bl21 de3 cells  (Thermo Fisher)


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    Structured Review

    Thermo Fisher bl21 de3 cells
    Bl21 De3 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bl21 de3 cells/product/Thermo Fisher
    Average 94 stars, based on 86 article reviews
    Price from $9.99 to $1999.99
    bl21 de3 cells - by Bioz Stars, 2020-07
    94/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Spindly, a novel protein essential for silencing the spindle assembly checkpoint, recruits dynein to the kinetochore
    Article Snippet: .. Antibody production and immunoblotting A region of the Drosophila Spindly gene corresponding to amino acids 451–780 was cloned into pET28a (Novagen), and protein expression was induced in BL21 DE3 cells (Invitrogen). .. Full-length Hs Spindly was also cloned into pET28a, and the protein was expressed in BL21 DE3 cells.

    Isolation:

    Article Title: The Multiple Roles of Mps1 in Drosophila Female Meiosis
    Article Snippet: .. This construct was transformed into BL21 (DE3) cells (Invitrogen), induced with IPTG, lysed with 8 M urea, and the expressed protein was isolated with ProBOND nickel bead resin (Invitrogen). .. Animals were injected by Cocalico Biologicals ( http://www.cocalicobiologicals.com ).

    Construct:

    Article Title: Giardia Cyst Wall Protein 1 Is a Lectin That Binds to Curled Fibrils of the GalNAc Homopolymer
    Article Snippet: .. Bl21-DE3 cells from Invitrogen were transformed with the pMAL-p2E- constructs, and recombinant proteins were induced with 0.1 mM IPTG. .. E. coli expressing MBP fusion proteins in the periplasm were suspended in 20% sucrose in PBS for 10 min in ice.

    Article Title: The Multiple Roles of Mps1 in Drosophila Female Meiosis
    Article Snippet: .. This construct was transformed into BL21 (DE3) cells (Invitrogen), induced with IPTG, lysed with 8 M urea, and the expressed protein was isolated with ProBOND nickel bead resin (Invitrogen). .. Animals were injected by Cocalico Biologicals ( http://www.cocalicobiologicals.com ).

    Article Title: Expression of a 28-Kilodalton Glutathione S-Transferase Antigen of Schistosoma mansoni on the Surface of Filamentous Phages and Evaluation of Its Vaccine Potential
    Article Snippet: .. The recombinant his-GST construct in pRSET B was transformed into BL21(DE3) cells containing pLysS (Invitrogen). .. Cells were grown to OD600 = 0.6 and induced with 1 mM isopropyl-β- d -thiogalactopyranoside for 3 h. The presence of recombinant protein was confirmed by immunoblot analysis using an anti-Xpress antibody that recognizes the 8-amino-acid Xpress epitope on the fusion protein.

    Purification:

    Article Title: A tale of three kingdoms: members of the Phylum Nematoda independently acquired the detoxifying enzyme cyanase through horizontal gene transfer from plants and bacteria
    Article Snippet: .. For protein expression, purified plasmid preparations of Tscyn-1(c33A) and Tscyn-1(c94.1) were each transformed into BL21 DE3 cells (Invitrogen, Thermo Fisher). ..

    Incubation:

    Article Title: Tissue-Specific Defects Are Caused by Loss of the Drosophila MAN1 LEM Domain Protein
    Article Snippet: .. This plasmid was transformed into BL21-DE3 cells (Invitrogen) and expression of the fusion protein was obtained by incubation with IPTG overnight at 18°. .. The resulting dMAN1-CTD-His fusion protein was purified over a Ni2+ column (QIAGEN, Valencia, CA) and used to immunize sheep (Elmira Biologicals, Iowa City, IA).

    Expressing:

    Article Title: Spindly, a novel protein essential for silencing the spindle assembly checkpoint, recruits dynein to the kinetochore
    Article Snippet: .. Antibody production and immunoblotting A region of the Drosophila Spindly gene corresponding to amino acids 451–780 was cloned into pET28a (Novagen), and protein expression was induced in BL21 DE3 cells (Invitrogen). .. Full-length Hs Spindly was also cloned into pET28a, and the protein was expressed in BL21 DE3 cells.

