nebuilder  (New England Biolabs)


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    New England Biolabs nebuilder
    Nebuilder, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nebuilder hifi assembly
    Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by <t>NEBuilder</t> <t>HiFi</t> assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.
    Nebuilder Hifi Assembly, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs nebuilder hifi dna assembly cloning kit
    Construction of the EVG 08/NC_USA/2015 infectious clone and rescue of recombinant viruses. (A) Strategy for assembling the full-length cDNA infectious clone of EVG 08/NC_USA/2015. Two RT-PCR-amplified genomic fragments and a synthesized HDV ribozyme gene were assembled into the pACYC177 vector by using the <t>NEBuilder</t> <t>HiFi</t> <t>DNA</t> assembly method. The CMV promoter sequence was inserted at the 5′ end of the genome. (B) Schematic diagram of the PLP knockout mutant (vPLP-KO). (C) Immunofluorescence detection of recombinant viruses rescued from full-length cDNA clones. BHK-21C cells were initially transfected with plasmid DNA of the full-length cDNA clone pEVG or its mutant. ST cells were subsequently infected with the cloned viruses rescued from BHK cells. The expression of ToV-PLP and the structural protein VP1 was detected by specific MAb 128-28 and MAb 115-5, respectively. The cell nucleus is stained with DAPI. (D) Western blot detection of PLP expression in recombinant virus-infected ST cells. The membrane was probed with ToV-PLP-specific MAb 128-28. The expression of the GAPDH housekeeping gene was detected as a loading control.
    Nebuilder Hifi Dna Assembly Cloning Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs pbelo sars cov 2
    Dose response of the <t>recSARS-CoV-2</t> reporter viruses to RDV and GC376. CaCo-2 cells were infected with the clinical isolate, the recSARS-CoV-2 WT, the N-3xFLAG marker virus, fluorescence reporter viruses expressing YFP instead of ORF6 (d6-YFP) or between ORF8 and N (after8-YFP), and the luciferase reporter virus d7-GLuc, which expresses gaussia luciferase instead of ORF7a (MOI = 0.005). At the same time, the cells were treated with 2-fold serial dilutions of remdesivir (RDV) ( A ) or GC376 ( B ). The cells were harvested at 30 hpi, and viral replication was determined by the quantification of spike expression (immunostaining with mAb against spikes), fluorescence reporter, or luciferase expression, respectively. The percentage of viral replication was determined relative to the solvent-treated cells. The 50% effective concentration (EC 50 ) was assessed by nonlinear four-parameter curve fitting using GraphPad Prism and are shown within the respective graphs. Mean values of quadruplicates relative to the solvent-treated cells ± SD are depicted.
    Pbelo Sars Cov 2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs hifi assembly master mix
    Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by <t>NEBuilder</t> <t>HiFi</t> assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.
    Hifi Assembly Master Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Article Snippet: In contrast to Golden Gate and derived methods, overlap-depended assembly methods, such as circular polymerase extension cloning (CPEC) [ ], uracil-specific excision reagent cloning (USER) [ ], Gibson assembly [ ], NeBuilder HiFi assembly (NEB), sequence and ligation independent cloning (SLIC) [ ], transformation-associated recombination (TAR) cloning [ ] and seamless ligation cloning extract (SLiCE) [ ], are sequence independent and therefore convenient and efficient when performing multigene cloning (see review [ ]).

    Techniques: Clone Assay, Expressing, Amplification, Plasmid Preparation, Polymerase Chain Reaction, Construct, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay

    Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with Bam HI and Hin dIII to obtain pCAMBIA_ASX. (B) T35S–AttR2– ccd B–AttR1 cassette was released from the Gateway plasmid backbone by digestion with Xba I and Sac I and replaced with a I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Spec: selection marker conferring spectinomycin resistance in E . coli and A . tumefaciens , Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, Hin dIII, I- Sce I, Bam HI, Xba I, Sac I: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with Bam HI and Hin dIII to obtain pCAMBIA_ASX. (B) T35S–AttR2– ccd B–AttR1 cassette was released from the Gateway plasmid backbone by digestion with Xba I and Sac I and replaced with a I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Spec: selection marker conferring spectinomycin resistance in E . coli and A . tumefaciens , Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, Hin dIII, I- Sce I, Bam HI, Xba I, Sac I: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method.

