bl21  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 76
    Name:
    BL21 Star (DE3)pLysS One Shot Chemically Competent E. coli
    Description:
    One Shot BL21 Star (DE3) pLysS E. coli are chemically competent cells designed for applications that require high-level expression of non-toxic recombinant proteins from high copy number, T7 promoter-based expression systems (e.g., pRSET T7 vectors). One Shot BL21 Star (DE3) pLysS Chemically Competent cells have a transformation efficiency of >1 x 108 cfu/ µg plasmid DNA.• High mRNA stability results in increased protein yield• Ideal for use with high copy number, T7 promoter-based plasmids• Low background expression in uninduced cellsIncreased mRNA Stability Improves Protein Yield One Shot BL21 Star (DE3) pLysS E. coli offers enhanced mRNA stability so that abundant mRNA is available for protein expression. This enhanced stability is due to a mutation in the RNaseE gene (rne131), which is involved in mRNA degradation.High Expression of Non-Toxic Recombinant Proteins One Shot BL21 Star (DE3) pLysS cells are ideal for high-level expression of non-toxic but potentially growth-inhibiting recombinant proteins from high copy number, T7 promoter-based expression systems. The pLysS CamR plasmid carried by the BL21 Star (DE3) pLysS strain expresses T7 lysozyme, a T7 RNA polymerase inhibitor that prevents leaky expression in uninduced cells. The p15a origin of pLysS makes this plasmid compatible with pUC or pBR322-derived plasmids. This strain also contains the DE3 lysogen that carries the T7 RNA polymerase gene under control of the lacUV5 promoter, and IPTG is required to induce expression of the T7 RNA polymerase. One Shot BL21 Star (DE3) pLysS cells do not contain the lon protease and are also deficient in the outer membrane protease, OmpT. The absence of these proteases reduces degradation of heterologous proteins. Note: The BL21 Star strains have higher basal expression of heterologous genes than BL21 strains, due to the increased stability of mRNA. Therefore, these strains may not be useful for expression of toxic genes. However, the BL21 Star (DE3) pLysS strains offer lower expression levels than the BL21 Star strains.Genotype: F-ompT hsdSB (rB-, mB-) galdcmrne131 (DE3) pLysS (CamR)Easy-to-use, Single Tube Format The single tube, single use, format allows for all steps of transformation up to plating to take place in the same tube, thereby helping to save time and prevent contamination.Find the Strain and Format That You Need We also offer many other different strains and formats of chemically competent cells and electrocompetent cells to help meet your specific transformation needs. For expression of toxic proteins, choose BL21-AI One Shot Chemically Competent E. coli. For expression of non-toxic recombinant proteins from low copy number (e.g. Champion pET vectors), T7 promoter-based expression system, choose our One Shot BL21 Star (DE3) chemically competent cells. For Research Use Only. Not intended for animal or human diagnostic or therapeutic use.
    Catalog Number:
    C602003
    Price:
    None
    Applications:
    Bacterial Expression|Bioproduction|Competent Cells for Bacterial Expression|Protein Biology|Protein Expression
    Size:
    20 x 50 µL
    Category:
    Competent Cells & Strains, Expression Competent Cells
    Score:
    85
    Buy from Supplier


    Structured Review

    Thermo Fisher bl21
    SDS-PAGE (12.5% polyacrylamide gel) of rATB. Lanes: 1: molecular size markers; 2: E. coli <t>BL21</t> (pET28arATB) prior to induction; 3: E. coli BL21 (pET28arATB) after induction; 4: 5 μ g denatured rATB in inclusion bodies; 5: 1 μ g refolded soluble rATB.
    One Shot BL21 Star (DE3) pLysS E. coli are chemically competent cells designed for applications that require high-level expression of non-toxic recombinant proteins from high copy number, T7 promoter-based expression systems (e.g., pRSET T7 vectors). One Shot BL21 Star (DE3) pLysS Chemically Competent cells have a transformation efficiency of >1 x 108 cfu/ µg plasmid DNA.• High mRNA stability results in increased protein yield• Ideal for use with high copy number, T7 promoter-based plasmids• Low background expression in uninduced cellsIncreased mRNA Stability Improves Protein Yield One Shot BL21 Star (DE3) pLysS E. coli offers enhanced mRNA stability so that abundant mRNA is available for protein expression. This enhanced stability is due to a mutation in the RNaseE gene (rne131), which is involved in mRNA degradation.High Expression of Non-Toxic Recombinant Proteins One Shot BL21 Star (DE3) pLysS cells are ideal for high-level expression of non-toxic but potentially growth-inhibiting recombinant proteins from high copy number, T7 promoter-based expression systems. The pLysS CamR plasmid carried by the BL21 Star (DE3) pLysS strain expresses T7 lysozyme, a T7 RNA polymerase inhibitor that prevents leaky expression in uninduced cells. The p15a origin of pLysS makes this plasmid compatible with pUC or pBR322-derived plasmids. This strain also contains the DE3 lysogen that carries the T7 RNA polymerase gene under control of the lacUV5 promoter, and IPTG is required to induce expression of the T7 RNA polymerase. One Shot BL21 Star (DE3) pLysS cells do not contain the lon protease and are also deficient in the outer membrane protease, OmpT. The absence of these proteases reduces degradation of heterologous proteins. Note: The BL21 Star strains have higher basal expression of heterologous genes than BL21 strains, due to the increased stability of mRNA. Therefore, these strains may not be useful for expression of toxic genes. However, the BL21 Star (DE3) pLysS strains offer lower expression levels than the BL21 Star strains.Genotype: F-ompT hsdSB (rB-, mB-) galdcmrne131 (DE3) pLysS (CamR)Easy-to-use, Single Tube Format The single tube, single use, format allows for all steps of transformation up to plating to take place in the same tube, thereby helping to save time and prevent contamination.Find the Strain and Format That You Need We also offer many other different strains and formats of chemically competent cells and electrocompetent cells to help meet your specific transformation needs. For expression of toxic proteins, choose BL21-AI One Shot Chemically Competent E. coli. For expression of non-toxic recombinant proteins from low copy number (e.g. Champion pET vectors), T7 promoter-based expression system, choose our One Shot BL21 Star (DE3) chemically competent cells. For Research Use Only. Not intended for animal or human diagnostic or therapeutic use.
    https://www.bioz.com/result/bl21/product/Thermo Fisher
    Average 76 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bl21 - by Bioz Stars, 2020-01
    76/100 stars

    Related Products / Commonly Used Together

    2xyt auto-induction

    Images

    1) Product Images from "Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells"

    Article Title: Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2011/283747

    SDS-PAGE (12.5% polyacrylamide gel) of rATB. Lanes: 1: molecular size markers; 2: E. coli BL21 (pET28arATB) prior to induction; 3: E. coli BL21 (pET28arATB) after induction; 4: 5 μ g denatured rATB in inclusion bodies; 5: 1 μ g refolded soluble rATB.
    Figure Legend Snippet: SDS-PAGE (12.5% polyacrylamide gel) of rATB. Lanes: 1: molecular size markers; 2: E. coli BL21 (pET28arATB) prior to induction; 3: E. coli BL21 (pET28arATB) after induction; 4: 5 μ g denatured rATB in inclusion bodies; 5: 1 μ g refolded soluble rATB.

    Techniques Used: SDS Page

    2) Product Images from "A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors"

    Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.02405

    SDS-PAGE (A) and Western Blot (B) analysis of the expression of E. coli cell surface display systems for HuNoV (GII.4) recombinant capsid proteins with (3) or without the linker fragment (2) and the thrombin-released P proteins (4). M: prestained protein ladder (Catalog No.: 26616, Thermo Fisher, Shanghai, China); (1) pET-28a/BL21; (2) pET28a-inaQn-P (GII.4)/BL21; (3) pET28a-inaQn-TB-P (GII.4) /BL21; (4) Thrombin-released P proteins.
    Figure Legend Snippet: SDS-PAGE (A) and Western Blot (B) analysis of the expression of E. coli cell surface display systems for HuNoV (GII.4) recombinant capsid proteins with (3) or without the linker fragment (2) and the thrombin-released P proteins (4). M: prestained protein ladder (Catalog No.: 26616, Thermo Fisher, Shanghai, China); (1) pET-28a/BL21; (2) pET28a-inaQn-P (GII.4)/BL21; (3) pET28a-inaQn-TB-P (GII.4) /BL21; (4) Thrombin-released P proteins.

    Techniques Used: SDS Page, Western Blot, Expressing, Recombinant, Positron Emission Tomography

    OD of type A HBGA bound to serially diluted thrombin-released P proteins (red) and the thrombin-released background proteins from pET28a-inaQn-TB/BL21 (black). Bars represent standard errors.
    Figure Legend Snippet: OD of type A HBGA bound to serially diluted thrombin-released P proteins (red) and the thrombin-released background proteins from pET28a-inaQn-TB/BL21 (black). Bars represent standard errors.

    Techniques Used:

    3) Product Images from "Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods"

    Article Title: Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods

    Journal: Iranian Biomedical Journal

    doi: 10.22034/ibj.22.4.283

    SDS-PAGE analysis of purified antigen and Western blot analysis of rDgK antigen. (a) Purified protein was loaded on 12% acrylamide gel, stained with Coomassie brilliant blue R-250. Lane M, protein molecular standard; lane 1, SDS-PAGE of purified recombinant protein (arrow). (b) Lane M, protein molecular standard; lane 1, Western blotting of expressed rDgK probed with (lane 1) the negative control pool sera, (lane 2) suspected dog pool sera, and (lane 3) the positive-control serum pools (arrows). Lane 4 indicates total protein of untransformed E. coli BL21 in Western blot with the positive-control serum pools.
    Figure Legend Snippet: SDS-PAGE analysis of purified antigen and Western blot analysis of rDgK antigen. (a) Purified protein was loaded on 12% acrylamide gel, stained with Coomassie brilliant blue R-250. Lane M, protein molecular standard; lane 1, SDS-PAGE of purified recombinant protein (arrow). (b) Lane M, protein molecular standard; lane 1, Western blotting of expressed rDgK probed with (lane 1) the negative control pool sera, (lane 2) suspected dog pool sera, and (lane 3) the positive-control serum pools (arrows). Lane 4 indicates total protein of untransformed E. coli BL21 in Western blot with the positive-control serum pools.

