bl21 star de3  (Thermo Fisher)


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    Structured Review

    Thermo Fisher bl21 star de3
    Systematic study of AND gate circuit overlap domain lengths and comparison of two-input AND ribocomputing devices in different strains a , An early two-input AND gate was constructed from a standard toehold switch by dividing the trigger evenly into two 15-nt domains A1 and A2. Overlap domains u and u* were designed to cause the two input RNAs to hybridize and form an active trigger. b , A domain u’ was used to vary the region complementary to u* and measure its effect on expression levels. ON/OFF GFP ratios (left axis) vary as a function of the u’ domain length. The onset of substantial GFP expression coincides with the melting temperature of u’-u* hybridization rising above 37°C (right axis). c-f , Comparison of two-input AND ribocomputing devices in RNase-deficient E. coli <t>BL21</t> Star <t>DE3</t> and non-RNase-deficient E. coli BL21 DE3. c-d , ON/OFF GFP on linear (c) and logarithmic (d) scales measured for the two-input AND gate from Figure 2e–h . e-f , ON/OFF GFP on linear (e) and logarithmic (f) scales measured for a second two-input AND gate with an identical design but different RNA sequences.
    Bl21 Star De3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bl21 star de3/product/Thermo Fisher
    Average 99 stars, based on 29 article reviews
    Price from $9.99 to $1999.99
    bl21 star de3 - by Bioz Stars, 2020-02
    99/100 stars

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    1) Product Images from "Complex cellular logic computation using ribocomputing devices"

    Article Title: Complex cellular logic computation using ribocomputing devices

    Journal: Nature

    doi: 10.1038/nature23271

    Systematic study of AND gate circuit overlap domain lengths and comparison of two-input AND ribocomputing devices in different strains a , An early two-input AND gate was constructed from a standard toehold switch by dividing the trigger evenly into two 15-nt domains A1 and A2. Overlap domains u and u* were designed to cause the two input RNAs to hybridize and form an active trigger. b , A domain u’ was used to vary the region complementary to u* and measure its effect on expression levels. ON/OFF GFP ratios (left axis) vary as a function of the u’ domain length. The onset of substantial GFP expression coincides with the melting temperature of u’-u* hybridization rising above 37°C (right axis). c-f , Comparison of two-input AND ribocomputing devices in RNase-deficient E. coli BL21 Star DE3 and non-RNase-deficient E. coli BL21 DE3. c-d , ON/OFF GFP on linear (c) and logarithmic (d) scales measured for the two-input AND gate from Figure 2e–h . e-f , ON/OFF GFP on linear (e) and logarithmic (f) scales measured for a second two-input AND gate with an identical design but different RNA sequences.
    Figure Legend Snippet: Systematic study of AND gate circuit overlap domain lengths and comparison of two-input AND ribocomputing devices in different strains a , An early two-input AND gate was constructed from a standard toehold switch by dividing the trigger evenly into two 15-nt domains A1 and A2. Overlap domains u and u* were designed to cause the two input RNAs to hybridize and form an active trigger. b , A domain u’ was used to vary the region complementary to u* and measure its effect on expression levels. ON/OFF GFP ratios (left axis) vary as a function of the u’ domain length. The onset of substantial GFP expression coincides with the melting temperature of u’-u* hybridization rising above 37°C (right axis). c-f , Comparison of two-input AND ribocomputing devices in RNase-deficient E. coli BL21 Star DE3 and non-RNase-deficient E. coli BL21 DE3. c-d , ON/OFF GFP on linear (c) and logarithmic (d) scales measured for the two-input AND gate from Figure 2e–h . e-f , ON/OFF GFP on linear (e) and logarithmic (f) scales measured for a second two-input AND gate with an identical design but different RNA sequences.

    Techniques Used: Construct, Expressing, Hybridization

    Comparison of six-input OR gate ribocomputing devices measured in RNase-deficient E. coli (BL21 Star DE3) and non-RNase-deficient E. coli (BL21 DE3, MG1655Pro) a-b , ON/OFF GFP ratios measured for the device using T7 RNA polymerase in BL21 Star DE3 and BL21 DE3 cells on linear (a) and logarithmic (b) scales. Gate and input RNAs were expressed using the T7 RNA polymerase and measured 4 hours after induction with IPTG. c-d , ON/OFF GFP ratios obtained from the OR gate using E. coli RNA polymerase in MG1655Pro cells on linear (c) and logarithmic (d) scales. Gate and input RNAs were expressed using the E. coli RNA polymerase and measured 4 hours after induction of the gate RNA with IPTG. Input and decoy RNAs were expressed using a constitutive PN25 promoter.
    Figure Legend Snippet: Comparison of six-input OR gate ribocomputing devices measured in RNase-deficient E. coli (BL21 Star DE3) and non-RNase-deficient E. coli (BL21 DE3, MG1655Pro) a-b , ON/OFF GFP ratios measured for the device using T7 RNA polymerase in BL21 Star DE3 and BL21 DE3 cells on linear (a) and logarithmic (b) scales. Gate and input RNAs were expressed using the T7 RNA polymerase and measured 4 hours after induction with IPTG. c-d , ON/OFF GFP ratios obtained from the OR gate using E. coli RNA polymerase in MG1655Pro cells on linear (c) and logarithmic (d) scales. Gate and input RNAs were expressed using the E. coli RNA polymerase and measured 4 hours after induction of the gate RNA with IPTG. Input and decoy RNAs were expressed using a constitutive PN25 promoter.

