nsp7 8  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 85
    Name:
    NEB Express Competent E coli High Efficiency
    Description:
    NEB Express Competent E coli High Efficiency 20x0 05 ml
    Catalog Number:
    c2523h
    Price:
    204
    Size:
    1 ml
    Category:
    Competent Bacteria
    Buy from Supplier


    Structured Review

    New England Biolabs nsp7 8
    NEB Express Competent E coli High Efficiency
    NEB Express Competent E coli High Efficiency 20x0 05 ml
    https://www.bioz.com/result/nsp7 8/product/New England Biolabs
    Average 85 stars, based on 149 article reviews
    Price from $9.99 to $1999.99
    nsp7 8 - by Bioz Stars, 2020-09
    85/100 stars

    Images

    1) Product Images from "The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension"

    Article Title: The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr893

    Purification and multimerization of recombinant SARS-CoV nsp7-8 precursor and different nsp8 variants. ( A ) Expression of nsp8 in the presence of the ubiquitin protease Ubp1 to liberate the natural N-terminal sequence (AIASEF), followed by purification and cleavage by recombinant SARS-CoV nsp5 main protease to remove the C-terminal His 6 -tag and its upstream GSSG linker. ( B ) Eighteen percent SDS-PAGE analysis of nsp5-treated, purified nsp8-His demonstrates near-complete release of the C-terminal His 6 -tag within 60 min. The maltose binding protein (MBP) was added to the reaction to serve as an independent loading control. Asterisks indicate non-specific bands. ( C ) In addition to the tag-less nps8 and nsp8-His, we also produced the N-terminally His 6 -tagged nsp8 (His-nsp8) used by Imbert et al . ( 12 ). ( D ) Comparative gel filtration analysis of nsp8 (22 kDa as a monomer) versus His-nsp8 and ( E ) nsp8 versus nsp8-His. In all three cases, nsp8 formed multimers in solution, but the apparent molecular mass of complexes formed by both nsp8 and nsp8-His was ∼2-fold higher than for complexes formed by His-nsp8. ( F ) Comparative analysis of nsp8, nsp(7+8), His-nsp8 and nsp7+nsp8-His. Only nsp(7+8) showed a molecular weight shift to the ∼225-kDa size range with a standard deviation of 15-kDa ( n = 3). This size is indicative of hexadecamer formation, whereas the analysis of nsp7+nsp8-His showed dominant peaks of nsp8-His and nsp7 (which is ∼10 kDa as a monomer).
    Figure Legend Snippet: Purification and multimerization of recombinant SARS-CoV nsp7-8 precursor and different nsp8 variants. ( A ) Expression of nsp8 in the presence of the ubiquitin protease Ubp1 to liberate the natural N-terminal sequence (AIASEF), followed by purification and cleavage by recombinant SARS-CoV nsp5 main protease to remove the C-terminal His 6 -tag and its upstream GSSG linker. ( B ) Eighteen percent SDS-PAGE analysis of nsp5-treated, purified nsp8-His demonstrates near-complete release of the C-terminal His 6 -tag within 60 min. The maltose binding protein (MBP) was added to the reaction to serve as an independent loading control. Asterisks indicate non-specific bands. ( C ) In addition to the tag-less nps8 and nsp8-His, we also produced the N-terminally His 6 -tagged nsp8 (His-nsp8) used by Imbert et al . ( 12 ). ( D ) Comparative gel filtration analysis of nsp8 (22 kDa as a monomer) versus His-nsp8 and ( E ) nsp8 versus nsp8-His. In all three cases, nsp8 formed multimers in solution, but the apparent molecular mass of complexes formed by both nsp8 and nsp8-His was ∼2-fold higher than for complexes formed by His-nsp8. ( F ) Comparative analysis of nsp8, nsp(7+8), His-nsp8 and nsp7+nsp8-His. Only nsp(7+8) showed a molecular weight shift to the ∼225-kDa size range with a standard deviation of 15-kDa ( n = 3). This size is indicative of hexadecamer formation, whereas the analysis of nsp7+nsp8-His showed dominant peaks of nsp8-His and nsp7 (which is ∼10 kDa as a monomer).

    Techniques Used: Purification, Recombinant, Expressing, Sequencing, SDS Page, Binding Assay, Produced, Filtration, Molecular Weight, Standard Deviation

    Influence of His 6 -tags and nsp7 on the RdRp activity of SARS-CoV nsp8. ( A ) The UC 10 template (see Figure 4 ) was incubated with 1 µM of wild-type nsp8 or either of three nsp8 variants to investigate the influence of the His 6 -tag. Samples were taken at the indicated time points and checked for [α- 32 P]AMP incorporation by 20% PAGE/7 M urea analysis. ( B ) Side-by-side comparison of the primer-extension activities of nps8 and nsp(7+8). Shorter incubations are shown to better demonstrate the difference in activity. ( C ) De novo activity of nsp8 and nsp(7+8) on template AFMB131 (see Table 1 ), using the synthesis of the first dinucleotide pppGpA, as previously described by Imbert et al . ( 12 ), as readout. Nsp8 template binding mutant K58A was used as negative control. The AMP contaminant present in the used [α- 32 P]ATP label is marked as loading control and size reference. ( D ) Side-by-side comparison of the de novo initiation activities of nps8, His-nsp8 and nsp7-8-His. ( E ) Elution profile of the nsp7-8-His fusion protein relative to nsp8-His. ( F ) Primer-extension activities of putative cleavage intermediate nsp7-8 on the U 20 template (see Figure 4 and Table 1 ).
    Figure Legend Snippet: Influence of His 6 -tags and nsp7 on the RdRp activity of SARS-CoV nsp8. ( A ) The UC 10 template (see Figure 4 ) was incubated with 1 µM of wild-type nsp8 or either of three nsp8 variants to investigate the influence of the His 6 -tag. Samples were taken at the indicated time points and checked for [α- 32 P]AMP incorporation by 20% PAGE/7 M urea analysis. ( B ) Side-by-side comparison of the primer-extension activities of nps8 and nsp(7+8). Shorter incubations are shown to better demonstrate the difference in activity. ( C ) De novo activity of nsp8 and nsp(7+8) on template AFMB131 (see Table 1 ), using the synthesis of the first dinucleotide pppGpA, as previously described by Imbert et al . ( 12 ), as readout. Nsp8 template binding mutant K58A was used as negative control. The AMP contaminant present in the used [α- 32 P]ATP label is marked as loading control and size reference. ( D ) Side-by-side comparison of the de novo initiation activities of nps8, His-nsp8 and nsp7-8-His. ( E ) Elution profile of the nsp7-8-His fusion protein relative to nsp8-His. ( F ) Primer-extension activities of putative cleavage intermediate nsp7-8 on the U 20 template (see Figure 4 and Table 1 ).

    Techniques Used: Activity Assay, Incubation, Polyacrylamide Gel Electrophoresis, Binding Assay, Mutagenesis, Negative Control

    2) Product Images from "The RNA polymerase activity of SARS-coronavirus nsp12 is primer dependent"

    Article Title: The RNA polymerase activity of SARS-coronavirus nsp12 is primer dependent

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp904

    Expression and purification of the SARS-CoV nsp12. ( A ) Expression of nsp12 was performed in the presence of the ubiquitin (Ub) protease Ubp1, which removed the N-terminal Ub fusion partner by cleaving after the LRGG signature that formed the junction between the Ub and nsp12 moieties. This in vivo cleavage created the native nsp12 amino-terminus with the sequence SADAS. ( B ) Western blotting using an nsp12-specific polyclonal antiserum identified a single band of the expected mass after purification of the in vivo cleaved expression product. Isolation of fusion proteins with different N-terminal tags resulted in common patterns of degradation. Similar results were reported by Cheng et al. ( 24 ) for a GST-nsp12 fusion protein. ( C ) Purified nsp12-CHis 6 was readily visualized by Coomassie staining as a ∼106-kDa protein product, in line with the expected mass. Inductions in the absence of Ubp1 expression, however, resulted in a mixture of full-length fusion protein and degradation products, likely as a result of improper folding. No RdRp activity was observed for these preparations. ( D ) Gel filtration analysis of purified nsp12 in assay buffer showed a single peak, corresponding to a globular molecular weight of 140 ± 10 kDa, suggesting that only the monomeric form of nsp12 is present under the conditions used.
    Figure Legend Snippet: Expression and purification of the SARS-CoV nsp12. ( A ) Expression of nsp12 was performed in the presence of the ubiquitin (Ub) protease Ubp1, which removed the N-terminal Ub fusion partner by cleaving after the LRGG signature that formed the junction between the Ub and nsp12 moieties. This in vivo cleavage created the native nsp12 amino-terminus with the sequence SADAS. ( B ) Western blotting using an nsp12-specific polyclonal antiserum identified a single band of the expected mass after purification of the in vivo cleaved expression product. Isolation of fusion proteins with different N-terminal tags resulted in common patterns of degradation. Similar results were reported by Cheng et al. ( 24 ) for a GST-nsp12 fusion protein. ( C ) Purified nsp12-CHis 6 was readily visualized by Coomassie staining as a ∼106-kDa protein product, in line with the expected mass. Inductions in the absence of Ubp1 expression, however, resulted in a mixture of full-length fusion protein and degradation products, likely as a result of improper folding. No RdRp activity was observed for these preparations. ( D ) Gel filtration analysis of purified nsp12 in assay buffer showed a single peak, corresponding to a globular molecular weight of 140 ± 10 kDa, suggesting that only the monomeric form of nsp12 is present under the conditions used.