    Techniques Used: SDS Page, Purification, Western Blot, Acrylamide Gel Assay, Staining, Recombinant, Negative Control, Positive Control

    4) Product Images from "Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42"

    Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42

    Journal: Virology Journal

    doi: 10.1186/1743-422X-7-99

    Expression and Identification of SNP22b (SARS-CoV NP) . ( A ) Supernatants (lanes 1 and 10) and pellets (lanes 2 and 6) of E. coli BL21 containing the pET22b-SNP22b vector expressing SARS-CoV NP, without IPTG induction. Supernatants (lanes 3 and 7) and pellets (lanes 4 and 8) of E. coli BL21 containing the pET22b-SNP22b vector and expressing SARS-CoV NP, induced with IPTG. Pellets of E . coli BL21 containing pET22b induced with IPTG (lane 5). Molecular weight markers (lane 9). The position of SARS-CoV NP (~47 kDa) is indicated by an arrow. ( B ) Western blot analysis of SARS-CoV NP using an anti-SARS-CoV NP horse polyclonal antibody. Lysate of E . coli BL21 expressing SARS-CoV NP, induced by IPTG (lane 1). Lysate of E . coli BL21 containing pET22b induced by IPTG (lane 2). Molecular weight markers (lane 3). The position of SARS-CoV NP (~47 kDa) is indicated by an arrow.
    Figure Legend Snippet: Expression and Identification of SNP22b (SARS-CoV NP) . ( A ) Supernatants (lanes 1 and 10) and pellets (lanes 2 and 6) of E. coli BL21 containing the pET22b-SNP22b vector expressing SARS-CoV NP, without IPTG induction. Supernatants (lanes 3 and 7) and pellets (lanes 4 and 8) of E. coli BL21 containing the pET22b-SNP22b vector and expressing SARS-CoV NP, induced with IPTG. Pellets of E . coli BL21 containing pET22b induced with IPTG (lane 5). Molecular weight markers (lane 9). The position of SARS-CoV NP (~47 kDa) is indicated by an arrow. ( B ) Western blot analysis of SARS-CoV NP using an anti-SARS-CoV NP horse polyclonal antibody. Lysate of E . coli BL21 expressing SARS-CoV NP, induced by IPTG (lane 1). Lysate of E . coli BL21 containing pET22b induced by IPTG (lane 2). Molecular weight markers (lane 3). The position of SARS-CoV NP (~47 kDa) is indicated by an arrow.

    Techniques Used: Expressing, Plasmid Preparation, Molecular Weight, Western Blot

    Expression and identification of GST and the p42-GST fusion protein . ( A ) Lysate of E . coli BL21 containing the pGEX-5X-1-p42 vector, expressing p42-GST fusion protein without IPTG induction (lanes 1, 5) or induced by IPTG (lanes 2, 4). Lysate of E . coli BL21 containing the pGEX-5X-1 vector expressing GST with IPTG induction (lane 6) or without IPTG (lane 7). Molecular weight markers (lane 3). ( B ) Western blot analysis of p42-GST fusion protein and GST expression using an anti-GST mAb. Lysates of GST-expressing E . coli BL21 with IPTG induction (lanes 1, 2). Lysates of p42-GST fusion protein-expressing E . coli BL21 with IPTG induction (lanes 6, 7). Supernatants of E . coli BL21 expressing GST lysed using lysozyme (lane 3). Supernatants of E . coli BL21 expressing p42-GST fusion protein lysed by lysozyme (lane 5). Molecular weight markers (lane 4).
    Figure Legend Snippet: Expression and identification of GST and the p42-GST fusion protein . ( A ) Lysate of E . coli BL21 containing the pGEX-5X-1-p42 vector, expressing p42-GST fusion protein without IPTG induction (lanes 1, 5) or induced by IPTG (lanes 2, 4). Lysate of E . coli BL21 containing the pGEX-5X-1 vector expressing GST with IPTG induction (lane 6) or without IPTG (lane 7). Molecular weight markers (lane 3). ( B ) Western blot analysis of p42-GST fusion protein and GST expression using an anti-GST mAb. Lysates of GST-expressing E . coli BL21 with IPTG induction (lanes 1, 2). Lysates of p42-GST fusion protein-expressing E . coli BL21 with IPTG induction (lanes 6, 7). Supernatants of E . coli BL21 expressing GST lysed using lysozyme (lane 3). Supernatants of E . coli BL21 expressing p42-GST fusion protein lysed by lysozyme (lane 5). Molecular weight markers (lane 4).

    Techniques Used: Expressing, Plasmid Preparation, Molecular Weight, Western Blot

    5) Product Images from "Biological Role of Trichoderma harzianum-Derived Platelet-Activating Factor Acetylhydrolase (PAF-AH) on Stress Response and Antagonism"

    Article Title: Biological Role of Trichoderma harzianum-Derived Platelet-Activating Factor Acetylhydrolase (PAF-AH) on Stress Response and Antagonism

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0100367

    Properties of the PAF-AH enzyme. (A) The protein was expressed in E. coli BL21 (DE3). M, protein marker. 1, supernatant of induced lysis cells; 2 and 3, purified PAF-AH from the elution buffer. (B) Immunoblot analysis of purified PAF-AH using anti-His tag antibodies as indicated. (C) The PAF-AH activity assay experiment was carried out as described in Materials and Methods . Error bars denote SD. Asterisks indicate significant differences in enzyme activity under the condition of no Ca 2+ compared with 1 mM Ca 2+ (Student's t -test, P
    Figure Legend Snippet: Properties of the PAF-AH enzyme. (A) The protein was expressed in E. coli BL21 (DE3). M, protein marker. 1, supernatant of induced lysis cells; 2 and 3, purified PAF-AH from the elution buffer. (B) Immunoblot analysis of purified PAF-AH using anti-His tag antibodies as indicated. (C) The PAF-AH activity assay experiment was carried out as described in Materials and Methods . Error bars denote SD. Asterisks indicate significant differences in enzyme activity under the condition of no Ca 2+ compared with 1 mM Ca 2+ (Student's t -test, P

    Techniques Used: Marker, Lysis, Purification, Activity Assay

    6) Product Images from "A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during Streptomyces Conjugation"

    Article Title: A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during Streptomyces Conjugation

    Journal: mBio

    doi: 10.1128/mBio.02559-14

    SpdB2-His promotes spheroplast formation by T7 lysozyme and forms unstable pores in planar lipid bilayers. (A) When expression of SpdB2-His was induced in BL21/pLysS, spheroplast (arrows) were formed. In contrast, spheroplasts were not observed in the absence of spdB2 (B) or when expressing spdB2 in BL21 lacking the T7 amidase (C). (D) When purified SpdB2-His was added to the cis side of a lipid bilayer, SpdB2-His inserted into the planar lipid bilayer, showing flickering pore structures. (E) The corresponding all-points conductance level histogram shows that there is no discrete conductance state of the open pore indicated by the long tail in the histogram.
    Figure Legend Snippet: SpdB2-His promotes spheroplast formation by T7 lysozyme and forms unstable pores in planar lipid bilayers. (A) When expression of SpdB2-His was induced in BL21/pLysS, spheroplast (arrows) were formed. In contrast, spheroplasts were not observed in the absence of spdB2 (B) or when expressing spdB2 in BL21 lacking the T7 amidase (C). (D) When purified SpdB2-His was added to the cis side of a lipid bilayer, SpdB2-His inserted into the planar lipid bilayer, showing flickering pore structures. (E) The corresponding all-points conductance level histogram shows that there is no discrete conductance state of the open pore indicated by the long tail in the histogram.

    Techniques Used: Expressing, Purification

    7) Product Images from "Effect of blood type on anti-α-Gal immunity and the incidence of infectious diseases"

    Article Title: Effect of blood type on anti-α-Gal immunity and the incidence of infectious diseases

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/emm.2016.164

    Correlation between anti-α-Gal antibodies and protection against malaria and tuberculosis. ( a ) Flow cytometry and immunofluorescence showing the presence of α-Gal on the surface of mycobacteria ( Mycobacterium marinum CECT 7091 reference strain). Escherichia coli O86:B7 (ATCC 12701) and BL21 (DE3) strains were included as positive and negative controls for α-Gal, respectively. For flow cytometry, cells were stained with BSI-IB 4 -FITC to visualize α-Gal and the viable cell population was gated according to forward scatter and side scatter parameters. Ten microliters of the fixed and stained samples were also used for immunofluorescence assays after air-drying and mounted in ProLong Antifade reagent containing DAPI. Representative immunofluorescence images are shown for bacterial cells stained with BSI-IB 4 -FITC (anti-α-Gal-FITC, green; blue, DAPI). ( b ) Anti-α-Gal antibody titers in patients with malaria or tuberculosis and healthy individuals. The levels of anti-α-Gal IgM and IgG antibodies were significantly higher in P. falciparum uninfected ( Pf− ) vs infected ( Pf +) individuals from Senegal. A similar pattern was observed in M. tuberculosis uninfected ( Mt −) vs infected ( Mt +) individuals from the Iberian Peninsula. The levels of anti-α-Gal IgE antibodies were lower in both Mt + and Pf + patients, when compared with Mt − and Pf − healthy individuals, respectively. The nonparametric Mann–Whitney U -test was used to compare values between infected and uninfected groups ( P =0.05).
    Figure Legend Snippet: Correlation between anti-α-Gal antibodies and protection against malaria and tuberculosis. ( a ) Flow cytometry and immunofluorescence showing the presence of α-Gal on the surface of mycobacteria ( Mycobacterium marinum CECT 7091 reference strain). Escherichia coli O86:B7 (ATCC 12701) and BL21 (DE3) strains were included as positive and negative controls for α-Gal, respectively. For flow cytometry, cells were stained with BSI-IB 4 -FITC to visualize α-Gal and the viable cell population was gated according to forward scatter and side scatter parameters. Ten microliters of the fixed and stained samples were also used for immunofluorescence assays after air-drying and mounted in ProLong Antifade reagent containing DAPI. Representative immunofluorescence images are shown for bacterial cells stained with BSI-IB 4 -FITC (anti-α-Gal-FITC, green; blue, DAPI). ( b ) Anti-α-Gal antibody titers in patients with malaria or tuberculosis and healthy individuals. The levels of anti-α-Gal IgM and IgG antibodies were significantly higher in P. falciparum uninfected ( Pf− ) vs infected ( Pf +) individuals from Senegal. A similar pattern was observed in M. tuberculosis uninfected ( Mt −) vs infected ( Mt +) individuals from the Iberian Peninsula. The levels of anti-α-Gal IgE antibodies were lower in both Mt + and Pf + patients, when compared with Mt − and Pf − healthy individuals, respectively. The nonparametric Mann–Whitney U -test was used to compare values between infected and uninfected groups ( P =0.05).

