Structured Review

Promega tnt coupled reticulate lysate system
Design and validation of rotavirus (RV) VP4 and nonstructural protein 3 (NSP3) plasmid constructs used to generate mutant viruses. (A) Schematic showing overall topology of SARS-CoV-2 spike protein in gray boxes: N-terminal domain (NTD), receptor binding domain (RBD), which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and the intracellular domain (IC) (adapted from Lan et al. ). Dashed lines represent selected regions of the spike protein (colored boxes) that were inserted into the hypervariable region of the SA11 viral protein 4 (VP4) gene (top) and the C terminus of the RF NSP3 gene (bottom). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2, or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without Thosea asigna virus 2A (T2A) (yellow box), represented by +/− sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (“HDV Rib,” green boxes) followed by T7 terminator (T7T). *Stop codons. Schematic not to scale. (B) Ribbon representation of VP4 (adapted from Settembre et al. ). Two orthogonal views are shown. The VP8* fragment is in magenta extending into the VP5* foot domains (in blue), the hypervariable region of VP4 is in gray and the region where SARS-CoV-2 peptides (omitted for clarity) were inserted is in green. VP5* β-barrel domains are in cyan and purple. (C and D) <t>Coupled</t> in vitro transcription and translation reactions of mutated SA11 VP4 (C) and RF NSP3 (D) segments were carried out using the <t>TnT</t> rabbit reticulocyte <t>lysate</t> <t>system</t> supplemented with [ 35 S]methionine. Samples were analyzed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In panel D, black asterisks indicate T2A read-through product and red asterisks identify separated products. WT, wild type.
Tnt Coupled Reticulate Lysate System, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/tnt coupled reticulate lysate system/product/Promega
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
tnt coupled reticulate lysate system - by Bioz Stars, 2023-02
86/100 stars

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1) Product Images from "Using Species a Rotavirus Reverse Genetics to Engineer Chimeric Viruses Expressing SARS-CoV-2 Spike Epitopes"

Article Title: Using Species a Rotavirus Reverse Genetics to Engineer Chimeric Viruses Expressing SARS-CoV-2 Spike Epitopes

Journal: Journal of Virology

doi: 10.1128/jvi.00488-22

Design and validation of rotavirus (RV) VP4 and nonstructural protein 3 (NSP3) plasmid constructs used to generate mutant viruses. (A) Schematic showing overall topology of SARS-CoV-2 spike protein in gray boxes: N-terminal domain (NTD), receptor binding domain (RBD), which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and the intracellular domain (IC) (adapted from Lan et al. ). Dashed lines represent selected regions of the spike protein (colored boxes) that were inserted into the hypervariable region of the SA11 viral protein 4 (VP4) gene (top) and the C terminus of the RF NSP3 gene (bottom). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2, or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without Thosea asigna virus 2A (T2A) (yellow box), represented by +/− sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (“HDV Rib,” green boxes) followed by T7 terminator (T7T). *Stop codons. Schematic not to scale. (B) Ribbon representation of VP4 (adapted from Settembre et al. ). Two orthogonal views are shown. The VP8* fragment is in magenta extending into the VP5* foot domains (in blue), the hypervariable region of VP4 is in gray and the region where SARS-CoV-2 peptides (omitted for clarity) were inserted is in green. VP5* β-barrel domains are in cyan and purple. (C and D) Coupled in vitro transcription and translation reactions of mutated SA11 VP4 (C) and RF NSP3 (D) segments were carried out using the TnT rabbit reticulocyte lysate system supplemented with [ 35 S]methionine. Samples were analyzed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In panel D, black asterisks indicate T2A read-through product and red asterisks identify separated products. WT, wild type.
Figure Legend Snippet: Design and validation of rotavirus (RV) VP4 and nonstructural protein 3 (NSP3) plasmid constructs used to generate mutant viruses. (A) Schematic showing overall topology of SARS-CoV-2 spike protein in gray boxes: N-terminal domain (NTD), receptor binding domain (RBD), which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and the intracellular domain (IC) (adapted from Lan et al. ). Dashed lines represent selected regions of the spike protein (colored boxes) that were inserted into the hypervariable region of the SA11 viral protein 4 (VP4) gene (top) and the C terminus of the RF NSP3 gene (bottom). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2, or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without Thosea asigna virus 2A (T2A) (yellow box), represented by +/− sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (“HDV Rib,” green boxes) followed by T7 terminator (T7T). *Stop codons. Schematic not to scale. (B) Ribbon representation of VP4 (adapted from Settembre et al. ). Two orthogonal views are shown. The VP8* fragment is in magenta extending into the VP5* foot domains (in blue), the hypervariable region of VP4 is in gray and the region where SARS-CoV-2 peptides (omitted for clarity) were inserted is in green. VP5* β-barrel domains are in cyan and purple. (C and D) Coupled in vitro transcription and translation reactions of mutated SA11 VP4 (C) and RF NSP3 (D) segments were carried out using the TnT rabbit reticulocyte lysate system supplemented with [ 35 S]methionine. Samples were analyzed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In panel D, black asterisks indicate T2A read-through product and red asterisks identify separated products. WT, wild type.

