bl21  (New England Biolabs)


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    Name:
    BL21 Competent E coli
    Description:
    BL21 Competent E coli 20x0 05 ml
    Catalog Number:
    C2530H
    Price:
    204
    Category:
    Competent Bacteria
    Size:
    1 ml
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    Structured Review

    New England Biolabs bl21
    BL21 Competent E coli
    BL21 Competent E coli 20x0 05 ml
    https://www.bioz.com/result/bl21/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bl21 - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) Product Images from "Redox environment is an intracellular factor to operate distinct pathways for aggregation of Cu,Zn-superoxide dismutase in amyotrophic lateral sclerosis"

    Article Title: Redox environment is an intracellular factor to operate distinct pathways for aggregation of Cu,Zn-superoxide dismutase in amyotrophic lateral sclerosis

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2013.00240

    Aggregation of disulfide-reduced SOD1 with fALS mutations in E. coli BL21 was not rescued by addition of ZnSO 4 . E. coli BL21(DE3) was transformed with pET15b harboring human SOD1 cDNA with indicated fALS mutations, and the protein expression was induced by IPTG in the presence of 1 mM ZnSO 4 . Cell lysates were fractionated into soluble supernatant (s) and insoluble pellets (p), treated with iodoacetamide, and then analyzed with non-reducing SDS-PAGE by using a 15% polyacrylamide gel. White (SOD1 S-S ) and black (SOD1 SH ) arrows at the right side of the gel image indicate positions of bands corresponding to SOD1 with and without a disulfide bond, respectively.
    Figure Legend Snippet: Aggregation of disulfide-reduced SOD1 with fALS mutations in E. coli BL21 was not rescued by addition of ZnSO 4 . E. coli BL21(DE3) was transformed with pET15b harboring human SOD1 cDNA with indicated fALS mutations, and the protein expression was induced by IPTG in the presence of 1 mM ZnSO 4 . Cell lysates were fractionated into soluble supernatant (s) and insoluble pellets (p), treated with iodoacetamide, and then analyzed with non-reducing SDS-PAGE by using a 15% polyacrylamide gel. White (SOD1 S-S ) and black (SOD1 SH ) arrows at the right side of the gel image indicate positions of bands corresponding to SOD1 with and without a disulfide bond, respectively.

    Techniques Used: Transformation Assay, Expressing, SDS Page

    Amyloid-like characters of SOD1 aggregates purified from insoluble inclusions in E. coli . (A) SOD1(G37R) aggregates purified from insoluble inclusions in E. coli were reacted with iodoacetamide for protection of free thiol groups and analyzed with reducing (+β-ME) and non-reducing (+IA) SDS-PAGE. Disulfide-crosslinked oligomers were identified in SOD1(G37R) aggregates purified from insoluble inclusions in E. coli SHuffle TM but not in BL21(DE3). (B, C) Tinctorial properties of SOD1(G37R) aggregates purified from E. coli BL21 and SHuffle TM (red and blue curves, respectively) were examined by (B) fluorescence of thioflavin T and (C) absorption of Congo red. Black curves represent (B) fluorescence spectrum of thioflavin T and (C) absorption spectrum of Congo red without addition of SOD1 aggregates. (D, E) Electron micrograms of SOD1(G37R) aggregates purified from E. coli (D) BL21 and (E) SHuffle TM . SOD1(G37R) aggregates exhibit large, amorphous morphologies (left panels), while fibrillar structures become evident after brief treatment of aggregates with Proteinase K (right panels). A bar represents 2 μm (left panels) or 50 nm (right panels).
    Figure Legend Snippet: Amyloid-like characters of SOD1 aggregates purified from insoluble inclusions in E. coli . (A) SOD1(G37R) aggregates purified from insoluble inclusions in E. coli were reacted with iodoacetamide for protection of free thiol groups and analyzed with reducing (+β-ME) and non-reducing (+IA) SDS-PAGE. Disulfide-crosslinked oligomers were identified in SOD1(G37R) aggregates purified from insoluble inclusions in E. coli SHuffle TM but not in BL21(DE3). (B, C) Tinctorial properties of SOD1(G37R) aggregates purified from E. coli BL21 and SHuffle TM (red and blue curves, respectively) were examined by (B) fluorescence of thioflavin T and (C) absorption of Congo red. Black curves represent (B) fluorescence spectrum of thioflavin T and (C) absorption spectrum of Congo red without addition of SOD1 aggregates. (D, E) Electron micrograms of SOD1(G37R) aggregates purified from E. coli (D) BL21 and (E) SHuffle TM . SOD1(G37R) aggregates exhibit large, amorphous morphologies (left panels), while fibrillar structures become evident after brief treatment of aggregates with Proteinase K (right panels). A bar represents 2 μm (left panels) or 50 nm (right panels).