    Techniques Used: Flow Cytometry, Cytometry, Immunofluorescence, Staining, Infection, MANN-WHITNEY

    8) Product Images from "Vaccination with Killed but Metabolically Active E. coli Over-expressing Hemagglutinin Elicits Neutralizing Antibodies to H1N1 Swine Origin Influenza A Virus"

    Article Title: Vaccination with Killed but Metabolically Active E. coli Over-expressing Hemagglutinin Elicits Neutralizing Antibodies to H1N1 Swine Origin Influenza A Virus

    Journal:

    doi:

    Estimation of the expression level of HA of H1N1-S-OIV in E. coli, BL21 (DE3) (1 × 10 8 CFU)
    Figure Legend Snippet: Estimation of the expression level of HA of H1N1-S-OIV in E. coli, BL21 (DE3) (1 × 10 8 CFU)

    Techniques Used: Expressing, Hemagglutination Assay

    9) Product Images from "High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development"

    Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

    Journal: Microbial Cell Factories

    doi: 10.1186/1475-2859-10-56

    Expression of recombinant GST-VP1 protein in three different E. coli strains . The GST-VP1 protein expression in three E. coli strains, BL21(DE3), BL21-CodonPlus(DE3)-RP and BL21(DE3)-pLysS all containing pGEX-4T-1-VP1 was examined. The GST-VP1 protein was examined and detected using SDS-PAGE (A) and Western-blotting (B). Anti-GST tag monoclonal antibody was used to recognize the VP1 protein. Lane M, pre-stained protein marker; lane 1, 4 and 7, before IPTG induction; lane 2, 5 and 8, after IPTG induction for 1 hrs cultivation; lane 3, 6 and 9, after IPTG induction for 4 h cultivation. The solid triangle pinpoints the expressed VP1 protein.
    Figure Legend Snippet: Expression of recombinant GST-VP1 protein in three different E. coli strains . The GST-VP1 protein expression in three E. coli strains, BL21(DE3), BL21-CodonPlus(DE3)-RP and BL21(DE3)-pLysS all containing pGEX-4T-1-VP1 was examined. The GST-VP1 protein was examined and detected using SDS-PAGE (A) and Western-blotting (B). Anti-GST tag monoclonal antibody was used to recognize the VP1 protein. Lane M, pre-stained protein marker; lane 1, 4 and 7, before IPTG induction; lane 2, 5 and 8, after IPTG induction for 1 hrs cultivation; lane 3, 6 and 9, after IPTG induction for 4 h cultivation. The solid triangle pinpoints the expressed VP1 protein.

    Techniques Used: Expressing, Recombinant, SDS Page, Western Blot, Staining, Marker

    Growth kinetics and production profiles of GST-VP1 protein in three E. coli strains . (A) Growth profile of BL21(DE3), BL21-CodonPlus(DE3)-RP and BL21(DE3)-pLysS in LB medium post-induction. The production profiles of the three E. coli strains, (B)BL21(DE3)-pLysS, (C)BL21(DE3) and (D)BL21-CodonPlus(DE3)-RP containing pGEX-4T-1-VP1 are shown over time course after IPTG induction.
    Figure Legend Snippet: Growth kinetics and production profiles of GST-VP1 protein in three E. coli strains . (A) Growth profile of BL21(DE3), BL21-CodonPlus(DE3)-RP and BL21(DE3)-pLysS in LB medium post-induction. The production profiles of the three E. coli strains, (B)BL21(DE3)-pLysS, (C)BL21(DE3) and (D)BL21-CodonPlus(DE3)-RP containing pGEX-4T-1-VP1 are shown over time course after IPTG induction.

    Techniques Used:

    Related Articles

    Clone Assay:

    Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
    Article Snippet: Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA). .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

    Article Title: Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods
    Article Snippet: The amplification of internal transcribed spacer region 2 (ITS2) was performed using specific primer DiF (5′-CATTATC GAGCTTCAACAAACAAC-3′) and DiR (TTCAGC GGGTAATCACGACTG). .. E. coli strains DH5α and BL21 (DE3) and the pTG19-T (Invitrogen, Barcelona, Spain) and pET-24a(+) (Biomatik, Canada) plasmids were used for cloning and recombinant protein expression, respectively. .. Bacterial cells were grown in LB agar and LB broth (Merck, Darmstadt, Germany) and incubated at 30°C overnight and 4 h, respectively.

    Article Title: Accurate de novo design of hyperstable constrained peptides
    Article Snippet: Genes of designed disulfide-rich peptides were cloned into the vector pCDB180 (which we have made available via Addgene) using Gibson Assembly . .. Designed proteins were expressed from BL21*(DE3) E. coli (Invitrogen), and expression cultures were grown overnight with incubation at 37 °C and shaking at 225 RPM.

    Article Title: Acoustically modulated magnetic resonance imaging of gas-filled protein nanostructures
    Article Snippet: Gas vesicle concentrations were determined using the relationships summarized in . .. For reporter gene experiments, the gene cluster encoding a hybrid GV variant named A2C was cloned into pET28a plasmid (Novagen, Temecula, CA), and the resulting plasmid was transformed into BL21(A1) cells (Thermo Fisher Scientific, Waltham, MA). .. The transformed cells were first grown overnight at 30°C in LB media supplemented with 1% glucose, and this starter culture was subsequently diluted 1:100 into LB media supplemented with 0.2% glucose.

    Article Title: Computational design of trimeric influenza neutralizing proteins targeting the hemagglutinin receptor binding site
    Article Snippet: The 400 mM elution fraction was subjected to further cleaning via SEC and further processed using bacterial endotoxin removal beads (Miltenyi Biotec). .. Protein Expression of Designs Design variants were cloned into pET29 and expressed in BL21* (Invitrogen), followed by purification using Ni-NTA (QIAgen). .. Recombinant HA was expressed and biotinylated as previously described .

    Article Title: A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites
    Article Snippet: The PCR product was cloned by ligation independent cloning into vector BG1861 , which introduces an N-terminal 6xHis tag and the construct was verified by Sanger sequencing. .. BG1861:6xHis-PfHAD1 was transformed into BL21(DE3)pLysS Escherichia coli cells (Life Technologies).

    Centrifugation:

    Article Title: Vaccination with Killed but Metabolically Active E. coli Over-expressing Hemagglutinin Elicits Neutralizing Antibodies to H1N1 Swine Origin Influenza A Virus
    Article Snippet: The amplified DNA products were inserted into the In-Fusion Ready pEcoli-Nterm 6×HN vector (Clontech Laboratories, Mountain View, CA) and transformed into competent cells [ (E. coli), BL21 (DE3), Invitrogen, Carlsbad, CA]. .. The amplified DNA products were inserted into the In-Fusion Ready pEcoli-Nterm 6×HN vector (Clontech Laboratories, Mountain View, CA) and transformed into competent cells [ (E. coli), BL21 (DE3), Invitrogen, Carlsbad, CA].

    Article Title: Maintenance of Neural Progenitor Cell Stemness in 3D Hydrogels Requires Matrix Remodeling
    Article Snippet: Synthesis and characterization of ELP hydrogels ELPs were expressed in BL21(DE3)pLysS Escherichia coli (Life Technologies), based on a previously published procedure . .. Bacteria containing the plasmids were cultured in Terrific Broth to an OD600 of 0.8, and expression was induced by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG).

    Amplification:

    Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
    Article Snippet: The p42 gene was amplified by RT-PCR using mRNA from 2BS cells with the following primers: p42 forward: 5'-GATGAATTCATGGCGGACCCTAGAGATAAGG-3', reverse: 5'-GATCTCGAGTTACACAGGTTTGTAGTCCAATTTAG-3'. .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

    Article Title: Vaccination with Killed but Metabolically Active E. coli Over-expressing Hemagglutinin Elicits Neutralizing Antibodies to H1N1 Swine Origin Influenza A Virus
    Article Snippet: The forward PCR primer (5′-AATAGTCGACATGAAGGCAATACTAGTAGTTCTGCTAT AT-3′) and the reverse PCR primer (5′-GATACAGCGGCCG CTTAAATACATATTCTACACTGTAGAGACCCA-3′) were designed for a PCR reaction. .. The amplified DNA products were inserted into the In-Fusion Ready pEcoli-Nterm 6×HN vector (Clontech Laboratories, Mountain View, CA) and transformed into competent cells [ (E. coli), BL21 (DE3), Invitrogen, Carlsbad, CA]. .. As a control, a pEcoli-Nterm-green fluorescent protein (GFP) plasmid (Clontech Laboratories Inc.) was transformed.

    Article Title: A Re-wired Green Fluorescent Protein: Folding and Function in a Non-sequential, Non-circular GFP Permutant
    Article Snippet: Of this material, 1 μL was used as the template for amplification PCR with 0.2 μM of the forward and reverse primers (the outermost oligomers on each DNA strand) each, annealing temperature of 62ºC and 35 cycles. .. Completed genes with included NheI and EcoRI (enzymes from New England Biosciences, NEB) restriction sites were inserted into pET28a (Novagen) using T4 ligase per manufacturers instructions (NEB) and transformed into XL-1Blue or BL21 for DNA sequencing ( ) and BL21 for expression (Invitrogen).

    Article Title: A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites
    Article Snippet: All PfHAD1 (PlasmoDB ID PF3D7_1033400) alleles were amplified from P. falciparum genomic DNA using the following primers: 5′-CTCACCACCACCACCACCATATGCACGAAATTGTAGATAAGAA-3′ and 5′-ATCCTATCTTACTCACTTATATGTCACAGAATGTCTTCA-3′. .. BG1861:6xHis-PfHAD1 was transformed into BL21(DE3)pLysS Escherichia coli cells (Life Technologies).

    Synthesized:

    Article Title: Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs
    Article Snippet: Genes for the five constructs were synthesized and subcloned into a pET26b(+) vector (Thermo Scientific, Chicago, IL, USA). .. The plasmids were transformed into BL21*(DE3) Escherichia coli ( E. coli) (Thermo Fisher Scientific, Chicago, IL, USA) using a standard heat-shock protocol.

    Construct:

    Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors
    Article Snippet: Competent E. coli DH5α and BL21 (Thermo Fisher, Shanghai, China) were used for recombinant plasmid construction and protein expression, respectively. .. After re-digestion of plasmids pET28a, pET28a-P (GII.4) and pUC57-inaQn-TB ( Table ), the inaQn -TB and P (GII.4) fragment were inserted into pET-28a to create recombinant plasmid pET28a-inaQn-TB-P (GII.4).

    Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
    Article Snippet: The plasmid pcDNA3.0-SNP22b was constructed by subcloning the SNP gene, released from pET22b-SNP22b by BamHI/EcoRI, into pcDNA3.0 (Invitrogen). .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

    Article Title: Acoustic reporter genes for noninvasive imaging of microbes in mammalian hosts
    Article Snippet: After 17 h at 37 °C, the plates were imaged with the Chemidoc MP instrument with blue transillumination, and unfiltered light was collected to form an image. .. ARG and GFP constructs were transformed into BL21(A1) one-shot competent cells (Thermo Fisher Scientific, Carlsbad, CA) and plated onto LB-Agar two-layer inducer plates as described above. .. The colonies were immobilized by depositing a 4 mm layer of 0.5% Agarose-PBS gently onto the plate surface.

    Article Title: A Re-wired Green Fluorescent Protein: Folding and Function in a Non-sequential, Non-circular GFP Permutant
    Article Snippet: Genes of circular permutants of rGFP3 were constructed by blunt end ligation (T4 kinase reaction to provide phosphate groups for ligation followed by T4 ligase reaction, NEB enzymes) of the rGFP3 gene to form a circular template for PCR. .. Completed genes with included NheI and EcoRI (enzymes from New England Biosciences, NEB) restriction sites were inserted into pET28a (Novagen) using T4 ligase per manufacturers instructions (NEB) and transformed into XL-1Blue or BL21 for DNA sequencing ( ) and BL21 for expression (Invitrogen).

    Article Title: A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites
    Article Snippet: The PCR product was cloned by ligation independent cloning into vector BG1861 , which introduces an N-terminal 6xHis tag and the construct was verified by Sanger sequencing. .. BG1861:6xHis-PfHAD1 was transformed into BL21(DE3)pLysS Escherichia coli cells (Life Technologies).

