nebuilder  (New England Biolabs)


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    Structured Review

    New England Biolabs nebuilder
    Nebuilder, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nebuilder/product/New England Biolabs
    Average 90 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    nebuilder - by Bioz Stars, 2020-05
    90/100 stars

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    Clone Assay:

    Article Title: COMPASS for rapid combinatorial optimization of biochemical pathways based on artificial transcription factors
    Article Snippet: .. Plasmids were constructed by NEBuilder HiFi DNA assembly (New England Biolabs, Frankfurt am Main, Germany) and SLiCE cloning . ..

    Electroporation:

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants
    Article Snippet: .. Appropriate volumes of the SLiCE, NEBuilder HiFi or Gibson DNA assembled products were transformed into TOP10 E . coli (Invitrogen) or NEB5α (NEB) by electroporation or heat shock according to the manufacturer’s protocols. .. Transformed cells were spread on plates containing appropriate antibiotic, ampicillin (100 μg/ml), spectinomycin (75 μg/ml), kanamycin (50 μg/ml) or rifampicin (20 μg/ml).

    Construct:

    Article Title: COMPASS for rapid combinatorial optimization of biochemical pathways based on artificial transcription factors
    Article Snippet: .. Plasmids were constructed by NEBuilder HiFi DNA assembly (New England Biolabs, Frankfurt am Main, Germany) and SLiCE cloning . ..

    Sequencing:

    Article Title: LF4/MOK and a CDK-related kinase regulate the number and length of cilia in Tetrahymena
    Article Snippet: .. To overexpress mCherry-LF4AF82A , the GFP-LF4A part of pNeo5_ovGFP-LF4A was replaced with two fragments that provide the sequence of mCherry-LF4AF82A using NEBuilder Hifi DNA Assembly. .. The point mutation was created at the junction between the two fragments, which were amplified with the following primer pairs: 5’-CTAAACTTAAAATAATGGCCAAGTCGACGGTTTCAAAAGGAGAAGAAG-3’, 5-CAATTCAGCCACTAGTGCCAAACGTCCTGTAG-3’, 5’- GCACTAGTGGCTGAATTGATGGATCAGAACC-3’ AND 5’-CAAAAGCTGGGTACCGGGCCCATATGGGTGGCGTG-3’ from pNeo5_ovmCherry-LF4.

    Polymerase Chain Reaction:

    Article Title: Mitochondrial type II NADH dehydrogenase of Plasmodium falciparum (PfNDH2) is dispensable in the asexual blood stages
    Article Snippet: .. The PCR product and the digested vector were then joined together using NEBuilder® HiFi DNA Assembly (New England Biolabs® , Inc). .. A colony PCR was performed to screen colonies using primers P1 and P2.

    Transformation Assay:

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants
    Article Snippet: .. Appropriate volumes of the SLiCE, NEBuilder HiFi or Gibson DNA assembled products were transformed into TOP10 E . coli (Invitrogen) or NEB5α (NEB) by electroporation or heat shock according to the manufacturer’s protocols. .. Transformed cells were spread on plates containing appropriate antibiotic, ampicillin (100 μg/ml), spectinomycin (75 μg/ml), kanamycin (50 μg/ml) or rifampicin (20 μg/ml).

    Plasmid Preparation:

    Article Title: Mitochondrial type II NADH dehydrogenase of Plasmodium falciparum (PfNDH2) is dispensable in the asexual blood stages
    Article Snippet: .. The PCR product and the digested vector were then joined together using NEBuilder® HiFi DNA Assembly (New England Biolabs® , Inc). .. A colony PCR was performed to screen colonies using primers P1 and P2.

    Article Title: A novel ER membrane protein Ehg1/May24 plays a critical role in maintaining multiple nutrient permeases in yeast under high-pressure perturbation
    Article Snippet: .. NEBuilder HiFi DNA assembly (New England Biolabs Japan Inc, Tokyo, Japan) was also used for plasmid constructions. .. To construct pUA51 (EHG1 -GFP, LEU2 , 2μ ), the EHG1 ORF was amplified using pUA36 as a template and primers 5′- TTGATATCGAATTCCTGCAGTACGTCACCCGCCTCTTCGCTGAT-3′ and 5′- TGCTCACCATGGATCCCATAACGGAACCAACCATGGAATAACTTAG-3′.

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  • 99
    New England Biolabs nebuilder hifi
    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.
    Nebuilder Hifi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nebuilder hifi/product/New England Biolabs
    Average 99 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
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    93
    New England Biolabs nebuilder assembly
    Workflow of the PASSPORT-seq bioassay. (A) 100 Reference and variant miRNA binding regions each with the same 15–20 bp flanking sequence was synthesized as an oligonucleotide pool. (B) Using the flanking universal sequences, the oligonucleotide pool was amplified and made double stranded by PCR. pIS-0 plasmid was linearized by restriction enzymes. (C) The double stranded oligonucleotides were inserted into the linear plasmid using the <t>NEBuilder</t> TM gene assembly system. (D) Chemically competent bacteria were transformed with the plasmid pool containing the test miRNA binding regions. Transformed bacteria were plated on four plates. (E) All colonies from the plates were harvested, combined and scaled up in liquid culture. Plasmids were isolated from the liquid culture. (F) Three cell lines were transfected with the plasmid pool and incubated for 48 h after which cDNA was prepared from total RNA. (G) miRNA binding regions were amplified using universal primers that were uniquely barcoded for replicates within cell lines and for the input plasmid pool. (H) The barcoded PCR products were combined to form the sequencing pool.
    Nebuilder Assembly, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs nebuilder hifi 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 15 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 15 article reviews
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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) 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 reaction contained 50–500 ng of linear vector, an appropriate amount of insert DNA in a 1: 2 to 1: 10 vector to insert molar ratio and 2x NEBuilder HiFi or Gibson DNA Assembly Master Mix.