    Techniques Used:

    Related Articles

    Clone Assay:

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    Centrifugation:

    Article Title: Structure of a patient-derived antibody in complex with allergen reveals simultaneous conventional and superantigen-like recognition
    Article Snippet: For expression, the plasmid was transformed into BL21 star DE3 (Life Technologies) and was plated on ampicillin overnight. .. The bacteria were harvested by centrifugation at 4,000 × g and then were lysed using BugBuster (Merck Millipore) according to the manufacturer’s instructions.

    Amplification:

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    Phosphatase Assay:

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    Article Snippet: Paragraph title: Phosphatase Assay. ... cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen).

    Synthesized:

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    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
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    Construct:

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
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    Article Title: An Efficient Strategy for Small-Scale Screening and Production of Archaeal Membrane Transport Proteins in Escherichia coli
    Article Snippet: .. Over-production of archaeal membrane transport proteins Expression constructs were used to transform two E. coli strains, BL21 Star (Invitrogen) and C43(DE3) [ ], containing the pRARE2 plasmid. .. Expression trials were performed as follows.

    Incubation:

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: .. Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C. .. All proteins had combined C-terminal Flag and His6 peptides for affinity purification.

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development
    Article Snippet: cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen). .. After eletrophoresis, the gel was incubated in 2.5% Triton X-100 for 30 min and then phosphatase assay buffer (50 mM Tris⋅HCl, pH 7.9/10% glycerol/10 mM MgCl2 /10 mM potassium acetate/1 mM ZnCl2 ) for 30 min.

    Expressing:

    Article Title: Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿ †
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    Article Title: A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity
    Article Snippet: .. Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific). .. Expression was confirmed by using anti-V5 monoclonal antibodies by western blotting and purified by 6xHis tag elution using nickel resins (Promega).

    Article Title: Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins
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    Article Title: Hetero-oligomer of dynamin-related proteins participates in the fission of highly divergent mitochondria from Entamoeba histolytica
    Article Snippet: .. These plasmids were transformed into BL21 Star™ (DE3) One Shot® Chemically Competent E . coli (Invitrogen) and expression of recombinant proteins was induced by low temperature (15 °C) with 1 mM IPTG. .. After induction, target proteins were purified from bacterial lysates by Ni-NTA system (QIAGEN GmbH, Hilden, Germany).

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: .. Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C. .. All proteins had combined C-terminal Flag and His6 peptides for affinity purification.

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: .. Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. Mammalian expression vectors encoding N-terminally FLAG-tagged OGG1s were constructed by cloning PCR amplified OGG1 genes into pCMV-2B expression vector (Stratagene).

    Article Title: An Efficient Strategy for Small-Scale Screening and Production of Archaeal Membrane Transport Proteins in Escherichia coli
    Article Snippet: .. Over-production of archaeal membrane transport proteins Expression constructs were used to transform two E. coli strains, BL21 Star (Invitrogen) and C43(DE3) [ ], containing the pRARE2 plasmid. .. Expression trials were performed as follows.

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development
    Article Snippet: cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen). .. For recombinant protein synthesis, BL21SI cells without and with pDCPL1 plasmid were induced for 10 h by adding 0.3 M NaCl into the medium. pDCPL3 was introduced into BL21 star because of low AtCPL3 expression in BL21SI, and cells were induced with 1 mM isopropyl β- d -thiogalactoside for 10 h. The recombinant AtCPL1 and AtCPL3 proteins were recovered in the insoluble fraction after sonication of bacterial cells in 50 mM Tris (pH 7.5) buffer and extensive washes with 1% Triton X-100.

    Article Title: Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
    Article Snippet: Paragraph title: Expression and purification of recombinant proteins. ... The pMW17 vector was transformed into BL21 Star(DE3) (Invitrogen, Carlsbad, CA) cells for protein production.

    Article Title: Structure of a patient-derived antibody in complex with allergen reveals simultaneous conventional and superantigen-like recognition
    Article Snippet: .. For expression, the plasmid was transformed into BL21 star DE3 (Life Technologies) and was plated on ampicillin overnight. ..

    Western Blot:

    Article Title: A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity
    Article Snippet: Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific). .. Expression was confirmed by using anti-V5 monoclonal antibodies by western blotting and purified by 6xHis tag elution using nickel resins (Promega).