    Techniques Used: Expressing, Purification, In Vivo, Sequencing, Western Blot, Isolation, Staining, Activity Assay, Filtration, Molecular Weight

    Nucleotide incorporation fidelity of nsp12. ( A ) Experimental set-up of pulse-chase experiments with different nucleotides and a primed poly(U) template (see Figure 2 A). The reactions were initiated with a limiting concentration of [α- 32 P]ATP to allow the incorporation of a first nucleotide and the formation of a stable polymerase-template complex. Subsequently, after 10 min, different unlabeled nucleotides were added to a final concentration of 50 μM to allow elongation for another 30 min. ( B ) SARS-CoV nsp12 allows only limited transversional and transitional misincorporations. Interestingly, also in the presence of dATP no significant activity was observed, implying that the SARS-CoV RdRp is capable of discriminating between ATP and dATP. ( C ) Pulse-chase experiments in the presence of 6 mM Mn 2+ show that manganese ions promote misincorporation of ribonucleotides (both transversions and transitions). The selection against dATP remained unaltered.
    Figure Legend Snippet: Nucleotide incorporation fidelity of nsp12. ( A ) Experimental set-up of pulse-chase experiments with different nucleotides and a primed poly(U) template (see Figure 2 A). The reactions were initiated with a limiting concentration of [α- 32 P]ATP to allow the incorporation of a first nucleotide and the formation of a stable polymerase-template complex. Subsequently, after 10 min, different unlabeled nucleotides were added to a final concentration of 50 μM to allow elongation for another 30 min. ( B ) SARS-CoV nsp12 allows only limited transversional and transitional misincorporations. Interestingly, also in the presence of dATP no significant activity was observed, implying that the SARS-CoV RdRp is capable of discriminating between ATP and dATP. ( C ) Pulse-chase experiments in the presence of 6 mM Mn 2+ show that manganese ions promote misincorporation of ribonucleotides (both transversions and transitions). The selection against dATP remained unaltered.

    Techniques Used: Pulse Chase, Concentration Assay, Activity Assay, Selection

    RNA binding affinity of nsp12. ( A ) A fixed concentration (0.2 nM) of radiolabeled dsRNA or ssRNA (data not shown) was titrated with purified nsp12 and the complexes formed were separated from unbound RNA on a native 8% polyacrylamide gel. ( B ) Free and bound RNA were quantified and fit to the Hill equation (see ‘Materials and Methods’ section), resulting in K d values of 0.13 ± 0.3 μM for dsRNA and 0.10 ± 0.2 μM for ssRNA binding. R 2 values of these Hill fits were 0.97 and 0.98 for dsRNA and ssRNA, respectively. Error bars represent standard error of the mean ( n = 3).
    Figure Legend Snippet: RNA binding affinity of nsp12. ( A ) A fixed concentration (0.2 nM) of radiolabeled dsRNA or ssRNA (data not shown) was titrated with purified nsp12 and the complexes formed were separated from unbound RNA on a native 8% polyacrylamide gel. ( B ) Free and bound RNA were quantified and fit to the Hill equation (see ‘Materials and Methods’ section), resulting in K d values of 0.13 ± 0.3 μM for dsRNA and 0.10 ± 0.2 μM for ssRNA binding. R 2 values of these Hill fits were 0.97 and 0.98 for dsRNA and ssRNA, respectively. Error bars represent standard error of the mean ( n = 3).

    Techniques Used: RNA Binding Assay, Concentration Assay, Purification, Binding Assay

    Biochemical determinants of SARS-CoV nsp12 activity. ( A and B ) Tests to determine the influence of temperature indicated that nsp12 incorporates ATP most efficiently at 37°C, although this effect mainly stemmed from increased initiation. Based on these results, 30°C was taken as the standard for subsequent experiments. ( C and D ) Titration of the Mg 2+ concentration showed that activity reaches its maximum at 6 mM. ( E and F ) The effect of pH on RdRp activity was evident as well, with lower pHs limiting the activity of the enzyme. All reactions were incubated for 60 min at 30°C, unless otherwise indicated. Error bars in A, C and E represent standard error of the mean ( n = 3).
    Figure Legend Snippet: Biochemical determinants of SARS-CoV nsp12 activity. ( A and B ) Tests to determine the influence of temperature indicated that nsp12 incorporates ATP most efficiently at 37°C, although this effect mainly stemmed from increased initiation. Based on these results, 30°C was taken as the standard for subsequent experiments. ( C and D ) Titration of the Mg 2+ concentration showed that activity reaches its maximum at 6 mM. ( E and F ) The effect of pH on RdRp activity was evident as well, with lower pHs limiting the activity of the enzyme. All reactions were incubated for 60 min at 30°C, unless otherwise indicated. Error bars in A, C and E represent standard error of the mean ( n = 3).

    Techniques Used: Activity Assay, Titration, Concentration Assay, Incubation

    SARS-CoV nsp12 primer extension assay and active-site mutant. ( A ) Schematic showing the structure of the partially double-stranded RNA template with 3′ U 10 stretch that served as template for primer extension in the initial nsp12-based nucleotide incorporation assay. ( B ) Comparison of the RdRp activity of wild-type nsp12 and a D618A active-site mutant, which displayed minimal activity after a 60-min incubation at 30°C. ( C ) A primer extension assay using a 5′ 32 P-labeled primer and ATP confirmed that wild-type nsp12, in contrast to the D618A mutant, was able to elongate the 20-mer primer. The slight reduction of input primer in the D618A lane probably resulted from degradation by RNase activity. The D618A mutant showed 8 ± 3% residual activity (mean of three independent experiments). In the loading control ( Figure 2 B and C, lower panels) the two nsp12 variants were visualized by silver staining of an SDS–PAGE gel.
    Figure Legend Snippet: SARS-CoV nsp12 primer extension assay and active-site mutant. ( A ) Schematic showing the structure of the partially double-stranded RNA template with 3′ U 10 stretch that served as template for primer extension in the initial nsp12-based nucleotide incorporation assay. ( B ) Comparison of the RdRp activity of wild-type nsp12 and a D618A active-site mutant, which displayed minimal activity after a 60-min incubation at 30°C. ( C ) A primer extension assay using a 5′ 32 P-labeled primer and ATP confirmed that wild-type nsp12, in contrast to the D618A mutant, was able to elongate the 20-mer primer. The slight reduction of input primer in the D618A lane probably resulted from degradation by RNase activity. The D618A mutant showed 8 ± 3% residual activity (mean of three independent experiments). In the loading control ( Figure 2 B and C, lower panels) the two nsp12 variants were visualized by silver staining of an SDS–PAGE gel.

    Techniques Used: Primer Extension Assay, Mutagenesis, Activity Assay, Incubation, Labeling, Silver Staining, SDS Page

    Analysis of the nucleotide incorporation rate of nsp12. Steady-state time courses were performed with 0.1 μM nsp12 as described in Figure 6 . Experimental data were subsequently fit to linear regression to obtain NMP incorporation rates. These clearly illustrated an increase of the incorporation velocity with template length ( V U10 = 1.2 ± 0.4 nM/min and V U20 = 27 ± 3 nM/min) and from homopolymeric template to copolymeric template ( V (CU)10 = 45 ± 2 nM/min). R 2 values were 0.97, 0.99 and 0.99, respectively, and error bars indicate standard error of the mean ( n = 3).
    Figure Legend Snippet: Analysis of the nucleotide incorporation rate of nsp12. Steady-state time courses were performed with 0.1 μM nsp12 as described in Figure 6 . Experimental data were subsequently fit to linear regression to obtain NMP incorporation rates. These clearly illustrated an increase of the incorporation velocity with template length ( V U10 = 1.2 ± 0.4 nM/min and V U20 = 27 ± 3 nM/min) and from homopolymeric template to copolymeric template ( V (CU)10 = 45 ± 2 nM/min). R 2 values were 0.97, 0.99 and 0.99, respectively, and error bars indicate standard error of the mean ( n = 3).

    Techniques Used:

    Analysis of nucleotide incorporation by nsp12. ( A ) Reactions with 0.1 μM nsp12, 1 μM template and 50 μM ATP were followed over time and quenched with 50 mM EDTA at the time points indicated. Gel analysis showed that, similar to the curves in Figure 3 , the major accumulating product is the primer (p) extended by 7 nt (i.e. p + 7) and not the expected full-length product of n + 10. ( B ) To assess whether nsp12 was capable of DNA-templated incorporation of dATP, reactions were performed as in Figure 6 A, but now using a DNA template. The time course illustrates robust [α- 32 P]dAMP incorporation, implying that the SARS-CoV RdRp is able to utilize both RNA and DNA templates. The incorporation of dAMP was 20% lower than that of AMP on an RNA template. ( C ) Time courses were additionally performed using an RNA template containing a U 20 template region. Similar to the shorter 10-nt template used in Figure 6 A, the major product here was around 2 nt shorter than the expected full-length product. ( D ) Changing the single-stranded template from U 20 to a heteromeric (CU) 10 not only increased the nucleotide incorporation rate by 34%, but also the behavior of nsp12 on the template. Products of the expected full-length size (p + 20) were observed, but also products longer than the template, hinting at, e.g. template switching by nsp12. Future research will address this observation in more detail. In this reaction both 50 μM ATP and GTP were present. The gel image represents the top half of a 20% denaturing PAGE gel.
    Figure Legend Snippet: Analysis of nucleotide incorporation by nsp12. ( A ) Reactions with 0.1 μM nsp12, 1 μM template and 50 μM ATP were followed over time and quenched with 50 mM EDTA at the time points indicated. Gel analysis showed that, similar to the curves in Figure 3 , the major accumulating product is the primer (p) extended by 7 nt (i.e. p + 7) and not the expected full-length product of n + 10. ( B ) To assess whether nsp12 was capable of DNA-templated incorporation of dATP, reactions were performed as in Figure 6 A, but now using a DNA template. The time course illustrates robust [α- 32 P]dAMP incorporation, implying that the SARS-CoV RdRp is able to utilize both RNA and DNA templates. The incorporation of dAMP was 20% lower than that of AMP on an RNA template. ( C ) Time courses were additionally performed using an RNA template containing a U 20 template region. Similar to the shorter 10-nt template used in Figure 6 A, the major product here was around 2 nt shorter than the expected full-length product. ( D ) Changing the single-stranded template from U 20 to a heteromeric (CU) 10 not only increased the nucleotide incorporation rate by 34%, but also the behavior of nsp12 on the template. Products of the expected full-length size (p + 20) were observed, but also products longer than the template, hinting at, e.g. template switching by nsp12. Future research will address this observation in more detail. In this reaction both 50 μM ATP and GTP were present. The gel image represents the top half of a 20% denaturing PAGE gel.

