e. coli top10 Search Results


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  • 94
    ATCC e coli top10
    Dot blot assay for individual MTase activity. Serially diluted DNA (450 ng–150 ng) was used to test the in vivo methylation by MTases. (A) DNA methylated by the MTases from B. amyloliquefaciens TA208 and B. cereus ATCC 10987. (B) DNA methylated by the MTases from N. hamburgensis X14. Antibodies against m6A, m4C, and m5C were used in the upper, middle, and lower panels, respectively. DNA from the E. coli EC135 strain harboring pBAD43 was used as negative control. The identical DNA, 150 ng DNA of the E. coli <t>TOP10</t> strain, 150 ng of E. coli EC135 DNA in vivo methylated by M.BamHI and 150 ng of E. coli EC135 DNA in vivo methylated by M.AluI (arranged from top to bottom), were used as controls in each “Control+” column for the m6A, m4C, and m5C experiments. All experiments were repeated at least three times, and representative results are shown.
    E Coli Top10, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher competent e coli top10 cells
    Overexpression and purification of the 45-kDa recombinant GDH protein. The protein was overexpressed and purified as described in Materials and Methods. An SDS-10% polyacrylamide gel stained with Coomassie brilliant blue R-250 is shown. Lanes: M, rainbow molecular size marker in kilodaltons; 1, whole-cell lysate of pOT411 transformant of E. coli <t>TOP10</t> uninduced; 2, whole-cell lysate of E. coli transformed with pOT411 and induced with arabinose; 3 and 4, different amounts of the recombinant protein purified from pOT411 transformant of E. coli TOP10.
    Competent E Coli Top10 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 898 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher e coli top10
    RT-PCR analysis of tet35 expression. (A) RT-PCR of total RNA from E. coli <t>TOP10</t> cells carrying pATJ1. Lanes: 1, 1-kb ladder; 2, 1.1-kb amplicon obtained with tet35F/R primers; 3, 900-base amplicon obtained with txrF/R primers; 4, positive control using 16S rRNA-specific primers, amplifying a 1.3-kb amplicon; 5, negative control (no RT). (B) RT-PCR of total DNA from E. coli TOP10 cells carrying pJKM115. Lanes 1, 1-kb ladder; 2, 1.1-kb PCR amplicon obtained with tet35F/R primers; 3, no detectable amplicon obtained with txrF/R primers; 4; positive control using 16S rRNA specific primers, amplifying a 1.3-kb amplicon; 5, negative control (no RT).
    E Coli Top10, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 4131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher e coli top10 cells
    Epifluorescent micrograph of GFP fusions. (A) E. coli pBAD-GFP. (B) Wild-type V. cholerae pBAD-GFP. (C) GFP fusion to the complete nqrA gene (GANC) in E. coli <t>Top10</t> cells. (D) GANC in wild-type V. cholerae . (E) GFP fusion to nqrD from the N terminus to
    E Coli Top10 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2331 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher electrocompetent e coli top10 cells
    Epifluorescent micrograph of GFP fusions. (A) E. coli pBAD-GFP. (B) Wild-type V. cholerae pBAD-GFP. (C) GFP fusion to the complete nqrA gene (GANC) in E. coli <t>Top10</t> cells. (D) GANC in wild-type V. cholerae . (E) GFP fusion to nqrD from the N terminus to
    Electrocompetent E Coli Top10 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co e coli top10
    Epifluorescent micrograph of GFP fusions. (A) E. coli pBAD-GFP. (B) Wild-type V. cholerae pBAD-GFP. (C) GFP fusion to the complete nqrA gene (GANC) in E. coli <t>Top10</t> cells. (D) GANC in wild-type V. cholerae . (E) GFP fusion to nqrD from the N terminus to
    E Coli Top10, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 92/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore e coli top10
    Monitoring cellular integrity of the E. coli host strain expressing AT chimeras. (A) SDS-PAGE analysis of OM (M) and culture supernatant (S) fractions derived from E. coli <t>TOP10</t> expressing Pet*, mCherry*and ESAT6*. Non cleaved species are denoted by arrows. Molecular weight markers (MWM, kDa) are indicated to the right of the panel. OM fractions demonstrating the presence and absence of the β-domain are shown in Additional file 6 : Figure S5. (B) E. coli cells expressing empty vector, Pet*, mCherry*, ESAT6* and their cleaved parents were harvested 2 h after induction and subjected to indirect immunofluorescence using the indicated antibody. For each population expressing a cleavage deficient variant, a sample was divided in two: one half was probed with an antibody to the periplasmic protein BamD, while the other half was permeabilised (−P) and subsequently probed with the same antibody. Corresponding fields are also shown by phase contrast microscopy. For the mCherry constructs panels showing mCherry derived fluorescence are shown. (C) The integrity of E. coli host cells expressing wild type Pet and secreted ESAT6-Pet fusions were assessed by staining with BOX and PI prior to and 2 h post induction. Q3 represents the population that is viable and healthy and did not label with either stain. Q2 represents cells that stain with both stains and are no longer viable. Q4 (BOX-positive) represents cells with impaired membrane potential suggesting compromised membrane integrity. (D) Periplasmic leakage from E. coli TOP10 cultures secreting Pet or ESAT6-Pet fusion proteins was assessed by measuring (2 h after induction) the activity in the culture medium of the periplasmic enzyme alkaline phosphatase. Clarified whole cell lysate was used as a positive control and the wild-type plasmid-free strain as a negative control. There is no significant difference between the negative control and culture medium derived from strains expressing Pet or ESAT6-Pet-BB. In contrast, culture medium from both constructs displayed activity significantly less than the positive control. Absorbance measurements are derived from equal volumes of culture and are normalised relative to positive control (in %). The error bars represent standard error for two independent data sets.