    Article Snippet: In contrast to Golden Gate and derived methods, overlap-depended assembly methods, such as circular polymerase extension cloning (CPEC) [ ], uracil-specific excision reagent cloning (USER) [ ], Gibson assembly [ ], NeBuilder HiFi assembly (NEB), sequence and ligation independent cloning (SLIC) [ ], transformation-associated recombination (TAR) cloning [ ] and seamless ligation cloning extract (SLiCE) [ ], are sequence independent and therefore convenient and efficient when performing multigene cloning (see review [ ]).

    Techniques: Expressing, Plasmid Preparation, Clone Assay, Selection, Marker, Ligation

    Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Article Snippet: In contrast to Golden Gate and derived methods, overlap-depended assembly methods, such as circular polymerase extension cloning (CPEC) [ ], uracil-specific excision reagent cloning (USER) [ ], Gibson assembly [ ], NeBuilder HiFi assembly (NEB), sequence and ligation independent cloning (SLIC) [ ], transformation-associated recombination (TAR) cloning [ ] and seamless ligation cloning extract (SLiCE) [ ], are sequence independent and therefore convenient and efficient when performing multigene cloning (see review [ ]).

    Techniques: Functional Assay, Construct, Clone Assay, Expressing, Amplification, Plasmid Preparation, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay, Polymerase Chain Reaction

    Construction of the EVG 08/NC_USA/2015 infectious clone and rescue of recombinant viruses. (A) Strategy for assembling the full-length cDNA infectious clone of EVG 08/NC_USA/2015. Two RT-PCR-amplified genomic fragments and a synthesized HDV ribozyme gene were assembled into the pACYC177 vector by using the NEBuilder HiFi DNA assembly method. The CMV promoter sequence was inserted at the 5′ end of the genome. (B) Schematic diagram of the PLP knockout mutant (vPLP-KO). (C) Immunofluorescence detection of recombinant viruses rescued from full-length cDNA clones. BHK-21C cells were initially transfected with plasmid DNA of the full-length cDNA clone pEVG or its mutant. ST cells were subsequently infected with the cloned viruses rescued from BHK cells. The expression of ToV-PLP and the structural protein VP1 was detected by specific MAb 128-28 and MAb 115-5, respectively. The cell nucleus is stained with DAPI. (D) Western blot detection of PLP expression in recombinant virus-infected ST cells. The membrane was probed with ToV-PLP-specific MAb 128-28. The expression of the GAPDH housekeeping gene was detected as a loading control.

    Journal: Journal of Virology

    Article Title: A Naturally Occurring Recombinant Enterovirus Expresses a Torovirus Deubiquitinase

    doi: 10.1128/JVI.00450-17

    Figure Lengend Snippet: Construction of the EVG 08/NC_USA/2015 infectious clone and rescue of recombinant viruses. (A) Strategy for assembling the full-length cDNA infectious clone of EVG 08/NC_USA/2015. Two RT-PCR-amplified genomic fragments and a synthesized HDV ribozyme gene were assembled into the pACYC177 vector by using the NEBuilder HiFi DNA assembly method. The CMV promoter sequence was inserted at the 5′ end of the genome. (B) Schematic diagram of the PLP knockout mutant (vPLP-KO). (C) Immunofluorescence detection of recombinant viruses rescued from full-length cDNA clones. BHK-21C cells were initially transfected with plasmid DNA of the full-length cDNA clone pEVG or its mutant. ST cells were subsequently infected with the cloned viruses rescued from BHK cells. The expression of ToV-PLP and the structural protein VP1 was detected by specific MAb 128-28 and MAb 115-5, respectively. The cell nucleus is stained with DAPI. (D) Western blot detection of PLP expression in recombinant virus-infected ST cells. The membrane was probed with ToV-PLP-specific MAb 128-28. The expression of the GAPDH housekeeping gene was detected as a loading control.