    Article Title: Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs
    Article Snippet: Paragraph title: 2.1. Production of Constructs, Chelator Conjugation, and Product Purification ... The plasmids were transformed into BL21*(DE3) Escherichia coli ( E. coli) (Thermo Fisher Scientific, Chicago, IL, USA) using a standard heat-shock protocol.

    Incubation:

    Article Title: Effect of blood type on anti-α-Gal immunity and the incidence of infectious diseases
    Article Snippet: Spectrophotometric absorbance was measured at 600 nm (OD600 ) and the concentration was adjusted to 108 –109 CFU ml−1 . .. The Escherichia coli O86:B7 (ATCC 12701) and BL21 (DE3; Invitrogen, Carlsbad, CA, USA) strains were included as positive and negative controls for α-Gal, respectively, inoculated into 50 ml of Luria Broth (LB), and incubated at 37 °C overnight. .. The E. coli O86:B7, E. coli BL21 (DE3) and M. marinum cell cultures were washed 2 × in PBS (4000 g , 5 min) and re-suspended in PBS.

    Article Title: Accurate de novo design of hyperstable constrained peptides
    Article Snippet: Protein expression from E. coli was carried out using a large N-terminal fusion domain consisting of: the native E. coli protein OsmY to direct periplasmic and extracellular localization , a deca-histidine tag for protein purification, and the SUMO protein Smt3 from Saccharomyces cerevisiae to chaperone folding and provide a mechanism for scarless cleavage of the fusion from the designed protein . .. Designed proteins were expressed from BL21*(DE3) E. coli (Invitrogen), and expression cultures were grown overnight with incubation at 37 °C and shaking at 225 RPM. .. Following expression via Studier autoinduction , a periplasmic extract was prepared by washing cells with: 20% sucrose, 30 mM Tris-HCl pH 8.0, 1 mM ethylenediaminetetraacetic acid pH 8.0, 1 mg/mL lysozyme.

    Expressing:

    Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors
    Article Snippet: In this study, we have surmounted the fore-mentioned hurdle to applying the bacterial-surface-P-protein-display system in a viral receptor isolation context by adding a thrombin-susceptible domain to the existing construct to facilitate release of the P-protein-candidate-receptor complex from bacteria to enable easy purification ( Figure ). .. Competent E. coli DH5α and BL21 (Thermo Fisher, Shanghai, China) were used for recombinant plasmid construction and protein expression, respectively. .. Oligonucleotide “TB” coding for the peptide sequence of Leu-Val-Pro-Arg-Gly-Ser, was synthesized by Suzhou GENEWIZ Bio-Technology, Co., Ltd. Then, the artificially synthesized inaQn-TB sequence was inserted into cloning vector pUC57 to create pUC57-inaQn-TB.

    Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
    Article Snippet: BL21 (DE3) was obtained from Invitrogen Life Technologies (Carlsbad, CA). .. BL21 (DE3) was obtained from Invitrogen Life Technologies (Carlsbad, CA).

    Article Title: A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during Streptomyces Conjugation
    Article Snippet: Proteins were purified from E. coli Rosetta 2 (Merck), BL21(DE3), or BL21(DE3)/pLysS (Invitrogen) and S. lividans TK23 ( ). .. Proteins were purified from E. coli Rosetta 2 (Merck), BL21(DE3), or BL21(DE3)/pLysS (Invitrogen) and S. lividans TK23 ( ).

    Article Title: Biological Role of Trichoderma harzianum-Derived Platelet-Activating Factor Acetylhydrolase (PAF-AH) on Stress Response and Antagonism
    Article Snippet: Wild type and transformants were maintained in PDA (Difco, Becton Dickinson & Co., USA) at 28°C until sporulation occurred. .. Escherichia coli DH5α and BL21 (DE3) were purchased from Invitrogen (Carlsbad, CA, USA) for plasmid propagation and protein expression, respectively. .. Bacterial strains were grown in Luria-Bertani (LB) broth or LB agar plates supplemented with kanamycin sulfate (0.1 mg/mL) or ampicillin (0.1 mg/mL).

    Article Title: Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods
    Article Snippet: The amplification of internal transcribed spacer region 2 (ITS2) was performed using specific primer DiF (5′-CATTATC GAGCTTCAACAAACAAC-3′) and DiR (TTCAGC GGGTAATCACGACTG). .. E. coli strains DH5α and BL21 (DE3) and the pTG19-T (Invitrogen, Barcelona, Spain) and pET-24a(+) (Biomatik, Canada) plasmids were used for cloning and recombinant protein expression, respectively. .. Bacterial cells were grown in LB agar and LB broth (Merck, Darmstadt, Germany) and incubated at 30°C overnight and 4 h, respectively.

    Article Title: Vaccination with Killed but Metabolically Active E. coli Over-expressing Hemagglutinin Elicits Neutralizing Antibodies to H1N1 Swine Origin Influenza A Virus
    Article Snippet: Molecular cloning, expression, and purification of recombinant HA of SD/H1N1-S-OIV The complementary DNA (cDNA) of SD/H1N1-S-OIV was used as a template ( ) to amplify a polymerase chain reaction (PCR) product encoding SD/H1N1-S-OIV HA (Accession number: ). .. The amplified DNA products were inserted into the In-Fusion Ready pEcoli-Nterm 6×HN vector (Clontech Laboratories, Mountain View, CA) and transformed into competent cells [ (E. coli), BL21 (DE3), Invitrogen, Carlsbad, CA].

    Article Title: Maintenance of Neural Progenitor Cell Stemness in 3D Hydrogels Requires Matrix Remodeling
    Article Snippet: Synthesis and characterization of ELP hydrogels ELPs were expressed in BL21(DE3)pLysS Escherichia coli (Life Technologies), based on a previously published procedure . .. Briefly, ELPs were cloned into pET15b plasmids and expressed under control of the T7 promoter.

    Article Title: Accurate de novo design of hyperstable constrained peptides
    Article Snippet: Protein expression from E. coli was carried out using a large N-terminal fusion domain consisting of: the native E. coli protein OsmY to direct periplasmic and extracellular localization , a deca-histidine tag for protein purification, and the SUMO protein Smt3 from Saccharomyces cerevisiae to chaperone folding and provide a mechanism for scarless cleavage of the fusion from the designed protein . .. Designed proteins were expressed from BL21*(DE3) E. coli (Invitrogen), and expression cultures were grown overnight with incubation at 37 °C and shaking at 225 RPM. .. Following expression via Studier autoinduction , a periplasmic extract was prepared by washing cells with: 20% sucrose, 30 mM Tris-HCl pH 8.0, 1 mM ethylenediaminetetraacetic acid pH 8.0, 1 mg/mL lysozyme.

    Article Title: A Re-wired Green Fluorescent Protein: Folding and Function in a Non-sequential, Non-circular GFP Permutant
    Article Snippet: New 5′ and 3′ primers were designed for each circular permutant to include new EcoRI and NheI sites as well as stop codon followed by PCR performed as above (Pfu Turbo from Stratagene). .. Completed genes with included NheI and EcoRI (enzymes from New England Biosciences, NEB) restriction sites were inserted into pET28a (Novagen) using T4 ligase per manufacturers instructions (NEB) and transformed into XL-1Blue or BL21 for DNA sequencing ( ) and BL21 for expression (Invitrogen). .. DNA was collected using a Sigma GeneElute Plasmid Miniprep kit and buffer exchanged using a Promega Wizard SV Gel and PCR-cleanup System kit.

    Article Title: Acoustically modulated magnetic resonance imaging of gas-filled protein nanostructures
    Article Snippet: Paragraph title: Reporter gene expression ... For reporter gene experiments, the gene cluster encoding a hybrid GV variant named A2C was cloned into pET28a plasmid (Novagen, Temecula, CA), and the resulting plasmid was transformed into BL21(A1) cells (Thermo Fisher Scientific, Waltham, MA).

    Article Title: Computational design of trimeric influenza neutralizing proteins targeting the hemagglutinin receptor binding site
    Article Snippet: The 400 mM elution fraction was subjected to further cleaning via SEC and further processed using bacterial endotoxin removal beads (Miltenyi Biotec). .. Protein Expression of Designs Design variants were cloned into pET29 and expressed in BL21* (Invitrogen), followed by purification using Ni-NTA (QIAgen). .. Recombinant HA was expressed and biotinylated as previously described .

    Article Title: A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites
    Article Snippet: Paragraph title: Recombinant expression and purification of PfHAD1 variants ... BG1861:6xHis-PfHAD1 was transformed into BL21(DE3)pLysS Escherichia coli cells (Life Technologies).

    Transformation Assay:

    Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
    Article Snippet: BL21 (DE3) was obtained from Invitrogen Life Technologies (Carlsbad, CA). .. BL21 (DE3) was obtained from Invitrogen Life Technologies (Carlsbad, CA).

    Article Title: Vaccination with Killed but Metabolically Active E. coli Over-expressing Hemagglutinin Elicits Neutralizing Antibodies to H1N1 Swine Origin Influenza A Virus
    Article Snippet: The forward PCR primer (5′-AATAGTCGACATGAAGGCAATACTAGTAGTTCTGCTAT AT-3′) and the reverse PCR primer (5′-GATACAGCGGCCG CTTAAATACATATTCTACACTGTAGAGACCCA-3′) were designed for a PCR reaction. .. The amplified DNA products were inserted into the In-Fusion Ready pEcoli-Nterm 6×HN vector (Clontech Laboratories, Mountain View, CA) and transformed into competent cells [ (E. coli), BL21 (DE3), Invitrogen, Carlsbad, CA]. .. As a control, a pEcoli-Nterm-green fluorescent protein (GFP) plasmid (Clontech Laboratories Inc.) was transformed.

    Article Title: Acoustic reporter genes for noninvasive imaging of microbes in mammalian hosts
    Article Snippet: After 17 h at 37 °C, the plates were imaged with the Chemidoc MP instrument with blue transillumination, and unfiltered light was collected to form an image. .. ARG and GFP constructs were transformed into BL21(A1) one-shot competent cells (Thermo Fisher Scientific, Carlsbad, CA) and plated onto LB-Agar two-layer inducer plates as described above. .. The colonies were immobilized by depositing a 4 mm layer of 0.5% Agarose-PBS gently onto the plate surface.

    Article Title: A Re-wired Green Fluorescent Protein: Folding and Function in a Non-sequential, Non-circular GFP Permutant
    Article Snippet: New 5′ and 3′ primers were designed for each circular permutant to include new EcoRI and NheI sites as well as stop codon followed by PCR performed as above (Pfu Turbo from Stratagene). .. Completed genes with included NheI and EcoRI (enzymes from New England Biosciences, NEB) restriction sites were inserted into pET28a (Novagen) using T4 ligase per manufacturers instructions (NEB) and transformed into XL-1Blue or BL21 for DNA sequencing ( ) and BL21 for expression (Invitrogen). .. DNA was collected using a Sigma GeneElute Plasmid Miniprep kit and buffer exchanged using a Promega Wizard SV Gel and PCR-cleanup System kit.

