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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
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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

Clone 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: Plasmid DNA from this entry clone was isolated and cloned into the pET-DEST42 Gateway vector (Life Technologies) in a Clonase recombination reaction. .. This DNA was then transformed into BL21-AI One Shot Chemically Competent E . coli (Life Technologies).

Article Title: A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration
Article Snippet: This fragment was cloned into pT7(-1) (pT7-7) ( ) with a one nucleotide deletion to change the reading frame. .. The resulting plasmid was sequenced to verify DNA sequence and orientation then introduced into BL21-AI (Invitrogen) for large scale expression.

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: Plasmid DNA from this entry clone was isolated and cloned into the pET-DEST42 Gateway vector (Life Technologies) in a Clonase recombination reaction. .. This DNA was then transformed into BL21-AI One Shot Chemically Competent E . coli (Life Technologies).

Centrifugation:

Article Title: Exposed Loop Domains of Complexed 14-3-3 Proteins Contribute to Structural Diversity and Functional Specificity 1
Article Snippet: The pET15b 14-3-3 expression vectors were transformed into Escherichia coli BL21DE3 or BL21-AI (Invitrogen). .. Bacteria were pelleted by centrifugation and protein extracted using the Ni-NTA HisBind purification kit (Novagen).

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). .. Prior to purification of the recombinant protein, E . coli were concentrated by centrifugation at 10 000 rpm for 20 minutes at 4°C.

Article Title: Functional characterization of Actinobacillus pleuropneumoniae urea transport protein, ApUT
Article Snippet: BL21-AI, E. coli strain (Invitrogen) was cultured at 37°C in Luria-Bertani broth at pH 7.4 supplemented with 50 μg/ml ampicillin when carrying the plasmid pRSET-A containing ApUT cDNA. .. After a 10-h induction period, cells were harvested by centrifugation for 10 min at 3,000 g .

Article Title: A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration
Article Snippet: The resulting plasmid was sequenced to verify DNA sequence and orientation then introduced into BL21-AI (Invitrogen) for large scale expression. .. After 5–6 hours of induction cells were harvested by centrifugation and stored as wet pellets at −20°C until processed.

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). .. Prior to purification of the recombinant protein, E . coli were concentrated by centrifugation at 10 000 rpm for 20 minutes at 4°C.

Amplification:

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: The coding region was amplified with Phusion high-fidelity DNA polymerase from a targeted DNA template in a pUC57 plasmid (synthesized by GenScript) and cloned into the pENTR/D-TOPO vector. .. This DNA was then transformed into BL21-AI One Shot Chemically Competent E . coli (Life Technologies).

Article Title: Docking-dependent Ubiquitination of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by the Ubiquitin Ligase CHIP *
Article Snippet: A BlpI-IRF-1(141–325)-BamHI fragment was amplified from FLAG-IRF-1 WT and ligated with FLAG-IRF-1 WT that was digested with BlpI and BamHI to give IRF-1 Δ106–140; this was inserted into the Gateway system by introducing attB1 and attB2 sites according to the manufacturer's instructions. pDEST53-IRF-1 (GFP-IRF-1) was as described previously ( ). pET15bmod-CHIP was a kind gift from Prof. Alicja Zylicz and Renata Filipek, and pMBC1-IRF-1(115–140) (GFP-IRF-1(115–140)) was from the Hauser group ( ). pcDNA3.1-His/Myc-CHIP WT and domains were kind gifts from Prof. Cam Patterson and Dr. Holly McDonough. .. GST-IRF-1 expressed in BL21-AI (Invitrogen) was purified using glutathione-Sepharose beads (GE Healthcare) according to the manufacturer's instructions.

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: The coding region was amplified with Phusion high-fidelity DNA polymerase from a targeted DNA template in a pUC57 plasmid (synthesized by GenScript) and cloned into the pENTR/D-TOPO vector. .. This DNA was then transformed into BL21-AI One Shot Chemically Competent E . coli (Life Technologies).

Stable Transfection:

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: 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.

Synthesized:

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: The coding region was amplified with Phusion high-fidelity DNA polymerase from a targeted DNA template in a pUC57 plasmid (synthesized by GenScript) and cloned into the pENTR/D-TOPO vector. .. This DNA was then transformed into BL21-AI One Shot Chemically Competent E . coli (Life Technologies).

