bl21 c3013 e coli cells  (New England Biolabs)

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

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

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.

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

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.

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

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.

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: Fluorescence, Negative Control, Expressing, Imaging

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

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

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