    Techniques Used: Polyacrylamide Gel Electrophoresis

    3) Product Images from "Lamina-associated Polypeptide-1 Interacts with the Muscular Dystrophy Protein Emerin and is Essential for Skeletal Muscle Maintenance"

    Article Title: Lamina-associated Polypeptide-1 Interacts with the Muscular Dystrophy Protein Emerin and is Essential for Skeletal Muscle Maintenance

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2013.08.012

    LAP1 and Emerin Co-immunoprecipitate from Cell Extracts (A) Endogenous emerin was co-immunopreciptated with overexpressed V5-tagged LAP1 in HEK 293 cells. Protein extracts from un-induced (− DOX) or induced (+ DOX) cells were collected and immunoprecipitated (IP) with anti-V5 antibodies. Protein inputs (1% of total lysate) and precipitated products were separated by SDS-PAGE, transferred to nitrocellulose and probed with antibodies against V5, emerin, Man1, LAP2β and lamin A, the migrations of which are indicated. Migrations of molecular mass standards in KDa are indicated at the left of the blots. (B) Protein extracts from HEK 293 cells were incubated with anti-emerin (4G5), isotype matched anti-immunoglobulin G (IgG) and agarose beads with no antibody for co-immunoprecipitation. LAP1 co-immunoprecipitated with emerin but LAP2β did not as indicated in the immunoblot. Double band of emerin may be due to differential phosphorylation. Bands corresponding to IgG heavy and light chains are indicated and migrations of molecular mass standards in KDa are indicated at the left of the blots. (C) In vitro pull-down assay showed that LAP1 binds to the nucleoplasmic domain of emerin. Immunoblot shows the input and GST-LAP1 and V5-tagged LAP1 purified from HEK 293 cells fused to agarose beads pulled down purified MBP-emerin fusion protein but not purified MBP. Analysis of bacterially expressed MBP-emerin showed intact protein (*) and smaller fragments (bracket). Migrations of molecular mass standards in KDa are indicated at the left of the blots. (D) Schematic diagram of full-length human LAP1 (LAP1-FL), the nucleoplasmic domain plus transmembrane segment (LAP1-Nuc) and luminal domain plus transmembrane segment (LAP1-Lum) expressed in HEK 293 cells for the co-immunoprecipitation experiments shown in panel E. Red indicated V5 epitope and blue the transmembrane segment; amino acid residues 1, 330, 360 and 584 are indicated. The blue mesh indicates the relative position of the nuclear lamins and green rectangles the inner (INM) and outer (ONM) nuclear membranes [not to scale]. (E) Full-length LAP1 and the nucleoplasmic domain plus transmembrane segment bind emerin but the luminal domain plus the transmembrane segment does not. HEK 293 cells were transfected with vector only or plasmids expressing V5 tagged fusions of LAP1 as indicated. Protein extracts were collected and immunoprecipitated (IP) with anti-V5 antibodies. Protein inputs (1% of total lysate) and precipitated products were separated by SDS-PAGE, transferred to nitrocellulose and probed with antibodies against V5 or emerin as indicated. Migrations of molecular mass standards in KDa are indicated at the left of the blots. .
    Figure Legend Snippet: LAP1 and Emerin Co-immunoprecipitate from Cell Extracts (A) Endogenous emerin was co-immunopreciptated with overexpressed V5-tagged LAP1 in HEK 293 cells. Protein extracts from un-induced (− DOX) or induced (+ DOX) cells were collected and immunoprecipitated (IP) with anti-V5 antibodies. Protein inputs (1% of total lysate) and precipitated products were separated by SDS-PAGE, transferred to nitrocellulose and probed with antibodies against V5, emerin, Man1, LAP2β and lamin A, the migrations of which are indicated. Migrations of molecular mass standards in KDa are indicated at the left of the blots. (B) Protein extracts from HEK 293 cells were incubated with anti-emerin (4G5), isotype matched anti-immunoglobulin G (IgG) and agarose beads with no antibody for co-immunoprecipitation. LAP1 co-immunoprecipitated with emerin but LAP2β did not as indicated in the immunoblot. Double band of emerin may be due to differential phosphorylation. Bands corresponding to IgG heavy and light chains are indicated and migrations of molecular mass standards in KDa are indicated at the left of the blots. (C) In vitro pull-down assay showed that LAP1 binds to the nucleoplasmic domain of emerin. Immunoblot shows the input and GST-LAP1 and V5-tagged LAP1 purified from HEK 293 cells fused to agarose beads pulled down purified MBP-emerin fusion protein but not purified MBP. Analysis of bacterially expressed MBP-emerin showed intact protein (*) and smaller fragments (bracket). Migrations of molecular mass standards in KDa are indicated at the left of the blots. (D) Schematic diagram of full-length human LAP1 (LAP1-FL), the nucleoplasmic domain plus transmembrane segment (LAP1-Nuc) and luminal domain plus transmembrane segment (LAP1-Lum) expressed in HEK 293 cells for the co-immunoprecipitation experiments shown in panel E. Red indicated V5 epitope and blue the transmembrane segment; amino acid residues 1, 330, 360 and 584 are indicated. The blue mesh indicates the relative position of the nuclear lamins and green rectangles the inner (INM) and outer (ONM) nuclear membranes [not to scale]. (E) Full-length LAP1 and the nucleoplasmic domain plus transmembrane segment bind emerin but the luminal domain plus the transmembrane segment does not. HEK 293 cells were transfected with vector only or plasmids expressing V5 tagged fusions of LAP1 as indicated. Protein extracts were collected and immunoprecipitated (IP) with anti-V5 antibodies. Protein inputs (1% of total lysate) and precipitated products were separated by SDS-PAGE, transferred to nitrocellulose and probed with antibodies against V5 or emerin as indicated. Migrations of molecular mass standards in KDa are indicated at the left of the blots. .

    Techniques Used: Immunoprecipitation, SDS Page, Incubation, In Vitro, Pull Down Assay, Purification, Transfection, Plasmid Preparation, Expressing

    4) Product Images from "A novel conjugal donor strain for improved DNA transfer into Clostridium spp."

    Article Title: A novel conjugal donor strain for improved DNA transfer into Clostridium spp.

    Journal: Anaerobe

    doi: 10.1016/j.anaerobe.2019.06.020

    Determination of conjugation efficiency into C. difficile R20291 using the Kirk et al. protocol. E. coli donors CA434 or Sexpress were used to transfer plasmid pMTL84151 into C. difficile R20291 following 8 or 24 h (h) incubation on mating plates, with (HT) or without (N) prior heat-treatment of recipient R20291 cells. This experiment utilised the Kirk et al. [ 31 ]method of conjugative transfer. Briefly, C. difficile R20291 recipient cells were grown overnight in TY broth, from which 200 μl aliquots were taken and either heat-treated at 52 °C for 5 min or not, before being used to resuspend 1 ml of E. coli donor cell pellets. The resulting suspension was spotted onto antibiotic-free Brain Heart Infusion (BHI) agar and incubated anaerobically for 8 or 24 h. Growth on mating plates was then harvested and diluted in TY broth and plated onto BHI agar plates supplemented with d -cycloserine, cefoxitin and thiamphenicol where appropriate. Conjugation efficiency was calculated as the ratio of thiamphenicol resistant CFU to total C. difficile CFU. Bars represent the means of three separate conjugations with error bars representing standard deviation. The two donor strains were compared for each condition and statistical significance was determined using multiple unpaired t-tests, with one asterisk denoting a (p
    Figure Legend Snippet: Determination of conjugation efficiency into C. difficile R20291 using the Kirk et al. protocol. E. coli donors CA434 or Sexpress were used to transfer plasmid pMTL84151 into C. difficile R20291 following 8 or 24 h (h) incubation on mating plates, with (HT) or without (N) prior heat-treatment of recipient R20291 cells. This experiment utilised the Kirk et al. [ 31 ]method of conjugative transfer. Briefly, C. difficile R20291 recipient cells were grown overnight in TY broth, from which 200 μl aliquots were taken and either heat-treated at 52 °C for 5 min or not, before being used to resuspend 1 ml of E. coli donor cell pellets. The resulting suspension was spotted onto antibiotic-free Brain Heart Infusion (BHI) agar and incubated anaerobically for 8 or 24 h. Growth on mating plates was then harvested and diluted in TY broth and plated onto BHI agar plates supplemented with d -cycloserine, cefoxitin and thiamphenicol where appropriate. Conjugation efficiency was calculated as the ratio of thiamphenicol resistant CFU to total C. difficile CFU. Bars represent the means of three separate conjugations with error bars representing standard deviation. The two donor strains were compared for each condition and statistical significance was determined using multiple unpaired t-tests, with one asterisk denoting a (p