    Transformation Assay:

    Article Title: Hetero-oligomer of dynamin-related proteins participates in the fission of highly divergent mitochondria from Entamoeba histolytica
    Article Snippet: .. These plasmids were transformed into BL21 Star™ (DE3) One Shot® Chemically Competent E . coli (Invitrogen) and expression of recombinant proteins was induced by low temperature (15 °C) with 1 mM IPTG. .. After induction, target proteins were purified from bacterial lysates by Ni-NTA system (QIAGEN GmbH, Hilden, Germany).

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development
    Article Snippet: .. cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen). .. For recombinant protein synthesis, BL21SI cells without and with pDCPL1 plasmid were induced for 10 h by adding 0.3 M NaCl into the medium. pDCPL3 was introduced into BL21 star because of low AtCPL3 expression in BL21SI, and cells were induced with 1 mM isopropyl β- d -thiogalactoside for 10 h. The recombinant AtCPL1 and AtCPL3 proteins were recovered in the insoluble fraction after sonication of bacterial cells in 50 mM Tris (pH 7.5) buffer and extensive washes with 1% Triton X-100.

    Article Title: Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
    Article Snippet: .. The pMW17 vector was transformed into BL21 Star(DE3) (Invitrogen, Carlsbad, CA) cells for protein production. ..

    Article Title: Structure of a patient-derived antibody in complex with allergen reveals simultaneous conventional and superantigen-like recognition
    Article Snippet: .. For expression, the plasmid was transformed into BL21 star DE3 (Life Technologies) and was plated on ampicillin overnight. ..

    Over Expression:

    Article Title: Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿ †
    Article Snippet: Paragraph title: Overexpression and purification of WXG100 proteins as heterodimers. ... A 5-ml starter culture of BL21 Star (Invitrogen) cells carrying the expression plasmid was inoculated into 400 ml of broth, grown at 37°C to an optical density at 600 nm of 0.7, and induced with isopropyl-β- d -thiogalactopyranoside (IPTG) (3 × 10−4 M).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: The primers for CFL2 amplification were as follows: 5′-ATG CGA ATT CAT GGC TTC TGG AGT TAC A-3′and 5′-ATG CCT CGA GTG GCA CTT GAC TGT CAT T-3′ for GST-CFL2 (aa 1 to 166), 5′-CCG TGA ATT CAC ATG GCT TCT GGA GTT-3′ and 5′-ACG TCT CGA GCA AGA TCT GCT TTG CTT C-3′ for GST-CFL2-A (aa 1 to 55), 5′-ACG TGA ATT CAA GTC ATC AAA GTT T-3′and 5′-ACG TCT CGA GCT ACT ACA TTG CCT C-3′ for GST-CFL2-B (aa 105 to 166), and 5′-ATG CGA ATT CAG ATC TTG GTG GGT GAC-3′ and 5′-ATG CCT CGA GAC TTT CAG GAG CCC A-3′ for GST-CFL2-C (aa 55 to 108). .. Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    High Performance Liquid Chromatography:

    Article Title: A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity
    Article Snippet: Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific). .. Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific).

    Chromatography:

    Article Title: Structure of a patient-derived antibody in complex with allergen reveals simultaneous conventional and superantigen-like recognition
    Article Snippet: For expression, the plasmid was transformed into BL21 star DE3 (Life Technologies) and was plated on ampicillin overnight. .. The soluble fraction had imidazole added to a final concentration of 10 mM and then was passed over a 1-mL HisTrap column (GE Healthcare) using an AKTA prime chromatography system (GE Healthcare Life Sciences) and was washed with Tris-buffered saline (TBS) buffer [25 mM Tris⋅HCl (pH 7.4), 300 mM NaCl, 4 mM CaCl2 ] containing 10 mM imidazole.

    Oligonucleotide Synthesis:

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: Oligonucleotides (79–80 bases) were synthesized by solid phase oligonucleotide synthesis (MerMade 192; BioAutomation) and assembled into full-length open reading frames (ORFs) by automation , which were reamplified by PCR using 5′-biotinylated primers. .. Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C.

    SPR Assay:

    Article Title: Complex cellular logic computation using ribocomputing devices
    Article Snippet: .. Strains and growth conditions The following E. coli strains were used in this study: BL21 Star DE3 (F− ompT hsdS B (rB − mB − ) gal dcm rne131 (DE3); Invitrogen), BL21 DE3 (F− ompT hsdS B (rB − mB − ) gal dcm (DE3); Invitrogen), MG1655Pro (F− λ− ilvG- rfb-50 rph-1 SpR lacR tetR ), and DH5α (endA1 recA1 gyrA96 thi-1 glnV44 relA1 hsdR17 (rK − mK + ) λ− ). ..

    DNA Sequencing:

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. All plasmid sequences were verified by bi-directional DNA sequencing.