    Article Title: Acoustically modulated magnetic resonance imaging of gas-filled protein nanostructures
    Article Snippet: Gas vesicle concentrations were determined using the relationships summarized in . .. For reporter gene experiments, the gene cluster encoding a hybrid GV variant named A2C was cloned into pET28a plasmid (Novagen, Temecula, CA), and the resulting plasmid was transformed into BL21(A1) cells (Thermo Fisher Scientific, Waltham, MA). .. The transformed cells were first grown overnight at 30°C in LB media supplemented with 1% glucose, and this starter culture was subsequently diluted 1:100 into LB media supplemented with 0.2% glucose.

    Article Title: A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites
    Article Snippet: The PCR product was cloned by ligation independent cloning into vector BG1861 , which introduces an N-terminal 6xHis tag and the construct was verified by Sanger sequencing. .. BG1861:6xHis-PfHAD1 was transformed into BL21(DE3)pLysS Escherichia coli cells (Life Technologies). .. Following induction with IPTG, cells were harvested by centrifugation and stored at −20 °C.

    Article Title: Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs
    Article Snippet: Genes for the five constructs were synthesized and subcloned into a pET26b(+) vector (Thermo Scientific, Chicago, IL, USA). .. The plasmids were transformed into BL21*(DE3) Escherichia coli ( E. coli) (Thermo Fisher Scientific, Chicago, IL, USA) using a standard heat-shock protocol. .. Protein production proceeded overnight at 25 °C after induced expression with 100 μM isopropyl β- d -1-thiogalactopyranoside (IPTG) at an optical density measured at a wavelength of 600 nm (OD600 ) of 0.8.

    Conjugation Assay:

    Article Title: Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs
    Article Snippet: Paragraph title: 2.1. Production of Constructs, Chelator Conjugation, and Product Purification ... The plasmids were transformed into BL21*(DE3) Escherichia coli ( E. coli) (Thermo Fisher Scientific, Chicago, IL, USA) using a standard heat-shock protocol.

    Chromatography:

    Article Title: Accurate de novo design of hyperstable constrained peptides
    Article Snippet: Designed proteins were expressed from BL21*(DE3) E. coli (Invitrogen), and expression cultures were grown overnight with incubation at 37 °C and shaking at 225 RPM. .. Following expression via Studier autoinduction , a periplasmic extract was prepared by washing cells with: 20% sucrose, 30 mM Tris-HCl pH 8.0, 1 mM ethylenediaminetetraacetic acid pH 8.0, 1 mg/mL lysozyme.

    Ligation:

    Article Title: A Re-wired Green Fluorescent Protein: Folding and Function in a Non-sequential, Non-circular GFP Permutant
    Article Snippet: Genes of circular permutants of rGFP3 were constructed by blunt end ligation (T4 kinase reaction to provide phosphate groups for ligation followed by T4 ligase reaction, NEB enzymes) of the rGFP3 gene to form a circular template for PCR. .. Completed genes with included NheI and EcoRI (enzymes from New England Biosciences, NEB) restriction sites were inserted into pET28a (Novagen) using T4 ligase per manufacturers instructions (NEB) and transformed into XL-1Blue or BL21 for DNA sequencing ( ) and BL21 for expression (Invitrogen).

    Article Title: A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites
    Article Snippet: The PCR product was cloned by ligation independent cloning into vector BG1861 , which introduces an N-terminal 6xHis tag and the construct was verified by Sanger sequencing. .. BG1861:6xHis-PfHAD1 was transformed into BL21(DE3)pLysS Escherichia coli cells (Life Technologies).

    Protease Inhibitor:

    Article Title: Maintenance of Neural Progenitor Cell Stemness in 3D Hydrogels Requires Matrix Remodeling
    Article Snippet: Synthesis and characterization of ELP hydrogels ELPs were expressed in BL21(DE3)pLysS Escherichia coli (Life Technologies), based on a previously published procedure . .. After 7 hours, the bacteria were harvested by centrifugation, resuspended in TEN buffer (10 mM Tris, 1 mM EDTA, and 100 mM NaCl, pH 8.0), and lysed by repetitive freeze-thaw cycles.

    Article Title: A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites
    Article Snippet: BG1861:6xHis-PfHAD1 was transformed into BL21(DE3)pLysS Escherichia coli cells (Life Technologies). .. Following induction with IPTG, cells were harvested by centrifugation and stored at −20 °C.

    Cell Culture:

    Article Title: Maintenance of Neural Progenitor Cell Stemness in 3D Hydrogels Requires Matrix Remodeling
    Article Snippet: Synthesis and characterization of ELP hydrogels ELPs were expressed in BL21(DE3)pLysS Escherichia coli (Life Technologies), based on a previously published procedure . .. Briefly, ELPs were cloned into pET15b plasmids and expressed under control of the T7 promoter.

    Hemagglutination Assay:

    Article Title: Vaccination with Killed but Metabolically Active E. coli Over-expressing Hemagglutinin Elicits Neutralizing Antibodies to H1N1 Swine Origin Influenza A Virus
    Article Snippet: Molecular cloning, expression, and purification of recombinant HA of SD/H1N1-S-OIV The complementary DNA (cDNA) of SD/H1N1-S-OIV was used as a template ( ) to amplify a polymerase chain reaction (PCR) product encoding SD/H1N1-S-OIV HA (Accession number: ). .. The amplified DNA products were inserted into the In-Fusion Ready pEcoli-Nterm 6×HN vector (Clontech Laboratories, Mountain View, CA) and transformed into competent cells [ (E. coli), BL21 (DE3), Invitrogen, Carlsbad, CA].

    other:

    Article Title: Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells
    Article Snippet: Escherichia coli XL1 blue (F− φ 80(lacZ )ΔM15 ΔlacX74 hsdR (rk − , mk + ) ΔrecA1398 endA1 tonA ) and BL21 [F− ompT hsdSB (rB − mB − ) gal dcm (DE3) pLysS(CamR )] were obtained from Invitrogen.

    DNA Sequencing:

    Article Title: A Re-wired Green Fluorescent Protein: Folding and Function in a Non-sequential, Non-circular GFP Permutant
    Article Snippet: New 5′ and 3′ primers were designed for each circular permutant to include new EcoRI and NheI sites as well as stop codon followed by PCR performed as above (Pfu Turbo from Stratagene). .. Completed genes with included NheI and EcoRI (enzymes from New England Biosciences, NEB) restriction sites were inserted into pET28a (Novagen) using T4 ligase per manufacturers instructions (NEB) and transformed into XL-1Blue or BL21 for DNA sequencing ( ) and BL21 for expression (Invitrogen). .. DNA was collected using a Sigma GeneElute Plasmid Miniprep kit and buffer exchanged using a Promega Wizard SV Gel and PCR-cleanup System kit.

    Protein Concentration:

    Article Title: Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs
    Article Snippet: The plasmids were transformed into BL21*(DE3) Escherichia coli ( E. coli) (Thermo Fisher Scientific, Chicago, IL, USA) using a standard heat-shock protocol. .. The plasmids were transformed into BL21*(DE3) Escherichia coli ( E. coli) (Thermo Fisher Scientific, Chicago, IL, USA) using a standard heat-shock protocol.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
    Article Snippet: The p42 gene was amplified by RT-PCR using mRNA from 2BS cells with the following primers: p42 forward: 5'-GATGAATTCATGGCGGACCCTAGAGATAAGG-3', reverse: 5'-GATCTCGAGTTACACAGGTTTGTAGTCCAATTTAG-3'. .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

    Binding Assay:

    Article Title: A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during Streptomyces Conjugation
    Article Snippet: Proteins were purified from E. coli Rosetta 2 (Merck), BL21(DE3), or BL21(DE3)/pLysS (Invitrogen) and S. lividans TK23 ( ). .. Proteins were purified from E. coli Rosetta 2 (Merck), BL21(DE3), or BL21(DE3)/pLysS (Invitrogen) and S. lividans TK23 ( ).

    Imaging:

    Article Title: Acoustic reporter genes for noninvasive imaging of microbes in mammalian hosts
    Article Snippet: ARG and GFP constructs were transformed into BL21(A1) one-shot competent cells (Thermo Fisher Scientific, Carlsbad, CA) and plated onto LB-Agar two-layer inducer plates as described above. .. ARG and GFP constructs were transformed into BL21(A1) one-shot competent cells (Thermo Fisher Scientific, Carlsbad, CA) and plated onto LB-Agar two-layer inducer plates as described above.

    Nucleic Acid Electrophoresis:

    Article Title: Vaccination with Killed but Metabolically Active E. coli Over-expressing Hemagglutinin Elicits Neutralizing Antibodies to H1N1 Swine Origin Influenza A Virus
    Article Snippet: The amplified DNA products were inserted into the In-Fusion Ready pEcoli-Nterm 6×HN vector (Clontech Laboratories, Mountain View, CA) and transformed into competent cells [ (E. coli), BL21 (DE3), Invitrogen, Carlsbad, CA]. .. As a control, a pEcoli-Nterm-green fluorescent protein (GFP) plasmid (Clontech Laboratories Inc.) was transformed.

    Subcloning:

    Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
    Article Snippet: The plasmid pcDNA3.0-SNP22b was constructed by subcloning the SNP gene, released from pET22b-SNP22b by BamHI/EcoRI, into pcDNA3.0 (Invitrogen). .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

    Polymerase Chain Reaction:

    Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
    Article Snippet: PCR products were purified and cloned into the pGEX-5X-1 plasmid (Pharmacia, GE) using XhoI/EcoRI, to generate pGEX-5X-1-p42. .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

    Article Title: A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during Streptomyces Conjugation
    Article Snippet: Proteins were purified from E. coli Rosetta 2 (Merck), BL21(DE3), or BL21(DE3)/pLysS (Invitrogen) and S. lividans TK23 ( ). .. Proteins were purified from E. coli Rosetta 2 (Merck), BL21(DE3), or BL21(DE3)/pLysS (Invitrogen) and S. lividans TK23 ( ).

    Article Title: Vaccination with Killed but Metabolically Active E. coli Over-expressing Hemagglutinin Elicits Neutralizing Antibodies to H1N1 Swine Origin Influenza A Virus
    Article Snippet: The forward PCR primer (5′-AATAGTCGACATGAAGGCAATACTAGTAGTTCTGCTAT AT-3′) and the reverse PCR primer (5′-GATACAGCGGCCG CTTAAATACATATTCTACACTGTAGAGACCCA-3′) were designed for a PCR reaction. .. The amplified DNA products were inserted into the In-Fusion Ready pEcoli-Nterm 6×HN vector (Clontech Laboratories, Mountain View, CA) and transformed into competent cells [ (E. coli), BL21 (DE3), Invitrogen, Carlsbad, CA].

    Article Title: A Re-wired Green Fluorescent Protein: Folding and Function in a Non-sequential, Non-circular GFP Permutant
    Article Snippet: New 5′ and 3′ primers were designed for each circular permutant to include new EcoRI and NheI sites as well as stop codon followed by PCR performed as above (Pfu Turbo from Stratagene). .. Completed genes with included NheI and EcoRI (enzymes from New England Biosciences, NEB) restriction sites were inserted into pET28a (Novagen) using T4 ligase per manufacturers instructions (NEB) and transformed into XL-1Blue or BL21 for DNA sequencing ( ) and BL21 for expression (Invitrogen).