    E Coli Top10, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher one shot top10 chemically competent e coli
    Monitoring cellular integrity of the E. coli host strain expressing AT chimeras. (A) SDS-PAGE analysis of OM (M) and culture supernatant (S) fractions derived from E. coli <t>TOP10</t> expressing Pet*, mCherry*and ESAT6*. Non cleaved species are denoted by arrows. Molecular weight markers (MWM, kDa) are indicated to the right of the panel. OM fractions demonstrating the presence and absence of the β-domain are shown in Additional file 6 : Figure S5. (B) E. coli cells expressing empty vector, Pet*, mCherry*, ESAT6* and their cleaved parents were harvested 2 h after induction and subjected to indirect immunofluorescence using the indicated antibody. For each population expressing a cleavage deficient variant, a sample was divided in two: one half was probed with an antibody to the periplasmic protein BamD, while the other half was permeabilised (−P) and subsequently probed with the same antibody. Corresponding fields are also shown by phase contrast microscopy. For the mCherry constructs panels showing mCherry derived fluorescence are shown. (C) The integrity of E. coli host cells expressing wild type Pet and secreted ESAT6-Pet fusions were assessed by staining with BOX and PI prior to and 2 h post induction. Q3 represents the population that is viable and healthy and did not label with either stain. Q2 represents cells that stain with both stains and are no longer viable. Q4 (BOX-positive) represents cells with impaired membrane potential suggesting compromised membrane integrity. (D) Periplasmic leakage from E. coli TOP10 cultures secreting Pet or ESAT6-Pet fusion proteins was assessed by measuring (2 h after induction) the activity in the culture medium of the periplasmic enzyme alkaline phosphatase. Clarified whole cell lysate was used as a positive control and the wild-type plasmid-free strain as a negative control. There is no significant difference between the negative control and culture medium derived from strains expressing Pet or ESAT6-Pet-BB. In contrast, culture medium from both constructs displayed activity significantly less than the positive control. Absorbance measurements are derived from equal volumes of culture and are normalised relative to positive control (in %). The error bars represent standard error for two independent data sets.
    One Shot Top10 Chemically Competent E Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 372 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher top10 electrocomp kit
    Monitoring cellular integrity of the E. coli host strain expressing AT chimeras. (A) SDS-PAGE analysis of OM (M) and culture supernatant (S) fractions derived from E. coli <t>TOP10</t> expressing Pet*, mCherry*and ESAT6*. Non cleaved species are denoted by arrows. Molecular weight markers (MWM, kDa) are indicated to the right of the panel. OM fractions demonstrating the presence and absence of the β-domain are shown in Additional file 6 : Figure S5. (B) E. coli cells expressing empty vector, Pet*, mCherry*, ESAT6* and their cleaved parents were harvested 2 h after induction and subjected to indirect immunofluorescence using the indicated antibody. For each population expressing a cleavage deficient variant, a sample was divided in two: one half was probed with an antibody to the periplasmic protein BamD, while the other half was permeabilised (−P) and subsequently probed with the same antibody. Corresponding fields are also shown by phase contrast microscopy. For the mCherry constructs panels showing mCherry derived fluorescence are shown. (C) The integrity of E. coli host cells expressing wild type Pet and secreted ESAT6-Pet fusions were assessed by staining with BOX and PI prior to and 2 h post induction. Q3 represents the population that is viable and healthy and did not label with either stain. Q2 represents cells that stain with both stains and are no longer viable. Q4 (BOX-positive) represents cells with impaired membrane potential suggesting compromised membrane integrity. (D) Periplasmic leakage from E. coli TOP10 cultures secreting Pet or ESAT6-Pet fusion proteins was assessed by measuring (2 h after induction) the activity in the culture medium of the periplasmic enzyme alkaline phosphatase. Clarified whole cell lysate was used as a positive control and the wild-type plasmid-free strain as a negative control. There is no significant difference between the negative control and culture medium derived from strains expressing Pet or ESAT6-Pet-BB. In contrast, culture medium from both constructs displayed activity significantly less than the positive control. Absorbance measurements are derived from equal volumes of culture and are normalised relative to positive control (in %). The error bars represent standard error for two independent data sets.
    Top10 Electrocomp Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Dot blot assay for individual MTase activity. Serially diluted DNA (450 ng–150 ng) was used to test the in vivo methylation by MTases. (A) DNA methylated by the MTases from B. amyloliquefaciens TA208 and B. cereus ATCC 10987. (B) DNA methylated by the MTases from N. hamburgensis X14. Antibodies against m6A, m4C, and m5C were used in the upper, middle, and lower panels, respectively. DNA from the E. coli EC135 strain harboring pBAD43 was used as negative control. The identical DNA, 150 ng DNA of the E. coli TOP10 strain, 150 ng of E. coli EC135 DNA in vivo methylated by M.BamHI and 150 ng of E. coli EC135 DNA in vivo methylated by M.AluI (arranged from top to bottom), were used as controls in each “Control+” column for the m6A, m4C, and m5C experiments. All experiments were repeated at least three times, and representative results are shown.