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: The coding region was amplified with Phusion high-fidelity DNA polymerase from a targeted DNA template in a pUC57 plasmid (synthesized by GenScript) and cloned into the pENTR/D-TOPO vector. .. This DNA was then transformed into BL21-AI One Shot Chemically Competent E . coli (Life Technologies).

Blocking Assay:

Article Title: Development of a fluorescent monoclonal antibody-based assay to measure the allosteric effects of synthetic peptides on self-oligomerization of AGR2 protein
Article Snippet: Human Reptin expressed in BL21 AI (Invitrogen) E. coli was purified as described. .. Following blocking with PBS containing 0.05% Tween-20 and 3% BSA, the indicated AGR2 proteins were titrated in triplicate and incubated for 1 h at room temperature followed by detection with an AGR2 monoclonal antibody (Abnova) and HRP conjugated rabbit anti-mouse secondary antibody (Dako).

Article Title: PKS-NRPS Enzymology and Structural Biology: Considerations in Protein Production
Article Snippet: Several features have been incorporated to reduce the problems caused by leakage transcription from the lac UV5 promoter, for example, insertion of lac repressor sites downstream of the T7 promoter in the expression vector to block progression of T7 RNA polymerase prior to induction. .. An alternative host strain for T7 vectors is BL21 AI (Invitrogen), which carries the gene for T7 RNA polymerase under the control of the promoter araBAD , derived from the arabinose metabolic pathway ( ).

Incubation:

Article Title: Use of FabV-Triclosan Plasmid Selection System for Efficient Expression and Production of Recombinant Proteins in Escherichia coli
Article Snippet: At 22°C and after 12 h incubation, plasmid loss from IPTG -induced bacteria was similar between the two selection systems. .. Certain bacterial host such as BL21(DE3)-pLysS (Novagen), BL21-AI (Invitrogen), Origami (Novagen) etc., are grown in presence of antibiotics such as Chloramphenicol, Kanamycin, or Tetracycline either for verification purposes or to maintain additional extra-chromosomal genetic elements such as pLysS vectors.

Article Title: Development of a fluorescent monoclonal antibody-based assay to measure the allosteric effects of synthetic peptides on self-oligomerization of AGR2 protein
Article Snippet: Human Reptin expressed in BL21 AI (Invitrogen) E. coli was purified as described. .. Following blocking with PBS containing 0.05% Tween-20 and 3% BSA, the indicated AGR2 proteins were titrated in triplicate and incubated for 1 h at room temperature followed by detection with an AGR2 monoclonal antibody (Abnova) and HRP conjugated rabbit anti-mouse secondary antibody (Dako).

Article Title: Vibrio Phage KVP40 Encodes a Functional NAD+ Salvage Pathway
Article Snippet: The bacteria used were Vibrio parahaemolyticus strain EB101 , E. coli strains TOP10, E. cloni (Lucigen, Inc.), and BL21-AI (Invitrogen, CA), and Salmonella enterica serovar Typhimurium LT2 strain TT13007 ( nadB499 ::MudJ pncA278 ::Tn 10 cam) (J. Roth, University of California, Davis, CA). .. Phage were added to early-log-phase EB101 cells at an MOI of 0.01 in 3 ml of 0.4% YP top agar, applied to 1% YP agar plates, and incubated overnight at 30°C.

Expressing:

Article Title: Use of FabV-Triclosan Plasmid Selection System for Efficient Expression and Production of Recombinant Proteins in Escherichia coli
Article Snippet: Unexpectedly, we did not observe any better yields of P24 and Nef proteins and expression of these proteins appeared to be independent of number of bacteria that retained the expression vectors. .. Certain bacterial host such as BL21(DE3)-pLysS (Novagen), BL21-AI (Invitrogen), Origami (Novagen) etc., are grown in presence of antibiotics such as Chloramphenicol, Kanamycin, or Tetracycline either for verification purposes or to maintain additional extra-chromosomal genetic elements such as pLysS vectors.

Article Title: Evaluation of recombinant invasive, non-pathogenic Eschericia coli as a vaccine vector against the intracellular pathogen, Brucella
Article Snippet: .. E. coli vector vaccines All Escherichia coli used in these studies were strain DH5α™ (Invitrogen) except for recombinants expressing pDEST17 vectors were we used BL21-AI™ (Invitrogen). .. Table describes the recombinant E. coli vector vaccines.

