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Thermo Fisher bl21 ai
Physiological effects of rate of membrane protein production. CX21-GFP was produced by rapid, restrained, and basal expression in <t>BL21-AI</t> cells. A: Biomass production. Growth was monitored via OD 600 nm of culture. B: Cell viability analysis using the
Bl21 Ai, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Restrained expression, a method to overproduce toxic membrane proteins by exploiting operator-repressor interactions"

Article Title: Restrained expression, a method to overproduce toxic membrane proteins by exploiting operator-repressor interactions

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1002/pro.535

Physiological effects of rate of membrane protein production. CX21-GFP was produced by rapid, restrained, and basal expression in BL21-AI cells. A: Biomass production. Growth was monitored via OD 600 nm of culture. B: Cell viability analysis using the
Figure Legend Snippet: Physiological effects of rate of membrane protein production. CX21-GFP was produced by rapid, restrained, and basal expression in BL21-AI cells. A: Biomass production. Growth was monitored via OD 600 nm of culture. B: Cell viability analysis using the

Techniques Used: Produced, Expressing

Validation of the restrained-expression method to produce toxic proteins. 6K protein, an ion channel from Sindibis virus, was expressed as a GFP fusion in BL21-AI and BL21(DE3) cells. Cell growth (A) was monitored at 0, 2, 6, and 20 h postinduction by
Figure Legend Snippet: Validation of the restrained-expression method to produce toxic proteins. 6K protein, an ion channel from Sindibis virus, was expressed as a GFP fusion in BL21-AI and BL21(DE3) cells. Cell growth (A) was monitored at 0, 2, 6, and 20 h postinduction by

Techniques Used: Expressing

Comparative analysis of overexpression methods. A: Application of the restrained-expression method to improve expression of CX homologs. Fluorescence of GFP fused to CX homologs expressed in BL21-AI and BL21(DE3) cells induced with 0.01% (w/v) arabinose
Figure Legend Snippet: Comparative analysis of overexpression methods. A: Application of the restrained-expression method to improve expression of CX homologs. Fluorescence of GFP fused to CX homologs expressed in BL21-AI and BL21(DE3) cells induced with 0.01% (w/v) arabinose

Techniques Used: Over Expression, Expressing, Fluorescence

Improving membrane protein expression. A and B: Tuning target gene expression in BL21-AI cells. Fluorescence (AFU/mL) of cultures expressing either GFP (A) or CX21-GFP (B) using restrained and rapid expression measured 20 h after addition of inducer(s)
Figure Legend Snippet: Improving membrane protein expression. A and B: Tuning target gene expression in BL21-AI cells. Fluorescence (AFU/mL) of cultures expressing either GFP (A) or CX21-GFP (B) using restrained and rapid expression measured 20 h after addition of inducer(s)

Techniques Used: Expressing, Fluorescence

2) Product Images from "Vibrio Phage KVP40 Encodes a Functional NAD+ Salvage Pathway"

Article Title: Vibrio Phage KVP40 Encodes a Functional NAD+ Salvage Pathway

Journal: Journal of Bacteriology

doi: 10.1128/JB.00855-16

Purified KVP40 NadV-His6 and NatV-His6 enzymes. Proteins expressed in E. coli were purified on Ni-Sepharose, dialyzed, and analyzed on SDS-PAGE as described in Materials and Methods. (A) NadV-His6 (55.5 kDa). Lanes: 1, protein markers; 2, induced BL21-AI/pZL405
Figure Legend Snippet: Purified KVP40 NadV-His6 and NatV-His6 enzymes. Proteins expressed in E. coli were purified on Ni-Sepharose, dialyzed, and analyzed on SDS-PAGE as described in Materials and Methods. (A) NadV-His6 (55.5 kDa). Lanes: 1, protein markers; 2, induced BL21-AI/pZL405

Techniques Used: Purification, SDS Page

3) Product Images from "The vapB–vapC Operon of Acidovorax citrulli Functions as a Bona-fide Toxin–Antitoxin Module"

Article Title: The vapB–vapC Operon of Acidovorax citrulli Functions as a Bona-fide Toxin–Antitoxin Module

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2015.01499

Growth curve of Escherichia coli BL21-AI transformed with pACYCDuet-1 plasmids expressing recombinant VapB or VapC separately, or VapB and VapC together. E. coli cells carrying the empty vector were grown as control. Cells were grown in LB media at 37°C, with linear shaking (1 mm) for 15 s every 15 min. Arrow indicates expression induction by 1 mM IPTG and 0.5% (w/v) arabinose. Growth curves were initiated by adding three colonies to each well, n = 12 (each replicate consisting of three pooled colonies); error bars are standard error of mean. Results from one representative experiment, out of three with similar results are shown.
Figure Legend Snippet: Growth curve of Escherichia coli BL21-AI transformed with pACYCDuet-1 plasmids expressing recombinant VapB or VapC separately, or VapB and VapC together. E. coli cells carrying the empty vector were grown as control. Cells were grown in LB media at 37°C, with linear shaking (1 mm) for 15 s every 15 min. Arrow indicates expression induction by 1 mM IPTG and 0.5% (w/v) arabinose. Growth curves were initiated by adding three colonies to each well, n = 12 (each replicate consisting of three pooled colonies); error bars are standard error of mean. Results from one representative experiment, out of three with similar results are shown.