    Article Snippet: The assembly of these DNA fragments was performed by using a NEBuilder HiFi DNA Assembly cloning kit (New England BioLabs, Ipswich, MA).

    Techniques: Recombinant, Reverse Transcription Polymerase Chain Reaction, Amplification, Synthesized, Plasmid Preparation, Sequencing, Plasmid Purification, Knock-Out, Mutagenesis, Immunofluorescence, Clone Assay, Transfection, Infection, Expressing, Staining, Western Blot

    Dose response of the recSARS-CoV-2 reporter viruses to RDV and GC376. CaCo-2 cells were infected with the clinical isolate, the recSARS-CoV-2 WT, the N-3xFLAG marker virus, fluorescence reporter viruses expressing YFP instead of ORF6 (d6-YFP) or between ORF8 and N (after8-YFP), and the luciferase reporter virus d7-GLuc, which expresses gaussia luciferase instead of ORF7a (MOI = 0.005). At the same time, the cells were treated with 2-fold serial dilutions of remdesivir (RDV) ( A ) or GC376 ( B ). The cells were harvested at 30 hpi, and viral replication was determined by the quantification of spike expression (immunostaining with mAb against spikes), fluorescence reporter, or luciferase expression, respectively. The percentage of viral replication was determined relative to the solvent-treated cells. The 50% effective concentration (EC 50 ) was assessed by nonlinear four-parameter curve fitting using GraphPad Prism and are shown within the respective graphs. Mean values of quadruplicates relative to the solvent-treated cells ± SD are depicted.

    Journal: International Journal of Molecular Sciences

    Article Title: Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination

    doi: 10.3390/ijms221910188

    Figure Lengend Snippet: Dose response of the recSARS-CoV-2 reporter viruses to RDV and GC376. CaCo-2 cells were infected with the clinical isolate, the recSARS-CoV-2 WT, the N-3xFLAG marker virus, fluorescence reporter viruses expressing YFP instead of ORF6 (d6-YFP) or between ORF8 and N (after8-YFP), and the luciferase reporter virus d7-GLuc, which expresses gaussia luciferase instead of ORF7a (MOI = 0.005). At the same time, the cells were treated with 2-fold serial dilutions of remdesivir (RDV) ( A ) or GC376 ( B ). The cells were harvested at 30 hpi, and viral replication was determined by the quantification of spike expression (immunostaining with mAb against spikes), fluorescence reporter, or luciferase expression, respectively. The percentage of viral replication was determined relative to the solvent-treated cells. The 50% effective concentration (EC 50 ) was assessed by nonlinear four-parameter curve fitting using GraphPad Prism and are shown within the respective graphs. Mean values of quadruplicates relative to the solvent-treated cells ± SD are depicted.

    Article Snippet: The four fragments covering the SARS-CoV-2 genome were then assembled with the Pac I-digested pBeloCoV vector according to the NEBuilder HiFi DNA Assembly Cloning Kit protocol (E5520, NEB) to obtain pBelo-SARS-CoV-2 (pBSCoV2).

    Techniques: Infection, Marker, Fluorescence, Expressing, Luciferase, Immunostaining, Concentration Assay

    Rescue of recSARS-CoV-2. ( A ) Western blot analysis of HEK293T cells stably expressing either T7 RNA polymerase (T7RNAP) and the viral nucleocapsid protein (N) or the ACE2 receptor upon lentiviral transduction. ( B ) Illustration of the approach followed to rescue recSARS-CoV-2. A coculture of HEK293T-ACE2 and HEK293T-T7RNAP/N cells was transfected with pBSCoV2. After three days, the supernatant was transferred on CaCo-2 or Vero E6 cells to obtain the first passage (P1) virus stocks. P1 stocks were used to infect CaCo-2 and Vero E6 cells for the P2 working stocks, respectively. The virus titers were determined by TCID 50 titration. ( C ) Visualization of the cytopathic effect (CPE) in recSARS-CoV-2 infected CaCo-2 and Vero E6 cells three days after infection. Mock-infected cells are shown as the control. ( D ) Growth kinetics of a SARS-CoV-2 clinical isolate compared to recSARS-CoV-2. CaCo-2 cells were infected with the clinical isolate or the recombinant SARS-CoV-2 virus at an MOI of 0.005. Cell culture supernatants were collected at the indicated time points post-infection, and viral copies were determined by RT-qPCR targeting the viral polymerase RdRp. Data are presented as the means of triplicates ± SD.