    Techniques Used: Conjugation Assay, Plasmid Preparation, Incubation, Standard Deviation

    Comparative efficiencies of conjugative plasmid transfer of various vectors from the E. coli donors CA434 and sExpress into the recipient strain C. difficile R20291. The indicated plasmids, along with a control pMTL80000 plasmid lacking a functional Gram-positive replicon, were transferred from either donor using the conjugation method outlined in the materials and methods section. Conjugation efficiency was calculated as the proportion of putative transconjugant thiamphenicol resistant colonies divided by the CFU of recipient cells. Bars represent the means of three separate conjugations with error bars representing standard deviation. Statistical significance was determined using multiple unpaired t-tests, with one asterisk denoting a (p
    Figure Legend Snippet: Comparative efficiencies of conjugative plasmid transfer of various vectors from the E. coli donors CA434 and sExpress into the recipient strain C. difficile R20291. The indicated plasmids, along with a control pMTL80000 plasmid lacking a functional Gram-positive replicon, were transferred from either donor using the conjugation method outlined in the materials and methods section. Conjugation efficiency was calculated as the proportion of putative transconjugant thiamphenicol resistant colonies divided by the CFU of recipient cells. Bars represent the means of three separate conjugations with error bars representing standard deviation. Statistical significance was determined using multiple unpaired t-tests, with one asterisk denoting a (p

    Techniques Used: Plasmid Preparation, Functional Assay, Conjugation Assay, Standard Deviation

    A comparison of conjugation efficiency between two donor strains (CA434 and sExpress) into two different C. sporogenes NCIMB 10696 recipient strains. C. sporogenes ΔCLSPO_c06750 is a knockout strain for a putative Type IV restriction system. Three recipient strain cultures were grown of both wild type and ΔCLSPO_c06750 and each of these strains was used in three separate conjugations from separate donor strain cultures. This gave a total of nine conjugations for each donor/recipient combination. Conjugation efficiency was calculated as the ratio of CFU on transconjugant selective plates to the CFU of donor cells going into each conjugation. There was a significant difference in conjugation efficiency between CA434 and sExpress in wild type C. sporogenes (p = 0.0003 ***, n = 9) and between the wild type and knockout strain when using CA434 (p = 0.0008 ***, n = 9) but not when using sExpress. There was no statistically significance difference in conjugation efficiency between CA434 and sExpress in the ΔCLSPO_c06750 strain (p = 0.0684, n = 9). Two-tailed T-tests using Welch's correction were used in each comparison.
    Figure Legend Snippet: A comparison of conjugation efficiency between two donor strains (CA434 and sExpress) into two different C. sporogenes NCIMB 10696 recipient strains. C. sporogenes ΔCLSPO_c06750 is a knockout strain for a putative Type IV restriction system. Three recipient strain cultures were grown of both wild type and ΔCLSPO_c06750 and each of these strains was used in three separate conjugations from separate donor strain cultures. This gave a total of nine conjugations for each donor/recipient combination. Conjugation efficiency was calculated as the ratio of CFU on transconjugant selective plates to the CFU of donor cells going into each conjugation. There was a significant difference in conjugation efficiency between CA434 and sExpress in wild type C. sporogenes (p = 0.0003 ***, n = 9) and between the wild type and knockout strain when using CA434 (p = 0.0008 ***, n = 9) but not when using sExpress. There was no statistically significance difference in conjugation efficiency between CA434 and sExpress in the ΔCLSPO_c06750 strain (p = 0.0684, n = 9). Two-tailed T-tests using Welch's correction were used in each comparison.

    Techniques Used: Conjugation Assay, Knock-Out, Two Tailed Test

    A comparison of conjugation efficiency using either the ΔCLAU_0514 strain of C. autoethanogenum DSM 10061 (IIG deficient) or the wild type strain as the recipient. sExpress was used as a conjugal donor in either case with the only difference in conjugation protocol being the choice of recipient strain. Error bars show the standard deviation. An unpaired two-tailed T-test was performed with Welch's correction to measure the significance of the difference between the two recipient strains giving a P value of
    Figure Legend Snippet: A comparison of conjugation efficiency using either the ΔCLAU_0514 strain of C. autoethanogenum DSM 10061 (IIG deficient) or the wild type strain as the recipient. sExpress was used as a conjugal donor in either case with the only difference in conjugation protocol being the choice of recipient strain. Error bars show the standard deviation. An unpaired two-tailed T-test was performed with Welch's correction to measure the significance of the difference between the two recipient strains giving a P value of

    Techniques Used: Conjugation Assay, Standard Deviation, Two Tailed Test

    5) Product Images from "Transcription factor YcjW controls the emergency H2S production in E. coli"

    Article Title: Transcription factor YcjW controls the emergency H2S production in E. coli

    Journal: Nature Communications

    doi: 10.1038/s41467-019-10785-x

    E. coli MG1655 lacking 3MSTA acquires phenotypic suppression and has increased H 2 S levels and tolerance to Gm and H 2 O 2 . a Δ mstA-sup has increased survival rate compared with Δ mstA when treated with 2 µg ml −1 gentamicin in a time-kill assay. Values correspond to colony-forming units (c.f.u). b Δ mstA-sup also has increased tolerance after exposure to 5 mM H 2 O 2 for 30 min. c H 2 S production as measured with fluorescent probe, WSP5. Relative fluorescent units are normalized to OD 600 and minus the background fluorescent of PBS buffer + 100 µM L-cysteine and WSP5. H 2 S reacts with lead acetate, leading to staining of strips (Sigma-Aldrich). Values are means ± SD ( n = 3). * p
    Figure Legend Snippet: E. coli MG1655 lacking 3MSTA acquires phenotypic suppression and has increased H 2 S levels and tolerance to Gm and H 2 O 2 . a Δ mstA-sup has increased survival rate compared with Δ mstA when treated with 2 µg ml −1 gentamicin in a time-kill assay. Values correspond to colony-forming units (c.f.u). b Δ mstA-sup also has increased tolerance after exposure to 5 mM H 2 O 2 for 30 min. c H 2 S production as measured with fluorescent probe, WSP5. Relative fluorescent units are normalized to OD 600 and minus the background fluorescent of PBS buffer + 100 µM L-cysteine and WSP5. H 2 S reacts with lead acetate, leading to staining of strips (Sigma-Aldrich). Values are means ± SD ( n = 3). * p

    Techniques Used: Time-Kill Assay, Staining

    6) Product Images from "In vivo photoacoustic imaging of a nonfluorescent E2 crimson genetic reporter in mammalian tissues"

    Article Title: In vivo photoacoustic imaging of a nonfluorescent E2 crimson genetic reporter in mammalian tissues

    Journal: Journal of Biomedical Optics

    doi: 10.1117/1.JBO.25.4.046004

    In vitro characterization of human colorectal tumor cells LS174T, expressing E2 crimson FP or NFA mutant. (a) Photograph of cell pellets after transduction. The contrast between the visual appearance of E2 crimson-expressing cells and the NFA mutant cells is similar to the purified proteins shown in Fig. 2(a) . (b) Normalized PA signal amplitude from cells under prolonged exposure to nanosecond laser pulses. The excitation wavelength was 610 nm for the FP and 585 nm for NFA, corresponding to the peak absorption wavelengths of each protein. The incident fluence on the cells was 2.67 and 2.93 mJ cm − 2 respectively. There is no reduction in the PA signal amplitude, indicating high photostability. (c) PA amplitude spectra of cells compared to the PA spectra of the purified protein for (c) E2 crimson FP and (d) NFA mutant. The NFA mutant shows better agreement between the cells and the purified protein. The PA signal amplitude from the NFA cells is also greater than the FP.
    Figure Legend Snippet: In vitro characterization of human colorectal tumor cells LS174T, expressing E2 crimson FP or NFA mutant. (a) Photograph of cell pellets after transduction. The contrast between the visual appearance of E2 crimson-expressing cells and the NFA mutant cells is similar to the purified proteins shown in Fig. 2(a) . (b) Normalized PA signal amplitude from cells under prolonged exposure to nanosecond laser pulses. The excitation wavelength was 610 nm for the FP and 585 nm for NFA, corresponding to the peak absorption wavelengths of each protein. The incident fluence on the cells was 2.67 and 2.93 mJ cm − 2 respectively. There is no reduction in the PA signal amplitude, indicating high photostability. (c) PA amplitude spectra of cells compared to the PA spectra of the purified protein for (c) E2 crimson FP and (d) NFA mutant. The NFA mutant shows better agreement between the cells and the purified protein. The PA signal amplitude from the NFA cells is also greater than the FP.