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

4) Product Images from "Evaluation of recombinant invasive, non-pathogenic Eschericia coli as a vaccine vector against the intracellular pathogen, Brucella"

Article Title: Evaluation of recombinant invasive, non-pathogenic Eschericia coli as a vaccine vector against the intracellular pathogen, Brucella

Journal: Journal of Immune Based Therapies and Vaccines

doi: 10.1186/1476-8518-7-1

Microfluorimetry of supernatant of HeLa cells expressing murine IL12 indicate efficient plasmid delivery after infection by recombinant invasive E. coli vaccine . FACS analysis showed greater than 70% of HeLa cells were expressing murine specific IL12 at 72 h after 3 h infection with the invasive E. coli vaccine. The positive control was endogenous IL12 produced by the mouse macrophage cell line RAW 264.7. Negative controls included invasive E.coli not carrying the murine IL12 expression vector ( E. coli BL21 infected HeLa supernatant) and uninfected HeLa cells supernatant.
Figure Legend Snippet: Microfluorimetry of supernatant of HeLa cells expressing murine IL12 indicate efficient plasmid delivery after infection by recombinant invasive E. coli vaccine . FACS analysis showed greater than 70% of HeLa cells were expressing murine specific IL12 at 72 h after 3 h infection with the invasive E. coli vaccine. The positive control was endogenous IL12 produced by the mouse macrophage cell line RAW 264.7. Negative controls included invasive E.coli not carrying the murine IL12 expression vector ( E. coli BL21 infected HeLa supernatant) and uninfected HeLa cells supernatant.

Techniques Used: Expressing, Plasmid Preparation, Infection, Recombinant, FACS, Positive Control, Produced

Related Articles

Over Expression:

Article Title: Antigenic Variation in Neisseria gonorrhoeae Occurs Independently of RecQ-Mediated Unwinding of the pilE G Quadruplex
Article Snippet: .. BL21-AI Escherichia coli cells were transformed with overexpression plasmids encoding N-terminally His-tagged NgRecQ or the NgRecQ Asp307Ala variant. .. Cells were grown at 37°C to an optical density at 600 nm of 0.6 before protein expression was induced with 0.2% (wt/vol) arabinose and 1 mM IPTG (NgRecQ) or 0.2% (wt/vol) arabinose (NgRecQ Asp307Ala).

Incubation:

Article Title: Immunogenicity of Non-Living Anthrax Vaccine Candidates in Cattle and Protective Efficacy of Immune Sera in A/J Mouse Model Compared to the Sterne Live Spore Vaccine
Article Snippet: .. Then, protein expression was induced with 0.3 mM of isopropyl-beta-D-thiogalactopyranoside (IPTG) and the E. coli SE1 were further incubated overnight at 25 °C before harvesting by centrifuging at 2500 xg for 35 min. ..

Expressing:

Article Title: Breakage of the Oligomeric CaMKII Hub by the Regulatory Segment of the Kinase
Article Snippet: .. Briefly, CaMKII-α hub protein expression was carried out in E. coli BL21 cells. ..

Article Title: Immunogenicity of Non-Living Anthrax Vaccine Candidates in Cattle and Protective Efficacy of Immune Sera in A/J Mouse Model Compared to the Sterne Live Spore Vaccine
Article Snippet: .. Then, protein expression was induced with 0.3 mM of isopropyl-beta-D-thiogalactopyranoside (IPTG) and the E. coli SE1 were further incubated overnight at 25 °C before harvesting by centrifuging at 2500 xg for 35 min. ..

Article Title: Evaluation of the functional role of the maize Glossy2 and Glossy2-like genes in cuticular lipid deposition
Article Snippet: .. The ZmGL2 protein was also expressed in E. coli BL21AI strain (Invitrogen, Carlsbad, CA, USA, www.thermofisher.com ) using the pDEST17 expression vector (Invitrogen). ..

Sequencing:

Article Title: Two serological approaches for detection of antibodies to SARS-CoV-2 in different scenarios: A screening tool and a point-of-care test
Article Snippet: .. Resulting plasmid was verified by sequence analysis and used to transform BL21-AI™ One Shot™ (Invitrogen; Thermo Fisher Scientific, Inc.). .. The N protein was produced in Escherichia coli ; an overnight culture was grown in lysogeny broth medium, supplemented with ampicillin (100 μg/mL) and incubated at 37 °C, 220 rpm.