    Journal: PLoS Genetics

    Article Title: A Mimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria

    doi: 10.1371/journal.pgen.1002987

    Figure Lengend Snippet: Dot blot assay for individual MTase activity. Serially diluted DNA (450 ng–150 ng) was used to test the in vivo methylation by MTases. (A) DNA methylated by the MTases from B. amyloliquefaciens TA208 and B. cereus ATCC 10987. (B) DNA methylated by the MTases from N. hamburgensis X14. Antibodies against m6A, m4C, and m5C were used in the upper, middle, and lower panels, respectively. DNA from the E. coli EC135 strain harboring pBAD43 was used as negative control. The identical DNA, 150 ng DNA of the E. coli TOP10 strain, 150 ng of E. coli EC135 DNA in vivo methylated by M.BamHI and 150 ng of E. coli EC135 DNA in vivo methylated by M.AluI (arranged from top to bottom), were used as controls in each “Control+” column for the m6A, m4C, and m5C experiments. All experiments were repeated at least three times, and representative results are shown.

    Article Snippet: The plasmids prepared from E. coli TOP10 and EC135 strains showed similar transformation efficiencies, indicating that the putative Type IV R-M systems (BCE_1016 and BCE_2317) in B. cereus ATCC 10987 may be inactive.

    Techniques: Dot Blot, Activity Assay, In Vivo, Methylation, Negative Control

    Transformation efficiency of B. amyloliquefaciens TA208 and B. cereus ATCC 10987 with various shuttle plasmids. (A) Transformation efficiency of the B. amyloliquefaciens TA208 strain with various shuttle plasmids prepared from the E. coli TOP10, EC135 and the EC135 harboring pM.Bam. (B) Transformation efficiency of the B. cereus ATCC 10987 strain with various shuttle plasmids prepared from the E. coli TOP10, EC135 and the EC135 harboring pM.Bce. Transformation efficiencies shown are averages of at least three replicates ± SD. * Not Detected.

    Journal: PLoS Genetics

    Article Title: A Mimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria

    doi: 10.1371/journal.pgen.1002987

    Figure Lengend Snippet: Transformation efficiency of B. amyloliquefaciens TA208 and B. cereus ATCC 10987 with various shuttle plasmids. (A) Transformation efficiency of the B. amyloliquefaciens TA208 strain with various shuttle plasmids prepared from the E. coli TOP10, EC135 and the EC135 harboring pM.Bam. (B) Transformation efficiency of the B. cereus ATCC 10987 strain with various shuttle plasmids prepared from the E. coli TOP10, EC135 and the EC135 harboring pM.Bce. Transformation efficiencies shown are averages of at least three replicates ± SD. * Not Detected.