    Techniques Used: In Vitro, Expressing, Mutagenesis, Transduction, Purification

    In vitro characterization of 1 mg mL − 1 solutions of purified proteins. (a) Photograph of purified proteins; the light blue color of the purified E2 crimson FP solution is in contrast to the light purple color of the NF mutants. (b) Fluorescence emission spectrum; the fluorescence of the NF mutants is at least 2 orders of magnitude lower than E2 crimson FP. (c) Absorbance spectrum; the absorbance peak of the NF mutants exhibits a small blue shift (585 nm) relative to E2 crimson FP (610 nm). (d) PA signal amplitude spectrum; all the NF mutants give higher PA signal amplitude than E2 crimson FP. The PA signal amplitude of mutant NFA is approximately three times higher than E2 crimson FP. (e) PA signal amplitude normalized by the peak absorption; the absorption-normalized PA signals from the NF mutants are approximately two to three times greater than the FP.
    Figure Legend Snippet: In vitro characterization of 1 mg mL − 1 solutions of purified proteins. (a) Photograph of purified proteins; the light blue color of the purified E2 crimson FP solution is in contrast to the light purple color of the NF mutants. (b) Fluorescence emission spectrum; the fluorescence of the NF mutants is at least 2 orders of magnitude lower than E2 crimson FP. (c) Absorbance spectrum; the absorbance peak of the NF mutants exhibits a small blue shift (585 nm) relative to E2 crimson FP (610 nm). (d) PA signal amplitude spectrum; all the NF mutants give higher PA signal amplitude than E2 crimson FP. The PA signal amplitude of mutant NFA is approximately three times higher than E2 crimson FP. (e) PA signal amplitude normalized by the peak absorption; the absorption-normalized PA signals from the NF mutants are approximately two to three times greater than the FP.

    Techniques Used: In Vitro, Purification, Fluorescence, Mutagenesis

    In vivo PA images of the flank of nude mice after subcutaneous injection of LS174T human colorectal tumor cells expressing E2 crimson FP or NFA mutant. The excitation wavelength was 600 nm. The images are horizontal x – y MIPs (a) MIP for z = 0 to 5 mm after injection of E2 crimson-expressing cells. The dashed rectangle shows the region, where the cells are located (b) MIP for z = 0.5 to 1.5 mm of the rectangular region from (a). The oval shape represents the boundary of where the cells are located. The E2 crimson-expressing cells are not visible. (c) MIP for z = 0 to 5 mm after injection of cells expressing the NFA mutant. The dashed rectangle shows the region, where the cells are located (d) MIP for z = 0.5 to 1.5 mm of the rectangular region from (c). The oval shape represents the boundary of the location of the NFA-expressing cells. The latter can be visualized within this region due to the generation of higher PA signals compared to the E2 crimson-expressing cells.
    Figure Legend Snippet: In vivo PA images of the flank of nude mice after subcutaneous injection of LS174T human colorectal tumor cells expressing E2 crimson FP or NFA mutant. The excitation wavelength was 600 nm. The images are horizontal x – y MIPs (a) MIP for z = 0 to 5 mm after injection of E2 crimson-expressing cells. The dashed rectangle shows the region, where the cells are located (b) MIP for z = 0.5 to 1.5 mm of the rectangular region from (a). The oval shape represents the boundary of where the cells are located. The E2 crimson-expressing cells are not visible. (c) MIP for z = 0 to 5 mm after injection of cells expressing the NFA mutant. The dashed rectangle shows the region, where the cells are located (d) MIP for z = 0.5 to 1.5 mm of the rectangular region from (c). The oval shape represents the boundary of the location of the NFA-expressing cells. The latter can be visualized within this region due to the generation of higher PA signals compared to the E2 crimson-expressing cells.

    Techniques Used: In Vivo, Mouse Assay, Injection, Expressing, Mutagenesis

    7) Product Images from "The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension"

    Article Title: The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr893

    The nsp(7+8) complex has primer extension activity. ( A ) Schematic presentation of the nsp8 primer extension assay, in which [α- 32 P]AMP and GMP are incorporated into a primed RNA template. In the figure, the initial RNA duplex is coloured red, the (UC) 10 template region blue and the newly incorporated nucleotides grey. ( B ) Incorporation of [α- 32 P]AMP by the nsp(7+8) complex. Samples were taken at the indicated time points and resolved on a 20% PAGE/7 M urea gel. ( C ) Schematic presentation of the single-cycle reaction. Template and nsp(7+8) complex were pre-incubated for 10 min before nucleotides were added. The mixture was then rapidly split into equal aliquots that were immediately mixed with heparin to trap unbound or released enzyme. ( D ) Samples were taken after 60 min and resolved on 20% PAGE/7 M Urea.
    Figure Legend Snippet: The nsp(7+8) complex has primer extension activity. ( A ) Schematic presentation of the nsp8 primer extension assay, in which [α- 32 P]AMP and GMP are incorporated into a primed RNA template. In the figure, the initial RNA duplex is coloured red, the (UC) 10 template region blue and the newly incorporated nucleotides grey. ( B ) Incorporation of [α- 32 P]AMP by the nsp(7+8) complex. Samples were taken at the indicated time points and resolved on a 20% PAGE/7 M urea gel. ( C ) Schematic presentation of the single-cycle reaction. Template and nsp(7+8) complex were pre-incubated for 10 min before nucleotides were added. The mixture was then rapidly split into equal aliquots that were immediately mixed with heparin to trap unbound or released enzyme. ( D ) Samples were taken after 60 min and resolved on 20% PAGE/7 M Urea.

    Techniques Used: Activity Assay, Primer Extension Assay, Polyacrylamide Gel Electrophoresis, Incubation

    Purification and multimerization of recombinant SARS-CoV nsp7-8 precursor and different nsp8 variants. ( A ) Expression of nsp8 in the presence of the ubiquitin protease Ubp1 to liberate the natural N-terminal sequence (AIASEF), followed by purification and cleavage by recombinant SARS-CoV nsp5 main protease to remove the C-terminal His 6 -tag and its upstream GSSG linker. ( B ) Eighteen percent SDS-PAGE analysis of nsp5-treated, purified nsp8-His demonstrates near-complete release of the C-terminal His 6 -tag within 60 min. The maltose binding protein (MBP) was added to the reaction to serve as an independent loading control. Asterisks indicate non-specific bands. ( C ) In addition to the tag-less nps8 and nsp8-His, we also produced the N-terminally His 6 -tagged nsp8 (His-nsp8) used by Imbert et al . ( 12 ). ( D ) Comparative gel filtration analysis of nsp8 (22 kDa as a monomer) versus His-nsp8 and ( E ) nsp8 versus nsp8-His. In all three cases, nsp8 formed multimers in solution, but the apparent molecular mass of complexes formed by both nsp8 and nsp8-His was ∼2-fold higher than for complexes formed by His-nsp8. ( F ) Comparative analysis of nsp8, nsp(7+8), His-nsp8 and nsp7+nsp8-His. Only nsp(7+8) showed a molecular weight shift to the ∼225-kDa size range with a standard deviation of 15-kDa ( n = 3). This size is indicative of hexadecamer formation, whereas the analysis of nsp7+nsp8-His showed dominant peaks of nsp8-His and nsp7 (which is ∼10 kDa as a monomer).
    Figure Legend Snippet: Purification and multimerization of recombinant SARS-CoV nsp7-8 precursor and different nsp8 variants. ( A ) Expression of nsp8 in the presence of the ubiquitin protease Ubp1 to liberate the natural N-terminal sequence (AIASEF), followed by purification and cleavage by recombinant SARS-CoV nsp5 main protease to remove the C-terminal His 6 -tag and its upstream GSSG linker. ( B ) Eighteen percent SDS-PAGE analysis of nsp5-treated, purified nsp8-His demonstrates near-complete release of the C-terminal His 6 -tag within 60 min. The maltose binding protein (MBP) was added to the reaction to serve as an independent loading control. Asterisks indicate non-specific bands. ( C ) In addition to the tag-less nps8 and nsp8-His, we also produced the N-terminally His 6 -tagged nsp8 (His-nsp8) used by Imbert et al . ( 12 ). ( D ) Comparative gel filtration analysis of nsp8 (22 kDa as a monomer) versus His-nsp8 and ( E ) nsp8 versus nsp8-His. In all three cases, nsp8 formed multimers in solution, but the apparent molecular mass of complexes formed by both nsp8 and nsp8-His was ∼2-fold higher than for complexes formed by His-nsp8. ( F ) Comparative analysis of nsp8, nsp(7+8), His-nsp8 and nsp7+nsp8-His. Only nsp(7+8) showed a molecular weight shift to the ∼225-kDa size range with a standard deviation of 15-kDa ( n = 3). This size is indicative of hexadecamer formation, whereas the analysis of nsp7+nsp8-His showed dominant peaks of nsp8-His and nsp7 (which is ∼10 kDa as a monomer).

