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You searched for: EV200158 (EV-TRACK ID)

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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV200158 2/4 Escherichia coli Cell culture supernatant (d)(U)C
Filtration
Tangential flow filtration
Zanella, Ilaria 2021 33%

Study summary

Full title
All authors
Ilaria Zanella, Enrico König, Michele Tomasi, Assunta Gagliardi, Luca Frattini, Laura Fantappiè, Carmela Irene, Francesca Zerbini, Elena Caproni, Samine J. Isaac, Martina Grigolato, Riccardo Corbellari, Silvia Valensin, Ilaria Ferlenghi, Fabiola Giusti, Luca Bini, Yaqoub Ashhab, Alberto Grandi, Guido Grandi
Journal
J Extracell Vesicles
Abstract
Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐nega (show more...)Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes‐specific antibody titers and high frequencies of epitope‐specific IFN‐γ‐producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV‐based vaccines (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
E. coli BL21(DE3)DompA
Sample origin
Control condition
Focus vesicles
Other / OMVsDompA
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Tangential flow filtration
Protein markers
EV: ompF
non-EV: None
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)/engineering
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
E. coli BL21(DE3)DompA
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
Detected EV-associated proteins
ompF
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
66.95
EV concentration
Yes
Particle yield
particle number per 100 ng of total OMV protein;Yes, other: 3.21E+08
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200158 4/4 Escherichia coli Cell culture supernatant (d)(U)C
Filtration
Tangential flow filtration
Zanella, Ilaria 2021 33%

Study summary

Full title
All authors
Ilaria Zanella, Enrico König, Michele Tomasi, Assunta Gagliardi, Luca Frattini, Laura Fantappiè, Carmela Irene, Francesca Zerbini, Elena Caproni, Samine J. Isaac, Martina Grigolato, Riccardo Corbellari, Silvia Valensin, Ilaria Ferlenghi, Fabiola Giusti, Luca Bini, Yaqoub Ashhab, Alberto Grandi, Guido Grandi
Journal
J Extracell Vesicles
Abstract
Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐nega (show more...)Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes‐specific antibody titers and high frequencies of epitope‐specific IFN‐γ‐producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV‐based vaccines (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
E. coli BL21(DE3)D60
Sample origin
genetically modified strain
Focus vesicles
Other / OMVsD60
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Tangential flow filtration
Protein markers
EV: ompF
non-EV: None
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)/engineering
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
Sample Condition
genetically modified strain
EV-producing cells
E. coli BL21(DE3)D60
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
Detected EV-associated proteins
ompF
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
69.9
EV concentration
Yes
Particle yield
particle number per 100 ng of total OMV protein;Yes, other: 3.09E+08
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200158 1/4 Escherichia coli Cell culture supernatant Filtration
(d)(U)C
Zanella, Ilaria 2021 14%

Study summary

Full title
All authors
Ilaria Zanella, Enrico König, Michele Tomasi, Assunta Gagliardi, Luca Frattini, Laura Fantappiè, Carmela Irene, Francesca Zerbini, Elena Caproni, Samine J. Isaac, Martina Grigolato, Riccardo Corbellari, Silvia Valensin, Ilaria Ferlenghi, Fabiola Giusti, Luca Bini, Yaqoub Ashhab, Alberto Grandi, Guido Grandi
Journal
J Extracell Vesicles
Abstract
Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐nega (show more...)Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes‐specific antibody titers and high frequencies of epitope‐specific IFN‐γ‐producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV‐based vaccines (hide)
EV-METRIC
14% (35th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
E. coli BL21(DE3)DompA
Sample origin
Control condition
Focus vesicles
Other / OMVsDompA
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Filtration
(Differential) (ultra)centrifugation
Protein markers
EV: ompF
non-EV: None
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)/engineering
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
E. coli BL21(DE3)DompA
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
132000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Flow cytometry
Hardware adjustments
Detected EV-associated proteins
ompF
Characterization: Particle analysis
EV200158 3/4 Escherichia coli Cell culture supernatant Filtration
(d)(U)C
Zanella, Ilaria 2021 14%

Study summary

Full title
All authors
Ilaria Zanella, Enrico König, Michele Tomasi, Assunta Gagliardi, Luca Frattini, Laura Fantappiè, Carmela Irene, Francesca Zerbini, Elena Caproni, Samine J. Isaac, Martina Grigolato, Riccardo Corbellari, Silvia Valensin, Ilaria Ferlenghi, Fabiola Giusti, Luca Bini, Yaqoub Ashhab, Alberto Grandi, Guido Grandi
Journal
J Extracell Vesicles
Abstract
Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐nega (show more...)Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes‐specific antibody titers and high frequencies of epitope‐specific IFN‐γ‐producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV‐based vaccines (hide)
EV-METRIC
14% (35th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
E. coli BL21(DE3)D60
Sample origin
genetically modified strain
Focus vesicles
Other / OMVsD60
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Filtration
(Differential) (ultra)centrifugation
Protein markers
EV: ompF
non-EV: None
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)/engineering
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
Sample Condition
genetically modified strain
EV-producing cells
E. coli BL21(DE3)D60
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
121
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
132001
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Flow cytometry
Hardware adjustments
Detected EV-associated proteins
ompF
Characterization: Particle analysis
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