    Techniques Used: Purification, IA, SDS Page, Fluorescence

    2) Product Images from "Safe Recombinant Outer Membrane Vesicles that Display M2e Elicit Heterologous Influenza Protection"

    Article Title: Safe Recombinant Outer Membrane Vesicles that Display M2e Elicit Heterologous Influenza Protection

    Journal: Molecular Therapy

    doi: 10.1016/j.ymthe.2017.01.010

    Pyrogenicity and TLR Activity Are rOMV Source Strain Dependent (A) Pyrogenicity (measured in endotoxin units) of CC, Nsl, and BL21 rOMVS determined using whole-blood pyrogenicity test. Groups were compared with Kruskal-Wallis test, followed by Mann-Whitney between pairs, using Bonferroni method to account for multiple comparisons (*p
    Figure Legend Snippet: Pyrogenicity and TLR Activity Are rOMV Source Strain Dependent (A) Pyrogenicity (measured in endotoxin units) of CC, Nsl, and BL21 rOMVS determined using whole-blood pyrogenicity test. Groups were compared with Kruskal-Wallis test, followed by Mann-Whitney between pairs, using Bonferroni method to account for multiple comparisons (*p

    Techniques Used: Activity Assay, MANN-WHITNEY

    rOMVs Produced from Three Different E. coli Strains Are Structurally Comparable (A–C) TEM images of rOMVs stained with uranyl acetate: CC rOMVs (A), BL21 rOMVs (B), and Nsl rOMVs (C). Scale bars represent 100 nm. (D and E) Total IgG (D) and isotypes IgG1 and IgG2a (E) anti-GFP titers from BALB/c mice 8 weeks post prime dose of ClyA-GFP-expressing CC or BL21 rOMVs. Titer error bars represent 95% confidence intervals (CI) of geometric mean. Log-transformed data analyzed using an unpaired Student’s t test to compare CC versus BL21 rOMV anti-GFP IgG levels and using paired Student’s t test to compare IgG1:IgG2a levels for each rOMV type. Dotted line indicates titer of sera from mice pre-vaccination.
    Figure Legend Snippet: rOMVs Produced from Three Different E. coli Strains Are Structurally Comparable (A–C) TEM images of rOMVs stained with uranyl acetate: CC rOMVs (A), BL21 rOMVs (B), and Nsl rOMVs (C). Scale bars represent 100 nm. (D and E) Total IgG (D) and isotypes IgG1 and IgG2a (E) anti-GFP titers from BALB/c mice 8 weeks post prime dose of ClyA-GFP-expressing CC or BL21 rOMVs. Titer error bars represent 95% confidence intervals (CI) of geometric mean. Log-transformed data analyzed using an unpaired Student’s t test to compare CC versus BL21 rOMV anti-GFP IgG levels and using paired Student’s t test to compare IgG1:IgG2a levels for each rOMV type. Dotted line indicates titer of sera from mice pre-vaccination.

