e coli bl21  (Millipore)


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    Name:
    E coli BL21
    Description:
    It is an exemplary host for the expression of recombinant proteins As an E coli B strain it further lacks the major protease encoded by the lon gene catalyzing the endoproteolytic cleavage of damaged and recombinant proteins in the cell
    Catalog Number:
    ge27-1542-01
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    Structured Review

    Millipore e coli bl21
    E coli BL21
    It is an exemplary host for the expression of recombinant proteins As an E coli B strain it further lacks the major protease encoded by the lon gene catalyzing the endoproteolytic cleavage of damaged and recombinant proteins in the cell
    https://www.bioz.com/result/e coli bl21/product/Millipore
    Average 99 stars, based on 1033 article reviews
    Price from $9.99 to $1999.99
    e coli bl21 - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Chemical complexity of protein determines optimal E. coli expression host; A comparative study using Erythropoietin, Streptokinase and Tumor Necrosis Factor Receptor"

    Article Title: Chemical complexity of protein determines optimal E. coli expression host; A comparative study using Erythropoietin, Streptokinase and Tumor Necrosis Factor Receptor

    Journal: Journal of Genetic Engineering & Biotechnology

    doi: 10.1016/j.jgeb.2016.12.006

    Comparison of expression levels of TNFR ED, EPO and SK in different hosts at 37 °C on induction with 1 mM IPTG for E. coli BL21 (DE3) and BL21 (DE3) pLys S strains and 0.3 M NaCl for E. coli GJ1158 strains at (A) 0.6 OD 600 , (B) 1.0 OD. Basal level expression was given a minimal value of 0.5%.
    Figure Legend Snippet: Comparison of expression levels of TNFR ED, EPO and SK in different hosts at 37 °C on induction with 1 mM IPTG for E. coli BL21 (DE3) and BL21 (DE3) pLys S strains and 0.3 M NaCl for E. coli GJ1158 strains at (A) 0.6 OD 600 , (B) 1.0 OD. Basal level expression was given a minimal value of 0.5%.

    Techniques Used: Expressing

    Immunoblot analysis of total protein of induced E. coli BL21 (DE3) cellsharboring the recombinant plasmids using anti-His antibodies . Lanes 1 – Protein marker, 2 – pRSET A vector control induced, 3 – pRSET-TNFR ED uninduced, 4 – pRSET-TNFR ED induced, 5 – pRSET EPO-uninduced, 6 – pRSET EPO-induced, 7 – pRSET SK uninduced, 8 – pRSET SK induced.
    Figure Legend Snippet: Immunoblot analysis of total protein of induced E. coli BL21 (DE3) cellsharboring the recombinant plasmids using anti-His antibodies . Lanes 1 – Protein marker, 2 – pRSET A vector control induced, 3 – pRSET-TNFR ED uninduced, 4 – pRSET-TNFR ED induced, 5 – pRSET EPO-uninduced, 6 – pRSET EPO-induced, 7 – pRSET SK uninduced, 8 – pRSET SK induced.

    Techniques Used: Recombinant, Marker, Plasmid Preparation

    2) Product Images from "Preparation and Antitumor Activity of CS5931, A Novel Polypeptide from Sea Squirt Ciona Savignyi"

    Article Title: Preparation and Antitumor Activity of CS5931, A Novel Polypeptide from Sea Squirt Ciona Savignyi

    Journal: Marine Drugs

    doi: 10.3390/md14030047

    ( a ) SDS-PAGE analysis of recombinant CS5931 expression in BL21 (DE3) cells. The plasmid pET28a/CS5931 was transformed into E. coli BL21 (DE3). After being cultured at 37 °C for 16 h, the whole cell extract was resolved using SDS-PAGE in the absence (lane 1) and presence of IPTG induction (lane 2, 3). Lane 4 represented the total protein in the supernatant and lane 5 indicated the purified protein with the AKTA; ( b ) Western blot analysis of the purified polypeptide.
    Figure Legend Snippet: ( a ) SDS-PAGE analysis of recombinant CS5931 expression in BL21 (DE3) cells. The plasmid pET28a/CS5931 was transformed into E. coli BL21 (DE3). After being cultured at 37 °C for 16 h, the whole cell extract was resolved using SDS-PAGE in the absence (lane 1) and presence of IPTG induction (lane 2, 3). Lane 4 represented the total protein in the supernatant and lane 5 indicated the purified protein with the AKTA; ( b ) Western blot analysis of the purified polypeptide.

    Techniques Used: SDS Page, Recombinant, Expressing, Plasmid Preparation, Transformation Assay, Cell Culture, Purification, Western Blot

    3) Product Images from "A new hybrid bacteriocin, Ent35-MccV, displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria"

    Article Title: A new hybrid bacteriocin, Ent35-MccV, displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria

    Journal: FEBS Open Bio

    doi: 10.1016/j.fob.2012.01.002

    RP-HPLC separation of the heated cellular extract. Cellular extract of E. coli BL21 [DE3] (pMA24) were treated as described in Section 2.4 . Chromatogram was monitored at (A) λ = 220 nm and (B) λ = 280 nm. Peaks were collected and individually analyzed by MALDI-TOF MS.
    Figure Legend Snippet: RP-HPLC separation of the heated cellular extract. Cellular extract of E. coli BL21 [DE3] (pMA24) were treated as described in Section 2.4 . Chromatogram was monitored at (A) λ = 220 nm and (B) λ = 280 nm. Peaks were collected and individually analyzed by MALDI-TOF MS.

    Techniques Used: High Performance Liquid Chromatography, Mass Spectrometry

    4) Product Images from "A simple vector system to improve performance and utilisation of recombinant antibodies"

    Article Title: A simple vector system to improve performance and utilisation of recombinant antibodies

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-6-46

    A comparison of scFv expression using auto-induction media and IPTG induction . A : ScFv expressed from pSANG10-3F; B : ScFv-AP expressed from pSANG14-3F. BL21 (DE3) were grown overnight at 30°C using either auto-induction media (Auto-Ind) or mid-log phase induction with IPTG. The bacterial cell pellet was lysed and analysed by western-blot (10 μl/lane) using an anti-histidine tag mouse monoclonal antibody and Cy5 labelled anti-mouse detection system.
    Figure Legend Snippet: A comparison of scFv expression using auto-induction media and IPTG induction . A : ScFv expressed from pSANG10-3F; B : ScFv-AP expressed from pSANG14-3F. BL21 (DE3) were grown overnight at 30°C using either auto-induction media (Auto-Ind) or mid-log phase induction with IPTG. The bacterial cell pellet was lysed and analysed by western-blot (10 μl/lane) using an anti-histidine tag mouse monoclonal antibody and Cy5 labelled anti-mouse detection system.

    Techniques Used: Expressing, Western Blot

    Construction of pSANG vectors . A . Sequence of the basic vector pSANG-10, which was created by inserting an oligo between a Bgl I site (GCCnnnnnGGC) and Xho I site (CTCGAG now destroyed) downstream of the promoter within the vector backbone derived from pET26 (+) plasmid (Novagen, San Diego, CA). The inserted sequence cloned between these restriction sites is shown. Translated sequences from the end of the signal peptide and hexa-histidine tag are indicated. The scFv encoding gene is sub-cloned at the Nco I /Not I site and the Hind III site is available for insertion of peptide tags and fusion partners. B. For comparison the sequence of pSANG10-3F is shown where a tri-FLAG tag has been inserted. An oligonucleotide encoding the tri-FLAG sequence was cloned into the Hind III site of pSANG10 destroying the site at 5' end and retaining it at 3' end for future use. C. Schematic view of the expression cassettes of the pSANG vectors. a-d : vectors used with BL21 (DE3) host strain for periplasmic expression. e-f : vectors used with Origami 2™ (DE3) host strain for cytoplasmic expression. MBP= Maltose Binding Protein; * = serine replaced by a cysteine.
    Figure Legend Snippet: Construction of pSANG vectors . A . Sequence of the basic vector pSANG-10, which was created by inserting an oligo between a Bgl I site (GCCnnnnnGGC) and Xho I site (CTCGAG now destroyed) downstream of the promoter within the vector backbone derived from pET26 (+) plasmid (Novagen, San Diego, CA). The inserted sequence cloned between these restriction sites is shown. Translated sequences from the end of the signal peptide and hexa-histidine tag are indicated. The scFv encoding gene is sub-cloned at the Nco I /Not I site and the Hind III site is available for insertion of peptide tags and fusion partners. B. For comparison the sequence of pSANG10-3F is shown where a tri-FLAG tag has been inserted. An oligonucleotide encoding the tri-FLAG sequence was cloned into the Hind III site of pSANG10 destroying the site at 5' end and retaining it at 3' end for future use. C. Schematic view of the expression cassettes of the pSANG vectors. a-d : vectors used with BL21 (DE3) host strain for periplasmic expression. e-f : vectors used with Origami 2™ (DE3) host strain for cytoplasmic expression. MBP= Maltose Binding Protein; * = serine replaced by a cysteine.

    Techniques Used: Sequencing, Plasmid Preparation, Derivative Assay, Clone Assay, FLAG-tag, Expressing, Binding Assay

    5) Product Images from "A novel HPV16 E7-affitoxin for targeted therapy of HPV16-induced human cervical cancer"

    Article Title: A novel HPV16 E7-affitoxin for targeted therapy of HPV16-induced human cervical cancer

    Journal: Theranostics

    doi: 10.7150/thno.24607

    Expression and purification of Z HPV16 E7 affitoxin 384 protein. The pET21a (+)/Z HPV16 E7 affitoxin384 plasmid was transformed into E. coli BL21 (DE3). The protein was expressed and purified by Ni-NTA agarose affinity chromatography. (A) Schematic structure of pET21a (+)/Z HPV16 E7 affitoxin384 plasmid. (B) Comassie blue-stained SDS-PAGE gel of the recombinant proteins. M, protein marker; 1, Empty E.coli BL21 (DE3); 2, E.coli BL21 (DE3) transformed with pET21a empty vector; 3, E.coli BL21 (DE3) transformed with pET21a (+)/Z HPV16 E7 affitoxin384 plasmid; 4-5, E.coli BL21 (DE3) transformed with pET21a (+)/Z HPV16 E7 affitoxin384 plasmid and induced by 1 mM IPTG; 6, E.coli BL21 (DE3) transformed with pET21a (+)/Z wt affitoxin; 7-8, E.coli BL21 (DE3) transformed with pET21a (+)/Z wt affitoxin and induced by 1 mM IPTG. (C) Analysis of the purified Z HPV16 E7 affitoxin384 and Z wt affitoxin recombinant proteins by SDS-PAGE. M, protein marker; 1, Z HPV16 E7 affitoxin384; 2, Z wt affitoxin. (D-F) Confirmation of the expression of Z HPV16 E7 affitoxin384 and Z wt affitoxin recombinant proteins by western blot using the primary antibodies against His tag, PE38KDEL and SPA-Z, respectively. 1, Empty E.coli BL21 (DE3); 2, E.coli BL21 (DE3) transformed with pET21a empty vector; 3, E.coli BL21 (DE3) transformed with pET21a (+)/Z HPV16 E7 affitoxin384 plasmid and induced by 1 mM IPTG; 4, E.coli BL21 (DE3) transformed with pET21a (+)/Z wt affitoxin and induced by 1 mM IPTG.
    Figure Legend Snippet: Expression and purification of Z HPV16 E7 affitoxin 384 protein. The pET21a (+)/Z HPV16 E7 affitoxin384 plasmid was transformed into E. coli BL21 (DE3). The protein was expressed and purified by Ni-NTA agarose affinity chromatography. (A) Schematic structure of pET21a (+)/Z HPV16 E7 affitoxin384 plasmid. (B) Comassie blue-stained SDS-PAGE gel of the recombinant proteins. M, protein marker; 1, Empty E.coli BL21 (DE3); 2, E.coli BL21 (DE3) transformed with pET21a empty vector; 3, E.coli BL21 (DE3) transformed with pET21a (+)/Z HPV16 E7 affitoxin384 plasmid; 4-5, E.coli BL21 (DE3) transformed with pET21a (+)/Z HPV16 E7 affitoxin384 plasmid and induced by 1 mM IPTG; 6, E.coli BL21 (DE3) transformed with pET21a (+)/Z wt affitoxin; 7-8, E.coli BL21 (DE3) transformed with pET21a (+)/Z wt affitoxin and induced by 1 mM IPTG. (C) Analysis of the purified Z HPV16 E7 affitoxin384 and Z wt affitoxin recombinant proteins by SDS-PAGE. M, protein marker; 1, Z HPV16 E7 affitoxin384; 2, Z wt affitoxin. (D-F) Confirmation of the expression of Z HPV16 E7 affitoxin384 and Z wt affitoxin recombinant proteins by western blot using the primary antibodies against His tag, PE38KDEL and SPA-Z, respectively. 1, Empty E.coli BL21 (DE3); 2, E.coli BL21 (DE3) transformed with pET21a empty vector; 3, E.coli BL21 (DE3) transformed with pET21a (+)/Z HPV16 E7 affitoxin384 plasmid and induced by 1 mM IPTG; 4, E.coli BL21 (DE3) transformed with pET21a (+)/Z wt affitoxin and induced by 1 mM IPTG.

