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  • 99
    Name:
    Esp3I
    Description:
    Esp3I 1 500 units
    Catalog Number:
    R0734L
    Price:
    337
    Size:
    1 500 units
    Category:
    Restriction Enzymes
    Score:
    85
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    New England Biolabs bsmbi
    Esp3I
    Esp3I 1 500 units
    https://www.bioz.com/result/bsmbi/product/New England Biolabs
    Average 99 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    bsmbi - by Bioz Stars, 2019-10
    99/100 stars

    Images

    1) Product Images from "A Rapid Cloning Method Employing Orthogonal End Protection"

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0037617

    Split-and-pool assembly of DNA synthons. (A) Entry synthons are flanked on both sides by recognition sequences for the type IIS endonucleases BsaI and BsmBI. Restriction by either BsaI or BsmBI selectively exposes user-definable 4-base cohesive overhang sequences (5′-XXXX vs. 5′-xxxx) at one end of the synthon, while maintaining orthogonal protection groups (with 5′-YYYY vs. 5′-zzzz overhangs) at the opposite end. (B) Schematic representation of the ‘split-and-pool’ assembly principle. Cohesive ends of entry synthons are selectively deprotected by digestion with either BsaI or BsmBI. Pooling of the deprotected synthons in the presence of ligase results in unidirectional assembly, affording an idempotent tandem repeat synthon by restoration of orthogonal protecting groups on opposite ends. Each product module can recursively enter the assembly cycle (left panel) N times to yield concatameric synthons with 2N elements. The same strategy can be applied to the assembly of heterosynthons (dashed box), which allows for the engineering of chimeric and multimodular proteins or polycistronic genes.
    Figure Legend Snippet: Split-and-pool assembly of DNA synthons. (A) Entry synthons are flanked on both sides by recognition sequences for the type IIS endonucleases BsaI and BsmBI. Restriction by either BsaI or BsmBI selectively exposes user-definable 4-base cohesive overhang sequences (5′-XXXX vs. 5′-xxxx) at one end of the synthon, while maintaining orthogonal protection groups (with 5′-YYYY vs. 5′-zzzz overhangs) at the opposite end. (B) Schematic representation of the ‘split-and-pool’ assembly principle. Cohesive ends of entry synthons are selectively deprotected by digestion with either BsaI or BsmBI. Pooling of the deprotected synthons in the presence of ligase results in unidirectional assembly, affording an idempotent tandem repeat synthon by restoration of orthogonal protecting groups on opposite ends. Each product module can recursively enter the assembly cycle (left panel) N times to yield concatameric synthons with 2N elements. The same strategy can be applied to the assembly of heterosynthons (dashed box), which allows for the engineering of chimeric and multimodular proteins or polycistronic genes.

    Techniques Used:

    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
    Figure Legend Snippet: Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.

    Techniques Used: Incubation, Plasmid Preparation, Clone Assay, Expressing, Construct

    2) Product Images from "Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae"

    Article Title: Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv466

    Yeast Golden Gate (yGG) to assemble transcription units (TUs) flanked by VEGAS adapters. ( A ) yGG reactions to build TUs destined for VEGAS pathway assembly in S. cerevisiae include five parts: a left VEGAS adapter (LVA), a promoter (PRO), a coding sequence (CDS), a terminator (TER) and a right VEGAS adapter (RVA). Each part is flanked by inwardly facing recognition sequences for the BsaI restriction enzyme, an ‘offset cutter’ which cuts outside its recognition sequence (at positions 1/5 bp downstream) to expose the indicated four base-pair overhangs. All parts are cloned into vectors encoding kanamycin resistance (KAN R ) and an E. coli replication origin (Ori). ( B ) The yGG acceptor vector for VEGAS is designed such that outwardly facing BsaI sites expose overhangs corresponding to the 5′ LVA and 3′ RVA overhangs to promote assembly of the TU in the vector during a one-pot restriction-digestion reaction. The RFP cassette, built for expression in E. coli , is cut out of the vector when a TU correctly assembles, enabling white–red screening. The yGG acceptor vector encodes resistance to ampicillin (AMP R ) ( C ) The structure of a VA-flanked TU assembled by yGG. An assembled TU plus the flanking VA sequences may be released from the yGG acceptor vector by digestion with BsmBI.
    Figure Legend Snippet: Yeast Golden Gate (yGG) to assemble transcription units (TUs) flanked by VEGAS adapters. ( A ) yGG reactions to build TUs destined for VEGAS pathway assembly in S. cerevisiae include five parts: a left VEGAS adapter (LVA), a promoter (PRO), a coding sequence (CDS), a terminator (TER) and a right VEGAS adapter (RVA). Each part is flanked by inwardly facing recognition sequences for the BsaI restriction enzyme, an ‘offset cutter’ which cuts outside its recognition sequence (at positions 1/5 bp downstream) to expose the indicated four base-pair overhangs. All parts are cloned into vectors encoding kanamycin resistance (KAN R ) and an E. coli replication origin (Ori). ( B ) The yGG acceptor vector for VEGAS is designed such that outwardly facing BsaI sites expose overhangs corresponding to the 5′ LVA and 3′ RVA overhangs to promote assembly of the TU in the vector during a one-pot restriction-digestion reaction. The RFP cassette, built for expression in E. coli , is cut out of the vector when a TU correctly assembles, enabling white–red screening. The yGG acceptor vector encodes resistance to ampicillin (AMP R ) ( C ) The structure of a VA-flanked TU assembled by yGG. An assembled TU plus the flanking VA sequences may be released from the yGG acceptor vector by digestion with BsmBI.

    Techniques Used: Sequencing, Clone Assay, Plasmid Preparation, Expressing

    VEGAS with adapter homology to assemble the carotenoid pathway in S. cerevisiae . ( A ) The four β-carotene pathway genes ( crtE, crtI, crtYB and tHMG1 ), assembled as TUs flanked by the indicated VAs (see Table 2 for PRO and TER parts), were released from the yGG acceptor vector with BsmBI digestion and co-transformed into yeast with the linearized VEGAS assembly vector. ( B ) S. cerevisiae colonies encoding assembled pathways develop a bright yellow color on medium lacking uracil (SC–Ura; left panel) as well as on YPD medium supplemented with G418 (right panel).
    Figure Legend Snippet: VEGAS with adapter homology to assemble the carotenoid pathway in S. cerevisiae . ( A ) The four β-carotene pathway genes ( crtE, crtI, crtYB and tHMG1 ), assembled as TUs flanked by the indicated VAs (see Table 2 for PRO and TER parts), were released from the yGG acceptor vector with BsmBI digestion and co-transformed into yeast with the linearized VEGAS assembly vector. ( B ) S. cerevisiae colonies encoding assembled pathways develop a bright yellow color on medium lacking uracil (SC–Ura; left panel) as well as on YPD medium supplemented with G418 (right panel).