    Techniques Used: Purification, Recombinant, Expressing, Sequencing, SDS Page, Binding Assay, Produced, Filtration, Molecular Weight, Standard Deviation

    Influence of divalent ions and pH on nsp(7+8) activity. ( A ) To test the influence of magnesium ions on nsp(7+8) activity, we performed nsp(7+8) primer extension reactions at 0–10 mM Mg 2+ . ( B ) Quantification of the results presented in Figure 6 A, presented as the amount of NTP incorporated per μM nsp8 monomer. Error bars represent standard deviations ( n = 3). ( C ) The influence of the pH on nsp(7+8) activity was tested for a pH range of 6–11. A clear optimum was observed around 9.5. ( D ) Quantification of the results in Figure 6 C, presented as the amount of NTP incorporated per μM nsp8 monomer. Error bars indicate standard deviations ( n = 3). ( E ) Schematic presentation of the pulse-chase experiment that was used to test the nsp(7+8) nucleotide incorporation specificity on a primed poly(U) template (see Table 1 ). The reactions were initiated with a limiting concentration of [α- 32 P]ATP to allow the formation of a stable polymerase-template complex. Unlabelled nucleotides were used at a final concentration of 50 μM. ( F ) SARS-CoV nsp(7+8) allowed only limited transversional and transitional mutations. Use of manganese ions as cofactor for polymerase activity resulted in a minor, though noticeable loss of fidelity. Lane 1 represents the input signal to which no unlabelled nucleotides were added. Nucleoside triphosphates are abbreviated to single letters (i.e. A for ATP, G for GTP, U for UTP, C for CTP and R for RTP).
    Figure Legend Snippet: Influence of divalent ions and pH on nsp(7+8) activity. ( A ) To test the influence of magnesium ions on nsp(7+8) activity, we performed nsp(7+8) primer extension reactions at 0–10 mM Mg 2+ . ( B ) Quantification of the results presented in Figure 6 A, presented as the amount of NTP incorporated per μM nsp8 monomer. Error bars represent standard deviations ( n = 3). ( C ) The influence of the pH on nsp(7+8) activity was tested for a pH range of 6–11. A clear optimum was observed around 9.5. ( D ) Quantification of the results in Figure 6 C, presented as the amount of NTP incorporated per μM nsp8 monomer. Error bars indicate standard deviations ( n = 3). ( E ) Schematic presentation of the pulse-chase experiment that was used to test the nsp(7+8) nucleotide incorporation specificity on a primed poly(U) template (see Table 1 ). The reactions were initiated with a limiting concentration of [α- 32 P]ATP to allow the formation of a stable polymerase-template complex. Unlabelled nucleotides were used at a final concentration of 50 μM. ( F ) SARS-CoV nsp(7+8) allowed only limited transversional and transitional mutations. Use of manganese ions as cofactor for polymerase activity resulted in a minor, though noticeable loss of fidelity. Lane 1 represents the input signal to which no unlabelled nucleotides were added. Nucleoside triphosphates are abbreviated to single letters (i.e. A for ATP, G for GTP, U for UTP, C for CTP and R for RTP).

    Techniques Used: Activity Assay, Pulse Chase, Concentration Assay

    Mutagenesis of SARS-CoV nsp8. ( A ) Alignment of nsp8 sequences from representative alpha-, beta- and gammacoronaviruses. Fully conserved residues are shaded red, while partially conserved residues are boxed. The residues targeted by mutagenesis are indicated with asterisks. Please see ‘Material and Methods’ section for the Genbank accession numbers associated with the presented sequences. ( B ) To verify that the observed extension activity was nsp8-dependent, we tested the incorporation of AMP into the primed U 20 template by 1, 5 or 10 µM of wild-type nsp8 or template-binding mutant K58A. Mutation of K58 resulted in a ∼95% reduction of AMP incorporation. ( C ) To assess the importance of the two D/ExD/E motifs in nsp8, we engineered alanine substitution mutants of these residues and tested their primer extension activity on the primed UC 10 template (see Figure 4 ). Reactions were stopped after 60 min and compared to the activity of the wild-type nsp(7+8) complex on a 20% PAGE/7 M urea gel. The bottom panel shows the nsp8 protein concentration present in each of the reactions. ( D ) Quantification of the primer extension activities on the CU 10 template of the D/ExD/E alanine substitution mutants and control substitutions K58A and K127A. Values are normalized to the protein concentration. Error bars represent standard deviations ( n = 3).
    Figure Legend Snippet: Mutagenesis of SARS-CoV nsp8. ( A ) Alignment of nsp8 sequences from representative alpha-, beta- and gammacoronaviruses. Fully conserved residues are shaded red, while partially conserved residues are boxed. The residues targeted by mutagenesis are indicated with asterisks. Please see ‘Material and Methods’ section for the Genbank accession numbers associated with the presented sequences. ( B ) To verify that the observed extension activity was nsp8-dependent, we tested the incorporation of AMP into the primed U 20 template by 1, 5 or 10 µM of wild-type nsp8 or template-binding mutant K58A. Mutation of K58 resulted in a ∼95% reduction of AMP incorporation. ( C ) To assess the importance of the two D/ExD/E motifs in nsp8, we engineered alanine substitution mutants of these residues and tested their primer extension activity on the primed UC 10 template (see Figure 4 ). Reactions were stopped after 60 min and compared to the activity of the wild-type nsp(7+8) complex on a 20% PAGE/7 M urea gel. The bottom panel shows the nsp8 protein concentration present in each of the reactions. ( D ) Quantification of the primer extension activities on the CU 10 template of the D/ExD/E alanine substitution mutants and control substitutions K58A and K127A. Values are normalized to the protein concentration. Error bars represent standard deviations ( n = 3).

    Techniques Used: Mutagenesis, Activity Assay, Binding Assay, Polyacrylamide Gel Electrophoresis, Protein Concentration

    Influence of His 6 -tags and nsp7 on the RdRp activity of SARS-CoV nsp8. ( A ) The UC 10 template (see Figure 4 ) was incubated with 1 µM of wild-type nsp8 or either of three nsp8 variants to investigate the influence of the His 6 -tag. Samples were taken at the indicated time points and checked for [α- 32 P]AMP incorporation by 20% PAGE/7 M urea analysis. ( B ) Side-by-side comparison of the primer-extension activities of nps8 and nsp(7+8). Shorter incubations are shown to better demonstrate the difference in activity. ( C ) De novo activity of nsp8 and nsp(7+8) on template AFMB131 (see Table 1 ), using the synthesis of the first dinucleotide pppGpA, as previously described by Imbert et al . ( 12 ), as readout. Nsp8 template binding mutant K58A was used as negative control. The AMP contaminant present in the used [α- 32 P]ATP label is marked as loading control and size reference. ( D ) Side-by-side comparison of the de novo initiation activities of nps8, His-nsp8 and nsp7-8-His. ( E ) Elution profile of the nsp7-8-His fusion protein relative to nsp8-His. ( F ) Primer-extension activities of putative cleavage intermediate nsp7-8 on the U 20 template (see Figure 4 and Table 1 ).
    Figure Legend Snippet: Influence of His 6 -tags and nsp7 on the RdRp activity of SARS-CoV nsp8. ( A ) The UC 10 template (see Figure 4 ) was incubated with 1 µM of wild-type nsp8 or either of three nsp8 variants to investigate the influence of the His 6 -tag. Samples were taken at the indicated time points and checked for [α- 32 P]AMP incorporation by 20% PAGE/7 M urea analysis. ( B ) Side-by-side comparison of the primer-extension activities of nps8 and nsp(7+8). Shorter incubations are shown to better demonstrate the difference in activity. ( C ) De novo activity of nsp8 and nsp(7+8) on template AFMB131 (see Table 1 ), using the synthesis of the first dinucleotide pppGpA, as previously described by Imbert et al . ( 12 ), as readout. Nsp8 template binding mutant K58A was used as negative control. The AMP contaminant present in the used [α- 32 P]ATP label is marked as loading control and size reference. ( D ) Side-by-side comparison of the de novo initiation activities of nps8, His-nsp8 and nsp7-8-His. ( E ) Elution profile of the nsp7-8-His fusion protein relative to nsp8-His. ( F ) Primer-extension activities of putative cleavage intermediate nsp7-8 on the U 20 template (see Figure 4 and Table 1 ).

    Techniques Used: Activity Assay, Incubation, Polyacrylamide Gel Electrophoresis, Binding Assay, Mutagenesis, Negative Control

    SARS-CoV nsp7 stimulates nsp8-dependent RNA binding. ( A ) Five prime 32 P-labelled dsRNA was incubated with increasing concentrations (0–5 µM) of wild-type nsp8, ( B ) nsp8 K58A, ( C ) or nsp8 D52A. Clearly, mutation of K58 to alanine significantly reduced the binding affinity of nsp8, whereas mutation of D52 to alanine did not. We also noted that the change in charge due to the mutation (up to 8-fold in the octamer) resulted in an upward shift of the dominant RNP band, relative to the dominant RNP in panel 3 A (labelled with black 1). ( D ) Five prime 32 P-labelled duplex RNA was incubated with a fixed concentration of nsp7 (5 µM) and increasing concentrations of wild-type nsp8 (0-5 µM). Note the migration shift of the dominant ribonucleotide-protein (RNP) complex in the presence of nsp7 (compare RNPs labelled with black 1 and grey 2). ( E ) Addition of an equimolar amount of nsp7 to the nsp8 mutants D52A and K58A stimulated binding of dsRNA. For reference, the 2:1 and 1:1 ratios of wild-type nsp8 and nsp7 are shown in the left panel. Asterisks indicate non-specific bands. ( F ) RNA-binding curves for nsp8 in the absence (grey triangles) or presence of a fixed (5 µM) concentration of nsp7 (black circles). Lines represent fits to the Hill equation, while error bars represent standard deviations ( n = 3).
    Figure Legend Snippet: SARS-CoV nsp7 stimulates nsp8-dependent RNA binding. ( A ) Five prime 32 P-labelled dsRNA was incubated with increasing concentrations (0–5 µM) of wild-type nsp8, ( B ) nsp8 K58A, ( C ) or nsp8 D52A. Clearly, mutation of K58 to alanine significantly reduced the binding affinity of nsp8, whereas mutation of D52 to alanine did not. We also noted that the change in charge due to the mutation (up to 8-fold in the octamer) resulted in an upward shift of the dominant RNP band, relative to the dominant RNP in panel 3 A (labelled with black 1). ( D ) Five prime 32 P-labelled duplex RNA was incubated with a fixed concentration of nsp7 (5 µM) and increasing concentrations of wild-type nsp8 (0-5 µM). Note the migration shift of the dominant ribonucleotide-protein (RNP) complex in the presence of nsp7 (compare RNPs labelled with black 1 and grey 2). ( E ) Addition of an equimolar amount of nsp7 to the nsp8 mutants D52A and K58A stimulated binding of dsRNA. For reference, the 2:1 and 1:1 ratios of wild-type nsp8 and nsp7 are shown in the left panel. Asterisks indicate non-specific bands. ( F ) RNA-binding curves for nsp8 in the absence (grey triangles) or presence of a fixed (5 µM) concentration of nsp7 (black circles). Lines represent fits to the Hill equation, while error bars represent standard deviations ( n = 3).