Article Title: Characterization of Three L-Asparaginases from Maritime Pine (Pinus pinaster Ait.)
Article Snippet: .. Biological Material Escherichia coli strains DH5α and BL21-AI (Invitrogen Corporation, Carlsbad, CA, United States) were used for plasmid propagation and expression of the protein in a prokaryotic system, respectively. ..

Article Title: Exposed Loop Domains of Complexed 14-3-3 Proteins Contribute to Structural Diversity and Functional Specificity 1
Article Snippet: .. The pET15b 14-3-3 expression vectors were transformed into Escherichia coli BL21DE3 or BL21-AI (Invitrogen). ..

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: Functional characterization of Actinobacillus pleuropneumoniae urea transport protein, ApUT
Article Snippet: Paragraph title: Bacterial expression studies. ... BL21-AI, E. coli strain (Invitrogen) was cultured at 37°C in Luria-Bertani broth at pH 7.4 supplemented with 50 μg/ml ampicillin when carrying the plasmid pRSET-A containing ApUT cDNA.

Article Title: Docking-dependent Ubiquitination of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by the Ubiquitin Ligase CHIP *
Article Snippet: The human IRF-1 sequence was codon-optimized for Escherichia coli expression (Genscript) and inserted into pDEST-15 using Gateway technology (Invitrogen) to generate GST-IRF-1. .. GST-IRF-1 expressed in BL21-AI (Invitrogen) was purified using glutathione-Sepharose beads (GE Healthcare) according to the manufacturer's instructions.

Article Title: PKS-NRPS Enzymology and Structural Biology: Considerations in Protein Production
Article Snippet: Several features have been incorporated to reduce the problems caused by leakage transcription from the lac UV5 promoter, for example, insertion of lac repressor sites downstream of the T7 promoter in the expression vector to block progression of T7 RNA polymerase prior to induction. .. An alternative host strain for T7 vectors is BL21 AI (Invitrogen), which carries the gene for T7 RNA polymerase under the control of the promoter araBAD , derived from the arabinose metabolic pathway ( ).

Article Title: A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration
Article Snippet: .. The resulting plasmid was sequenced to verify DNA sequence and orientation then introduced into BL21-AI (Invitrogen) for large scale expression. .. Protein expression was induced by the addition of 0.5 g of arabinose to 500 ml of exponentially growing E. coli at an OD600 ∼0.8.

Article Title: Myosin 7 and its adaptors link cadherins to actin
Article Snippet: .. Protein expression and purification All 6xHis fusion proteins were expressed in either BL21-AI, BL21 (DE3) Rosetta (Invitrogen) or BL21-Gold (DE3) (Agilent) Escherichia coli at 20 °C after induction with 0.2 mM IPTG with or without 0.2% L -arabinose, accordingly. .. Selenomethionine derivatives were prepared using B834 (DE3) E. coli (Agilent) and Selenomethionine medium from Molecular Dimensions.

Article Title: Restrained expression, a method to overproduce toxic membrane proteins by exploiting operator-repressor interactions
Article Snippet: The following E. coli strains were used to express proteins from T7 lac promoter based vectors: BL21(DE3) and BL21(DE3) pLysS (Novagen, CA), C43(DE3) (Lucigen, WI), Lemo21(DE3) (NEB, MA), and BL21-AI (Invitrogen, CA). .. The Top10 strain (Invitrogen, CA) was used in cases where pBAD/HisA or pTetGFPHis8 vectors were used for protein expression.

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.

Western Blot:

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). .. Optimal expression of protein after the incorporation of L-arabinose and IPTG was then quantified by SDS-PAGE and Western blot using antibodies against the 6xHis tag and V5 epitope (Life Technologies) on the recombinant protein.

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). .. Optimal expression of protein after the incorporation of L-arabinose and IPTG was then quantified by SDS-PAGE and Western blot using antibodies against the 6xHis tag and V5 epitope (Life Technologies) on the recombinant protein.

Transformation Assay:

Article Title: Exposed Loop Domains of Complexed 14-3-3 Proteins Contribute to Structural Diversity and Functional Specificity 1
Article Snippet: .. The pET15b 14-3-3 expression vectors were transformed into Escherichia coli BL21DE3 or BL21-AI (Invitrogen). ..

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: 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.

