pet21 (New England Biolabs)

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New England Biolabs pet21
Functional analysis of mutated MukB neck variants. in vivo complementation in cells lacking chromosomal mukB gene by variants expressed from <t>pET21</t> plasmid. Growth of material streaked from 6 colonies of each variant was compared to growth of cells carrying WT MukB construct at permissive, 22°C, and non-permissive, 37°C; ϕ - a negative control, empty vector.

Functional analysis of mutated MukB neck variants. in vivo complementation in cells lacking chromosomal mukB gene by variants expressed from <t>pET21</t> plasmid. Growth of material streaked from 6 colonies of each variant was compared to growth of cells carrying WT MukB construct at permissive, 22°C, and non-permissive, 37°C; ϕ - a negative control, empty vector.

Pet21, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 95 stars, based on 1 article reviews
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pet21 - by Bioz Stars, 2022-07

95/100 stars

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1) Product Images from "MukB ATPases are regulated independently by the N- and C-terminal domains of MukF kleisin"

Article Title: MukB ATPases are regulated independently by the N- and C-terminal domains of MukF kleisin

Journal: eLife

doi: 10.7554/eLife.31522

Figure Legend Snippet: Functional analysis of mutated MukB neck variants. in vivo complementation in cells lacking chromosomal mukB gene by variants expressed from pET21 plasmid. Growth of material streaked from 6 colonies of each variant was compared to growth of cells carrying WT MukB construct at permissive, 22°C, and non-permissive, 37°C; ϕ - a negative control, empty vector.

Techniques Used: Functional Assay, In Vivo, Plasmid Preparation, Variant Assay, Construct, Negative Control

Functional analysis of mutated MukF helix9 variants. in vivo complementation in strain lacking chromosomal mukF gene by variants expressed from pET21 was assessed in the absence of IPTG (constitutive leaky expression). Growth material from colonies of each variant and MukF was streaked on LB and incubated at permissive, 22°C, and non-permissive, 37°C temperature and compared to negative control, empty vector.

Figure Legend Snippet: Functional analysis of mutated MukF helix9 variants. in vivo complementation in strain lacking chromosomal mukF gene by variants expressed from pET21 was assessed in the absence of IPTG (constitutive leaky expression). Growth material from colonies of each variant and MukF was streaked on LB and incubated at permissive, 22°C, and non-permissive, 37°C temperature and compared to negative control, empty vector.

Techniques Used: Functional Assay, In Vivo, Expressing, Variant Assay, Incubation, Negative Control, Plasmid Preparation

Similar Products

t7 express lysy iq competent e coli (New England Biolabs)

New England Biolabs t7 express lysy iq competent e coli

SAMP-Dep enhanced intracellular and extracellular activity. a , Clonal growth:induction slope was well correlated with SAMP-Dep slope for 36 mutants from Gen. 1 (grey) and Gen. 2 (blue). Negative controls (orange) and oncocin (black) were assessed from both generations. Points represent mean, and the error bars represent slope standard deviation from three replicates for both SAMP-Dep and clonal experiments. DNA sequences with corresponding SAMP-Dep and clonal slopes and errors were tabulated in Supplementary Table 1 . A cluster of three first-generation mutants was expected to enhance activity but was inactive. One second-generation mutant expected to enhance activity did not, but the mutant retained clonal activity comparable to first-generation oncocin. Some false positives were expected given the high-throughput nature of SAMP-Dep. Deviation from the correlation was weakly correlated with both SAMP-Dep slope error and uninduced read count but did not completely explain the false positive ( Supplementary Figs. 5c,d ). b , Clonal mutants had a range of intracellular activities. Bars represent mean, and the error bars represent slope standard deviation from three SAMP-Dep replicates. • indicates a degenerate position for second-generation negative control. Colors represent the same categories as Figure 4a. c , Synthetic peptides with enhanced SAMP-Dep activity displayed antimicrobial activity against <t>T7</t> Express LysY/I q host. * indicates p