    Techniques Used: RNA Binding Assay, Incubation, Mutagenesis, Binding Assay, Concentration Assay, Migration

    SARS-coronavirus genome organization and structure of the nsp7+nsp8 hexadecamer. ( A ) The coronavirus genome contains two large 5′-proximal ORFs (ORF1a and 1 b) that encode the two replicase polyproteins, whose mature products assemble into the viral replication and transcription complex. Both polyproteins are cleaved (cleavage sites indicated with arrow heads) by the proteinase activities of nsp3 (black lines) and nsp5 (red lines), which releases the mature nsps. Also indicated are the 5′ cap structure and the 3′ polyA tail (A n ). ( B ) The SARS-CoV nsp8 crystal structure (pdb 2AHM) resembles a ‘golf club-like’ shape, as presented by the yellow ribbon structure. This nsp8 conformation connects to a much larger, hexadecameric structure that is composed of seven additional nsp8 subunits (grey) and eight nsp7 subunits (green). The hollow hexadecameric ring structure has a positively charged channel (blue background shading) that was proposed to mediate RNA binding. The outside of the structure is predominantly negatively charged (red background shading).
    Figure Legend Snippet: SARS-coronavirus genome organization and structure of the nsp7+nsp8 hexadecamer. ( A ) The coronavirus genome contains two large 5′-proximal ORFs (ORF1a and 1 b) that encode the two replicase polyproteins, whose mature products assemble into the viral replication and transcription complex. Both polyproteins are cleaved (cleavage sites indicated with arrow heads) by the proteinase activities of nsp3 (black lines) and nsp5 (red lines), which releases the mature nsps. Also indicated are the 5′ cap structure and the 3′ polyA tail (A n ). ( B ) The SARS-CoV nsp8 crystal structure (pdb 2AHM) resembles a ‘golf club-like’ shape, as presented by the yellow ribbon structure. This nsp8 conformation connects to a much larger, hexadecameric structure that is composed of seven additional nsp8 subunits (grey) and eight nsp7 subunits (green). The hollow hexadecameric ring structure has a positively charged channel (blue background shading) that was proposed to mediate RNA binding. The outside of the structure is predominantly negatively charged (red background shading).

    Techniques Used: RNA Binding Assay

    Related Articles

    Clone Assay:

    Article Title: Transcription factor YcjW controls the emergency H2S production in E. coli
    Article Snippet: .. To generate pLLY1, ycjW was PCR amplified from E. coli MG1655 using primers LL10 and LL11 and cloned into pACYC184 plasmid (NEB) using the Gibson Assembly Mastermix, according to the manufacturer’s protocol (NEB). .. Plasmid pLLSN3 was generated as above, except ycjW was PCR amplified from mstA-sup.

    Article Title: Hamster Melatonin Receptors: Cloning and Binding Characterization of MT1 and Attempt to Clone MT2
    Article Snippet: .. Eluted DNA was inserted into the blunt pJET vector using the CloneJET PCR Cloning Kit (Thermo Fisher Scientific) and transformed into DH10β chemically competent Escherichia coli cells (NEB). .. Forward and reverse sequencing reactions were performed with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA) using the same primers used for amplification or vector primers.

    Article Title: Lactobacillus fermentum 3872 as a potential tool for combatting Campylobacter jejuni infections
    Article Snippet: .. The PCR product was purified using the QIAquick PCR purification kit (Qiagen), digested with enzymes Xba I and Sph I (NEB) and cloned into expression vector pBAD33 using Quick Ligation kit (NEB) and E. coli Express competent cells (NEB). .. Sanger sequencing, conducted by GENEWIZ revealed no errors in the cloned fragment.

    Amplification:

    Article Title: Transcription factor YcjW controls the emergency H2S production in E. coli
    Article Snippet: .. To generate pLLY1, ycjW was PCR amplified from E. coli MG1655 using primers LL10 and LL11 and cloned into pACYC184 plasmid (NEB) using the Gibson Assembly Mastermix, according to the manufacturer’s protocol (NEB). .. Plasmid pLLSN3 was generated as above, except ycjW was PCR amplified from mstA-sup.

    Ligation:

    Article Title: Lactobacillus fermentum 3872 as a potential tool for combatting Campylobacter jejuni infections
    Article Snippet: .. The PCR product was purified using the QIAquick PCR purification kit (Qiagen), digested with enzymes Xba I and Sph I (NEB) and cloned into expression vector pBAD33 using Quick Ligation kit (NEB) and E. coli Express competent cells (NEB). .. Sanger sequencing, conducted by GENEWIZ revealed no errors in the cloned fragment.

    Cell Culture:

    Article Title: Novel structural arrangement of nematode cystathionine ?-synthases: characterization of Caenorhabditis elegans CBS-1
    Article Snippet: .. Express Competent Escherichia coli cells (New England Biolabs) were transformed with the plasmid that encodes double-tagged CBS-1 (GST–CBS–1–His6 ) and cultured in the presence of 100 μM IPTG (isopropyl β-D -thiogalactopyranoside) at 18°C for 24 h. The GST–CBS-1 fusion protein was purified according to the purification protocol for human CBS described previously [ ] with the following modifications: after cleavage by the PreScission protease (GE Healthcare), recombinant CBS-1 was loaded on to a Ni-Sepharose column that had been equilibrated with IMAC buffer [20 mM phosphate (pH 7.5), containing 0.5 M NaCl, 20 mM imidazole and 1 mM DTT (dithiothreitol)]. ..

    Purification:

    Article Title: Lactobacillus fermentum 3872 as a potential tool for combatting Campylobacter jejuni infections
    Article Snippet: .. The PCR product was purified using the QIAquick PCR purification kit (Qiagen), digested with enzymes Xba I and Sph I (NEB) and cloned into expression vector pBAD33 using Quick Ligation kit (NEB) and E. coli Express competent cells (NEB). .. Sanger sequencing, conducted by GENEWIZ revealed no errors in the cloned fragment.

    Article Title: Novel structural arrangement of nematode cystathionine ?-synthases: characterization of Caenorhabditis elegans CBS-1
    Article Snippet: .. Express Competent Escherichia coli cells (New England Biolabs) were transformed with the plasmid that encodes double-tagged CBS-1 (GST–CBS–1–His6 ) and cultured in the presence of 100 μM IPTG (isopropyl β-D -thiogalactopyranoside) at 18°C for 24 h. The GST–CBS-1 fusion protein was purified according to the purification protocol for human CBS described previously [ ] with the following modifications: after cleavage by the PreScission protease (GE Healthcare), recombinant CBS-1 was loaded on to a Ni-Sepharose column that had been equilibrated with IMAC buffer [20 mM phosphate (pH 7.5), containing 0.5 M NaCl, 20 mM imidazole and 1 mM DTT (dithiothreitol)]. ..

    Article Title: DNA supercoiling enables the Type IIS restriction enzyme BspMI to recognise the relative orientation of two DNA sequences
    Article Snippet: .. The BspMI endonuclease was purified from an over- producing strain of Escherichia coli (from R. Morgan, New England Biolabs) and its concentration (given in terms of its tetrameric form) determined as before ( ). ..

    Concentration Assay:

    Article Title: DNA supercoiling enables the Type IIS restriction enzyme BspMI to recognise the relative orientation of two DNA sequences
    Article Snippet: .. The BspMI endonuclease was purified from an over- producing strain of Escherichia coli (from R. Morgan, New England Biolabs) and its concentration (given in terms of its tetrameric form) determined as before ( ). ..

    Expressing:

    Article Title: Lactobacillus fermentum 3872 as a potential tool for combatting Campylobacter jejuni infections
    Article Snippet: .. The PCR product was purified using the QIAquick PCR purification kit (Qiagen), digested with enzymes Xba I and Sph I (NEB) and cloned into expression vector pBAD33 using Quick Ligation kit (NEB) and E. coli Express competent cells (NEB). .. Sanger sequencing, conducted by GENEWIZ revealed no errors in the cloned fragment.

    Polymerase Chain Reaction:

    Article Title: Transcription factor YcjW controls the emergency H2S production in E. coli
    Article Snippet: .. To generate pLLY1, ycjW was PCR amplified from E. coli MG1655 using primers LL10 and LL11 and cloned into pACYC184 plasmid (NEB) using the Gibson Assembly Mastermix, according to the manufacturer’s protocol (NEB). .. Plasmid pLLSN3 was generated as above, except ycjW was PCR amplified from mstA-sup.

    Article Title: Hamster Melatonin Receptors: Cloning and Binding Characterization of MT1 and Attempt to Clone MT2
    Article Snippet: .. Eluted DNA was inserted into the blunt pJET vector using the CloneJET PCR Cloning Kit (Thermo Fisher Scientific) and transformed into DH10β chemically competent Escherichia coli cells (NEB). .. Forward and reverse sequencing reactions were performed with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA) using the same primers used for amplification or vector primers.