    Techniques Used: Expressing, Purification, Plasmid Preparation, Transformation Assay, Affinity Chromatography, Staining, SDS Page, Recombinant, Marker, Western Blot

    6) Product Images from "Germacrene A synthase in yarrow (Achillea millefolium) is an enzyme with mixed substrate specificity: gene cloning, functional characterization and expression analysis"

    Article Title: Germacrene A synthase in yarrow (Achillea millefolium) is an enzyme with mixed substrate specificity: gene cloning, functional characterization and expression analysis

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2015.00111

    Heterologus expression of AmGAS . (A) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the numbers indicate—1: protein marker, 2: induced recombinant strain containing pET-26b (+) plus AmGAS gene after 6 h, 3, after 4 h; 4, after 2 h; 5, non-induced and 6, E. coli BL21 (DE3) without plasmid, and (B) western blotting, the numbers denote—1, non-induced recombinant strain containing pET-26b (+) plus AmGAS gene; 2, induced protein after 2 h; 3, after 4 h; 4, after 6 h and 5, protein marker. Arrows show the band that belongs to the recombinant protein of AmGAS.
    Figure Legend Snippet: Heterologus expression of AmGAS . (A) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the numbers indicate—1: protein marker, 2: induced recombinant strain containing pET-26b (+) plus AmGAS gene after 6 h, 3, after 4 h; 4, after 2 h; 5, non-induced and 6, E. coli BL21 (DE3) without plasmid, and (B) western blotting, the numbers denote—1, non-induced recombinant strain containing pET-26b (+) plus AmGAS gene; 2, induced protein after 2 h; 3, after 4 h; 4, after 6 h and 5, protein marker. Arrows show the band that belongs to the recombinant protein of AmGAS.

    Techniques Used: Expressing, Polyacrylamide Gel Electrophoresis, SDS Page, Marker, Recombinant, Positron Emission Tomography, Plasmid Preparation, Western Blot

    7) Product Images from "T-Cell Expression Cloning of Porphyromonas gingivalis Genes Coding for T Helper-Biased Immune Responses during Infection "

    Article Title: T-Cell Expression Cloning of Porphyromonas gingivalis Genes Coding for T Helper-Biased Immune Responses during Infection

    Journal:

    doi: 10.1128/IAI.02029-05

    Expression of P. gingivalis thiol peroxidase gene as purified recombinant protein. E. coli BL21/pLysS transformed with the expression vector pET17b containing the thiol peroxidase gene was grown and induced with IPTG. The cells were lysed, and the thiol
    Figure Legend Snippet: Expression of P. gingivalis thiol peroxidase gene as purified recombinant protein. E. coli BL21/pLysS transformed with the expression vector pET17b containing the thiol peroxidase gene was grown and induced with IPTG. The cells were lysed, and the thiol

    Techniques Used: Expressing, Purification, Recombinant, Transformation Assay, Plasmid Preparation

    8) Product Images from "The non-typeable Haemophilus influenzae major adhesin Hia is a dual function lectin that binds to human-specific respiratory tract sialic acid glycan receptors"

    Article Title: The non-typeable Haemophilus influenzae major adhesin Hia is a dual function lectin that binds to human-specific respiratory tract sialic acid glycan receptors

    Journal: bioRxiv

    doi: 10.1101/2020.05.08.084038

    Percent adherence to Chang cells of E. coli BL21 strains expressing wild-type Hia or isogenic mutant variants. Percent adherence of each strain is calculated as adherent cfu following 2 hrs incubation / total input cfu. All raw data is presented in Supplementary Data 1. * = P-value =
    Figure Legend Snippet: Percent adherence to Chang cells of E. coli BL21 strains expressing wild-type Hia or isogenic mutant variants. Percent adherence of each strain is calculated as adherent cfu following 2 hrs incubation / total input cfu. All raw data is presented in Supplementary Data 1. * = P-value =

    Techniques Used: Expressing, Mutagenesis, Incubation

    9) Product Images from "Molecular Cloning and Characterization of P4 Nuclease from Leishmania infantum"

    Article Title: Molecular Cloning and Characterization of P4 Nuclease from Leishmania infantum

    Journal: Enzyme Research

    doi: 10.4061/2011/970983

    SDS-PAGE analysis of recombinant Li-P4 produced in BL21, Lane 1: bacterial lysate before induction, Lane 2: bacterial lysate after induction with IPTG, Lane 3: purified recombinant Li-P4, and Lane 4: molecular weight marker.
    Figure Legend Snippet: SDS-PAGE analysis of recombinant Li-P4 produced in BL21, Lane 1: bacterial lysate before induction, Lane 2: bacterial lysate after induction with IPTG, Lane 3: purified recombinant Li-P4, and Lane 4: molecular weight marker.

    Techniques Used: SDS Page, Recombinant, Produced, Purification, Molecular Weight, Marker

    10) Product Images from "Discovery of Glycoside Hydrolase Enzymes in an Avicel-Adapted Forest Soil Fungal Community by a Metatranscriptomic Approach"

    Article Title: Discovery of Glycoside Hydrolase Enzymes in an Avicel-Adapted Forest Soil Fungal Community by a Metatranscriptomic Approach

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0055485

    Synergism of cellulase in the presence of the CelA enzyme. The reaction mixture (200 µl final volume) contained 0–1 mg Accellerase 1500 protein per g of Avicel, 1.0% (w/v) Avicel, and 0–10 mg CelA protein per g of Avicel in 0.05M acetate buffer (pH 5.0). The mixture was shaken at 50°C for 1 h before quantification of reducing sugars; sample 1, cell lysate of the E. coli BL21 cells harboring plasmid pET28, sample 2, cell lysate of the E. coli BL21 cells producing recombinant FX003685 enzyme.
    Figure Legend Snippet: Synergism of cellulase in the presence of the CelA enzyme. The reaction mixture (200 µl final volume) contained 0–1 mg Accellerase 1500 protein per g of Avicel, 1.0% (w/v) Avicel, and 0–10 mg CelA protein per g of Avicel in 0.05M acetate buffer (pH 5.0). The mixture was shaken at 50°C for 1 h before quantification of reducing sugars; sample 1, cell lysate of the E. coli BL21 cells harboring plasmid pET28, sample 2, cell lysate of the E. coli BL21 cells producing recombinant FX003685 enzyme.

    Techniques Used: Plasmid Preparation, Recombinant

    11) Product Images from "High-throughput mapping of the phage resistance landscape in E. coli"

    Article Title: High-throughput mapping of the phage resistance landscape in E. coli

    Journal: bioRxiv

    doi: 10.1101/2020.02.15.951020

    Overview of high-throughput genome-wide screens. We used barcoded loss-of-function (LOF) technologies (RB-TnSeq and CRISPRi) and a gain-of-function (GOF) technology (Dub-seq) in E. coli K-12 (BW25113 and MG1655) to screen for host factors important in phage infection and resistance. In E. coli BL21, we performed RB-TnSeq and Dub-seq (but not CRISPRi). We sourced 14 diverse E. coli phages with double-stranded DNA genomes, belonging to Myoviridae, Podoviridae and Siphoviridae families, and performed pooled fitness screens in both planktonic and solid agar formats. Disruption or overexpression of certain genes provide fitness to host in the presence of phages and we monitor these changes by quantifying the abundance of the DNA barcode or sgRNA associated with each strain. The individual strain abundances are then converted to gene fitness scores (normalized log2 change in the abundance of mutants in that gene).
    Figure Legend Snippet: Overview of high-throughput genome-wide screens. We used barcoded loss-of-function (LOF) technologies (RB-TnSeq and CRISPRi) and a gain-of-function (GOF) technology (Dub-seq) in E. coli K-12 (BW25113 and MG1655) to screen for host factors important in phage infection and resistance. In E. coli BL21, we performed RB-TnSeq and Dub-seq (but not CRISPRi). We sourced 14 diverse E. coli phages with double-stranded DNA genomes, belonging to Myoviridae, Podoviridae and Siphoviridae families, and performed pooled fitness screens in both planktonic and solid agar formats. Disruption or overexpression of certain genes provide fitness to host in the presence of phages and we monitor these changes by quantifying the abundance of the DNA barcode or sgRNA associated with each strain. The individual strain abundances are then converted to gene fitness scores (normalized log2 change in the abundance of mutants in that gene).

    Techniques Used: High Throughput Screening Assay, Genome Wide, Infection, Over Expression

    Description of E. coli BL21 Dub-seq library (A) The fragment insert size distribution in the E. coli BL21 Dub-seq library. (B) Cumulative distribution plot showing the percentage of genes in the E. coli BL21 genome (y-axis) covered by a number of independent genomic fragments (x-axis). (C) The distribution of the number of genes that are completely covered (start to stop codon) per genomic fragment in the E. coli BL21 Dub-seq library. (D) Genome coverage of E . coli BL21 Dub-seq library in 10,000 kB windows mapped to E. coli BL21-DE3
    Figure Legend Snippet: Description of E. coli BL21 Dub-seq library (A) The fragment insert size distribution in the E. coli BL21 Dub-seq library. (B) Cumulative distribution plot showing the percentage of genes in the E. coli BL21 genome (y-axis) covered by a number of independent genomic fragments (x-axis). (C) The distribution of the number of genes that are completely covered (start to stop codon) per genomic fragment in the E. coli BL21 Dub-seq library. (D) Genome coverage of E . coli BL21 Dub-seq library in 10,000 kB windows mapped to E. coli BL21-DE3

    Techniques Used:

    Genome-wide screens in E. coli BL21 strain (A) Heatmap of BL21 LOF RB-TnSeq data for 12 dsDNA phages at a single MOI, and selected genes with high-confidence fitness benefits are shown. Additional data is provided in the Supplementary Table S8. (B) Heatmap of GOF BL21 Dub-seq data for 12 dsDNA phages with high-confidence fitness fitness benefit. Fitness scores of > =4 in at least one phage assay are shown. These assays were performed in planktonic culture. Additional data is provided in the Supplementary Table S9.
    Figure Legend Snippet: Genome-wide screens in E. coli BL21 strain (A) Heatmap of BL21 LOF RB-TnSeq data for 12 dsDNA phages at a single MOI, and selected genes with high-confidence fitness benefits are shown. Additional data is provided in the Supplementary Table S8. (B) Heatmap of GOF BL21 Dub-seq data for 12 dsDNA phages with high-confidence fitness fitness benefit. Fitness scores of > =4 in at least one phage assay are shown. These assays were performed in planktonic culture. Additional data is provided in the Supplementary Table S9.

    Techniques Used: Genome Wide

    12) Product Images from "Immunization with H7-HCP-Tir-Intimin Significantly Reduces Colonization and Shedding of Escherichia coli O157:H7 in Goats"

    Article Title: Immunization with H7-HCP-Tir-Intimin Significantly Reduces Colonization and Shedding of Escherichia coli O157:H7 in Goats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0091632

    His-H7-HCP-Tir-Intimin was assayed by SAS-PAGE and Western blot. A: Lane 1, BL21 (Pcold I- fliC-hcpA - tir - eae ) uninduced. Lane 2 shows the precipitation of BL21 (Pcold I- fliC-hcpA - tir - eae ) induced by IPTG; lane 3 BL21 (Pcold I- fliC-hcpA - tir - eae ) supernatant. Lane M protein marker. B: Purified His-H7-HCP-Tir-Intimin was identified using rabbit anti-H7, anti-HCP, anti-Tir, anti-Intimin, and pre-immunization sera. Lanes 1–5, purified His-H7-HCP-Tir-Intimin; lanes 6–10, immunoblots with anti-H7, anti-HCP, anti-Tir, anti-Intimin, and pre-immunization sera.
    Figure Legend Snippet: His-H7-HCP-Tir-Intimin was assayed by SAS-PAGE and Western blot. A: Lane 1, BL21 (Pcold I- fliC-hcpA - tir - eae ) uninduced. Lane 2 shows the precipitation of BL21 (Pcold I- fliC-hcpA - tir - eae ) induced by IPTG; lane 3 BL21 (Pcold I- fliC-hcpA - tir - eae ) supernatant. Lane M protein marker. B: Purified His-H7-HCP-Tir-Intimin was identified using rabbit anti-H7, anti-HCP, anti-Tir, anti-Intimin, and pre-immunization sera. Lanes 1–5, purified His-H7-HCP-Tir-Intimin; lanes 6–10, immunoblots with anti-H7, anti-HCP, anti-Tir, anti-Intimin, and pre-immunization sera.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Western Blot, Marker, Purification

    13) Product Images from "A Comparative Analysis of Industrial Escherichia coli K-12 and B Strains in High-Glucose Batch Cultivations on Process-, Transcriptome- and Proteome Level"

    Article Title: A Comparative Analysis of Industrial Escherichia coli K-12 and B Strains in High-Glucose Batch Cultivations on Process-, Transcriptome- and Proteome Level

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0070516

    Batch growth and acetate data. Bacterial growth (cell dry mass; CDM) and acetate production in the supernatant of the three replicate cultivations of HMS174 (A), RV308 (B) and BL21 (C) showing the mean values including standard error of the mean.
    Figure Legend Snippet: Batch growth and acetate data. Bacterial growth (cell dry mass; CDM) and acetate production in the supernatant of the three replicate cultivations of HMS174 (A), RV308 (B) and BL21 (C) showing the mean values including standard error of the mean.

