lemo21  (New England Biolabs)


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    Name:
    Lemo21 DE3 Competent E coli
    Description:
    Lemo21 DE3 Competent E coli 12x0 05 ml
    Catalog Number:
    c2528j
    Price:
    228
    Size:
    12x0 05 ml
    Category:
    Competent Bacteria
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    Structured Review

    New England Biolabs lemo21
    Lemo21 DE3 Competent E coli
    Lemo21 DE3 Competent E coli 12x0 05 ml
    https://www.bioz.com/result/lemo21/product/New England Biolabs
    Average 95 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    lemo21 - by Bioz Stars, 2020-02
    95/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Bacterial Polysaccharide Specificity of the Pattern Recognition Receptor Langerin Is Highly Species-dependent *
    Article Snippet: Paragraph title: Cloning, Protein Expression, and Purification ... Murine CRD was expressed in E. coli Lemo21(DE3) (New England Biolabs); solubilized in 6 m guanidine HCl; refolded by rapid dilution into 0.8 m l -arginine in TBS, pH 7.5, containing 2.5 m m reduced and 0.5 m m oxidized glutathione; and purified via a StrepTactin column followed by dialysis against 25 m m MES, 40 m m NaCl, pH 6.

    Article Title: Discovery of Polyesterases from Moss-Associated Microorganisms
    Article Snippet: Paragraph title: Subcloning, sequencing of fosmids, and cloning of esterases. ... Ligated/hybridized plasmids were transformed into chemically competent E. coli BL21(DE3) cells (Invitrogen, USA) and E. coli Lemo21(DE3) cells (New England BioLabs, Germany).

    Article Title: De Novo Design of Bioactive Protein Switches
    Article Snippet: For pET29b+ constructs, the synthesized DNA was cloned in frame with the C-terminal hexahistidine tag. .. Plasmids were transformed into chemically competent E. coli BL21(DE3)Star, BL21(DE3)Star-pLysS cells (Invitrogen), or Lemo21(DE3) cells (NEB) for protein expression.

    Article Title: SLC38A9 is a component of the lysosomal amino acid-sensing machinery that controls mTORC1
    Article Snippet: Paragraph title: Cloning, expression and purification of recombinant human SLC38A9 ... The plasmid was used to transform E. coli Lemo21(DE3)pLysS (NEB).

    Article Title: Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family
    Article Snippet: Paragraph title: Cloning of ActTBEA6 . ... After selection of transformants using LB medium containing ampicillin, the hybrid plasmids were isolated, analyzed by sequencing, and used for transformation of CaCl2 -competent cells of E. coli Lemo21(DE3) (New England BioLabs, Inc., Ipswich, MA).

    Amplification:

    Article Title: Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family
    Article Snippet: act TBEA6 was amplified from total genomic DNA of V. paradoxus strain TBEA6 by PCR using Platinum Taq DNA polymerase (Invitrogen, Karlsruhe, Germany) and the following oligonucleotides: act _ Hind III_For and act _ Xho I_Rev_oS (see Table S1 in the supplemental material). .. After selection of transformants using LB medium containing ampicillin, the hybrid plasmids were isolated, analyzed by sequencing, and used for transformation of CaCl2 -competent cells of E. coli Lemo21(DE3) (New England BioLabs, Inc., Ipswich, MA).

    Filtration:

    Article Title: IP6K Structure and the Molecular Determinants of Catalytic Specificity in an Inositol Phosphate Kinase Family
    Article Snippet: As a final step, a Superdex™ 200 gel filtration column (GE Healthcare) was used with a running buffer of 50 mM NaCl and 20 mM Tris-HCl, pH 7.5. .. Lemo21(DE3) Competent E. coli cells (New England Biolabs) were transformed with the resultant plasmid.

    Synthesized:

    Article Title: De Novo Design of Bioactive Protein Switches
    Article Snippet: For pET29b+ constructs, the synthesized DNA was cloned in frame with the C-terminal hexahistidine tag. .. Plasmids were transformed into chemically competent E. coli BL21(DE3)Star, BL21(DE3)Star-pLysS cells (Invitrogen), or Lemo21(DE3) cells (NEB) for protein expression.

    Construct:

    Article Title: An engineered thermal-shift screen reveals specific lipid preferences of eukaryotic and prokaryotic membrane proteins
    Article Snippet: Bacterial membrane protein–GFP fusions: The bacterial membrane protein–GFP8His fusions had been previously constructed into the expression vector, pWaldo GFPd , . .. For overexpression, a single colony of freshly transformed E. coli Lemo21 (DE3) cells from New England Biolabs harboring a pWaldo membrane protein–GFP8His fusion on a Luria Bertani (LB) agar/kanamycin plate, was used to inoculate 20 mL of LB broth supplemented with 50 μg mL−1 kanamycin and grown at 37 °C with shaking at 200 rpm using Innova 44 Incubator shaker (NEW BRUNSWICK) for 16 h. The cultures were then used to inoculate 2 × 1 L of MemStar medium and grown to an OD600 of 0.5.

    Article Title: Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon
    Article Snippet: Preparation of fluorescence-labeled SecYEG SecYEG for fluorescence labeling was expressed from a pTrc99a construct containing N-terminally His6 -tagged SecE. .. The protein was expressed in Lemo21(DE3) E.coli strain (New England Biolabs), expression was induced at OD600 of 0.6 by 0.4 mM IPTG for 4 h. SecY(S111C)EG was purified as described previously ( ) by metal-affinity chromatography, followed by cation-exchange chromatography.

    Article Title: De Novo Design of Bioactive Protein Switches
    Article Snippet: For pET29b+ constructs, the synthesized DNA was cloned in frame with the C-terminal hexahistidine tag. .. Plasmids were transformed into chemically competent E. coli BL21(DE3)Star, BL21(DE3)Star-pLysS cells (Invitrogen), or Lemo21(DE3) cells (NEB) for protein expression.