    Polymerase Chain Reaction:

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: For the generation of the CFL2 constructs, PCR products were digested with EcoRI/XhoI (Takara Bio, Otsu, Shiga, Japan) and subcloned into the pGEX-5X vector (Amersham Biosciences Europe). .. Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: .. Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C. .. All proteins had combined C-terminal Flag and His6 peptides for affinity purification.

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: .. Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. Mammalian expression vectors encoding N-terminally FLAG-tagged OGG1s were constructed by cloning PCR amplified OGG1 genes into pCMV-2B expression vector (Stratagene).

    Article Title: Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH
    Article Snippet: Strains and plasmids The E. coli strains used were JM109 from Promega (Madison, WI, USA), BL21 (DE3) and Rosetta 2 (DE3) from Novagen (VWR International GmbH, Vienna, Austria) and BL21 star (DE3) from Invitrogen (Carlsbad, California, USA). .. The Cb FDH gene was amplified from pETDuet_XR_FDH by a PCR using Pfu DNA polymerase and primers providing Pag I (compatible ends to Nco I) and Avr II restriction sites.

    Article Title: An evolutionary route to xylanase process fitness
    Article Snippet: The PCR products were cloned into pTrcHis2 as described above. .. Overnight cultures of BL21 Star (Invitrogen) cells carrying the appropriate gene in pTrcHis2 were used to inoculate 1.5 L of Terrific broth (12 g/liter Bactotryptone, 24 g/L yeast extract, 4 ml/L glycerol, 12.5 g/L K2 HPO4 , 2.3 g/L KH2 PO4 ) containing 100 μg/mL ampicillin in a six-liter shake flask.

    Sonication:

    Article Title: Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿ †
    Article Snippet: A 5-ml starter culture of BL21 Star (Invitrogen) cells carrying the expression plasmid was inoculated into 400 ml of broth, grown at 37°C to an optical density at 600 nm of 0.7, and induced with isopropyl-β- d -thiogalactopyranoside (IPTG) (3 × 10−4 M). .. Cell pellets were resuspended in 15 ml of a buffer containing urea (8 M), Tris-HCl (0.01 M), and NaH2 PO4 (0.1 M) and were lysed by sonication with 10 to 15 bursts (10 s) at maximum power.

    Article Title: Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins
    Article Snippet: TOP10 (Invitrogen) and BL21 Star (Invitrogen) were used for protein expression. .. H. pylori CCUG 17874 DNA was sonicated (10 sec on, then 10 sec off over 3–5 min) at maximum power.

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development
    Article Snippet: cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen). .. For recombinant protein synthesis, BL21SI cells without and with pDCPL1 plasmid were induced for 10 h by adding 0.3 M NaCl into the medium. pDCPL3 was introduced into BL21 star because of low AtCPL3 expression in BL21SI, and cells were induced with 1 mM isopropyl β- d -thiogalactoside for 10 h. The recombinant AtCPL1 and AtCPL3 proteins were recovered in the insoluble fraction after sonication of bacterial cells in 50 mM Tris (pH 7.5) buffer and extensive washes with 1% Triton X-100.

    Affinity Purification:

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C. .. All proteins had combined C-terminal Flag and His6 peptides for affinity purification.

    Binding Assay:

    Article Title: A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity
    Article Snippet: Immunizations to induce CD4+ T-cell responses were performed with 20-mers containing the core 11 amino acids because MHC class II molecules have open-ended binding pockets and additional amino acids on either side enhances immunoreactivity . .. Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific).

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: The following primer sets were used for amplification: 5′-ATC GGA ATT CGT CTT CAA GAT GCC AAA C-3′ and 5′-ATC GGT CGA CTC TGT GCA GGA TTA CTT G-3′ for maltose binding protein (MBP)-MLP (amino acids [aa] 1 to 195), 5′-ATG CGA ATT CAA ATG TGG AGC CTG TGA A-3′and 5′-ACT GGT CGA CGA CTG TTG GAA CTG CAG G-3′ for MBP-MLP-LIM1 (aa 9 to 94), 5′-ACG TGA ATT CAG CAA CCC TTC CAA ATT C-3′ and 5′-ACG TGT CGA CTG TTG TGT AAG GCC TCC A-3′ for MBP-MLP-LIM2 (aa 105 to 188), and 5′-ATG CGA ATT CCG CAG ATA TGG CCC CAA A-3′and 5′-ATC GGT CGA CGG ACT CTC CAA CTT CGC-3′ for MBP-MLP-inter-LIM (aa 64 to 117). .. Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    Cellular Antioxidant Activity Assay:

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: The primers for CFL2 amplification were as follows: 5′-ATG CGA ATT CAT GGC TTC TGG AGT TAC A-3′and 5′-ATG CCT CGA GTG GCA CTT GAC TGT CAT T-3′ for GST-CFL2 (aa 1 to 166), 5′-CCG TGA ATT CAC ATG GCT TCT GGA GTT-3′ and 5′-ACG TCT CGA GCA AGA TCT GCT TTG CTT C-3′ for GST-CFL2-A (aa 1 to 55), 5′-ACG TGA ATT CAA GTC ATC AAA GTT T-3′and 5′-ACG TCT CGA GCT ACT ACA TTG CCT C-3′ for GST-CFL2-B (aa 105 to 166), and 5′-ATG CGA ATT CAG ATC TTG GTG GGT GAC-3′ and 5′-ATG CCT CGA GAC TTT CAG GAG CCC A-3′ for GST-CFL2-C (aa 55 to 108). .. Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    Countercurrent Chromatography:

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: The primers for CFL2 amplification were as follows: 5′-ATG CGA ATT CAT GGC TTC TGG AGT TAC A-3′and 5′-ATG CCT CGA GTG GCA CTT GAC TGT CAT T-3′ for GST-CFL2 (aa 1 to 166), 5′-CCG TGA ATT CAC ATG GCT TCT GGA GTT-3′ and 5′-ACG TCT CGA GCA AGA TCT GCT TTG CTT C-3′ for GST-CFL2-A (aa 1 to 55), 5′-ACG TGA ATT CAA GTC ATC AAA GTT T-3′and 5′-ACG TCT CGA GCT ACT ACA TTG CCT C-3′ for GST-CFL2-B (aa 105 to 166), and 5′-ATG CGA ATT CAG ATC TTG GTG GGT GAC-3′ and 5′-ATG CCT CGA GAC TTT CAG GAG CCC A-3′ for GST-CFL2-C (aa 55 to 108). .. Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    Mutagenesis:

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: .. Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. Mammalian expression vectors encoding N-terminally FLAG-tagged OGG1s were constructed by cloning PCR amplified OGG1 genes into pCMV-2B expression vector (Stratagene).

    Article Title: An evolutionary route to xylanase process fitness
    Article Snippet: Paragraph title: Construction and purification of wild-type and mutant enzymes ... Overnight cultures of BL21 Star (Invitrogen) cells carrying the appropriate gene in pTrcHis2 were used to inoculate 1.5 L of Terrific broth (12 g/liter Bactotryptone, 24 g/L yeast extract, 4 ml/L glycerol, 12.5 g/L K2 HPO4 , 2.3 g/L KH2 PO4 ) containing 100 μg/mL ampicillin in a six-liter shake flask.

    Size-exclusion Chromatography:

    Article Title: Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins
    Article Snippet: TOP10 (Invitrogen) and BL21 Star (Invitrogen) were used for protein expression. .. H. pylori CCUG 17874 DNA was sonicated (10 sec on, then 10 sec off over 3–5 min) at maximum power.

    Purification:

    Article Title: Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿ †
    Article Snippet: Paragraph title: Overexpression and purification of WXG100 proteins as heterodimers. ... A 5-ml starter culture of BL21 Star (Invitrogen) cells carrying the expression plasmid was inoculated into 400 ml of broth, grown at 37°C to an optical density at 600 nm of 0.7, and induced with isopropyl-β- d -thiogalactopyranoside (IPTG) (3 × 10−4 M).

    Article Title: A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity
    Article Snippet: Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific). .. Expression was confirmed by using anti-V5 monoclonal antibodies by western blotting and purified by 6xHis tag elution using nickel resins (Promega).

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains. .. The recombinant proteins were purified by affinity chromatography using glutathione Sepharose 4B beads (Amersham Biosciences) for GST fusion proteins or amylose resin beads for MBP fusion proteins (New England Biolabs), according to the manufacturer's instructions.

    Article Title: Hetero-oligomer of dynamin-related proteins participates in the fission of highly divergent mitochondria from Entamoeba histolytica
    Article Snippet: These plasmids were transformed into BL21 Star™ (DE3) One Shot® Chemically Competent E . coli (Invitrogen) and expression of recombinant proteins was induced by low temperature (15 °C) with 1 mM IPTG. .. After induction, target proteins were purified from bacterial lysates by Ni-NTA system (QIAGEN GmbH, Hilden, Germany).

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C. .. Proteins were purified at 4°C from TnT reactions using anti-Flag M2 agarose (Sigma) and Cl− -free buffer: four (15 min) washes of agarose-bound FPs with 20 m m HEPES, pH 7.1, removed Cl− before elution with 120 μl of 3×Flag (Sigma).

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: .. Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. Mammalian expression vectors encoding N-terminally FLAG-tagged OGG1s were constructed by cloning PCR amplified OGG1 genes into pCMV-2B expression vector (Stratagene).

    Article Title: Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
    Article Snippet: Paragraph title: Expression and purification of recombinant proteins. ... The pMW17 vector was transformed into BL21 Star(DE3) (Invitrogen, Carlsbad, CA) cells for protein production.