    Journal: International Journal of Molecular Sciences

    Article Title: Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination

    doi: 10.3390/ijms221910188

    Figure Lengend Snippet: Rescue of recSARS-CoV-2. ( A ) Western blot analysis of HEK293T cells stably expressing either T7 RNA polymerase (T7RNAP) and the viral nucleocapsid protein (N) or the ACE2 receptor upon lentiviral transduction. ( B ) Illustration of the approach followed to rescue recSARS-CoV-2. A coculture of HEK293T-ACE2 and HEK293T-T7RNAP/N cells was transfected with pBSCoV2. After three days, the supernatant was transferred on CaCo-2 or Vero E6 cells to obtain the first passage (P1) virus stocks. P1 stocks were used to infect CaCo-2 and Vero E6 cells for the P2 working stocks, respectively. The virus titers were determined by TCID 50 titration. ( C ) Visualization of the cytopathic effect (CPE) in recSARS-CoV-2 infected CaCo-2 and Vero E6 cells three days after infection. Mock-infected cells are shown as the control. ( D ) Growth kinetics of a SARS-CoV-2 clinical isolate compared to recSARS-CoV-2. CaCo-2 cells were infected with the clinical isolate or the recombinant SARS-CoV-2 virus at an MOI of 0.005. Cell culture supernatants were collected at the indicated time points post-infection, and viral copies were determined by RT-qPCR targeting the viral polymerase RdRp. Data are presented as the means of triplicates ± SD.

    Article Snippet: The four fragments covering the SARS-CoV-2 genome were then assembled with the Pac I-digested pBeloCoV vector according to the NEBuilder HiFi DNA Assembly Cloning Kit protocol (E5520, NEB) to obtain pBelo-SARS-CoV-2 (pBSCoV2).

    Techniques: Western Blot, Stable Transfection, Expressing, Transduction, Transfection, Titration, Infection, Recombinant, Cell Culture, Quantitative RT-PCR

    Visualization of the incoming SARS-CoV-2 nucleocapsids and newly synthesized viral RNA. ( A , B ) Vero E6 cells were infected with a recSARS-CoV-2 marker virus expressing a 3x-FLAG-tagged nucleoprotein (N-3xFLAG) under high MOI conditions (MOI~5). After one hour, the virus inoculum was replaced by a cell culture medium containing 20-µg/mL actinomycin D to inhibit cellular transcription. One hour later, 5-ethynyl uridine (5-EU) at a final concentration of 1 mM was added to the cells. The cells were fixed at the indicated time points post-infection, blocked, and permeabilized. A copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction was performed with Alexa 488-conjugated picolyl azide to label a newly synthesized viral RNA (green). Afterwards, the cells were stained with antibodies against FLAG-tag to detect N-3xFLAG (red) and dsRNA to visualize the viral replication intermediates (cyan). Hoechst 33342 was used for nuclear staining (blue). The cells were analyzed by confocal laser scanning microscopy. Representative Z stacks and overlays are shown. ( B ) Enlargement of the overlapping signals (white dotted square in ( A ), 8 hpi). White arrows indicate overlapping signals for N-3xFLAG and dsRNA (visible as a white signal), white asterisks (*) imply merging signals of N-3xFLAG and dsRNA (visible as a yellow signal).