    Article Title: An engineered thermal-shift screen reveals specific lipid preferences of eukaryotic and prokaryotic membrane proteins
    Article Snippet: Isolation of membrane protein–GFP fusion membranes Bacterial membrane protein–GFP fusions: The bacterial membrane protein–GFP8His fusions had been previously constructed into the expression vector, pWaldo GFPd , . .. For overexpression, a single colony of freshly transformed E. coli Lemo21 (DE3) cells from New England Biolabs harboring a pWaldo membrane protein–GFP8His fusion on a Luria Bertani (LB) agar/kanamycin plate, was used to inoculate 20 mL of LB broth supplemented with 50 μg mL−1 kanamycin and grown at 37 °C with shaking at 200 rpm using Innova 44 Incubator shaker (NEW BRUNSWICK) for 16 h. The cultures were then used to inoculate 2 × 1 L of MemStar medium and grown to an OD600 of 0.5.

    Incubation:

    Article Title: Programmable design of orthogonal protein heterodimers
    Article Snippet: Plasmids were transformed into chemically competent E. coli expression strains BL21(DE3)Star (Invitrogen) or Lemo21(DE3) (New England Biolabs) for protein expression. .. Starter cultures were diluted into 500 ml TBM-5052 containing 100 μg/mL carbenicillin or kanamycin, and incubated with shaking at 225 rpm for 24 hours at 37°C.

    Expressing:

    Article Title: Bacterial Polysaccharide Specificity of the Pattern Recognition Receptor Langerin Is Highly Species-dependent *
    Article Snippet: Paragraph title: Cloning, Protein Expression, and Purification ... Murine CRD was expressed in E. coli Lemo21(DE3) (New England Biolabs); solubilized in 6 m guanidine HCl; refolded by rapid dilution into 0.8 m l -arginine in TBS, pH 7.5, containing 2.5 m m reduced and 0.5 m m oxidized glutathione; and purified via a StrepTactin column followed by dialysis against 25 m m MES, 40 m m NaCl, pH 6.

    Article Title: IP6K Structure and the Molecular Determinants of Catalytic Specificity in an Inositol Phosphate Kinase Family
    Article Snippet: The Gateway expression system (Invitrogen) was used to subclone the kinase into the pDest-566 vector. .. Lemo21(DE3) Competent E. coli cells (New England Biolabs) were transformed with the resultant plasmid.

    Article Title: An engineered thermal-shift screen reveals specific lipid preferences of eukaryotic and prokaryotic membrane proteins
    Article Snippet: Bacterial membrane protein–GFP fusions: The bacterial membrane protein–GFP8His fusions had been previously constructed into the expression vector, pWaldo GFPd , . .. For overexpression, a single colony of freshly transformed E. coli Lemo21 (DE3) cells from New England Biolabs harboring a pWaldo membrane protein–GFP8His fusion on a Luria Bertani (LB) agar/kanamycin plate, was used to inoculate 20 mL of LB broth supplemented with 50 μg mL−1 kanamycin and grown at 37 °C with shaking at 200 rpm using Innova 44 Incubator shaker (NEW BRUNSWICK) for 16 h. The cultures were then used to inoculate 2 × 1 L of MemStar medium and grown to an OD600 of 0.5.

    Article Title: Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon
    Article Snippet: .. The protein was expressed in Lemo21(DE3) E.coli strain (New England Biolabs), expression was induced at OD600 of 0.6 by 0.4 mM IPTG for 4 h. SecY(S111C)EG was purified as described previously ( ) by metal-affinity chromatography, followed by cation-exchange chromatography. .. Labeling with MDCC maleimide was performed by following the protocol provided by the manufacturer (Invitrogen), using labeling buffer (20 mM Hepes, pH 7.0, 150 mM KCl, 10% [wt/vol] glycerol, and 0.03% DDM).

    Article Title: De Novo Design of Bioactive Protein Switches
    Article Snippet: .. Sequence verified plasmid was purified using Qiagen miniprep kit and transformed into chemically competent E. coli BL21(DE3)Star, BL21(DE3)Star-pLysS cells (Invitrogen), or Lemo21(DE3) cells (NEB) for protein expression. .. Synthetic gene construction Synthetic genes were ordered from Genscript Inc. (Piscataway, NJ, USA) and delivered in pET 28b+, pET21b+, or pET29b+ E. coli expression vectors, inserted at the NdeI and XhoI sites of each vector.

    Article Title: Programmable design of orthogonal protein heterodimers
    Article Snippet: .. Plasmids were transformed into chemically competent E. coli expression strains BL21(DE3)Star (Invitrogen) or Lemo21(DE3) (New England Biolabs) for protein expression. .. Single colonies were picked from agar plates following transformation and growth overnight, and 5 ml starter cultures were grown at 37°C in Luria-Bertani (LB) medium containing 100 μg/mL carbenicillin (for pET21-NESG vectors) or kanamycin (for pRSFDuet-1 vectors) with shaking at 225 rpm for 18 hours at 37°C.

    Article Title: De Novo Design of Bioactive Protein Switches
    Article Snippet: .. Plasmids were transformed into chemically competent E. coli BL21(DE3)Star, BL21(DE3)Star-pLysS cells (Invitrogen), or Lemo21(DE3) cells (NEB) for protein expression. .. Bacterial protein expression and purification Starter cultures were grown in Lysogeny Broth (LB) or Terrific Broth II (TBII) overnight in the presence of 50 μg/mL carbenicillin (pET21b+) or 30 μg/mL (for LB) to 60 μg/mL (for TBII) kanamycin (pET28b+ and pET29b+).

    Article Title: Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic β-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity
    Article Snippet: Paragraph title: Expression and purification of IP6K1 ... Lemo21 (DE3) Competent E. coli cells (New England Biolabs, Massachusetts, USA) were transformed with the resultant plasmid.

    Article Title: SLC38A9 is a component of the lysosomal amino acid-sensing machinery that controls mTORC1
    Article Snippet: Paragraph title: Cloning, expression and purification of recombinant human SLC38A9 ... The plasmid was used to transform E. coli Lemo21(DE3)pLysS (NEB).

    Article Title: Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family
    Article Snippet: After ligation with the expression vector pET22b(+), which was linearized with the same restriction endonucleases, the ligation product, pET22b(+):: act TBEA6 (see Fig. S1 in the supplemental material), was used for transformation of CaCl2 -competent cells of E. coli Top10. .. After selection of transformants using LB medium containing ampicillin, the hybrid plasmids were isolated, analyzed by sequencing, and used for transformation of CaCl2 -competent cells of E. coli Lemo21(DE3) (New England BioLabs, Inc., Ipswich, MA).