    Article Title: An evolutionary route to xylanase process fitness
    Article Snippet: Paragraph title: Construction and purification of wild-type and mutant enzymes ... Overnight cultures of BL21 Star (Invitrogen) cells carrying the appropriate gene in pTrcHis2 were used to inoculate 1.5 L of Terrific broth (12 g/liter Bactotryptone, 24 g/L yeast extract, 4 ml/L glycerol, 12.5 g/L K2 HPO4 , 2.3 g/L KH2 PO4 ) containing 100 μg/mL ampicillin in a six-liter shake flask.

    Sequencing:

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: .. Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. Mammalian expression vectors encoding N-terminally FLAG-tagged OGG1s were constructed by cloning PCR amplified OGG1 genes into pCMV-2B expression vector (Stratagene).

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development
    Article Snippet: .. cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen). .. For recombinant protein synthesis, BL21SI cells without and with pDCPL1 plasmid were induced for 10 h by adding 0.3 M NaCl into the medium. pDCPL3 was introduced into BL21 star because of low AtCPL3 expression in BL21SI, and cells were induced with 1 mM isopropyl β- d -thiogalactoside for 10 h. The recombinant AtCPL1 and AtCPL3 proteins were recovered in the insoluble fraction after sonication of bacterial cells in 50 mM Tris (pH 7.5) buffer and extensive washes with 1% Triton X-100.

    Article Title: Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
    Article Snippet: To purify the kinase domain of SrrB, primers srrBfwd-NdeI and srrBrev-XhoI were used to amplify the sequence from S. aureus LAC genomic DNA that was cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA). .. The pMW17 vector was transformed into BL21 Star(DE3) (Invitrogen, Carlsbad, CA) cells for protein production.

    Article Title: Structure of a patient-derived antibody in complex with allergen reveals simultaneous conventional and superantigen-like recognition
    Article Snippet: The DNA was cloned into a pET151 vector, which includes an N-terminal cleavable V5- and His-tag, according to the manufacturer’s instructions (Life Technologies), and the sequence was verified. .. For expression, the plasmid was transformed into BL21 star DE3 (Life Technologies) and was plated on ampicillin overnight.

    Affinity Chromatography:

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains. .. The recombinant proteins were purified by affinity chromatography using glutathione Sepharose 4B beads (Amersham Biosciences) for GST fusion proteins or amylose resin beads for MBP fusion proteins (New England Biolabs), according to the manufacturer's instructions.

    Recombinant:

    Article Title: A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity
    Article Snippet: .. Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific). .. Expression was confirmed by using anti-V5 monoclonal antibodies by western blotting and purified by 6xHis tag elution using nickel resins (Promega).

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: Paragraph title: Construction of recombinant proteins. ... Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    Article Title: Hetero-oligomer of dynamin-related proteins participates in the fission of highly divergent mitochondria from Entamoeba histolytica
    Article Snippet: .. These plasmids were transformed into BL21 Star™ (DE3) One Shot® Chemically Competent E . coli (Invitrogen) and expression of recombinant proteins was induced by low temperature (15 °C) with 1 mM IPTG. .. After induction, target proteins were purified from bacterial lysates by Ni-NTA system (QIAGEN GmbH, Hilden, Germany).

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: .. Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. Mammalian expression vectors encoding N-terminally FLAG-tagged OGG1s were constructed by cloning PCR amplified OGG1 genes into pCMV-2B expression vector (Stratagene).

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development
    Article Snippet: cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen). .. For recombinant protein synthesis, BL21SI cells without and with pDCPL1 plasmid were induced for 10 h by adding 0.3 M NaCl into the medium. pDCPL3 was introduced into BL21 star because of low AtCPL3 expression in BL21SI, and cells were induced with 1 mM isopropyl β- d -thiogalactoside for 10 h. The recombinant AtCPL1 and AtCPL3 proteins were recovered in the insoluble fraction after sonication of bacterial cells in 50 mM Tris (pH 7.5) buffer and extensive washes with 1% Triton X-100.

    Article Title: Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
    Article Snippet: Paragraph title: Expression and purification of recombinant proteins. ... The pMW17 vector was transformed into BL21 Star(DE3) (Invitrogen, Carlsbad, CA) cells for protein production.

    Lysis:

    Article Title: An evolutionary route to xylanase process fitness
    Article Snippet: Overnight cultures of BL21 Star (Invitrogen) cells carrying the appropriate gene in pTrcHis2 were used to inoculate 1.5 L of Terrific broth (12 g/liter Bactotryptone, 24 g/L yeast extract, 4 ml/L glycerol, 12.5 g/L K2 HPO4 , 2.3 g/L KH2 PO4 ) containing 100 μg/mL ampicillin in a six-liter shake flask. .. The pellet was resuspended in lysis buffer (50 mM potassium phosphate [pH 7.0], 100 mM KCl, 7.5 mM β-mercaptoethanol) and cells were lysed by French press (15,000 psi).

