vls cassette region  (New England Biolabs)


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    Structured Review

    New England Biolabs vls cassette region
    Vls Cassette Region, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vls cassette region/product/New England Biolabs
    Average 78 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    vls cassette region - by Bioz Stars, 2020-01
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    Related Products / Commonly Used Together

    bigeasy v2.0 linear cloning kit
    genomic dna
    drai

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    Related Articles

    Clone Assay:

    Article Title: Natural Selection Promotes Antigenic Evolvability
    Article Snippet: .. The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit (Lucigen; Middleton, WI) by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector (isolates cN40, 36B, and 39B). .. Long-range PCR amplification was conducted using the primers N40-vlsLR-R (5′ Phos - GCT GGA CTT GAA TTT GGT AGG GAT TC 3′ ) and N40-vlsLR-F (5′ Phos - GGT GAT GGT GCC GAT TCA AAA TCT GG 3′ ) which anneal to the unique conserved regions flanking the unexpressed cassettes.

    Amplification:

    Article Title: Natural Selection Promotes Antigenic Evolvability
    Article Snippet: The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit (Lucigen; Middleton, WI) by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector (isolates cN40, 36B, and 39B). .. Long-range PCR amplification was conducted using the primers N40-vlsLR-R (5′ Phos - GCT GGA CTT GAA TTT GGT AGG GAT TC 3′ ) and N40-vlsLR-F (5′ Phos - GGT GAT GGT GCC GAT TCA AAA TCT GG 3′ ) which anneal to the unique conserved regions flanking the unexpressed cassettes.

    Isolation:

    Article Title: Natural Selection Promotes Antigenic Evolvability
    Article Snippet: The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit (Lucigen; Middleton, WI) by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector (isolates cN40, 36B, and 39B). .. BigEasy vectors containing an intact cassette region were amplified in E.coli and isolated using a Qiagen Mini-prep kit.

    Mouse Assay:

    Article Title: Natural Selection Promotes Antigenic Evolvability
    Article Snippet: Experimental evolution A clonal isolate of B. burgdorferi strain N40 was intradermally inoculated into three C3H/HeN mice and re-isolated after 12 months from the blood (derived isolate 1(36B), derived isolate 2(44B), and derived isolate 3(39B)) as previously described . .. The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit (Lucigen; Middleton, WI) by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector (isolates cN40, 36B, and 39B).

    Derivative Assay:

    Article Title: Natural Selection Promotes Antigenic Evolvability
    Article Snippet: .. The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit (Lucigen; Middleton, WI) by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector (isolates cN40, 36B, and 39B). .. Long-range PCR amplification was conducted using the primers N40-vlsLR-R (5′ Phos - GCT GGA CTT GAA TTT GGT AGG GAT TC 3′ ) and N40-vlsLR-F (5′ Phos - GGT GAT GGT GCC GAT TCA AAA TCT GG 3′ ) which anneal to the unique conserved regions flanking the unexpressed cassettes.

    Polymerase Chain Reaction:

    Article Title: Natural Selection Promotes Antigenic Evolvability
    Article Snippet: .. The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit (Lucigen; Middleton, WI) by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector (isolates cN40, 36B, and 39B). .. Long-range PCR amplification was conducted using the primers N40-vlsLR-R (5′ Phos - GCT GGA CTT GAA TTT GGT AGG GAT TC 3′ ) and N40-vlsLR-F (5′ Phos - GGT GAT GGT GCC GAT TCA AAA TCT GG 3′ ) which anneal to the unique conserved regions flanking the unexpressed cassettes.

    Purification:

    Article Title: Natural Selection Promotes Antigenic Evolvability
    Article Snippet: The clonal N40 parent isolate (cN40) and each of the derived isolates were grown from frozen stocks at 34°C in BSK-H medium supplemented with 6% rabbit serum (Sigma Aldrich) to a density of ∼5*107 cells/ml and the genomic DNA was purified using the DNeasy Blood and Tissue Kit protocol for gram negative bacteria (Qiagen; Valencia, CA). .. The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit (Lucigen; Middleton, WI) by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector (isolates cN40, 36B, and 39B).

    Plasmid Preparation:

    Article Title: Natural Selection Promotes Antigenic Evolvability
    Article Snippet: .. The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit (Lucigen; Middleton, WI) by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector (isolates cN40, 36B, and 39B). .. Long-range PCR amplification was conducted using the primers N40-vlsLR-R (5′ Phos - GCT GGA CTT GAA TTT GGT AGG GAT TC 3′ ) and N40-vlsLR-F (5′ Phos - GGT GAT GGT GCC GAT TCA AAA TCT GG 3′ ) which anneal to the unique conserved regions flanking the unexpressed cassettes.