    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 reaction contained 50–500 ng of linear vector, an appropriate amount of insert DNA in a 1: 2 to 1: 10 vector to insert molar ratio and 2x NEBuilder HiFi or Gibson DNA Assembly Master Mix.

    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 reaction contained 50–500 ng of linear vector, an appropriate amount of insert DNA in a 1: 2 to 1: 10 vector to insert molar ratio and 2x NEBuilder HiFi or Gibson DNA Assembly Master Mix.

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

    Workflow of the PASSPORT-seq bioassay. (A) 100 Reference and variant miRNA binding regions each with the same 15–20 bp flanking sequence was synthesized as an oligonucleotide pool. (B) Using the flanking universal sequences, the oligonucleotide pool was amplified and made double stranded by PCR. pIS-0 plasmid was linearized by restriction enzymes. (C) The double stranded oligonucleotides were inserted into the linear plasmid using the NEBuilder TM gene assembly system. (D) Chemically competent bacteria were transformed with the plasmid pool containing the test miRNA binding regions. Transformed bacteria were plated on four plates. (E) All colonies from the plates were harvested, combined and scaled up in liquid culture. Plasmids were isolated from the liquid culture. (F) Three cell lines were transfected with the plasmid pool and incubated for 48 h after which cDNA was prepared from total RNA. (G) miRNA binding regions were amplified using universal primers that were uniquely barcoded for replicates within cell lines and for the input plasmid pool. (H) The barcoded PCR products were combined to form the sequencing pool.

    Journal: Frontiers in Genetics

    Article Title: PASSPORT-seq: A Novel High-Throughput Bioassay to Functionally Test Polymorphisms in Micro-RNA Target Sites

    doi: 10.3389/fgene.2018.00219

    Figure Lengend Snippet: Workflow of the PASSPORT-seq bioassay. (A) 100 Reference and variant miRNA binding regions each with the same 15–20 bp flanking sequence was synthesized as an oligonucleotide pool. (B) Using the flanking universal sequences, the oligonucleotide pool was amplified and made double stranded by PCR. pIS-0 plasmid was linearized by restriction enzymes. (C) The double stranded oligonucleotides were inserted into the linear plasmid using the NEBuilder TM gene assembly system. (D) Chemically competent bacteria were transformed with the plasmid pool containing the test miRNA binding regions. Transformed bacteria were plated on four plates. (E) All colonies from the plates were harvested, combined and scaled up in liquid culture. Plasmids were isolated from the liquid culture. (F) Three cell lines were transfected with the plasmid pool and incubated for 48 h after which cDNA was prepared from total RNA. (G) miRNA binding regions were amplified using universal primers that were uniquely barcoded for replicates within cell lines and for the input plasmid pool. (H) The barcoded PCR products were combined to form the sequencing pool.

    Article Snippet: The universal primers used to amplify the test-oligonucleotide pool also served as the flanking homology regions for the NEBuilder® assembly.

    Techniques: Variant Assay, Binding Assay, Sequencing, Synthesized, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Transformation Assay, Isolation, Transfection, Incubation

    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: We subsequently assembled both expression cassettes in Level 1 vector pL1A-hc / pL1A-lc (A0/AR) [ ] by TAR and NEBuilder HiFi assembly ( ).

    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: We subsequently assembled both expression cassettes in Level 1 vector pL1A-hc / pL1A-lc (A0/AR) [ ] by TAR and NEBuilder HiFi assembly ( ).

    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: We subsequently assembled both expression cassettes in Level 1 vector pL1A-hc / pL1A-lc (A0/AR) [ ] by TAR and NEBuilder HiFi assembly ( ).

    Techniques: Functional Assay, Construct, Clone Assay, Expressing, Amplification, Plasmid Preparation, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation 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: Following isolation from the gel using Wizard® SV Gel and PCR Clean-Up System (Promega), inserts were assembled into Hin dIII linearized Plant X-tender expression vectors by NEBuilder HiFi or SLiCE assembly method.

    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: Following isolation from the gel using Wizard® SV Gel and PCR Clean-Up System (Promega), inserts were assembled into Hin dIII linearized Plant X-tender expression vectors by NEBuilder HiFi or SLiCE assembly method.

    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: Following isolation from the gel using Wizard® SV Gel and PCR Clean-Up System (Promega), inserts were assembled into Hin dIII linearized Plant X-tender expression vectors by NEBuilder HiFi or SLiCE assembly method.

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