nebuilder  (New England Biolabs)


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    Name:
    NEBuilder HiFi DNA Assembly Master Mix
    Description:

    Catalog Number:
    E2621
    Price:
    630
    Category:
    DNA Modifying Enzymes
    Applications:
    DNA Manipulation
    Size:
    50 reactions
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    Structured Review

    New England Biolabs nebuilder
    NEBuilder HiFi DNA Assembly Master Mix

    https://www.bioz.com/result/nebuilder/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nebuilder - by Bioz Stars, 2021-07
    99/100 stars

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    Related Articles

    Plasmid Preparation:

    Article Title: Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries
    Article Snippet: .. The resulting DNA would be mixed with the NEBuilder HiFi assembly master mix, resulting in 3’ overhangs able to hybridize to complementary sequences on linearized vector. .. Following hybridization, NEBuilder HiFi assembly master mix polymerase and ligase fill gaps and ligate nicks, respectively, to produce a covalently sealed construct for transformation .

    Article Title: Antagonism of PP2A is an independent and conserved function of HIV-1 Vif and causes cell cycle arrest
    Article Snippet: These mutation-specific primers were used in pairwise PCR reactions in conjunction with common primers complementary to the vector backbone, which was cut between XhoI/KpnI sites. .. The two PCR products were then assembled into the vector using the NEBuilder HiFi DNA Assembly Master Mix (NEB). .. Sequences for all primers used are tabulated in (PCR primers for Vif mutant library construction).

    Polymerase Chain Reaction:

    Article Title: Antagonism of PP2A is an independent and conserved function of HIV-1 Vif and causes cell cycle arrest
    Article Snippet: These mutation-specific primers were used in pairwise PCR reactions in conjunction with common primers complementary to the vector backbone, which was cut between XhoI/KpnI sites. .. The two PCR products were then assembled into the vector using the NEBuilder HiFi DNA Assembly Master Mix (NEB). .. Sequences for all primers used are tabulated in (PCR primers for Vif mutant library construction).

    Protein Purification:

    Article Title: Disruption of ATRX-RNA interactions uncovers roles in ATRX localization and PRC2 function
    Article Snippet: .. Protein purification Full-length FLAG-ATRX-HA, FLAG-ATRXΔRBR-HA, and FLAG-ATRX helicase domain were generated using NEBuilder (NEB #E2621S). .. Proteins were purified from Sf9 cells using M2 agarose beads (Sigma #M8823-1ML).

    Generated:

    Article Title: Disruption of ATRX-RNA interactions uncovers roles in ATRX localization and PRC2 function
    Article Snippet: .. Protein purification Full-length FLAG-ATRX-HA, FLAG-ATRXΔRBR-HA, and FLAG-ATRX helicase domain were generated using NEBuilder (NEB #E2621S). .. Proteins were purified from Sf9 cells using M2 agarose beads (Sigma #M8823-1ML).

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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
    https://www.bioz.com/result/nebuilder hifi assembly/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nebuilder hifi assembly - by Bioz Stars, 2021-07
    99/100 stars
      Buy from Supplier

    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
    https://www.bioz.com/result/nebuilder hifi dna assembly cloning kit/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nebuilder hifi dna assembly cloning kit - by Bioz Stars, 2021-07
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs nebuilder hifi assembly master mix
    Colony PCR to determine library size and average insert length of mixed genome library prepared by METa assembly or blunt cloning Lanes 1, 25, 45, and 69: DNA ladders with top and brightest bands corresponding to 20 kb and 1.5 kb, respectively. Lanes 2-24, 26-44: Colonies from three replicate METa assemblies using <t>NEBuilder</t> <t>HiFi</t> (13 colonies per replicate, lanes 15, 30, and 44 from negative control sham colonies). Lanes 46-68, 70-88: Colonies from triplicate blunt ligation cloning reactions (13 colonies per replicate, lanes 59, 74, and 88 from negative control sham colonies). Reactions resulting in 500 bp amplicons indicate carriage of a no inserts vector. Reactions resulting in no visible band indicate technical failure of the colony PCR reaction.
    Nebuilder 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
    https://www.bioz.com/result/nebuilder hifi assembly master mix/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nebuilder hifi assembly master mix - by Bioz Stars, 2021-07
    99/100 stars
      Buy from Supplier

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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

    Colony PCR to determine library size and average insert length of mixed genome library prepared by METa assembly or blunt cloning Lanes 1, 25, 45, and 69: DNA ladders with top and brightest bands corresponding to 20 kb and 1.5 kb, respectively. Lanes 2-24, 26-44: Colonies from three replicate METa assemblies using NEBuilder HiFi (13 colonies per replicate, lanes 15, 30, and 44 from negative control sham colonies). Lanes 46-68, 70-88: Colonies from triplicate blunt ligation cloning reactions (13 colonies per replicate, lanes 59, 74, and 88 from negative control sham colonies). Reactions resulting in 500 bp amplicons indicate carriage of a no inserts vector. Reactions resulting in no visible band indicate technical failure of the colony PCR reaction.

