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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200158	2/4	Escherichia coli	E. coli BL21(DE3)DompA	(d)(U)C Filtration Tangential flow filtration	Zanella, Ilaria	2021	33%
Study summary Full title Proteome‐minimized outer membrane vesicles from Escherichia coli as a generalized vaccine platform 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% (75th 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. For the quantitative method, the reporting of measured EV concentration is expected. 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 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. dUC = (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 EV-producing cells E. coli BL21(DE3)DompA EV-harvesting Medium Serum free medium Cell count OD600nm: 6.46 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Other Name other separation method Tangential flow filtration Characterization: Protein analysis Protein Concentration Method Lowry-based assay 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	E. coli BL21(DE3)D60	(d)(U)C Filtration Tangential flow filtration	Zanella, Ilaria	2021	33%
Study summary Full title Proteome‐minimized outer membrane vesicles from Escherichia coli as a generalized vaccine platform 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% (75th 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. For the quantitative method, the reporting of measured EV concentration is expected. 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 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. dUC = (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 EV-producing cells E. coli BL21(DE3)D60 EV-harvesting Medium Serum free medium Cell count OD600nm: 7.0 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Other Name other separation method Tangential flow filtration Characterization: Protein analysis Protein Concentration Method Lowry-based assay 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	E. coli BL21(DE3)DompA	Filtration (d)(U)C	Zanella, Ilaria	2021	14%
Study summary Full title Proteome‐minimized outer membrane vesicles from Escherichia coli as a generalized vaccine platform 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% (44th 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. For the quantitative method, the reporting of measured EV concentration is expected. 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 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. dUC = (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 EV-producing cells E. coli BL21(DE3)DompA EV-harvesting Medium Serum free medium Cell count OD600nm: 6.46 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed 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 Detected EV-associated proteins ompF Characterization: Lipid analysis No Characterization: Particle analysis None

	EV200158	3/4	Escherichia coli	E. coli BL21(DE3)D60	Filtration (d)(U)C	Zanella, Ilaria	2021	14%
Study summary Full title Proteome‐minimized outer membrane vesicles from Escherichia coli as a generalized vaccine platform 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% (44th 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. For the quantitative method, the reporting of measured EV concentration is expected. 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 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. dUC = (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 EV-producing cells E. coli BL21(DE3)D60 EV-harvesting Medium Serum free medium Cell count OD600nm: 7.0 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed 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 Detected EV-associated proteins ompF Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV200158
species	Escherichia coli
sample type	Cell culture
cell type	E. coli BL21(DE3)DompA	E. coli BL21(DE3)D60	E. coli BL21(DE3)DompA	E. coli BL21(DE3)D60
condition	Control condition	genetically modified strain	Control condition	genetically modified strain
separation protocol	dUC Filtration Tangential flow filtration	dUC Filtration Tangential flow filtration	Filtration dUC	Filtration dUC
vesicle related term	Other OMVsDompA	Other OMVsD60	Other OMVsDompA	Other OMVsD60
Exp. nr.	2	4	1	3
EV-METRIC %	33	33	14	14