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    New England Biolabs a13 vl
    Competition ELISA for comparison of relative affinities of anti-EGFR IgGs. ER414 is a fully human IgG1 originated from the mAb <t>A13</t> by guided panning. Binding inhibition of anti-EGFR IgGs to EGFR was analyzed on EGFR-coated plates with different concentrations
    A13 Vl, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/a13 vl/product/New England Biolabs
    Average 78 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    77
    New England Biolabs vl damaged dna
    Repair of UVC-induced CPDs from the 3-kb <t>EcoRI</t> fragment of the Ad-encoded lacZ gene in human and rodent cells measured by loss of T4pdg-sensitive sites. ( A ) Southern blot analysis of the repair of UVC-induced CPDs in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad <t>DNA,</t> while lanes 3 and 4 contain Ad DNA exposed to 240 J/m 2 UVC. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with T4pdg (lanes 1 and 3) represent ssDNA breaks generated by other sources. Following UVC exposure, a large number of T4pdg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, NER removes CPDs resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as CPDs persist in the lacZ DNA, T4pdg will induce ssDNA breaks resulting in less full-length signal compared to the control. ( B ) Quantification of the percent removal of CPDs from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents an arithmetic mean ± SE of the percent removal of UVC-induced T4pdg-sensitive sites from three independent experiments. A significant increase in the percent removal of UVC-induced T4pdg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of T4pdg-sensitive sites was observed between GM637F and CHO-AA8 24 h (indicated by a cross/plus sign).
    Vl Damaged Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    70
    New England Biolabs hel23 vl gene
    Affinity maturation by PnP screening of a random mutagenesis library. (a) Schematic shows the workflow to generate a random mutagenesis library by error-prone (EP) PCR of the V H region of <t>HEL23,</t> expression in hybridoma cells and screening for affinity maturation. (b) Mutation rate varies based on amount of template used in the EP-PCR reaction. 100 ng of plasmid template produced the highest mutation rate and was selected for cloning of final library. (c) Flow cytometry dot plots show the frequency of antibody-expressing cells following transfection with the wild-type HEL23 plasmid (left) and the random mutagenesis library (right). (d) Flow cytometry dot plots show library screening and enrichment. From left to right: control transfection (wild-type HEL23) after enrichment of all antibody-expressing cells; EP-PCR library after enrichment of all antibody-expressing cells, but prior to any antigen enrichment; outputs of the first and second rounds of antigen enrichment. Each of the three screening steps of the EP-PCR library is shown at two different antigen staining concentrations to highlight affinity maturation. Representative flow cytometry gates do not correspond to actual sorting gates but are drawn to illustrate successful enrichment.
    Hel23 Vl Gene, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 70/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    New England Biolabs vl pcr
    One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and <t>PCR</t> amplicons contain or are flanked by <t>Bsa</t> I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping
    Vl Pcr, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Competition ELISA for comparison of relative affinities of anti-EGFR IgGs. ER414 is a fully human IgG1 originated from the mAb A13 by guided panning. Binding inhibition of anti-EGFR IgGs to EGFR was analyzed on EGFR-coated plates with different concentrations

    Journal: Experimental & Molecular Medicine

    Article Title: Conversion of a murine monoclonal antibody A13 targeting epidermal growth factor receptor to a human monoclonal antibody by guided selection

    doi: 10.3858/emm.2012.44.1.005

    Figure Lengend Snippet: Competition ELISA for comparison of relative affinities of anti-EGFR IgGs. ER414 is a fully human IgG1 originated from the mAb A13 by guided panning. Binding inhibition of anti-EGFR IgGs to EGFR was analyzed on EGFR-coated plates with different concentrations

    Article Snippet: Again the PCR DNAs of A13 VH and A13 VL were amplified with primers H-extension(F)/H-extension(R) and K-extension(F)/K-extension(R) ( ) and inserted to pSC73His vector ( ) by digestion with Sfi I/BstE II (New England Bio Labs, Beverly, MA) for VH and BstE II/Not I (New England Bio Lab) for VL , which was designated as pSC73His-13VH -13VL .