    Journal: bioRxiv

    Article Title: Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries

    doi: 10.1101/2021.02.01.429292

    Figure Lengend Snippet: Colony PCR to determine library size and average insert length of mixed genome library prepared by METa assembly or blunt cloning Lanes 1, 25, 45, and 69: DNA ladders with top and brightest bands corresponding to 20 kb and 1.5 kb, respectively. Lanes 2-24, 26-44: Colonies from three replicate METa assemblies using NEBuilder HiFi (13 colonies per replicate, lanes 15, 30, and 44 from negative control sham colonies). Lanes 46-68, 70-88: Colonies from triplicate blunt ligation cloning reactions (13 colonies per replicate, lanes 59, 74, and 88 from negative control sham colonies). Reactions resulting in 500 bp amplicons indicate carriage of a no inserts vector. Reactions resulting in no visible band indicate technical failure of the colony PCR reaction.

    Article Snippet: The resulting DNA would be mixed with the NEBuilder HiFi assembly master mix, resulting in 3’ overhangs able to hybridize to complementary sequences on linearized vector.

    Techniques: Polymerase Chain Reaction, Clone Assay, Negative Control, Ligation, Plasmid Preparation

    Colony PCR to determine library size and average insert length of soil metagenomic DNA prepared by METa assembly (In-Fusion and NEBuilder HiFi) or blunt cloning Lanes 1, 17, 33, and 49: DNA ladders with brightest bands corresponding to 5 kb, 1.5 kb, and 500 bp. Lane 2: Single clone resulting from In-Fusion mediated METa assembly. Lanes 3-11, 12-21, and 22-31: Colonies from three replicate METa assemblies using NEBuilder HiFi. Lanes 32-41, 42-51, and 52-60: Colonies from triplicate blunt ligation cloning reactions. Lanes with bands at 500 bp correspond to amplification of vector backbone only (no inserts) while lanes with no band indicate colony PCR reaction failure

    Journal: bioRxiv

    Article Title: Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries

    doi: 10.1101/2021.02.01.429292

    Figure Lengend Snippet: Colony PCR to determine library size and average insert length of soil metagenomic DNA prepared by METa assembly (In-Fusion and NEBuilder HiFi) or blunt cloning Lanes 1, 17, 33, and 49: DNA ladders with brightest bands corresponding to 5 kb, 1.5 kb, and 500 bp. Lane 2: Single clone resulting from In-Fusion mediated METa assembly. Lanes 3-11, 12-21, and 22-31: Colonies from three replicate METa assemblies using NEBuilder HiFi. Lanes 32-41, 42-51, and 52-60: Colonies from triplicate blunt ligation cloning reactions. Lanes with bands at 500 bp correspond to amplification of vector backbone only (no inserts) while lanes with no band indicate colony PCR reaction failure

    Article Snippet: The resulting DNA would be mixed with the NEBuilder HiFi assembly master mix, resulting in 3’ overhangs able to hybridize to complementary sequences on linearized vector.

    Techniques: Polymerase Chain Reaction, Clone Assay, Ligation, Amplification, Plasmid Preparation

    Blunt cloning protocol compared to METa assembly with NEBuilder HiFi or In-Fusion a) Transposase enzyme fragments DNA with 5’ mosaic end oligos. Inserts can be used as input for all three methods. All three protocols are compatible with linear pZE21-ME vector prepared by inverse PCR. b) Blunt cloning via end-repair and ligase. 5’ overhangs must be resolved by gap filling and phosphorylation using end-repair enzyme mixes. Blunt ended inserts can be ligated to blunt ended vector. c) METa assembly via In-Fusion enzyme mix. In-Fusion 3’ exonuclease activity is directly compatible with transposase fragments. Single stranded DNA overhangs on inserts and vector hybridize into a stable complex that can be transformed without filling gaps or covalently sealing nicks. d) METa assembly via NEBuilder HiFi enzyme mix. 5’ overhangs must be resolved by DNA polymerase gap filling. NEBuilder HiFi enzyme mix includes 5’ exonuclease to create 3’ overhangs which hybridize with target pZE21-ME. DNA polymerase fills in gaps and ligase seals nicks. e) pZE21-ME is prepared and linearized by inverse PCR and is compatible with all three DNA pipelines.