Techniques Used: Plasmid Preparation, Construct, Mutagenesis, Binding Assay, Sequencing, In Vitro, SDS Page, Autoradiography, Molecular Weight, Marker, Negative Control


Structured Review

Promega tnt coupled reticulate lysate system
(A) Schematic showing overall topology of SARS-CoV-2 spike protein in grey boxes: N-terminal domain (NTD), receptor binding domain (RBD) which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM) and the intracellular domain (IC) (Adapted from Lan et al., 2020) . Dashed lines represent selected regions of the spike protein (coloured boxes with their relative nucleotide positions) that were inserted into the hypervariable region of the SA11 VP4 gene (panel above) and the C-terminus of the RF NSP3 gene (panel below). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2 or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without T2A (yellow box), represented by +/- sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic HDV ribozyme (‘HDV Rib’, green boxes) followed by T7 terminator (T7T). Asterisks (*) represent stop codons. Schematic not to scale. <t>Coupled</t> in vitro transcription and translation reactions of mutated SA11 VP4 (B) and RF NSP3 (C) segments were carried out using the <t>TnT</t> rabbit reticulocyte <t>lysate</t> <t>system</t> supplemented with [ 35 S] methionine. Samples were analysed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In (C) , black asterisks indicate T2A read-through product and red asterisks identify separated products.
Tnt Coupled Reticulate Lysate System, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/tnt coupled reticulate lysate system/product/Promega
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
tnt coupled reticulate lysate system - by Bioz Stars, 2023-02
86/100 stars

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1) Product Images from "Engineering a Vaccine Platform using Rotavirus A to Express SARS-CoV-2 Spike Epitopes"

Article Title: Engineering a Vaccine Platform using Rotavirus A to Express SARS-CoV-2 Spike Epitopes