Derivative Assay:

Article Title: PKS-NRPS Enzymology and Structural Biology: Considerations in Protein Production
Article Snippet: .. An alternative host strain for T7 vectors is BL21 AI (Invitrogen), which carries the gene for T7 RNA polymerase under the control of the promoter araBAD , derived from the arabinose metabolic pathway ( ). .. The araBAD promoter is more tightly controlled than lac UV5 with a significantly lower level of leakage transcription but still capable of high-level expression from a T7 target plasmid when induced by the addition of arabinose ( ).

Cell Culture:

Article Title: Functional characterization of Actinobacillus pleuropneumoniae urea transport protein, ApUT
Article Snippet: .. BL21-AI, E. coli strain (Invitrogen) was cultured at 37°C in Luria-Bertani broth at pH 7.4 supplemented with 50 μg/ml ampicillin when carrying the plasmid pRSET-A containing ApUT cDNA. ..

Generated:

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: Recombinant SmLeish was generated by using the Gateway cloning system according to the manufacturer’s instructions (Life Technologies). .. This DNA was then transformed into BL21-AI One Shot Chemically Competent E . coli (Life Technologies).

Article Title: Docking-dependent Ubiquitination of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by the Ubiquitin Ligase CHIP *
Article Snippet: FLAG-IRF-1 was generated by amplifying an EcoRI-IRF-1-BamHI fragment from pcDNA3-IRF-1 ( ) and ligating it into p3xFLAG-Myc-CMV-24 (Sigma). .. GST-IRF-1 expressed in BL21-AI (Invitrogen) was purified using glutathione-Sepharose beads (GE Healthcare) according to the manufacturer's instructions.

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: Production of recombinant SmLeish Recombinant SmLeish was generated by using the Gateway cloning system according to the manufacturer’s instructions (Life Technologies). .. This DNA was then transformed into BL21-AI One Shot Chemically Competent E . coli (Life Technologies).

Sequencing:

Article Title: Docking-dependent Ubiquitination of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by the Ubiquitin Ligase CHIP *
Article Snippet: The human IRF-1 sequence was codon-optimized for Escherichia coli expression (Genscript) and inserted into pDEST-15 using Gateway technology (Invitrogen) to generate GST-IRF-1. .. GST-IRF-1 expressed in BL21-AI (Invitrogen) was purified using glutathione-Sepharose beads (GE Healthcare) according to the manufacturer's instructions.

Article Title: A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration
Article Snippet: .. The resulting plasmid was sequenced to verify DNA sequence and orientation then introduced into BL21-AI (Invitrogen) for large scale expression. .. Protein expression was induced by the addition of 0.5 g of arabinose to 500 ml of exponentially growing E. coli at an OD600 ∼0.8.

Article Title: Vibrio Phage KVP40 Encodes a Functional NAD+ Salvage Pathway
Article Snippet: The bacteria used were Vibrio parahaemolyticus strain EB101 , E. coli strains TOP10, E. cloni (Lucigen, Inc.), and BL21-AI (Invitrogen, CA), and Salmonella enterica serovar Typhimurium LT2 strain TT13007 ( nadB499 ::MudJ pncA278 ::Tn 10 cam) (J. Roth, University of California, Davis, CA). .. Vibrio phage KVP40 ( ) and its genome sequence ( ) have been described previously.

Binding Assay:

Article Title: Development of a fluorescent monoclonal antibody-based assay to measure the allosteric effects of synthetic peptides on self-oligomerization of AGR2 protein
Article Snippet: Human Reptin expressed in BL21 AI (Invitrogen) E. coli was purified as described. .. To measure the binding of AGR2 (and mutants), purified Reptin (100 ng per well) was adsorbed to the well in carbonate buffer (0.1 M pH 9.0) overnight.

Isolation:

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: Plasmid DNA from this entry clone was isolated and cloned into the pET-DEST42 Gateway vector (Life Technologies) in a Clonase recombination reaction. .. This DNA was then transformed into BL21-AI One Shot Chemically Competent E . coli (Life Technologies).

Article Title: The vapB–vapC Operon of Acidovorax citrulli Functions as a Bona-fide Toxin–Antitoxin Module
Article Snippet: Therefore, the study was performed using A. citrulli strain 7a1, a group II strain isolated in Israel , in which the vapBC operon is 100% identical to that of strain AAC00-1 (see Results). .. 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.

