was assessed using one-way analysis of variance (ANOVA) with Dunnett’s post hoc test. * * , P < 0.01; ** * , P < 0.001; *** * , P < 0.0001; ns, nonsignificant.

Techniques Used: Over Expression, Plasmid Preparation, Fluorescence, Cell Culture

Lon overexpression rescues the colony-forming ability of Δ lon cells exposed to UV. (A) WT and Δ lon E. coli containing pMSs201-P sulA -gfp were grown to stationary phase, diluted 100-fold in fresh LB medium, and exposed to UV for the indicated times. After exposure, cells were cultured in a shaker for 2 h, and phase-contrast and fluorescence (GFP) micrographs of cells were obtained. Images from a representative replicate are shown, but similar data were obtained for four biological replicates. n = 4. Scale bar, 25 μm. (B) WT and Δ lon E. coli were exposed to UV as described in panel A and spotted onto LB agar plates to enumerate CFU. n = 4. (C to E) Cells containing the inducible lon overexpression plasmid (pUA66- lon ) or empty vector (pUA66-EV) control were exposed to UV for the indicated times and transferred to agar plates supplemented with IPTG at indicated concentrations to enumerate CFU. n = 4. Statistical significance for pairwise comparisons was assessed using one-way ANOVA with Dunnett’s post hoc test. ** * , P < 0.001; *** * , P < 0.0001; ns, nonsignificant.
Figure Legend Snippet: Lon overexpression rescues the colony-forming ability of Δ lon cells exposed to UV. (A) WT and Δ lon E. coli containing pMSs201-P sulA -gfp were grown to stationary phase, diluted 100-fold in fresh LB medium, and exposed to UV for the indicated times. After exposure, cells were cultured in a shaker for 2 h, and phase-contrast and fluorescence (GFP) micrographs of cells were obtained. Images from a representative replicate are shown, but similar data were obtained for four biological replicates. n = 4. Scale bar, 25 μm. (B) WT and Δ lon E. coli were exposed to UV as described in panel A and spotted onto LB agar plates to enumerate CFU. n = 4. (C to E) Cells containing the inducible lon overexpression plasmid (pUA66- lon ) or empty vector (pUA66-EV) control were exposed to UV for the indicated times and transferred to agar plates supplemented with IPTG at indicated concentrations to enumerate CFU. n = 4. Statistical significance for pairwise comparisons was assessed using one-way ANOVA with Dunnett’s post hoc test. ** * , P < 0.001; *** * , P < 0.0001; ns, nonsignificant.

Techniques Used: Over Expression, Cell Culture, Fluorescence, Plasmid Preparation

Starvation in PBS solution rescues Δ lon persisters. (A) WT and Δ lon E. coli with or without the inducible lon overexpression plasmid (pUA66- lon ) or empty vector control (pUA66-EV) were grown to stationary phase and diluted 100-fold in fresh LB medium. Diluted cells were then treated with OFX for 6 h. After treatment, cells were washed, serially diluted, and spotted onto LB agar plates with or without IPTG (1 mM). Plates were incubated for 48 h, and images were captured at indicated time points. A representative replicate is shown, but similar data were obtained for four biological replicates. n = 4. (B) WT and Δ lon E. coli with or without pUA66- lon or pUA66-EV were treated with OFX for 6 h, transferred to sterile PBS solution with or without 1 mM IPTG, and incubated at 37°C in a shaker for 7 days. Samples were collected at the indicated time points and spotted onto agar plates to enumerate surviving cells. n = 4. Statistical significance for pairwise comparison was assessed using one-way ANOVA with Dunnett’s post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant.
Figure Legend Snippet: Starvation in PBS solution rescues Δ lon persisters. (A) WT and Δ lon E. coli with or without the inducible lon overexpression plasmid (pUA66- lon ) or empty vector control (pUA66-EV) were grown to stationary phase and diluted 100-fold in fresh LB medium. Diluted cells were then treated with OFX for 6 h. After treatment, cells were washed, serially diluted, and spotted onto LB agar plates with or without IPTG (1 mM). Plates were incubated for 48 h, and images were captured at indicated time points. A representative replicate is shown, but similar data were obtained for four biological replicates. n = 4. (B) WT and Δ lon E. coli with or without pUA66- lon or pUA66-EV were treated with OFX for 6 h, transferred to sterile PBS solution with or without 1 mM IPTG, and incubated at 37°C in a shaker for 7 days. Samples were collected at the indicated time points and spotted onto agar plates to enumerate surviving cells. n = 4. Statistical significance for pairwise comparison was assessed using one-way ANOVA with Dunnett’s post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant.

Techniques Used: Over Expression, Plasmid Preparation, Incubation


Structured Review

Addgene inc e coli mg1655
(A) Hexbin density plot showing the relative distributions of expression changes for yigI and tesB in response to the range of conditions represented in the COLOMBOS database. (B) As in A , comparing yigI and fadM expression. (C) Structural (lower triangle) and sequence (upper triangle) similarities between the four <t>E.</t> <t>coli</t> thioesterase domains considered here; TM scores are averaged across normalizations by the longer and shorter protein in each case. (D) Sequence identities of the best hit (identified via psiblast) in the landmark database for each of the E. coli <t>MG1655</t> thioesterase domains considered here. “0” is shown in cases where no detectable homologs were present passing our E-value threshold.
E Coli Mg1655, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Escherichia coli YigI is a conserved ɣ-proteobacterial acyl-CoA thioesterase permitting metabolism of unusual fatty acid substrates"

Article Title: Escherichia coli YigI is a conserved ɣ-proteobacterial acyl-CoA thioesterase permitting metabolism of unusual fatty acid substrates

Journal: bioRxiv

doi: 10.1101/2022.01.10.475765

(A) Hexbin density plot showing the relative distributions of expression changes for yigI and tesB in response to the range of conditions represented in the COLOMBOS database. (B) As in A , comparing yigI and fadM expression. (C) Structural (lower triangle) and sequence (upper triangle) similarities between the four E. coli thioesterase domains considered here; TM scores are averaged across normalizations by the longer and shorter protein in each case. (D) Sequence identities of the best hit (identified via psiblast) in the landmark database for each of the E. coli MG1655 thioesterase domains considered here. “0” is shown in cases where no detectable homologs were present passing our E-value threshold.
Figure Legend Snippet: (A) Hexbin density plot showing the relative distributions of expression changes for yigI and tesB in response to the range of conditions represented in the COLOMBOS database. (B) As in A , comparing yigI and fadM expression. (C) Structural (lower triangle) and sequence (upper triangle) similarities between the four E. coli thioesterase domains considered here; TM scores are averaged across normalizations by the longer and shorter protein in each case. (D) Sequence identities of the best hit (identified via psiblast) in the landmark database for each of the E. coli MG1655 thioesterase domains considered here. “0” is shown in cases where no detectable homologs were present passing our E-value threshold.