    Techniques Used: Plasmid Preparation, Transformation Assay

    VEGAS with adapter homology to assemble a five-gene pathway. ( A ) The pathway consisting of VA-flanked TUs assembled by yGG may be released in one piece from the yGG acceptor vector by digestion with BsmBI (scissors). ( B ) A genetic pathway may be assembled into the linearized VEGAS assembly vector in S. cerevisiae by homologous recombination between VAs that flank TUs (TU1–5). X's indicate homologous recombination.
    Figure Legend Snippet: VEGAS with adapter homology to assemble a five-gene pathway. ( A ) The pathway consisting of VA-flanked TUs assembled by yGG may be released in one piece from the yGG acceptor vector by digestion with BsmBI (scissors). ( B ) A genetic pathway may be assembled into the linearized VEGAS assembly vector in S. cerevisiae by homologous recombination between VAs that flank TUs (TU1–5). X's indicate homologous recombination.

    Techniques Used: Plasmid Preparation, Homologous Recombination

    3) Product Images from "Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments"

    Article Title: Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments

    Journal: Scientific Reports

    doi: 10.1038/srep10655

    Lambda phage genome construction. ( A ) Design of the OGAB blocks. A total of 48.5 kb in length of lambda phage genome was divided into 50 fragments, each of which was cloned into cloning vector pMD19. Three restriction enzyme sites, BbsI (green), AarI (red), and BsmBI (blue), at least one of which did not appear in each OGAB block, were used. ( B ) The 50 OGAB block mixtures before (blue) and after (red) size selection by electrophoresis were compared relative to the population by quantitative PCR. The apparent CV mol values for the OGAB blocks before and after size selection were 7.4% and 7.0%; however, due to the 3.6% measurement error of the PCR machine, the error-corrected CV mol values were 7.0% and 6.6%, respectively. The population profile after size selection was almost the same as that before size selection. We performed two additional OGAB block preparations using independently measured and mixed OGAB block plasmids and determined CV mol (%), resulting in a similar value to that of the initial experiment (CV mol (%)(error-corrected) = 6.8% and 7.3%). All raw data are indicated in Supplemental Table S8 . ( C ) Restriction digestion pattern of plasmids from 12 randomly selected transformants. HindIII and SfiI were used for double digestion. In the case of four clones (numbers circled), except for the 15 kb of the assembly vector pGET118-AarI, all of the bands were the same as the commercial size marker λ/HindIII. ( D ) Plaque formation assay of correctly assembled plasmid. The four plasmids were digested with lambda terminase, packed into lambda phage extract, and used to infect E. coli . There were no differences in features between the assembled plasmid-born plaque and the authentic lambda phage DNA-born plaque. ( E ) Confirmation of the plaque as designed. Due to an intended synonymous codon mutation in OGAB block 10, a restriction enzyme AvaI site appeared at the largest fragment (14,678 bp) of the wild type, generating two fragments (9,885 and 4,793 bp). All of the clones had restriction patterns at most large AvaI fragments distinct from those of the wild type, indicating that these phages originated from assembled DNAs.
    Figure Legend Snippet: Lambda phage genome construction. ( A ) Design of the OGAB blocks. A total of 48.5 kb in length of lambda phage genome was divided into 50 fragments, each of which was cloned into cloning vector pMD19. Three restriction enzyme sites, BbsI (green), AarI (red), and BsmBI (blue), at least one of which did not appear in each OGAB block, were used. ( B ) The 50 OGAB block mixtures before (blue) and after (red) size selection by electrophoresis were compared relative to the population by quantitative PCR. The apparent CV mol values for the OGAB blocks before and after size selection were 7.4% and 7.0%; however, due to the 3.6% measurement error of the PCR machine, the error-corrected CV mol values were 7.0% and 6.6%, respectively. The population profile after size selection was almost the same as that before size selection. We performed two additional OGAB block preparations using independently measured and mixed OGAB block plasmids and determined CV mol (%), resulting in a similar value to that of the initial experiment (CV mol (%)(error-corrected) = 6.8% and 7.3%). All raw data are indicated in Supplemental Table S8 . ( C ) Restriction digestion pattern of plasmids from 12 randomly selected transformants. HindIII and SfiI were used for double digestion. In the case of four clones (numbers circled), except for the 15 kb of the assembly vector pGET118-AarI, all of the bands were the same as the commercial size marker λ/HindIII. ( D ) Plaque formation assay of correctly assembled plasmid. The four plasmids were digested with lambda terminase, packed into lambda phage extract, and used to infect E. coli . There were no differences in features between the assembled plasmid-born plaque and the authentic lambda phage DNA-born plaque. ( E ) Confirmation of the plaque as designed. Due to an intended synonymous codon mutation in OGAB block 10, a restriction enzyme AvaI site appeared at the largest fragment (14,678 bp) of the wild type, generating two fragments (9,885 and 4,793 bp). All of the clones had restriction patterns at most large AvaI fragments distinct from those of the wild type, indicating that these phages originated from assembled DNAs.

    Techniques Used: Clone Assay, Plasmid Preparation, Blocking Assay, Selection, Electrophoresis, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Marker, Plaque Formation Assay, Mutagenesis

    4) Product Images from "Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants"