    Article Title: A tale of three kingdoms: members of the Phylum Nematoda independently acquired the detoxifying enzyme cyanase through horizontal gene transfer from plants and bacteria
    Article Snippet: .. For protein expression, purified plasmid preparations of Tscyn-1(c33A) and Tscyn-1(c94.1) were each transformed into BL21 DE3 cells (Invitrogen, Thermo Fisher). ..

    Article Title: Tissue-Specific Defects Are Caused by Loss of the Drosophila MAN1 LEM Domain Protein
    Article Snippet: .. This plasmid was transformed into BL21-DE3 cells (Invitrogen) and expression of the fusion protein was obtained by incubation with IPTG overnight at 18°. .. The resulting dMAN1-CTD-His fusion protein was purified over a Ni2+ column (QIAGEN, Valencia, CA) and used to immunize sheep (Elmira Biologicals, Iowa City, IA).

    Transformation Assay:

    Article Title: Giardia Cyst Wall Protein 1 Is a Lectin That Binds to Curled Fibrils of the GalNAc Homopolymer
    Article Snippet: .. Bl21-DE3 cells from Invitrogen were transformed with the pMAL-p2E- constructs, and recombinant proteins were induced with 0.1 mM IPTG. .. E. coli expressing MBP fusion proteins in the periplasm were suspended in 20% sucrose in PBS for 10 min in ice.

    Article Title: Cardiomyopathy mutation (F88L) in troponin T abolishes length dependency of myofilament Ca2+ sensitivity
    Article Snippet: .. Recombinant DNA was transformed and expressed in BL21*DE3 cells (Invitrogen). .. In brief, TnTWT and TnTF88L were purified by ion-exchange chromatography on a DEAE-Fast Sepharose column (GE Healthcare Biosciences).

    Article Title: The Multiple Roles of Mps1 in Drosophila Female Meiosis
    Article Snippet: .. This construct was transformed into BL21 (DE3) cells (Invitrogen), induced with IPTG, lysed with 8 M urea, and the expressed protein was isolated with ProBOND nickel bead resin (Invitrogen). .. Animals were injected by Cocalico Biologicals ( http://www.cocalicobiologicals.com ).

    Article Title: A tale of three kingdoms: members of the Phylum Nematoda independently acquired the detoxifying enzyme cyanase through horizontal gene transfer from plants and bacteria
    Article Snippet: .. For protein expression, purified plasmid preparations of Tscyn-1(c33A) and Tscyn-1(c94.1) were each transformed into BL21 DE3 cells (Invitrogen, Thermo Fisher). ..

    Article Title: Tissue-Specific Defects Are Caused by Loss of the Drosophila MAN1 LEM Domain Protein
    Article Snippet: .. This plasmid was transformed into BL21-DE3 cells (Invitrogen) and expression of the fusion protein was obtained by incubation with IPTG overnight at 18°. .. The resulting dMAN1-CTD-His fusion protein was purified over a Ni2+ column (QIAGEN, Valencia, CA) and used to immunize sheep (Elmira Biologicals, Iowa City, IA).

    Article Title: Expression of a 28-Kilodalton Glutathione S-Transferase Antigen of Schistosoma mansoni on the Surface of Filamentous Phages and Evaluation of Its Vaccine Potential
    Article Snippet: .. The recombinant his-GST construct in pRSET B was transformed into BL21(DE3) cells containing pLysS (Invitrogen). .. Cells were grown to OD600 = 0.6 and induced with 1 mM isopropyl-β- d -thiogalactopyranoside for 3 h. The presence of recombinant protein was confirmed by immunoblot analysis using an anti-Xpress antibody that recognizes the 8-amino-acid Xpress epitope on the fusion protein.