T7 Express Lysy Iq Competent E Coli, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 96 stars, based on 1 article reviews
Price from $9.99 to $1999.99

t7 express lysy iq competent e coli - by Bioz Stars, 2022-07

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Journal: bioRxiv

Article Title: A platform for deep sequence-activity mapping and engineering antimicrobial peptides

doi: 10.1101/2021.05.13.444096

Figure Lengend Snippet: SAMP-Dep enhanced intracellular and extracellular activity. a , Clonal growth:induction slope was well correlated with SAMP-Dep slope for 36 mutants from Gen. 1 (grey) and Gen. 2 (blue). Negative controls (orange) and oncocin (black) were assessed from both generations. Points represent mean, and the error bars represent slope standard deviation from three replicates for both SAMP-Dep and clonal experiments. DNA sequences with corresponding SAMP-Dep and clonal slopes and errors were tabulated in Supplementary Table 1 . A cluster of three first-generation mutants was expected to enhance activity but was inactive. One second-generation mutant expected to enhance activity did not, but the mutant retained clonal activity comparable to first-generation oncocin. Some false positives were expected given the high-throughput nature of SAMP-Dep. Deviation from the correlation was weakly correlated with both SAMP-Dep slope error and uninduced read count but did not completely explain the false positive ( Supplementary Figs. 5c,d ). b , Clonal mutants had a range of intracellular activities. Bars represent mean, and the error bars represent slope standard deviation from three SAMP-Dep replicates. • indicates a degenerate position for second-generation negative control. Colors represent the same categories as Figure 4a. c , Synthetic peptides with enhanced SAMP-Dep activity displayed antimicrobial activity against T7 Express LysY/I q host. * indicates p

Article Snippet: Given the T7 LysY/Iq E. coli transformation efficiency was approximately 560,000 CFU per 50 μL competent cells transformed with 80 ng pETh plasmid, sixteen 50 μL reactions of T7 LysY/Iq competent E. coli were transformed to yield a hypothetical 9 million CFU per replicate, providing adequate coverage of the first-generation library.

Techniques: Activity Assay, Standard Deviation, Mutagenesis, High Throughput Screening Assay, Negative Control

$Purification of RNase I (6xHis) and RNase activity assay. A. Purification of RNase I (6xHis) from Nickel-NTA agarose column. Lane 1, RNase I (6xHis) pooled fractions from a nickel column (purified from T7 Express cell extract). Arrows indicate the cytoplasmic RNase I precursor (cRNase I) with the signal peptide (predicted MW 30.7 kDa), and the periplasmic RNase I with the signal peptide removed (predicted MW 27.0 kDa). B. RNase activity on a FAM-labeled COVID-19 RNA (60 mer). S = substrate; P = cleavage product(s). Positive controls, 50 and 5 U of RNase I f (MBP-RNase I fusion, NEB). RNase I (6xHis) enzyme titration (2 μg to 25 ng protein) was used in the activity assay to digest fixed amount of RNA (16 nM) in NEB buffer 3 at 37°C for 1 h. Proteinase K (1.6 U) was added to remove RNase I. The final cleavage products were analyzed by capillary electrophoresis (CE) and peaks were visualized by PeakScan.$

Journal: bioRxiv

Article Title: Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A

doi: 10.1101/2021.01.31.429007

Figure Lengend Snippet: Purification of RNase I (6xHis) and RNase activity assay. A. Purification of RNase I (6xHis) from Nickel-NTA agarose column. Lane 1, RNase I (6xHis) pooled fractions from a nickel column (purified from T7 Express cell extract). Arrows indicate the cytoplasmic RNase I precursor (cRNase I) with the signal peptide (predicted MW 30.7 kDa), and the periplasmic RNase I with the signal peptide removed (predicted MW 27.0 kDa). B. RNase activity on a FAM-labeled COVID-19 RNA (60 mer). S = substrate; P = cleavage product(s). Positive controls, 50 and 5 U of RNase I f (MBP-RNase I fusion, NEB). RNase I (6xHis) enzyme titration (2 μg to 25 ng protein) was used in the activity assay to digest fixed amount of RNA (16 nM) in NEB buffer 3 at 37°C for 1 h. Proteinase K (1.6 U) was added to remove RNase I. The final cleavage products were analyzed by capillary electrophoresis (CE) and peaks were visualized by PeakScan.