    Techniques Used: Produced, Transmission Electron Microscopy, Staining, Mouse Assay, Expressing, Transformation Assay

    3) Product Images from "A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha"

    Article Title: A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha

    Journal: Plant and Cell Physiology

    doi: 10.1093/pcp/pcv160

    Detection of chloroplast-expressed mTurquoise2 in transgenic lines of Marchantia polymorpha. (A) M. polymorpha protein extracts separated by SDS–PAGE. Protein extracts from wild type (WT) and transplastomic (CL0*b) lines of M. polymorpha were separated by 4–12% SDS–PAGE, and visualized by in-gel fluorescence (left) and subsequent Coomassie stain (right). The fluorescence image was generated using a custom imaging device described in the Materials and Methods for visualization of CFP bands (emission 486/10 nm, green) and marker (emission 540/10 nm, red). (B) Untagged and His 6 -tagged mTurquoise2 separated by SDS–PAGE. Untagged mTurquoise2 was expressed from plasmids pCS CL0*b ( psbA promoter) in BL21 E. coli and pCRB SREI (T7 promoter) in T7 Express E. coli. His 6 -tagged mTurquoise2 was expressed from plasmid pCRB SREI 6his (T7 promoter) and purified by affinity chromatography. The rightmost lanes contain 50 ng of unboiled and boiled purified protein, respectively. Visualization by in-gel fluorescence and Coomassie stain was conducted as described above. (C) Standard curve for quantification of mTurquoise2 based on in-gel fluorescence. Serial dilutions of 1 µg purified of mTurquoise2 were separated by 4–12% SDS–PAGE and the band intensity quantified by in-gel fluorescence as described in the Materials and Methods. Levels of mTurquoise2 extracted from transplastomic (CL0*b) M. polymorpha (red cross) were estimated by linear regression analysis on densities of fluorescent target bands as shown in (A). Error bars represent the SD of average band density between three different experiments.
    Figure Legend Snippet: Detection of chloroplast-expressed mTurquoise2 in transgenic lines of Marchantia polymorpha. (A) M. polymorpha protein extracts separated by SDS–PAGE. Protein extracts from wild type (WT) and transplastomic (CL0*b) lines of M. polymorpha were separated by 4–12% SDS–PAGE, and visualized by in-gel fluorescence (left) and subsequent Coomassie stain (right). The fluorescence image was generated using a custom imaging device described in the Materials and Methods for visualization of CFP bands (emission 486/10 nm, green) and marker (emission 540/10 nm, red). (B) Untagged and His 6 -tagged mTurquoise2 separated by SDS–PAGE. Untagged mTurquoise2 was expressed from plasmids pCS CL0*b ( psbA promoter) in BL21 E. coli and pCRB SREI (T7 promoter) in T7 Express E. coli. His 6 -tagged mTurquoise2 was expressed from plasmid pCRB SREI 6his (T7 promoter) and purified by affinity chromatography. The rightmost lanes contain 50 ng of unboiled and boiled purified protein, respectively. Visualization by in-gel fluorescence and Coomassie stain was conducted as described above. (C) Standard curve for quantification of mTurquoise2 based on in-gel fluorescence. Serial dilutions of 1 µg purified of mTurquoise2 were separated by 4–12% SDS–PAGE and the band intensity quantified by in-gel fluorescence as described in the Materials and Methods. Levels of mTurquoise2 extracted from transplastomic (CL0*b) M. polymorpha (red cross) were estimated by linear regression analysis on densities of fluorescent target bands as shown in (A). Error bars represent the SD of average band density between three different experiments.

    Techniques Used: Transgenic Assay, SDS Page, Fluorescence, Staining, Generated, Imaging, Marker, Plasmid Preparation, Purification, Affinity Chromatography

    Related Articles

    Expressing:

    Article Title: Novel Characteristics of Succinate Coenzyme A (Succinate-CoA) Ligases: Conversion of Malate to Malyl-CoA and CoA-Thioester Formation of Succinate Analogues In Vitro
    Article Snippet: Expression of the relevant genes sucC and sucD in about equimolar amounts in a successful purification protocol was observed to require 68 or 135 bp of the corresponding sucC upstream regions in the expression vector pBluescriptSK(−) for sucCD Am and sucCD BL21 , respectively. .. During the experiments, the best expression was obtained when the sucCD genes of A. mimigardefordensis DPN7T and E. coli BL21 were each applied in one bicistronic operon that included the strain-specific Shine-Dalgarno sequence upstream of sucC . ..