    Article Title: An engineered thermal-shift screen reveals specific lipid preferences of eukaryotic and prokaryotic membrane proteins
    Article Snippet: Isolation of membrane protein–GFP fusion membranes Bacterial membrane protein–GFP fusions: The bacterial membrane protein–GFP8His fusions had been previously constructed into the expression vector, pWaldo GFPd , . .. For overexpression, a single colony of freshly transformed E. coli Lemo21 (DE3) cells from New England Biolabs harboring a pWaldo membrane protein–GFP8His fusion on a Luria Bertani (LB) agar/kanamycin plate, was used to inoculate 20 mL of LB broth supplemented with 50 μg mL−1 kanamycin and grown at 37 °C with shaking at 200 rpm using Innova 44 Incubator shaker (NEW BRUNSWICK) for 16 h. The cultures were then used to inoculate 2 × 1 L of MemStar medium and grown to an OD600 of 0.5.

    Transformation Assay:

    Article Title: IP6K Structure and the Molecular Determinants of Catalytic Specificity in an Inositol Phosphate Kinase Family
    Article Snippet: .. Lemo21(DE3) Competent E. coli cells (New England Biolabs) were transformed with the resultant plasmid. ..

    Article Title: An engineered thermal-shift screen reveals specific lipid preferences of eukaryotic and prokaryotic membrane proteins
    Article Snippet: .. For overexpression, a single colony of freshly transformed E. coli Lemo21 (DE3) cells from New England Biolabs harboring a pWaldo membrane protein–GFP8His fusion on a Luria Bertani (LB) agar/kanamycin plate, was used to inoculate 20 mL of LB broth supplemented with 50 μg mL−1 kanamycin and grown at 37 °C with shaking at 200 rpm using Innova 44 Incubator shaker (NEW BRUNSWICK) for 16 h. The cultures were then used to inoculate 2 × 1 L of MemStar medium and grown to an OD600 of 0.5. .. The cells were then induced with 0.4 mM IPTG (Sigma) and the temperature of the shaker dropped from 37 to 25 °C and grown further for 16 h. Cells were harvested by centrifuging at 10,000 g for 10 min at 4 °C, resuspended in 200 mL of ice-cold 1× phosphate-buffered saline (PBS) buffer and flash-frozen in liquid nitrogen before storing at −80 °C until use.

    Article Title: Discovery of Polyesterases from Moss-Associated Microorganisms
    Article Snippet: .. Ligated/hybridized plasmids were transformed into chemically competent E. coli BL21(DE3) cells (Invitrogen, USA) and E. coli Lemo21(DE3) cells (New England BioLabs, Germany). .. The strains employed for expression of the six cloned esterases in pET28a(+) were E. coli Lemo21(DE3) for EstB3, EstB11, EstC5, EstC9, and EstG4 and E. coli BL21(DE3) for EstC7.

    Article Title: De Novo Design of Bioactive Protein Switches
    Article Snippet: .. Sequence verified plasmid was purified using Qiagen miniprep kit and transformed into chemically competent E. coli BL21(DE3)Star, BL21(DE3)Star-pLysS cells (Invitrogen), or Lemo21(DE3) cells (NEB) for protein expression. .. Synthetic gene construction Synthetic genes were ordered from Genscript Inc. (Piscataway, NJ, USA) and delivered in pET 28b+, pET21b+, or pET29b+ E. coli expression vectors, inserted at the NdeI and XhoI sites of each vector.

    Article Title: Programmable design of orthogonal protein heterodimers
    Article Snippet: .. Plasmids were transformed into chemically competent E. coli expression strains BL21(DE3)Star (Invitrogen) or Lemo21(DE3) (New England Biolabs) for protein expression. .. Single colonies were picked from agar plates following transformation and growth overnight, and 5 ml starter cultures were grown at 37°C in Luria-Bertani (LB) medium containing 100 μg/mL carbenicillin (for pET21-NESG vectors) or kanamycin (for pRSFDuet-1 vectors) with shaking at 225 rpm for 18 hours at 37°C.

    Article Title: De Novo Design of Bioactive Protein Switches
    Article Snippet: .. Plasmids were transformed into chemically competent E. coli BL21(DE3)Star, BL21(DE3)Star-pLysS cells (Invitrogen), or Lemo21(DE3) cells (NEB) for protein expression. .. Bacterial protein expression and purification Starter cultures were grown in Lysogeny Broth (LB) or Terrific Broth II (TBII) overnight in the presence of 50 μg/mL carbenicillin (pET21b+) or 30 μg/mL (for LB) to 60 μg/mL (for TBII) kanamycin (pET28b+ and pET29b+).

    Article Title: Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic β-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity
    Article Snippet: .. Lemo21 (DE3) Competent E. coli cells (New England Biolabs, Massachusetts, USA) were transformed with the resultant plasmid. .. An overnight culture of the transformed E. coli cells was inoculated into nutrient-rich 2xYT medium supplemented with 0.6 mM l -rhamnose at pH 7.5 and grown at 37 °C to A 595 = 0.7.

    Article Title: Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family
    Article Snippet: .. After selection of transformants using LB medium containing ampicillin, the hybrid plasmids were isolated, analyzed by sequencing, and used for transformation of CaCl2 -competent cells of E. coli Lemo21(DE3) (New England BioLabs, Inc., Ipswich, MA). .. The 526- and 691-bp fragments upstream and downstream of act TBEA6 were amplified by using the primers Xba I_upAct/ Nde I_upAct or Nde I_downAct/ Xba I_downAct, respectively.

    Article Title: Substrate-bound outward-open structure of a Na+-coupled sialic acid symporter reveals a new Na+ site
    Article Snippet: .. Protein production and purification The pSiaT1 plasmid was transformed into the E. coli Lemo21(DE3) strain (NEB). ..