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: Plasmid DNA from this entry clone was isolated and cloned into the pET-DEST42 Gateway vector (Life Technologies) in a Clonase recombination reaction. .. This DNA was then transformed into BL21-AI One Shot Chemically Competent E . coli (Life Technologies).

Purification:

Article Title: Exposed Loop Domains of Complexed 14-3-3 Proteins Contribute to Structural Diversity and Functional Specificity 1
Article Snippet: Paragraph title: Recombinant 14-3-3 Protein Expression and Purification ... The pET15b 14-3-3 expression vectors were transformed into Escherichia coli BL21DE3 or BL21-AI (Invitrogen).

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). .. Prior to purification of the recombinant protein, E . coli were concentrated by centrifugation at 10 000 rpm for 20 minutes at 4°C.

Article Title: Development of a fluorescent monoclonal antibody-based assay to measure the allosteric effects of synthetic peptides on self-oligomerization of AGR2 protein
Article Snippet: .. Human Reptin expressed in BL21 AI (Invitrogen) E. coli was purified as described. .. To measure the binding of AGR2 (and mutants), purified Reptin (100 ng per well) was adsorbed to the well in carbonate buffer (0.1 M pH 9.0) overnight.

Article Title: Docking-dependent Ubiquitination of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by the Ubiquitin Ligase CHIP *
Article Snippet: .. GST-IRF-1 expressed in BL21-AI (Invitrogen) was purified using glutathione-Sepharose beads (GE Healthcare) according to the manufacturer's instructions. .. His-CHIP was expressed in BL21-DE3 and purified using Ni2+ -NTA-agarose (Qiagen) according to the manufacturer's instructions.

Article Title: A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration
Article Snippet: The resulting plasmid was sequenced to verify DNA sequence and orientation then introduced into BL21-AI (Invitrogen) for large scale expression. .. The recombinant protein readily formed inclusion bodies that were purified using lysozyme/detergent lysis with DNase/RNase digestion followed by high salt and low salt washes ( ).

Article Title: Myosin 7 and its adaptors link cadherins to actin
Article Snippet: .. Protein expression and purification All 6xHis fusion proteins were expressed in either BL21-AI, BL21 (DE3) Rosetta (Invitrogen) or BL21-Gold (DE3) (Agilent) Escherichia coli at 20 °C after induction with 0.2 mM IPTG with or without 0.2% L -arabinose, accordingly. .. Selenomethionine derivatives were prepared using B834 (DE3) E. coli (Agilent) and Selenomethionine medium from Molecular Dimensions.

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). .. Prior to purification of the recombinant protein, E . coli were concentrated by centrifugation at 10 000 rpm for 20 minutes at 4°C.

Protein Purification:

Article Title: Docking-dependent Ubiquitination of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by the Ubiquitin Ligase CHIP *
Article Snippet: Paragraph title: Plasmids and Protein Purification ... GST-IRF-1 expressed in BL21-AI (Invitrogen) was purified using glutathione-Sepharose beads (GE Healthcare) according to the manufacturer's instructions.

Article Title: PKS-NRPS Enzymology and Structural Biology: Considerations in Protein Production
Article Snippet: The lysozyme can assist in cell lysis prior to protein purification, but may also cause unexpected cell lysis. .. An alternative host strain for T7 vectors is BL21 AI (Invitrogen), which carries the gene for T7 RNA polymerase under the control of the promoter araBAD , derived from the arabinose metabolic pathway ( ).

Selection:

Article Title: Use of FabV-Triclosan Plasmid Selection System for Efficient Expression and Production of Recombinant Proteins in Escherichia coli
Article Snippet: At 22°C and after 12 h incubation, plasmid loss from IPTG -induced bacteria was similar between the two selection systems. .. Certain bacterial host such as BL21(DE3)-pLysS (Novagen), BL21-AI (Invitrogen), Origami (Novagen) etc., are grown in presence of antibiotics such as Chloramphenicol, Kanamycin, or Tetracycline either for verification purposes or to maintain additional extra-chromosomal genetic elements such as pLysS vectors.

Recombinant:

Article Title: Evaluation of recombinant invasive, non-pathogenic Eschericia coli as a vaccine vector against the intracellular pathogen, Brucella
Article Snippet: E. coli vector vaccines All Escherichia coli used in these studies were strain DH5α™ (Invitrogen) except for recombinants expressing pDEST17 vectors were we used BL21-AI™ (Invitrogen). .. Table describes the recombinant E. coli vector vaccines.