    Article Title: A human monocytic NF-κB fluorescent reporter cell line for detection of microbial contaminants in biological samples
    Article Snippet: Bacterial protein expression A construct encoding human complement split product C4dg fused to a C-terminal 6xHIS Tag was cloned into the IPTG-inducible bacterial expression vector pET21a(+) (EMD Millipore, Billerica, MA). .. The electrocompetent E. coli protein expression strain ClearColi BL21 was purchased from Lucigen (Middleton, WI) and standard E. coli BL21 were obtained from New England Biolabs (Ipswich, MA). .. Protein expression was performed in standard LB (for BL21) or LB-Miller (10 g/L NaCl, for ClearColi BL21).

    Sequencing:

    Article Title: Novel Characteristics of Succinate Coenzyme A (Succinate-CoA) Ligases: Conversion of Malate to Malyl-CoA and CoA-Thioester Formation of Succinate Analogues In Vitro
    Article Snippet: Expression of the relevant genes sucC and sucD in about equimolar amounts in a successful purification protocol was observed to require 68 or 135 bp of the corresponding sucC upstream regions in the expression vector pBluescriptSK(−) for sucCD Am and sucCD BL21 , respectively. .. During the experiments, the best expression was obtained when the sucCD genes of A. mimigardefordensis DPN7T and E. coli BL21 were each applied in one bicistronic operon that included the strain-specific Shine-Dalgarno sequence upstream of sucC . ..

    Over Expression:

    Article Title: Amino-Terminal Processing of Helicobacter pylori Serine Protease HtrA: Role in Oligomerization and Activity Regulation
    Article Snippet: As control, E. coli BL21 without a plasmid exhibited no expression of HtrAHp or GST-tagged protein, respectively ( Figures ). .. Together, these findings confirmed the successful overexpression of HtrAHp variants in E. coli BL21, useful for further analysis of HtrA activity. .. As next, the above generated E. coli BL21 lysates were subjected to casein zymography to investigate an effect of the shortened amino-terminus of HtrAHp on the proteolytic activity.

    Article Title: HMD-ARG: hierarchical multi-task deep learning for annotating antibiotic resistance genes
    Article Snippet: After ligation using the Quick LigationTM Kit (NEB, USA) and verification by PCR and DNA sequencing (BGI, China), the resulting plasmids were transformed into E. coli BL21 for antibiotic sensitivity analysis. .. Overnight culture of E. coli BL21 strains containing the plasmids for overexpression of the predicted genes were 1:100 diluted and grown in LB medium supplemented with kanamycin (20 μg/ml) and Isopropyl β-D-1-thiogalactopyranoside (IPTG) (0.5 mM). .. After incubation at 37 °C with 220-rpm agitation for 90 min, bacterial cultures were transferred to a 24-well plate.

    Activity Assay:

    Article Title: Amino-Terminal Processing of Helicobacter pylori Serine Protease HtrA: Role in Oligomerization and Activity Regulation
    Article Snippet: As control, E. coli BL21 without a plasmid exhibited no expression of HtrAHp or GST-tagged protein, respectively ( Figures ). .. Together, these findings confirmed the successful overexpression of HtrAHp variants in E. coli BL21, useful for further analysis of HtrA activity. .. As next, the above generated E. coli BL21 lysates were subjected to casein zymography to investigate an effect of the shortened amino-terminus of HtrAHp on the proteolytic activity.

    Cell Culture:

    Article Title: Improve Protein Solubility and Activity based on Machine Learning Models
    Article Snippet: Plasmid construction All the plasmids used in this work were constructed by using GT DNA standard (Supplementary Table S7). .. Cell culture and SDS-PAGE analysis of protein solubility Each of constructed plasmid was introduced into E. coli BL21 (DE3) (C2530H, New England Biolabs) for SDS-PAGE analysis by using standard heat shock protocol. ..

    SDS Page:

    Article Title: Improve Protein Solubility and Activity based on Machine Learning Models
    Article Snippet: Plasmid construction All the plasmids used in this work were constructed by using GT DNA standard (Supplementary Table S7). .. Cell culture and SDS-PAGE analysis of protein solubility Each of constructed plasmid was introduced into E. coli BL21 (DE3) (C2530H, New England Biolabs) for SDS-PAGE analysis by using standard heat shock protocol. ..