    Article Snippet: The plasmids prepared from E. coli TOP10 and EC135 strains showed similar transformation efficiencies, indicating that the putative Type IV R-M systems (BCE_1016 and BCE_2317) in B. cereus ATCC 10987 may be inactive.

    Techniques: Transformation Assay

    Overexpression and purification of the 45-kDa recombinant GDH protein. The protein was overexpressed and purified as described in Materials and Methods. An SDS-10% polyacrylamide gel stained with Coomassie brilliant blue R-250 is shown. Lanes: M, rainbow molecular size marker in kilodaltons; 1, whole-cell lysate of pOT411 transformant of E. coli TOP10 uninduced; 2, whole-cell lysate of E. coli transformed with pOT411 and induced with arabinose; 3 and 4, different amounts of the recombinant protein purified from pOT411 transformant of E. coli TOP10.

    Journal: Clinical and Diagnostic Laboratory Immunology

    Article Title: Cloning and Characterization of the Gene Encoding the Glutamate Dehydrogenase of Streptococcus suis Serotype 2

    doi: 10.1128/CDLI.8.2.251-257.2001

    Figure Lengend Snippet: Overexpression and purification of the 45-kDa recombinant GDH protein. The protein was overexpressed and purified as described in Materials and Methods. An SDS-10% polyacrylamide gel stained with Coomassie brilliant blue R-250 is shown. Lanes: M, rainbow molecular size marker in kilodaltons; 1, whole-cell lysate of pOT411 transformant of E. coli TOP10 uninduced; 2, whole-cell lysate of E. coli transformed with pOT411 and induced with arabinose; 3 and 4, different amounts of the recombinant protein purified from pOT411 transformant of E. coli TOP10.

    Article Snippet: The DNA insert in pOT410 was cloned in frame into a pBAD/ Myc -His version B expression vector to create pOT411. pOT411 was transformed into E. coli TOP10-competent cells and overexpressed by following the manufacturer's protocol (Invitrogen).

    Techniques: Over Expression, Purification, Recombinant, Staining, Marker, Transformation Assay

    RT-PCR analysis of tet35 expression. (A) RT-PCR of total RNA from E. coli TOP10 cells carrying pATJ1. Lanes: 1, 1-kb ladder; 2, 1.1-kb amplicon obtained with tet35F/R primers; 3, 900-base amplicon obtained with txrF/R primers; 4, positive control using 16S rRNA-specific primers, amplifying a 1.3-kb amplicon; 5, negative control (no RT). (B) RT-PCR of total DNA from E. coli TOP10 cells carrying pJKM115. Lanes 1, 1-kb ladder; 2, 1.1-kb PCR amplicon obtained with tet35F/R primers; 3, no detectable amplicon obtained with txrF/R primers; 4; positive control using 16S rRNA specific primers, amplifying a 1.3-kb amplicon; 5, negative control (no RT).

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Genetic Determinants of Tetracycline Resistance in Vibrio harveyi

    doi: 10.1128/AAC.46.4.1038-1045.2002

    Figure Lengend Snippet: RT-PCR analysis of tet35 expression. (A) RT-PCR of total RNA from E. coli TOP10 cells carrying pATJ1. Lanes: 1, 1-kb ladder; 2, 1.1-kb amplicon obtained with tet35F/R primers; 3, 900-base amplicon obtained with txrF/R primers; 4, positive control using 16S rRNA-specific primers, amplifying a 1.3-kb amplicon; 5, negative control (no RT). (B) RT-PCR of total DNA from E. coli TOP10 cells carrying pJKM115. Lanes 1, 1-kb ladder; 2, 1.1-kb PCR amplicon obtained with tet35F/R primers; 3, no detectable amplicon obtained with txrF/R primers; 4; positive control using 16S rRNA specific primers, amplifying a 1.3-kb amplicon; 5, negative control (no RT).

    Article Snippet: E. coli TOP10 (Invitrogen Corp., Carlsbad, Calif.) was used as the host strain for the recombinant plasmids.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Positive Control, Negative Control, Polymerase Chain Reaction

    Accumulation of tetracycline by susceptible E. coli TOP10 cells and resistant clones. [ 3 H]tetracycline was added to cells at time zero. (A) Accumulation in E. coli carrying no plasmid (▪), pATJ1 (•), or pCT71 (▴); (B) effect of CCCP (0.2 mM) on accumulation in E. coli with no plasmid (▪) or pATJ1 (•). The graphs are typical of at least two independent experiments.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Genetic Determinants of Tetracycline Resistance in Vibrio harveyi

    doi: 10.1128/AAC.46.4.1038-1045.2002

    Figure Lengend Snippet: Accumulation of tetracycline by susceptible E. coli TOP10 cells and resistant clones. [ 3 H]tetracycline was added to cells at time zero. (A) Accumulation in E. coli carrying no plasmid (▪), pATJ1 (•), or pCT71 (▴); (B) effect of CCCP (0.2 mM) on accumulation in E. coli with no plasmid (▪) or pATJ1 (•). The graphs are typical of at least two independent experiments.