Article Title: Exposed Loop Domains of Complexed 14-3-3 Proteins Contribute to Structural Diversity and Functional Specificity 1
Article Snippet: Paragraph title: Recombinant 14-3-3 Protein Expression and Purification ... The pET15b 14-3-3 expression vectors were transformed into Escherichia coli BL21DE3 or BL21-AI (Invitrogen).

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: Paragraph title: Production of recombinant SmLeish ... This DNA was then transformed into BL21-AI One Shot Chemically Competent E . coli (Life Technologies).

Article Title: A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration
Article Snippet: Protein expression and antibody production Plasmid NvTSP168100N2 (pTSPN) was the source for DNA sequences used to express a recombinant partial N-terminal thrombospondin domain of NvTSP168100 for antibody production. pTSPN was digested with Eco RI to release an ∼1.1 kb fragment encoding aa 67 to aa 437 of the presumptive protein. .. The resulting plasmid was sequenced to verify DNA sequence and orientation then introduced into BL21-AI (Invitrogen) for large scale expression.

SDS Page:

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). .. Optimal expression of protein after the incorporation of L-arabinose and IPTG was then quantified by SDS-PAGE and Western blot using antibodies against the 6xHis tag and V5 epitope (Life Technologies) on the recombinant protein.

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). .. Optimal expression of protein after the incorporation of L-arabinose and IPTG was then quantified by SDS-PAGE and Western blot using antibodies against the 6xHis tag and V5 epitope (Life Technologies) on the recombinant protein.

Plasmid Preparation:

Article Title: Use of FabV-Triclosan Plasmid Selection System for Efficient Expression and Production of Recombinant Proteins in Escherichia coli
Article Snippet: Interestingly, compared to P24 and Nef, when Vif was expresses from Bla or FabV plasmids, FabV-Triclosan resulted in no better plasmid stability under all three culture conditions. .. Certain bacterial host such as BL21(DE3)-pLysS (Novagen), BL21-AI (Invitrogen), Origami (Novagen) etc., are grown in presence of antibiotics such as Chloramphenicol, Kanamycin, or Tetracycline either for verification purposes or to maintain additional extra-chromosomal genetic elements such as pLysS vectors.

Article Title: Evaluation of recombinant invasive, non-pathogenic Eschericia coli as a vaccine vector against the intracellular pathogen, Brucella
Article Snippet: .. E. coli vector vaccines All Escherichia coli used in these studies were strain DH5α™ (Invitrogen) except for recombinants expressing pDEST17 vectors were we used BL21-AI™ (Invitrogen). .. Table describes the recombinant E. coli vector vaccines.

Article Title: Characterization of Three L-Asparaginases from Maritime Pine (Pinus pinaster Ait.)
Article Snippet: .. Biological Material Escherichia coli strains DH5α and BL21-AI (Invitrogen Corporation, Carlsbad, CA, United States) were used for plasmid propagation and expression of the protein in a prokaryotic system, respectively. ..

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: Plasmid DNA from this entry clone was isolated and cloned into the pET-DEST42 Gateway vector (Life Technologies) in a Clonase recombination reaction. .. This DNA was then transformed into BL21-AI One Shot Chemically Competent E . coli (Life Technologies).

Article Title: Functional characterization of Actinobacillus pleuropneumoniae urea transport protein, ApUT
Article Snippet: .. BL21-AI, E. coli strain (Invitrogen) was cultured at 37°C in Luria-Bertani broth at pH 7.4 supplemented with 50 μg/ml ampicillin when carrying the plasmid pRSET-A containing ApUT cDNA. ..

Article Title: PKS-NRPS Enzymology and Structural Biology: Considerations in Protein Production
Article Snippet: Another level of repression can be achieved with a secondary plasmid (pLysS or E) ( ) carrying a constitutively expressed copy of the T7 gene for lysozyme, which binds T7 RNA polymerase and inhibits transcription. .. An alternative host strain for T7 vectors is BL21 AI (Invitrogen), which carries the gene for T7 RNA polymerase under the control of the promoter araBAD , derived from the arabinose metabolic pathway ( ).