    Article Title: An engineered thermal-shift screen reveals specific lipid preferences of eukaryotic and prokaryotic membrane proteins
    Article Snippet: .. For overexpression, a single colony of freshly transformed E. coli Lemo21 (DE3) cells from New England Biolabs harboring a pWaldo membrane protein–GFP8His fusion on a Luria Bertani (LB) agar/kanamycin plate, was used to inoculate 20 mL of LB broth supplemented with 50 μg mL−1 kanamycin and grown at 37 °C with shaking at 200 rpm using Innova 44 Incubator shaker (NEW BRUNSWICK) for 16 h. The cultures were then used to inoculate 2 × 1 L of MemStar medium and grown to an OD600 of 0.5. .. The cells were then induced with 0.4 mM IPTG (Sigma) and the temperature of the shaker dropped from 37 to 25 °C and grown further for 16 h. Cells were harvested by centrifuging at 10,000g for 10 min at 4 °C, resuspended in 200 mL of ice-cold 1× phosphate-buffered saline (PBS) buffer and flash-frozen in liquid nitrogen before storing at −80 °C until use.

    Article Title: Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic β-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity
    Article Snippet: .. Lemo21 (DE3) Competent E. coli cells (New England Biolabs, Massachusetts, USA) were transformed with the resultant plasmid. .. An overnight culture of the transformed E. coli cells was inoculated into nutrient-rich 2xYT medium supplemented with 0.6 mM l -rhamnose at pH 7.5 and grown at 37 °C to A 595 = 0.7.

    Over Expression:

    Article Title: An engineered thermal-shift screen reveals specific lipid preferences of eukaryotic and prokaryotic membrane proteins
    Article Snippet: .. For overexpression, a single colony of freshly transformed E. coli Lemo21 (DE3) cells from New England Biolabs harboring a pWaldo membrane protein–GFP8His fusion on a Luria Bertani (LB) agar/kanamycin plate, was used to inoculate 20 mL of LB broth supplemented with 50 μg mL−1 kanamycin and grown at 37 °C with shaking at 200 rpm using Innova 44 Incubator shaker (NEW BRUNSWICK) for 16 h. The cultures were then used to inoculate 2 × 1 L of MemStar medium and grown to an OD600 of 0.5. .. The cells were then induced with 0.4 mM IPTG (Sigma) and the temperature of the shaker dropped from 37 to 25 °C and grown further for 16 h. Cells were harvested by centrifuging at 10,000 g for 10 min at 4 °C, resuspended in 200 mL of ice-cold 1× phosphate-buffered saline (PBS) buffer and flash-frozen in liquid nitrogen before storing at −80 °C until use.

    Article Title: An engineered thermal-shift screen reveals specific lipid preferences of eukaryotic and prokaryotic membrane proteins
    Article Snippet: .. For overexpression, a single colony of freshly transformed E. coli Lemo21 (DE3) cells from New England Biolabs harboring a pWaldo membrane protein–GFP8His fusion on a Luria Bertani (LB) agar/kanamycin plate, was used to inoculate 20 mL of LB broth supplemented with 50 μg mL−1 kanamycin and grown at 37 °C with shaking at 200 rpm using Innova 44 Incubator shaker (NEW BRUNSWICK) for 16 h. The cultures were then used to inoculate 2 × 1 L of MemStar medium and grown to an OD600 of 0.5. .. The cells were then induced with 0.4 mM IPTG (Sigma) and the temperature of the shaker dropped from 37 to 25 °C and grown further for 16 h. Cells were harvested by centrifuging at 10,000g for 10 min at 4 °C, resuspended in 200 mL of ice-cold 1× phosphate-buffered saline (PBS) buffer and flash-frozen in liquid nitrogen before storing at −80 °C until use.

    Chromatography:

    Article Title: Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon
    Article Snippet: .. The protein was expressed in Lemo21(DE3) E.coli strain (New England Biolabs), expression was induced at OD600 of 0.6 by 0.4 mM IPTG for 4 h. SecY(S111C)EG was purified as described previously ( ) by metal-affinity chromatography, followed by cation-exchange chromatography. .. Labeling with MDCC maleimide was performed by following the protocol provided by the manufacturer (Invitrogen), using labeling buffer (20 mM Hepes, pH 7.0, 150 mM KCl, 10% [wt/vol] glycerol, and 0.03% DDM).

    Ligation:

    Article Title: Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family
    Article Snippet: After ligation with the expression vector pET22b(+), which was linearized with the same restriction endonucleases, the ligation product, pET22b(+):: act TBEA6 (see Fig. S1 in the supplemental material), was used for transformation of CaCl2 -competent cells of E. coli Top10. .. After selection of transformants using LB medium containing ampicillin, the hybrid plasmids were isolated, analyzed by sequencing, and used for transformation of CaCl2 -competent cells of E. coli Lemo21(DE3) (New England BioLabs, Inc., Ipswich, MA).

    Protease Inhibitor:

    Article Title: Role of SpaO in the assembly of the sorting platform of a Salmonella type III secretion system
    Article Snippet: Purification of His-tagged SpaO A 50 ml overnight culture of E . coli Lemo21(DE3) (New England Biolabs C2528J) harboring plasmid pSB3775 in LB containing 50 μg/ml of kanamycin and 30 μg/ml of chloramphenicol was diluted in 1 L of LB containing the same antibiotics. .. Cells were pelleted at 6,000 rpm and the pellet resuspended in 10 ml of lysis buffer (50 mM NaH2 PO4 , 300 mM NaCl, 10 mM imidazole, 1mM Mg2 Cl, 2.5U of DNAse, and cOmplete™ Protease Inhibitor Cocktail [Sigma 11697498001]).

    Article Title: Substrate-bound outward-open structure of a Na+-coupled sialic acid symporter reveals a new Na+ site
    Article Snippet: Protein production and purification The pSiaT1 plasmid was transformed into the E. coli Lemo21(DE3) strain (NEB). .. Cells were solubilised in phosphate-buffered saline (PBS) supplemented with cOmplete™ EDTA-free protease inhibitor tablets (Roche), lysozyme (0.5 mg/mL), DNaseI (5 µg/mL), MgCl2 (2 mM) and disrupted using an EmulsiFlex-C3 (AVESTIN) at 20,000 psi.

    Generated:

    Article Title: Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon
    Article Snippet: SecY(S111C)EG was generated by using the same protocol. .. The protein was expressed in Lemo21(DE3) E.coli strain (New England Biolabs), expression was induced at OD600 of 0.6 by 0.4 mM IPTG for 4 h. SecY(S111C)EG was purified as described previously ( ) by metal-affinity chromatography, followed by cation-exchange chromatography.