    Journal: bioRxiv

    Article Title: Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries

    doi: 10.1101/2021.02.01.429292

    Figure Lengend Snippet: Blunt cloning protocol compared to METa assembly with NEBuilder HiFi or In-Fusion a) Transposase enzyme fragments DNA with 5’ mosaic end oligos. Inserts can be used as input for all three methods. All three protocols are compatible with linear pZE21-ME vector prepared by inverse PCR. b) Blunt cloning via end-repair and ligase. 5’ overhangs must be resolved by gap filling and phosphorylation using end-repair enzyme mixes. Blunt ended inserts can be ligated to blunt ended vector. c) METa assembly via In-Fusion enzyme mix. In-Fusion 3’ exonuclease activity is directly compatible with transposase fragments. Single stranded DNA overhangs on inserts and vector hybridize into a stable complex that can be transformed without filling gaps or covalently sealing nicks. d) METa assembly via NEBuilder HiFi enzyme mix. 5’ overhangs must be resolved by DNA polymerase gap filling. NEBuilder HiFi enzyme mix includes 5’ exonuclease to create 3’ overhangs which hybridize with target pZE21-ME. DNA polymerase fills in gaps and ligase seals nicks. e) pZE21-ME is prepared and linearized by inverse PCR and is compatible with all three DNA pipelines.

    Article Snippet: The resulting DNA would be mixed with the NEBuilder HiFi assembly master mix, resulting in 3’ overhangs able to hybridize to complementary sequences on linearized vector.

    Techniques: Clone Assay, Plasmid Preparation, Inverse PCR, Activity Assay, Transformation Assay

    Comparing metagenomic libraries prepared by assembly or blunt ligation Functional metagenomic libraries were created using METa assembly via In-Fusion assembly or NEBuilder HiFi assembly and compared to a library constructed using blunt ligation. All libraries used the same input DNA and were each prepared in triplicate. Error bars represent standard deviation of n=3 experiments. Comparisons between blunt ligation and NEBuilder HiFi METa assembly were made using unpaired two tailed t tests. Only a single In-Fusion colony was isolated and therefore we did not perform statistical tests on that method. a) Post-transformation culture titers normalized to the quantity of insert DNA used in the assembly or cloning reaction itself. b) Average insert size for plasmids containing an insert. Colony PCR was performed on the single In-Fusion colony, and 9 colonies were analyzed and averaged for each replicate NEBuilder HiFi assembly or blunt ligation reaction to give three data points per method. c) Final total library size or each replicate measured in gigabase pairs (Gb) of captured metagenomic DNA normalized to the amount of insert (μg) used during cloning or assembly.

    Journal: bioRxiv

    Article Title: Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries

    doi: 10.1101/2021.02.01.429292

    Figure Lengend Snippet: Comparing metagenomic libraries prepared by assembly or blunt ligation Functional metagenomic libraries were created using METa assembly via In-Fusion assembly or NEBuilder HiFi assembly and compared to a library constructed using blunt ligation. All libraries used the same input DNA and were each prepared in triplicate. Error bars represent standard deviation of n=3 experiments. Comparisons between blunt ligation and NEBuilder HiFi METa assembly were made using unpaired two tailed t tests. Only a single In-Fusion colony was isolated and therefore we did not perform statistical tests on that method. a) Post-transformation culture titers normalized to the quantity of insert DNA used in the assembly or cloning reaction itself. b) Average insert size for plasmids containing an insert. Colony PCR was performed on the single In-Fusion colony, and 9 colonies were analyzed and averaged for each replicate NEBuilder HiFi assembly or blunt ligation reaction to give three data points per method. c) Final total library size or each replicate measured in gigabase pairs (Gb) of captured metagenomic DNA normalized to the amount of insert (μg) used during cloning or assembly.

    Article Snippet: The resulting DNA would be mixed with the NEBuilder HiFi assembly master mix, resulting in 3’ overhangs able to hybridize to complementary sequences on linearized vector.

    Techniques: Ligation, Functional Assay, Construct, Standard Deviation, Two Tailed Test, Isolation, Transformation Assay, Clone Assay, Polymerase Chain Reaction

    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