    Solubility:

    Article Title: Improve Protein Solubility and Activity based on Machine Learning Models
    Article Snippet: Plasmid construction All the plasmids used in this work were constructed by using GT DNA standard (Supplementary Table S7). .. Cell culture and SDS-PAGE analysis of protein solubility Each of constructed plasmid was introduced into E. coli BL21 (DE3) (C2530H, New England Biolabs) for SDS-PAGE analysis by using standard heat shock protocol. ..

    Construct:

    Article Title: Improve Protein Solubility and Activity based on Machine Learning Models
    Article Snippet: Plasmid construction All the plasmids used in this work were constructed by using GT DNA standard (Supplementary Table S7). .. Cell culture and SDS-PAGE analysis of protein solubility Each of constructed plasmid was introduced into E. coli BL21 (DE3) (C2530H, New England Biolabs) for SDS-PAGE analysis by using standard heat shock protocol. ..

    Plasmid Preparation:

    Article Title: Improve Protein Solubility and Activity based on Machine Learning Models
    Article Snippet: Plasmid construction All the plasmids used in this work were constructed by using GT DNA standard (Supplementary Table S7). .. Cell culture and SDS-PAGE analysis of protein solubility Each of constructed plasmid was introduced into E. coli BL21 (DE3) (C2530H, New England Biolabs) for SDS-PAGE analysis by using standard heat shock protocol. ..

    Article Title: Characterization of diverse homoserine lactone synthases in Escherichia coli
    Article Snippet: Graphs and heat maps were generated in Microsoft Excel 2016. .. Cultures of E . coli BL21 (New England Biolabs) transformed with Sender plasmids, a Receiver plasmid, a negative receiver plasmid, and a GFP positive plasmid were grown in 3 mL of LB with 5 ug/mL ampicillin for 16 hours at 37°C and shaking at 220 rpm. .. Bacterial culture was subsequently spread onto LB agar supplemented with 5 ug/mL ampicillin with sterile disposable plastic micropipette tip, such that a central spot of Sender culture would evenly diffuse towards proximal Receiver and Control-EGFP positive control cultures.

    Staining:

    Article Title: Amino-Terminal Processing of Helicobacter pylori Serine Protease HtrA: Role in Oligomerization and Activity Regulation
    Article Snippet: To investigate the importance of the amino-terminus on the proteolytic activity of HtrAHp in more detail, wt HtrAHp , ΔN1 or ΔN2 were expressed as GST-tagged variants in E. coli strain BL21 (Supplementary Figure ). .. E. coli BL21 were induced for 4 h with IPTG and the resulting bacterial lysates were subjected to Coomassie staining, confirming that equal amounts of protein were present ( Figure ). .. HtrAHp wt missing only the signal peptide (ΔSP) revealed a strong overexpression of the fusion protein (p55 HtrAHp with GST-tag of ∼70 kDa) as detected by Coomassie-staining ( Figure , black asterisk).

    Transformation Assay:

    Article Title: Characterization of diverse homoserine lactone synthases in Escherichia coli
    Article Snippet: Graphs and heat maps were generated in Microsoft Excel 2016. .. Cultures of E . coli BL21 (New England Biolabs) transformed with Sender plasmids, a Receiver plasmid, a negative receiver plasmid, and a GFP positive plasmid were grown in 3 mL of LB with 5 ug/mL ampicillin for 16 hours at 37°C and shaking at 220 rpm. .. Bacterial culture was subsequently spread onto LB agar supplemented with 5 ug/mL ampicillin with sterile disposable plastic micropipette tip, such that a central spot of Sender culture would evenly diffuse towards proximal Receiver and Control-EGFP positive control cultures.