Journal: bioRxiv

doi: 10.1101/2022.03.23.485570

(A) Schematic showing overall topology of SARS-CoV-2 spike protein in grey boxes: N-terminal domain (NTD), receptor binding domain (RBD) which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM) and the intracellular domain (IC) (Adapted from Lan et al., 2020) . Dashed lines represent selected regions of the spike protein (coloured boxes with their relative nucleotide positions) that were inserted into the hypervariable region of the SA11 VP4 gene (panel above) and the C-terminus of the RF NSP3 gene (panel below). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2 or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without T2A (yellow box), represented by +/- sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic HDV ribozyme (‘HDV Rib’, green boxes) followed by T7 terminator (T7T). Asterisks (*) represent stop codons. Schematic not to scale. Coupled in vitro transcription and translation reactions of mutated SA11 VP4 (B) and RF NSP3 (C) segments were carried out using the TnT rabbit reticulocyte lysate system supplemented with [ 35 S] methionine. Samples were analysed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In (C) , black asterisks indicate T2A read-through product and red asterisks identify separated products.
Figure Legend Snippet: (A) Schematic showing overall topology of SARS-CoV-2 spike protein in grey boxes: N-terminal domain (NTD), receptor binding domain (RBD) which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM) and the intracellular domain (IC) (Adapted from Lan et al., 2020) . Dashed lines represent selected regions of the spike protein (coloured boxes with their relative nucleotide positions) that were inserted into the hypervariable region of the SA11 VP4 gene (panel above) and the C-terminus of the RF NSP3 gene (panel below). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2 or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without T2A (yellow box), represented by +/- sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic HDV ribozyme (‘HDV Rib’, green boxes) followed by T7 terminator (T7T). Asterisks (*) represent stop codons. Schematic not to scale. Coupled in vitro transcription and translation reactions of mutated SA11 VP4 (B) and RF NSP3 (C) segments were carried out using the TnT rabbit reticulocyte lysate system supplemented with [ 35 S] methionine. Samples were analysed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In (C) , black asterisks indicate T2A read-through product and red asterisks identify separated products.

Techniques Used: Binding Assay, Sequencing, In Vitro, SDS Page, Autoradiography, Molecular Weight, Marker, Plasmid Preparation, Negative Control

rabbit reticulate lysate tnt quick coupled transcription translation system  (Promega)

 
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    Promega rabbit reticulate lysate tnt quick coupled transcription translation system
    Rabbit Reticulate Lysate Tnt Quick Coupled Transcription Translation System, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit reticulate lysate tnt quick coupled transcription translation system  (Promega)

     
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    Promega rabbit reticulate lysate tnt quick coupled transcription translation system
    Rabbit Reticulate Lysate Tnt Quick Coupled Transcription Translation System, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit reticulate lysate tnt quick coupled transcription translation system  (Promega)

     
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    Promega rabbit reticulate lysate tnt quick coupled transcription translation system
    Rabbit Reticulate Lysate Tnt Quick Coupled Transcription Translation System, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit reticulate lysate tnt quick coupled transcription translation system  (Promega)