    Recombinant:

    Article Title: Giardia Cyst Wall Protein 1 Is a Lectin That Binds to Curled Fibrils of the GalNAc Homopolymer
    Article Snippet: .. Bl21-DE3 cells from Invitrogen were transformed with the pMAL-p2E- constructs, and recombinant proteins were induced with 0.1 mM IPTG. .. E. coli expressing MBP fusion proteins in the periplasm were suspended in 20% sucrose in PBS for 10 min in ice.

    Article Title: Cardiomyopathy mutation (F88L) in troponin T abolishes length dependency of myofilament Ca2+ sensitivity
    Article Snippet: .. Recombinant DNA was transformed and expressed in BL21*DE3 cells (Invitrogen). .. In brief, TnTWT and TnTF88L were purified by ion-exchange chromatography on a DEAE-Fast Sepharose column (GE Healthcare Biosciences).

    Article Title: Expression of a 28-Kilodalton Glutathione S-Transferase Antigen of Schistosoma mansoni on the Surface of Filamentous Phages and Evaluation of Its Vaccine Potential
    Article Snippet: .. The recombinant his-GST construct in pRSET B was transformed into BL21(DE3) cells containing pLysS (Invitrogen). .. Cells were grown to OD600 = 0.6 and induced with 1 mM isopropyl-β- d -thiogalactopyranoside for 3 h. The presence of recombinant protein was confirmed by immunoblot analysis using an anti-Xpress antibody that recognizes the 8-amino-acid Xpress epitope on the fusion protein.

    Plasmid Preparation:

    Article Title: A tale of three kingdoms: members of the Phylum Nematoda independently acquired the detoxifying enzyme cyanase through horizontal gene transfer from plants and bacteria
    Article Snippet: .. For protein expression, purified plasmid preparations of Tscyn-1(c33A) and Tscyn-1(c94.1) were each transformed into BL21 DE3 cells (Invitrogen, Thermo Fisher). ..

    Article Title: Tissue-Specific Defects Are Caused by Loss of the Drosophila MAN1 LEM Domain Protein
    Article Snippet: .. This plasmid was transformed into BL21-DE3 cells (Invitrogen) and expression of the fusion protein was obtained by incubation with IPTG overnight at 18°. .. The resulting dMAN1-CTD-His fusion protein was purified over a Ni2+ column (QIAGEN, Valencia, CA) and used to immunize sheep (Elmira Biologicals, Iowa City, IA).

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  • 99
    Thermo Fisher sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page
    <t>SDS-PAGE</t> ( A ) showing non-induced control (lane 1), expressed protein in pellet (lane 2), and supernatant/soluble fraction (lanes 3); SDS-PAGE ( B ) for purified GST-BmLDH protein (lanes 4) and PreScission ™ Protease cleaved BmLDH (lane 5).
    Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Sds Page, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 871 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page/product/Thermo Fisher
    Average 99 stars, based on 871 article reviews
    Price from $9.99 to $1999.99
    sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page - by Bioz Stars, 2020-07
    99/100 stars
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    92
    Thermo Fisher e coli bl21 cells
    SDS-PAGE analysis and activity of nitrite reductase expressed by E. coli <t>BL21.</t> (a) M: protein marker; Lane 1: protein product. (b) Nitrite reductase activity of E. coli BL21 transformed via OMVs under different temperatures.
    E Coli Bl21 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli bl21 cells/product/Thermo Fisher
    Average 92 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    e coli bl21 cells - by Bioz Stars, 2020-07
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    Image Search Results


    SDS-PAGE ( A ) showing non-induced control (lane 1), expressed protein in pellet (lane 2), and supernatant/soluble fraction (lanes 3); SDS-PAGE ( B ) for purified GST-BmLDH protein (lanes 4) and PreScission ™ Protease cleaved BmLDH (lane 5).