Techniques Used: Expressing, Sequencing


Structured Review

Addgene inc e coli mg1655
(A) DSAD1 inhibits DSR2 defense. Liquid culture growth of B. subtilis BEST7003 cells expressing either DSR2 alone, or DSR2 and DSAD1, or control cells expressing neither gene, infected by phage SPR at 30 °C. Bacteria were infected at time 0 at an MOI of 0.04. Three independent replicates are shown for each MOI, and each curve shows an individual replicate. (B) Pulldown assays of the DSR2-DSAD1 and DSR2-tail tube complexes. DSAD1 and the tail tube protein of phage SPR were C-terminally tagged with TwinStrep tag. DSR2 in this experiment was mutated (H171A) to avoid toxicity. Shown is an SDS-PAGE gel. Lane A, cells expressing DSR2. Lane B, cells expressing tagged DSAD1. Lane C, cells expressing tagged tail tube protein from phage SPR. Lane D, cells co-expressing DSR2 and tagged DSAD1. Lane E, cells co-expressing DSR2 and tagged tail tube protein from SPR. (C) Transformation efficiencies of a vector containing the SPR tail tube protein or GFP control were measured for cells containing either WT DSR2 or two inactive DSR2 mutants. Y-axis represents the number of colonyforming units per milliliter. Bar graphs represent average of three replicates, with individual data points overlaid. (D) Liquid culture growth of <t>E.</t> <t>coli</t> that contains DSR2 and the tail tube gene of phage SPR, each under the control of an inducible promoter, and control E. coli that contains inducible GFP and DSR2 genes. Expression was induced at time 0 by adding 0.2% arabinose (for the tail tube gene) and 1 mM IPTG (for DSR2). Three independent replicates are shown, and each curve shows an individual replicate. (E-F) Concentrations of NAD + and ADPR in cell lysates extracted from E. coli co-expressing DSR2 and SPR tail tube. X-axis represents minutes post expression induction, with zero representing non-induced cells. Control cells in this experiment express RFP and DSR2. Bar graphs represent the average of two biological replicates, with individual data points overlaid. (G) A model for the mechanism of action of DSR2. Phage infection is sensed by the recognition of the phage tail tube protein through direct binding to DSR2. This triggers the enzymatic activity of the SIR2 domain to deplete the cell of NAD + thereby causing abortive infection. The phage anti-DSR2 protein DSAD1 inhibits DSR2 by direct binding.
E Coli Mg1655, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 1 article reviews
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e coli mg1655 - by Bioz Stars, 2023-01
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1) Product Images from "Multiple phage resistance systems inhibit infection via SIR2-dependent NAD + depletion"

Article Title: Multiple phage resistance systems inhibit infection via SIR2-dependent NAD + depletion

Journal: bioRxiv

doi: 10.1101/2021.12.14.472415

(A) DSAD1 inhibits DSR2 defense. Liquid culture growth of B. subtilis BEST7003 cells expressing either DSR2 alone, or DSR2 and DSAD1, or control cells expressing neither gene, infected by phage SPR at 30 °C. Bacteria were infected at time 0 at an MOI of 0.04. Three independent replicates are shown for each MOI, and each curve shows an individual replicate. (B) Pulldown assays of the DSR2-DSAD1 and DSR2-tail tube complexes. DSAD1 and the tail tube protein of phage SPR were C-terminally tagged with TwinStrep tag. DSR2 in this experiment was mutated (H171A) to avoid toxicity. Shown is an SDS-PAGE gel. Lane A, cells expressing DSR2. Lane B, cells expressing tagged DSAD1. Lane C, cells expressing tagged tail tube protein from phage SPR. Lane D, cells co-expressing DSR2 and tagged DSAD1. Lane E, cells co-expressing DSR2 and tagged tail tube protein from SPR. (C) Transformation efficiencies of a vector containing the SPR tail tube protein or GFP control were measured for cells containing either WT DSR2 or two inactive DSR2 mutants. Y-axis represents the number of colonyforming units per milliliter. Bar graphs represent average of three replicates, with individual data points overlaid. (D) Liquid culture growth of E. coli that contains DSR2 and the tail tube gene of phage SPR, each under the control of an inducible promoter, and control E. coli that contains inducible GFP and DSR2 genes. Expression was induced at time 0 by adding 0.2% arabinose (for the tail tube gene) and 1 mM IPTG (for DSR2). Three independent replicates are shown, and each curve shows an individual replicate. (E-F) Concentrations of NAD + and ADPR in cell lysates extracted from E. coli co-expressing DSR2 and SPR tail tube. X-axis represents minutes post expression induction, with zero representing non-induced cells. Control cells in this experiment express RFP and DSR2. Bar graphs represent the average of two biological replicates, with individual data points overlaid. (G) A model for the mechanism of action of DSR2. Phage infection is sensed by the recognition of the phage tail tube protein through direct binding to DSR2. This triggers the enzymatic activity of the SIR2 domain to deplete the cell of NAD + thereby causing abortive infection. The phage anti-DSR2 protein DSAD1 inhibits DSR2 by direct binding.
Figure Legend Snippet: (A) DSAD1 inhibits DSR2 defense. Liquid culture growth of B. subtilis BEST7003 cells expressing either DSR2 alone, or DSR2 and DSAD1, or control cells expressing neither gene, infected by phage SPR at 30 °C. Bacteria were infected at time 0 at an MOI of 0.04. Three independent replicates are shown for each MOI, and each curve shows an individual replicate. (B) Pulldown assays of the DSR2-DSAD1 and DSR2-tail tube complexes. DSAD1 and the tail tube protein of phage SPR were C-terminally tagged with TwinStrep tag. DSR2 in this experiment was mutated (H171A) to avoid toxicity. Shown is an SDS-PAGE gel. Lane A, cells expressing DSR2. Lane B, cells expressing tagged DSAD1. Lane C, cells expressing tagged tail tube protein from phage SPR. Lane D, cells co-expressing DSR2 and tagged DSAD1. Lane E, cells co-expressing DSR2 and tagged tail tube protein from SPR. (C) Transformation efficiencies of a vector containing the SPR tail tube protein or GFP control were measured for cells containing either WT DSR2 or two inactive DSR2 mutants. Y-axis represents the number of colonyforming units per milliliter. Bar graphs represent average of three replicates, with individual data points overlaid. (D) Liquid culture growth of E. coli that contains DSR2 and the tail tube gene of phage SPR, each under the control of an inducible promoter, and control E. coli that contains inducible GFP and DSR2 genes. Expression was induced at time 0 by adding 0.2% arabinose (for the tail tube gene) and 1 mM IPTG (for DSR2). Three independent replicates are shown, and each curve shows an individual replicate. (E-F) Concentrations of NAD + and ADPR in cell lysates extracted from E. coli co-expressing DSR2 and SPR tail tube. X-axis represents minutes post expression induction, with zero representing non-induced cells. Control cells in this experiment express RFP and DSR2. Bar graphs represent the average of two biological replicates, with individual data points overlaid. (G) A model for the mechanism of action of DSR2. Phage infection is sensed by the recognition of the phage tail tube protein through direct binding to DSR2. This triggers the enzymatic activity of the SIR2 domain to deplete the cell of NAD + thereby causing abortive infection. The phage anti-DSR2 protein DSAD1 inhibits DSR2 by direct binding.