     
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    Promega rabbit reticulate lysate tnt quick coupled transcription translation system
    Rabbit Reticulate Lysate Tnt Quick Coupled Transcription Translation System, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega tnt coupled reticulate lysate system
    Tnt Coupled Reticulate Lysate System, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega tnt t7 coupled rabbit reticulate lysate system
    Tnt T7 Coupled Rabbit Reticulate Lysate System, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega tnt coupled reticulate lysate system
    Tnt Coupled Reticulate Lysate System, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega tnt coupled reticulate lysate system
    Tnt Coupled Reticulate Lysate System, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega tnt coupled reticulate lysate system
    Design and validation of rotavirus (RV) VP4 and nonstructural protein 3 (NSP3) plasmid constructs used to generate mutant viruses. (A) Schematic showing overall topology of SARS-CoV-2 spike protein in gray boxes: N-terminal domain (NTD), receptor binding domain (RBD), which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and the intracellular domain (IC) (adapted from Lan et al. ). Dashed lines represent selected regions of the spike protein (colored boxes) that were inserted into the hypervariable region of the SA11 viral protein 4 (VP4) gene (top) and the C terminus of the RF NSP3 gene (bottom). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2, or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without Thosea asigna virus 2A (T2A) (yellow box), represented by +/− sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (“HDV Rib,” green boxes) followed by T7 terminator (T7T). *Stop codons. Schematic not to scale. (B) Ribbon representation of VP4 (adapted from Settembre et al. ). Two orthogonal views are shown. The VP8* fragment is in magenta extending into the VP5* foot domains (in blue), the hypervariable region of VP4 is in gray and the region where SARS-CoV-2 peptides (omitted for clarity) were inserted is in green. VP5* β-barrel domains are in cyan and purple. (C and D) <t>Coupled</t> in vitro transcription and translation reactions of mutated SA11 VP4 (C) and RF NSP3 (D) segments were carried out using the <t>TnT</t> rabbit reticulocyte <t>lysate</t> <t>system</t> supplemented with [ 35 S]methionine. Samples were analyzed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In panel D, black asterisks indicate T2A read-through product and red asterisks identify separated products. WT, wild type.
    Tnt Coupled Reticulate Lysate System, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tnt coupled reticulate lysate system/product/Promega
    Average 86 stars, based on 1 article reviews
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    tnt coupled reticulate lysate system - by Bioz Stars, 2023-02
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    Promega rabbit reticulate lysate tnt quick coupled transcription translation system
    Design and validation of rotavirus (RV) VP4 and nonstructural protein 3 (NSP3) plasmid constructs used to generate mutant viruses. (A) Schematic showing overall topology of SARS-CoV-2 spike protein in gray boxes: N-terminal domain (NTD), receptor binding domain (RBD), which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and the intracellular domain (IC) (adapted from Lan et al. ). Dashed lines represent selected regions of the spike protein (colored boxes) that were inserted into the hypervariable region of the SA11 viral protein 4 (VP4) gene (top) and the C terminus of the RF NSP3 gene (bottom). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2, or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without Thosea asigna virus 2A (T2A) (yellow box), represented by +/− sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (“HDV Rib,” green boxes) followed by T7 terminator (T7T). *Stop codons. Schematic not to scale. (B) Ribbon representation of VP4 (adapted from Settembre et al. ). Two orthogonal views are shown. The VP8* fragment is in magenta extending into the VP5* foot domains (in blue), the hypervariable region of VP4 is in gray and the region where SARS-CoV-2 peptides (omitted for clarity) were inserted is in green. VP5* β-barrel domains are in cyan and purple. (C and D) <t>Coupled</t> in vitro transcription and translation reactions of mutated SA11 VP4 (C) and RF NSP3 (D) segments were carried out using the <t>TnT</t> rabbit reticulocyte <t>lysate</t> <t>system</t> supplemented with [ 35 S]methionine. Samples were analyzed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In panel D, black asterisks indicate T2A read-through product and red asterisks identify separated products. WT, wild type.
    Rabbit Reticulate Lysate Tnt Quick Coupled Transcription Translation System, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit reticulate lysate tnt quick coupled transcription translation system/product/Promega
    Average 86 stars, based on 1 article reviews
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    Promega tnt t7 coupled rabbit reticulate lysate system
    Design and validation of rotavirus (RV) VP4 and nonstructural protein 3 (NSP3) plasmid constructs used to generate mutant viruses. (A) Schematic showing overall topology of SARS-CoV-2 spike protein in gray boxes: N-terminal domain (NTD), receptor binding domain (RBD), which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and the intracellular domain (IC) (adapted from Lan et al. ). Dashed lines represent selected regions of the spike protein (colored boxes) that were inserted into the hypervariable region of the SA11 viral protein 4 (VP4) gene (top) and the C terminus of the RF NSP3 gene (bottom). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2, or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without Thosea asigna virus 2A (T2A) (yellow box), represented by +/− sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (“HDV Rib,” green boxes) followed by T7 terminator (T7T). *Stop codons. Schematic not to scale. (B) Ribbon representation of VP4 (adapted from Settembre et al. ). Two orthogonal views are shown. The VP8* fragment is in magenta extending into the VP5* foot domains (in blue), the hypervariable region of VP4 is in gray and the region where SARS-CoV-2 peptides (omitted for clarity) were inserted is in green. VP5* β-barrel domains are in cyan and purple. (C and D) <t>Coupled</t> in vitro transcription and translation reactions of mutated SA11 VP4 (C) and RF NSP3 (D) segments were carried out using the <t>TnT</t> rabbit reticulocyte <t>lysate</t> <t>system</t> supplemented with [ 35 S]methionine. Samples were analyzed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In panel D, black asterisks indicate T2A read-through product and red asterisks identify separated products. WT, wild type.
    Tnt T7 Coupled Rabbit Reticulate Lysate System, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Design and validation of rotavirus (RV) VP4 and nonstructural protein 3 (NSP3) plasmid constructs used to generate mutant viruses. (A) Schematic showing overall topology of SARS-CoV-2 spike protein in gray boxes: N-terminal domain (NTD), receptor binding domain (RBD), which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and the intracellular domain (IC) (adapted from Lan et al. ). Dashed lines represent selected regions of the spike protein (colored boxes) that were inserted into the hypervariable region of the SA11 viral protein 4 (VP4) gene (top) and the C terminus of the RF NSP3 gene (bottom). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2, or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without Thosea asigna virus 2A (T2A) (yellow box), represented by +/− sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (“HDV Rib,” green boxes) followed by T7 terminator (T7T). *Stop codons. Schematic not to scale. (B) Ribbon representation of VP4 (adapted from Settembre et al. ). Two orthogonal views are shown. The VP8* fragment is in magenta extending into the VP5* foot domains (in blue), the hypervariable region of VP4 is in gray and the region where SARS-CoV-2 peptides (omitted for clarity) were inserted is in green. VP5* β-barrel domains are in cyan and purple. (C and D) Coupled in vitro transcription and translation reactions of mutated SA11 VP4 (C) and RF NSP3 (D) segments were carried out using the TnT rabbit reticulocyte lysate system supplemented with [ 35 S]methionine. Samples were analyzed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In panel D, black asterisks indicate T2A read-through product and red asterisks identify separated products. WT, wild type.