    Techniques Used:

    Differential responses: fli -operon of K–12 strains. Log-fold changes of HMS174 and RV308 for all genes of the fli -operon. These genes are absent on the BL21 genome because of an insertion element and consequently not in the commonset. Therefore, no BL21 mRNA expression data were available.
    Figure Legend Snippet: Differential responses: fli -operon of K–12 strains. Log-fold changes of HMS174 and RV308 for all genes of the fli -operon. These genes are absent on the BL21 genome because of an insertion element and consequently not in the commonset. Therefore, no BL21 mRNA expression data were available.

    Techniques Used: Expressing

    Motility test of the three hosts. Semi-synthetic agar plate (A, glucose present) with colonies of RV308 (top), HMS174 (bottom left), and BL21 (bottom right). None of the three strains showed swarming on the plate. Lysogeny broth agar plate (B, no glucose present) with colonies of RV308 (top), HMS174 (bottom left), and BL21 (bottom right). Only RV308 showed motile behavior by swarming on the plate.
    Figure Legend Snippet: Motility test of the three hosts. Semi-synthetic agar plate (A, glucose present) with colonies of RV308 (top), HMS174 (bottom left), and BL21 (bottom right). None of the three strains showed swarming on the plate. Lysogeny broth agar plate (B, no glucose present) with colonies of RV308 (top), HMS174 (bottom left), and BL21 (bottom right). Only RV308 showed motile behavior by swarming on the plate.

    Techniques Used:

    14) Product Images from "Development of a sandwich ELISA to detect Leishmania 40S ribosomal protein S12 antigen from blood samples of visceral leishmaniasis patients"

    Article Title: Development of a sandwich ELISA to detect Leishmania 40S ribosomal protein S12 antigen from blood samples of visceral leishmaniasis patients

    Journal: BMC Infectious Diseases

    doi: 10.1186/s12879-018-3420-2

    Production and purification of the recombinant abundant Leishmania proteins. The eight abundant Leishmania protein genes were PCR amplified from L. donovani and cloned into the pET29w expression vector. Recombinant proteins containing an N-terminal His-tag were expressed in E. coli BL21 and purified with Ni-NTA agarose. Purity was evaluated by SDS-PAGE and Coomassie blue staining. These recombinant Leishmania proteins were sent out for rabbit polyclonal antibody production
    Figure Legend Snippet: Production and purification of the recombinant abundant Leishmania proteins. The eight abundant Leishmania protein genes were PCR amplified from L. donovani and cloned into the pET29w expression vector. Recombinant proteins containing an N-terminal His-tag were expressed in E. coli BL21 and purified with Ni-NTA agarose. Purity was evaluated by SDS-PAGE and Coomassie blue staining. These recombinant Leishmania proteins were sent out for rabbit polyclonal antibody production

    Techniques Used: Purification, Recombinant, Polymerase Chain Reaction, Amplification, Clone Assay, Expressing, Plasmid Preparation, SDS Page, Staining

    15) Product Images from "Isolation and Characterization of the First Xylanolytic Hyperthermophilic Euryarchaeon Thermococcus sp. Strain 2319x1 and Its Unusual Multidomain Glycosidase"

    Article Title: Isolation and Characterization of the First Xylanolytic Hyperthermophilic Euryarchaeon Thermococcus sp. Strain 2319x1 and Its Unusual Multidomain Glycosidase

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2016.00552

    Cellulolytic activities of full length and truncated MDG proteins. Hydrolytic activities were analyzed on CMC-agar-plates (A) and in CMC-zymogram gels (B) . For CMC screening plates 10 μL (5–10 μg of total protein after heat precipitation at 60°C) and for zymogram gels 20 μL (3 μg of protein after heat precipitation at 60°C (GH5 and GH5-12) or 2 μg of solubilized membrane fraction (GH5-12-12 and MDG full length)) were applied. As negative control, crude extract of E. coli BL21 (DE3) pRIL with vector pET24a without insert was used. Marker, M (B) : prestained protein standard (Fermentas), GH5 (∼46 kDa) and GH5-12 (∼88 kDa), GH5-12-12 (∼125 kDa) and MDG full length (∼143 kDa).
    Figure Legend Snippet: Cellulolytic activities of full length and truncated MDG proteins. Hydrolytic activities were analyzed on CMC-agar-plates (A) and in CMC-zymogram gels (B) . For CMC screening plates 10 μL (5–10 μg of total protein after heat precipitation at 60°C) and for zymogram gels 20 μL (3 μg of protein after heat precipitation at 60°C (GH5 and GH5-12) or 2 μg of solubilized membrane fraction (GH5-12-12 and MDG full length)) were applied. As negative control, crude extract of E. coli BL21 (DE3) pRIL with vector pET24a without insert was used. Marker, M (B) : prestained protein standard (Fermentas), GH5 (∼46 kDa) and GH5-12 (∼88 kDa), GH5-12-12 (∼125 kDa) and MDG full length (∼143 kDa).

    Techniques Used: Negative Control, Plasmid Preparation, Marker

    16) Product Images from "Identification of Antigenic Proteins in Trichomonas vaginalis"

    Article Title: Identification of Antigenic Proteins in Trichomonas vaginalis

    Journal: The Korean Journal of Parasitology

    doi: 10.3347/kjp.2011.49.1.79

    Immunolocalization of AP65-1 adhesin in T. vaginalis trophozoites. (A) Expression of rAP65-1 in E. coli BL21 (DE3). E. coli expressing rAP65-1 (with 1 mM IPTG), was lysed by sonication, fractionated into cytoplasmic (C) and membrane (M) proteins. The resultant proteins were separated by 12% SDS-PAGE. (B) Reactivity of anti-AP65-1 antibodies against E. coli extracts expressing rAP65-1. (C) Detection of AP65-1 in T. vaginalis extracts by western blot analysis using anti-rAP65-1 antibodies. (D) T. vaginalis incubated with rat anti-rAP65-1 antibodies and mouse anti-tubulin antibodies. The slides were then incubated with TRITC-conjugated anti-rat IgG and FITC-conjugated anti-mouse IgG as secondary antibodies. To visualize nuclei, the cells were treated with 1 µg/ml-1 4'6-diamidino-2-phenylindole, mounted with an anti-fade mounting medium, and then observed with an immunofluorescence microscope. The bars represent 5 µm. (E) T. vaginalis trophozoites incubated with rat pre-immune serum. (a) a differential interference contrast (DIC) image, (b) a fluorescence image at a wavelength of 345 nm to detect nuclei, (c) a fluorescence image at a wavelength of 494 nm to detect flagella, (d) a fluorescence image at a wavelength of 547 nm to detect rAP65-1, (e) a combined fluorescence image, and (f) a combined fluorescence and DIC image.
    Figure Legend Snippet: Immunolocalization of AP65-1 adhesin in T. vaginalis trophozoites. (A) Expression of rAP65-1 in E. coli BL21 (DE3). E. coli expressing rAP65-1 (with 1 mM IPTG), was lysed by sonication, fractionated into cytoplasmic (C) and membrane (M) proteins. The resultant proteins were separated by 12% SDS-PAGE. (B) Reactivity of anti-AP65-1 antibodies against E. coli extracts expressing rAP65-1. (C) Detection of AP65-1 in T. vaginalis extracts by western blot analysis using anti-rAP65-1 antibodies. (D) T. vaginalis incubated with rat anti-rAP65-1 antibodies and mouse anti-tubulin antibodies. The slides were then incubated with TRITC-conjugated anti-rat IgG and FITC-conjugated anti-mouse IgG as secondary antibodies. To visualize nuclei, the cells were treated with 1 µg/ml-1 4'6-diamidino-2-phenylindole, mounted with an anti-fade mounting medium, and then observed with an immunofluorescence microscope. The bars represent 5 µm. (E) T. vaginalis trophozoites incubated with rat pre-immune serum. (a) a differential interference contrast (DIC) image, (b) a fluorescence image at a wavelength of 345 nm to detect nuclei, (c) a fluorescence image at a wavelength of 494 nm to detect flagella, (d) a fluorescence image at a wavelength of 547 nm to detect rAP65-1, (e) a combined fluorescence image, and (f) a combined fluorescence and DIC image.

    Techniques Used: Expressing, Sonication, SDS Page, Western Blot, Incubation, Immunofluorescence, Microscopy, Fluorescence

    17) Product Images from "Characterization of a New Cyclohexylamine Oxidase From Acinetobacter sp. YT-02"

    Article Title: Characterization of a New Cyclohexylamine Oxidase From Acinetobacter sp. YT-02

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02848

    SDS-PAGE [stacking gel (5%, w/v) and separating gel (10%, w/v)] analysis of CHAO YT–02 . Lanes: M, molecular standard; 1, purified recombinant CHAO YT–02 ; 2, E. coli BL21 (DE3) harboring pET-28b- chao after induction; 3, E. coli BL21 (DE3) harboring pET-28b- chao without induction; 4, E. coli BL21 (DE3) harboring pET-28b without induction; 5, E. coli BL21 (DE3) harboring pET-28b after induction.
    Figure Legend Snippet: SDS-PAGE [stacking gel (5%, w/v) and separating gel (10%, w/v)] analysis of CHAO YT–02 . Lanes: M, molecular standard; 1, purified recombinant CHAO YT–02 ; 2, E. coli BL21 (DE3) harboring pET-28b- chao after induction; 3, E. coli BL21 (DE3) harboring pET-28b- chao without induction; 4, E. coli BL21 (DE3) harboring pET-28b without induction; 5, E. coli BL21 (DE3) harboring pET-28b after induction.

    Techniques Used: SDS Page, Purification, Recombinant, Positron Emission Tomography

    18) Product Images from "Heterologous expression of a truncated form of human recombinant vascular endothelial growth factor-A and its biological activity in wound healing"

    Article Title: Heterologous expression of a truncated form of human recombinant vascular endothelial growth factor-A and its biological activity in wound healing

    Journal: Iranian Journal of Basic Medical Sciences

    doi: 10.22038/IJBMS.2017.9011

    Approved purified proteins in pLysS and E. coli BL21 (DE3) (Line 1: Marker, Line 2: pLysS, Line 3:DE3)
    Figure Legend Snippet: Approved purified proteins in pLysS and E. coli BL21 (DE3) (Line 1: Marker, Line 2: pLysS, Line 3:DE3)

    Techniques Used: Purification, Marker

    19) Product Images from "Metagenomic approach for the isolation of a thermostable ?-galactosidase with high tolerance of galactose and glucose from soil samples of Turpan Basin"

    Article Title: Metagenomic approach for the isolation of a thermostable ?-galactosidase with high tolerance of galactose and glucose from soil samples of Turpan Basin

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-13-237

    SDS-PAGE analysis of recombinant Gal308 from supernatant of E. coli BL21 (DE3) cell lysates and purified Gal308 by affinity chromatography. Lanes: M, standard protein molecular mass markers (sizes in kilodaltons are indicated on the left); 1, recombinant Gal308 from supernatant of E. coli BL21 (DE3) cell lysates; 2, recombinant Gal308 purified by His•Bind® Purification Kit. The sizes in kilodaltons of protein marker were listed as follows: porcine heart myosin (200,000 Da), E. coli β-galactosidase (116,000 Da), rabbit muscle phosphorylase B (97,200 Da), bovine serum albumin (66,409 Da), ovalbumin (44,287 Da), carbonic anhydrase (29,000 Da).
    Figure Legend Snippet: SDS-PAGE analysis of recombinant Gal308 from supernatant of E. coli BL21 (DE3) cell lysates and purified Gal308 by affinity chromatography. Lanes: M, standard protein molecular mass markers (sizes in kilodaltons are indicated on the left); 1, recombinant Gal308 from supernatant of E. coli BL21 (DE3) cell lysates; 2, recombinant Gal308 purified by His•Bind® Purification Kit. The sizes in kilodaltons of protein marker were listed as follows: porcine heart myosin (200,000 Da), E. coli β-galactosidase (116,000 Da), rabbit muscle phosphorylase B (97,200 Da), bovine serum albumin (66,409 Da), ovalbumin (44,287 Da), carbonic anhydrase (29,000 Da).

    Techniques Used: SDS Page, Recombinant, Purification, Affinity Chromatography, Marker

    20) Product Images from "Chemical complexity of protein determines optimal E. coli expression host; A comparative study using Erythropoietin, Streptokinase and Tumor Necrosis Factor Receptor"

    Article Title: Chemical complexity of protein determines optimal E. coli expression host; A comparative study using Erythropoietin, Streptokinase and Tumor Necrosis Factor Receptor

    Journal: Journal of Genetic Engineering & Biotechnology

    doi: 10.1016/j.jgeb.2016.12.006

    Comparison of expression levels of TNFR ED, EPO and SK in different hosts at 37 °C on induction with 1 mM IPTG for E. coli BL21 (DE3) and BL21 (DE3) pLys S strains and 0.3 M NaCl for E. coli GJ1158 strains at (A) 0.6 OD 600 , (B) 1.0 OD. Basal level expression was given a minimal value of 0.5%.
    Figure Legend Snippet: Comparison of expression levels of TNFR ED, EPO and SK in different hosts at 37 °C on induction with 1 mM IPTG for E. coli BL21 (DE3) and BL21 (DE3) pLys S strains and 0.3 M NaCl for E. coli GJ1158 strains at (A) 0.6 OD 600 , (B) 1.0 OD. Basal level expression was given a minimal value of 0.5%.