Transformation Assay:

Article Title: A metalloprotease produced by larval Schistosoma mansoni facilitates infection establishment and maintenance in the snail host by interfering with immune cell function
Article Snippet: .. This DNA was then transformed into BL21-AI One Shot Chemically Competent E . coli (Life Technologies). .. E . coli stably expressing SmLeish were then selected and grown up at 37°C in 100μg/mL ampicillin LB medium.

Article Title: Antigenic Variation in Neisseria gonorrhoeae Occurs Independently of RecQ-Mediated Unwinding of the pilE G Quadruplex
Article Snippet: .. BL21-AI Escherichia coli cells were transformed with overexpression plasmids encoding N-terminally His-tagged NgRecQ or the NgRecQ Asp307Ala variant. .. Cells were grown at 37°C to an optical density at 600 nm of 0.6 before protein expression was induced with 0.2% (wt/vol) arabinose and 1 mM IPTG (NgRecQ) or 0.2% (wt/vol) arabinose (NgRecQ Asp307Ala).

Article Title: A metalloprotease produced by larval Schistosoma mansoni facilitates infection establishment and maintenance in the snail host by interfering with immune cell function
Article Snippet: .. This DNA was then transformed into BL21-AI One Shot Chemically Competent E . coli (Life Technologies). .. E . coli stably expressing SmLeish were then selected and grown up at 37°C in 100μg/mL ampicillin LB medium.

Variant Assay:

Article Title: Antigenic Variation in Neisseria gonorrhoeae Occurs Independently of RecQ-Mediated Unwinding of the pilE G Quadruplex
Article Snippet: .. BL21-AI Escherichia coli cells were transformed with overexpression plasmids encoding N-terminally His-tagged NgRecQ or the NgRecQ Asp307Ala variant. .. Cells were grown at 37°C to an optical density at 600 nm of 0.6 before protein expression was induced with 0.2% (wt/vol) arabinose and 1 mM IPTG (NgRecQ) or 0.2% (wt/vol) arabinose (NgRecQ Asp307Ala).

Plasmid Preparation:

Article Title: The alcohol dehydrogenase gene family in sugarcane and its involvement in cold stress regulation
Article Snippet: .. E. coli BL21 with the empty vector pEZYHb was used as the control. .. When E. coli BL21 cells containing different vectors were grown to OD600 of 0.6, 1.0 mmol L− 1 isopropyl β-D-thiogalactoside (IPTG) was added to induce protein production.

Article Title: Evaluation of the functional role of the maize Glossy2 and Glossy2-like genes in cuticular lipid deposition
Article Snippet: .. The ZmGL2 protein was also expressed in E. coli BL21AI strain (Invitrogen, Carlsbad, CA, USA, www.thermofisher.com ) using the pDEST17 expression vector (Invitrogen). ..

Article Title: Two serological approaches for detection of antibodies to SARS-CoV-2 in different scenarios: A screening tool and a point-of-care test
Article Snippet: .. Resulting plasmid was verified by sequence analysis and used to transform BL21-AI™ One Shot™ (Invitrogen; Thermo Fisher Scientific, Inc.). .. The N protein was produced in Escherichia coli ; an overnight culture was grown in lysogeny broth medium, supplemented with ampicillin (100 μg/mL) and incubated at 37 °C, 220 rpm.

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  • 99
    Thermo Fisher e coli bl21
    Spot assays used for monitoring the growth performance of <t>BL21/pEZYHb</t> and BL21/pEZYHb- ScADH3 transformed E. coli cells on LB plates under cold stress. The growth performance of BL21/pEZYHb and BL21/ pEZYHb- ScADH3 cells on LB plates without supplements or stresses were used as controls. After spotting the sample on LB agar plates, incubation at 4 °C in darkness for 3 days, 7 days, and 14 days was used to study the tolerance of BL21/pEZYHb and BL21/ pEZYHb- ScADH3 cells under cold stress
    E Coli Bl21, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli bl21/product/Thermo Fisher
    Average 99 stars, based on 151 article reviews
    Price from $9.99 to $1999.99
    e coli bl21 - by Bioz Stars, 2020-09
    99/100 stars
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    91
    Thermo Fisher plasmid interference assay bl21 ai
    Transcription-dependent loss of the target plasmid. <t>BL21-AI</t> strain with the STH Csm module plasmid was challenged with plasmids with STH target sequence or without a target sequence with or without IPTG (1μM) as indicated. (A) Plasmid targeting assay was performed as in Fig 2 (without arabinose). Corresponding Csm modules and targets are indicated with green boxes. (B) Presence of plasmids was assessed by plasmid extraction following overnight growth in medium with or without 1mM IPTG as indicated. Plasmid DNA was linearized, separated by agarose gel electrophoresis and visualized with ethidium bromide. Plasmid DNA from 4 individual colonies is shown for each condition. Positions of linearized Csm module and target plasmids are indicated. Dashed lines indicate rearranged non-contiguous sections of single original gel.
    Plasmid Interference Assay Bl21 Ai, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid interference assay bl21 ai/product/Thermo Fisher
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    plasmid interference assay bl21 ai - by Bioz Stars, 2020-09
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    90
    Thermo Fisher bl21 ai
    Growth curve of Escherichia coli <t>BL21-AI</t> transformed with pACYCDuet-1 plasmids expressing recombinant VapB or VapC separately, or VapB and VapC together. E. coli cells carrying the empty vector were grown as control. Cells were grown in LB media at 37°C, with linear shaking (1 mm) for 15 s every 15 min. Arrow indicates expression induction by 1 mM IPTG and 0.5% (w/v) arabinose. Growth curves were initiated by adding three colonies to each well, n = 12 (each replicate consisting of three pooled colonies); error bars are standard error of mean. Results from one representative experiment, out of three with similar results are shown.
    Bl21 Ai, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bl21 ai/product/Thermo Fisher
    Average 90 stars, based on 38 article reviews
    Price from $9.99 to $1999.99
    bl21 ai - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    Image Search Results