    Article Snippet: E. coli TOP10 (Invitrogen Corp., Carlsbad, Calif.) was used as the host strain for the recombinant plasmids.

    Techniques: Clone Assay, Plasmid Preparation

    Regulated transcription of asa . ( A ) Promoter activity of the cloned putative promoter region (black letters in Fig. 4A ) was measured as fluorescence intensity of E. coli Top-10 + pProbe-NT:: asa P (OD 600 = 0.8) after growth in LB (grey), in LB + 450 mM NaCl (blue) or in LB + 10 mM L-arginine (orange); NC I: negative control using the terminator region of the gene EDL933_1236 (violet letters in Fig. 4A ), NC II: pProbe-NT without insert. The mean values and standard deviations of all three replicates are shown here. ( B ) RT-qPCR threshold cycles of asa normalized to the 16S rRNA gene (∆cq). Lower ∆cq values indicate higher amounts of mRNA and vice versa . EHEC was grown in LB medium (left columns) or LB + 450 mM NaCl (right columns). Approximately 10 8 cells were harvested for total RNA extraction either at exponential phase (OD 600 = 0.2–0.3, blue) or at late exponential phase (OD 600 = 0.7 – 0.8, orange). The RNA was reverse transcribed into cDNA and quantified using RT-qPCR. The mean values and standard deviations of all three replicates are shown here.

    Journal: Scientific Reports

    Article Title: The novel EHEC gene asa overlaps the TEGT transporter gene in antisense and is regulated by NaCl and growth phase

    doi: 10.1038/s41598-018-35756-y

    Figure Lengend Snippet: Regulated transcription of asa . ( A ) Promoter activity of the cloned putative promoter region (black letters in Fig. 4A ) was measured as fluorescence intensity of E. coli Top-10 + pProbe-NT:: asa P (OD 600 = 0.8) after growth in LB (grey), in LB + 450 mM NaCl (blue) or in LB + 10 mM L-arginine (orange); NC I: negative control using the terminator region of the gene EDL933_1236 (violet letters in Fig. 4A ), NC II: pProbe-NT without insert. The mean values and standard deviations of all three replicates are shown here. ( B ) RT-qPCR threshold cycles of asa normalized to the 16S rRNA gene (∆cq). Lower ∆cq values indicate higher amounts of mRNA and vice versa . EHEC was grown in LB medium (left columns) or LB + 450 mM NaCl (right columns). Approximately 10 8 cells were harvested for total RNA extraction either at exponential phase (OD 600 = 0.2–0.3, blue) or at late exponential phase (OD 600 = 0.7 – 0.8, orange). The RNA was reverse transcribed into cDNA and quantified using RT-qPCR. The mean values and standard deviations of all three replicates are shown here.

    Article Snippet: List of bacteria and incubation conditions The following bacterial strains were used: E. coli O157:H7 strain EDL933 (Collection de l’Institute Pasteur: CIP 106327, GenBank accession number CP008957.1), E. coli O157:H7 strain Sakai (Weihenstephan strain collection WS 4518, GenBank accession number NC_002695.1), E. coli LF82 (kindly provided by R. Balfour Sartor, GenBank accession number NC_011993.1), E. coli TOP-10 (Invitrogen, Paisley, UK).

    Techniques: Activity Assay, Clone Assay, Fluorescence, Negative Control, Quantitative RT-PCR, RNA Extraction

    Epifluorescent micrograph of GFP fusions. (A) E. coli pBAD-GFP. (B) Wild-type V. cholerae pBAD-GFP. (C) GFP fusion to the complete nqrA gene (GANC) in E. coli Top10 cells. (D) GANC in wild-type V. cholerae . (E) GFP fusion to nqrD from the N terminus to

    Journal:

    Article Title: Membrane Topology Mapping of the Na+-Pumping NADH: Quinone Oxidoreductase from Vibrio cholerae by PhoA- Green Fluorescent Protein Fusion Analysis ▿

    doi: 10.1128/JB.01383-06

    Figure Lengend Snippet: Epifluorescent micrograph of GFP fusions. (A) E. coli pBAD-GFP. (B) Wild-type V. cholerae pBAD-GFP. (C) GFP fusion to the complete nqrA gene (GANC) in E. coli Top10 cells. (D) GANC in wild-type V. cholerae . (E) GFP fusion to nqrD from the N terminus to

    Article Snippet: Fragments were first cloned into pCR2.1-TOPO TA vectors (Invitrogen) and transformed into E. coli Top10 cells (Invitrogen).