    Journal: Drug Target Insights

    Article Title: Molecular and Kinetic Characterization of Babesia microti Gray Strain Lactate Dehydrogenase as a Potential Drug Target

    doi: 10.4137/DTI.S16504

    Figure Lengend Snippet: SDS-PAGE ( A ) showing non-induced control (lane 1), expressed protein in pellet (lane 2), and supernatant/soluble fraction (lanes 3); SDS-PAGE ( B ) for purified GST-BmLDH protein (lanes 4) and PreScission ™ Protease cleaved BmLDH (lane 5).

    Article Snippet: The purity and concentration of the purified protein were determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Lowry protein assay kit (Thermo Scientific, USA).

    Techniques: SDS Page, Purification

    SDS-PAGE analysis and activity of nitrite reductase expressed by E. coli BL21. (a) M: protein marker; Lane 1: protein product. (b) Nitrite reductase activity of E. coli BL21 transformed via OMVs under different temperatures.

    Journal: bioRxiv

    Article Title: Outer Membrane Vesicles Mediated Horizontal Transfer of an Aerobic Denitrification Gene between Escherichia coli

    doi: 10.1101/835694

    Figure Lengend Snippet: SDS-PAGE analysis and activity of nitrite reductase expressed by E. coli BL21. (a) M: protein marker; Lane 1: protein product. (b) Nitrite reductase activity of E. coli BL21 transformed via OMVs under different temperatures.

    Article Snippet: E. coli BL21 cells were stained with VybrantTM DiO live cell tracer (ThermoFisher).

    Techniques: SDS Page, Activity Assay, Marker, Transformation Assay

    The LCSM images of E. coli BL21 induced by IPTG. PH: phase contrast image; Green fluorescence: green fluorescence image; Overlay: image of phase contrast image and green fluorescence image superimposed.

    Journal: bioRxiv

    Article Title: Outer Membrane Vesicles Mediated Horizontal Transfer of an Aerobic Denitrification Gene between Escherichia coli

    doi: 10.1101/835694

    Figure Lengend Snippet: The LCSM images of E. coli BL21 induced by IPTG. PH: phase contrast image; Green fluorescence: green fluorescence image; Overlay: image of phase contrast image and green fluorescence image superimposed.

    Article Snippet: E. coli BL21 cells were stained with VybrantTM DiO live cell tracer (ThermoFisher).

    Techniques: Confocal Laser Scanning Microscopy, Fluorescence

    Detection of recombinant plasmids and expression and purification of recombinant proteins M1: DNA marker (DL2000); lane 1: PCR products of candidate antigen genes; lane 2: enzyme digestion of recombinant plasmids with Bam HI + Xho I; lane 3: enzyme digestion of recombinant plasmids with Bam HI; M2: DNA marker (DL1000); M3: DNA marker (DL10000); M4: DNA marker (DL15000). M: Protein markers. A1~A9: uninduced BL21 (pET32a) supernatant, uninduced BL21 (pET32a) sediment, induced BL21 (pET32a) supernatant, induced BL21 (pET32a) sediment, uninduced BL21 (P-Srr) supernatant, uninduced BL21 (P-Srr) sediment, induced BL21 (P-Srr) supernatant, induced BL21 (P-Srr) sediment, purification of recombinant rSrr. B1~B7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-NeuA), induced BL21 (P-NeuA), induced BL21 (P-NeuA) supernatant, induced BL21 (P-NeuA) sediment, purification of recombinant rNeuA. C1~C7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-Hsp), induced BL21 (P-Hsp), induced BL21 (P-Hsp) supernatant, induced BL21 (P-Hsp) sediment, purification of recombinant rHsp.