    Article Title: A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites
    Article Snippet: The PCR product was cloned by ligation independent cloning into vector BG1861 , which introduces an N-terminal 6xHis tag and the construct was verified by Sanger sequencing. .. BG1861:6xHis-PfHAD1 was transformed into BL21(DE3)pLysS Escherichia coli cells (Life Technologies).

    Purification:

    Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
    Article Snippet: PCR products were purified and cloned into the pGEX-5X-1 plasmid (Pharmacia, GE) using XhoI/EcoRI, to generate pGEX-5X-1-p42. .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

    Article Title: A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during Streptomyces Conjugation
    Article Snippet: Cultivation of strains and procedures for DNA manipulation were performed as previously described ( , ). .. Proteins were purified from E. coli Rosetta 2 (Merck), BL21(DE3), or BL21(DE3)/pLysS (Invitrogen) and S. lividans TK23 ( ). .. S. lividans strains TK54 ( ) and TK64 ( ) were used for mating experiments.

    Article Title: Vaccination with Killed but Metabolically Active E. coli Over-expressing Hemagglutinin Elicits Neutralizing Antibodies to H1N1 Swine Origin Influenza A Virus
    Article Snippet: Molecular cloning, expression, and purification of recombinant HA of SD/H1N1-S-OIV The complementary DNA (cDNA) of SD/H1N1-S-OIV was used as a template ( ) to amplify a polymerase chain reaction (PCR) product encoding SD/H1N1-S-OIV HA (Accession number: ). .. The amplified DNA products were inserted into the In-Fusion Ready pEcoli-Nterm 6×HN vector (Clontech Laboratories, Mountain View, CA) and transformed into competent cells [ (E. coli), BL21 (DE3), Invitrogen, Carlsbad, CA].

    Article Title: Accurate de novo design of hyperstable constrained peptides
    Article Snippet: Designed proteins were expressed from BL21*(DE3) E. coli (Invitrogen), and expression cultures were grown overnight with incubation at 37 °C and shaking at 225 RPM. .. Following expression via Studier autoinduction , a periplasmic extract was prepared by washing cells with: 20% sucrose, 30 mM Tris-HCl pH 8.0, 1 mM ethylenediaminetetraacetic acid pH 8.0, 1 mg/mL lysozyme.

    Article Title: Computational design of trimeric influenza neutralizing proteins targeting the hemagglutinin receptor binding site
    Article Snippet: The 400 mM elution fraction was subjected to further cleaning via SEC and further processed using bacterial endotoxin removal beads (Miltenyi Biotec). .. Protein Expression of Designs Design variants were cloned into pET29 and expressed in BL21* (Invitrogen), followed by purification using Ni-NTA (QIAgen). .. Recombinant HA was expressed and biotinylated as previously described .

    Article Title: A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites
    Article Snippet: Paragraph title: Recombinant expression and purification of PfHAD1 variants ... BG1861:6xHis-PfHAD1 was transformed into BL21(DE3)pLysS Escherichia coli cells (Life Technologies).

    Article Title: Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs
    Article Snippet: Paragraph title: 2.1. Production of Constructs, Chelator Conjugation, and Product Purification ... The plasmids were transformed into BL21*(DE3) Escherichia coli ( E. coli) (Thermo Fisher Scientific, Chicago, IL, USA) using a standard heat-shock protocol.

    Protein Purification:

    Article Title: Accurate de novo design of hyperstable constrained peptides
    Article Snippet: Paragraph title: Protein purification of genetically-encodable disulfide-rich peptides ... Designed proteins were expressed from BL21*(DE3) E. coli (Invitrogen), and expression cultures were grown overnight with incubation at 37 °C and shaking at 225 RPM.

    Sequencing:

    Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
    Article Snippet: Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA). .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

    Article Title: A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites
    Article Snippet: The PCR product was cloned by ligation independent cloning into vector BG1861 , which introduces an N-terminal 6xHis tag and the construct was verified by Sanger sequencing. .. BG1861:6xHis-PfHAD1 was transformed into BL21(DE3)pLysS Escherichia coli cells (Life Technologies).

    Positron Emission Tomography:

    Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors
    Article Snippet: Competent E. coli DH5α and BL21 (Thermo Fisher, Shanghai, China) were used for recombinant plasmid construction and protein expression, respectively. .. Oligonucleotide “TB” coding for the peptide sequence of Leu-Val-Pro-Arg-Gly-Ser, was synthesized by Suzhou GENEWIZ Bio-Technology, Co., Ltd. Then, the artificially synthesized inaQn-TB sequence was inserted into cloning vector pUC57 to create pUC57-inaQn-TB.

    Article Title: Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods
    Article Snippet: The amplification of internal transcribed spacer region 2 (ITS2) was performed using specific primer DiF (5′-CATTATC GAGCTTCAACAAACAAC-3′) and DiR (TTCAGC GGGTAATCACGACTG). .. E. coli strains DH5α and BL21 (DE3) and the pTG19-T (Invitrogen, Barcelona, Spain) and pET-24a(+) (Biomatik, Canada) plasmids were used for cloning and recombinant protein expression, respectively. .. Bacterial cells were grown in LB agar and LB broth (Merck, Darmstadt, Germany) and incubated at 30°C overnight and 4 h, respectively.

    Recombinant:

    Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors
    Article Snippet: In this study, we have surmounted the fore-mentioned hurdle to applying the bacterial-surface-P-protein-display system in a viral receptor isolation context by adding a thrombin-susceptible domain to the existing construct to facilitate release of the P-protein-candidate-receptor complex from bacteria to enable easy purification ( Figure ). .. Competent E. coli DH5α and BL21 (Thermo Fisher, Shanghai, China) were used for recombinant plasmid construction and protein expression, respectively. .. Oligonucleotide “TB” coding for the peptide sequence of Leu-Val-Pro-Arg-Gly-Ser, was synthesized by Suzhou GENEWIZ Bio-Technology, Co., Ltd. Then, the artificially synthesized inaQn-TB sequence was inserted into cloning vector pUC57 to create pUC57-inaQn-TB.

    Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
    Article Snippet: Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA). .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

    Article Title: Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods
    Article Snippet: The amplification of internal transcribed spacer region 2 (ITS2) was performed using specific primer DiF (5′-CATTATC GAGCTTCAACAAACAAC-3′) and DiR (TTCAGC GGGTAATCACGACTG). .. E. coli strains DH5α and BL21 (DE3) and the pTG19-T (Invitrogen, Barcelona, Spain) and pET-24a(+) (Biomatik, Canada) plasmids were used for cloning and recombinant protein expression, respectively. .. Bacterial cells were grown in LB agar and LB broth (Merck, Darmstadt, Germany) and incubated at 30°C overnight and 4 h, respectively.

    Article Title: Vaccination with Killed but Metabolically Active E. coli Over-expressing Hemagglutinin Elicits Neutralizing Antibodies to H1N1 Swine Origin Influenza A Virus
    Article Snippet: Molecular cloning, expression, and purification of recombinant HA of SD/H1N1-S-OIV The complementary DNA (cDNA) of SD/H1N1-S-OIV was used as a template ( ) to amplify a polymerase chain reaction (PCR) product encoding SD/H1N1-S-OIV HA (Accession number: ). .. The amplified DNA products were inserted into the In-Fusion Ready pEcoli-Nterm 6×HN vector (Clontech Laboratories, Mountain View, CA) and transformed into competent cells [ (E. coli), BL21 (DE3), Invitrogen, Carlsbad, CA].

    Article Title: A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites
    Article Snippet: Paragraph title: Recombinant expression and purification of PfHAD1 variants ... BG1861:6xHis-PfHAD1 was transformed into BL21(DE3)pLysS Escherichia coli cells (Life Technologies).

    Lysis:

    Article Title: A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites
    Article Snippet: BG1861:6xHis-PfHAD1 was transformed into BL21(DE3)pLysS Escherichia coli cells (Life Technologies). .. Following induction with IPTG, cells were harvested by centrifugation and stored at −20 °C.

    Article Title: Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs
    Article Snippet: The plasmids were transformed into BL21*(DE3) Escherichia coli ( E. coli) (Thermo Fisher Scientific, Chicago, IL, USA) using a standard heat-shock protocol. .. Protein production proceeded overnight at 25 °C after induced expression with 100 μM isopropyl β- d -1-thiogalactopyranoside (IPTG) at an optical density measured at a wavelength of 600 nm (OD600 ) of 0.8.

    Concentration Assay:

    Article Title: Effect of blood type on anti-α-Gal immunity and the incidence of infectious diseases
    Article Snippet: Spectrophotometric absorbance was measured at 600 nm (OD600 ) and the concentration was adjusted to 108 –109 CFU ml−1 . .. The Escherichia coli O86:B7 (ATCC 12701) and BL21 (DE3; Invitrogen, Carlsbad, CA, USA) strains were included as positive and negative controls for α-Gal, respectively, inoculated into 50 ml of Luria Broth (LB), and incubated at 37 °C overnight.

    Article Title: A Re-wired Green Fluorescent Protein: Folding and Function in a Non-sequential, Non-circular GFP Permutant
    Article Snippet: Overlapping oligomers for each gene were designed using the DNAworks 2.0 program ( ) with the following parameters: 40nt oligo length, codon frequency threshold of 20, 50 mM Na+ /K+ , 200 nM oligo concentration, annealing temperature of 64ºC, number of solutions of 1, and 2 mM Mg2+ . .. Completed genes with included NheI and EcoRI (enzymes from New England Biosciences, NEB) restriction sites were inserted into pET28a (Novagen) using T4 ligase per manufacturers instructions (NEB) and transformed into XL-1Blue or BL21 for DNA sequencing ( ) and BL21 for expression (Invitrogen).

    Article Title: Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs
    Article Snippet: The plasmids were transformed into BL21*(DE3) Escherichia coli ( E. coli) (Thermo Fisher Scientific, Chicago, IL, USA) using a standard heat-shock protocol. .. The plasmids were transformed into BL21*(DE3) Escherichia coli ( E. coli) (Thermo Fisher Scientific, Chicago, IL, USA) using a standard heat-shock protocol.

    Polymerase Cycling Assembly:

    Article Title: A Re-wired Green Fluorescent Protein: Folding and Function in a Non-sequential, Non-circular GFP Permutant
    Article Snippet: Assembly PCR was performed first with 0.2 ng/mL of each oligomer , Pfu Turbo (Stratagene), with an annealing temperature of 59ºC (5º below set point temperature in DNAworks) and 35 cycles. .. Completed genes with included NheI and EcoRI (enzymes from New England Biosciences, NEB) restriction sites were inserted into pET28a (Novagen) using T4 ligase per manufacturers instructions (NEB) and transformed into XL-1Blue or BL21 for DNA sequencing ( ) and BL21 for expression (Invitrogen).