    Journal: International Journal of Molecular Sciences

    Article Title: Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination

    doi: 10.3390/ijms221910188

    Figure Lengend Snippet: Visualization of the incoming SARS-CoV-2 nucleocapsids and newly synthesized viral RNA. ( A , B ) Vero E6 cells were infected with a recSARS-CoV-2 marker virus expressing a 3x-FLAG-tagged nucleoprotein (N-3xFLAG) under high MOI conditions (MOI~5). After one hour, the virus inoculum was replaced by a cell culture medium containing 20-µg/mL actinomycin D to inhibit cellular transcription. One hour later, 5-ethynyl uridine (5-EU) at a final concentration of 1 mM was added to the cells. The cells were fixed at the indicated time points post-infection, blocked, and permeabilized. A copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction was performed with Alexa 488-conjugated picolyl azide to label a newly synthesized viral RNA (green). Afterwards, the cells were stained with antibodies against FLAG-tag to detect N-3xFLAG (red) and dsRNA to visualize the viral replication intermediates (cyan). Hoechst 33342 was used for nuclear staining (blue). The cells were analyzed by confocal laser scanning microscopy. Representative Z stacks and overlays are shown. ( B ) Enlargement of the overlapping signals (white dotted square in ( A ), 8 hpi). White arrows indicate overlapping signals for N-3xFLAG and dsRNA (visible as a white signal), white asterisks (*) imply merging signals of N-3xFLAG and dsRNA (visible as a yellow signal).

    Article Snippet: The four fragments covering the SARS-CoV-2 genome were then assembled with the Pac I-digested pBeloCoV vector according to the NEBuilder HiFi DNA Assembly Cloning Kit protocol (E5520, NEB) to obtain pBelo-SARS-CoV-2 (pBSCoV2).

    Techniques: Synthesized, Infection, Marker, Expressing, Cell Culture, Concentration Assay, Staining, FLAG-tag, Confocal Laser Scanning Microscopy

    Cloning of a passage-free SARS-CoV-2 genome into a bacterial artificial chromosome (BAC). ( A ) Amplification of four overlapping fragments covering the entire SARS-CoV-2 genome. The open reading frames (ORFs) and structural and accessory proteins are indicated. Extracted viral nucleic acids of a respiratory swab sample served as the template. The genome structure is not to scale. ( B ) Schematic depiction of the modified pBeloBAC11 backbone pBeloCoV. Human cytomegalovirus (CMV) and T7 RNA polymerase (T7) promoters, as well as the bovine growth hormone polyA tail (bGH), were amplified from pcDNA4 and cloned into pBeloBAC11 together with gene blocks encoding the 5′ and 3′ ends of the SARS-CoV-2 genome with a flanking Pac I site and the hepatitis delta virus (HDV) ribozyme (Rz). ( C ) Schematic illustration of pBelo-SARS-CoV-2 (pBSCoV2). The four amplified fragments were assembled with the Pac I-linearized pBeloCoV backbone.

    Journal: International Journal of Molecular Sciences

    Article Title: Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination

    doi: 10.3390/ijms221910188

    Figure Lengend Snippet: Cloning of a passage-free SARS-CoV-2 genome into a bacterial artificial chromosome (BAC). ( A ) Amplification of four overlapping fragments covering the entire SARS-CoV-2 genome. The open reading frames (ORFs) and structural and accessory proteins are indicated. Extracted viral nucleic acids of a respiratory swab sample served as the template. The genome structure is not to scale. ( B ) Schematic depiction of the modified pBeloBAC11 backbone pBeloCoV. Human cytomegalovirus (CMV) and T7 RNA polymerase (T7) promoters, as well as the bovine growth hormone polyA tail (bGH), were amplified from pcDNA4 and cloned into pBeloBAC11 together with gene blocks encoding the 5′ and 3′ ends of the SARS-CoV-2 genome with a flanking Pac I site and the hepatitis delta virus (HDV) ribozyme (Rz). ( C ) Schematic illustration of pBelo-SARS-CoV-2 (pBSCoV2). The four amplified fragments were assembled with the Pac I-linearized pBeloCoV backbone.