    Journal: Oncotarget

    Article Title: Identification and screening of effective protective antigens for channel catfish against Streptococcus iniae

    doi: 10.18632/oncotarget.16475

    Figure Lengend Snippet: Detection of recombinant plasmids and expression and purification of recombinant proteins M1: DNA marker (DL2000); lane 1: PCR products of candidate antigen genes; lane 2: enzyme digestion of recombinant plasmids with Bam HI + Xho I; lane 3: enzyme digestion of recombinant plasmids with Bam HI; M2: DNA marker (DL1000); M3: DNA marker (DL10000); M4: DNA marker (DL15000). M: Protein markers. A1~A9: uninduced BL21 (pET32a) supernatant, uninduced BL21 (pET32a) sediment, induced BL21 (pET32a) supernatant, induced BL21 (pET32a) sediment, uninduced BL21 (P-Srr) supernatant, uninduced BL21 (P-Srr) sediment, induced BL21 (P-Srr) supernatant, induced BL21 (P-Srr) sediment, purification of recombinant rSrr. B1~B7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-NeuA), induced BL21 (P-NeuA), induced BL21 (P-NeuA) supernatant, induced BL21 (P-NeuA) sediment, purification of recombinant rNeuA. C1~C7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-Hsp), induced BL21 (P-Hsp), induced BL21 (P-Hsp) supernatant, induced BL21 (P-Hsp) sediment, purification of recombinant rHsp.

    Article Snippet: The plasmids were then transformed into E. coli BL21 cells and induced by adding 1.0 mM IPTG at 37°C for 4 h. The cells were then centrifuged at 8000 × g for 10 min at 4°C, suspended in sterile phosphate-buffered saline (PBS), sonicated with a Sonic Dismembrator (model 500; Thermo Fisher Scientific, Waltham, MA, USA), and examined by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

    Techniques: Recombinant, Expressing, Purification, Marker, Polymerase Chain Reaction

    Spacer-seq identifies hundreds of off-target spacer integration sites within the E. coli genome a , Schematic of the Spacer-seq workflow. Fragmentation of isolated genomic DNA containing DSA events (step 1). Ligation of adaptor sequences onto fragment ends (step 2). PCR amplification using the defined spacer sequence and adaptor sequence as primers (step 3) for specific enrichment of fragments containing spacer insertions (step 4). High-throughput sequencing of enriched fragments and mapping of reads to the reference genome (step 5). b , The diagram of the genome shows an example of a single Spacer-seq experiment with the number of reads mapped to the E. coli BL21 genome (binned per 10 kb). Dashed lines represent 100 reads. c , The percentage of Spacer-seq reads mapped to a CRISPR array, or to off-target sites within the genome or expression plasmid. Error bars represent mean ± s.d., n = 3 biological replicates. d , Comparison between the average number of off-target integration events mapped to the genome or plasmid, normalized by the total DNA content within the cell (assuming ~30 plasmids per cell). Error bars represent mean ± s.d., n = 3 biological replicates. e , WebLogo of the ~700 unique off-target integration sites identified by Spacer-seq, aligned to the BL21 CRISPR1 array leader and the repeat sequence. f , The percentage of expanded arrays after the DSA experiment. Plasmid containing the minimal version of the K12 CRISPR1 array (native repeat) is compared to a mutant version with repeat mutations C14G and A15C (OTCR). Error bars represent mean ± s.e.m. n = 3 biological replicates. * P = 0.04 calculated with a two-sample unpaired t -test. g , The percentage of expanded arrays after the DSA experiment. The genomic CRISPR1 array (native repeat) is compared to a strain in which the entire CRISPR1 locus is replaced with a minimal array consisting of a 100-nt leader and a single mutant repeat (OTCR). Error bars represent mean ± s.e.m. n = 3 biological replicates. ** P = 0.002 calculated with a two-sample unpaired t -test. Open circles represent individual replicate data points.