    Activated Clotting Time Assay:

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: The primers for CFL2 amplification were as follows: 5′-ATG CGA ATT CAT GGC TTC TGG AGT TAC A-3′and 5′-ATG CCT CGA GTG GCA CTT GAC TGT CAT T-3′ for GST-CFL2 (aa 1 to 166), 5′-CCG TGA ATT CAC ATG GCT TCT GGA GTT-3′ and 5′-ACG TCT CGA GCA AGA TCT GCT TTG CTT C-3′ for GST-CFL2-A (aa 1 to 55), 5′-ACG TGA ATT CAA GTC ATC AAA GTT T-3′and 5′-ACG TCT CGA GCT ACT ACA TTG CCT C-3′ for GST-CFL2-B (aa 105 to 166), and 5′-ATG CGA ATT CAG ATC TTG GTG GGT GAC-3′ and 5′-ATG CCT CGA GAC TTT CAG GAG CCC A-3′ for GST-CFL2-C (aa 55 to 108). .. Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    SDS Page:

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development
    Article Snippet: cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen). .. AtCPL proteins were prepared in nonreducing SDS/PAGE loading buffer and size-fractioned in a 6.25% SDS/PAGE gel ( ).

    Plasmid Preparation:

    Article Title: Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿ †
    Article Snippet: .. A 5-ml starter culture of BL21 Star (Invitrogen) cells carrying the expression plasmid was inoculated into 400 ml of broth, grown at 37°C to an optical density at 600 nm of 0.7, and induced with isopropyl-β- d -thiogalactopyranoside (IPTG) (3 × 10−4 M). ..

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: For the generation of the CFL2 constructs, PCR products were digested with EcoRI/XhoI (Takara Bio, Otsu, Shiga, Japan) and subcloned into the pGEX-5X vector (Amersham Biosciences Europe). .. Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    Article Title: Hetero-oligomer of dynamin-related proteins participates in the fission of highly divergent mitochondria from Entamoeba histolytica
    Article Snippet: Fragments were digested by appropriate sets of restriction enzymes and ligated into pCold™ I plasmid (TaKaRa). .. These plasmids were transformed into BL21 Star™ (DE3) One Shot® Chemically Competent E . coli (Invitrogen) and expression of recombinant proteins was induced by low temperature (15 °C) with 1 mM IPTG.

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: .. Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. Mammalian expression vectors encoding N-terminally FLAG-tagged OGG1s were constructed by cloning PCR amplified OGG1 genes into pCMV-2B expression vector (Stratagene).

    Article Title: An Efficient Strategy for Small-Scale Screening and Production of Archaeal Membrane Transport Proteins in Escherichia coli
    Article Snippet: .. Over-production of archaeal membrane transport proteins Expression constructs were used to transform two E. coli strains, BL21 Star (Invitrogen) and C43(DE3) [ ], containing the pRARE2 plasmid. .. Expression trials were performed as follows.

    Article Title: Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH
    Article Snippet: Strains and plasmids The E. coli strains used were JM109 from Promega (Madison, WI, USA), BL21 (DE3) and Rosetta 2 (DE3) from Novagen (VWR International GmbH, Vienna, Austria) and BL21 star (DE3) from Invitrogen (Carlsbad, California, USA). .. The construction of the E. coli BL21 (DE3) (BL21_XR_FDH) harbouring Ct XR and Cb FDH genes on a pETDuet-1 vector (pETDuet_XR_FDH) was described elsewhere [ ].

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development
    Article Snippet: cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen). .. For recombinant protein synthesis, BL21SI cells without and with pDCPL1 plasmid were induced for 10 h by adding 0.3 M NaCl into the medium. pDCPL3 was introduced into BL21 star because of low AtCPL3 expression in BL21SI, and cells were induced with 1 mM isopropyl β- d -thiogalactoside for 10 h. The recombinant AtCPL1 and AtCPL3 proteins were recovered in the insoluble fraction after sonication of bacterial cells in 50 mM Tris (pH 7.5) buffer and extensive washes with 1% Triton X-100.

    Article Title: Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
    Article Snippet: .. The pMW17 vector was transformed into BL21 Star(DE3) (Invitrogen, Carlsbad, CA) cells for protein production. ..

    Article Title: Structure of a patient-derived antibody in complex with allergen reveals simultaneous conventional and superantigen-like recognition
    Article Snippet: .. For expression, the plasmid was transformed into BL21 star DE3 (Life Technologies) and was plated on ampicillin overnight. ..

    Software:

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C. .. Protein expression and purity relative to a sample of the Clomeleon chloride-sensitive YFP Topaz domain (CT) was determined by densitometry of the YFP band in protein gels stained with GelCode Blue (Thermo Scientific) using ImageJ (release 1.42q) software (good category of expression > 90% pure; poorly expressed proteins were less pure).