    Protein Concentration:

    Article Title: Bacterial Polysaccharide Specificity of the Pattern Recognition Receptor Langerin Is Highly Species-dependent *
    Article Snippet: Murine CRD was expressed in E. coli Lemo21(DE3) (New England Biolabs); solubilized in 6 m guanidine HCl; refolded by rapid dilution into 0.8 m l -arginine in TBS, pH 7.5, containing 2.5 m m reduced and 0.5 m m oxidized glutathione; and purified via a StrepTactin column followed by dialysis against 25 m m MES, 40 m m NaCl, pH 6. .. Protein concentration was determined by A 280 nm using the calculated molar extinction coefficients of 56,170 m −1 cm−1 for the human langerin ECD and CRD and 56,170 m −1 cm−1 and 54,680 m −1 cm−1 for the murine ECD and CRD, respectively.

    Polymerase Chain Reaction:

    Article Title: Discovery of Polyesterases from Moss-Associated Microorganisms
    Article Snippet: The PCR primers employed are listed in Table S1. .. Ligated/hybridized plasmids were transformed into chemically competent E. coli BL21(DE3) cells (Invitrogen, USA) and E. coli Lemo21(DE3) cells (New England BioLabs, Germany).

    Article Title: De Novo Design of Bioactive Protein Switches
    Article Snippet: Paragraph title: PCR mutagenesis and isothermal assembly ... Sequence verified plasmid was purified using Qiagen miniprep kit and transformed into chemically competent E. coli BL21(DE3)Star, BL21(DE3)Star-pLysS cells (Invitrogen), or Lemo21(DE3) cells (NEB) for protein expression.

    Article Title: Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family
    Article Snippet: PCR products were isolated from agarose gels using the peqGOLD GelExtraction Kit (PEQLAB Biotechnologie GmbH, Erlangen, Germany) and ligated with pCR2.1-TOPO DNA (Invitrogen, Carlsbad, CA). .. After selection of transformants using LB medium containing ampicillin, the hybrid plasmids were isolated, analyzed by sequencing, and used for transformation of CaCl2 -competent cells of E. coli Lemo21(DE3) (New England BioLabs, Inc., Ipswich, MA).

    Recombinant:

    Article Title: Bacterial Polysaccharide Specificity of the Pattern Recognition Receptor Langerin Is Highly Species-dependent *
    Article Snippet: Recombinant human and murine ECDs were insolubly expressed in E. coli BL21(DE3), refolded, and purified as described before ( ). .. Murine CRD was expressed in E. coli Lemo21(DE3) (New England Biolabs); solubilized in 6 m guanidine HCl; refolded by rapid dilution into 0.8 m l -arginine in TBS, pH 7.5, containing 2.5 m m reduced and 0.5 m m oxidized glutathione; and purified via a StrepTactin column followed by dialysis against 25 m m MES, 40 m m NaCl, pH 6.

    Article Title: SLC38A9 is a component of the lysosomal amino acid-sensing machinery that controls mTORC1
    Article Snippet: Paragraph title: Cloning, expression and purification of recombinant human SLC38A9 ... The plasmid was used to transform E. coli Lemo21(DE3)pLysS (NEB).

    Fluorescence:

    Article Title: Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon
    Article Snippet: Paragraph title: Preparation of fluorescence-labeled SecYEG ... The protein was expressed in Lemo21(DE3) E.coli strain (New England Biolabs), expression was induced at OD600 of 0.6 by 0.4 mM IPTG for 4 h. SecY(S111C)EG was purified as described previously ( ) by metal-affinity chromatography, followed by cation-exchange chromatography.

    Mutagenesis:

    Article Title: Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon
    Article Snippet: All native cysteine residues were mutated to serine by site-directed mutagenesis using the Phusion polymerase protocol (Thermo Fisher Scientific). .. The protein was expressed in Lemo21(DE3) E.coli strain (New England Biolabs), expression was induced at OD600 of 0.6 by 0.4 mM IPTG for 4 h. SecY(S111C)EG was purified as described previously ( ) by metal-affinity chromatography, followed by cation-exchange chromatography.

    Article Title: De Novo Design of Bioactive Protein Switches
    Article Snippet: Paragraph title: PCR mutagenesis and isothermal assembly ... Sequence verified plasmid was purified using Qiagen miniprep kit and transformed into chemically competent E. coli BL21(DE3)Star, BL21(DE3)Star-pLysS cells (Invitrogen), or Lemo21(DE3) cells (NEB) for protein expression.

    Isolation:

    Article Title: An engineered thermal-shift screen reveals specific lipid preferences of eukaryotic and prokaryotic membrane proteins
    Article Snippet: Paragraph title: Isolation of membrane protein–GFP fusion membranes ... For overexpression, a single colony of freshly transformed E. coli Lemo21 (DE3) cells from New England Biolabs harboring a pWaldo membrane protein–GFP8His fusion on a Luria Bertani (LB) agar/kanamycin plate, was used to inoculate 20 mL of LB broth supplemented with 50 μg mL−1 kanamycin and grown at 37 °C with shaking at 200 rpm using Innova 44 Incubator shaker (NEW BRUNSWICK) for 16 h. The cultures were then used to inoculate 2 × 1 L of MemStar medium and grown to an OD600 of 0.5.

    Article Title: Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family
    Article Snippet: .. After selection of transformants using LB medium containing ampicillin, the hybrid plasmids were isolated, analyzed by sequencing, and used for transformation of CaCl2 -competent cells of E. coli Lemo21(DE3) (New England BioLabs, Inc., Ipswich, MA). .. The 526- and 691-bp fragments upstream and downstream of act TBEA6 were amplified by using the primers Xba I_upAct/ Nde I_upAct or Nde I_downAct/ Xba I_downAct, respectively.

    Subcloning:

    Article Title: Discovery of Polyesterases from Moss-Associated Microorganisms
    Article Snippet: Paragraph title: Subcloning, sequencing of fosmids, and cloning of esterases. ... Ligated/hybridized plasmids were transformed into chemically competent E. coli BL21(DE3) cells (Invitrogen, USA) and E. coli Lemo21(DE3) cells (New England BioLabs, Germany).