Techniques Used: Expressing, Infection, SDS Page, Transformation Assay, Plasmid Preparation, Binding Assay, Activity Assay

(A) Domain organization of three defense systems that contain SIR2 domains. Protein accessions in NCBI are indicated. (B) Efficiency of plating for phages infecting defense-system-containing strains and control strains. SIR2-HerA and SIR2/pAgo were cloned into E. coli MG 1655, and DSR1 was cloned into B. subtilis BEST7003. Bar graphs are average of three biological replicates, with individual data points overlaid. KAW1E185 is short for vB_EcoM-KAW1E185, a T4-like phage. (C-E) Concentrations of NAD + in cell lysates extracted from infected cells as measured by targeted LC-MS with synthesized standards. X-axis represents minutes post infection, with zero representing non-infected cells. “No system” are control cells that contain an empty vector instead of the defense system. Bar graphs represent the average of two biological replicates, with individual data points overlaid. (F-H). Liquid culture growth of bacteria that contain the respective defense system and control bacteria that contain an empty vector (no system). Bacteria were infected at time 0 at low or high MOIs, as indicated. Three independent replicates are shown for each MOI, and each curve shows an individual replicate.
Figure Legend Snippet: (A) Domain organization of three defense systems that contain SIR2 domains. Protein accessions in NCBI are indicated. (B) Efficiency of plating for phages infecting defense-system-containing strains and control strains. SIR2-HerA and SIR2/pAgo were cloned into E. coli MG 1655, and DSR1 was cloned into B. subtilis BEST7003. Bar graphs are average of three biological replicates, with individual data points overlaid. KAW1E185 is short for vB_EcoM-KAW1E185, a T4-like phage. (C-E) Concentrations of NAD + in cell lysates extracted from infected cells as measured by targeted LC-MS with synthesized standards. X-axis represents minutes post infection, with zero representing non-infected cells. “No system” are control cells that contain an empty vector instead of the defense system. Bar graphs represent the average of two biological replicates, with individual data points overlaid. (F-H). Liquid culture growth of bacteria that contain the respective defense system and control bacteria that contain an empty vector (no system). Bacteria were infected at time 0 at low or high MOIs, as indicated. Three independent replicates are shown for each MOI, and each curve shows an individual replicate.

Techniques Used: Clone Assay, Infection, Liquid Chromatography with Mass Spectroscopy, Synthesized, Plasmid Preparation


Structured Review

Addgene inc e coli mg1655
Purified MgtA solubilized in 3x CMC DDM was analysed by native MS (a) in the absence or (b) presence of total <t>E.</t> <t>coli</t> lipid extract. Total E. coli lipid extract contains only 9.8 % CL, while the remaining lipids are 57.5 % PE, 15.1 % PG and 17.6 % unknown (Avanti Polar Lipids). The left spectrum shows the most intense charge states 22+ and 21+ of purified MgtA after lipid removal by high CMC DDM. The right spectrum exhibits MgtA as seen after incubation with E. coli lipid mixture. This sample was washed with 3x CMC DDM to remove weakly bound lipids leaving only the strongest interacting lipids. The spectrum on the right reveals additions of approximately 1400 and 2800 Da (red and green) which correspond to the molecular weight of one and two cardiolipin molecules, respectively. The spectra were recorded in TOF mode and the key MS settings are the voltages (sampling cone, trap collision, transfer collision and trap bias) and pressures (backing pressure, trap cell pressure). These values were 200 V, 150 V, 150 V, 45 V, 6.41 mbar and 2.00×10 −2 mbar respectively.
E Coli Mg1655, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "The bacterial magnesium transporter MgtA reveals highly selective interaction with specific cardiolipin species"

Article Title: The bacterial magnesium transporter MgtA reveals highly selective interaction with specific cardiolipin species

Journal: bioRxiv

doi: 10.1101/2021.09.27.462033

Purified MgtA solubilized in 3x CMC DDM was analysed by native MS (a) in the absence or (b) presence of total E. coli lipid extract. Total E. coli lipid extract contains only 9.8 % CL, while the remaining lipids are 57.5 % PE, 15.1 % PG and 17.6 % unknown (Avanti Polar Lipids). The left spectrum shows the most intense charge states 22+ and 21+ of purified MgtA after lipid removal by high CMC DDM. The right spectrum exhibits MgtA as seen after incubation with E. coli lipid mixture. This sample was washed with 3x CMC DDM to remove weakly bound lipids leaving only the strongest interacting lipids. The spectrum on the right reveals additions of approximately 1400 and 2800 Da (red and green) which correspond to the molecular weight of one and two cardiolipin molecules, respectively. The spectra were recorded in TOF mode and the key MS settings are the voltages (sampling cone, trap collision, transfer collision and trap bias) and pressures (backing pressure, trap cell pressure). These values were 200 V, 150 V, 150 V, 45 V, 6.41 mbar and 2.00×10 −2 mbar respectively.
Figure Legend Snippet: Purified MgtA solubilized in 3x CMC DDM was analysed by native MS (a) in the absence or (b) presence of total E. coli lipid extract. Total E. coli lipid extract contains only 9.8 % CL, while the remaining lipids are 57.5 % PE, 15.1 % PG and 17.6 % unknown (Avanti Polar Lipids). The left spectrum shows the most intense charge states 22+ and 21+ of purified MgtA after lipid removal by high CMC DDM. The right spectrum exhibits MgtA as seen after incubation with E. coli lipid mixture. This sample was washed with 3x CMC DDM to remove weakly bound lipids leaving only the strongest interacting lipids. The spectrum on the right reveals additions of approximately 1400 and 2800 Da (red and green) which correspond to the molecular weight of one and two cardiolipin molecules, respectively. The spectra were recorded in TOF mode and the key MS settings are the voltages (sampling cone, trap collision, transfer collision and trap bias) and pressures (backing pressure, trap cell pressure). These values were 200 V, 150 V, 150 V, 45 V, 6.41 mbar and 2.00×10 −2 mbar respectively.