    Journal: Journal of Virology

    Article Title: Using Species a Rotavirus Reverse Genetics to Engineer Chimeric Viruses Expressing SARS-CoV-2 Spike Epitopes

    doi: 10.1128/jvi.00488-22

    Figure Lengend Snippet: Design and validation of rotavirus (RV) VP4 and nonstructural protein 3 (NSP3) plasmid constructs used to generate mutant viruses. (A) Schematic showing overall topology of SARS-CoV-2 spike protein in gray boxes: N-terminal domain (NTD), receptor binding domain (RBD), which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and the intracellular domain (IC) (adapted from Lan et al. ). Dashed lines represent selected regions of the spike protein (colored boxes) that were inserted into the hypervariable region of the SA11 viral protein 4 (VP4) gene (top) and the C terminus of the RF NSP3 gene (bottom). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2, or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without Thosea asigna virus 2A (T2A) (yellow box), represented by +/− sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (“HDV Rib,” green boxes) followed by T7 terminator (T7T). *Stop codons. Schematic not to scale. (B) Ribbon representation of VP4 (adapted from Settembre et al. ). Two orthogonal views are shown. The VP8* fragment is in magenta extending into the VP5* foot domains (in blue), the hypervariable region of VP4 is in gray and the region where SARS-CoV-2 peptides (omitted for clarity) were inserted is in green. VP5* β-barrel domains are in cyan and purple. (C and D) Coupled in vitro transcription and translation reactions of mutated SA11 VP4 (C) and RF NSP3 (D) segments were carried out using the TnT rabbit reticulocyte lysate system supplemented with [ 35 S]methionine. Samples were analyzed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In panel D, black asterisks indicate T2A read-through product and red asterisks identify separated products. WT, wild type.

    Article Snippet: Coupled in vitro transcription and translation reactions were carried out using the Promega TnT Coupled Reticulate Lysate System labeled with radioactive [ 35 S]methionine (PerkinElmer Inc.) according to the manufacturer’s protocol.

    Techniques: Plasmid Preparation, Construct, Mutagenesis, Binding Assay, Sequencing, In Vitro, SDS Page, Autoradiography, Molecular Weight, Marker, Negative Control