    Article Title: Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms14036205

    Assembly process of the secretory immunoglobulin A (sIgA). ( a ) Collection of basic parts necessary to construct a secretory IgA. Each basic part is cloned in a pGem-T vector. 35S, SP, VH-CH, VL-CL, SC, JC, Tnos, correspond, respectively, to the 35s CMV promoter, pectate lyase signal peptide, variable and constant regions of the heavy chain, variable and constant regions of the light chain, secretory component, J-chain and nopaline synthase terminator; ( b ) Example of domestication of a basic part. The 35s promoter is flanked by fixed BsmbI recognition-cleavage sites. The overhangs left by the BsmbI restriction enzyme converge with GB pDGB vectors on 5′ and on 3′, with the next basic part to assemble; ( c ) Multipartite assembly of the basic parts to form the four different transcriptional units: heavy chain (HC), light chain (LC), secretory component (SC) and J-chain (JC), into level Ω-GB destiny vectors (pDGB_1AB3 and pDGB_3AB2); ( d ) Binary assembly of transcriptional units in level α-GB destination vectors (pDGB_C12B and pDGB_A12C), in order to construct two different composite parts—IgA and JC-SC; ( e ) Last construct of sIgA by binary assembly of two composite parts in a final pDGB; ( f ) Example of restriction analysis of four colonies of each construct: left, BglII (expected bands of 2825, 1886 and 1197) and BglI (expected bands of 2345, 1790, 1498 and 275) restriction analysis of the HC transcriptional unit; middle, BglII (expected bands of 4183, 2495 and 1228 kDa) restriction analysis of IgA; right, BamHI (expected bands of 6815, 5857 and 913 kDa) and BsaI (expected bands of 10,664 + 2921 kDa) restriction analysis of sIgA.
    Figure Legend Snippet: Assembly process of the secretory immunoglobulin A (sIgA). ( a ) Collection of basic parts necessary to construct a secretory IgA. Each basic part is cloned in a pGem-T vector. 35S, SP, VH-CH, VL-CL, SC, JC, Tnos, correspond, respectively, to the 35s CMV promoter, pectate lyase signal peptide, variable and constant regions of the heavy chain, variable and constant regions of the light chain, secretory component, J-chain and nopaline synthase terminator; ( b ) Example of domestication of a basic part. The 35s promoter is flanked by fixed BsmbI recognition-cleavage sites. The overhangs left by the BsmbI restriction enzyme converge with GB pDGB vectors on 5′ and on 3′, with the next basic part to assemble; ( c ) Multipartite assembly of the basic parts to form the four different transcriptional units: heavy chain (HC), light chain (LC), secretory component (SC) and J-chain (JC), into level Ω-GB destiny vectors (pDGB_1AB3 and pDGB_3AB2); ( d ) Binary assembly of transcriptional units in level α-GB destination vectors (pDGB_C12B and pDGB_A12C), in order to construct two different composite parts—IgA and JC-SC; ( e ) Last construct of sIgA by binary assembly of two composite parts in a final pDGB; ( f ) Example of restriction analysis of four colonies of each construct: left, BglII (expected bands of 2825, 1886 and 1197) and BglI (expected bands of 2345, 1790, 1498 and 275) restriction analysis of the HC transcriptional unit; middle, BglII (expected bands of 4183, 2495 and 1228 kDa) restriction analysis of IgA; right, BamHI (expected bands of 6815, 5857 and 913 kDa) and BsaI (expected bands of 10,664 + 2921 kDa) restriction analysis of sIgA.

    Techniques Used: Construct, Clone Assay, Plasmid Preparation, Liquid Chromatography

    5) Product Images from "Comparing CDRH3 diversity captured from secondary lymphoid organs for the generation of recombinant human antibodies"

    Article Title: Comparing CDRH3 diversity captured from secondary lymphoid organs for the generation of recombinant human antibodies

    Journal:

    doi: 10.4161/mabs.25592

    Figure 1. Capture of murine CDRH3 into a library of human scFv. (A) BalbC mice, divided in three groups, were either kept naïve or immunized with hIFNγ or hCCL5. (B) After sacrifice, the spleen and the lymph nodes were kept separated and VH repertoires were recovered by PCR. CDRH3 were then amplified from the VH pool by PCR, along with recognition sites for FokI, a type IIS restriction enzyme, were added to the inserts. CDR3 are represented in gray, CDR1 and 2 in white. (C) Murine inserts were then digested with FokI and (D) ligated to the human acceptor scFv library, itself digested with BsmBI. This second type IIS enzyme permitted the removal of a non-diversified stuffer sequence (“S”) at the location of CDRH3 and the generation of compatible cohesive ends for the incorporation of CDRH3. The acceptor library also contains synthetic diversity at the location of the CDRL3 (“L3”) and tags (“T,” a c-myc and a His tag) at the scFv C-terminal for purification purposes. Libraries featuring mouse CDRH3 were analyzed by NGS, which covered the CDRH3 and part of VH framework 3 (“NGS”). (E) The fusion to gIII allowed the expression of scFv at the surface of M13 phage for phage display selection.
    Figure Legend Snippet: Figure 1. Capture of murine CDRH3 into a library of human scFv. (A) BalbC mice, divided in three groups, were either kept naïve or immunized with hIFNγ or hCCL5. (B) After sacrifice, the spleen and the lymph nodes were kept separated and VH repertoires were recovered by PCR. CDRH3 were then amplified from the VH pool by PCR, along with recognition sites for FokI, a type IIS restriction enzyme, were added to the inserts. CDR3 are represented in gray, CDR1 and 2 in white. (C) Murine inserts were then digested with FokI and (D) ligated to the human acceptor scFv library, itself digested with BsmBI. This second type IIS enzyme permitted the removal of a non-diversified stuffer sequence (“S”) at the location of CDRH3 and the generation of compatible cohesive ends for the incorporation of CDRH3. The acceptor library also contains synthetic diversity at the location of the CDRL3 (“L3”) and tags (“T,” a c-myc and a His tag) at the scFv C-terminal for purification purposes. Libraries featuring mouse CDRH3 were analyzed by NGS, which covered the CDRH3 and part of VH framework 3 (“NGS”). (E) The fusion to gIII allowed the expression of scFv at the surface of M13 phage for phage display selection.

    Techniques Used: Mouse Assay, Polymerase Chain Reaction, Amplification, Sequencing, Purification, Next-Generation Sequencing, Expressing, Selection

    6) Product Images from "Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality"

    Article Title: Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005310

    Construction of the mutant libraries. (A) A schematic representation of the fitness profiling experiment is shown. A 240 bp insert was generated by error-prone PCR and BsaI digestion. The corresponding vector was generated by high-fidelity PCR and BsmBI digestion. Each of the nine plasmid libraries in this study consist of ∼ 50,000 clones. Each viral mutant library was rescued by transfecting ∼ 35 million 293T cells. Each infection was performed with ∼ 10 million A549 cells. (B) A schematic representation of the sequencing library preparation is shown. DNA plasmid mutant library or viral cDNA was used for PCR. This PCR amplified the 240 bp randomized region. The amplicon product was then digested with BpmI, end-repaired, dA-tailed, ligated to sequencing adapters, and sequenced using the Illumina MiSeq platform. BpmI digestion removed the primer region in the amplicon PCR, resulting in sequencing reads covering only the barcode for multiplex sequencing and the 240 bp region that was randomized in the mutant library. With this experimental design, the number of mutations carried by individual genomes in the mutant libraries could be precisely determined.
    Figure Legend Snippet: Construction of the mutant libraries. (A) A schematic representation of the fitness profiling experiment is shown. A 240 bp insert was generated by error-prone PCR and BsaI digestion. The corresponding vector was generated by high-fidelity PCR and BsmBI digestion. Each of the nine plasmid libraries in this study consist of ∼ 50,000 clones. Each viral mutant library was rescued by transfecting ∼ 35 million 293T cells. Each infection was performed with ∼ 10 million A549 cells. (B) A schematic representation of the sequencing library preparation is shown. DNA plasmid mutant library or viral cDNA was used for PCR. This PCR amplified the 240 bp randomized region. The amplicon product was then digested with BpmI, end-repaired, dA-tailed, ligated to sequencing adapters, and sequenced using the Illumina MiSeq platform. BpmI digestion removed the primer region in the amplicon PCR, resulting in sequencing reads covering only the barcode for multiplex sequencing and the 240 bp region that was randomized in the mutant library. With this experimental design, the number of mutations carried by individual genomes in the mutant libraries could be precisely determined.