Techniques Used: Purification, Incubation, Molecular Weight, Sampling

Purified MgtA, solubilized in 3x CMC DDM, was analysed by native MS ( a, b ) in the absence or ( c, d ) presence of total E. coli lipid extract. Total E. coli lipid extract contains only 9.8 % CL, while the remaining lipids are 57.5 % PE, 15.1 % PG and 17.6 % unknown (Avanti Polar Lipids). The upper spectrum shows delipidated MgtA in DDM (19+ to 24+) with DDM clusters in the lower m/z region, while the lower spectra exhibits MgtA with E. coli lipid mixture (18+ to 24+) with a mixture of DDM clusters and lipids in the lower m/z region. Close-up on MgtA spectra (right) reveals additions of approximately 1400 and 2800 Da (red and green arrows) which correspond to the molecular weight of one and two cardiolipin molecules, respectively. The spectra were recorded in TOF mode and the key MS settings used to record these spectra are the voltages (sampling cone, trap collision, transfer collision and trap bias) and pressures (backing pressure, trap cell pressure). These values were 200 V, 150 V, 150 V, 45 V, 6.41 mbar and 2.00×10 −2 mbar respectively.
Figure Legend Snippet: Purified MgtA, solubilized in 3x CMC DDM, was analysed by native MS ( a, b ) in the absence or ( c, d ) presence of total E. coli lipid extract. Total E. coli lipid extract contains only 9.8 % CL, while the remaining lipids are 57.5 % PE, 15.1 % PG and 17.6 % unknown (Avanti Polar Lipids). The upper spectrum shows delipidated MgtA in DDM (19+ to 24+) with DDM clusters in the lower m/z region, while the lower spectra exhibits MgtA with E. coli lipid mixture (18+ to 24+) with a mixture of DDM clusters and lipids in the lower m/z region. Close-up on MgtA spectra (right) reveals additions of approximately 1400 and 2800 Da (red and green arrows) which correspond to the molecular weight of one and two cardiolipin molecules, respectively. The spectra were recorded in TOF mode and the key MS settings used to record these spectra are the voltages (sampling cone, trap collision, transfer collision and trap bias) and pressures (backing pressure, trap cell pressure). These values were 200 V, 150 V, 150 V, 45 V, 6.41 mbar and 2.00×10 −2 mbar respectively.

Techniques Used: Purification, Molecular Weight, Sampling

To determine binding of cardiolipin to MgtA dependent on the stages of transport cycle, lipid binding of MgtA was determined in the presence of AlF 4 - and ADP-AlF 4 - , which lock MgtA in the E2 and E1 state, respectively. (A) Mg 2+ -transport cycle of by MgtA is described by the Post-Albers cycle, in which MgtA alternates between different conformational states, termed E1 and E2 state. The E1 state has high affinity and is open for ions binding from the cytoplasm. The ion transported from the cytoplasm to the periplasm in the case of MgtA remains unknown. Upon ion binding, autophosphorylation of MgtA by ATP hydrolysis is induced, forming the E1P state. The phosphorylation induces domain rearrangements leading to the E2P state, which is now open to the periplasmic side. E2P has low affinity for the bound ions and high affinity for the counter ion, Mg 2+. The exchange of counter ions dephosphorylates the enzyme and forms the E2 state. Upon further conformational changes the enzyme returns to the E1 state and the counterions are released into the cytoplasm. MgtA inhibitors, AlF 4 - and ADP-AlF 4 - , lock the enzyme in the indicated conformational steps. (B) MgtA was incubated with inhibitors in 1:100 molar ratio for two hours, following incubation with total E. coli lipid extract in 1:100 molar ratio. Spectra of MgtA lipidated with cardiolipin in the absence (violet, blue) and in the presence of inhibitors (dark blue, yellow) are shown. A control spectrum of unlipidated MgtA (black) is included.
Figure Legend Snippet: To determine binding of cardiolipin to MgtA dependent on the stages of transport cycle, lipid binding of MgtA was determined in the presence of AlF 4 - and ADP-AlF 4 - , which lock MgtA in the E2 and E1 state, respectively. (A) Mg 2+ -transport cycle of by MgtA is described by the Post-Albers cycle, in which MgtA alternates between different conformational states, termed E1 and E2 state. The E1 state has high affinity and is open for ions binding from the cytoplasm. The ion transported from the cytoplasm to the periplasm in the case of MgtA remains unknown. Upon ion binding, autophosphorylation of MgtA by ATP hydrolysis is induced, forming the E1P state. The phosphorylation induces domain rearrangements leading to the E2P state, which is now open to the periplasmic side. E2P has low affinity for the bound ions and high affinity for the counter ion, Mg 2+. The exchange of counter ions dephosphorylates the enzyme and forms the E2 state. Upon further conformational changes the enzyme returns to the E1 state and the counterions are released into the cytoplasm. MgtA inhibitors, AlF 4 - and ADP-AlF 4 - , lock the enzyme in the indicated conformational steps. (B) MgtA was incubated with inhibitors in 1:100 molar ratio for two hours, following incubation with total E. coli lipid extract in 1:100 molar ratio. Spectra of MgtA lipidated with cardiolipin in the absence (violet, blue) and in the presence of inhibitors (dark blue, yellow) are shown. A control spectrum of unlipidated MgtA (black) is included.