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    New England Biolabs standard e coli bl21
    Use of THP-1 TLR4-CD14 NF-κB-eGFP reporter cells for the detection of microbial contaminations in recombinant protein preparations. (A) THP-1 TLR4-CD14 NF-κB-eGFP reporter cells were incubated with the indicated serial dilutions of ultrapure LPS for 24 h. NF-κB-driven eGFP expression was assessed by flow cytometry. Bar graphs show geometric mean of fluorescence intensity (gMFI). eGFP expression of the parental THP-1 reporter cells treated with 300 ng/ml ultrapure LPS is shown for comparison (grey bar). (B) mRNA expression of TLR4, CD14 and MD2 was measured in parental and TLR4-CD14 THP-1 reporter cells by real-time qPCR. Data are presented as mean ± SEM of expression values normalized against the housekeeping gene GAPDH (n = 4). (C) Recombinant human split product C4dg produced in three different expression systems (HEK293-6E, <t>standard</t> E . coli <t>BL21</t> and E . coli ClearColi BL21) was tested for TLR4-agonist contaminations using HEK293 hTLR4A-MD2-CD14 cells. Following 24 h of incubation the IL-8 content in the culture supernatants was measured by ELISA. (D) Parental and TLR4-CD14 THP-1 reporters were incubated with the protein preparations described in (C) and NF-κB-driven eGFP expression was assessed by flow cytometry 24 h later. Bar graphs show geometric mean of fluorescence intensity (gMFI). Mean and SE were calculated from duplicates of four independently performed experiments (n = 4) (E) Immature human moDCs were incubated with standard LPS, E . coli BL21 or E . coli ClearColi BL21 expressed C4dg protein at the indicated concentrations for 24 h or were left untreated. Expression of maturation markers CD83 and CD86 was assessed by flow cytometry. (F) E . coli ClearColi BL21-expressed C4dg protein was subjected to a single round of bulk chromatography using a polymyxin resin (see Material and Methods ). Samples before and after chromatography were tested using the THP-1 TLR4-CD14 reporters. Mean and SE were calculated from duplicates of four independently performed experiments (n = 4).
    Standard E Coli Bl21, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/standard e coli bl21/product/New England Biolabs
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    Use of THP-1 TLR4-CD14 NF-κB-eGFP reporter cells for the detection of microbial contaminations in recombinant protein preparations. (A) THP-1 TLR4-CD14 NF-κB-eGFP reporter cells were incubated with the indicated serial dilutions of ultrapure LPS for 24 h. NF-κB-driven eGFP expression was assessed by flow cytometry. Bar graphs show geometric mean of fluorescence intensity (gMFI). eGFP expression of the parental THP-1 reporter cells treated with 300 ng/ml ultrapure LPS is shown for comparison (grey bar). (B) mRNA expression of TLR4, CD14 and MD2 was measured in parental and TLR4-CD14 THP-1 reporter cells by real-time qPCR. Data are presented as mean ± SEM of expression values normalized against the housekeeping gene GAPDH (n = 4). (C) Recombinant human split product C4dg produced in three different expression systems (HEK293-6E, standard E . coli BL21 and E . coli ClearColi BL21) was tested for TLR4-agonist contaminations using HEK293 hTLR4A-MD2-CD14 cells. Following 24 h of incubation the IL-8 content in the culture supernatants was measured by ELISA. (D) Parental and TLR4-CD14 THP-1 reporters were incubated with the protein preparations described in (C) and NF-κB-driven eGFP expression was assessed by flow cytometry 24 h later. Bar graphs show geometric mean of fluorescence intensity (gMFI). Mean and SE were calculated from duplicates of four independently performed experiments (n = 4) (E) Immature human moDCs were incubated with standard LPS, E . coli BL21 or E . coli ClearColi BL21 expressed C4dg protein at the indicated concentrations for 24 h or were left untreated. Expression of maturation markers CD83 and CD86 was assessed by flow cytometry. (F) E . coli ClearColi BL21-expressed C4dg protein was subjected to a single round of bulk chromatography using a polymyxin resin (see Material and Methods ). Samples before and after chromatography were tested using the THP-1 TLR4-CD14 reporters. Mean and SE were calculated from duplicates of four independently performed experiments (n = 4).