    Techniques Used: Expressing

    Immunoblot analysis of total protein of induced E. coli BL21 (DE3) cellsharboring the recombinant plasmids using anti-His antibodies . Lanes 1 – Protein marker, 2 – pRSET A vector control induced, 3 – pRSET-TNFR ED uninduced, 4 – pRSET-TNFR ED induced, 5 – pRSET EPO-uninduced, 6 – pRSET EPO-induced, 7 – pRSET SK uninduced, 8 – pRSET SK induced.
    Figure Legend Snippet: Immunoblot analysis of total protein of induced E. coli BL21 (DE3) cellsharboring the recombinant plasmids using anti-His antibodies . Lanes 1 – Protein marker, 2 – pRSET A vector control induced, 3 – pRSET-TNFR ED uninduced, 4 – pRSET-TNFR ED induced, 5 – pRSET EPO-uninduced, 6 – pRSET EPO-induced, 7 – pRSET SK uninduced, 8 – pRSET SK induced.

    Techniques Used: Recombinant, Marker, Plasmid Preparation

    21) Product Images from "A Highly-Conserved Single-Stranded DNA-Binding Protein in Xanthomonas Functions as a Harpin-Like Protein to Trigger Plant Immunity"

    Article Title: A Highly-Conserved Single-Stranded DNA-Binding Protein in Xanthomonas Functions as a Harpin-Like Protein to Trigger Plant Immunity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056240

    A highly conserved single-stranded DNA-binding protein (SSB) triggers a HR in tobacco. (A) HR induction by the SSB Xoc protein of X. oryzae pv. oryzicola . A. tumefaciens GV3101 (OD 600 = 0.5) containing hpa1 , ssb X and bax genes in the PVX vector pgR107 was inoculated into N. benthamiana tobacco leaves with a needleless syringe. Hpa1 and Bax were used as positive controls, and A. tumefaciens containing the empty vector PVX was used as a negative control. (B) Concentration of SSB Xoc required for HR induction in tobacco cv. Xanthi. Purified SSB Xoc was diluted in PBS buffer and inoculated into tobacco with needleless syringes. Hpa1, which functions as a harpin of X. oryzae pv. oryzicola , was used as a positive control and EVP as a negative control. (C) HR assays in tobacco inoculated with SSB X homologues obtained from various bacterial species. SSB proteins were overproduced in E. coli , purified (see Methods), and diluted in PBS buffer to different concentrations from 0.01 to 50 µM. A typical image of HRs on tobacco leaves caused by the proteins at 1 µM was taken in this report. Numbers represent sections of leaves inoculated with the following: 1, EVP; 2, SSB Xoc from X. oryzae pv. oryzicola RS105; 3, SSB Xoo from X. oryzae pv. oryzae PXO99 A ; 4, SSB Xac from X. axonopodis pv. citri 306; 5, SSB Xcv from X. campestris pv. vesicatoria 85-10; 6, SSB Xcc from X. campestris pv. campestris 8004; 7, SSB Pf from P. fluorescens Pf-5; 8, Hpa1 Xoc , from X. oryzae pv. oryzicola RS105; 9, SSB Ea from E. amylovora 0065; 10, SSB Ec from E. coli BL21 (DES); 11, SSB Rs from R. solanacearum ZJ2731; and 12, SSB Pst from P. syringae pv. tomato DC3000. (D) Assays for SSBx and Hpa1-induced HR in response to various metabolic inhibitors. Tobacco plants were infiltrated with SSB X (1 µM) or Hpa1 (0.5 µM), which was heat-treated or incubated (see methods) with one of the following: 1 µM LaCl 3 , 0.71 µM actinomycin D, 0.1 µM cycloheximide or protease K (0.5 U/ml). Leaf panels: 1, Hpa1; 2, heat-treated Hpa1; 3, protease K-treated Hpa1; 4, Hpa1 plus 1 µM LaCl 3 ; 5, Hpa1 plus 0.71 µM actinomycin D; 6, Hpa1 plus 0.1 µM cycloheximide; 7, SSB X ; 8, heat-treated SSB Xoc ; 9, protease K-treated SSB Xoc ; 10, SSB Xoc plus LaCl 3 ; 11, SSB Xoc plus 0.1 µM actinomycin D; 12, SSB Xoc plus cycloheximide; 13, distilled water; and 14, EVP. Leaves in panels A to D were photographed 24–48 h after infiltration. (E) Analysis of DNA laddering in SSB Xoc -treated tobacco leaves. Total genomic DNA was isolated from tobacco leaves 3, 6, 12, 24, 36 and 48 hpi with Hpa1 (0.5 µM), SSB Xoc (1 µM) and EVP. DNA laddering was evaluated in 2% agarose gels. (F) Northern blot analysis in tobacco inoculated with SSB X , Hpa1, or EVP. The marker genes, HIN1 , HSR203J , PR1a and PR1b , were chosen as the targets. Total RNAs were extracted from tobacco leaves infiltrated with SSB Xoc (1 µM), Hpa1 (0.5 µM), or PBS buffer. Aliquots (10 µg each) of the extracted RNAs were separated in 1% agarose gels, transferred onto membranes, and analyzed by northern blotting. Blots were hybridized with digoxigenin-labeled HIN1 , HSR203J , PR1a and PR1b cDNA. The experiment was conducted twice with similar results.
    Figure Legend Snippet: A highly conserved single-stranded DNA-binding protein (SSB) triggers a HR in tobacco. (A) HR induction by the SSB Xoc protein of X. oryzae pv. oryzicola . A. tumefaciens GV3101 (OD 600 = 0.5) containing hpa1 , ssb X and bax genes in the PVX vector pgR107 was inoculated into N. benthamiana tobacco leaves with a needleless syringe. Hpa1 and Bax were used as positive controls, and A. tumefaciens containing the empty vector PVX was used as a negative control. (B) Concentration of SSB Xoc required for HR induction in tobacco cv. Xanthi. Purified SSB Xoc was diluted in PBS buffer and inoculated into tobacco with needleless syringes. Hpa1, which functions as a harpin of X. oryzae pv. oryzicola , was used as a positive control and EVP as a negative control. (C) HR assays in tobacco inoculated with SSB X homologues obtained from various bacterial species. SSB proteins were overproduced in E. coli , purified (see Methods), and diluted in PBS buffer to different concentrations from 0.01 to 50 µM. A typical image of HRs on tobacco leaves caused by the proteins at 1 µM was taken in this report. Numbers represent sections of leaves inoculated with the following: 1, EVP; 2, SSB Xoc from X. oryzae pv. oryzicola RS105; 3, SSB Xoo from X. oryzae pv. oryzae PXO99 A ; 4, SSB Xac from X. axonopodis pv. citri 306; 5, SSB Xcv from X. campestris pv. vesicatoria 85-10; 6, SSB Xcc from X. campestris pv. campestris 8004; 7, SSB Pf from P. fluorescens Pf-5; 8, Hpa1 Xoc , from X. oryzae pv. oryzicola RS105; 9, SSB Ea from E. amylovora 0065; 10, SSB Ec from E. coli BL21 (DES); 11, SSB Rs from R. solanacearum ZJ2731; and 12, SSB Pst from P. syringae pv. tomato DC3000. (D) Assays for SSBx and Hpa1-induced HR in response to various metabolic inhibitors. Tobacco plants were infiltrated with SSB X (1 µM) or Hpa1 (0.5 µM), which was heat-treated or incubated (see methods) with one of the following: 1 µM LaCl 3 , 0.71 µM actinomycin D, 0.1 µM cycloheximide or protease K (0.5 U/ml). Leaf panels: 1, Hpa1; 2, heat-treated Hpa1; 3, protease K-treated Hpa1; 4, Hpa1 plus 1 µM LaCl 3 ; 5, Hpa1 plus 0.71 µM actinomycin D; 6, Hpa1 plus 0.1 µM cycloheximide; 7, SSB X ; 8, heat-treated SSB Xoc ; 9, protease K-treated SSB Xoc ; 10, SSB Xoc plus LaCl 3 ; 11, SSB Xoc plus 0.1 µM actinomycin D; 12, SSB Xoc plus cycloheximide; 13, distilled water; and 14, EVP. Leaves in panels A to D were photographed 24–48 h after infiltration. (E) Analysis of DNA laddering in SSB Xoc -treated tobacco leaves. Total genomic DNA was isolated from tobacco leaves 3, 6, 12, 24, 36 and 48 hpi with Hpa1 (0.5 µM), SSB Xoc (1 µM) and EVP. DNA laddering was evaluated in 2% agarose gels. (F) Northern blot analysis in tobacco inoculated with SSB X , Hpa1, or EVP. The marker genes, HIN1 , HSR203J , PR1a and PR1b , were chosen as the targets. Total RNAs were extracted from tobacco leaves infiltrated with SSB Xoc (1 µM), Hpa1 (0.5 µM), or PBS buffer. Aliquots (10 µg each) of the extracted RNAs were separated in 1% agarose gels, transferred onto membranes, and analyzed by northern blotting. Blots were hybridized with digoxigenin-labeled HIN1 , HSR203J , PR1a and PR1b cDNA. The experiment was conducted twice with similar results.

    Techniques Used: Binding Assay, Plasmid Preparation, Negative Control, Concentration Assay, Purification, Positive Control, Incubation, DNA Laddering, Isolation, Northern Blot, Marker, Labeling

    22) Product Images from "A novel HPV16 E7-affitoxin for targeted therapy of HPV16-induced human cervical cancer"

    Article Title: A novel HPV16 E7-affitoxin for targeted therapy of HPV16-induced human cervical cancer

    Journal: Theranostics

    doi: 10.7150/thno.24607

    Expression and purification of Z HPV16 E7 affitoxin 384 protein. The pET21a (+)/Z HPV16 E7 affitoxin384 plasmid was transformed into E. coli BL21 (DE3). The protein was expressed and purified by Ni-NTA agarose affinity chromatography. (A) Schematic structure of pET21a (+)/Z HPV16 E7 affitoxin384 plasmid. (B) Comassie blue-stained SDS-PAGE gel of the recombinant proteins. M, protein marker; 1, Empty E.coli BL21 (DE3); 2, E.coli BL21 (DE3) transformed with pET21a empty vector; 3, E.coli BL21 (DE3) transformed with pET21a (+)/Z HPV16 E7 affitoxin384 plasmid; 4-5, E.coli BL21 (DE3) transformed with pET21a (+)/Z HPV16 E7 affitoxin384 plasmid and induced by 1 mM IPTG; 6, E.coli BL21 (DE3) transformed with pET21a (+)/Z wt affitoxin; 7-8, E.coli BL21 (DE3) transformed with pET21a (+)/Z wt affitoxin and induced by 1 mM IPTG. (C) Analysis of the purified Z HPV16 E7 affitoxin384 and Z wt affitoxin recombinant proteins by SDS-PAGE. M, protein marker; 1, Z HPV16 E7 affitoxin384; 2, Z wt affitoxin. (D-F) Confirmation of the expression of Z HPV16 E7 affitoxin384 and Z wt affitoxin recombinant proteins by western blot using the primary antibodies against His tag, PE38KDEL and SPA-Z, respectively. 1, Empty E.coli BL21 (DE3); 2, E.coli BL21 (DE3) transformed with pET21a empty vector; 3, E.coli BL21 (DE3) transformed with pET21a (+)/Z HPV16 E7 affitoxin384 plasmid and induced by 1 mM IPTG; 4, E.coli BL21 (DE3) transformed with pET21a (+)/Z wt affitoxin and induced by 1 mM IPTG.
    Figure Legend Snippet: Expression and purification of Z HPV16 E7 affitoxin 384 protein. The pET21a (+)/Z HPV16 E7 affitoxin384 plasmid was transformed into E. coli BL21 (DE3). The protein was expressed and purified by Ni-NTA agarose affinity chromatography. (A) Schematic structure of pET21a (+)/Z HPV16 E7 affitoxin384 plasmid. (B) Comassie blue-stained SDS-PAGE gel of the recombinant proteins. M, protein marker; 1, Empty E.coli BL21 (DE3); 2, E.coli BL21 (DE3) transformed with pET21a empty vector; 3, E.coli BL21 (DE3) transformed with pET21a (+)/Z HPV16 E7 affitoxin384 plasmid; 4-5, E.coli BL21 (DE3) transformed with pET21a (+)/Z HPV16 E7 affitoxin384 plasmid and induced by 1 mM IPTG; 6, E.coli BL21 (DE3) transformed with pET21a (+)/Z wt affitoxin; 7-8, E.coli BL21 (DE3) transformed with pET21a (+)/Z wt affitoxin and induced by 1 mM IPTG. (C) Analysis of the purified Z HPV16 E7 affitoxin384 and Z wt affitoxin recombinant proteins by SDS-PAGE. M, protein marker; 1, Z HPV16 E7 affitoxin384; 2, Z wt affitoxin. (D-F) Confirmation of the expression of Z HPV16 E7 affitoxin384 and Z wt affitoxin recombinant proteins by western blot using the primary antibodies against His tag, PE38KDEL and SPA-Z, respectively. 1, Empty E.coli BL21 (DE3); 2, E.coli BL21 (DE3) transformed with pET21a empty vector; 3, E.coli BL21 (DE3) transformed with pET21a (+)/Z HPV16 E7 affitoxin384 plasmid and induced by 1 mM IPTG; 4, E.coli BL21 (DE3) transformed with pET21a (+)/Z wt affitoxin and induced by 1 mM IPTG.