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Inhibition

    Competition ELISA to analyze whether the ER414 has the same binding site with the murine counterpart A13 mAb. The competition was performed on the EGFR-coated plates with 1 µg of biotinylated ER414 and different concentrations of free ER414, A13

    Journal: Experimental & Molecular Medicine

    Article Title: Conversion of a murine monoclonal antibody A13 targeting epidermal growth factor receptor to a human monoclonal antibody by guided selection

    doi: 10.3858/emm.2012.44.1.005

    Figure Lengend Snippet: Competition ELISA to analyze whether the ER414 has the same binding site with the murine counterpart A13 mAb. The competition was performed on the EGFR-coated plates with 1 µg of biotinylated ER414 and different concentrations of free ER414, A13

    Article Snippet: Again the PCR DNAs of A13 VH and A13 VL were amplified with primers H-extension(F)/H-extension(R) and K-extension(F)/K-extension(R) ( ) and inserted to pSC73His vector ( ) by digestion with Sfi I/BstE II (New England Bio Labs, Beverly, MA) for VH and BstE II/Not I (New England Bio Lab) for VL , which was designated as pSC73His-13VH -13VL .

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

    Competition ELISA for measuring relative affinities of anti-EGFR human scFvs which were isolated by guided panning of mAb A13. The inhibition of binding of anti-EGFR human scFvs to EGFR was analyzed on EGFR-coated plates with different concentrations

    Journal: Experimental & Molecular Medicine

    Article Title: Conversion of a murine monoclonal antibody A13 targeting epidermal growth factor receptor to a human monoclonal antibody by guided selection

    doi: 10.3858/emm.2012.44.1.005

    Figure Lengend Snippet: Competition ELISA for measuring relative affinities of anti-EGFR human scFvs which were isolated by guided panning of mAb A13. The inhibition of binding of anti-EGFR human scFvs to EGFR was analyzed on EGFR-coated plates with different concentrations

    Article Snippet: Again the PCR DNAs of A13 VH and A13 VL were amplified with primers H-extension(F)/H-extension(R) and K-extension(F)/K-extension(R) ( ) and inserted to pSC73His vector ( ) by digestion with Sfi I/BstE II (New England Bio Labs, Beverly, MA) for VH and BstE II/Not I (New England Bio Lab) for VL , which was designated as pSC73His-13VH -13VL .

    Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Inhibition, Binding Assay

    FACS analysis for binding activity of anti-EGFR human scFvs to cell surface-expressed EGFR, in which the scFvs were isolated by guided panning of mAb A13. Differential binding of scFvs to EGFR-positive A431 tumor cells and EGFR-negative HL60 cells (indicated

    Journal: Experimental & Molecular Medicine

    Article Title: Conversion of a murine monoclonal antibody A13 targeting epidermal growth factor receptor to a human monoclonal antibody by guided selection

    doi: 10.3858/emm.2012.44.1.005

    Figure Lengend Snippet: FACS analysis for binding activity of anti-EGFR human scFvs to cell surface-expressed EGFR, in which the scFvs were isolated by guided panning of mAb A13. Differential binding of scFvs to EGFR-positive A431 tumor cells and EGFR-negative HL60 cells (indicated

    Article Snippet: Again the PCR DNAs of A13 VH and A13 VL were amplified with primers H-extension(F)/H-extension(R) and K-extension(F)/K-extension(R) ( ) and inserted to pSC73His vector ( ) by digestion with Sfi I/BstE II (New England Bio Labs, Beverly, MA) for VH and BstE II/Not I (New England Bio Lab) for VL , which was designated as pSC73His-13VH -13VL .

    Techniques: FACS, Binding Assay, Activity Assay, Isolation

    Repair of UVC-induced CPDs from the 3-kb EcoRI fragment of the Ad-encoded lacZ gene in human and rodent cells measured by loss of T4pdg-sensitive sites. ( A ) Southern blot analysis of the repair of UVC-induced CPDs in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 240 J/m 2 UVC. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with T4pdg (lanes 1 and 3) represent ssDNA breaks generated by other sources. Following UVC exposure, a large number of T4pdg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, NER removes CPDs resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as CPDs persist in the lacZ DNA, T4pdg will induce ssDNA breaks resulting in less full-length signal compared to the control. ( B ) Quantification of the percent removal of CPDs from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents an arithmetic mean ± SE of the percent removal of UVC-induced T4pdg-sensitive sites from three independent experiments. A significant increase in the percent removal of UVC-induced T4pdg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of T4pdg-sensitive sites was observed between GM637F and CHO-AA8 24 h (indicated by a cross/plus sign).