    Techniques Used: Mutagenesis, Generated, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay, Infection, Sequencing, Amplification, Multiplex Assay

    7) Product Images from "Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9"

    Article Title: Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9

    Journal: ACS Synthetic Biology

    doi: 10.1021/acssynbio.6b00339

    Schematic overview of the basic pWUR_Cas9nt construct. (A) The non-codon-optimized cas9 sp gene was employed for the construction of the pWUR_Cas9nt vector, since S. pyogenes and B. smithii GC content and codon usage are highly similar. In the pNW33n-based basic construct, s pCas9 was placed under the control of P xynA . A Rho-independent terminator from B. subtilis ( 59 ) was introduced after the stop codon of the gene. The spCas9 module is followed by an sgRNA-expressing module that encompasses a spacer which does not target the genome of ET 138. The sgRNA module was placed under the transcriptional control of P pta from B. coagulans (without its RBS), which was followed by a second Rho-independent terminator from B. subtilis . 15 , 49 The spCas9 and sgRNA modules were synthesized as one fragment, which was subsequently cloned into pNW33n through the BspHI and HindIII restriction sites. (B) To prevent double restriction sites and create a modular system, five silent point mutations (C192A, T387C, T1011A, C3126A, G354A) were introduced to the gene (depicted as *). The depicted restriction sites are unique in the construct and introduced to facilitate the exchange of genetic parts. The spacer was easily exchanged to targeting spacers via BsmBI restriction digestion or Gibson assembly. The basic construct did not contain any HR templates, but in cases where these were added, they were always inserted immediately upstream of the spCas9 module and downstream of the origin of replication. (C) Total RNA was isolated from ET 138 wild-type cells transformed with pWUR_Cas9nt or pNW33n and grown at 55, 45, and 37 °C. Six cDNA libraries were produced with rt-PCR and used as templates for PCR with cas9sp-specific primers that amplify a 255 bp region. The PCR results are depicted as follows: lane 1 corresponds to the marker (1kb+ DNA ladder, ThermoFisher), lanes 2–4 correspond to ET 138 wild-type cultures transformed with pWUR_Cas9nt and grown at 55, 42, or 37 °C, respectively, lanes 5–7 correspond to ET 138 wild-type cultures transformed with pNW33n and grown at 55, 42, or 37 °C, respectively, lanes 7, 8, 9, 11, 12 correspond to different negative controls, and lane 10 corresponds to the positive control, for which pWUR_Cas9nt was used as the PCR template.
    Figure Legend Snippet: Schematic overview of the basic pWUR_Cas9nt construct. (A) The non-codon-optimized cas9 sp gene was employed for the construction of the pWUR_Cas9nt vector, since S. pyogenes and B. smithii GC content and codon usage are highly similar. In the pNW33n-based basic construct, s pCas9 was placed under the control of P xynA . A Rho-independent terminator from B. subtilis ( 59 ) was introduced after the stop codon of the gene. The spCas9 module is followed by an sgRNA-expressing module that encompasses a spacer which does not target the genome of ET 138. The sgRNA module was placed under the transcriptional control of P pta from B. coagulans (without its RBS), which was followed by a second Rho-independent terminator from B. subtilis . 15 , 49 The spCas9 and sgRNA modules were synthesized as one fragment, which was subsequently cloned into pNW33n through the BspHI and HindIII restriction sites. (B) To prevent double restriction sites and create a modular system, five silent point mutations (C192A, T387C, T1011A, C3126A, G354A) were introduced to the gene (depicted as *). The depicted restriction sites are unique in the construct and introduced to facilitate the exchange of genetic parts. The spacer was easily exchanged to targeting spacers via BsmBI restriction digestion or Gibson assembly. The basic construct did not contain any HR templates, but in cases where these were added, they were always inserted immediately upstream of the spCas9 module and downstream of the origin of replication. (C) Total RNA was isolated from ET 138 wild-type cells transformed with pWUR_Cas9nt or pNW33n and grown at 55, 45, and 37 °C. Six cDNA libraries were produced with rt-PCR and used as templates for PCR with cas9sp-specific primers that amplify a 255 bp region. The PCR results are depicted as follows: lane 1 corresponds to the marker (1kb+ DNA ladder, ThermoFisher), lanes 2–4 correspond to ET 138 wild-type cultures transformed with pWUR_Cas9nt and grown at 55, 42, or 37 °C, respectively, lanes 5–7 correspond to ET 138 wild-type cultures transformed with pNW33n and grown at 55, 42, or 37 °C, respectively, lanes 7, 8, 9, 11, 12 correspond to different negative controls, and lane 10 corresponds to the positive control, for which pWUR_Cas9nt was used as the PCR template.

    Techniques Used: Construct, Plasmid Preparation, Gas Chromatography, Expressing, Synthesized, Clone Assay, Isolation, Transformation Assay, Produced, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Marker, Positive Control

    Related Articles

    Clone Assay:

    Article Title: Inheritable and Precise Large Genomic Deletions of Non-Coding RNA Genes in Zebrafish Using TALENs
    Article Snippet: The second digestion and ligation step was performed in 10 ul volumes containing 60 ng of each array plasmid, pCS2-TALEN-KKR or pCS2-TALEN-ELD, the last repeat plasmid, 1 ul of Esp3I buffer (NEB buffer 3), 0.6 ul of Esp3I (6 U, NEB), 0.4 ul of T4 ligase (800 U, NEB), and 0.4 ul of 25 mM ATP. .. The second digestion and ligation step was performed in 10 ul volumes containing 60 ng of each array plasmid, pCS2-TALEN-KKR or pCS2-TALEN-ELD, the last repeat plasmid, 1 ul of Esp3I buffer (NEB buffer 3), 0.6 ul of Esp3I (6 U, NEB), 0.4 ul of T4 ligase (800 U, NEB), and 0.4 ul of 25 mM ATP.