    Techniques Used: Expressing, Purification, Plasmid Preparation, Transformation Assay, Affinity Chromatography, Staining, SDS Page, Recombinant, Marker, Western Blot

    23) Product Images from "The non-typeable Haemophilus influenzae major adhesin Hia is a dual function lectin that binds to human-specific respiratory tract sialic acid glycan receptors"

    Article Title: The non-typeable Haemophilus influenzae major adhesin Hia is a dual function lectin that binds to human-specific respiratory tract sialic acid glycan receptors

    Journal: bioRxiv

    doi: 10.1101/2020.05.08.084038

    Percent adherence to Chang cells of E. coli BL21 strains expressing wild-type Hia or isogenic mutant variants. Percent adherence of each strain is calculated as adherent cfu following 2 hrs incubation / total input cfu. All raw data is presented in Supplementary Data 1. * = P-value =
    Figure Legend Snippet: Percent adherence to Chang cells of E. coli BL21 strains expressing wild-type Hia or isogenic mutant variants. Percent adherence of each strain is calculated as adherent cfu following 2 hrs incubation / total input cfu. All raw data is presented in Supplementary Data 1. * = P-value =

    Techniques Used: Expressing, Mutagenesis, Incubation

    24) Product Images from "Cloning, Expression and Characterization of a Lipase Encoding Gene from Human Oral Metagenome"

    Article Title: Cloning, Expression and Characterization of a Lipase Encoding Gene from Human Oral Metagenome

    Journal: Indian Journal of Microbiology

    doi: 10.1007/s12088-014-0455-y

    PAGE analysis. a Native PAGE. Lane M molecular weight marker, Lane 1 purified OMLip1. b SDS-PAGE. Lane M molecular weight marker, Lane 1 intracellular lysate of E. coli BL21 (DE3) transformed with pET-30b, Lane 2 intracellular lysate of E. coli BL21 (DE3) transformed with pETOMLip1, Lane 3 purified fraction from Ni–NTA chromatography, Lane 4 zymogram of purified fraction with tributyrin as substrate
    Figure Legend Snippet: PAGE analysis. a Native PAGE. Lane M molecular weight marker, Lane 1 purified OMLip1. b SDS-PAGE. Lane M molecular weight marker, Lane 1 intracellular lysate of E. coli BL21 (DE3) transformed with pET-30b, Lane 2 intracellular lysate of E. coli BL21 (DE3) transformed with pETOMLip1, Lane 3 purified fraction from Ni–NTA chromatography, Lane 4 zymogram of purified fraction with tributyrin as substrate

    Techniques Used: Polyacrylamide Gel Electrophoresis, Clear Native PAGE, Molecular Weight, Marker, Purification, SDS Page, Transformation Assay, Positron Emission Tomography, Chromatography

    25) Product Images from "Identification of defensin-encoding genes of Picea glauca: characterization of PgD5, a conserved spruce defensin with strong antifungal activity"

    Article Title: Identification of defensin-encoding genes of Picea glauca: characterization of PgD5, a conserved spruce defensin with strong antifungal activity

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-12-180

    Purification of recombinant PgD5 produced by E. coli BL21 (Origami pLys S) DE3. ( A ) RP-HPLC chromatography of the His6-SUMO-PgD5 fusion protein after cleavage with the SUMO protease. Elution times are marked. ( B ) SDS-PAGE analysis of the His6-SUMO-PgD5 fusion before and after SUMO protease treatment. Lane M, low molecular weight marker (New England Biolabs). Lane 1, Purified fusion protein by Ni-NTA column. Lane 2, SUMO protease cleavage products after 60 min. Lane 3, Purified PgD5 peptide after SUMO protease digestion and reverse-phase chromatography. ( C ) Mass spectrometric analysis of recombinant PgD5 after separation from the His6-SUMO tag using reverse-phase chromatography.
    Figure Legend Snippet: Purification of recombinant PgD5 produced by E. coli BL21 (Origami pLys S) DE3. ( A ) RP-HPLC chromatography of the His6-SUMO-PgD5 fusion protein after cleavage with the SUMO protease. Elution times are marked. ( B ) SDS-PAGE analysis of the His6-SUMO-PgD5 fusion before and after SUMO protease treatment. Lane M, low molecular weight marker (New England Biolabs). Lane 1, Purified fusion protein by Ni-NTA column. Lane 2, SUMO protease cleavage products after 60 min. Lane 3, Purified PgD5 peptide after SUMO protease digestion and reverse-phase chromatography. ( C ) Mass spectrometric analysis of recombinant PgD5 after separation from the His6-SUMO tag using reverse-phase chromatography.

    Techniques Used: Purification, Recombinant, Produced, High Performance Liquid Chromatography, Chromatography, SDS Page, Molecular Weight, Marker, Reversed-phase Chromatography

    26) Product Images from "The Oxidative Stress Agent Hypochlorite Stimulates c-di-GMP Synthesis and Biofilm Formation in Pseudomonas aeruginosa"

    Article Title: The Oxidative Stress Agent Hypochlorite Stimulates c-di-GMP Synthesis and Biofilm Formation in Pseudomonas aeruginosa

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.02311

    Characterization of the DGC PA3177. (A) E. coli BL21-pET3177 and the vector control strain BL21-pET23a were grown in LB broth supplemented with 0.4 mM IPTG for 5 h at 37°C followed by nucleotide extraction and quantification of intracellular c-di-GMP by LC-MS. Levels of c-di-GMP were normalized to the total protein content of the respective sample and compared to c-di-GMP levels in empty vector control strain BL21-pET23a ( n = 6). The chromatogram shows c-di-GMP peaks of one representative measurement. (B) Attachment of P. aeruginosa during PA3177 overexpression was evaluated by crystal violet staining. PAO1-pJN3177 and empty vector control strain PAO1-pJN105 were incubated in 96-well microtiter plates for 2 h at 37°C followed by biomass quantification. For recombinant gene expression, BM2 was supplemented with 0.1% (w/v) arabinose (+Ara). Cultures without arabinose (–Ara) served as negative controls. Experiments were carried out in triplicate, each with six wells per strain and condition ( n = 18). (C) Planktonic growth during 5 h at 37°C. (D) Overnight cultures of PA3177-overexpressing strain P. aeruginosa PA01-pJN3177 and vector control PAO1-pJN105 were diluted in LB broth supplemented with 0.1% (w/v) arabinose and grown for 5 h at 37°C followed by evaluation of swimming, swarming and twitching motility. Assays were carried out with three independent bacterial cultures and at least four agar plates per experiment ( n ≥ 12). In all experiments statistical significance was evaluated by the Mann-Whitney test ( ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.
    Figure Legend Snippet: Characterization of the DGC PA3177. (A) E. coli BL21-pET3177 and the vector control strain BL21-pET23a were grown in LB broth supplemented with 0.4 mM IPTG for 5 h at 37°C followed by nucleotide extraction and quantification of intracellular c-di-GMP by LC-MS. Levels of c-di-GMP were normalized to the total protein content of the respective sample and compared to c-di-GMP levels in empty vector control strain BL21-pET23a ( n = 6). The chromatogram shows c-di-GMP peaks of one representative measurement. (B) Attachment of P. aeruginosa during PA3177 overexpression was evaluated by crystal violet staining. PAO1-pJN3177 and empty vector control strain PAO1-pJN105 were incubated in 96-well microtiter plates for 2 h at 37°C followed by biomass quantification. For recombinant gene expression, BM2 was supplemented with 0.1% (w/v) arabinose (+Ara). Cultures without arabinose (–Ara) served as negative controls. Experiments were carried out in triplicate, each with six wells per strain and condition ( n = 18). (C) Planktonic growth during 5 h at 37°C. (D) Overnight cultures of PA3177-overexpressing strain P. aeruginosa PA01-pJN3177 and vector control PAO1-pJN105 were diluted in LB broth supplemented with 0.1% (w/v) arabinose and grown for 5 h at 37°C followed by evaluation of swimming, swarming and twitching motility. Assays were carried out with three independent bacterial cultures and at least four agar plates per experiment ( n ≥ 12). In all experiments statistical significance was evaluated by the Mann-Whitney test ( ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.

    Techniques Used: Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Over Expression, Staining, Incubation, Recombinant, Expressing, Acetylene Reduction Assay, MANN-WHITNEY

    27) Product Images from "Characterization of a New Cyclohexylamine Oxidase From Acinetobacter sp. YT-02"

    Article Title: Characterization of a New Cyclohexylamine Oxidase From Acinetobacter sp. YT-02

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02848

    SDS-PAGE [stacking gel (5%, w/v) and separating gel (10%, w/v)] analysis of CHAO YT–02 . Lanes: M, molecular standard; 1, purified recombinant CHAO YT–02 ; 2, E. coli BL21 (DE3) harboring pET-28b- chao after induction; 3, E. coli BL21 (DE3) harboring pET-28b- chao without induction; 4, E. coli BL21 (DE3) harboring pET-28b without induction; 5, E. coli BL21 (DE3) harboring pET-28b after induction.
    Figure Legend Snippet: SDS-PAGE [stacking gel (5%, w/v) and separating gel (10%, w/v)] analysis of CHAO YT–02 . Lanes: M, molecular standard; 1, purified recombinant CHAO YT–02 ; 2, E. coli BL21 (DE3) harboring pET-28b- chao after induction; 3, E. coli BL21 (DE3) harboring pET-28b- chao without induction; 4, E. coli BL21 (DE3) harboring pET-28b without induction; 5, E. coli BL21 (DE3) harboring pET-28b after induction.

    Techniques Used: SDS Page, Purification, Recombinant, Positron Emission Tomography

    28) Product Images from "Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae"

    Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-12-16

    Purification of SXT-Exo and lambda-Exo, and determination of their multimericity by size exclusion chromatography . Panel A : Size exclusion chromatogram of purified SXT-Exo protein expressed from plasmid pEA1-1. Panel B : Size exclusion chromatogram of purified lambda-Exo protein expressed from plasmid pEE4. Panel C : 12% polyacrylamide gel (SDS-PAGE) analysis of the SXT-Exo purification procedure and purified SXT-Bet, SXT-Ssb, lambda-Bet and lambda-Exo proteins; lane 1: Benchmark protein ladder (Invitrogen); lane 2: pEA1-1/ E. coli BL21 (DE3) pLysS Rosetta whole cell extract immediately prior to induction; lane 3: whole cell extract 6 hours after induction with IPTG; lane 4: supernatant from cell extract 6 hours post induction; lane 5: purified SXT-Exo; lane 6: purified SXT-Bet expressed from pX28-1; lane 7: purified SXT-Ssb expressed from pSB2; lane 8: purified lambda-Bet expressed from p1DB; lane 9: purified lambda-Exo expressed from pEE4.
    Figure Legend Snippet: Purification of SXT-Exo and lambda-Exo, and determination of their multimericity by size exclusion chromatography . Panel A : Size exclusion chromatogram of purified SXT-Exo protein expressed from plasmid pEA1-1. Panel B : Size exclusion chromatogram of purified lambda-Exo protein expressed from plasmid pEE4. Panel C : 12% polyacrylamide gel (SDS-PAGE) analysis of the SXT-Exo purification procedure and purified SXT-Bet, SXT-Ssb, lambda-Bet and lambda-Exo proteins; lane 1: Benchmark protein ladder (Invitrogen); lane 2: pEA1-1/ E. coli BL21 (DE3) pLysS Rosetta whole cell extract immediately prior to induction; lane 3: whole cell extract 6 hours after induction with IPTG; lane 4: supernatant from cell extract 6 hours post induction; lane 5: purified SXT-Exo; lane 6: purified SXT-Bet expressed from pX28-1; lane 7: purified SXT-Ssb expressed from pSB2; lane 8: purified lambda-Bet expressed from p1DB; lane 9: purified lambda-Exo expressed from pEE4.