    Techniques:

    (A) Construction of plasmid pCMPG10652. Constitutive promoter P A , gene hpaA , and 4-Hydroxyphenylacetic acid (4-HPA) responsive promoter P BC were copied from plasmid pRA 2 (Prieto and García, 1997 ) and placed in plasmid pFPV25 (Valdivia and Falkow, 1996 ), upstream of gene gfp . This yields GFP production controlled by 4-HPA and 2-Phenylacetic Acid (PAA) concentrations, detected by HpaA. (B) Fluorescence measurements of GFP production of strain pCMPG10652/ E. coli TOP10 in response to a range of 4-HPA concentrations. (C) Fluorescence measurements of GFP production of strain pCMPG10652/ E. coli TOP10 in response to a range of PAA concentrations. Auxin concentrations used in fluorescence measurements: 3 mM, 2 mM, 1.5 mM, 1 mM, 500 μM, 250 μM, 125 μM, 62 μM, 31.25 μM, 15.625 μM, and 0M. n = 8 per measured concentration.

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Design and Construction of a Whole Cell Bacterial 4-Hydroxyphenylacetic Acid and 2-Phenylacetic Acid Bioassay

    doi: 10.3389/fbioe.2015.00088

    Figure Lengend Snippet: (A) Construction of plasmid pCMPG10652. Constitutive promoter P A , gene hpaA , and 4-Hydroxyphenylacetic acid (4-HPA) responsive promoter P BC were copied from plasmid pRA 2 (Prieto and García, 1997 ) and placed in plasmid pFPV25 (Valdivia and Falkow, 1996 ), upstream of gene gfp . This yields GFP production controlled by 4-HPA and 2-Phenylacetic Acid (PAA) concentrations, detected by HpaA. (B) Fluorescence measurements of GFP production of strain pCMPG10652/ E. coli TOP10 in response to a range of 4-HPA concentrations. (C) Fluorescence measurements of GFP production of strain pCMPG10652/ E. coli TOP10 in response to a range of PAA concentrations. Auxin concentrations used in fluorescence measurements: 3 mM, 2 mM, 1.5 mM, 1 mM, 500 μM, 250 μM, 125 μM, 62 μM, 31.25 μM, 15.625 μM, and 0M. n = 8 per measured concentration.

    Article Snippet: All experiments were performed with E. coli TOP10 strains, purchased from Life Technologies Europe.

    Techniques: Plasmid Preparation, Fluorescence, Concentration Assay

    Monitoring cellular integrity of the E. coli host strain expressing AT chimeras. (A) SDS-PAGE analysis of OM (M) and culture supernatant (S) fractions derived from E. coli TOP10 expressing Pet*, mCherry*and ESAT6*. Non cleaved species are denoted by arrows. Molecular weight markers (MWM, kDa) are indicated to the right of the panel. OM fractions demonstrating the presence and absence of the β-domain are shown in Additional file 6 : Figure S5. (B) E. coli cells expressing empty vector, Pet*, mCherry*, ESAT6* and their cleaved parents were harvested 2 h after induction and subjected to indirect immunofluorescence using the indicated antibody. For each population expressing a cleavage deficient variant, a sample was divided in two: one half was probed with an antibody to the periplasmic protein BamD, while the other half was permeabilised (−P) and subsequently probed with the same antibody. Corresponding fields are also shown by phase contrast microscopy. For the mCherry constructs panels showing mCherry derived fluorescence are shown. (C) The integrity of E. coli host cells expressing wild type Pet and secreted ESAT6-Pet fusions were assessed by staining with BOX and PI prior to and 2 h post induction. Q3 represents the population that is viable and healthy and did not label with either stain. Q2 represents cells that stain with both stains and are no longer viable. Q4 (BOX-positive) represents cells with impaired membrane potential suggesting compromised membrane integrity. (D) Periplasmic leakage from E. coli TOP10 cultures secreting Pet or ESAT6-Pet fusion proteins was assessed by measuring (2 h after induction) the activity in the culture medium of the periplasmic enzyme alkaline phosphatase. Clarified whole cell lysate was used as a positive control and the wild-type plasmid-free strain as a negative control. There is no significant difference between the negative control and culture medium derived from strains expressing Pet or ESAT6-Pet-BB. In contrast, culture medium from both constructs displayed activity significantly less than the positive control. Absorbance measurements are derived from equal volumes of culture and are normalised relative to positive control (in %). The error bars represent standard error for two independent data sets.

    Journal: Microbial Cell Factories

    Article Title: A generalised module for the selective extracellular accumulation of recombinant proteins

    doi: 10.1186/1475-2859-11-69

    Figure Lengend Snippet: Monitoring cellular integrity of the E. coli host strain expressing AT chimeras. (A) SDS-PAGE analysis of OM (M) and culture supernatant (S) fractions derived from E. coli TOP10 expressing Pet*, mCherry*and ESAT6*. Non cleaved species are denoted by arrows. Molecular weight markers (MWM, kDa) are indicated to the right of the panel. OM fractions demonstrating the presence and absence of the β-domain are shown in Additional file 6 : Figure S5. (B) E. coli cells expressing empty vector, Pet*, mCherry*, ESAT6* and their cleaved parents were harvested 2 h after induction and subjected to indirect immunofluorescence using the indicated antibody. For each population expressing a cleavage deficient variant, a sample was divided in two: one half was probed with an antibody to the periplasmic protein BamD, while the other half was permeabilised (−P) and subsequently probed with the same antibody. Corresponding fields are also shown by phase contrast microscopy. For the mCherry constructs panels showing mCherry derived fluorescence are shown. (C) The integrity of E. coli host cells expressing wild type Pet and secreted ESAT6-Pet fusions were assessed by staining with BOX and PI prior to and 2 h post induction. Q3 represents the population that is viable and healthy and did not label with either stain. Q2 represents cells that stain with both stains and are no longer viable. Q4 (BOX-positive) represents cells with impaired membrane potential suggesting compromised membrane integrity. (D) Periplasmic leakage from E. coli TOP10 cultures secreting Pet or ESAT6-Pet fusion proteins was assessed by measuring (2 h after induction) the activity in the culture medium of the periplasmic enzyme alkaline phosphatase. Clarified whole cell lysate was used as a positive control and the wild-type plasmid-free strain as a negative control. There is no significant difference between the negative control and culture medium derived from strains expressing Pet or ESAT6-Pet-BB. In contrast, culture medium from both constructs displayed activity significantly less than the positive control. Absorbance measurements are derived from equal volumes of culture and are normalised relative to positive control (in %). The error bars represent standard error for two independent data sets.

    Article Snippet: E. coli TOP10, TOP10 dsbA [ ] and BL21* (Novagen) and S. enterica SL1344 and SL3261 strains [ ] were used for protein expression and secretion.

    Techniques: Expressing, SDS Page, Derivative Assay, Positron Emission Tomography, Molecular Weight, Plasmid Preparation, Immunofluorescence, Variant Assay, Microscopy, Construct, Fluorescence, Staining, Activity Assay, Positive Control, Negative Control

    Modification of the Pet-AT secretion platform. (A) SapA, Pmp17, Pet and a Pet derivative (PetΔD1) lacking the serine protease domain were modified to add a His 6 -Tag to the N-terminus of the secreted passenger domain. The His-tagged Pmp17 protein was expressed in an E. coli TOP10 dsbA strain and the rest of proteins were produced in the wild-type E. coli TOP10. In all cases the proteins are well secreted. The cysteine-containing Pmp17 was expressed in E. coli TOP10 (wt) and a dsbA - derivative. A full length protein is present in the E. coli TOP10 dsbA - derivative but not the E. coli TOP10 parent strain. Break down products are apparent and correspond to proteins with a truncated N-terminus. A multicomponent construct was created by fusing DNA encoding Ag85 to ESAT-6 and Pet (see Figure 1A) to encode a single polypeptide chain contiguous with the AT-translocation unit. This latter chimera was detected in the culture supernatant with antibodies directed at Pet and ESAT-6. Equivalent amounts of culture supernatant fractions were analysed by SDS-PAGE. (B) Secretion of heterologous fusions from S. Typhimurium. Culture medium from S. enterica SL1344 strains expressing ESAT6-Pet-BP and ESAT6-PetΔ*6 (see Figure 4 ) were harvested and analysed by SDS-PAGE and detected by immunoblotting with a polyclonal antibody to ESAT-6. In all SDS-PAGE gels the positions of the molecular weight markers (MWM, kDa) are depicted at the right side of the panel. The equivalent OM fractions demonstrating the presence of the cleaved β-barrel are shown in Additional file 5 : Figure S4.

    Journal: Microbial Cell Factories

    Article Title: A generalised module for the selective extracellular accumulation of recombinant proteins

    doi: 10.1186/1475-2859-11-69

    Figure Lengend Snippet: Modification of the Pet-AT secretion platform. (A) SapA, Pmp17, Pet and a Pet derivative (PetΔD1) lacking the serine protease domain were modified to add a His 6 -Tag to the N-terminus of the secreted passenger domain. The His-tagged Pmp17 protein was expressed in an E. coli TOP10 dsbA strain and the rest of proteins were produced in the wild-type E. coli TOP10. In all cases the proteins are well secreted. The cysteine-containing Pmp17 was expressed in E. coli TOP10 (wt) and a dsbA - derivative. A full length protein is present in the E. coli TOP10 dsbA - derivative but not the E. coli TOP10 parent strain. Break down products are apparent and correspond to proteins with a truncated N-terminus. A multicomponent construct was created by fusing DNA encoding Ag85 to ESAT-6 and Pet (see Figure 1A) to encode a single polypeptide chain contiguous with the AT-translocation unit. This latter chimera was detected in the culture supernatant with antibodies directed at Pet and ESAT-6. Equivalent amounts of culture supernatant fractions were analysed by SDS-PAGE. (B) Secretion of heterologous fusions from S. Typhimurium. Culture medium from S. enterica SL1344 strains expressing ESAT6-Pet-BP and ESAT6-PetΔ*6 (see Figure 4 ) were harvested and analysed by SDS-PAGE and detected by immunoblotting with a polyclonal antibody to ESAT-6. In all SDS-PAGE gels the positions of the molecular weight markers (MWM, kDa) are depicted at the right side of the panel. The equivalent OM fractions demonstrating the presence of the cleaved β-barrel are shown in Additional file 5 : Figure S4.

    Article Snippet: E. coli TOP10, TOP10 dsbA [ ] and BL21* (Novagen) and S. enterica SL1344 and SL3261 strains [ ] were used for protein expression and secretion.

    Techniques: Modification, Positron Emission Tomography, Produced, Construct, Translocation Assay, SDS Page, Expressing, Molecular Weight

    Identification of the minimal AT module permitting secretion of heterologous proteins to the culture supernatant fraction. (A) Schematic of ESAT6-Pet-BP protein fusion and some truncations created to determine the minimal C-terminal Pet fragment capable of ESAT-6 secretion. The Δ*1, Δ*2, Δ*6, Δ*17 and Δ*20 Pet truncations are shown while for simplicity the intermediate variants Δ*3–Δ*5, Δ*7–Δ*16, Δ*18 and Δ*19 are omitted. Abbreviations are the same as in Figure 1 . (B and C) Detection by western immunoblotting of ESAT6-Pet (B) and ESAT6-Pic (C) chimeras expressed in E. coli TOP10. The TCA-precipitated culture supernatants were analysed by SDS-PAGE and probed with polyclonal anti-ESAT6. The equivalent OM fractions demonstrating the presence of the cleaved β-barrel in the OM are shown in Additional file 6 : Figure S5.

    Journal: Microbial Cell Factories

    Article Title: A generalised module for the selective extracellular accumulation of recombinant proteins

    doi: 10.1186/1475-2859-11-69

    Figure Lengend Snippet: Identification of the minimal AT module permitting secretion of heterologous proteins to the culture supernatant fraction. (A) Schematic of ESAT6-Pet-BP protein fusion and some truncations created to determine the minimal C-terminal Pet fragment capable of ESAT-6 secretion. The Δ*1, Δ*2, Δ*6, Δ*17 and Δ*20 Pet truncations are shown while for simplicity the intermediate variants Δ*3–Δ*5, Δ*7–Δ*16, Δ*18 and Δ*19 are omitted. Abbreviations are the same as in Figure 1 . (B and C) Detection by western immunoblotting of ESAT6-Pet (B) and ESAT6-Pic (C) chimeras expressed in E. coli TOP10. The TCA-precipitated culture supernatants were analysed by SDS-PAGE and probed with polyclonal anti-ESAT6. The equivalent OM fractions demonstrating the presence of the cleaved β-barrel in the OM are shown in Additional file 6 : Figure S5.

    Article Snippet: E. coli TOP10, TOP10 dsbA [ ] and BL21* (Novagen) and S. enterica SL1344 and SL3261 strains [ ] were used for protein expression and secretion.

    Techniques: Positron Emission Tomography, Western Blot, SDS Page