Techniques Used: Binding Assay, Incubation

(A) Cardiolipins 18:1 and 16:0 contain the same polar hydrophilic headgroup (red), but exhibit differences in the length and saturation degree of their acyl chains. (B) Concentration-dependent activation of MgtA ATPase activity by E. coli extracted cardiolipin (black), a 1:1 mixture of cardiolipin 18:1 and 16:0 (green) or individual cardiolipin species. Lipids are prepared as described in the Materials and Methods section; specific activity is determined by measuring phosphate release. Curves are representatives from three independent experiments, showing mean +/- SD. Enzymatic activation curves were fitted to an allosteric sigmoidal model (GraphPad, Prism8). Fitting curves are shown (lines) for data with least squares fit of 0.95 or above with parameters described in . Dotted lines represent activation curves that could not be fitted with the allosteric sigmoidal model.
Figure Legend Snippet: (A) Cardiolipins 18:1 and 16:0 contain the same polar hydrophilic headgroup (red), but exhibit differences in the length and saturation degree of their acyl chains. (B) Concentration-dependent activation of MgtA ATPase activity by E. coli extracted cardiolipin (black), a 1:1 mixture of cardiolipin 18:1 and 16:0 (green) or individual cardiolipin species. Lipids are prepared as described in the Materials and Methods section; specific activity is determined by measuring phosphate release. Curves are representatives from three independent experiments, showing mean +/- SD. Enzymatic activation curves were fitted to an allosteric sigmoidal model (GraphPad, Prism8). Fitting curves are shown (lines) for data with least squares fit of 0.95 or above with parameters described in . Dotted lines represent activation curves that could not be fitted with the allosteric sigmoidal model.

Techniques Used: Concentration Assay, Activation Assay, Activity Assay

To assess whether cardiolipin functions as the main signal for polar MgtA localization in E. coli , in vivo imaging studies were performed using a GFP-tagged MgtA construct. (A) Schematic representation of the Clover-MgtA expression plasmid. The expression is controlled by the arabinose-inducible P ara promoter. (B) The localization of MgtA-GFP was assessed in wild-type E. coli strain MG1655, cardiolipin-deficient E. coli MG1655 Δcls , a knockout strain of cardiolipin synthase (Δ cls ), and wild type Vibrio cholerae strain C6706. The phase contrast channel (PH) and GFP channel (Clover-MgtA) of bacteria grown in the presence of 0.2 % arabinose (induced) or in its absence (uninduced) are shown. White scale bars are 5 μm. (C) Intensity profiles of single, representative cells (highlighted by a dotted white box in (B)). The intensities are normalized to the brightest area in each cell, respectively. (D) Clover-MgtA localization profiles of cells sorted by their cell length. This representation approximates the localization patterns through the cell cycle with small cells approximating newborn cells and long cells approximating cells immediately prior to division. The cell lengths are normalized to the longest cell in each analysis and the fluorescent intensities are normalized to the brightest area in all cells from each strain. E. coli MG1655 (n=171 cells), E. coli MG1655 Δ cls (n=75 cells), and V. cholerae C6706 (n=125 cells).
Figure Legend Snippet: To assess whether cardiolipin functions as the main signal for polar MgtA localization in E. coli , in vivo imaging studies were performed using a GFP-tagged MgtA construct. (A) Schematic representation of the Clover-MgtA expression plasmid. The expression is controlled by the arabinose-inducible P ara promoter. (B) The localization of MgtA-GFP was assessed in wild-type E. coli strain MG1655, cardiolipin-deficient E. coli MG1655 Δcls , a knockout strain of cardiolipin synthase (Δ cls ), and wild type Vibrio cholerae strain C6706. The phase contrast channel (PH) and GFP channel (Clover-MgtA) of bacteria grown in the presence of 0.2 % arabinose (induced) or in its absence (uninduced) are shown. White scale bars are 5 μm. (C) Intensity profiles of single, representative cells (highlighted by a dotted white box in (B)). The intensities are normalized to the brightest area in each cell, respectively. (D) Clover-MgtA localization profiles of cells sorted by their cell length. This representation approximates the localization patterns through the cell cycle with small cells approximating newborn cells and long cells approximating cells immediately prior to division. The cell lengths are normalized to the longest cell in each analysis and the fluorescent intensities are normalized to the brightest area in all cells from each strain. E. coli MG1655 (n=171 cells), E. coli MG1655 Δ cls (n=75 cells), and V. cholerae C6706 (n=125 cells).

Techniques Used: In Vivo Imaging, Construct, Expressing, Plasmid Preparation, Knock-Out

( A ) Western blot analysis of E. coli MG1655 and V. cholerae C6706 expressing Clover-fusion to MgtA. E. coli and V. cholerae containing either a plasmid encoding a translational fusion of Clover-MgtA under control of the arabinose inducible BAD promoter (pClover-MgtA) or the empty pBAD33.1 plasmid (pCTRL) were grown in the presence (0.2%) or absence (Ø) of arabinose. Samples of these cultures were analyzed by Western blot with a monoclonal antibody directed against GFP. ( B ) Quantification of Clover-MgtA fluorescence by flow cytometry. E. coli containing the MgtA-Clover fusion plasmid (pClover-MgtA) or no plasmid were induced with different arabinose concentrations. The fluorescence intensity was quantified after excitation at 488 nm. The bars depict the mean fluorescence, the error bars the standard deviation of at least 450,000 cells.
Figure Legend Snippet: ( A ) Western blot analysis of E. coli MG1655 and V. cholerae C6706 expressing Clover-fusion to MgtA. E. coli and V. cholerae containing either a plasmid encoding a translational fusion of Clover-MgtA under control of the arabinose inducible BAD promoter (pClover-MgtA) or the empty pBAD33.1 plasmid (pCTRL) were grown in the presence (0.2%) or absence (Ø) of arabinose. Samples of these cultures were analyzed by Western blot with a monoclonal antibody directed against GFP. ( B ) Quantification of Clover-MgtA fluorescence by flow cytometry. E. coli containing the MgtA-Clover fusion plasmid (pClover-MgtA) or no plasmid were induced with different arabinose concentrations. The fluorescence intensity was quantified after excitation at 488 nm. The bars depict the mean fluorescence, the error bars the standard deviation of at least 450,000 cells.

Techniques Used: Western Blot, Expressing, Plasmid Preparation, Fluorescence, Flow Cytometry, Standard Deviation

MgtA protein localizes in the inner membrane at the E. coli cell poles, which represent cardiolipin-rich regions. Association of MgtA with cardiolipin is essential for MgtA activity and stability, but MgtA shows high selectivity for specific cardiolipin species. We propose specific interaction between MgtA and two CL 18:1 molecules, affecting MgtA thermal stabilization. Further, CL18:1 is required for MgtA ATPase activity. CL 16:0 is needed for maximal MgtA activation but does not affect protein stabilization significantly.
Figure Legend Snippet: MgtA protein localizes in the inner membrane at the E. coli cell poles, which represent cardiolipin-rich regions. Association of MgtA with cardiolipin is essential for MgtA activity and stability, but MgtA shows high selectivity for specific cardiolipin species. We propose specific interaction between MgtA and two CL 18:1 molecules, affecting MgtA thermal stabilization. Further, CL18:1 is required for MgtA ATPase activity. CL 16:0 is needed for maximal MgtA activation but does not affect protein stabilization significantly.