    Techniques Used: Purification, Size-exclusion Chromatography, Plasmid Preparation, SDS Page

    29) Product Images from "A novel esterase from a marine mud metagenomic library for biocatalytic synthesis of short-chain flavor esters"

    Article Title: A novel esterase from a marine mud metagenomic library for biocatalytic synthesis of short-chain flavor esters

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-016-0435-5

    SDS-PAGE analysis of soluble lysates of lipolytic enzymes and the purified esterase EST4. Lane M, molecular weight protein marker (Thermo Scientific, Cat. No: 26610); lane 1 , soluble lysates of E. coli Top10Fʹ/pLLP-OmpA, as negative control; lane 2 , soluble lysates of E. coli BL21/pET28a, as negative control; lane 3 , EST1; lane 4 , EST2; lane 5 , EST3; lane 6 , EST4; lane 7 , EST5; lane 8 , purified EST4 (34 kDa). The arrows indicate expected proteins from recombinant E. coli
    Figure Legend Snippet: SDS-PAGE analysis of soluble lysates of lipolytic enzymes and the purified esterase EST4. Lane M, molecular weight protein marker (Thermo Scientific, Cat. No: 26610); lane 1 , soluble lysates of E. coli Top10Fʹ/pLLP-OmpA, as negative control; lane 2 , soluble lysates of E. coli BL21/pET28a, as negative control; lane 3 , EST1; lane 4 , EST2; lane 5 , EST3; lane 6 , EST4; lane 7 , EST5; lane 8 , purified EST4 (34 kDa). The arrows indicate expected proteins from recombinant E. coli

    Techniques Used: SDS Page, Purification, Molecular Weight, Marker, Negative Control, Recombinant

    30) Product Images from "Cloning, Expression and Purification of Pseudomonas putida ATCC12633 Creatinase"

    Article Title: Cloning, Expression and Purification of Pseudomonas putida ATCC12633 Creatinase

    Journal: Avicenna Journal of Medical Biotechnology

    doi:

    SDS-PAGE analysis of recombinant creatinase expression. Lane 1 and 2 are soluble fractions of induced BL21 (containing pET28a-cre) for 4 and 16 hr and 3 is soluble fraction of induced negative control. Lane 4 is the protein ladder. Lane 5 and 6 are crude extract of total protein of induced BL21 (containing pET28a-cre), and lane 7 is the negative control.
    Figure Legend Snippet: SDS-PAGE analysis of recombinant creatinase expression. Lane 1 and 2 are soluble fractions of induced BL21 (containing pET28a-cre) for 4 and 16 hr and 3 is soluble fraction of induced negative control. Lane 4 is the protein ladder. Lane 5 and 6 are crude extract of total protein of induced BL21 (containing pET28a-cre), and lane 7 is the negative control.

    Techniques Used: SDS Page, Recombinant, Expressing, Negative Control

    31) Product Images from "Chemical complexity of protein determines optimal E. coli expression host; A comparative study using Erythropoietin, Streptokinase and Tumor Necrosis Factor Receptor"

    Article Title: Chemical complexity of protein determines optimal E. coli expression host; A comparative study using Erythropoietin, Streptokinase and Tumor Necrosis Factor Receptor

    Journal: Journal of Genetic Engineering & Biotechnology

    doi: 10.1016/j.jgeb.2016.12.006

    Comparison of expression levels of TNFR ED, EPO and SK in different hosts at 37 °C on induction with 1 mM IPTG for E. coli BL21 (DE3) and BL21 (DE3) pLys S strains and 0.3 M NaCl for E. coli GJ1158 strains at (A) 0.6 OD 600 , (B) 1.0 OD. Basal level expression was given a minimal value of 0.5%.
    Figure Legend Snippet: Comparison of expression levels of TNFR ED, EPO and SK in different hosts at 37 °C on induction with 1 mM IPTG for E. coli BL21 (DE3) and BL21 (DE3) pLys S strains and 0.3 M NaCl for E. coli GJ1158 strains at (A) 0.6 OD 600 , (B) 1.0 OD. Basal level expression was given a minimal value of 0.5%.

    Techniques Used: Expressing

    Immunoblot analysis of total protein of induced E. coli BL21 (DE3) cellsharboring the recombinant plasmids using anti-His antibodies . Lanes 1 – Protein marker, 2 – pRSET A vector control induced, 3 – pRSET-TNFR ED uninduced, 4 – pRSET-TNFR ED induced, 5 – pRSET EPO-uninduced, 6 – pRSET EPO-induced, 7 – pRSET SK uninduced, 8 – pRSET SK induced.
    Figure Legend Snippet: Immunoblot analysis of total protein of induced E. coli BL21 (DE3) cellsharboring the recombinant plasmids using anti-His antibodies . Lanes 1 – Protein marker, 2 – pRSET A vector control induced, 3 – pRSET-TNFR ED uninduced, 4 – pRSET-TNFR ED induced, 5 – pRSET EPO-uninduced, 6 – pRSET EPO-induced, 7 – pRSET SK uninduced, 8 – pRSET SK induced.

    Techniques Used: Recombinant, Marker, Plasmid Preparation

    32) Product Images from "FAM122A, a new endogenous inhibitor of protein phosphatase 2A"

    Article Title: FAM122A, a new endogenous inhibitor of protein phosphatase 2A

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11698

    FAM122A interacts directly with PP2A-Aα and B55α subunits ( A ) The graph indicats PP2A heterotrimer. ( B ) E. coli BL21 expressing GST and GST-FAM122A proteins were purified and incubated with His-tagged PP2A subunits. The GST pull down complexes were blotted with anti-His (top right panel) or anti-GST antibody (bottom).
    Figure Legend Snippet: FAM122A interacts directly with PP2A-Aα and B55α subunits ( A ) The graph indicats PP2A heterotrimer. ( B ) E. coli BL21 expressing GST and GST-FAM122A proteins were purified and incubated with His-tagged PP2A subunits. The GST pull down complexes were blotted with anti-His (top right panel) or anti-GST antibody (bottom).

    Techniques Used: Expressing, Purification, Incubation

    33) Product Images from "Immunoprotective potential of BamA, the outer membrane protein assembly factor, against MDR Acinetobacter baumannii"

    Article Title: Immunoprotective potential of BamA, the outer membrane protein assembly factor, against MDR Acinetobacter baumannii

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-12789-3

    SDS PAGE (12%): Lane 1: purified BamA; lane 2, 3 induced and uninduced E. coli BL21 (pET28-a- bamA ), lane 4: pink plus protein marker.
    Figure Legend Snippet: SDS PAGE (12%): Lane 1: purified BamA; lane 2, 3 induced and uninduced E. coli BL21 (pET28-a- bamA ), lane 4: pink plus protein marker.

    Techniques Used: SDS Page, Purification, Marker

    34) Product Images from "Yersinia pestis YscG Protein Is a Syc-Like Chaperone That Directly Binds YscE"

    Article Title: Yersinia pestis YscG Protein Is a Syc-Like Chaperone That Directly Binds YscE

    Journal: Infection and Immunity

    doi:

    Binding of YscG-FLAG to MBP-YscE. (A) Cell pellet fractions from E. coli BL21 expressing MBP, MBP-YscE, MBP-YscG, MBP-YscH, MBP-YscI, MBP-YscB, or MBP-YscF were separated by SDS-PAGE and transferred to Immobilon-P membranes. (B) Immobilon-P membranes containing cell pellet fractions from strain BL21 expressing MBP, MBP-YscE, or truncated MBP-YscE Δ57–66 , MBP-YscE Δ47–66 , MBP-YscE Δ1–9 or MBP-YscE Δ1–19 . MBP migrated more slowly than expected due to an in-frame fusion between malE and the vector LacZ α-peptide encoding sequences (MBP-α). (A and B) A cytoplasmic extract from BL21 carrying pYSCG-FLAG was used to probe the Immobilon-P membranes containing the SDS-PAGE-separated MBP and MBP derivatives. Bound YscG-FLAG was detected with the FLAG M2 monoclonal antibody. MBP and the MBP derivatives were detected with a polyclonal antiserum specific for the MBP portion of the hybrid proteins.
    Figure Legend Snippet: Binding of YscG-FLAG to MBP-YscE. (A) Cell pellet fractions from E. coli BL21 expressing MBP, MBP-YscE, MBP-YscG, MBP-YscH, MBP-YscI, MBP-YscB, or MBP-YscF were separated by SDS-PAGE and transferred to Immobilon-P membranes. (B) Immobilon-P membranes containing cell pellet fractions from strain BL21 expressing MBP, MBP-YscE, or truncated MBP-YscE Δ57–66 , MBP-YscE Δ47–66 , MBP-YscE Δ1–9 or MBP-YscE Δ1–19 . MBP migrated more slowly than expected due to an in-frame fusion between malE and the vector LacZ α-peptide encoding sequences (MBP-α). (A and B) A cytoplasmic extract from BL21 carrying pYSCG-FLAG was used to probe the Immobilon-P membranes containing the SDS-PAGE-separated MBP and MBP derivatives. Bound YscG-FLAG was detected with the FLAG M2 monoclonal antibody. MBP and the MBP derivatives were detected with a polyclonal antiserum specific for the MBP portion of the hybrid proteins.

    Techniques Used: Binding Assay, Expressing, SDS Page, Plasmid Preparation

    35) Product Images from "Soluble Human Leukocyte Antigen-G5 Activates Extracellular Signal-Regulated Protein Kinase Signaling and Stimulates Trophoblast Invasion"

    Article Title: Soluble Human Leukocyte Antigen-G5 Activates Extracellular Signal-Regulated Protein Kinase Signaling and Stimulates Trophoblast Invasion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076023

    Expression and purification of HLA-G5 protein from E. coli . (A) Recombinant HLA-G5 protein was purified from E. coli BL21 cells and stained with Coomassie brilliant blue. HLA-G5 protein was expressed in total bacterial lysate after 3 hours of IPTG induction (Total, 3 hrs). The HLA-G5 protein was further purified from the inclusion bodies (pellet) of the lysate and refolded (refolded HLA-G5) in native dialysis buffer. (B) Western blot analysis of IPTG induced (0 – 5 hrs) recombinant HLA-G5 protein from the total bacterial lysate probed with His-Tag (Anti-6xHis) and HLA-G5 (5A6G7) antibodies. (C) The identity of the purified HLA-G5 protein was confirmed by mass spectrometry (MS/MS). The peptide score for the HLA-G5 protein was 613.
    Figure Legend Snippet: Expression and purification of HLA-G5 protein from E. coli . (A) Recombinant HLA-G5 protein was purified from E. coli BL21 cells and stained with Coomassie brilliant blue. HLA-G5 protein was expressed in total bacterial lysate after 3 hours of IPTG induction (Total, 3 hrs). The HLA-G5 protein was further purified from the inclusion bodies (pellet) of the lysate and refolded (refolded HLA-G5) in native dialysis buffer. (B) Western blot analysis of IPTG induced (0 – 5 hrs) recombinant HLA-G5 protein from the total bacterial lysate probed with His-Tag (Anti-6xHis) and HLA-G5 (5A6G7) antibodies. (C) The identity of the purified HLA-G5 protein was confirmed by mass spectrometry (MS/MS). The peptide score for the HLA-G5 protein was 613.

    Techniques Used: Expressing, Purification, Recombinant, Staining, Western Blot, Mass Spectrometry

    Related Articles

    Transfection:

    Article Title: Preparation and Antitumor Activity of CS5931, A Novel Polypeptide from Sea Squirt Ciona Savignyi
    Article Snippet: .. Recombinant Expression of CS5931 from E. coli BL21 (DE3) The expression vector, pET28a/CS5931 synthesized by our lab previously [ ], was transfected into E. coli BL21 (DE3) cells (Novagen, Darmstadt, Germany). .. Cells were incubated in low-salt LB medium (containing 100 µg/mL Kanamycin) at 37 °C with shaking at 200 rpm.

    Positron Emission Tomography:

    Article Title: A Single Codon Optimization Enhances Recombinant Human TNF-α Vaccine Expression in Escherichia coli
    Article Snippet: .. The vector pET-22b and E. coli strain BL21 (DE3) were purchased from Novagen (San Diego, CA). .. Isopropyl β -D-1-thiogalactopyranoside (IPTG) was from Sigma-Aldrich (St. Louis, MO).

    Article Title: Characterization of the universal stress protein F from atypical enteropathogenic Escherichia coli and its prevalence in Enterobacteriaceae
    Article Snippet: .. The recombinant plasmid, named pET_UspF, was further purified and transformed into the E. coli BL21 (DE3) pLyS strain (Novagen). .. One recombinant clone was chosen at random among the recombinant colonies and selected for further analysis for protein expression and purification.

    Synthesized:

    Article Title: Preparation and Antitumor Activity of CS5931, A Novel Polypeptide from Sea Squirt Ciona Savignyi
    Article Snippet: .. Recombinant Expression of CS5931 from E. coli BL21 (DE3) The expression vector, pET28a/CS5931 synthesized by our lab previously [ ], was transfected into E. coli BL21 (DE3) cells (Novagen, Darmstadt, Germany). .. Cells were incubated in low-salt LB medium (containing 100 µg/mL Kanamycin) at 37 °C with shaking at 200 rpm.

    Construct:

    Article Title: Hypoallergenic Der p 1/Der p 2 combination vaccines for immunotherapy of house dust mite allergy
    Article Snippet: .. Expression plasmids containing the Der p 2/1 constructs were transformed into E coli strain BL21 (DE3; Novagen, Merck). .. Protein expression was performed in 250 mL of liquid culture by means of induction with 0.5 mmol/L isopropyl-β-D-1-thiogalactopyranoside at an OD600 of 0.8 for 4 hours at 37°C, and cells were harvested by means of centrifugation at 4000 g for 15 minutes at 4°C.

    Purification:

    Article Title: A new hybrid bacteriocin, Ent35-MccV, displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria
    Article Snippet: .. 2.3 Gene expression and bacteriocins purification for activity determinations Ent35–MccV was expressed from pMA24 in E. coli BL21 [DE3] (pLysS) grown in LB (Sigma) at 37 °C. ..