Article Snippet: The protein expression level was evaluated in four T7 expression strains: T7 Express (C2566, NEB), T7 Express with LysY and lacIq (C3013, NEB), T7 SHuffle (C3026, E. coli K strain, NEB) and Nico (λDE3) with reduced histidine-rich background proteins (NEB).

Techniques: Purification, Activity Assay, Nickel Column, Labeling, Titration, Electrophoresis

Expression and purification of E. coli RNase III and RNase activity assay on dsRNA. A. RNase III expression level in three E. coli T7 strains: T7 Shuffle (C3026), T7 Express with lacI q and LysY (C3013), and Nico (λDE3). B. Purified RNase III from nickel-NTA agarose column chromatography and Ni magnetic beads. C. Ribonuclease activity assay on dsRNA.

Journal: bioRxiv

Article Title: Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A

doi: 10.1101/2021.01.31.429007

Figure Lengend Snippet: Expression and purification of E. coli RNase III and RNase activity assay on dsRNA. A. RNase III expression level in three E. coli T7 strains: T7 Shuffle (C3026), T7 Express with lacI q and LysY (C3013), and Nico (λDE3). B. Purified RNase III from nickel-NTA agarose column chromatography and Ni magnetic beads. C. Ribonuclease activity assay on dsRNA.

Techniques: Expressing, Purification, Activity Assay, Column Chromatography, Magnetic Beads

$Expression of hRNase A-ACE2NTD150 in T7 Express LysY/lacI q (C3013). A. A schematic diagram of the fusion protein. B. SDS-PAGE analysis of five isolates of hRNase A-ACE2NTD150 (five isolates with MBP signal peptide, five clones without MBP signal peptide). Total cell lysate of pET21b serves as a negative control. C and D. Western blot analysis of the fusion proteins (soluble fraction/supernatant) by anti-6xHis Ab and anti-ACE2 monoclonal Ab.$

Journal: bioRxiv

Article Title: Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A

doi: 10.1101/2021.01.31.429007

Figure Lengend Snippet: Expression of hRNase A-ACE2NTD150 in T7 Express LysY/lacI q (C3013). A. A schematic diagram of the fusion protein. B. SDS-PAGE analysis of five isolates of hRNase A-ACE2NTD150 (five isolates with MBP signal peptide, five clones without MBP signal peptide). Total cell lysate of pET21b serves as a negative control. C and D. Western blot analysis of the fusion proteins (soluble fraction/supernatant) by anti-6xHis Ab and anti-ACE2 monoclonal Ab.

Techniques: Expressing, SDS Page, Clone Assay, Negative Control, Western Blot

Green fluorescence detection for GFP and ACE2NTD-GFP fusion protein. A. T7 Express (LysY/lacI q ) cells carrying pBR322 (a negative control) and pBR322-P T5 - lacO -DasherGFP visualized under long UV light. B. E. coli colonies (Amp R transformants) of NEB SHuffle and T7 Express cells expressing GFP or ACE2NTD-GFP fusion visualized under long UV light. GFP expressing colonies show bright green color. T7 Express [ACE2NTD-GFP] shows no green fluorescence. NEB SHuffle [ACE2NTD-GFP] shows a weak green fluorescence. C. Total proteins from cell lysates of NEB SHuffle [ACE2NTD-GFP] (K and B strains) detected by fluorescence imaging at 520 nm (Cy2 channel). Arrows indicate GFP and ACE2NTD-GFP fusion, respectively. The Protein ladder shows no fluorescence and was not detected in the imager. D. Cells suspension of C3013 [pBR322] and C3013 [GFP]. E. T7 Express (C2566) cell lysates of ACE2NTD-GFP fusion and GFP. GFP expressing cells and cell lysate show yellow/green color under normal light as visualized by naked eyes.