    Journal: Mutagenesis

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine

    doi: 10.1093/mutage/get027

    Figure Lengend Snippet: Repair of UVC-induced CPDs from the 3-kb EcoRI fragment of the Ad-encoded lacZ gene in human and rodent cells measured by loss of T4pdg-sensitive sites. ( A ) Southern blot analysis of the repair of UVC-induced CPDs in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 240 J/m 2 UVC. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with T4pdg (lanes 1 and 3) represent ssDNA breaks generated by other sources. Following UVC exposure, a large number of T4pdg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, NER removes CPDs resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as CPDs persist in the lacZ DNA, T4pdg will induce ssDNA breaks resulting in less full-length signal compared to the control. ( B ) Quantification of the percent removal of CPDs from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents an arithmetic mean ± SE of the percent removal of UVC-induced T4pdg-sensitive sites from three independent experiments. A significant increase in the percent removal of UVC-induced T4pdg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of T4pdg-sensitive sites was observed between GM637F and CHO-AA8 24 h (indicated by a cross/plus sign).

    Article Snippet: For endonuclease treatment of MB + VL-damaged DNA, the EcoRI-digested samples were divided in half and incubated with or without 16 units of Fpg (1× NEB buffer 1, 100 µg/ml BSA) for 5 h. Following incubation, the samples were loaded directly into a 0.8% alkaline agarose gel and separated by electrophoresis (20–24 h at 10 V).

    Techniques: Southern Blot, Generated, Incubation

    Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of Fpg-sensitive sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).

    Journal: Mutagenesis

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine

    doi: 10.1093/mutage/get027

    Figure Lengend Snippet: Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of Fpg-sensitive sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).

    Article Snippet: For endonuclease treatment of MB + VL-damaged DNA, the EcoRI-digested samples were divided in half and incubated with or without 16 units of Fpg (1× NEB buffer 1, 100 µg/ml BSA) for 5 h. Following incubation, the samples were loaded directly into a 0.8% alkaline agarose gel and separated by electrophoresis (20–24 h at 10 V).

    Techniques: Southern Blot, Incubation, Generated

    Affinity maturation by PnP screening of a random mutagenesis library. (a) Schematic shows the workflow to generate a random mutagenesis library by error-prone (EP) PCR of the V H region of HEL23, expression in hybridoma cells and screening for affinity maturation. (b) Mutation rate varies based on amount of template used in the EP-PCR reaction. 100 ng of plasmid template produced the highest mutation rate and was selected for cloning of final library. (c) Flow cytometry dot plots show the frequency of antibody-expressing cells following transfection with the wild-type HEL23 plasmid (left) and the random mutagenesis library (right). (d) Flow cytometry dot plots show library screening and enrichment. From left to right: control transfection (wild-type HEL23) after enrichment of all antibody-expressing cells; EP-PCR library after enrichment of all antibody-expressing cells, but prior to any antigen enrichment; outputs of the first and second rounds of antigen enrichment. Each of the three screening steps of the EP-PCR library is shown at two different antigen staining concentrations to highlight affinity maturation. Representative flow cytometry gates do not correspond to actual sorting gates but are drawn to illustrate successful enrichment.

    Journal: mAbs

    Article Title: Antibody discovery and engineering by enhanced CRISPR-Cas9 integration of variable gene cassette libraries in mammalian cells

    doi: 10.1080/19420862.2019.1662691

    Figure Lengend Snippet: Affinity maturation by PnP screening of a random mutagenesis library. (a) Schematic shows the workflow to generate a random mutagenesis library by error-prone (EP) PCR of the V H region of HEL23, expression in hybridoma cells and screening for affinity maturation. (b) Mutation rate varies based on amount of template used in the EP-PCR reaction. 100 ng of plasmid template produced the highest mutation rate and was selected for cloning of final library. (c) Flow cytometry dot plots show the frequency of antibody-expressing cells following transfection with the wild-type HEL23 plasmid (left) and the random mutagenesis library (right). (d) Flow cytometry dot plots show library screening and enrichment. From left to right: control transfection (wild-type HEL23) after enrichment of all antibody-expressing cells; EP-PCR library after enrichment of all antibody-expressing cells, but prior to any antigen enrichment; outputs of the first and second rounds of antigen enrichment. Each of the three screening steps of the EP-PCR library is shown at two different antigen staining concentrations to highlight affinity maturation. Representative flow cytometry gates do not correspond to actual sorting gates but are drawn to illustrate successful enrichment.