    Article Title: Characterization of the universal stress protein F from atypical enteropathogenic Escherichia coli and its prevalence in Enterobacteriaceae
    Article Snippet: .. The recombinant plasmid, named pET_UspF, was further purified and transformed into the E. coli BL21 (DE3) pLyS strain (Novagen). .. One recombinant clone was chosen at random among the recombinant colonies and selected for further analysis for protein expression and purification.

    Activity Assay:

    Article Title: A new hybrid bacteriocin, Ent35-MccV, displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria
    Article Snippet: .. 2.3 Gene expression and bacteriocins purification for activity determinations Ent35–MccV was expressed from pMA24 in E. coli BL21 [DE3] (pLysS) grown in LB (Sigma) at 37 °C. ..

    Expressing:

    Article Title: A new hybrid bacteriocin, Ent35-MccV, displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria
    Article Snippet: .. 2.3 Gene expression and bacteriocins purification for activity determinations Ent35–MccV was expressed from pMA24 in E. coli BL21 [DE3] (pLysS) grown in LB (Sigma) at 37 °C. ..

    Article Title: Hypoallergenic Der p 1/Der p 2 combination vaccines for immunotherapy of house dust mite allergy
    Article Snippet: .. Expression plasmids containing the Der p 2/1 constructs were transformed into E coli strain BL21 (DE3; Novagen, Merck). .. Protein expression was performed in 250 mL of liquid culture by means of induction with 0.5 mmol/L isopropyl-β-D-1-thiogalactopyranoside at an OD600 of 0.8 for 4 hours at 37°C, and cells were harvested by means of centrifugation at 4000 g for 15 minutes at 4°C.

    Article Title: Preparation and Antitumor Activity of CS5931, A Novel Polypeptide from Sea Squirt Ciona Savignyi
    Article Snippet: .. Recombinant Expression of CS5931 from E. coli BL21 (DE3) The expression vector, pET28a/CS5931 synthesized by our lab previously [ ], was transfected into E. coli BL21 (DE3) cells (Novagen, Darmstadt, Germany). .. Cells were incubated in low-salt LB medium (containing 100 µg/mL Kanamycin) at 37 °C with shaking at 200 rpm.

    Transformation Assay:

    Article Title: Hypoallergenic Der p 1/Der p 2 combination vaccines for immunotherapy of house dust mite allergy
    Article Snippet: .. Expression plasmids containing the Der p 2/1 constructs were transformed into E coli strain BL21 (DE3; Novagen, Merck). .. Protein expression was performed in 250 mL of liquid culture by means of induction with 0.5 mmol/L isopropyl-β-D-1-thiogalactopyranoside at an OD600 of 0.8 for 4 hours at 37°C, and cells were harvested by means of centrifugation at 4000 g for 15 minutes at 4°C.

    Article Title: Characterization of the universal stress protein F from atypical enteropathogenic Escherichia coli and its prevalence in Enterobacteriaceae
    Article Snippet: .. The recombinant plasmid, named pET_UspF, was further purified and transformed into the E. coli BL21 (DE3) pLyS strain (Novagen). .. One recombinant clone was chosen at random among the recombinant colonies and selected for further analysis for protein expression and purification.

    Article Title: Design and evaluation of a recombinant multi-epitope antigen for serodiagnosis of Toxoplasma gondii infection in humans
    Article Snippet: .. Consequently, the recombinant plasmid was transformed into E. coli BL21 (DE3) plysS competent cells (Novagen, USA). .. Following the confirmation of the sequences of inserts by DNA sequencing (IDT, Singapore), the protein expression was induced by isopropyl-D thiogalactopyranoside (IPTG) with a final concentration of 1 mM.

    Recombinant:

    Article Title: Characterization of the universal stress protein F from atypical enteropathogenic Escherichia coli and its prevalence in Enterobacteriaceae
    Article Snippet: .. The recombinant plasmid, named pET_UspF, was further purified and transformed into the E. coli BL21 (DE3) pLyS strain (Novagen). .. One recombinant clone was chosen at random among the recombinant colonies and selected for further analysis for protein expression and purification.

    Article Title: Design and evaluation of a recombinant multi-epitope antigen for serodiagnosis of Toxoplasma gondii infection in humans
    Article Snippet: .. Consequently, the recombinant plasmid was transformed into E. coli BL21 (DE3) plysS competent cells (Novagen, USA). .. Following the confirmation of the sequences of inserts by DNA sequencing (IDT, Singapore), the protein expression was induced by isopropyl-D thiogalactopyranoside (IPTG) with a final concentration of 1 mM.

    Article Title: Preparation and Antitumor Activity of CS5931, A Novel Polypeptide from Sea Squirt Ciona Savignyi
    Article Snippet: .. Recombinant Expression of CS5931 from E. coli BL21 (DE3) The expression vector, pET28a/CS5931 synthesized by our lab previously [ ], was transfected into E. coli BL21 (DE3) cells (Novagen, Darmstadt, Germany). .. Cells were incubated in low-salt LB medium (containing 100 µg/mL Kanamycin) at 37 °C with shaking at 200 rpm.

    Plasmid Preparation:

    Article Title: A simple vector system to improve performance and utilisation of recombinant antibodies
    Article Snippet: .. E. coli BL21 (DE3) and Origami 2™ (DE3) cells and the pET26b(+) plasmid were from Novagen (San Diego, CA). ..

    Article Title: A Single Codon Optimization Enhances Recombinant Human TNF-α Vaccine Expression in Escherichia coli
    Article Snippet: .. The vector pET-22b and E. coli strain BL21 (DE3) were purchased from Novagen (San Diego, CA). .. Isopropyl β -D-1-thiogalactopyranoside (IPTG) was from Sigma-Aldrich (St. Louis, MO).

    Article Title: Characterization of the universal stress protein F from atypical enteropathogenic Escherichia coli and its prevalence in Enterobacteriaceae
    Article Snippet: .. The recombinant plasmid, named pET_UspF, was further purified and transformed into the E. coli BL21 (DE3) pLyS strain (Novagen). .. One recombinant clone was chosen at random among the recombinant colonies and selected for further analysis for protein expression and purification.

    Article Title: Design and evaluation of a recombinant multi-epitope antigen for serodiagnosis of Toxoplasma gondii infection in humans
    Article Snippet: .. Consequently, the recombinant plasmid was transformed into E. coli BL21 (DE3) plysS competent cells (Novagen, USA). .. Following the confirmation of the sequences of inserts by DNA sequencing (IDT, Singapore), the protein expression was induced by isopropyl-D thiogalactopyranoside (IPTG) with a final concentration of 1 mM.

    Article Title: Preparation and Antitumor Activity of CS5931, A Novel Polypeptide from Sea Squirt Ciona Savignyi
    Article Snippet: .. Recombinant Expression of CS5931 from E. coli BL21 (DE3) The expression vector, pET28a/CS5931 synthesized by our lab previously [ ], was transfected into E. coli BL21 (DE3) cells (Novagen, Darmstadt, Germany). .. Cells were incubated in low-salt LB medium (containing 100 µg/mL Kanamycin) at 37 °C with shaking at 200 rpm.

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  • 94
    Millipore gst prescission protease
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    Gst Prescission Protease, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore non induced transformed bl21
    SDS-PAGE gel showing protein expression of Vip3 . Lane 1–7: Total protein extracted from induced colonies at 16 h post-inoculation. C: Negative control of <t>Bl21</t> (DE3) transformed using P QE-30 alone. M: Page Ruler Protein ladder. The arrow shows the putative vegetative insecticidal protein (VIP3) of ~88 kDa.
    Non Induced Transformed Bl21, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore bl21
    Overview of high-throughput genome-wide screens. We used barcoded loss-of-function (LOF) technologies (RB-TnSeq and CRISPRi) and a gain-of-function (GOF) technology (Dub-seq) in E. coli K-12 (BW25113 and MG1655) to screen for host factors important in phage infection and resistance. In E. coli <t>BL21,</t> we performed RB-TnSeq and Dub-seq (but not CRISPRi). We sourced 14 diverse E. coli phages with double-stranded DNA genomes, belonging to Myoviridae, Podoviridae and Siphoviridae families, and performed pooled fitness screens in both planktonic and solid agar formats. Disruption or overexpression of certain genes provide fitness to host in the presence of phages and we monitor these changes by quantifying the abundance of the DNA barcode or sgRNA associated with each strain. The individual strain abundances are then converted to gene fitness scores (normalized log2 change in the abundance of mutants in that gene).
    Bl21, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 65 article reviews
    Price from $9.99 to $1999.99
    bl21 - by Bioz Stars, 2020-07
    93/100 stars
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    91
    Millipore lsrb bl21 ∆ luxs protein
    Binding of <t>LsrB</t> to endogenous AI-2. Purified LsrB (BL21) and LsrB <t>(BL21∆luxS)</t> proteins (5 mg/ml) were incubated for 10 min at 37, 50 and 60 °C to release endogenous AI-2, respectively. After incubation, the LsrB proteins were removed by ultrafiltration (10,000-Da cut-off; EMD Millipore), and the filtered reaction products were tested for AI-2 activity using a V. harveyi BB170 bioassay. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C, respectively ( a ). However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS) ( b ). Moreover, the recombinant LuxS protein, which was expressed in strain BL21 (pColdTF-lsrB) as a negative control, also showed no AI-2 binding activity ( c ). AI-2 (10 μM) was used as a positive control
    Lsrb Bl21 ∆ Luxs Protein, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 91 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    lsrb bl21 ∆ luxs protein - by Bioz Stars, 2020-07
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    Image Search Results


    Accessible hydrophobic residues in the predicted coiled coil are critical for Rab binding. (A) Helical wheel projection of a coiled coil predicted for GCC185 residues 1579−1606. Residues in registers “a-f” were predicted by the Paircoil program. Residues at positions “a” and “d” lie in the dimer interface. Boxed residues are candidates for binding interactions with Rab GTPases. (B, C) Effect of alanine substitutions on Rab binding. Reactions contained wild type or mutant GST-C-110 (B; 3 μM, C; 2 μM) and 35 S-GTPγS-preloaded GTPases (B; 170 pmol Rab9-His, C; 190 pmol His-Rab6). Data are mean ± SD. (D) Mass determination of untagged RBD-87 I1588A/L1595A by multiple angle static light scattering. The gel filtration elution profile of the protein (black line) and molecular mass (grey line) are shown. Polydispersity of the peak was 1.001.

    Journal: Cell

    Article Title: Dual GTPase regulation of the GCC185 Golgin: Communication between adjacent Rab6 and Arl1 binding sites

    doi: 10.1016/j.cell.2007.11.048

    Figure Lengend Snippet: Accessible hydrophobic residues in the predicted coiled coil are critical for Rab binding. (A) Helical wheel projection of a coiled coil predicted for GCC185 residues 1579−1606. Residues in registers “a-f” were predicted by the Paircoil program. Residues at positions “a” and “d” lie in the dimer interface. Boxed residues are candidates for binding interactions with Rab GTPases. (B, C) Effect of alanine substitutions on Rab binding. Reactions contained wild type or mutant GST-C-110 (B; 3 μM, C; 2 μM) and 35 S-GTPγS-preloaded GTPases (B; 170 pmol Rab9-His, C; 190 pmol His-Rab6). Data are mean ± SD. (D) Mass determination of untagged RBD-87 I1588A/L1595A by multiple angle static light scattering. The gel filtration elution profile of the protein (black line) and molecular mass (grey line) are shown. Polydispersity of the peak was 1.001.

    Article Snippet: RBD-87 was then separated from GST, GST-Prescission Protease and glutathione beads over a solid support and concentrated (Amicon Ultra, 5,000 MWCO, Millipore).

    Techniques: Binding Assay, Mutagenesis, Filtration

    Identification of a GCC185 Rab-binding domain. (A) Constructs used to map Rab-GCC185 interactions; numbers represent amino acid residues. The GRIP domain and a Rab binding domain (RBD) are shown. At right: summary of binding to Rab6 or Rab9. (B) GCC185 preferentially binds Rab6-GTP via residues upstream of the GRIP domain. Reactions contained 50 pmol His-Rab6 (left) or 1.2 nmol His-Rab6, or Rab9-His (right) using 35 S-GTPγS or 3 H-GDP-preloaded GTPase and either GST-C110 or GST-RBD-87 (2.8 μM). (C) The GRIP domain is not sufficient for Rab binding. GST-C-110, C-110 Y/A , or C-72 (2 μM) was incubated with 35 S-GTPγS-preloaded GTPases (∼500 pmol) (as in B. except Rab1 and Rab9 were untagged and or ArlQ71L-His was used). Untagged Rab9/1 and His-Rab6 1−174 were employed. (D) Rab6 specifically competes with Rab9 for GCC185 binding. GST-C-110:Rab9 complexes (pair of lanes in the center) were incubated for 3 min with ten fold excess competitor. Rab9-His was detected by immunoblot using a monoclonal anti-Rab9 antibody that did not cross react with Rab6 (see pair of lanes at far left). Lower panel, same as upper panel using indicated amounts of competitor Rab9. Data are mean ± SD.