    Labeling:

    Article Title: Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon
    Article Snippet: Preparation of fluorescence-labeled SecYEG SecYEG for fluorescence labeling was expressed from a pTrc99a construct containing N-terminally His6 -tagged SecE. .. The protein was expressed in Lemo21(DE3) E.coli strain (New England Biolabs), expression was induced at OD600 of 0.6 by 0.4 mM IPTG for 4 h. SecY(S111C)EG was purified as described previously ( ) by metal-affinity chromatography, followed by cation-exchange chromatography.

    Purification:

    Article Title: Bacterial Polysaccharide Specificity of the Pattern Recognition Receptor Langerin Is Highly Species-dependent *
    Article Snippet: .. Murine CRD was expressed in E. coli Lemo21(DE3) (New England Biolabs); solubilized in 6 m guanidine HCl; refolded by rapid dilution into 0.8 m l -arginine in TBS, pH 7.5, containing 2.5 m m reduced and 0.5 m m oxidized glutathione; and purified via a StrepTactin column followed by dialysis against 25 m m MES, 40 m m NaCl, pH 6. .. Protein concentration was determined by A 280 nm using the calculated molar extinction coefficients of 56,170 m −1 cm−1 for the human langerin ECD and CRD and 56,170 m −1 cm−1 and 54,680 m −1 cm−1 for the murine ECD and CRD, respectively.

    Article Title: Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon
    Article Snippet: .. The protein was expressed in Lemo21(DE3) E.coli strain (New England Biolabs), expression was induced at OD600 of 0.6 by 0.4 mM IPTG for 4 h. SecY(S111C)EG was purified as described previously ( ) by metal-affinity chromatography, followed by cation-exchange chromatography. .. Labeling with MDCC maleimide was performed by following the protocol provided by the manufacturer (Invitrogen), using labeling buffer (20 mM Hepes, pH 7.0, 150 mM KCl, 10% [wt/vol] glycerol, and 0.03% DDM).

    Article Title: De Novo Design of Bioactive Protein Switches
    Article Snippet: .. Sequence verified plasmid was purified using Qiagen miniprep kit and transformed into chemically competent E. coli BL21(DE3)Star, BL21(DE3)Star-pLysS cells (Invitrogen), or Lemo21(DE3) cells (NEB) for protein expression. .. Synthetic gene construction Synthetic genes were ordered from Genscript Inc. (Piscataway, NJ, USA) and delivered in pET 28b+, pET21b+, or pET29b+ E. coli expression vectors, inserted at the NdeI and XhoI sites of each vector.

    Article Title: Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic β-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity
    Article Snippet: Paragraph title: Expression and purification of IP6K1 ... Lemo21 (DE3) Competent E. coli cells (New England Biolabs, Massachusetts, USA) were transformed with the resultant plasmid.

    Article Title: SLC38A9 is a component of the lysosomal amino acid-sensing machinery that controls mTORC1
    Article Snippet: Paragraph title: Cloning, expression and purification of recombinant human SLC38A9 ... The plasmid was used to transform E. coli Lemo21(DE3)pLysS (NEB).

    Article Title: Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family
    Article Snippet: For heterologous expression in the T7 promoter/polymerase-based expression vector pET22b(+) (Novagen, Madison, WI), act TBEA6 was obtained by digestion of hybrid plasmid pCR2.1-TOPO:: act TBEA6 with restriction endonucleases HindIII and XhoI and purified from an agarose gel using the peqGOLD GelExtraction kit (PEQLAB Biotechnologie GmbH, Erlangen, Germany). .. After selection of transformants using LB medium containing ampicillin, the hybrid plasmids were isolated, analyzed by sequencing, and used for transformation of CaCl2 -competent cells of E. coli Lemo21(DE3) (New England BioLabs, Inc., Ipswich, MA).

    Article Title: Role of SpaO in the assembly of the sorting platform of a Salmonella type III secretion system
    Article Snippet: .. Purification of His-tagged SpaO A 50 ml overnight culture of E . coli Lemo21(DE3) (New England Biolabs C2528J) harboring plasmid pSB3775 in LB containing 50 μg/ml of kanamycin and 30 μg/ml of chloramphenicol was diluted in 1 L of LB containing the same antibiotics. ..

    Article Title: Substrate-bound outward-open structure of a Na+-coupled sialic acid symporter reveals a new Na+ site
    Article Snippet: .. Protein production and purification The pSiaT1 plasmid was transformed into the E. coli Lemo21(DE3) strain (NEB). ..

    Sequencing:

    Article Title: Discovery of Polyesterases from Moss-Associated Microorganisms
    Article Snippet: Paragraph title: Subcloning, sequencing of fosmids, and cloning of esterases. ... Ligated/hybridized plasmids were transformed into chemically competent E. coli BL21(DE3) cells (Invitrogen, USA) and E. coli Lemo21(DE3) cells (New England BioLabs, Germany).

    Article Title: De Novo Design of Bioactive Protein Switches
    Article Snippet: .. Sequence verified plasmid was purified using Qiagen miniprep kit and transformed into chemically competent E. coli BL21(DE3)Star, BL21(DE3)Star-pLysS cells (Invitrogen), or Lemo21(DE3) cells (NEB) for protein expression. .. Synthetic gene construction Synthetic genes were ordered from Genscript Inc. (Piscataway, NJ, USA) and delivered in pET 28b+, pET21b+, or pET29b+ E. coli expression vectors, inserted at the NdeI and XhoI sites of each vector.

    Article Title: Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family
    Article Snippet: .. After selection of transformants using LB medium containing ampicillin, the hybrid plasmids were isolated, analyzed by sequencing, and used for transformation of CaCl2 -competent cells of E. coli Lemo21(DE3) (New England BioLabs, Inc., Ipswich, MA). .. The 526- and 691-bp fragments upstream and downstream of act TBEA6 were amplified by using the primers Xba I_upAct/ Nde I_upAct or Nde I_downAct/ Xba I_downAct, respectively.

    Selection:

    Article Title: Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family
    Article Snippet: .. After selection of transformants using LB medium containing ampicillin, the hybrid plasmids were isolated, analyzed by sequencing, and used for transformation of CaCl2 -competent cells of E. coli Lemo21(DE3) (New England BioLabs, Inc., Ipswich, MA). .. The 526- and 691-bp fragments upstream and downstream of act TBEA6 were amplified by using the primers Xba I_upAct/ Nde I_upAct or Nde I_downAct/ Xba I_downAct, respectively.