    Article Snippet: The four fragments covering the SARS-CoV-2 genome were then assembled with the Pac I-digested pBeloCoV vector according to the NEBuilder HiFi DNA Assembly Cloning Kit protocol (E5520, NEB) to obtain pBelo-SARS-CoV-2 (pBSCoV2).

    Techniques: Clone Assay, BAC Assay, Amplification, Modification

    Generation and characterization of recSARS-CoV-2 reporter viruses. ( A ) Schematic depiction of recSARS-CoV-2 reporter and marker viruses. The illustration is not to scale. ( B ) Replication kinetics of luciferase reporter viruses. CaCo-2 cells were infected with recSARS-CoV-2 WT, as well as reporter viruses expressing gaussia luciferase (GLuc) instead of viral ORF7a (d7) and ORF8 (d8), respectively (MOI = 0.005). Cell culture supernatants were collected at the indicated time points post-infection, and viral RNA was quantified by RT-qPCR targeting the viral polymerase RdRp. ( C ) Luciferase assay for the detection of viral replication. HEK293T cells were transfected with pBSCoV2 (WT), pBSCoV2-d7-GLuc, or pBSCoV2-d8-GLuc. After 3 days, the supernatant was passaged on to CaCo-2 or Vero E6 cells. Cell culture supernatants were analyzed for GLuc activity three days post-transfection and post-infection, respectively. Luciferase activity is depicted as relative light units per second (RLU/s). ( D ) Replication kinetics of fluorescence reporter viruses. CaCo-2 cells were infected at an MOI of 0.005 with recSARS-CoV-2 WT and different reporter viruses expressing either GFP or YFP instead of viral ORF6 (d6), ORF7a (d7), and ORF8 (d8), or as an additional gene between ORF8 and N (after8). Cell culture supernatants were collected at the indicated time points, and viral RNA was quantified by RT-qPCR. ( E ) Fluorescence reporter expression. CaCo-2 cells were infected with the indicated fluorescence reporter viruses (MOI = 0.005). Cells were fixed at 12, 24, 48, and 72 hpi; permeabilized; immunostained with mAb against the spike protein (red); and visualized for reporter expression (green). Hoechst 33342 was used for nuclear staining (blue). Imaging was performed with an ImmunoSpot Image Analyzer, and representative overlays of the respective signals are depicted. ( F ) Replication kinetics of the N-3xFLAG marker virus. CaCo-2 cells were infected with recSARS-CoV-2 WT and a marker virus that expresses 3xFLAG-tagged N (MOI = 0.005). Cell culture supernatants were collected at the indicated time points post-infection, and viral RNA was quantified by RT-qPCR. ( G ) Western blot analysis of N-3xFLAG. Cell lysates of Vero E6 cells infected with the N-3xFLAG marker virus were analyzed for the expression of 3xFLAG-tagged nucleoprotein using an antibody specifically recognizing the tag sequence. Uninfected (mock) and HEK293T cells transfected with a N-3xFLAG expression vector served as the negative and positive controls, respectively.