Techniques Used: Activity Assay, Activation Assay


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Addgene inc k 12 series
Screening of 14 cell extracts. In this experiment, pPaF was selected for the UNAA embedding screening of 14 kinds of cell extracts. A total of 14 extracts were selected, including <t>K-12</t> series (K-12, K-12 ΔtnaA, K-12 ΔtnaAΔsdaB), commercial series (BL21 (DE3), BL21 ΔserB, Rosetta (DE3), Rosetta-gami B (DE3), Origami, Origami B), rEc series (rEc. 13, rEc. 13. ΔA), EcAR7 series (EcAR7 ΔA ΔSer), C321 series (C321, C321. ΔA).
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Article Title: A linear DNA template-based framework for site-specific unnatural amino acid incorporation

Journal: Synthetic and Systems Biotechnology

doi: 10.1016/j.synbio.2021.07.003

Screening of 14 cell extracts. In this experiment, pPaF was selected for the UNAA embedding screening of 14 kinds of cell extracts. A total of 14 extracts were selected, including K-12 series (K-12, K-12 ΔtnaA, K-12 ΔtnaAΔsdaB), commercial series (BL21 (DE3), BL21 ΔserB, Rosetta (DE3), Rosetta-gami B (DE3), Origami, Origami B), rEc series (rEc. 13, rEc. 13. ΔA), EcAR7 series (EcAR7 ΔA ΔSer), C321 series (C321, C321. ΔA).
Figure Legend Snippet: Screening of 14 cell extracts. In this experiment, pPaF was selected for the UNAA embedding screening of 14 kinds of cell extracts. A total of 14 extracts were selected, including K-12 series (K-12, K-12 ΔtnaA, K-12 ΔtnaAΔsdaB), commercial series (BL21 (DE3), BL21 ΔserB, Rosetta (DE3), Rosetta-gami B (DE3), Origami, Origami B), rEc series (rEc. 13, rEc. 13. ΔA), EcAR7 series (EcAR7 ΔA ΔSer), C321 series (C321, C321. ΔA).

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Addgene inc e coli mg1655
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    NT-seq simultaneously detects three types of methylation motifs in bacteria. a A to G frequency at known 6mA sites (G6mATC, A6mACN 6 GTGC, and GC6mACN 6 GTT) and unmethylated A sites in E. coli <t>MG1655</t> genome from native and PCR amplified DNA. b C to T frequency at known 4mC sites (T4mCTTC and 4mCCGG) and unmethylated C sites in H. pylori JP26 genome from native and PCR amplified DNA. c–e Negative log normalized A to G ratio of different 6mA motifs in E. coli WT strain MG1655 ( a ), hsdM deleted strain ( b ), and dam/dcm/hsdR mutated strain ( c ). 6mA position was underlined. f Negative log normalized C to T ratio of different 4mC motifs in H. pylori JP26. g Negative log normalized C to T ratio of 4mC sites identified by SMRT-seq in E. coli strain MG1655. h Negative log normalized C to T ratio of different 5mC motifs in H. pylori JP26. Only motifs with sequencing depth larger than 25× were considered for violin plots. Statistic test were performed by two-sided Mann-Whitney-Wilcoxon (MWW) test with Bonferroni correction (ns: P > 1.0e-3; ****: P ≤ 1.0e−6)
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    Lon overexpression rescues colony formation in OFX-treated Δ lon cells. (A and B) WT <t>E.</t> <t>coli</t> <t>MG1655</t> (A) and Δ lon E. coli MG1655 (B) containing the sulA-gfp reporter plasmid (pMSs201-P sulA -gfp ) were grown to stationary phase and diluted 100-fold in fresh LB medium. Diluted cells were then treated with 5 μg/mL OFX for 6 h. After treatment, phase-contrast and fluorescence (GFP) micrographs of cells were obtained. Images at 0 ( t = 0) and 6 h ( t = 6 h) from a representative replicate are shown, but similar data were obtained from four biological replicates ( n = 4). Scale bar, 25 μm. (C and D) E. coli cells (WT and Δ lon ) containing an inducible lon overexpression plasmid (pUA66- lon ) or empty vector control (pUA66-EV) were treated with OFX (5 μg/mL) for 6 h and transferred to LB agar plates supplemented with isopropyl β- d -1-thiogalactopyranoside (IPTG) at the indicated concentrations. CFU were measured for each strain before and after treatment. n = 4. (E) WT and Δ lon E. coli containing pUA66- lon were cultured with 1 mM IPTG before and/or during OFX treatment. After treatment, cells were transferred to agar plates with or without 1 mM IPTG. CFU were measured for each strain before and after treatment. n = 4. Statistical significance for pairwise comparisons was assessed using one-way analysis of variance (ANOVA) with Dunnett’s post hoc test. * * , P < 0.01; ** * , P < 0.001; *** * , P < 0.0001; ns, nonsignificant.
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    NT-seq simultaneously detects three types of methylation motifs in bacteria. a A to G frequency at known 6mA sites (G6mATC, A6mACN 6 GTGC, and GC6mACN 6 GTT) and unmethylated A sites in E. coli MG1655 genome from native and PCR amplified DNA. b C to T frequency at known 4mC sites (T4mCTTC and 4mCCGG) and unmethylated C sites in H. pylori JP26 genome from native and PCR amplified DNA. c–e Negative log normalized A to G ratio of different 6mA motifs in E. coli WT strain MG1655 ( a ), hsdM deleted strain ( b ), and dam/dcm/hsdR mutated strain ( c ). 6mA position was underlined. f Negative log normalized C to T ratio of different 4mC motifs in H. pylori JP26. g Negative log normalized C to T ratio of 4mC sites identified by SMRT-seq in E. coli strain MG1655. h Negative log normalized C to T ratio of different 5mC motifs in H. pylori JP26. Only motifs with sequencing depth larger than 25× were considered for violin plots. Statistic test were performed by two-sided Mann-Whitney-Wilcoxon (MWW) test with Bonferroni correction (ns: P > 1.0e-3; ****: P ≤ 1.0e−6)