    Activated Clotting Time Assay:

    Article Title: Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family
    Article Snippet: After ligation with the expression vector pET22b(+), which was linearized with the same restriction endonucleases, the ligation product, pET22b(+):: act TBEA6 (see Fig. S1 in the supplemental material), was used for transformation of CaCl2 -competent cells of E. coli Top10. .. After selection of transformants using LB medium containing ampicillin, the hybrid plasmids were isolated, analyzed by sequencing, and used for transformation of CaCl2 -competent cells of E. coli Lemo21(DE3) (New England BioLabs, Inc., Ipswich, MA).

    Plasmid Preparation:

    Article Title: Bacterial Polysaccharide Specificity of the Pattern Recognition Receptor Langerin Is Highly Species-dependent *
    Article Snippet: Codon-optimized human langerin ECD (residues 148–328) with the same tags was cloned into a pET30a expression vector (EMD Millipore). .. Murine CRD was expressed in E. coli Lemo21(DE3) (New England Biolabs); solubilized in 6 m guanidine HCl; refolded by rapid dilution into 0.8 m l -arginine in TBS, pH 7.5, containing 2.5 m m reduced and 0.5 m m oxidized glutathione; and purified via a StrepTactin column followed by dialysis against 25 m m MES, 40 m m NaCl, pH 6.

    Article Title: IP6K Structure and the Molecular Determinants of Catalytic Specificity in an Inositol Phosphate Kinase Family
    Article Snippet: .. Lemo21(DE3) Competent E. coli cells (New England Biolabs) were transformed with the resultant plasmid. ..

    Article Title: An engineered thermal-shift screen reveals specific lipid preferences of eukaryotic and prokaryotic membrane proteins
    Article Snippet: Bacterial membrane protein–GFP fusions: The bacterial membrane protein–GFP8His fusions had been previously constructed into the expression vector, pWaldo GFPd , . .. For overexpression, a single colony of freshly transformed E. coli Lemo21 (DE3) cells from New England Biolabs harboring a pWaldo membrane protein–GFP8His fusion on a Luria Bertani (LB) agar/kanamycin plate, was used to inoculate 20 mL of LB broth supplemented with 50 μg mL−1 kanamycin and grown at 37 °C with shaking at 200 rpm using Innova 44 Incubator shaker (NEW BRUNSWICK) for 16 h. The cultures were then used to inoculate 2 × 1 L of MemStar medium and grown to an OD600 of 0.5.

    Article Title: Discovery of Polyesterases from Moss-Associated Microorganisms
    Article Snippet: A standard PCR mixture (50 μl) contained 1× Phusion HF buffer, 1 U of Phusion DNA polymerase (New England BioLabs), 0.2 mM deoxynucleoside triphosphates, 0.2 μM each primer, and 10 ng of plasmid DNA. .. Ligated/hybridized plasmids were transformed into chemically competent E. coli BL21(DE3) cells (Invitrogen, USA) and E. coli Lemo21(DE3) cells (New England BioLabs, Germany).

    Article Title: De Novo Design of Bioactive Protein Switches
    Article Snippet: .. Sequence verified plasmid was purified using Qiagen miniprep kit and transformed into chemically competent E. coli BL21(DE3)Star, BL21(DE3)Star-pLysS cells (Invitrogen), or Lemo21(DE3) cells (NEB) for protein expression. .. Synthetic gene construction Synthetic genes were ordered from Genscript Inc. (Piscataway, NJ, USA) and delivered in pET 28b+, pET21b+, or pET29b+ E. coli expression vectors, inserted at the NdeI and XhoI sites of each vector.

    Article Title: De Novo Design of Bioactive Protein Switches
    Article Snippet: Synthetic gene construction Synthetic genes were ordered from Genscript Inc. (Piscataway, NJ, USA) and delivered in pET 28b+, pET21b+, or pET29b+ E. coli expression vectors, inserted at the NdeI and XhoI sites of each vector. .. Plasmids were transformed into chemically competent E. coli BL21(DE3)Star, BL21(DE3)Star-pLysS cells (Invitrogen), or Lemo21(DE3) cells (NEB) for protein expression.

    Article Title: Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic β-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity
    Article Snippet: .. Lemo21 (DE3) Competent E. coli cells (New England Biolabs, Massachusetts, USA) were transformed with the resultant plasmid. .. An overnight culture of the transformed E. coli cells was inoculated into nutrient-rich 2xYT medium supplemented with 0.6 mM l -rhamnose at pH 7.5 and grown at 37 °C to A 595 = 0.7.

    Article Title: SLC38A9 is a component of the lysosomal amino acid-sensing machinery that controls mTORC1
    Article Snippet: .. The plasmid was used to transform E. coli Lemo21(DE3)pLysS (NEB). ..

    Article Title: Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family
    Article Snippet: After ligation with the expression vector pET22b(+), which was linearized with the same restriction endonucleases, the ligation product, pET22b(+):: act TBEA6 (see Fig. S1 in the supplemental material), was used for transformation of CaCl2 -competent cells of E. coli Top10. .. After selection of transformants using LB medium containing ampicillin, the hybrid plasmids were isolated, analyzed by sequencing, and used for transformation of CaCl2 -competent cells of E. coli Lemo21(DE3) (New England BioLabs, Inc., Ipswich, MA).

    Article Title: Role of SpaO in the assembly of the sorting platform of a Salmonella type III secretion system
    Article Snippet: .. Purification of His-tagged SpaO A 50 ml overnight culture of E . coli Lemo21(DE3) (New England Biolabs C2528J) harboring plasmid pSB3775 in LB containing 50 μg/ml of kanamycin and 30 μg/ml of chloramphenicol was diluted in 1 L of LB containing the same antibiotics. ..

    Article Title: Substrate-bound outward-open structure of a Na+-coupled sialic acid symporter reveals a new Na+ site
    Article Snippet: .. Protein production and purification The pSiaT1 plasmid was transformed into the E. coli Lemo21(DE3) strain (NEB). ..