Journal: bioRxiv

Article Title: Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A

doi: 10.1101/2021.01.31.429007

Figure Lengend Snippet: Green fluorescence detection for GFP and ACE2NTD-GFP fusion protein. A. T7 Express (LysY/lacI q ) cells carrying pBR322 (a negative control) and pBR322-P T5 - lacO -DasherGFP visualized under long UV light. B. E. coli colonies (Amp R transformants) of NEB SHuffle and T7 Express cells expressing GFP or ACE2NTD-GFP fusion visualized under long UV light. GFP expressing colonies show bright green color. T7 Express [ACE2NTD-GFP] shows no green fluorescence. NEB SHuffle [ACE2NTD-GFP] shows a weak green fluorescence. C. Total proteins from cell lysates of NEB SHuffle [ACE2NTD-GFP] (K and B strains) detected by fluorescence imaging at 520 nm (Cy2 channel). Arrows indicate GFP and ACE2NTD-GFP fusion, respectively. The Protein ladder shows no fluorescence and was not detected in the imager. D. Cells suspension of C3013 [pBR322] and C3013 [GFP]. E. T7 Express (C2566) cell lysates of ACE2NTD-GFP fusion and GFP. GFP expressing cells and cell lysate show yellow/green color under normal light as visualized by naked eyes.

Techniques: Fluorescence, Negative Control, Expressing, Imaging

In vitro antimicrobial activity assay of recombinant antilisterial proteins. Each crude recombinant protein was obtained from three different cultures and assayed in triplicates. (i) (A) P1_1, P1_2, and P1_3, (B) P2_1, P2_2, P2_3, (C) P3_1, P3_2, P3_3, and (D) P4_1, P4_2, P4_3 using agar well diffusion assay. C: crude protein of untransformed E. coli T7 Express LysY/Iq (New England BioLabs, Inc). S: cell-free culture supernatant of P. polymyxa Kp10. Inhibition zones were obtained in all of the recombinant proteins, thus confirming their antilisterial activity.

Journal: Frontiers in Microbiology

Article Title: The Discovery of New Antilisterial Proteins From Paenibacillus polymyxa Kp10 via Genome Mining and Mass Spectrometry

doi: 10.3389/fmicb.2020.00960

Figure Lengend Snippet: In vitro antimicrobial activity assay of recombinant antilisterial proteins. Each crude recombinant protein was obtained from three different cultures and assayed in triplicates. (i) (A) P1_1, P1_2, and P1_3, (B) P2_1, P2_2, P2_3, (C) P3_1, P3_2, P3_3, and (D) P4_1, P4_2, P4_3 using agar well diffusion assay. C: crude protein of untransformed E. coli T7 Express LysY/Iq (New England BioLabs, Inc). S: cell-free culture supernatant of P. polymyxa Kp10. Inhibition zones were obtained in all of the recombinant proteins, thus confirming their antilisterial activity.

Article Snippet: The genes encoding for P1, P2, P3, and P4 were separately cloned into pET28b+ for expression in E. coli T7 Express LysY/Iq (New England BioLabs).

Techniques: In Vitro, Activity Assay, Recombinant, Diffusion-based Assay, Inhibition