    Article Title: A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells
    Article Snippet: Two unique Esp3I restriction sites flanking the gRNA sequence were consecutively introduced using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent). .. The LacZα fragment was then cloned into the plasmid using BsmBI (an isoschizomer of Esp3I) (NEB), replacing the original gRNA. .. The All-in-one CRISPR/Cas9 LacZ plasmid and complete sequence information is available from Addgene.

    Article Title: The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia
    Article Snippet: They were maintained in RPMI 1640 (Life Technologies) supplemented with 10% FBS and 1% penicillin/streptomycin (Life Technologies). .. The constitutive Cas9 expression vector (plasmid LentiCas9-Blast, a gift from Feng Zhang, Addgene plasmid # 52962) [ ] was used to generate the Boff-p210 cell line with stable Cas9 expression. pLKO5.sgRNA.EFS.GFP (Addgene plasmid # 57822) [ ] containing the coding sequence of GFP and a cloning site for sgRNA sequence was digested with Esp3I (BsmBI) (NEB). .. To clone the sgRNAs into the pLKO5 vector, two complementary oligos for each sgRNA were designed including two 4bp-overhang sequences (Table ).

    TALENs:

    Article Title: Inheritable and Precise Large Genomic Deletions of Non-Coding RNA Genes in Zebrafish Using TALENs
    Article Snippet: Paragraph title: Construction of Customized TALENs ... The second digestion and ligation step was performed in 10 ul volumes containing 60 ng of each array plasmid, pCS2-TALEN-KKR or pCS2-TALEN-ELD, the last repeat plasmid, 1 ul of Esp3I buffer (NEB buffer 3), 0.6 ul of Esp3I (6 U, NEB), 0.4 ul of T4 ligase (800 U, NEB), and 0.4 ul of 25 mM ATP.

    Ligation:

    Article Title: Inheritable and Precise Large Genomic Deletions of Non-Coding RNA Genes in Zebrafish Using TALENs
    Article Snippet: The assembled array plasmids were isolated from the correct clones. .. The second digestion and ligation step was performed in 10 ul volumes containing 60 ng of each array plasmid, pCS2-TALEN-KKR or pCS2-TALEN-ELD, the last repeat plasmid, 1 ul of Esp3I buffer (NEB buffer 3), 0.6 ul of Esp3I (6 U, NEB), 0.4 ul of T4 ligase (800 U, NEB), and 0.4 ul of 25 mM ATP. .. The reaction was performed on a PCR machine for 6 cycles of 20 min at 37C and 10 min at 16°C, followed by heating to 50°C for 5 min and then to 80°C for 5 min. Five ul of the final products were used to transform competent cells.

    Mutagenesis:

    Article Title: A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells
    Article Snippet: Two unique Esp3I restriction sites flanking the gRNA sequence were consecutively introduced using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent). .. The LacZα fragment was then cloned into the plasmid using BsmBI (an isoschizomer of Esp3I) (NEB), replacing the original gRNA.

    Isolation:

    Article Title: Inheritable and Precise Large Genomic Deletions of Non-Coding RNA Genes in Zebrafish Using TALENs
    Article Snippet: The second digestion and ligation step was performed in 10 ul volumes containing 60 ng of each array plasmid, pCS2-TALEN-KKR or pCS2-TALEN-ELD, the last repeat plasmid, 1 ul of Esp3I buffer (NEB buffer 3), 0.6 ul of Esp3I (6 U, NEB), 0.4 ul of T4 ligase (800 U, NEB), and 0.4 ul of 25 mM ATP. .. The second digestion and ligation step was performed in 10 ul volumes containing 60 ng of each array plasmid, pCS2-TALEN-KKR or pCS2-TALEN-ELD, the last repeat plasmid, 1 ul of Esp3I buffer (NEB buffer 3), 0.6 ul of Esp3I (6 U, NEB), 0.4 ul of T4 ligase (800 U, NEB), and 0.4 ul of 25 mM ATP.

    Construct:

    Article Title: The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia
    Article Snippet: Paragraph title: Lentiviral constructs, sgRNA design and sgRNA cloning ... The constitutive Cas9 expression vector (plasmid LentiCas9-Blast, a gift from Feng Zhang, Addgene plasmid # 52962) [ ] was used to generate the Boff-p210 cell line with stable Cas9 expression. pLKO5.sgRNA.EFS.GFP (Addgene plasmid # 57822) [ ] containing the coding sequence of GFP and a cloning site for sgRNA sequence was digested with Esp3I (BsmBI) (NEB).

    Polymerase Chain Reaction:

    Article Title: Inheritable and Precise Large Genomic Deletions of Non-Coding RNA Genes in Zebrafish Using TALENs
    Article Snippet: The reaction was performed on a PCR machine for 6 cycles of 20 min at 37°C and 10 min at 16°C, followed by heating to 50°C for 5 min and then to 80°C for 5 min. .. The second digestion and ligation step was performed in 10 ul volumes containing 60 ng of each array plasmid, pCS2-TALEN-KKR or pCS2-TALEN-ELD, the last repeat plasmid, 1 ul of Esp3I buffer (NEB buffer 3), 0.6 ul of Esp3I (6 U, NEB), 0.4 ul of T4 ligase (800 U, NEB), and 0.4 ul of 25 mM ATP.

    Generated:

    Article Title: A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells
    Article Snippet: “All-in-one CRISPR/Cas9 LacZ” was generated from the “scrambled sgRNA control for pCRISPR-CG01” plasmid (Genecopedia). .. The LacZα fragment was then cloned into the plasmid using BsmBI (an isoschizomer of Esp3I) (NEB), replacing the original gRNA.

    DNA Sequencing:

    Article Title: Inheritable and Precise Large Genomic Deletions of Non-Coding RNA Genes in Zebrafish Using TALENs
    Article Snippet: The second digestion and ligation step was performed in 10 ul volumes containing 60 ng of each array plasmid, pCS2-TALEN-KKR or pCS2-TALEN-ELD, the last repeat plasmid, 1 ul of Esp3I buffer (NEB buffer 3), 0.6 ul of Esp3I (6 U, NEB), 0.4 ul of T4 ligase (800 U, NEB), and 0.4 ul of 25 mM ATP. .. The reaction was performed on a PCR machine for 6 cycles of 20 min at 37C and 10 min at 16°C, followed by heating to 50°C for 5 min and then to 80°C for 5 min. Five ul of the final products were used to transform competent cells.