    Journal: Genome Biology

    Article Title: NT-seq: a chemical-based sequencing method for genomic methylome profiling

    doi: 10.1186/s13059-022-02689-9

    Figure Lengend Snippet: NT-seq simultaneously detects three types of methylation motifs in bacteria. a A to G frequency at known 6mA sites (G6mATC, A6mACN 6 GTGC, and GC6mACN 6 GTT) and unmethylated A sites in E. coli MG1655 genome from native and PCR amplified DNA. b C to T frequency at known 4mC sites (T4mCTTC and 4mCCGG) and unmethylated C sites in H. pylori JP26 genome from native and PCR amplified DNA. c–e Negative log normalized A to G ratio of different 6mA motifs in E. coli WT strain MG1655 ( a ), hsdM deleted strain ( b ), and dam/dcm/hsdR mutated strain ( c ). 6mA position was underlined. f Negative log normalized C to T ratio of different 4mC motifs in H. pylori JP26. g Negative log normalized C to T ratio of 4mC sites identified by SMRT-seq in E. coli strain MG1655. h Negative log normalized C to T ratio of different 5mC motifs in H. pylori JP26. Only motifs with sequencing depth larger than 25× were considered for violin plots. Statistic test were performed by two-sided Mann-Whitney-Wilcoxon (MWW) test with Bonferroni correction (ns: P > 1.0e-3; ****: P ≤ 1.0e−6)

    Article Snippet: WT E. coli strain MG1655 and hsdM deletion K12 strain were kindly provided by Dr. Susan M Rosenberg’s lab (Baylor College of Medicine). dam/dcm mutant E. coli strain JM110 was purchased from Addgene (Bacterial strain #49763).

    Techniques: Methylation, Amplification, Sequencing, MANN-WHITNEY

    Journal: iScience

    Article Title: Biosensor and chemo-enzymatic one-pot cascade applications to detect and transform PET-derived terephthalic acid in living cells

    doi: 10.1016/j.isci.2022.104326

    Figure Lengend Snippet:

    Article Snippet: E. coli K-12 MG1655 RARE , Prof. K.L.J. Prather ( ) , Addgene Bacterial strain #61440.

    Techniques: Recombinant, Purification, Variant Assay, Mutagenesis, Plasmid Preparation, Software

    Lon overexpression rescues colony formation in OFX-treated Δ lon cells. (A and B) WT E. coli MG1655 (A) and Δ lon E. coli MG1655 (B) containing the sulA-gfp reporter plasmid (pMSs201-P sulA -gfp ) were grown to stationary phase and diluted 100-fold in fresh LB medium. Diluted cells were then treated with 5 μg/mL OFX for 6 h. After treatment, phase-contrast and fluorescence (GFP) micrographs of cells were obtained. Images at 0 ( t = 0) and 6 h ( t = 6 h) from a representative replicate are shown, but similar data were obtained from four biological replicates ( n = 4). Scale bar, 25 μm. (C and D) E. coli cells (WT and Δ lon ) containing an inducible lon overexpression plasmid (pUA66- lon ) or empty vector control (pUA66-EV) were treated with OFX (5 μg/mL) for 6 h and transferred to LB agar plates supplemented with isopropyl β- d -1-thiogalactopyranoside (IPTG) at the indicated concentrations. CFU were measured for each strain before and after treatment. n = 4. (E) WT and Δ lon E. coli containing pUA66- lon were cultured with 1 mM IPTG before and/or during OFX treatment. After treatment, cells were transferred to agar plates with or without 1 mM IPTG. CFU were measured for each strain before and after treatment. n = 4. Statistical significance for pairwise comparisons was assessed using one-way analysis of variance (ANOVA) with Dunnett’s post hoc test. * * , P < 0.01; ** * , P < 0.001; *** * , P < 0.0001; ns, nonsignificant.

    Journal: mBio

    Article Title: lon Deletion Impairs Persister Cell Resuscitation in Escherichia coli

    doi: 10.1128/mbio.02187-21

    Figure Lengend Snippet: Lon overexpression rescues colony formation in OFX-treated Δ lon cells. (A and B) WT E. coli MG1655 (A) and Δ lon E. coli MG1655 (B) containing the sulA-gfp reporter plasmid (pMSs201-P sulA -gfp ) were grown to stationary phase and diluted 100-fold in fresh LB medium. Diluted cells were then treated with 5 μg/mL OFX for 6 h. After treatment, phase-contrast and fluorescence (GFP) micrographs of cells were obtained. Images at 0 ( t = 0) and 6 h ( t = 6 h) from a representative replicate are shown, but similar data were obtained from four biological replicates ( n = 4). Scale bar, 25 μm. (C and D) E. coli cells (WT and Δ lon ) containing an inducible lon overexpression plasmid (pUA66- lon ) or empty vector control (pUA66-EV) were treated with OFX (5 μg/mL) for 6 h and transferred to LB agar plates supplemented with isopropyl β- d -1-thiogalactopyranoside (IPTG) at the indicated concentrations. CFU were measured for each strain before and after treatment. n = 4. (E) WT and Δ lon E. coli containing pUA66- lon were cultured with 1 mM IPTG before and/or during OFX treatment. After treatment, cells were transferred to agar plates with or without 1 mM IPTG. CFU were measured for each strain before and after treatment. n = 4. Statistical significance for pairwise comparisons was assessed using one-way analysis of variance (ANOVA) with Dunnett’s post hoc test. * * , P < 0.01; ** * , P < 0.001; *** * , P < 0.0001; ns, nonsignificant.

    Article Snippet: E. coli MG1655 and the pQE-80L and pUA66 plasmids were obtained from Mark P. Brynildsen at Princeton University, and pXY027 was a gift from Jie Xiao (plasmid no. 98915; Addgene, Watertown, MA, USA) ( ).

    Techniques: Over Expression, Plasmid Preparation, Fluorescence, Cell Culture

    Lon overexpression rescues the colony-forming ability of Δ lon cells exposed to UV. (A) WT and Δ lon E. coli containing pMSs201-P sulA -gfp were grown to stationary phase, diluted 100-fold in fresh LB medium, and exposed to UV for the indicated times. After exposure, cells were cultured in a shaker for 2 h, and phase-contrast and fluorescence (GFP) micrographs of cells were obtained. Images from a representative replicate are shown, but similar data were obtained for four biological replicates. n = 4. Scale bar, 25 μm. (B) WT and Δ lon E. coli were exposed to UV as described in panel A and spotted onto LB agar plates to enumerate CFU. n = 4. (C to E) Cells containing the inducible lon overexpression plasmid (pUA66- lon ) or empty vector (pUA66-EV) control were exposed to UV for the indicated times and transferred to agar plates supplemented with IPTG at indicated concentrations to enumerate CFU. n = 4. Statistical significance for pairwise comparisons was assessed using one-way ANOVA with Dunnett’s post hoc test. ** * , P < 0.001; *** * , P < 0.0001; ns, nonsignificant.