    Article Title: Lactobacillus fermentum 3872 as a potential tool for combatting Campylobacter jejuni infections
    Article Snippet: .. The PCR product was purified using the QIAquick PCR purification kit (Qiagen), digested with enzymes Xba I and Sph I (NEB) and cloned into expression vector pBAD33 using Quick Ligation kit (NEB) and E. coli Express competent cells (NEB). .. Sanger sequencing, conducted by GENEWIZ revealed no errors in the cloned fragment.

    Transformation Assay:

    Article Title: Hamster Melatonin Receptors: Cloning and Binding Characterization of MT1 and Attempt to Clone MT2
    Article Snippet: .. Eluted DNA was inserted into the blunt pJET vector using the CloneJET PCR Cloning Kit (Thermo Fisher Scientific) and transformed into DH10β chemically competent Escherichia coli cells (NEB). .. Forward and reverse sequencing reactions were performed with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA) using the same primers used for amplification or vector primers.

    Article Title: Novel structural arrangement of nematode cystathionine ?-synthases: characterization of Caenorhabditis elegans CBS-1
    Article Snippet: .. Express Competent Escherichia coli cells (New England Biolabs) were transformed with the plasmid that encodes double-tagged CBS-1 (GST–CBS–1–His6 ) and cultured in the presence of 100 μM IPTG (isopropyl β-D -thiogalactopyranoside) at 18°C for 24 h. The GST–CBS-1 fusion protein was purified according to the purification protocol for human CBS described previously [ ] with the following modifications: after cleavage by the PreScission protease (GE Healthcare), recombinant CBS-1 was loaded on to a Ni-Sepharose column that had been equilibrated with IMAC buffer [20 mM phosphate (pH 7.5), containing 0.5 M NaCl, 20 mM imidazole and 1 mM DTT (dithiothreitol)]. ..

    Recombinant:

    Article Title: In Silico Design and Validation of OvMANE1, a Chimeric Antigen for Human Onchocerciasis Diagnosis
    Article Snippet: .. The recombinant OvMANE1_PMAL-c5X plasmid was used to transform NEB® Express Competent E. coli cells (New England Biolabs, Ipswich, MA, USA). .. Expression was induced using 0.3 mM Isopropyl β-d -1-thiogalactopyranoside at 37 °C for 2 h with shaking at 200 rpm and the protein was expressed as a fusion with MBP.

    Article Title: Novel structural arrangement of nematode cystathionine ?-synthases: characterization of Caenorhabditis elegans CBS-1
    Article Snippet: .. Express Competent Escherichia coli cells (New England Biolabs) were transformed with the plasmid that encodes double-tagged CBS-1 (GST–CBS–1–His6 ) and cultured in the presence of 100 μM IPTG (isopropyl β-D -thiogalactopyranoside) at 18°C for 24 h. The GST–CBS-1 fusion protein was purified according to the purification protocol for human CBS described previously [ ] with the following modifications: after cleavage by the PreScission protease (GE Healthcare), recombinant CBS-1 was loaded on to a Ni-Sepharose column that had been equilibrated with IMAC buffer [20 mM phosphate (pH 7.5), containing 0.5 M NaCl, 20 mM imidazole and 1 mM DTT (dithiothreitol)]. ..

    Plasmid Preparation:

    Article Title: Transcription factor YcjW controls the emergency H2S production in E. coli
    Article Snippet: .. To generate pLLY1, ycjW was PCR amplified from E. coli MG1655 using primers LL10 and LL11 and cloned into pACYC184 plasmid (NEB) using the Gibson Assembly Mastermix, according to the manufacturer’s protocol (NEB). .. Plasmid pLLSN3 was generated as above, except ycjW was PCR amplified from mstA-sup.

    Article Title: Signatures and mechanisms of efficacious therapeutic ribonucleotides against SARS-CoV-2 revealed by analysis of its replicase using magnetic tweezers
    Article Snippet: .. Cofactors were expressed in NEB Express C2523 (New England Biolabs) cells carrying the pRare2LacI (Novagen) plasmid in the presence of Ampicillin (100 μM/mL) and Chloramphenicol (17 μg/mL). .. Protein expression was induced with 100 μM IPTG once the OD600 = 0.5–0.6, and expressed overnight at 17°C.

    Article Title: Hamster Melatonin Receptors: Cloning and Binding Characterization of MT1 and Attempt to Clone MT2
    Article Snippet: .. Eluted DNA was inserted into the blunt pJET vector using the CloneJET PCR Cloning Kit (Thermo Fisher Scientific) and transformed into DH10β chemically competent Escherichia coli cells (NEB). .. Forward and reverse sequencing reactions were performed with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA) using the same primers used for amplification or vector primers.

    Article Title: In Silico Design and Validation of OvMANE1, a Chimeric Antigen for Human Onchocerciasis Diagnosis
    Article Snippet: .. The recombinant OvMANE1_PMAL-c5X plasmid was used to transform NEB® Express Competent E. coli cells (New England Biolabs, Ipswich, MA, USA). .. Expression was induced using 0.3 mM Isopropyl β-d -1-thiogalactopyranoside at 37 °C for 2 h with shaking at 200 rpm and the protein was expressed as a fusion with MBP.

    Article Title: Lactobacillus fermentum 3872 as a potential tool for combatting Campylobacter jejuni infections
    Article Snippet: .. The PCR product was purified using the QIAquick PCR purification kit (Qiagen), digested with enzymes Xba I and Sph I (NEB) and cloned into expression vector pBAD33 using Quick Ligation kit (NEB) and E. coli Express competent cells (NEB). .. Sanger sequencing, conducted by GENEWIZ revealed no errors in the cloned fragment.

    Article Title: Novel structural arrangement of nematode cystathionine ?-synthases: characterization of Caenorhabditis elegans CBS-1
    Article Snippet: .. Express Competent Escherichia coli cells (New England Biolabs) were transformed with the plasmid that encodes double-tagged CBS-1 (GST–CBS–1–His6 ) and cultured in the presence of 100 μM IPTG (isopropyl β-D -thiogalactopyranoside) at 18°C for 24 h. The GST–CBS-1 fusion protein was purified according to the purification protocol for human CBS described previously [ ] with the following modifications: after cleavage by the PreScission protease (GE Healthcare), recombinant CBS-1 was loaded on to a Ni-Sepharose column that had been equilibrated with IMAC buffer [20 mM phosphate (pH 7.5), containing 0.5 M NaCl, 20 mM imidazole and 1 mM DTT (dithiothreitol)]. ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs e coli mg1655
    E. coli <t>MG1655</t> lacking 3MSTA acquires phenotypic suppression and has increased H 2 S levels and tolerance to Gm and H 2 O 2 . a Δ mstA-sup has increased survival rate compared with Δ mstA when treated with 2 µg ml −1 gentamicin in a time-kill assay. Values correspond to colony-forming units (c.f.u). b Δ mstA-sup also has increased tolerance after exposure to 5 mM H 2 O 2 for 30 min. c H 2 S production as measured with fluorescent probe, WSP5. Relative fluorescent units are normalized to OD 600 and minus the background fluorescent of PBS buffer + 100 µM L-cysteine and WSP5. H 2 S reacts with lead acetate, leading to staining of strips (Sigma-Aldrich). Values are means ± SD ( n = 3). * p
    E Coli Mg1655, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli mg1655/product/New England Biolabs
    Average 99 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    e coli mg1655 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    E. coli MG1655 lacking 3MSTA acquires phenotypic suppression and has increased H 2 S levels and tolerance to Gm and H 2 O 2 . a Δ mstA-sup has increased survival rate compared with Δ mstA when treated with 2 µg ml −1 gentamicin in a time-kill assay. Values correspond to colony-forming units (c.f.u). b Δ mstA-sup also has increased tolerance after exposure to 5 mM H 2 O 2 for 30 min. c H 2 S production as measured with fluorescent probe, WSP5. Relative fluorescent units are normalized to OD 600 and minus the background fluorescent of PBS buffer + 100 µM L-cysteine and WSP5. H 2 S reacts with lead acetate, leading to staining of strips (Sigma-Aldrich). Values are means ± SD ( n = 3). * p

    Journal: Nature Communications

    Article Title: Transcription factor YcjW controls the emergency H2S production in E. coli

    doi: 10.1038/s41467-019-10785-x

    Figure Lengend Snippet: E. coli MG1655 lacking 3MSTA acquires phenotypic suppression and has increased H 2 S levels and tolerance to Gm and H 2 O 2 . a Δ mstA-sup has increased survival rate compared with Δ mstA when treated with 2 µg ml −1 gentamicin in a time-kill assay. Values correspond to colony-forming units (c.f.u). b Δ mstA-sup also has increased tolerance after exposure to 5 mM H 2 O 2 for 30 min. c H 2 S production as measured with fluorescent probe, WSP5. Relative fluorescent units are normalized to OD 600 and minus the background fluorescent of PBS buffer + 100 µM L-cysteine and WSP5. H 2 S reacts with lead acetate, leading to staining of strips (Sigma-Aldrich). Values are means ± SD ( n = 3). * p

    Article Snippet: To generate pLLY1, ycjW was PCR amplified from E. coli MG1655 using primers LL10 and LL11 and cloned into pACYC184 plasmid (NEB) using the Gibson Assembly Mastermix, according to the manufacturer’s protocol (NEB).

    Techniques: Time-Kill Assay, Staining