    Expressing:

    Article Title: The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia
    Article Snippet: They were maintained in RPMI 1640 (Life Technologies) supplemented with 10% FBS and 1% penicillin/streptomycin (Life Technologies). .. The constitutive Cas9 expression vector (plasmid LentiCas9-Blast, a gift from Feng Zhang, Addgene plasmid # 52962) [ ] was used to generate the Boff-p210 cell line with stable Cas9 expression. pLKO5.sgRNA.EFS.GFP (Addgene plasmid # 57822) [ ] containing the coding sequence of GFP and a cloning site for sgRNA sequence was digested with Esp3I (BsmBI) (NEB). .. To clone the sgRNAs into the pLKO5 vector, two complementary oligos for each sgRNA were designed including two 4bp-overhang sequences (Table ).

    CRISPR:

    Article Title: A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells
    Article Snippet: “All-in-one CRISPR/Cas9 LacZ” was generated from the “scrambled sgRNA control for pCRISPR-CG01” plasmid (Genecopedia). .. The LacZα fragment was then cloned into the plasmid using BsmBI (an isoschizomer of Esp3I) (NEB), replacing the original gRNA.

    Article Title: The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia
    Article Snippet: The constitutive Cas9 expression vector (plasmid LentiCas9-Blast, a gift from Feng Zhang, Addgene plasmid # 52962) [ ] was used to generate the Boff-p210 cell line with stable Cas9 expression. pLKO5.sgRNA.EFS.GFP (Addgene plasmid # 57822) [ ] containing the coding sequence of GFP and a cloning site for sgRNA sequence was digested with Esp3I (BsmBI) (NEB). .. To clone the sgRNAs into the pLKO5 vector, two complementary oligos for each sgRNA were designed including two 4bp-overhang sequences (Table ).

    Sequencing:

    Article Title: Inheritable and Precise Large Genomic Deletions of Non-Coding RNA Genes in Zebrafish Using TALENs
    Article Snippet: The second digestion and ligation step was performed in 10 ul volumes containing 60 ng of each array plasmid, pCS2-TALEN-KKR or pCS2-TALEN-ELD, the last repeat plasmid, 1 ul of Esp3I buffer (NEB buffer 3), 0.6 ul of Esp3I (6 U, NEB), 0.4 ul of T4 ligase (800 U, NEB), and 0.4 ul of 25 mM ATP. .. The second digestion and ligation step was performed in 10 ul volumes containing 60 ng of each array plasmid, pCS2-TALEN-KKR or pCS2-TALEN-ELD, the last repeat plasmid, 1 ul of Esp3I buffer (NEB buffer 3), 0.6 ul of Esp3I (6 U, NEB), 0.4 ul of T4 ligase (800 U, NEB), and 0.4 ul of 25 mM ATP.

    Article Title: A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells
    Article Snippet: Two unique Esp3I restriction sites flanking the gRNA sequence were consecutively introduced using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent). .. The LacZα fragment was then cloned into the plasmid using BsmBI (an isoschizomer of Esp3I) (NEB), replacing the original gRNA.

    Article Title: The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia
    Article Snippet: They were maintained in RPMI 1640 (Life Technologies) supplemented with 10% FBS and 1% penicillin/streptomycin (Life Technologies). .. The constitutive Cas9 expression vector (plasmid LentiCas9-Blast, a gift from Feng Zhang, Addgene plasmid # 52962) [ ] was used to generate the Boff-p210 cell line with stable Cas9 expression. pLKO5.sgRNA.EFS.GFP (Addgene plasmid # 57822) [ ] containing the coding sequence of GFP and a cloning site for sgRNA sequence was digested with Esp3I (BsmBI) (NEB). .. To clone the sgRNAs into the pLKO5 vector, two complementary oligos for each sgRNA were designed including two 4bp-overhang sequences (Table ).

    Plasmid Preparation:

    Article Title: Inheritable and Precise Large Genomic Deletions of Non-Coding RNA Genes in Zebrafish Using TALENs
    Article Snippet: The assembled array plasmids were isolated from the correct clones. .. The second digestion and ligation step was performed in 10 ul volumes containing 60 ng of each array plasmid, pCS2-TALEN-KKR or pCS2-TALEN-ELD, the last repeat plasmid, 1 ul of Esp3I buffer (NEB buffer 3), 0.6 ul of Esp3I (6 U, NEB), 0.4 ul of T4 ligase (800 U, NEB), and 0.4 ul of 25 mM ATP. .. The reaction was performed on a PCR machine for 6 cycles of 20 min at 37C and 10 min at 16°C, followed by heating to 50°C for 5 min and then to 80°C for 5 min. Five ul of the final products were used to transform competent cells.

    Article Title: A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells
    Article Snippet: Two unique Esp3I restriction sites flanking the gRNA sequence were consecutively introduced using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent). .. The LacZα fragment was then cloned into the plasmid using BsmBI (an isoschizomer of Esp3I) (NEB), replacing the original gRNA. .. The All-in-one CRISPR/Cas9 LacZ plasmid and complete sequence information is available from Addgene.

    Article Title: The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia
    Article Snippet: They were maintained in RPMI 1640 (Life Technologies) supplemented with 10% FBS and 1% penicillin/streptomycin (Life Technologies). .. The constitutive Cas9 expression vector (plasmid LentiCas9-Blast, a gift from Feng Zhang, Addgene plasmid # 52962) [ ] was used to generate the Boff-p210 cell line with stable Cas9 expression. pLKO5.sgRNA.EFS.GFP (Addgene plasmid # 57822) [ ] containing the coding sequence of GFP and a cloning site for sgRNA sequence was digested with Esp3I (BsmBI) (NEB). .. To clone the sgRNAs into the pLKO5 vector, two complementary oligos for each sgRNA were designed including two 4bp-overhang sequences (Table ).

    Software:

    Article Title: The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia
    Article Snippet: The constitutive Cas9 expression vector (plasmid LentiCas9-Blast, a gift from Feng Zhang, Addgene plasmid # 52962) [ ] was used to generate the Boff-p210 cell line with stable Cas9 expression. pLKO5.sgRNA.EFS.GFP (Addgene plasmid # 57822) [ ] containing the coding sequence of GFP and a cloning site for sgRNA sequence was digested with Esp3I (BsmBI) (NEB). .. To clone the sgRNAs into the pLKO5 vector, two complementary oligos for each sgRNA were designed including two 4bp-overhang sequences (Table ).