    Spot assays used for monitoring the growth performance of BL21/pEZYHb and BL21/pEZYHb- ScADH3 transformed E. coli cells on LB plates under cold stress. The growth performance of BL21/pEZYHb and BL21/ pEZYHb- ScADH3 cells on LB plates without supplements or stresses were used as controls. After spotting the sample on LB agar plates, incubation at 4 °C in darkness for 3 days, 7 days, and 14 days was used to study the tolerance of BL21/pEZYHb and BL21/ pEZYHb- ScADH3 cells under cold stress

    Journal: BMC Genomics

    Article Title: The alcohol dehydrogenase gene family in sugarcane and its involvement in cold stress regulation

    doi: 10.1186/s12864-020-06929-9

    Figure Lengend Snippet: Spot assays used for monitoring the growth performance of BL21/pEZYHb and BL21/pEZYHb- ScADH3 transformed E. coli cells on LB plates under cold stress. The growth performance of BL21/pEZYHb and BL21/ pEZYHb- ScADH3 cells on LB plates without supplements or stresses were used as controls. After spotting the sample on LB agar plates, incubation at 4 °C in darkness for 3 days, 7 days, and 14 days was used to study the tolerance of BL21/pEZYHb and BL21/ pEZYHb- ScADH3 cells under cold stress

    Article Snippet: E. coli BL21 with the empty vector pEZYHb was used as the control.

    Techniques: Transformation Assay, Incubation

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0176221

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

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

    Techniques: Plasmid Preparation, Sequencing, Agarose Gel Electrophoresis

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0176221

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

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

    Techniques: Plasmid Preparation, Transformation Assay

    Growth curve of Escherichia coli BL21-AI transformed with pACYCDuet-1 plasmids expressing recombinant VapB or VapC separately, or VapB and VapC together. E. coli cells carrying the empty vector were grown as control. Cells were grown in LB media at 37°C, with linear shaking (1 mm) for 15 s every 15 min. Arrow indicates expression induction by 1 mM IPTG and 0.5% (w/v) arabinose. Growth curves were initiated by adding three colonies to each well, n = 12 (each replicate consisting of three pooled colonies); error bars are standard error of mean. Results from one representative experiment, out of three with similar results are shown.

    Journal: Frontiers in Microbiology

    Article Title: The vapB–vapC Operon of Acidovorax citrulli Functions as a Bona-fide Toxin–Antitoxin Module

    doi: 10.3389/fmicb.2015.01499

    Figure Lengend Snippet: Growth curve of Escherichia coli BL21-AI transformed with pACYCDuet-1 plasmids expressing recombinant VapB or VapC separately, or VapB and VapC together. E. coli cells carrying the empty vector were grown as control. Cells were grown in LB media at 37°C, with linear shaking (1 mm) for 15 s every 15 min. Arrow indicates expression induction by 1 mM IPTG and 0.5% (w/v) arabinose. Growth curves were initiated by adding three colonies to each well, n = 12 (each replicate consisting of three pooled colonies); error bars are standard error of mean. Results from one representative experiment, out of three with similar results are shown.

    Article Snippet: Escherichia coli strains DH5α , BL21(DE3) and BL21-AI (Thermo Fisher ScientificTM , Waltham, MA, USA) were routinely grown in Lysogeny broth (LB; Difco) under constant shaking (150 rpm) or LB agar (LB containing 15 g/l agar) at 37°C.

    Techniques: Transformation Assay, Expressing, Recombinant, Plasmid Preparation

    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