Article Title: A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration
Article Snippet: .. The resulting plasmid was sequenced to verify DNA sequence and orientation then introduced into BL21-AI (Invitrogen) for large scale expression. .. Protein expression was induced by the addition of 0.5 g of arabinose to 500 ml of exponentially growing E. coli at an OD600 ∼0.8.

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: Plasmid DNA from this entry clone was isolated and cloned into the pET-DEST42 Gateway vector (Life Technologies) in a Clonase recombination reaction. .. This DNA was then transformed into BL21-AI One Shot Chemically Competent E . coli (Life Technologies).

Positron Emission Tomography:

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: Plasmid DNA from this entry clone was isolated and cloned into the pET-DEST42 Gateway vector (Life Technologies) in a Clonase recombination reaction. .. This DNA was then transformed into BL21-AI One Shot Chemically Competent E . coli (Life Technologies).

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: Plasmid DNA from this entry clone was isolated and cloned into the pET-DEST42 Gateway vector (Life Technologies) in a Clonase recombination reaction. .. This DNA was then transformed into BL21-AI One Shot Chemically Competent E . coli (Life Technologies).

In Vitro:

Article Title: Docking-dependent Ubiquitination of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by the Ubiquitin Ligase CHIP *
Article Snippet: GST-IRF-1 expressed in BL21-AI (Invitrogen) was purified using glutathione-Sepharose beads (GE Healthcare) according to the manufacturer's instructions. .. Additionally, untagged human IRF-1 was expressed and purified in a cell-free environment using the PURExpress in vitro protein synthesis kit (New England Biolabs) according to the manufacturer's instructions.

Lysis:

Article Title: PKS-NRPS Enzymology and Structural Biology: Considerations in Protein Production
Article Snippet: The lysozyme can assist in cell lysis prior to protein purification, but may also cause unexpected cell lysis. .. An alternative host strain for T7 vectors is BL21 AI (Invitrogen), which carries the gene for T7 RNA polymerase under the control of the promoter araBAD , derived from the arabinose metabolic pathway ( ).

Article Title: A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration
Article Snippet: The resulting plasmid was sequenced to verify DNA sequence and orientation then introduced into BL21-AI (Invitrogen) for large scale expression. .. The recombinant protein readily formed inclusion bodies that were purified using lysozyme/detergent lysis with DNase/RNase digestion followed by high salt and low salt washes ( ).

Chick Chorioallantoic Membrane Assay:

Article Title: Docking-dependent Ubiquitination of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by the Ubiquitin Ligase CHIP *
Article Snippet: A BlpI-IRF-1(141–325)-BamHI fragment was amplified from FLAG-IRF-1 WT and ligated with FLAG-IRF-1 WT that was digested with BlpI and BamHI to give IRF-1 Δ106–140; this was inserted into the Gateway system by introducing attB1 and attB2 sites according to the manufacturer's instructions. pDEST53-IRF-1 (GFP-IRF-1) was as described previously ( ). pET15bmod-CHIP was a kind gift from Prof. Alicja Zylicz and Renata Filipek, and pMBC1-IRF-1(115–140) (GFP-IRF-1(115–140)) was from the Hauser group ( ). pcDNA3.1-His/Myc-CHIP WT and domains were kind gifts from Prof. Cam Patterson and Dr. Holly McDonough. .. GST-IRF-1 expressed in BL21-AI (Invitrogen) was purified using glutathione-Sepharose beads (GE Healthcare) according to the manufacturer's instructions.

Article Title: Vibrio Phage KVP40 Encodes a Functional NAD+ Salvage Pathway
Article Snippet: .. The bacteria used were Vibrio parahaemolyticus strain EB101 , E. coli strains TOP10, E. cloni (Lucigen, Inc.), and BL21-AI (Invitrogen, CA), and Salmonella enterica serovar Typhimurium LT2 strain TT13007 ( nadB499 ::MudJ pncA278 ::Tn 10 cam) (J. Roth, University of California, Davis, CA). .. Vibrio phage KVP40 ( ) and its genome sequence ( ) have been described previously.

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    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: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 78 stars, based on 1 article reviews
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    plasmid interference assay bl21 ai - by Bioz Stars, 2020-02
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    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: 89/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bl21 ai/product/Thermo Fisher
    Average 89 stars, based on 32 article reviews
    Price from $9.99 to $1999.99
    bl21 ai - by Bioz Stars, 2020-02
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    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