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    New England Biolabs bsmbi
    Yeast Golden Gate <t>(yGG)</t> to assemble transcription units (TUs) flanked by VEGAS adapters. ( A ) yGG reactions to build TUs destined for VEGAS pathway assembly in S. cerevisiae include five parts: a left VEGAS adapter (LVA), a promoter (PRO), a coding sequence (CDS), a terminator (TER) and a right VEGAS adapter (RVA). Each part is flanked by inwardly facing recognition sequences for the BsaI restriction enzyme, an ‘offset cutter’ which cuts outside its recognition sequence (at positions 1/5 bp downstream) to expose the indicated four base-pair overhangs. All parts are cloned into vectors encoding kanamycin resistance (KAN R ) and an E. coli replication origin (Ori). ( B ) The yGG acceptor vector for VEGAS is designed such that outwardly facing BsaI sites expose overhangs corresponding to the 5′ LVA and 3′ RVA overhangs to promote assembly of the TU in the vector during a one-pot restriction-digestion reaction. The RFP cassette, built for expression in E. coli , is cut out of the vector when a TU correctly assembles, enabling white–red screening. The yGG acceptor vector encodes resistance to ampicillin (AMP R ) ( C ) The structure of a VA-flanked TU assembled by yGG. An assembled TU plus the flanking VA sequences may be released from the yGG acceptor vector by digestion with <t>BsmBI.</t>
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    Yeast Golden Gate (yGG) to assemble transcription units (TUs) flanked by VEGAS adapters. ( A ) yGG reactions to build TUs destined for VEGAS pathway assembly in S. cerevisiae include five parts: a left VEGAS adapter (LVA), a promoter (PRO), a coding sequence (CDS), a terminator (TER) and a right VEGAS adapter (RVA). Each part is flanked by inwardly facing recognition sequences for the BsaI restriction enzyme, an ‘offset cutter’ which cuts outside its recognition sequence (at positions 1/5 bp downstream) to expose the indicated four base-pair overhangs. All parts are cloned into vectors encoding kanamycin resistance (KAN R ) and an E. coli replication origin (Ori). ( B ) The yGG acceptor vector for VEGAS is designed such that outwardly facing BsaI sites expose overhangs corresponding to the 5′ LVA and 3′ RVA overhangs to promote assembly of the TU in the vector during a one-pot restriction-digestion reaction. The RFP cassette, built for expression in E. coli , is cut out of the vector when a TU correctly assembles, enabling white–red screening. The yGG acceptor vector encodes resistance to ampicillin (AMP R ) ( C ) The structure of a VA-flanked TU assembled by yGG. An assembled TU plus the flanking VA sequences may be released from the yGG acceptor vector by digestion with BsmBI.

    Journal: Nucleic Acids Research

    Article Title: Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae

    doi: 10.1093/nar/gkv466

    Figure Lengend Snippet: Yeast Golden Gate (yGG) to assemble transcription units (TUs) flanked by VEGAS adapters. ( A ) yGG reactions to build TUs destined for VEGAS pathway assembly in S. cerevisiae include five parts: a left VEGAS adapter (LVA), a promoter (PRO), a coding sequence (CDS), a terminator (TER) and a right VEGAS adapter (RVA). Each part is flanked by inwardly facing recognition sequences for the BsaI restriction enzyme, an ‘offset cutter’ which cuts outside its recognition sequence (at positions 1/5 bp downstream) to expose the indicated four base-pair overhangs. All parts are cloned into vectors encoding kanamycin resistance (KAN R ) and an E. coli replication origin (Ori). ( B ) The yGG acceptor vector for VEGAS is designed such that outwardly facing BsaI sites expose overhangs corresponding to the 5′ LVA and 3′ RVA overhangs to promote assembly of the TU in the vector during a one-pot restriction-digestion reaction. The RFP cassette, built for expression in E. coli , is cut out of the vector when a TU correctly assembles, enabling white–red screening. The yGG acceptor vector encodes resistance to ampicillin (AMP R ) ( C ) The structure of a VA-flanked TU assembled by yGG. An assembled TU plus the flanking VA sequences may be released from the yGG acceptor vector by digestion with BsmBI.

    Article Snippet: ∼1 μg of yGG-assembled, VA-flanked TU constructs were digested with BsmBI (New England Biolabs, R0580) in a final volume of 20 μl.

    Techniques: Sequencing, Clone Assay, Plasmid Preparation, Expressing

    VEGAS with adapter homology to assemble the carotenoid pathway in S. cerevisiae . ( A ) The four β-carotene pathway genes ( crtE, crtI, crtYB and tHMG1 ), assembled as TUs flanked by the indicated VAs (see Table 2 for PRO and TER parts), were released from the yGG acceptor vector with BsmBI digestion and co-transformed into yeast with the linearized VEGAS assembly vector. ( B ) S. cerevisiae colonies encoding assembled pathways develop a bright yellow color on medium lacking uracil (SC–Ura; left panel) as well as on YPD medium supplemented with G418 (right panel).

    Journal: Nucleic Acids Research

    Article Title: Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae

    doi: 10.1093/nar/gkv466

    Figure Lengend Snippet: VEGAS with adapter homology to assemble the carotenoid pathway in S. cerevisiae . ( A ) The four β-carotene pathway genes ( crtE, crtI, crtYB and tHMG1 ), assembled as TUs flanked by the indicated VAs (see Table 2 for PRO and TER parts), were released from the yGG acceptor vector with BsmBI digestion and co-transformed into yeast with the linearized VEGAS assembly vector. ( B ) S. cerevisiae colonies encoding assembled pathways develop a bright yellow color on medium lacking uracil (SC–Ura; left panel) as well as on YPD medium supplemented with G418 (right panel).

    Article Snippet: ∼1 μg of yGG-assembled, VA-flanked TU constructs were digested with BsmBI (New England Biolabs, R0580) in a final volume of 20 μl.

    Techniques: Plasmid Preparation, Transformation Assay

    VEGAS with adapter homology to assemble a five-gene pathway. ( A ) The pathway consisting of VA-flanked TUs assembled by yGG may be released in one piece from the yGG acceptor vector by digestion with BsmBI (scissors). ( B ) A genetic pathway may be assembled into the linearized VEGAS assembly vector in S. cerevisiae by homologous recombination between VAs that flank TUs (TU1–5). X's indicate homologous recombination.