    SDS Page:

    Article Title: Vaccination with Killed but Metabolically Active E. coli Over-expressing Hemagglutinin Elicits Neutralizing Antibodies to H1N1 Swine Origin Influenza A Virus
    Article Snippet: The amplified DNA products were inserted into the In-Fusion Ready pEcoli-Nterm 6×HN vector (Clontech Laboratories, Mountain View, CA) and transformed into competent cells [ (E. coli), BL21 (DE3), Invitrogen, Carlsbad, CA]. .. As a control, a pEcoli-Nterm-green fluorescent protein (GFP) plasmid (Clontech Laboratories Inc.) was transformed.

    Plasmid Preparation:

    Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors
    Article Snippet: In this study, we have surmounted the fore-mentioned hurdle to applying the bacterial-surface-P-protein-display system in a viral receptor isolation context by adding a thrombin-susceptible domain to the existing construct to facilitate release of the P-protein-candidate-receptor complex from bacteria to enable easy purification ( Figure ). .. Competent E. coli DH5α and BL21 (Thermo Fisher, Shanghai, China) were used for recombinant plasmid construction and protein expression, respectively. .. Oligonucleotide “TB” coding for the peptide sequence of Leu-Val-Pro-Arg-Gly-Ser, was synthesized by Suzhou GENEWIZ Bio-Technology, Co., Ltd. Then, the artificially synthesized inaQn-TB sequence was inserted into cloning vector pUC57 to create pUC57-inaQn-TB.

    Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
    Article Snippet: The plasmid pcDNA3.0-SNP22b was constructed by subcloning the SNP gene, released from pET22b-SNP22b by BamHI/EcoRI, into pcDNA3.0 (Invitrogen). .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

    Article Title: Biological Role of Trichoderma harzianum-Derived Platelet-Activating Factor Acetylhydrolase (PAF-AH) on Stress Response and Antagonism
    Article Snippet: Wild type and transformants were maintained in PDA (Difco, Becton Dickinson & Co., USA) at 28°C until sporulation occurred. .. Escherichia coli DH5α and BL21 (DE3) were purchased from Invitrogen (Carlsbad, CA, USA) for plasmid propagation and protein expression, respectively. .. Bacterial strains were grown in Luria-Bertani (LB) broth or LB agar plates supplemented with kanamycin sulfate (0.1 mg/mL) or ampicillin (0.1 mg/mL).

    Article Title: Vaccination with Killed but Metabolically Active E. coli Over-expressing Hemagglutinin Elicits Neutralizing Antibodies to H1N1 Swine Origin Influenza A Virus
    Article Snippet: The forward PCR primer (5′-AATAGTCGACATGAAGGCAATACTAGTAGTTCTGCTAT AT-3′) and the reverse PCR primer (5′-GATACAGCGGCCG CTTAAATACATATTCTACACTGTAGAGACCCA-3′) were designed for a PCR reaction. .. The amplified DNA products were inserted into the In-Fusion Ready pEcoli-Nterm 6×HN vector (Clontech Laboratories, Mountain View, CA) and transformed into competent cells [ (E. coli), BL21 (DE3), Invitrogen, Carlsbad, CA]. .. As a control, a pEcoli-Nterm-green fluorescent protein (GFP) plasmid (Clontech Laboratories Inc.) was transformed.

    Article Title: Accurate de novo design of hyperstable constrained peptides
    Article Snippet: Genes of designed disulfide-rich peptides were cloned into the vector pCDB180 (which we have made available via Addgene) using Gibson Assembly . .. Designed proteins were expressed from BL21*(DE3) E. coli (Invitrogen), and expression cultures were grown overnight with incubation at 37 °C and shaking at 225 RPM.

    Article Title: Acoustically modulated magnetic resonance imaging of gas-filled protein nanostructures
    Article Snippet: Gas vesicle concentrations were determined using the relationships summarized in . .. For reporter gene experiments, the gene cluster encoding a hybrid GV variant named A2C was cloned into pET28a plasmid (Novagen, Temecula, CA), and the resulting plasmid was transformed into BL21(A1) cells (Thermo Fisher Scientific, Waltham, MA). .. The transformed cells were first grown overnight at 30°C in LB media supplemented with 1% glucose, and this starter culture was subsequently diluted 1:100 into LB media supplemented with 0.2% glucose.

    Article Title: A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites
    Article Snippet: The PCR product was cloned by ligation independent cloning into vector BG1861 , which introduces an N-terminal 6xHis tag and the construct was verified by Sanger sequencing. .. BG1861:6xHis-PfHAD1 was transformed into BL21(DE3)pLysS Escherichia coli cells (Life Technologies).

    Article Title: Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs
    Article Snippet: Genes for the five constructs were synthesized and subcloned into a pET26b(+) vector (Thermo Scientific, Chicago, IL, USA). .. The plasmids were transformed into BL21*(DE3) Escherichia coli ( E. coli) (Thermo Fisher Scientific, Chicago, IL, USA) using a standard heat-shock protocol.

    Software:

    Article Title: Vaccination with Killed but Metabolically Active E. coli Over-expressing Hemagglutinin Elicits Neutralizing Antibodies to H1N1 Swine Origin Influenza A Virus
    Article Snippet: The amplified DNA products were inserted into the In-Fusion Ready pEcoli-Nterm 6×HN vector (Clontech Laboratories, Mountain View, CA) and transformed into competent cells [ (E. coli), BL21 (DE3), Invitrogen, Carlsbad, CA]. .. After isopropyl β-D-thiogalactopyranoside (IPTG) (1 mM) induction and centrifugation at 3,000 × g at 4°C for 5 min, bacterial pellets were re-suspended with sodium dodecyl sulfate (SDS) loading buffer [125 mM Tris-HCl buffer, pH 6.8, containing 4% (w/v) SDS, 10% (w/v) glycerol, 5% (v/v) 2-mercaptoethanol and 0.002% (w/v) bromophenol blue] and then boiled for 5 min. SDS-polyacrylamide gel electrophoresis (SDS-PAGE, 10%) and subsequent gel staining with coomassie blue were used for detection of protein expression.

    Negative Control:

    Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors
    Article Snippet: Competent E. coli DH5α and BL21 (Thermo Fisher, Shanghai, China) were used for recombinant plasmid construction and protein expression, respectively. .. After re-digestion of plasmids pET28a, pET28a-P (GII.4) and pUC57-inaQn-TB ( Table ), the inaQn -TB and P (GII.4) fragment were inserted into pET-28a to create recombinant plasmid pET28a-inaQn-TB-P (GII.4).

    Affinity Chromatography:

    Article Title: Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs
    Article Snippet: The plasmids were transformed into BL21*(DE3) Escherichia coli ( E. coli) (Thermo Fisher Scientific, Chicago, IL, USA) using a standard heat-shock protocol. .. Protein production proceeded overnight at 25 °C after induced expression with 100 μM isopropyl β- d -1-thiogalactopyranoside (IPTG) at an optical density measured at a wavelength of 600 nm (OD600 ) of 0.8.

    Activation Assay:

    Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
    Article Snippet: BL21 (DE3) was obtained from Invitrogen Life Technologies (Carlsbad, CA). .. BL21 (DE3) was obtained from Invitrogen Life Technologies (Carlsbad, CA).

    Staining:

    Article Title: Vaccination with Killed but Metabolically Active E. coli Over-expressing Hemagglutinin Elicits Neutralizing Antibodies to H1N1 Swine Origin Influenza A Virus
    Article Snippet: The amplified DNA products were inserted into the In-Fusion Ready pEcoli-Nterm 6×HN vector (Clontech Laboratories, Mountain View, CA) and transformed into competent cells [ (E. coli), BL21 (DE3), Invitrogen, Carlsbad, CA]. .. As a control, a pEcoli-Nterm-green fluorescent protein (GFP) plasmid (Clontech Laboratories Inc.) was transformed.

    Molecular Cloning:

    Article Title: Vaccination with Killed but Metabolically Active E. coli Over-expressing Hemagglutinin Elicits Neutralizing Antibodies to H1N1 Swine Origin Influenza A Virus
    Article Snippet: Molecular cloning, expression, and purification of recombinant HA of SD/H1N1-S-OIV The complementary DNA (cDNA) of SD/H1N1-S-OIV was used as a template ( ) to amplify a polymerase chain reaction (PCR) product encoding SD/H1N1-S-OIV HA (Accession number: ). .. The amplified DNA products were inserted into the In-Fusion Ready pEcoli-Nterm 6×HN vector (Clontech Laboratories, Mountain View, CA) and transformed into competent cells [ (E. coli), BL21 (DE3), Invitrogen, Carlsbad, CA].

    Variant Assay:

    Article Title: Acoustically modulated magnetic resonance imaging of gas-filled protein nanostructures
    Article Snippet: Gas vesicle concentrations were determined using the relationships summarized in . .. For reporter gene experiments, the gene cluster encoding a hybrid GV variant named A2C was cloned into pET28a plasmid (Novagen, Temecula, CA), and the resulting plasmid was transformed into BL21(A1) cells (Thermo Fisher Scientific, Waltham, MA). .. The transformed cells were first grown overnight at 30°C in LB media supplemented with 1% glucose, and this starter culture was subsequently diluted 1:100 into LB media supplemented with 0.2% glucose.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 76
    Thermo Fisher bl21
    SDS-PAGE (A) and Western Blot (B) analysis of the expression of E. coli cell surface display systems for HuNoV (GII.4) recombinant capsid proteins with (3) or without the linker fragment (2) and the thrombin-released P proteins (4). M: prestained protein ladder (Catalog No.: 26616, Thermo Fisher, Shanghai, China); (1) <t>pET-28a/BL21;</t> (2) pET28a-inaQn-P (GII.4)/BL21; (3) pET28a-inaQn-TB-P (GII.4) /BL21; (4) Thrombin-released P proteins.
    Bl21, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 76/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bl21/product/Thermo Fisher
    Average 76 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    bl21 - by Bioz Stars, 2020-01
    76/100 stars
      Buy from Supplier

    99
    Thermo Fisher bl21 ai
    Transcription-dependent loss of the target plasmid. <t>BL21-AI</t> strain with the STH Csm module plasmid was challenged with plasmids with STH target sequence or without a target sequence with or without IPTG (1μM) as indicated. (A) Plasmid targeting assay was performed as in Fig 2 (without arabinose). Corresponding Csm modules and targets are indicated with green boxes. (B) Presence of plasmids was assessed by plasmid extraction following overnight growth in medium with or without 1mM IPTG as indicated. Plasmid DNA was linearized, separated by agarose gel electrophoresis and visualized with ethidium bromide. Plasmid DNA from 4 individual colonies is shown for each condition. Positions of linearized Csm module and target plasmids are indicated. Dashed lines indicate rearranged non-contiguous sections of single original gel.
    Bl21 Ai, supplied by Thermo Fisher, 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/bl21 ai/product/Thermo Fisher
    Average 99 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    bl21 ai - by Bioz Stars, 2020-01
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher escherichia coli bl21 de3 cells
    : expression, purification, and detection of the recombinant argenine transcriptional regulator arginine catabolism mobile element (rArcRACME) protein. (A) The amount of the rArcRACME protein in the insoluble fraction of a total protein extract from <t>Escherichia</t> coli <t>BL21-arcR,</t> induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37ºC at different time points. Lane 1, E. coli BL21 (DE3) without recombinant plasmid pET303/CT-His. Lane 2, E. coli BL21 (DE3) with recombinant plasmid pET303/CT-His, not induced. rArcRACME (~27 kDa) was purified from overnight (O.N.) culture of E. coli BL21-arcR by electroelution and visualised by SDS-PAGE (B) and western blotting (C) using an anti-6x-His antibody. Lane C in the western blot is a positive control for the anti-His antibody. NI: not induced.
    Escherichia Coli Bl21 De3 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/escherichia coli bl21 de3 cells/product/Thermo Fisher
    Average 99 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    escherichia coli bl21 de3 cells - by Bioz Stars, 2020-01
    99/100 stars
      Buy from Supplier

    Image Search Results


    SDS-PAGE (A) and Western Blot (B) analysis of the expression of E. coli cell surface display systems for HuNoV (GII.4) recombinant capsid proteins with (3) or without the linker fragment (2) and the thrombin-released P proteins (4). M: prestained protein ladder (Catalog No.: 26616, Thermo Fisher, Shanghai, China); (1) pET-28a/BL21; (2) pET28a-inaQn-P (GII.4)/BL21; (3) pET28a-inaQn-TB-P (GII.4) /BL21; (4) Thrombin-released P proteins.