    Positron Emission Tomography:

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: .. Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. Mammalian expression vectors encoding N-terminally FLAG-tagged OGG1s were constructed by cloning PCR amplified OGG1 genes into pCMV-2B expression vector (Stratagene).

    In Vitro:

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: .. Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C. .. All proteins had combined C-terminal Flag and His6 peptides for affinity purification.

    Produced:

    Article Title: A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity
    Article Snippet: .. Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific). .. Expression was confirmed by using anti-V5 monoclonal antibodies by western blotting and purified by 6xHis tag elution using nickel resins (Promega).

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: .. Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C. .. All proteins had combined C-terminal Flag and His6 peptides for affinity purification.

    Concentration Assay:

    Article Title: l-Arabinose Isomerase and d-Xylose Isomerase from Lactobacillus reuteri: Characterization, Coexpression in the Food Grade Host Lactobacillus plantarum, and Application in the Conversion of d-Galactose and d-Glucose
    Article Snippet: E. coli strain NEB5α (New England Biolabs, Ipswich, MA, USA), MB2159 (d -alanine auxotrophe), and BL21 Star (DE3) (Invitrogen, Carlsbad, CA, USA) were grown in Luria–Bertani (LB) medium at 37 °C with shaking at 125 rpm. .. When needed, media were supplemented with ampicillin at a concentration of 100 μg/mL and erythromycin at concentrations of 5 and 200 μg/mL for L. plantarum and E. coli , respectively.

    Article Title: Structure of a patient-derived antibody in complex with allergen reveals simultaneous conventional and superantigen-like recognition
    Article Snippet: For expression, the plasmid was transformed into BL21 star DE3 (Life Technologies) and was plated on ampicillin overnight. .. The soluble fraction had imidazole added to a final concentration of 10 mM and then was passed over a 1-mL HisTrap column (GE Healthcare) using an AKTA prime chromatography system (GE Healthcare Life Sciences) and was washed with Tris-buffered saline (TBS) buffer [25 mM Tris⋅HCl (pH 7.4), 300 mM NaCl, 4 mM CaCl2 ] containing 10 mM imidazole.

    CTG Assay:

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: The following primer sets were used for amplification: 5′-ATC GGA ATT CGT CTT CAA GAT GCC AAA C-3′ and 5′-ATC GGT CGA CTC TGT GCA GGA TTA CTT G-3′ for maltose binding protein (MBP)-MLP (amino acids [aa] 1 to 195), 5′-ATG CGA ATT CAA ATG TGG AGC CTG TGA A-3′and 5′-ACT GGT CGA CGA CTG TTG GAA CTG CAG G-3′ for MBP-MLP-LIM1 (aa 9 to 94), 5′-ACG TGA ATT CAG CAA CCC TTC CAA ATT C-3′ and 5′-ACG TGT CGA CTG TTG TGT AAG GCC TCC A-3′ for MBP-MLP-LIM2 (aa 105 to 188), and 5′-ATG CGA ATT CCG CAG ATA TGG CCC CAA A-3′and 5′-ATC GGT CGA CGG ACT CTC CAA CTT CGC-3′ for MBP-MLP-inter-LIM (aa 64 to 117). .. Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    Staining:

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C. .. Protein expression and purity relative to a sample of the Clomeleon chloride-sensitive YFP Topaz domain (CT) was determined by densitometry of the YFP band in protein gels stained with GelCode Blue (Thermo Scientific) using ImageJ (release 1.42q) software (good category of expression > 90% pure; poorly expressed proteins were less pure).

    Variant Assay:

    Article Title: A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity
    Article Snippet: The residue numbers, in subscript, and peptide sequences, in brackets, of the peptides used are: mouse MPO409–428 peptide (PRWNGEKLYQEARKIVGAMV), Treponema vincentii hypothetical protein167–186 (LRKQLKRLYKEARKIQKCIP), Aspergillus fumigatus HEAT repeat protein825–844 (ISALPQRWYQEARKIIFEAA), Helicobacter pylori RNA polymerase factor sigma-54163–182 (RELDNNELYEEARKIILNLE), Bacteroides sp . chloramphenicol O-acetyltransferase104–123 (YHEDFETFYQEARKIIDSIP), S. aureus pSJH101-derived 6PGD391–410 (YFKNIVTDYQEALRDVVATG), S. aureus 6PGD391–410 Variant 1 (YFKNIVTDYQDALRDVVATG), S. aureus 6PGD391–410 Variant 2 (YFKNIVTNYQEALRDVVATG), S. aureus 6PGD391–410 Variant 3 (YFKNIVTEYQDALRDVVATG), S. aureus 6PGD391–410 Variant 4 (YFKNIVTNYQDALRDVVATG), Mus musculus 6PGD394–413 (FFKSAVDNCQDSWRRVISTGV), and control OVA323–339 peptide (ISQAVHAAHAEINEAGR). .. Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific).

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