    Journal: Nature microbiology

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

    doi: 10.1038/s41564-017-0097-z

    Figure Lengend Snippet: Spacer-seq identifies hundreds of off-target spacer integration sites within the E. coli genome a , Schematic of the Spacer-seq workflow. Fragmentation of isolated genomic DNA containing DSA events (step 1). Ligation of adaptor sequences onto fragment ends (step 2). PCR amplification using the defined spacer sequence and adaptor sequence as primers (step 3) for specific enrichment of fragments containing spacer insertions (step 4). High-throughput sequencing of enriched fragments and mapping of reads to the reference genome (step 5). b , The diagram of the genome shows an example of a single Spacer-seq experiment with the number of reads mapped to the E. coli BL21 genome (binned per 10 kb). Dashed lines represent 100 reads. c , The percentage of Spacer-seq reads mapped to a CRISPR array, or to off-target sites within the genome or expression plasmid. Error bars represent mean ± s.d., n = 3 biological replicates. d , Comparison between the average number of off-target integration events mapped to the genome or plasmid, normalized by the total DNA content within the cell (assuming ~30 plasmids per cell). Error bars represent mean ± s.d., n = 3 biological replicates. e , WebLogo of the ~700 unique off-target integration sites identified by Spacer-seq, aligned to the BL21 CRISPR1 array leader and the repeat sequence. f , The percentage of expanded arrays after the DSA experiment. Plasmid containing the minimal version of the K12 CRISPR1 array (native repeat) is compared to a mutant version with repeat mutations C14G and A15C (OTCR). Error bars represent mean ± s.e.m. n = 3 biological replicates. * P = 0.04 calculated with a two-sample unpaired t -test. g , The percentage of expanded arrays after the DSA experiment. The genomic CRISPR1 array (native repeat) is compared to a strain in which the entire CRISPR1 locus is replaced with a minimal array consisting of a 100-nt leader and a single mutant repeat (OTCR). Error bars represent mean ± s.e.m. n = 3 biological replicates. ** P = 0.002 calculated with a two-sample unpaired t -test. Open circles represent individual replicate data points.

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

    Techniques: Isolation, Ligation, Polymerase Chain Reaction, Amplification, Sequencing, Next-Generation Sequencing, CRISPR, Expressing, Plasmid Preparation, Mutagenesis

    Effects of genomic knockouts of IHF and the CRISPR1 locus on off-target spacer integration activity a , The percentage of oligo integrations into the CRISPR1 locus or the off-target sites normalized per cell (array) following DSA in the BL21-AI strain (WT), or the BL21-AI strain with either the IHF-α or the IHF-β subunits knocked out ΔIHF-α and ΔIHF-β, respectively). Error bars represent mean ± s.d. b , The percentage of Spacer-seq reads aligned on-target to the CRISPR1 locus or to other regions in the genome (off-target) in the WT, ΔIHF-α, and ΔIHF-β strains. Error bars represent mean ± s.d. c , Pearson correlation coefficient (r) of the off-target site identities between the WT versus ΔIHF-α/β strain, WT versus CRISPR1 deletion (ΔCRISPRI) strain and WT versus WT replicates. Error bars represent mean ± s.d. Open circles represent individual replicate data points. d , The percentage of Spacer-seq reads aligned to unique off-target sites within the genome. The knockout strain percentages ( y axis) of the ΔIHF-α/β strains and the ΔCRISPR1 strain are compared to those of WT ( x axis). Each point represents a unique genomic site. For all panels, n = 4 (for WT) and n = 3 (for knockout strains) biological replicates.

    Journal: Nature microbiology

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

    doi: 10.1038/s41564-017-0097-z

    Figure Lengend Snippet: Effects of genomic knockouts of IHF and the CRISPR1 locus on off-target spacer integration activity a , The percentage of oligo integrations into the CRISPR1 locus or the off-target sites normalized per cell (array) following DSA in the BL21-AI strain (WT), or the BL21-AI strain with either the IHF-α or the IHF-β subunits knocked out ΔIHF-α and ΔIHF-β, respectively). Error bars represent mean ± s.d. b , The percentage of Spacer-seq reads aligned on-target to the CRISPR1 locus or to other regions in the genome (off-target) in the WT, ΔIHF-α, and ΔIHF-β strains. Error bars represent mean ± s.d. c , Pearson correlation coefficient (r) of the off-target site identities between the WT versus ΔIHF-α/β strain, WT versus CRISPR1 deletion (ΔCRISPRI) strain and WT versus WT replicates. Error bars represent mean ± s.d. Open circles represent individual replicate data points. d , The percentage of Spacer-seq reads aligned to unique off-target sites within the genome. The knockout strain percentages ( y axis) of the ΔIHF-α/β strains and the ΔCRISPR1 strain are compared to those of WT ( x axis). Each point represents a unique genomic site. For all panels, n = 4 (for WT) and n = 3 (for knockout strains) biological replicates.