    Journal: Frontiers in Microbiology

    Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors

    doi: 10.3389/fmicb.2017.02405

    Figure Lengend Snippet: SDS-PAGE (A) and Western Blot (B) analysis of the expression of E. coli cell surface display systems for HuNoV (GII.4) recombinant capsid proteins with (3) or without the linker fragment (2) and the thrombin-released P proteins (4). M: prestained protein ladder (Catalog No.: 26616, Thermo Fisher, Shanghai, China); (1) pET-28a/BL21; (2) pET28a-inaQn-P (GII.4)/BL21; (3) pET28a-inaQn-TB-P (GII.4) /BL21; (4) Thrombin-released P proteins.

    Article Snippet: Competent E. coli DH5α and BL21 (Thermo Fisher, Shanghai, China) were used for recombinant plasmid construction and protein expression, respectively.

    Techniques: SDS Page, Western Blot, Expressing, Recombinant, Positron Emission Tomography

    OD of type A HBGA bound to serially diluted thrombin-released P proteins (red) and the thrombin-released background proteins from pET28a-inaQn-TB/BL21 (black). Bars represent standard errors.

    Journal: Frontiers in Microbiology

    Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors

    doi: 10.3389/fmicb.2017.02405

    Figure Lengend Snippet: OD of type A HBGA bound to serially diluted thrombin-released P proteins (red) and the thrombin-released background proteins from pET28a-inaQn-TB/BL21 (black). Bars represent standard errors.

    Article Snippet: Competent E. coli DH5α and BL21 (Thermo Fisher, Shanghai, China) were used for recombinant plasmid construction and protein expression, respectively.

    Techniques:

    Whole-genome deep sequencing reveals off-target spacer integration events within the E. coli genome a , Schematic of the experimental workflow. A culture of E. coli BL21 expressing Cas1 and Cas2 is electroporated with a 35-bp oligo protospacer that includes a 5′-AAG PAM. Following electroporation and outgrowth, the total DNA content of the cells is isolated, fragmented and shotgun sequenced on an Illumina high-throughput sequencing machine. Reads are mapped back to the BL21 reference genome. Spacer integration events are identified as an ~61-bp insertion, which includes the spacer sequence (33 bp) and the duplicated target site (~28-bp repeat). b , Eight of the off-target integration sites discovered within the genome, shown in the diagram labelled as the gene in which they were inserted. The origin of the dashed arrows indicates the site of the genome-derived spacer and point towards the site of integration. Note that off-target integration events within the lacI gene are not shown because they cannot be unambiguously mapped to the genome or plasmid. c , Comparison between the number of on-target integrations into the first position of the CRISPR1 array and off-target integrations elsewhere in the genome outside of the CRISPR1 array. Data represent the results from a single WGS experiment.

    Journal:

    Article Title: Spontaneous CRISPR loci generation in vivo by non-canonical spacer integration

    doi: 10.1038/s41564-017-0097-z

    Figure Lengend Snippet: Whole-genome deep sequencing reveals off-target spacer integration events within the E. coli genome a , Schematic of the experimental workflow. A culture of E. coli BL21 expressing Cas1 and Cas2 is electroporated with a 35-bp oligo protospacer that includes a 5′-AAG PAM. Following electroporation and outgrowth, the total DNA content of the cells is isolated, fragmented and shotgun sequenced on an Illumina high-throughput sequencing machine. Reads are mapped back to the BL21 reference genome. Spacer integration events are identified as an ~61-bp insertion, which includes the spacer sequence (33 bp) and the duplicated target site (~28-bp repeat). b , Eight of the off-target integration sites discovered within the genome, shown in the diagram labelled as the gene in which they were inserted. The origin of the dashed arrows indicates the site of the genome-derived spacer and point towards the site of integration. Note that off-target integration events within the lacI gene are not shown because they cannot be unambiguously mapped to the genome or plasmid. c , Comparison between the number of on-target integrations into the first position of the CRISPR1 array and off-target integrations elsewhere in the genome outside of the CRISPR1 array. Data represent the results from a single WGS experiment.

    Article Snippet: Briefly, liquid cultures of E. coli BL21-AI cells (Thermo) harbouring a plasmid expressing Cas1 and Cas2 under the control of a T7-lac promoter (pWUR 1 + 2, which was a generous gift from U. Qimron) were started from plates and grown overnight in LB.

    Techniques: Sequencing, Expressing, Electroporation, Isolation, Next-Generation Sequencing, Derivative Assay, Plasmid Preparation

    Transcription-dependent loss of the target plasmid. BL21-AI strain with the STH Csm module plasmid was challenged with plasmids with STH target sequence or without a target sequence with or without IPTG (1μM) as indicated. (A) Plasmid targeting assay was performed as in Fig 2 (without arabinose). Corresponding Csm modules and targets are indicated with green boxes. (B) Presence of plasmids was assessed by plasmid extraction following overnight growth in medium with or without 1mM IPTG as indicated. Plasmid DNA was linearized, separated by agarose gel electrophoresis and visualized with ethidium bromide. Plasmid DNA from 4 individual colonies is shown for each condition. Positions of linearized Csm module and target plasmids are indicated. Dashed lines indicate rearranged non-contiguous sections of single original gel.

    Journal: PLoS ONE

    Article Title: Programmable type III-A CRISPR-Cas DNA targeting modules

    doi: 10.1371/journal.pone.0176221

    Figure Lengend Snippet: Transcription-dependent loss of the target plasmid. BL21-AI strain with the STH Csm module plasmid was challenged with plasmids with STH target sequence or without a target sequence with or without IPTG (1μM) as indicated. (A) Plasmid targeting assay was performed as in Fig 2 (without arabinose). Corresponding Csm modules and targets are indicated with green boxes. (B) Presence of plasmids was assessed by plasmid extraction following overnight growth in medium with or without 1mM IPTG as indicated. Plasmid DNA was linearized, separated by agarose gel electrophoresis and visualized with ethidium bromide. Plasmid DNA from 4 individual colonies is shown for each condition. Positions of linearized Csm module and target plasmids are indicated. Dashed lines indicate rearranged non-contiguous sections of single original gel.

    Article Snippet: BL21-AI (Thermo Fisher Scientific) was transformed with a Csm module plasmid to generate a host strain.

    Techniques: Plasmid Preparation, Sequencing, Agarose Gel Electrophoresis

    Csm modules prevent plasmid invasion in E . coli . The BL21-AI strains containing the indicated Csm module plasmids (first column; LLA, SEP or STH) were each transformed with four target plasmids (second column; LLA, SEP, STH or vector). A series of 10-fold dilutions (10 0 to 10 6 ) of transformed cells were plated on a LB agar containing 50 μg/ml ampicillin and 34 μg/ml chloramphenicol with or without 0.2% arabinose as indicated. Representative results of 3 experiments are shown. Corresponding Csm modules and targets are indicated with green boxes. Dashed lines separate data from different plates.

    Journal: PLoS ONE

    Article Title: Programmable type III-A CRISPR-Cas DNA targeting modules

    doi: 10.1371/journal.pone.0176221

    Figure Lengend Snippet: Csm modules prevent plasmid invasion in E . coli . The BL21-AI strains containing the indicated Csm module plasmids (first column; LLA, SEP or STH) were each transformed with four target plasmids (second column; LLA, SEP, STH or vector). A series of 10-fold dilutions (10 0 to 10 6 ) of transformed cells were plated on a LB agar containing 50 μg/ml ampicillin and 34 μg/ml chloramphenicol with or without 0.2% arabinose as indicated. Representative results of 3 experiments are shown. Corresponding Csm modules and targets are indicated with green boxes. Dashed lines separate data from different plates.

    Article Snippet: BL21-AI (Thermo Fisher Scientific) was transformed with a Csm module plasmid to generate a host strain.

    Techniques: Plasmid Preparation, Transformation Assay

    : expression, purification, and detection of the recombinant argenine transcriptional regulator arginine catabolism mobile element (rArcRACME) protein. (A) The amount of the rArcRACME protein in the insoluble fraction of a total protein extract from Escherichia coli BL21-arcR, induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37ºC at different time points. Lane 1, E. coli BL21 (DE3) without recombinant plasmid pET303/CT-His. Lane 2, E. coli BL21 (DE3) with recombinant plasmid pET303/CT-His, not induced. rArcRACME (~27 kDa) was purified from overnight (O.N.) culture of E. coli BL21-arcR by electroelution and visualised by SDS-PAGE (B) and western blotting (C) using an anti-6x-His antibody. Lane C in the western blot is a positive control for the anti-His antibody. NI: not induced.

    Journal: Memórias do Instituto Oswaldo Cruz

    Article Title: Participation of the arcRACME protein in self-activation of the arc operon located in the arginine catabolism mobile element in pandemic clone USA300

    doi: 10.1590/0074-02760160424

    Figure Lengend Snippet: : expression, purification, and detection of the recombinant argenine transcriptional regulator arginine catabolism mobile element (rArcRACME) protein. (A) The amount of the rArcRACME protein in the insoluble fraction of a total protein extract from Escherichia coli BL21-arcR, induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37ºC at different time points. Lane 1, E. coli BL21 (DE3) without recombinant plasmid pET303/CT-His. Lane 2, E. coli BL21 (DE3) with recombinant plasmid pET303/CT-His, not induced. rArcRACME (~27 kDa) was purified from overnight (O.N.) culture of E. coli BL21-arcR by electroelution and visualised by SDS-PAGE (B) and western blotting (C) using an anti-6x-His antibody. Lane C in the western blot is a positive control for the anti-His antibody. NI: not induced.

    Article Snippet: Additionally, to test whether the ArcRACME protein binds to the regulatory region of the arc ACME operon, rArcRACME (recombinant ArcRACME protein) was produced and purified from Escherichia coli BL21 (DE3) cells following the manufacturer recommendations (Thermo Scientific) (Supplementary data, Table I and methods).

    Techniques: Expressing, Purification, Recombinant, Plasmid Preparation, SDS Page, Western Blot, Positive Control