    Journal: mBio

    Article Title: lon Deletion Impairs Persister Cell Resuscitation in Escherichia coli

    doi: 10.1128/mbio.02187-21

    Figure Lengend Snippet: Lon overexpression rescues the colony-forming ability of Δ lon cells exposed to UV. (A) WT and Δ lon E. coli containing pMSs201-P sulA -gfp were grown to stationary phase, diluted 100-fold in fresh LB medium, and exposed to UV for the indicated times. After exposure, cells were cultured in a shaker for 2 h, and phase-contrast and fluorescence (GFP) micrographs of cells were obtained. Images from a representative replicate are shown, but similar data were obtained for four biological replicates. n = 4. Scale bar, 25 μm. (B) WT and Δ lon E. coli were exposed to UV as described in panel A and spotted onto LB agar plates to enumerate CFU. n = 4. (C to E) Cells containing the inducible lon overexpression plasmid (pUA66- lon ) or empty vector (pUA66-EV) control were exposed to UV for the indicated times and transferred to agar plates supplemented with IPTG at indicated concentrations to enumerate CFU. n = 4. Statistical significance for pairwise comparisons was assessed using one-way ANOVA with Dunnett’s post hoc test. ** * , P < 0.001; *** * , P < 0.0001; ns, nonsignificant.

    Article Snippet: E. coli MG1655 and the pQE-80L and pUA66 plasmids were obtained from Mark P. Brynildsen at Princeton University, and pXY027 was a gift from Jie Xiao (plasmid no. 98915; Addgene, Watertown, MA, USA) ( ).

    Techniques: Over Expression, Cell Culture, Fluorescence, Plasmid Preparation

    Starvation in PBS solution rescues Δ lon persisters. (A) WT and Δ lon E. coli with or without the inducible lon overexpression plasmid (pUA66- lon ) or empty vector control (pUA66-EV) were grown to stationary phase and diluted 100-fold in fresh LB medium. Diluted cells were then treated with OFX for 6 h. After treatment, cells were washed, serially diluted, and spotted onto LB agar plates with or without IPTG (1 mM). Plates were incubated for 48 h, and images were captured at indicated time points. A representative replicate is shown, but similar data were obtained for four biological replicates. n = 4. (B) WT and Δ lon E. coli with or without pUA66- lon or pUA66-EV were treated with OFX for 6 h, transferred to sterile PBS solution with or without 1 mM IPTG, and incubated at 37°C in a shaker for 7 days. Samples were collected at the indicated time points and spotted onto agar plates to enumerate surviving cells. n = 4. Statistical significance for pairwise comparison was assessed using one-way ANOVA with Dunnett’s post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant.

    Journal: mBio

    Article Title: lon Deletion Impairs Persister Cell Resuscitation in Escherichia coli

    doi: 10.1128/mbio.02187-21

    Figure Lengend Snippet: Starvation in PBS solution rescues Δ lon persisters. (A) WT and Δ lon E. coli with or without the inducible lon overexpression plasmid (pUA66- lon ) or empty vector control (pUA66-EV) were grown to stationary phase and diluted 100-fold in fresh LB medium. Diluted cells were then treated with OFX for 6 h. After treatment, cells were washed, serially diluted, and spotted onto LB agar plates with or without IPTG (1 mM). Plates were incubated for 48 h, and images were captured at indicated time points. A representative replicate is shown, but similar data were obtained for four biological replicates. n = 4. (B) WT and Δ lon E. coli with or without pUA66- lon or pUA66-EV were treated with OFX for 6 h, transferred to sterile PBS solution with or without 1 mM IPTG, and incubated at 37°C in a shaker for 7 days. Samples were collected at the indicated time points and spotted onto agar plates to enumerate surviving cells. n = 4. Statistical significance for pairwise comparison was assessed using one-way ANOVA with Dunnett’s post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant.

    Article Snippet: E. coli MG1655 and the pQE-80L and pUA66 plasmids were obtained from Mark P. Brynildsen at Princeton University, and pXY027 was a gift from Jie Xiao (plasmid no. 98915; Addgene, Watertown, MA, USA) ( ).

    Techniques: Over Expression, Plasmid Preparation, Incubation

    Screening of 14 cell extracts. In this experiment, pPaF was selected for the UNAA embedding screening of 14 kinds of cell extracts. A total of 14 extracts were selected, including K-12 series (K-12, K-12 ΔtnaA, K-12 ΔtnaAΔsdaB), commercial series (BL21 (DE3), BL21 ΔserB, Rosetta (DE3), Rosetta-gami B (DE3), Origami, Origami B), rEc series (rEc. 13, rEc. 13. ΔA), EcAR7 series (EcAR7 ΔA ΔSer), C321 series (C321, C321. ΔA).

    Journal: Synthetic and Systems Biotechnology

    Article Title: A linear DNA template-based framework for site-specific unnatural amino acid incorporation

    doi: 10.1016/j.synbio.2021.07.003

    Figure Lengend Snippet: Screening of 14 cell extracts. In this experiment, pPaF was selected for the UNAA embedding screening of 14 kinds of cell extracts. A total of 14 extracts were selected, including K-12 series (K-12, K-12 ΔtnaA, K-12 ΔtnaAΔsdaB), commercial series (BL21 (DE3), BL21 ΔserB, Rosetta (DE3), Rosetta-gami B (DE3), Origami, Origami B), rEc series (rEc. 13, rEc. 13. ΔA), EcAR7 series (EcAR7 ΔA ΔSer), C321 series (C321, C321. ΔA).

    Article Snippet: A total of 14 E. coli extracts were selected in this study, including K-12 series (K-12 [ ] (Addgene #61440), K-12 ΔtnaA [ ], K-12 ΔtnaAΔsdaB [ ]), commercial series (BL21 (DE3) (Biomed, China), BL21 ΔserB [ ] (Addgene #34929), Rosetta (DE3), Rosetta-gami B (DE3), Origami, Origami B) (Biomed, China), rEc series (rEc.

    Techniques: