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  • 99
    New England Biolabs e coli bl21
    Importance of auto-processing for oligomerization and caseinolytic activity of HtrA Hp expressed in E. coli . The amino-terminus of HtrA Hp was mutagenized by generating deletion variants and expressed as GST-tagged variants in E. coli <t>BL21</t> (compare Supplementary Figure 1B ). E. coli BL21 expressing GST-tagged HtrA Hp without the signal peptide (ΔSP) and empty vector E. coli BL21 were used as controls. (A) After induction of protein expression by IPTG, the protein lysates were subjected to Coomassie staining. Overexpression of the HtrA Hp p55 monomers and the GST-tagged variants was observed (arrows). The amino-terminal deletion variants (ΔN1 and ΔN2, yellow asterisks) exhibit a lower molecular weight compared to HtrA Hp ΔSP (black asterisk). (B) The bacterial lysates were subjected to immunoblotting against HtrA Hp and GST. Overexpression of the GST-tagged HtrA Hp p55 variants was confirmed by detecting the HtrA Hp or GST protein, respectively (arrows). In addition, immunoblotting against HtrA Hp showed the presence of HtrA Hp monomers without GST-tag (arrow). The p52 form is migrating slightly below p55 (red asterisks). (C) Finally, the bacterial lysates were analyzed by casein zymography. For wt HtrA Hp ΔSP, a strong caseinolytic activity of the HtrA Hp monomers and its GST-tagged variants was observed (arrows). The p52 form is migrating slightly below p55 (red asterisks). Moreover, caseinolytic activity for the HtrA Hp trimer and its GST-tagged variant was shown (arrows). The ΔN1 and ΔN2 variants revealed no proteolytic activity (yellow asterisks). All assays were done in triplicates.
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    99
    Thermo Fisher competent e coli
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    93
    Thermo Fisher competent escherichia coli cells
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    99
    Thermo Fisher one shot top10
    AQUA Cloning conditions. (a) The DNA fragment encoding the red fluorescent protein mCherry was PCR amplified with flanking extensions of 16, 24, or 32 bp of homologous sequence overlaps to an SV40 promoter-driven mammalian expression vector which was digested, or PCR amplified. AQUA Cloning was performed with pre-incubations on ice, at room temperature (RT), or at 50°C. DNA mixtures were transformed into chemically competent E . coli <t>Top10</t> cells and colonies were obtained the next day. (b) Typical numbers of colony forming units (CFU) obtained from each condition. The highest number of CFU was derived from a pre-incubation at room temperature and with 32 bp of shared homology with PCR originating DNA fragments (arrow). The accuracies for each condition were determined by analytical colony PCR from eight selected clones and were extrapolated to the total number of CFU (grey). Abbreviations: P SV40 , simian virus 40 early promoter.
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    Thermo Fisher competent escherichia coli top10 cells
    AQUA Cloning conditions. (a) The DNA fragment encoding the red fluorescent protein mCherry was PCR amplified with flanking extensions of 16, 24, or 32 bp of homologous sequence overlaps to an SV40 promoter-driven mammalian expression vector which was digested, or PCR amplified. AQUA Cloning was performed with pre-incubations on ice, at room temperature (RT), or at 50°C. DNA mixtures were transformed into chemically competent E . coli <t>Top10</t> cells and colonies were obtained the next day. (b) Typical numbers of colony forming units (CFU) obtained from each condition. The highest number of CFU was derived from a pre-incubation at room temperature and with 32 bp of shared homology with PCR originating DNA fragments (arrow). The accuracies for each condition were determined by analytical colony PCR from eight selected clones and were extrapolated to the total number of CFU (grey). Abbreviations: P SV40 , simian virus 40 early promoter.
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    91
    Thermo Fisher one shot bl21 de3 chemically competent e coli
    AQUA Cloning conditions. (a) The DNA fragment encoding the red fluorescent protein mCherry was PCR amplified with flanking extensions of 16, 24, or 32 bp of homologous sequence overlaps to an SV40 promoter-driven mammalian expression vector which was digested, or PCR amplified. AQUA Cloning was performed with pre-incubations on ice, at room temperature (RT), or at 50°C. DNA mixtures were transformed into chemically competent E . coli <t>Top10</t> cells and colonies were obtained the next day. (b) Typical numbers of colony forming units (CFU) obtained from each condition. The highest number of CFU was derived from a pre-incubation at room temperature and with 32 bp of shared homology with PCR originating DNA fragments (arrow). The accuracies for each condition were determined by analytical colony PCR from eight selected clones and were extrapolated to the total number of CFU (grey). Abbreviations: P SV40 , simian virus 40 early promoter.
    One Shot Bl21 De3 Chemically Competent E Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 194 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher e coli dh5α competent cells
    Immunoprecipitation to determine the specificity of DivIVA interactions. (A) Immunoprecipitation of GFP-DivIVA-cI-Spo0J complex (lane 1), GFP-Spo0J-cI-DivIVA complex (lane 2), GFP-FtsZ-cI-DivIVA complex (lane 3), and GFP-DivIVA-cI-FtsZ complex (lane 4). (B) Immunoprecipitation of cI-DivIVA-GFP-PrfA complex (lane 1). Protein extracts were immunoprecipitated with anti-GFP antibodies and were detected with anti-cI rabbit polyclonal antibodies by Western blotting as described in Materials and Methods. Lane M contained molecular weight markers. In panel B lane 2 contained crude extract of E. coli strain <t>DH5α</t> expressing cI-DivIVA, detected with anti-cI antibodies.
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    99
    New England Biolabs bl21 de3 competent e coli
    Immunoprecipitation to determine the specificity of DivIVA interactions. (A) Immunoprecipitation of GFP-DivIVA-cI-Spo0J complex (lane 1), GFP-Spo0J-cI-DivIVA complex (lane 2), GFP-FtsZ-cI-DivIVA complex (lane 3), and GFP-DivIVA-cI-FtsZ complex (lane 4). (B) Immunoprecipitation of cI-DivIVA-GFP-PrfA complex (lane 1). Protein extracts were immunoprecipitated with anti-GFP antibodies and were detected with anti-cI rabbit polyclonal antibodies by Western blotting as described in Materials and Methods. Lane M contained molecular weight markers. In panel B lane 2 contained crude extract of E. coli strain <t>DH5α</t> expressing cI-DivIVA, detected with anti-cI antibodies.
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    99
    Promega escherichia coli jm109 competent cells
    Immunoprecipitation to determine the specificity of DivIVA interactions. (A) Immunoprecipitation of GFP-DivIVA-cI-Spo0J complex (lane 1), GFP-Spo0J-cI-DivIVA complex (lane 2), GFP-FtsZ-cI-DivIVA complex (lane 3), and GFP-DivIVA-cI-FtsZ complex (lane 4). (B) Immunoprecipitation of cI-DivIVA-GFP-PrfA complex (lane 1). Protein extracts were immunoprecipitated with anti-GFP antibodies and were detected with anti-cI rabbit polyclonal antibodies by Western blotting as described in Materials and Methods. Lane M contained molecular weight markers. In panel B lane 2 contained crude extract of E. coli strain <t>DH5α</t> expressing cI-DivIVA, detected with anti-cI antibodies.
    Escherichia Coli Jm109 Competent Cells, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 533 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs neb 5 alpha competent e coli high efficiency
    Immunoprecipitation to determine the specificity of DivIVA interactions. (A) Immunoprecipitation of GFP-DivIVA-cI-Spo0J complex (lane 1), GFP-Spo0J-cI-DivIVA complex (lane 2), GFP-FtsZ-cI-DivIVA complex (lane 3), and GFP-DivIVA-cI-FtsZ complex (lane 4). (B) Immunoprecipitation of cI-DivIVA-GFP-PrfA complex (lane 1). Protein extracts were immunoprecipitated with anti-GFP antibodies and were detected with anti-cI rabbit polyclonal antibodies by Western blotting as described in Materials and Methods. Lane M contained molecular weight markers. In panel B lane 2 contained crude extract of E. coli strain <t>DH5α</t> expressing cI-DivIVA, detected with anti-cI antibodies.
    Neb 5 Alpha Competent E Coli High Efficiency, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 410 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore bl21 de3 chemically competent cells
    Immunoprecipitation to determine the specificity of DivIVA interactions. (A) Immunoprecipitation of GFP-DivIVA-cI-Spo0J complex (lane 1), GFP-Spo0J-cI-DivIVA complex (lane 2), GFP-FtsZ-cI-DivIVA complex (lane 3), and GFP-DivIVA-cI-FtsZ complex (lane 4). (B) Immunoprecipitation of cI-DivIVA-GFP-PrfA complex (lane 1). Protein extracts were immunoprecipitated with anti-GFP antibodies and were detected with anti-cI rabbit polyclonal antibodies by Western blotting as described in Materials and Methods. Lane M contained molecular weight markers. In panel B lane 2 contained crude extract of E. coli strain <t>DH5α</t> expressing cI-DivIVA, detected with anti-cI antibodies.
    Bl21 De3 Chemically Competent Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs escherichia coli
    Immunoprecipitation to determine the specificity of DivIVA interactions. (A) Immunoprecipitation of GFP-DivIVA-cI-Spo0J complex (lane 1), GFP-Spo0J-cI-DivIVA complex (lane 2), GFP-FtsZ-cI-DivIVA complex (lane 3), and GFP-DivIVA-cI-FtsZ complex (lane 4). (B) Immunoprecipitation of cI-DivIVA-GFP-PrfA complex (lane 1). Protein extracts were immunoprecipitated with anti-GFP antibodies and were detected with anti-cI rabbit polyclonal antibodies by Western blotting as described in Materials and Methods. Lane M contained molecular weight markers. In panel B lane 2 contained crude extract of E. coli strain <t>DH5α</t> expressing cI-DivIVA, detected with anti-cI antibodies.
    Escherichia Coli, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1484 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher multishot stripwell dh5α t1r competent cells
    Expression of different glycosyl hydrolase (GH) genes from a common expression vector backbone and test of their effects on growth of E. coli <t>DH5α</t> in MOPS minimal medium ( 17 ) and Z. mobilis ZM4 in Zymomonas minimal medium ( 18 ) with glucose or cellobiose as a carbon source. (A) Expression of GH genes in a pVector backbone and a summary of the constructs and their effects on growth in minimal medium supplemented with cellobiose. (B-D) Growth of E. coli DH5α containing plasmids pVector or pCel3A or pGH3 in MOPS minimal medium supplied with 0.4% glucose or cellobiose. (E-G) Growth of Z. mobilis ZM4 containing plasmids pVector or pCel3A or pGH3 in a Zymomonas minimal medium containing 2% glucose or 2% cellobiose. The growth curves are averages of three replicates. *Gene from Cellvibrio japonicus , # Gene from Caulobacter crescentus .
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    Importance of auto-processing for oligomerization and caseinolytic activity of HtrA Hp expressed in E. coli . The amino-terminus of HtrA Hp was mutagenized by generating deletion variants and expressed as GST-tagged variants in E. coli BL21 (compare Supplementary Figure 1B ). E. coli BL21 expressing GST-tagged HtrA Hp without the signal peptide (ΔSP) and empty vector E. coli BL21 were used as controls. (A) After induction of protein expression by IPTG, the protein lysates were subjected to Coomassie staining. Overexpression of the HtrA Hp p55 monomers and the GST-tagged variants was observed (arrows). The amino-terminal deletion variants (ΔN1 and ΔN2, yellow asterisks) exhibit a lower molecular weight compared to HtrA Hp ΔSP (black asterisk). (B) The bacterial lysates were subjected to immunoblotting against HtrA Hp and GST. Overexpression of the GST-tagged HtrA Hp p55 variants was confirmed by detecting the HtrA Hp or GST protein, respectively (arrows). In addition, immunoblotting against HtrA Hp showed the presence of HtrA Hp monomers without GST-tag (arrow). The p52 form is migrating slightly below p55 (red asterisks). (C) Finally, the bacterial lysates were analyzed by casein zymography. For wt HtrA Hp ΔSP, a strong caseinolytic activity of the HtrA Hp monomers and its GST-tagged variants was observed (arrows). The p52 form is migrating slightly below p55 (red asterisks). Moreover, caseinolytic activity for the HtrA Hp trimer and its GST-tagged variant was shown (arrows). The ΔN1 and ΔN2 variants revealed no proteolytic activity (yellow asterisks). All assays were done in triplicates.

    Journal: Frontiers in Microbiology

    Article Title: Amino-Terminal Processing of Helicobacter pylori Serine Protease HtrA: Role in Oligomerization and Activity Regulation

    doi: 10.3389/fmicb.2018.00642

    Figure Lengend Snippet: Importance of auto-processing for oligomerization and caseinolytic activity of HtrA Hp expressed in E. coli . The amino-terminus of HtrA Hp was mutagenized by generating deletion variants and expressed as GST-tagged variants in E. coli BL21 (compare Supplementary Figure 1B ). E. coli BL21 expressing GST-tagged HtrA Hp without the signal peptide (ΔSP) and empty vector E. coli BL21 were used as controls. (A) After induction of protein expression by IPTG, the protein lysates were subjected to Coomassie staining. Overexpression of the HtrA Hp p55 monomers and the GST-tagged variants was observed (arrows). The amino-terminal deletion variants (ΔN1 and ΔN2, yellow asterisks) exhibit a lower molecular weight compared to HtrA Hp ΔSP (black asterisk). (B) The bacterial lysates were subjected to immunoblotting against HtrA Hp and GST. Overexpression of the GST-tagged HtrA Hp p55 variants was confirmed by detecting the HtrA Hp or GST protein, respectively (arrows). In addition, immunoblotting against HtrA Hp showed the presence of HtrA Hp monomers without GST-tag (arrow). The p52 form is migrating slightly below p55 (red asterisks). (C) Finally, the bacterial lysates were analyzed by casein zymography. For wt HtrA Hp ΔSP, a strong caseinolytic activity of the HtrA Hp monomers and its GST-tagged variants was observed (arrows). The p52 form is migrating slightly below p55 (red asterisks). Moreover, caseinolytic activity for the HtrA Hp trimer and its GST-tagged variant was shown (arrows). The ΔN1 and ΔN2 variants revealed no proteolytic activity (yellow asterisks). All assays were done in triplicates.

    Article Snippet: Overexpression of HtrA in E. coli For the overexpression of the GST-tagged H. pylori 26695 HtrA variants, the constructs (see mutagenesis of H. pylori htrA ) have been transformed into E. coli BL21 (NEB).

    Techniques: Activity Assay, Expressing, Plasmid Preparation, Staining, Over Expression, Molecular Weight, Zymography, Variant Assay

    Detection of chloroplast-expressed mTurquoise2 in transgenic lines of Marchantia polymorpha. (A) M. polymorpha protein extracts separated by SDS–PAGE. Protein extracts from wild type (WT) and transplastomic (CL0*b) lines of M. polymorpha were separated by 4–12% SDS–PAGE, and visualized by in-gel fluorescence (left) and subsequent Coomassie stain (right). The fluorescence image was generated using a custom imaging device described in the Materials and Methods for visualization of CFP bands (emission 486/10 nm, green) and marker (emission 540/10 nm, red). (B) Untagged and His 6 -tagged mTurquoise2 separated by SDS–PAGE. Untagged mTurquoise2 was expressed from plasmids pCS CL0*b ( psbA promoter) in BL21 E. coli and pCRB SREI (T7 promoter) in T7 Express E. coli. His 6 -tagged mTurquoise2 was expressed from plasmid pCRB SREI 6his (T7 promoter) and purified by affinity chromatography. The rightmost lanes contain 50 ng of unboiled and boiled purified protein, respectively. Visualization by in-gel fluorescence and Coomassie stain was conducted as described above. (C) Standard curve for quantification of mTurquoise2 based on in-gel fluorescence. Serial dilutions of 1 µg purified of mTurquoise2 were separated by 4–12% SDS–PAGE and the band intensity quantified by in-gel fluorescence as described in the Materials and Methods. Levels of mTurquoise2 extracted from transplastomic (CL0*b) M. polymorpha (red cross) were estimated by linear regression analysis on densities of fluorescent target bands as shown in (A). Error bars represent the SD of average band density between three different experiments.

    Journal: Plant and Cell Physiology

    Article Title: A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha

    doi: 10.1093/pcp/pcv160

    Figure Lengend Snippet: Detection of chloroplast-expressed mTurquoise2 in transgenic lines of Marchantia polymorpha. (A) M. polymorpha protein extracts separated by SDS–PAGE. Protein extracts from wild type (WT) and transplastomic (CL0*b) lines of M. polymorpha were separated by 4–12% SDS–PAGE, and visualized by in-gel fluorescence (left) and subsequent Coomassie stain (right). The fluorescence image was generated using a custom imaging device described in the Materials and Methods for visualization of CFP bands (emission 486/10 nm, green) and marker (emission 540/10 nm, red). (B) Untagged and His 6 -tagged mTurquoise2 separated by SDS–PAGE. Untagged mTurquoise2 was expressed from plasmids pCS CL0*b ( psbA promoter) in BL21 E. coli and pCRB SREI (T7 promoter) in T7 Express E. coli. His 6 -tagged mTurquoise2 was expressed from plasmid pCRB SREI 6his (T7 promoter) and purified by affinity chromatography. The rightmost lanes contain 50 ng of unboiled and boiled purified protein, respectively. Visualization by in-gel fluorescence and Coomassie stain was conducted as described above. (C) Standard curve for quantification of mTurquoise2 based on in-gel fluorescence. Serial dilutions of 1 µg purified of mTurquoise2 were separated by 4–12% SDS–PAGE and the band intensity quantified by in-gel fluorescence as described in the Materials and Methods. Levels of mTurquoise2 extracted from transplastomic (CL0*b) M. polymorpha (red cross) were estimated by linear regression analysis on densities of fluorescent target bands as shown in (A). Error bars represent the SD of average band density between three different experiments.

    Article Snippet: Visualization of proteins by in-gel fluorescence and coomassie staining For total protein extraction from M. polymorpha tissues, 50 mg (fresh weight) of thallus were ground by liquid nitrogen and the resulting powder vortexed in 200 µl of 2 × Tris–glycine for 30 s. For total protein extraction from BL21 or T7 Express competent E. coli (New England Biolabs), an aliquot of overnight culture containing approximately 0.5–1 × 109 cells was pelleted by centrifugation at 14,000 r.p.m. and 4°C for 1 min, resuspended in 50 µl of 2 × Tris–glycine SDS sample buffer and vortexed for 30 s. In both cases, 2-fold dilutions of crude extracts were centrifuged at 14,000 r.p.m. and 4°C for 20 min to remove cell debris, and unboiled (unless indicated otherwise) supernatants were directly loaded onto a NuPAGE Novex 4–12% Bis–Tris protein minigel (Life Technologies).

    Techniques: Transgenic Assay, SDS Page, Fluorescence, Staining, Generated, Imaging, Marker, Plasmid Preparation, Purification, Affinity Chromatography

    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.

    Journal: Blood Transfusion

    Article Title: Expression and purification of recombinant human coagulation factor VII fused to a histidine tag using Gateway technology

    doi: 10.2450/2009.0081-08

    Figure Lengend Snippet: A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent E. coli and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.

    Article Snippet: The reaction was then placed on ice and the pENTR TOPO/D-FVII construct was transformed to competent E. coli according to the manufacturer’s protocol (Invitrogen, USA).

    Techniques: Expressing, Clone Assay, Polymerase Chain Reaction, Plasmid Preparation, Construct, Recombinant, Transfection, Sequencing

    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

    AQUA Cloning conditions. (a) The DNA fragment encoding the red fluorescent protein mCherry was PCR amplified with flanking extensions of 16, 24, or 32 bp of homologous sequence overlaps to an SV40 promoter-driven mammalian expression vector which was digested, or PCR amplified. AQUA Cloning was performed with pre-incubations on ice, at room temperature (RT), or at 50°C. DNA mixtures were transformed into chemically competent E . coli Top10 cells and colonies were obtained the next day. (b) Typical numbers of colony forming units (CFU) obtained from each condition. The highest number of CFU was derived from a pre-incubation at room temperature and with 32 bp of shared homology with PCR originating DNA fragments (arrow). The accuracies for each condition were determined by analytical colony PCR from eight selected clones and were extrapolated to the total number of CFU (grey). Abbreviations: P SV40 , simian virus 40 early promoter.

    Journal: PLoS ONE

    Article Title: AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach

    doi: 10.1371/journal.pone.0137652

    Figure Lengend Snippet: AQUA Cloning conditions. (a) The DNA fragment encoding the red fluorescent protein mCherry was PCR amplified with flanking extensions of 16, 24, or 32 bp of homologous sequence overlaps to an SV40 promoter-driven mammalian expression vector which was digested, or PCR amplified. AQUA Cloning was performed with pre-incubations on ice, at room temperature (RT), or at 50°C. DNA mixtures were transformed into chemically competent E . coli Top10 cells and colonies were obtained the next day. (b) Typical numbers of colony forming units (CFU) obtained from each condition. The highest number of CFU was derived from a pre-incubation at room temperature and with 32 bp of shared homology with PCR originating DNA fragments (arrow). The accuracies for each condition were determined by analytical colony PCR from eight selected clones and were extrapolated to the total number of CFU (grey). Abbreviations: P SV40 , simian virus 40 early promoter.

    Article Snippet: E . coli strains The following commercially available strains of E . coli were also used for AQUA Cloning: One Shot TOP10 (Invitrogen cat. No. C4040-03, competency: > 109 CFU/μg), NEB5α (NEB, cat. No. C2987I, competency: 1–3 x 109 CFU/μg), NEB10β (NEB, cat. No. C3019I, competency: 1–3 x 109 CFU/μg), BL21 (DE3) (NEB, cat. No. C2527I, competency: 1–5 x 107 CFU/μg), JM109 (Promega, cat. No. L2005, competency: 108 CFU/μg).

    Techniques: Clone Assay, Polymerase Chain Reaction, Amplification, Sequencing, Expressing, Plasmid Preparation, Transformation Assay, Derivative Assay, Incubation

    AQUA Expression—combined cloning and protein expression. (a) Timeline for AQUA Expression. Cloning and production of recombinant protein in E . coli may be performed within 24 h starting with the PCR until the bacteria are harvested the next day. (b) A 3-DNA fragment cloning was performed by inserting the coding sequence for the red fluorescent protein mCherry into a bacterial T7 promoter-driven expression vector. The vector was split into two parts within the resistance gene for the antibiotic spectinomycin. Therefore, only correctly assembled fragments allow cell growth. (c) AQUA Expression in the expression strain BL21 (DE3) results in red colored bacteria due to mCherry protein production, while the TOP10 strain—lacking the required T7 RNA polymerase—remains colorless.

    Journal: PLoS ONE

    Article Title: AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach

    doi: 10.1371/journal.pone.0137652

    Figure Lengend Snippet: AQUA Expression—combined cloning and protein expression. (a) Timeline for AQUA Expression. Cloning and production of recombinant protein in E . coli may be performed within 24 h starting with the PCR until the bacteria are harvested the next day. (b) A 3-DNA fragment cloning was performed by inserting the coding sequence for the red fluorescent protein mCherry into a bacterial T7 promoter-driven expression vector. The vector was split into two parts within the resistance gene for the antibiotic spectinomycin. Therefore, only correctly assembled fragments allow cell growth. (c) AQUA Expression in the expression strain BL21 (DE3) results in red colored bacteria due to mCherry protein production, while the TOP10 strain—lacking the required T7 RNA polymerase—remains colorless.

    Article Snippet: E . coli strains The following commercially available strains of E . coli were also used for AQUA Cloning: One Shot TOP10 (Invitrogen cat. No. C4040-03, competency: > 109 CFU/μg), NEB5α (NEB, cat. No. C2987I, competency: 1–3 x 109 CFU/μg), NEB10β (NEB, cat. No. C3019I, competency: 1–3 x 109 CFU/μg), BL21 (DE3) (NEB, cat. No. C2527I, competency: 1–5 x 107 CFU/μg), JM109 (Promega, cat. No. L2005, competency: 108 CFU/μg).

    Techniques: Expressing, Clone Assay, Recombinant, Polymerase Chain Reaction, Sequencing, Plasmid Preparation

    AQUA Cloning: a dvanced qu ick a ssembly cloning. (a) DNA parts are produced by PCR, or restriction digest (or both). Oligonucleotides are designed to contribute flanking homologous regions to adjacent DNA fragments of optimally 32 bp in length. DNA parts are assembled into a circular plasmid by sequence-determined directionality. (b) AQUA Cloning work-flow. (1) DNA parts are generated by PCR amplification, or derived from an enzymatic digestion. (2) Next, DNA parts are purified by gel-electrophoresis and (3) mixed and simply incubated in water prior to transformation into chemically competent E . coli Top10 cells for in vivo assembly. (4) Finally, obtained colonies are confirmed for correct assembly by standard methods such as analytical PCR, restriction digest, or comprehensive sequencing.

    Journal: PLoS ONE

    Article Title: AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach

    doi: 10.1371/journal.pone.0137652

    Figure Lengend Snippet: AQUA Cloning: a dvanced qu ick a ssembly cloning. (a) DNA parts are produced by PCR, or restriction digest (or both). Oligonucleotides are designed to contribute flanking homologous regions to adjacent DNA fragments of optimally 32 bp in length. DNA parts are assembled into a circular plasmid by sequence-determined directionality. (b) AQUA Cloning work-flow. (1) DNA parts are generated by PCR amplification, or derived from an enzymatic digestion. (2) Next, DNA parts are purified by gel-electrophoresis and (3) mixed and simply incubated in water prior to transformation into chemically competent E . coli Top10 cells for in vivo assembly. (4) Finally, obtained colonies are confirmed for correct assembly by standard methods such as analytical PCR, restriction digest, or comprehensive sequencing.

    Article Snippet: E . coli strains The following commercially available strains of E . coli were also used for AQUA Cloning: One Shot TOP10 (Invitrogen cat. No. C4040-03, competency: > 109 CFU/μg), NEB5α (NEB, cat. No. C2987I, competency: 1–3 x 109 CFU/μg), NEB10β (NEB, cat. No. C3019I, competency: 1–3 x 109 CFU/μg), BL21 (DE3) (NEB, cat. No. C2527I, competency: 1–5 x 107 CFU/μg), JM109 (Promega, cat. No. L2005, competency: 108 CFU/μg).

    Techniques: Clone Assay, Produced, Polymerase Chain Reaction, Plasmid Preparation, Sequencing, Flow Cytometry, Generated, Amplification, Derivative Assay, Purification, Nucleic Acid Electrophoresis, Incubation, Transformation Assay, In Vivo

    Immunoprecipitation to determine the specificity of DivIVA interactions. (A) Immunoprecipitation of GFP-DivIVA-cI-Spo0J complex (lane 1), GFP-Spo0J-cI-DivIVA complex (lane 2), GFP-FtsZ-cI-DivIVA complex (lane 3), and GFP-DivIVA-cI-FtsZ complex (lane 4). (B) Immunoprecipitation of cI-DivIVA-GFP-PrfA complex (lane 1). Protein extracts were immunoprecipitated with anti-GFP antibodies and were detected with anti-cI rabbit polyclonal antibodies by Western blotting as described in Materials and Methods. Lane M contained molecular weight markers. In panel B lane 2 contained crude extract of E. coli strain DH5α expressing cI-DivIVA, detected with anti-cI antibodies.

    Journal: Journal of Bacteriology

    Article Title: Streptococcus pneumoniae DivIVA: Localization and Interactions in a MinCD-Free Context ▿ DivIVA: Localization and Interactions in a MinCD-Free Context ▿ †

    doi: 10.1128/JB.01168-06

    Figure Lengend Snippet: Immunoprecipitation to determine the specificity of DivIVA interactions. (A) Immunoprecipitation of GFP-DivIVA-cI-Spo0J complex (lane 1), GFP-Spo0J-cI-DivIVA complex (lane 2), GFP-FtsZ-cI-DivIVA complex (lane 3), and GFP-DivIVA-cI-FtsZ complex (lane 4). (B) Immunoprecipitation of cI-DivIVA-GFP-PrfA complex (lane 1). Protein extracts were immunoprecipitated with anti-GFP antibodies and were detected with anti-cI rabbit polyclonal antibodies by Western blotting as described in Materials and Methods. Lane M contained molecular weight markers. In panel B lane 2 contained crude extract of E. coli strain DH5α expressing cI-DivIVA, detected with anti-cI antibodies.

    Article Snippet: The ligation mixture was transformed into E. coli DH5α competent cells (Invitrogen), and transformants were selected after overnight growth on LB medium plates with ampicillin.

    Techniques: Immunoprecipitation, Western Blot, Molecular Weight, Expressing

    Expression of different glycosyl hydrolase (GH) genes from a common expression vector backbone and test of their effects on growth of E. coli DH5α in MOPS minimal medium ( 17 ) and Z. mobilis ZM4 in Zymomonas minimal medium ( 18 ) with glucose or cellobiose as a carbon source. (A) Expression of GH genes in a pVector backbone and a summary of the constructs and their effects on growth in minimal medium supplemented with cellobiose. (B-D) Growth of E. coli DH5α containing plasmids pVector or pCel3A or pGH3 in MOPS minimal medium supplied with 0.4% glucose or cellobiose. (E-G) Growth of Z. mobilis ZM4 containing plasmids pVector or pCel3A or pGH3 in a Zymomonas minimal medium containing 2% glucose or 2% cellobiose. The growth curves are averages of three replicates. *Gene from Cellvibrio japonicus , # Gene from Caulobacter crescentus .

    Journal: bioRxiv

    Article Title: Heterologous glycosyl hydrolase expression and cellular reprogramming resembling sucrose-induction enable Zymomonas mobilis growth on cellobiose

    doi: 10.1101/854646

    Figure Lengend Snippet: Expression of different glycosyl hydrolase (GH) genes from a common expression vector backbone and test of their effects on growth of E. coli DH5α in MOPS minimal medium ( 17 ) and Z. mobilis ZM4 in Zymomonas minimal medium ( 18 ) with glucose or cellobiose as a carbon source. (A) Expression of GH genes in a pVector backbone and a summary of the constructs and their effects on growth in minimal medium supplemented with cellobiose. (B-D) Growth of E. coli DH5α containing plasmids pVector or pCel3A or pGH3 in MOPS minimal medium supplied with 0.4% glucose or cellobiose. (E-G) Growth of Z. mobilis ZM4 containing plasmids pVector or pCel3A or pGH3 in a Zymomonas minimal medium containing 2% glucose or 2% cellobiose. The growth curves are averages of three replicates. *Gene from Cellvibrio japonicus , # Gene from Caulobacter crescentus .

    Article Snippet: The E. coli DH10B strain was used for cloning and E. coli DH5α was used for expressing the recombinant plasmids.

    Techniques: Expressing, Plasmid Preparation, Construct