    Journal: International Journal of Molecular Sciences

    Article Title: Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination

    doi: 10.3390/ijms221910188

    Figure Lengend Snippet: Generation and characterization of recSARS-CoV-2 reporter viruses. ( A ) Schematic depiction of recSARS-CoV-2 reporter and marker viruses. The illustration is not to scale. ( B ) Replication kinetics of luciferase reporter viruses. CaCo-2 cells were infected with recSARS-CoV-2 WT, as well as reporter viruses expressing gaussia luciferase (GLuc) instead of viral ORF7a (d7) and ORF8 (d8), respectively (MOI = 0.005). Cell culture supernatants were collected at the indicated time points post-infection, and viral RNA was quantified by RT-qPCR targeting the viral polymerase RdRp. ( C ) Luciferase assay for the detection of viral replication. HEK293T cells were transfected with pBSCoV2 (WT), pBSCoV2-d7-GLuc, or pBSCoV2-d8-GLuc. After 3 days, the supernatant was passaged on to CaCo-2 or Vero E6 cells. Cell culture supernatants were analyzed for GLuc activity three days post-transfection and post-infection, respectively. Luciferase activity is depicted as relative light units per second (RLU/s). ( D ) Replication kinetics of fluorescence reporter viruses. CaCo-2 cells were infected at an MOI of 0.005 with recSARS-CoV-2 WT and different reporter viruses expressing either GFP or YFP instead of viral ORF6 (d6), ORF7a (d7), and ORF8 (d8), or as an additional gene between ORF8 and N (after8). Cell culture supernatants were collected at the indicated time points, and viral RNA was quantified by RT-qPCR. ( E ) Fluorescence reporter expression. CaCo-2 cells were infected with the indicated fluorescence reporter viruses (MOI = 0.005). Cells were fixed at 12, 24, 48, and 72 hpi; permeabilized; immunostained with mAb against the spike protein (red); and visualized for reporter expression (green). Hoechst 33342 was used for nuclear staining (blue). Imaging was performed with an ImmunoSpot Image Analyzer, and representative overlays of the respective signals are depicted. ( F ) Replication kinetics of the N-3xFLAG marker virus. CaCo-2 cells were infected with recSARS-CoV-2 WT and a marker virus that expresses 3xFLAG-tagged N (MOI = 0.005). Cell culture supernatants were collected at the indicated time points post-infection, and viral RNA was quantified by RT-qPCR. ( G ) Western blot analysis of N-3xFLAG. Cell lysates of Vero E6 cells infected with the N-3xFLAG marker virus were analyzed for the expression of 3xFLAG-tagged nucleoprotein using an antibody specifically recognizing the tag sequence. Uninfected (mock) and HEK293T cells transfected with a N-3xFLAG expression vector served as the negative and positive controls, respectively.

    Article Snippet: The four fragments covering the SARS-CoV-2 genome were then assembled with the Pac I-digested pBeloCoV vector according to the NEBuilder HiFi DNA Assembly Cloning Kit protocol (E5520, NEB) to obtain pBelo-SARS-CoV-2 (pBSCoV2).

    Techniques: Marker, Luciferase, Infection, Expressing, Cell Culture, Quantitative RT-PCR, Transfection, Activity Assay, Fluorescence, Staining, Imaging, Western Blot, Sequencing, Plasmid Preparation

    Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Article Snippet: Assembly with NEBuilder HiFi assembly master mix or Gibson assembly master mix was performed according to manufacturer’s recommendations (NEB).

    Techniques: Clone Assay, Expressing, Amplification, Plasmid Preparation, Polymerase Chain Reaction, Construct, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay

    Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with Bam HI and Hin dIII to obtain pCAMBIA_ASX. (B) T35S–AttR2– ccd B–AttR1 cassette was released from the Gateway plasmid backbone by digestion with Xba I and Sac I and replaced with a I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Spec: selection marker conferring spectinomycin resistance in E . coli and A . tumefaciens , Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, Hin dIII, I- Sce I, Bam HI, Xba I, Sac I: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with Bam HI and Hin dIII to obtain pCAMBIA_ASX. (B) T35S–AttR2– ccd B–AttR1 cassette was released from the Gateway plasmid backbone by digestion with Xba I and Sac I and replaced with a I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Spec: selection marker conferring spectinomycin resistance in E . coli and A . tumefaciens , Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, Hin dIII, I- Sce I, Bam HI, Xba I, Sac I: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method.

    Article Snippet: Assembly with NEBuilder HiFi assembly master mix or Gibson assembly master mix was performed according to manufacturer’s recommendations (NEB).

    Techniques: Expressing, Plasmid Preparation, Clone Assay, Selection, Marker, Ligation

    Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Article Snippet: Assembly with NEBuilder HiFi assembly master mix or Gibson assembly master mix was performed according to manufacturer’s recommendations (NEB).

    Techniques: Functional Assay, Construct, Clone Assay, Expressing, Amplification, Plasmid Preparation, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay, Polymerase Chain Reaction