    Journal: PLoS ONE

    Article Title: A human monocytic NF-κB fluorescent reporter cell line for detection of microbial contaminants in biological samples

    doi: 10.1371/journal.pone.0178220

    Figure Lengend Snippet: Use of THP-1 TLR4-CD14 NF-κB-eGFP reporter cells for the detection of microbial contaminations in recombinant protein preparations. (A) THP-1 TLR4-CD14 NF-κB-eGFP reporter cells were incubated with the indicated serial dilutions of ultrapure LPS for 24 h. NF-κB-driven eGFP expression was assessed by flow cytometry. Bar graphs show geometric mean of fluorescence intensity (gMFI). eGFP expression of the parental THP-1 reporter cells treated with 300 ng/ml ultrapure LPS is shown for comparison (grey bar). (B) mRNA expression of TLR4, CD14 and MD2 was measured in parental and TLR4-CD14 THP-1 reporter cells by real-time qPCR. Data are presented as mean ± SEM of expression values normalized against the housekeeping gene GAPDH (n = 4). (C) Recombinant human split product C4dg produced in three different expression systems (HEK293-6E, standard E . coli BL21 and E . coli ClearColi BL21) was tested for TLR4-agonist contaminations using HEK293 hTLR4A-MD2-CD14 cells. Following 24 h of incubation the IL-8 content in the culture supernatants was measured by ELISA. (D) Parental and TLR4-CD14 THP-1 reporters were incubated with the protein preparations described in (C) and NF-κB-driven eGFP expression was assessed by flow cytometry 24 h later. Bar graphs show geometric mean of fluorescence intensity (gMFI). Mean and SE were calculated from duplicates of four independently performed experiments (n = 4) (E) Immature human moDCs were incubated with standard LPS, E . coli BL21 or E . coli ClearColi BL21 expressed C4dg protein at the indicated concentrations for 24 h or were left untreated. Expression of maturation markers CD83 and CD86 was assessed by flow cytometry. (F) E . coli ClearColi BL21-expressed C4dg protein was subjected to a single round of bulk chromatography using a polymyxin resin (see Material and Methods ). Samples before and after chromatography were tested using the THP-1 TLR4-CD14 reporters. Mean and SE were calculated from duplicates of four independently performed experiments (n = 4).

    Article Snippet: The electrocompetent E. coli protein expression strain ClearColi BL21 was purchased from Lucigen (Middleton, WI) and standard E. coli BL21 were obtained from New England Biolabs (Ipswich, MA).

    Techniques: Recombinant, Incubation, Expressing, Flow Cytometry, Cytometry, Fluorescence, Real-time Polymerase Chain Reaction, Produced, Enzyme-linked Immunosorbent Assay, Chromatography

    Sender plasmids expressed in Escherichia coli BL21. (A) The modular Sender vector allows facile cloning of any EcoRI , XbaI ] glyphs (top) and a scaled circular map (bottom). (B) Optical density (OD) readings for E . coli cultures expressing each of the Sender plasmids. (C) mCherry expression is shown as end-point RFP signal normalized to OD 600 after an 8 hour growth period.

    Journal: PLoS ONE

    Article Title: Characterization of diverse homoserine lactone synthases in Escherichia coli

    doi: 10.1371/journal.pone.0202294

    Figure Lengend Snippet: Sender plasmids expressed in Escherichia coli BL21. (A) The modular Sender vector allows facile cloning of any EcoRI , XbaI ] glyphs (top) and a scaled circular map (bottom). (B) Optical density (OD) readings for E . coli cultures expressing each of the Sender plasmids. (C) mCherry expression is shown as end-point RFP signal normalized to OD 600 after an 8 hour growth period.

    Article Snippet: Cultures of E . coli BL21 (New England Biolabs) transformed with Sender plasmids, a Receiver plasmid, a negative receiver plasmid, and a GFP positive plasmid were grown in 3 mL of LB with 5 ug/mL ampicillin for 16 hours at 37°C and shaking at 220 rpm.

    Techniques: Plasmid Preparation, Clone Assay, Expressing

    Functional validation of the predicted ARGs and structural investigation of conserved sites detected by HMD-ARG. a Left figure: A diagram showing the procedure of heterologous expression and functional analysis of the predicted ARGs from PA150567 in E. coli BL21 host. Middle figure : Growth curves of E. coli hosts with expression of the predicted β -lactamases that inactivate β -lactam antibiotics in the presence of 2 μg/ml meropenem. Right figure : Growth curves of E. coli hosts with expression of the predicted acetyltransferases that inactivate aminoglycoside antibiotics in the presence of 4 μg/ml amikacin. b The HMD-ARG predicted conserved sites and the corresponding sequence logo from 319 to 393 in AXX01_04100. c After we mutated the conserved site (346) from T to N, the mutated protein’s (colored in red) local structure (predicted by RaptorX) changed significantly from the wild type (colored in light blue). Moreover, the binding affinity (predicted by AutoDock) between the mutated protein and the ligand (antibiotics) also reduced, as illustrated in the middle (wild type) and right (mutated protein) figures