    Article Title: An engineered thermal-shift screen reveals specific lipid preferences of eukaryotic and prokaryotic membrane proteins
    Article Snippet: Isolation of membrane protein–GFP fusion membranes Bacterial membrane protein–GFP fusions: The bacterial membrane protein–GFP8His fusions had been previously constructed into the expression vector, pWaldo GFPd , . .. For overexpression, a single colony of freshly transformed E. coli Lemo21 (DE3) cells from New England Biolabs harboring a pWaldo membrane protein–GFP8His fusion on a Luria Bertani (LB) agar/kanamycin plate, was used to inoculate 20 mL of LB broth supplemented with 50 μg mL−1 kanamycin and grown at 37 °C with shaking at 200 rpm using Innova 44 Incubator shaker (NEW BRUNSWICK) for 16 h. The cultures were then used to inoculate 2 × 1 L of MemStar medium and grown to an OD600 of 0.5.

    Article Title: Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic β-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity
    Article Snippet: .. Lemo21 (DE3) Competent E. coli cells (New England Biolabs, Massachusetts, USA) were transformed with the resultant plasmid. .. An overnight culture of the transformed E. coli cells was inoculated into nutrient-rich 2xYT medium supplemented with 0.6 mM l -rhamnose at pH 7.5 and grown at 37 °C to A 595 = 0.7.

    Software:

    Article Title: Discovery of Polyesterases from Moss-Associated Microorganisms
    Article Snippet: CLC Main Workbench software (Qiagen) was employed for sequence assembly, BLASTX analysis, and identification of ORFs. .. Ligated/hybridized plasmids were transformed into chemically competent E. coli BL21(DE3) cells (Invitrogen, USA) and E. coli Lemo21(DE3) cells (New England BioLabs, Germany).

    Binding Assay:

    Article Title: Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic β-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity
    Article Snippet: This vector encodes a 6 × His tag, a maltose binding protein tag and TEV protease cleavage site at the N-terminus. .. Lemo21 (DE3) Competent E. coli cells (New England Biolabs, Massachusetts, USA) were transformed with the resultant plasmid.

    Article Title: SLC38A9 is a component of the lysosomal amino acid-sensing machinery that controls mTORC1
    Article Snippet: In this optimized gene, the Codon Adaptation Index (CAI) was upgraded from 0.63 (wild type) to 0.87, the GC content and unfavourable peaks were optimized to prolong the half-life of the mRNA and a ribosome binding site was removed. .. The plasmid was used to transform E. coli Lemo21(DE3)pLysS (NEB).

    Article Title: Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic β-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity
    Article Snippet: This vector encodes a 6 × His tag, a maltose binding protein tag and TEV protease cleavage site at the N-terminus. .. Lemo21 (DE3) Competent E. coli cells (New England Biolabs, Massachusetts, USA) were transformed with the resultant plasmid.

    Agarose Gel Electrophoresis:

    Article Title: Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family
    Article Snippet: For heterologous expression in the T7 promoter/polymerase-based expression vector pET22b(+) (Novagen, Madison, WI), act TBEA6 was obtained by digestion of hybrid plasmid pCR2.1-TOPO:: act TBEA6 with restriction endonucleases HindIII and XhoI and purified from an agarose gel using the peqGOLD GelExtraction kit (PEQLAB Biotechnologie GmbH, Erlangen, Germany). .. After selection of transformants using LB medium containing ampicillin, the hybrid plasmids were isolated, analyzed by sequencing, and used for transformation of CaCl2 -competent cells of E. coli Lemo21(DE3) (New England BioLabs, Inc., Ipswich, MA).

    Produced:

    Article Title: Substrate-bound outward-open structure of a Na+-coupled sialic acid symporter reveals a new Na+ site
    Article Snippet: Protein production and purification The pSiaT1 plasmid was transformed into the E. coli Lemo21(DE3) strain (NEB). .. Selenomethionine-derivatised (SeMet) protein was produced using PASM-5052 auto-induction media .

    Strep-tag:

    Article Title: Bacterial Polysaccharide Specificity of the Pattern Recognition Receptor Langerin Is Highly Species-dependent *
    Article Snippet: Codon-optimized truncated murine langerin ECD (residues 150–331) was cloned into a pUC19-derived expression vector containing a C-terminal TEV cleavage site and a Strep-tag II. .. Murine CRD was expressed in E. coli Lemo21(DE3) (New England Biolabs); solubilized in 6 m guanidine HCl; refolded by rapid dilution into 0.8 m l -arginine in TBS, pH 7.5, containing 2.5 m m reduced and 0.5 m m oxidized glutathione; and purified via a StrepTactin column followed by dialysis against 25 m m MES, 40 m m NaCl, pH 6.

    Lysis:

    Article Title: Role of SpaO in the assembly of the sorting platform of a Salmonella type III secretion system
    Article Snippet: Purification of His-tagged SpaO A 50 ml overnight culture of E . coli Lemo21(DE3) (New England Biolabs C2528J) harboring plasmid pSB3775 in LB containing 50 μg/ml of kanamycin and 30 μg/ml of chloramphenicol was diluted in 1 L of LB containing the same antibiotics. .. Cells were pelleted at 6,000 rpm and the pellet resuspended in 10 ml of lysis buffer (50 mM NaH2 PO4 , 300 mM NaCl, 10 mM imidazole, 1mM Mg2 Cl, 2.5U of DNAse, and cOmplete™ Protease Inhibitor Cocktail [Sigma 11697498001]).

    Positron Emission Tomography:

    Article Title: De Novo Design of Bioactive Protein Switches
    Article Snippet: PCR was used to create fragments upstream and downstream of the mutation site with > 20bp overlap with the desired pET vector. .. Sequence verified plasmid was purified using Qiagen miniprep kit and transformed into chemically competent E. coli BL21(DE3)Star, BL21(DE3)Star-pLysS cells (Invitrogen), or Lemo21(DE3) cells (NEB) for protein expression.

    Article Title: De Novo Design of Bioactive Protein Switches
    Article Snippet: Synthetic gene construction Synthetic genes were ordered from Genscript Inc. (Piscataway, NJ, USA) and delivered in pET 28b+, pET21b+, or pET29b+ E. coli expression vectors, inserted at the NdeI and XhoI sites of each vector. .. Plasmids were transformed into chemically competent E. coli BL21(DE3)Star, BL21(DE3)Star-pLysS cells (Invitrogen), or Lemo21(DE3) cells (NEB) for protein expression.

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    New England Biolabs lemo21 de3 competent e coli cells
    Lemo21 De3 Competent E Coli Cells, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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