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

    Techniques: Immunohistofluorescence, Activity Assay, Knock-Out

    Comparison of three different NCA sequences and their activity in target interference and primed acquisition a , Schematic of the plasmid-based NCAs that were used in the primed acquisition assays. The arrays contain an inducible promoter that drives the expression of 60 nt of the off-target leader (leader NCA ) along with a 33-nt spacer that matches the M13 bacteriophage genome (spacer M13 ) that is flanked by the 28-nt off-target repeat sequences (repeat NCA ). b , Multiple sequence alignment of the NCA repeats aligned to the BL21 CRISPR repeat sequence. Residues conserved with the BL21 repeat are shown in black. c , Results of the plasmid interference assay with the strains harbouring the plasmids that encode either WT (BL21) or NCAs containing the M13 spacer, or a strain with no plasmid-based array. n = 4 biological replicates (circles). Error bars represent mean ± s.d. c.f.u., colony-forming unit. d , Results of the primed acquisition assay with the strains harbouring the plasmids that encode either WT (BL21) or NCAs containing strains. Black bars correspond to the percentage of total arrays containing new M13-derived spacers. The red bars correspond to the percentage of newly expanded arrays containing M13-derived spacers. n = 3 biological replicates. Error bars represent mean ± s.d. e , Comparison of plasmid-based NCA expansion frequencies following DSA. Expansion frequencies for each NCA were quantified by high-throughput sequencing of the plasmid-based arrays. Each point represents the percentage of expansions detected for each array. We did not detect any expansions for NCAs that do not display a point (fic, potG and yfic) , indicating integration efficiencies of

    Journal: Nature microbiology

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

    doi: 10.1038/s41564-017-0097-z

    Figure Lengend Snippet: Comparison of three different NCA sequences and their activity in target interference and primed acquisition a , Schematic of the plasmid-based NCAs that were used in the primed acquisition assays. The arrays contain an inducible promoter that drives the expression of 60 nt of the off-target leader (leader NCA ) along with a 33-nt spacer that matches the M13 bacteriophage genome (spacer M13 ) that is flanked by the 28-nt off-target repeat sequences (repeat NCA ). b , Multiple sequence alignment of the NCA repeats aligned to the BL21 CRISPR repeat sequence. Residues conserved with the BL21 repeat are shown in black. c , Results of the plasmid interference assay with the strains harbouring the plasmids that encode either WT (BL21) or NCAs containing the M13 spacer, or a strain with no plasmid-based array. n = 4 biological replicates (circles). Error bars represent mean ± s.d. c.f.u., colony-forming unit. d , Results of the primed acquisition assay with the strains harbouring the plasmids that encode either WT (BL21) or NCAs containing strains. Black bars correspond to the percentage of total arrays containing new M13-derived spacers. The red bars correspond to the percentage of newly expanded arrays containing M13-derived spacers. n = 3 biological replicates. Error bars represent mean ± s.d. e , Comparison of plasmid-based NCA expansion frequencies following DSA. Expansion frequencies for each NCA were quantified by high-throughput sequencing of the plasmid-based arrays. Each point represents the percentage of expansions detected for each array. We did not detect any expansions for NCAs that do not display a point (fic, potG and yfic) , indicating integration efficiencies of

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

    Techniques: Activity Assay, Plasmid Preparation, Expressing, Sequencing, CRISPR, Derivative Assay, Next-Generation Sequencing