    Article Snippet: In order to obtain the library scaffold plasmid pPnP-lin5ʹ-AarI, two consecutive stop-codons were inserted into the beginning of the HEL23 VL gene in order to prevent background expression and an AarI restriction site was cloned in place of the VH gene in the HEL23 pPnP-lin5ʹ plasmid using the Gibson Assembly® Master Mix (NEB, E2611S).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Expressing, Plasmid Preparation, Produced, Clone Assay, Flow Cytometry, Cytometry, Transfection, Library Screening, Staining

    Characterization of antibody variants selected for affinity maturation. (a) The table shows the coding mutations of each of the five unique clones (HEL23v1-5) retrieved after single-cell sorting of the antigen-enriched libraries. (b) Flow cytometry dot plots show the surface expression and HEL-binding profile of the five HEL23 variants (green) and wild-type HEL23 (black). The ratio of HEL over antibody signal for each clone is shown in Figure S5a. (c) Supernatant ELISA comparing HEL binding for the five isolated variants (green) with wild-type HEL23 (black). Supernatants were adjusted to equal IgG-concentration (Fig. S5b). Two technical replicates were included for each of the mutated variants and one for the controls (PnP-HEL23 and PnP-mRuby-Cas9), and a five-parameter logistical curve was fitted to the data by nonlinear regression. For each data point, the mean is represented and the error bars indicate standard deviation. PnP-mRuby-Cas9 cell supernatant was used as negative control. (d) Affinity values of wild-type HEL23 and the five isolated variants obtained by bio-layer interferometry (BLI). The curves and fitting values are reported in Figure S6. (e) Heatmap shows enrichment of all possible substitutions for each WT residue (x-axis) in the HEL + library compared to IgG + library in a log2-fold change scale. Red substitutions are enriched in the HEL + library, while blue substitutions are depleted. White squares (log2 ratio = 0) indicate neutral substitutions not impacted by enrichment, or residues in which the mutation rate is negligible. The mutation rate for each position was calculated by summing the mutation frequency of all replicates (n = 3) for each condition. Key residues found in affinity-matured clones are indicated with black boxes.

    Journal: mAbs

    Article Title: Antibody discovery and engineering by enhanced CRISPR-Cas9 integration of variable gene cassette libraries in mammalian cells

    doi: 10.1080/19420862.2019.1662691

    Figure Lengend Snippet: Characterization of antibody variants selected for affinity maturation. (a) The table shows the coding mutations of each of the five unique clones (HEL23v1-5) retrieved after single-cell sorting of the antigen-enriched libraries. (b) Flow cytometry dot plots show the surface expression and HEL-binding profile of the five HEL23 variants (green) and wild-type HEL23 (black). The ratio of HEL over antibody signal for each clone is shown in Figure S5a. (c) Supernatant ELISA comparing HEL binding for the five isolated variants (green) with wild-type HEL23 (black). Supernatants were adjusted to equal IgG-concentration (Fig. S5b). Two technical replicates were included for each of the mutated variants and one for the controls (PnP-HEL23 and PnP-mRuby-Cas9), and a five-parameter logistical curve was fitted to the data by nonlinear regression. For each data point, the mean is represented and the error bars indicate standard deviation. PnP-mRuby-Cas9 cell supernatant was used as negative control. (d) Affinity values of wild-type HEL23 and the five isolated variants obtained by bio-layer interferometry (BLI). The curves and fitting values are reported in Figure S6. (e) Heatmap shows enrichment of all possible substitutions for each WT residue (x-axis) in the HEL + library compared to IgG + library in a log2-fold change scale. Red substitutions are enriched in the HEL + library, while blue substitutions are depleted. White squares (log2 ratio = 0) indicate neutral substitutions not impacted by enrichment, or residues in which the mutation rate is negligible. The mutation rate for each position was calculated by summing the mutation frequency of all replicates (n = 3) for each condition. Key residues found in affinity-matured clones are indicated with black boxes.

    Article Snippet: In order to obtain the library scaffold plasmid pPnP-lin5ʹ-AarI, two consecutive stop-codons were inserted into the beginning of the HEL23 VL gene in order to prevent background expression and an AarI restriction site was cloned in place of the VH gene in the HEL23 pPnP-lin5ʹ plasmid using the Gibson Assembly® Master Mix (NEB, E2611S).

    Techniques: Clone Assay, FACS, Flow Cytometry, Cytometry, Expressing, Binding Assay, Enzyme-linked Immunosorbent Assay, Isolation, Concentration Assay, Standard Deviation, Negative Control, Mutagenesis

    One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping

    Journal: Microbial Cell Factories

    Article Title: A novel one-step approach for the construction of yeast surface display Fab antibody libraries

    doi: 10.1186/s12934-017-0853-z

    Figure Lengend Snippet: One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping

    Article Snippet: 160 ng of VH PCR product and 160 ng VL PCR product as well as 200 U Bsa I (New England Biolabs), 800 U T4 DNA ligase (New England Biolabs) and 10 µL 10× T4 Ligase buffer (New England Biolabs).

    Techniques: Clone Assay, Polymerase Chain Reaction, Expressing, Sequencing, Generated, Plasmid Preparation