$Purification of RNase I (6×His) and RNase activity assay. (A) Purification of RNase I (6×His) from Nickel-NTA agarose column. Lane 1, RNase I (6×His) pooled fractions from a nickel column (purified from T7 Express cell extract). Arrows indicate the cytoplasmic RNase I precursor (cRNase I) with the signal peptide (predicted MW 30.7 kDa), and the periplasmic RNase I with the signal peptide removed (predicted MW 27.0 kDa). (B) RNase activity on a FAM-labeled SARS-CoV-2 RNA (50 mer). S = substrate; P = cleavage product(s). Positive controls, 50 and 5 U of RNase I f (NEB). RNase I (6×His) enzyme titration (2 μg to 25 ng protein) was used in the activity assay to digest fixed amount of RNA (16 nM) in NEB buffer 3 at 37°C for 1 h. Proteinase K (1.6 U) was added to remove RNase I. The final cleavage products were analyzed by capillary electrophoresis (CE), and peaks were visualized by PeakScan.$

Journal: Frontiers in Microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: Purification of RNase I (6×His) and RNase activity assay. (A) Purification of RNase I (6×His) from Nickel-NTA agarose column. Lane 1, RNase I (6×His) pooled fractions from a nickel column (purified from T7 Express cell extract). Arrows indicate the cytoplasmic RNase I precursor (cRNase I) with the signal peptide (predicted MW 30.7 kDa), and the periplasmic RNase I with the signal peptide removed (predicted MW 27.0 kDa). (B) RNase activity on a FAM-labeled SARS-CoV-2 RNA (50 mer). S = substrate; P = cleavage product(s). Positive controls, 50 and 5 U of RNase I f (NEB). RNase I (6×His) enzyme titration (2 μg to 25 ng protein) was used in the activity assay to digest fixed amount of RNA (16 nM) in NEB buffer 3 at 37°C for 1 h. Proteinase K (1.6 U) was added to remove RNase I. The final cleavage products were analyzed by capillary electrophoresis (CE), and peaks were visualized by PeakScan.

Article Snippet: The protein expression level was evaluated in three T7 expression strains: T7 Express with LysY and lacI q (C3013), T7 SHuffle (C3026, E. coli K strain), and Nico (λDE3).

Techniques: Purification, Activity Assay, Nickel Column, Labeling, Titration, Electrophoresis

Journal: Frontiers in Microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: Expression and purification of E. coli RNase III and RNase activity assay on dsRNA. (A) RNase III expression level in three E. coli T7 strains: T7 Shuffle (C3026, K strain), T7 Express with lacI q and LysY (C3013), and Nico (λDE3). (B) Purified RNase III from nickel-NTA agarose column chromatography and Ni magnetic beads. (C) Ribonuclease activity assay on dsRNA (40 mer duplex and dsRNA ladder).

Article Snippet: The protein expression level was evaluated in three T7 expression strains: T7 Express with LysY and lacI q (C3013), T7 SHuffle (C3026, E. coli K strain), and Nico (λDE3).

Techniques: Expressing, Purification, Activity Assay, Column Chromatography, Magnetic Beads

$Expression of hRNase A-ACE2NTD150 in T7 Express LysY/ lacI q (C3013). (A) Schematic diagram of the fusion protein. (B) SDS-PAGE analysis of five isolates of hRNase A-ACE2NTD150 (five isolates with MBP signal peptide, five clones without MBP signal peptide). Total cell lysate of pET21b serves as a negative control. (C,D) Western blot analysis of the fusion proteins (soluble fraction/supernatant) by anti-His mAb and anti-ACE2 mAb.$

Journal: Frontiers in Microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: Expression of hRNase A-ACE2NTD150 in T7 Express LysY/ lacI q (C3013). (A) Schematic diagram of the fusion protein. (B) SDS-PAGE analysis of five isolates of hRNase A-ACE2NTD150 (five isolates with MBP signal peptide, five clones without MBP signal peptide). Total cell lysate of pET21b serves as a negative control. (C,D) Western blot analysis of the fusion proteins (soluble fraction/supernatant) by anti-His mAb and anti-ACE2 mAb.

Article Snippet: The protein expression level was evaluated in three T7 expression strains: T7 Express with LysY and lacI q (C3013), T7 SHuffle (C3026, E. coli K strain), and Nico (λDE3).

Techniques: Expressing, SDS Page, Clone Assay, Negative Control, Western Blot