    Journal: Microbiome

    Article Title: HMD-ARG: hierarchical multi-task deep learning for annotating antibiotic resistance genes

    doi: 10.1186/s40168-021-01002-3

    Figure Lengend Snippet: Functional validation of the predicted ARGs and structural investigation of conserved sites detected by HMD-ARG. a Left figure: A diagram showing the procedure of heterologous expression and functional analysis of the predicted ARGs from PA150567 in E. coli BL21 host. Middle figure : Growth curves of E. coli hosts with expression of the predicted β -lactamases that inactivate β -lactam antibiotics in the presence of 2 μg/ml meropenem. Right figure : Growth curves of E. coli hosts with expression of the predicted acetyltransferases that inactivate aminoglycoside antibiotics in the presence of 4 μg/ml amikacin. b The HMD-ARG predicted conserved sites and the corresponding sequence logo from 319 to 393 in AXX01_04100. c After we mutated the conserved site (346) from T to N, the mutated protein’s (colored in red) local structure (predicted by RaptorX) changed significantly from the wild type (colored in light blue). Moreover, the binding affinity (predicted by AutoDock) between the mutated protein and the ligand (antibiotics) also reduced, as illustrated in the middle (wild type) and right (mutated protein) figures

    Article Snippet: Overnight culture of E. coli BL21 strains containing the plasmids for overexpression of the predicted genes were 1:100 diluted and grown in LB medium supplemented with kanamycin (20 μg/ml) and Isopropyl β-D-1-thiogalactopyranoside (IPTG) (0.5 mM).

    Techniques: Functional Assay, Expressing, Sequencing, Binding Assay

    ( a) The SDS-PAGE analysis of protein tal and dxs expressed in E. coli with and without tags designed by our optimization algorithm. “+” and “-” represented expressed proteins with and without peptide tags respectively. “P” and “S” represented the pellet fraction (insoluble) and supernatant fraction (soluble), respectively. The oval shapes highlight the bands of dxs and tal proteins. Protein tal and dxs were expressed in K3 medium with 20 g/L glucose at 30 °C. ( b) Quantitative presentation of the SDS-PAGE images in a . The protein solubility was the ratio of soluble protein amount to the total protein amount. The protein amount was estimated by using band intensity on SDS-PAGE images. The sequences of the designed tags for N-terminal and C-terminal were shown. The amino acid S and G on the two ends of the tags were the linkers for GT DNA assembly standard, which was used to construct the plasmids in this study 40 .

    Journal: bioRxiv

    Article Title: Improve Protein Solubility and Activity based on Machine Learning Models

    doi: 10.1101/817890

    Figure Lengend Snippet: ( a) The SDS-PAGE analysis of protein tal and dxs expressed in E. coli with and without tags designed by our optimization algorithm. “+” and “-” represented expressed proteins with and without peptide tags respectively. “P” and “S” represented the pellet fraction (insoluble) and supernatant fraction (soluble), respectively. The oval shapes highlight the bands of dxs and tal proteins. Protein tal and dxs were expressed in K3 medium with 20 g/L glucose at 30 °C. ( b) Quantitative presentation of the SDS-PAGE images in a . The protein solubility was the ratio of soluble protein amount to the total protein amount. The protein amount was estimated by using band intensity on SDS-PAGE images. The sequences of the designed tags for N-terminal and C-terminal were shown. The amino acid S and G on the two ends of the tags were the linkers for GT DNA assembly standard, which was used to construct the plasmids in this study 40 .

    Article Snippet: Cell culture and SDS-PAGE analysis of protein solubility Each of constructed plasmid was introduced into E. coli BL21 (DE3) (C2530H, New England Biolabs) for SDS-PAGE analysis by using standard heat shock protocol.

    Techniques: SDS Page, Solubility, Construct