    Journal: Cell

    Article Title: Dual GTPase regulation of the GCC185 Golgin: Communication between adjacent Rab6 and Arl1 binding sites

    doi: 10.1016/j.cell.2007.11.048

    Figure Lengend Snippet: Identification of a GCC185 Rab-binding domain. (A) Constructs used to map Rab-GCC185 interactions; numbers represent amino acid residues. The GRIP domain and a Rab binding domain (RBD) are shown. At right: summary of binding to Rab6 or Rab9. (B) GCC185 preferentially binds Rab6-GTP via residues upstream of the GRIP domain. Reactions contained 50 pmol His-Rab6 (left) or 1.2 nmol His-Rab6, or Rab9-His (right) using 35 S-GTPγS or 3 H-GDP-preloaded GTPase and either GST-C110 or GST-RBD-87 (2.8 μM). (C) The GRIP domain is not sufficient for Rab binding. GST-C-110, C-110 Y/A , or C-72 (2 μM) was incubated with 35 S-GTPγS-preloaded GTPases (∼500 pmol) (as in B. except Rab1 and Rab9 were untagged and or ArlQ71L-His was used). Untagged Rab9/1 and His-Rab6 1−174 were employed. (D) Rab6 specifically competes with Rab9 for GCC185 binding. GST-C-110:Rab9 complexes (pair of lanes in the center) were incubated for 3 min with ten fold excess competitor. Rab9-His was detected by immunoblot using a monoclonal anti-Rab9 antibody that did not cross react with Rab6 (see pair of lanes at far left). Lower panel, same as upper panel using indicated amounts of competitor Rab9. Data are mean ± SD.

    Article Snippet: RBD-87 was then separated from GST, GST-Prescission Protease and glutathione beads over a solid support and concentrated (Amicon Ultra, 5,000 MWCO, Millipore).

    Techniques: Binding Assay, Construct, Incubation

    SDS-PAGE gel showing protein expression of Vip3 . Lane 1–7: Total protein extracted from induced colonies at 16 h post-inoculation. C: Negative control of Bl21 (DE3) transformed using P QE-30 alone. M: Page Ruler Protein ladder. The arrow shows the putative vegetative insecticidal protein (VIP3) of ~88 kDa.

    Journal: Saudi Journal of Biological Sciences

    Article Title: Characterization, cloning, expression and bioassay of vip3 gene isolated from an Egyptian Bacillus thuringiensis against whiteflies

    doi: 10.1016/j.sjbs.2019.12.013

    Figure Lengend Snippet: SDS-PAGE gel showing protein expression of Vip3 . Lane 1–7: Total protein extracted from induced colonies at 16 h post-inoculation. C: Negative control of Bl21 (DE3) transformed using P QE-30 alone. M: Page Ruler Protein ladder. The arrow shows the putative vegetative insecticidal protein (VIP3) of ~88 kDa.

    Article Snippet: 2.4 Western blotting Proteins extracted from induced and non-induced transformed BL21 as previously described ( , ) were transferred onto an Immobilon® PVDF membrane (Millipore) using a trans-blot apparatus (Bio-Rad, USA).

    Techniques: SDS Page, Expressing, Negative Control, Transformation Assay, Polyacrylamide Gel Electrophoresis

    Overview of high-throughput genome-wide screens. We used barcoded loss-of-function (LOF) technologies (RB-TnSeq and CRISPRi) and a gain-of-function (GOF) technology (Dub-seq) in E. coli K-12 (BW25113 and MG1655) to screen for host factors important in phage infection and resistance. In E. coli BL21, we performed RB-TnSeq and Dub-seq (but not CRISPRi). We sourced 14 diverse E. coli phages with double-stranded DNA genomes, belonging to Myoviridae, Podoviridae and Siphoviridae families, and performed pooled fitness screens in both planktonic and solid agar formats. Disruption or overexpression of certain genes provide fitness to host in the presence of phages and we monitor these changes by quantifying the abundance of the DNA barcode or sgRNA associated with each strain. The individual strain abundances are then converted to gene fitness scores (normalized log2 change in the abundance of mutants in that gene).

    Journal: bioRxiv

    Article Title: High-throughput mapping of the phage resistance landscape in E. coli

    doi: 10.1101/2020.02.15.951020

    Figure Lengend Snippet: Overview of high-throughput genome-wide screens. We used barcoded loss-of-function (LOF) technologies (RB-TnSeq and CRISPRi) and a gain-of-function (GOF) technology (Dub-seq) in E. coli K-12 (BW25113 and MG1655) to screen for host factors important in phage infection and resistance. In E. coli BL21, we performed RB-TnSeq and Dub-seq (but not CRISPRi). We sourced 14 diverse E. coli phages with double-stranded DNA genomes, belonging to Myoviridae, Podoviridae and Siphoviridae families, and performed pooled fitness screens in both planktonic and solid agar formats. Disruption or overexpression of certain genes provide fitness to host in the presence of phages and we monitor these changes by quantifying the abundance of the DNA barcode or sgRNA associated with each strain. The individual strain abundances are then converted to gene fitness scores (normalized log2 change in the abundance of mutants in that gene).

    Article Snippet: We grew E. coli BL21 at 30°C to mid-log-phase and combined equal cell numbers of BL21 and donor strain APA752, conjugated them for 5 hrs at 30°C on 0.45-m nitrocellulose filters (Millipore) overlaid on LB agar plates containing diaminopimelic acid (DAP) (Sigma).

    Techniques: High Throughput Screening Assay, Genome Wide, Infection, Over Expression

    Description of E. coli BL21 Dub-seq library (A) The fragment insert size distribution in the E. coli BL21 Dub-seq library. (B) Cumulative distribution plot showing the percentage of genes in the E. coli BL21 genome (y-axis) covered by a number of independent genomic fragments (x-axis). (C) The distribution of the number of genes that are completely covered (start to stop codon) per genomic fragment in the E. coli BL21 Dub-seq library. (D) Genome coverage of E . coli BL21 Dub-seq library in 10,000 kB windows mapped to E. coli BL21-DE3

    Journal: bioRxiv

    Article Title: High-throughput mapping of the phage resistance landscape in E. coli

    doi: 10.1101/2020.02.15.951020

    Figure Lengend Snippet: Description of E. coli BL21 Dub-seq library (A) The fragment insert size distribution in the E. coli BL21 Dub-seq library. (B) Cumulative distribution plot showing the percentage of genes in the E. coli BL21 genome (y-axis) covered by a number of independent genomic fragments (x-axis). (C) The distribution of the number of genes that are completely covered (start to stop codon) per genomic fragment in the E. coli BL21 Dub-seq library. (D) Genome coverage of E . coli BL21 Dub-seq library in 10,000 kB windows mapped to E. coli BL21-DE3

    Article Snippet: We grew E. coli BL21 at 30°C to mid-log-phase and combined equal cell numbers of BL21 and donor strain APA752, conjugated them for 5 hrs at 30°C on 0.45-m nitrocellulose filters (Millipore) overlaid on LB agar plates containing diaminopimelic acid (DAP) (Sigma).

    Techniques:

    Genome-wide screens in E. coli BL21 strain (A) Heatmap of BL21 LOF RB-TnSeq data for 12 dsDNA phages at a single MOI, and selected genes with high-confidence fitness benefits are shown. Additional data is provided in the Supplementary Table S8. (B) Heatmap of GOF BL21 Dub-seq data for 12 dsDNA phages with high-confidence fitness fitness benefit. Fitness scores of > =4 in at least one phage assay are shown. These assays were performed in planktonic culture. Additional data is provided in the Supplementary Table S9.

    Journal: bioRxiv

    Article Title: High-throughput mapping of the phage resistance landscape in E. coli

    doi: 10.1101/2020.02.15.951020

    Figure Lengend Snippet: Genome-wide screens in E. coli BL21 strain (A) Heatmap of BL21 LOF RB-TnSeq data for 12 dsDNA phages at a single MOI, and selected genes with high-confidence fitness benefits are shown. Additional data is provided in the Supplementary Table S8. (B) Heatmap of GOF BL21 Dub-seq data for 12 dsDNA phages with high-confidence fitness fitness benefit. Fitness scores of > =4 in at least one phage assay are shown. These assays were performed in planktonic culture. Additional data is provided in the Supplementary Table S9.

    Article Snippet: We grew E. coli BL21 at 30°C to mid-log-phase and combined equal cell numbers of BL21 and donor strain APA752, conjugated them for 5 hrs at 30°C on 0.45-m nitrocellulose filters (Millipore) overlaid on LB agar plates containing diaminopimelic acid (DAP) (Sigma).

    Techniques: Genome Wide

    Binding of LsrB to endogenous AI-2. Purified LsrB (BL21) and LsrB (BL21∆luxS) proteins (5 mg/ml) were incubated for 10 min at 37, 50 and 60 °C to release endogenous AI-2, respectively. After incubation, the LsrB proteins were removed by ultrafiltration (10,000-Da cut-off; EMD Millipore), and the filtered reaction products were tested for AI-2 activity using a V. harveyi BB170 bioassay. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C, respectively ( a ). However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS) ( b ). Moreover, the recombinant LuxS protein, which was expressed in strain BL21 (pColdTF-lsrB) as a negative control, also showed no AI-2 binding activity ( c ). AI-2 (10 μM) was used as a positive control

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: Binding of LsrB to endogenous AI-2. Purified LsrB (BL21) and LsrB (BL21∆luxS) proteins (5 mg/ml) were incubated for 10 min at 37, 50 and 60 °C to release endogenous AI-2, respectively. After incubation, the LsrB proteins were removed by ultrafiltration (10,000-Da cut-off; EMD Millipore), and the filtered reaction products were tested for AI-2 activity using a V. harveyi BB170 bioassay. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C, respectively ( a ). However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS) ( b ). Moreover, the recombinant LuxS protein, which was expressed in strain BL21 (pColdTF-lsrB) as a negative control, also showed no AI-2 binding activity ( c ). AI-2 (10 μM) was used as a positive control

    Article Snippet: After incubation, the LsrB (BL21∆luxS) protein was removed by ultrafiltration (10,000-Da cut-off, EMD Millipore) and the filtered reaction products were tested for AI-2 activity by investigating the binding activity of LsrB (BL21∆luxS) to exogenous AI-2.

    Techniques: Binding Assay, Purification, Incubation, Activity Assay, Recombinant, Produced, Mutagenesis, Negative Control, Positive Control

    Schematic chart of strategy for binding of LsrB to endogenous AI-2 or exogenous AI-2. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C. However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS). The combination of endogenous AI-2 produced by BL21 (DE3), which is known to interfere with the binding of AI-2 to LsrB (BL21), resulted in the loss in the ability of LsrB (BL21) to bind to exogenous AI-2. Hence, the BL21 (DE3) luxS mutant, which was incapable of producing endogenous AI-2, but could bind to exogenous AI-2

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: Schematic chart of strategy for binding of LsrB to endogenous AI-2 or exogenous AI-2. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C. However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS). The combination of endogenous AI-2 produced by BL21 (DE3), which is known to interfere with the binding of AI-2 to LsrB (BL21), resulted in the loss in the ability of LsrB (BL21) to bind to exogenous AI-2. Hence, the BL21 (DE3) luxS mutant, which was incapable of producing endogenous AI-2, but could bind to exogenous AI-2

    Article Snippet: After incubation, the LsrB (BL21∆luxS) protein was removed by ultrafiltration (10,000-Da cut-off, EMD Millipore) and the filtered reaction products were tested for AI-2 activity by investigating the binding activity of LsrB (BL21∆luxS) to exogenous AI-2.

    Techniques: Binding Assay, Recombinant, Produced, Mutagenesis

    SDS-PAGE analysis of total cellular proteins and purified fusion proteins from BL21∆luxS cells. Lane 1 and lane 5: SDS-PAGE analysis of total cellular proteins from BL21∆luxS containing pCold TF (serve as negative control). Lane 2 and lane 6: SDS-PAGE analysis of total cellular proteins containing expression plasmids pCold-TF-lsrB ( a ) and pCold-TF-luxS ( b ), respectively. Lane 3 and lane 7: SDS-PAGE analysis of total precipitation proteins containing expression plasmids pCold-TF-lsrB and pCold-TF-luxS, respectively. Lane 4 and lane 8: elution of the LsrB and LuxS purified fusion protein from the affinity column, respectively

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: SDS-PAGE analysis of total cellular proteins and purified fusion proteins from BL21∆luxS cells. Lane 1 and lane 5: SDS-PAGE analysis of total cellular proteins from BL21∆luxS containing pCold TF (serve as negative control). Lane 2 and lane 6: SDS-PAGE analysis of total cellular proteins containing expression plasmids pCold-TF-lsrB ( a ) and pCold-TF-luxS ( b ), respectively. Lane 3 and lane 7: SDS-PAGE analysis of total precipitation proteins containing expression plasmids pCold-TF-lsrB and pCold-TF-luxS, respectively. Lane 4 and lane 8: elution of the LsrB and LuxS purified fusion protein from the affinity column, respectively

    Article Snippet: After incubation, the LsrB (BL21∆luxS) protein was removed by ultrafiltration (10,000-Da cut-off, EMD Millipore) and the filtered reaction products were tested for AI-2 activity by investigating the binding activity of LsrB (BL21∆luxS) to exogenous AI-2.

    Techniques: SDS Page, Purification, Negative Control, Expressing, Affinity Column