    Journal: Nucleic Acids Research

    Article Title: Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae

    doi: 10.1093/nar/gkv466

    Figure Lengend Snippet: VEGAS with adapter homology to assemble a five-gene pathway. ( A ) The pathway consisting of VA-flanked TUs assembled by yGG may be released in one piece from the yGG acceptor vector by digestion with BsmBI (scissors). ( B ) A genetic pathway may be assembled into the linearized VEGAS assembly vector in S. cerevisiae by homologous recombination between VAs that flank TUs (TU1–5). X's indicate homologous recombination.

    Article Snippet: ∼1 μg of yGG-assembled, VA-flanked TU constructs were digested with BsmBI (New England Biolabs, R0580) in a final volume of 20 μl.

    Techniques: Plasmid Preparation, Homologous Recombination

    Split-and-pool assembly of DNA synthons. (A) Entry synthons are flanked on both sides by recognition sequences for the type IIS endonucleases BsaI and BsmBI. Restriction by either BsaI or BsmBI selectively exposes user-definable 4-base cohesive overhang sequences (5′-XXXX vs. 5′-xxxx) at one end of the synthon, while maintaining orthogonal protection groups (with 5′-YYYY vs. 5′-zzzz overhangs) at the opposite end. (B) Schematic representation of the ‘split-and-pool’ assembly principle. Cohesive ends of entry synthons are selectively deprotected by digestion with either BsaI or BsmBI. Pooling of the deprotected synthons in the presence of ligase results in unidirectional assembly, affording an idempotent tandem repeat synthon by restoration of orthogonal protecting groups on opposite ends. Each product module can recursively enter the assembly cycle (left panel) N times to yield concatameric synthons with 2N elements. The same strategy can be applied to the assembly of heterosynthons (dashed box), which allows for the engineering of chimeric and multimodular proteins or polycistronic genes.

    Journal: PLoS ONE

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection

    doi: 10.1371/journal.pone.0037617

    Figure Lengend Snippet: Split-and-pool assembly of DNA synthons. (A) Entry synthons are flanked on both sides by recognition sequences for the type IIS endonucleases BsaI and BsmBI. Restriction by either BsaI or BsmBI selectively exposes user-definable 4-base cohesive overhang sequences (5′-XXXX vs. 5′-xxxx) at one end of the synthon, while maintaining orthogonal protection groups (with 5′-YYYY vs. 5′-zzzz overhangs) at the opposite end. (B) Schematic representation of the ‘split-and-pool’ assembly principle. Cohesive ends of entry synthons are selectively deprotected by digestion with either BsaI or BsmBI. Pooling of the deprotected synthons in the presence of ligase results in unidirectional assembly, affording an idempotent tandem repeat synthon by restoration of orthogonal protecting groups on opposite ends. Each product module can recursively enter the assembly cycle (left panel) N times to yield concatameric synthons with 2N elements. The same strategy can be applied to the assembly of heterosynthons (dashed box), which allows for the engineering of chimeric and multimodular proteins or polycistronic genes.

    Article Snippet: 50–100 µg DNA were mixed with 10x NEB reaction buffer 3 and either 5 µl BsaI or BsmBI (New England Biolabs) in a total volume of 50–100 µl and incubated at 50°C for 1 h on a thermal cycler (Bio-Rad S1000).

    Techniques:

    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.

    Journal: PLoS ONE

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection

    doi: 10.1371/journal.pone.0037617

    Figure Lengend Snippet: Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.

    Article Snippet: 50–100 µg DNA were mixed with 10x NEB reaction buffer 3 and either 5 µl BsaI or BsmBI (New England Biolabs) in a total volume of 50–100 µl and incubated at 50°C for 1 h on a thermal cycler (Bio-Rad S1000).

    Techniques: Incubation, Plasmid Preparation, Clone Assay, Expressing, Construct

    Figure 1. Capture of murine CDRH3 into a library of human scFv. (A) BalbC mice, divided in three groups, were either kept naïve or immunized with hIFNγ or hCCL5. (B) After sacrifice, the spleen and the lymph nodes were kept separated and VH repertoires were recovered by PCR. CDRH3 were then amplified from the VH pool by PCR, along with recognition sites for FokI, a type IIS restriction enzyme, were added to the inserts. CDR3 are represented in gray, CDR1 and 2 in white. (C) Murine inserts were then digested with FokI and (D) ligated to the human acceptor scFv library, itself digested with BsmBI. This second type IIS enzyme permitted the removal of a non-diversified stuffer sequence (“S”) at the location of CDRH3 and the generation of compatible cohesive ends for the incorporation of CDRH3. The acceptor library also contains synthetic diversity at the location of the CDRL3 (“L3”) and tags (“T,” a c-myc and a His tag) at the scFv C-terminal for purification purposes. Libraries featuring mouse CDRH3 were analyzed by NGS, which covered the CDRH3 and part of VH framework 3 (“NGS”). (E) The fusion to gIII allowed the expression of scFv at the surface of M13 phage for phage display selection.

    Journal:

    Article Title: Comparing CDRH3 diversity captured from secondary lymphoid organs for the generation of recombinant human antibodies

    doi: 10.4161/mabs.25592

    Figure Lengend Snippet: Figure 1. Capture of murine CDRH3 into a library of human scFv. (A) BalbC mice, divided in three groups, were either kept naïve or immunized with hIFNγ or hCCL5. (B) After sacrifice, the spleen and the lymph nodes were kept separated and VH repertoires were recovered by PCR. CDRH3 were then amplified from the VH pool by PCR, along with recognition sites for FokI, a type IIS restriction enzyme, were added to the inserts. CDR3 are represented in gray, CDR1 and 2 in white. (C) Murine inserts were then digested with FokI and (D) ligated to the human acceptor scFv library, itself digested with BsmBI. This second type IIS enzyme permitted the removal of a non-diversified stuffer sequence (“S”) at the location of CDRH3 and the generation of compatible cohesive ends for the incorporation of CDRH3. The acceptor library also contains synthetic diversity at the location of the CDRL3 (“L3”) and tags (“T,” a c-myc and a His tag) at the scFv C-terminal for purification purposes. Libraries featuring mouse CDRH3 were analyzed by NGS, which covered the CDRH3 and part of VH framework 3 (“NGS”). (E) The fusion to gIII allowed the expression of scFv at the surface of M13 phage for phage display selection.

    Article Snippet: The acceptor library and the CDRH3 inserts were digested with BsmBI and FokI (both from New England Biolabs), respectively, which generated compatible cohesive ends, and purified with Chroma Spin T1000 columns (Clontech) and Dynabeads (M280 Streptavidin from Invitrogen), respectively.

    Techniques: Mouse Assay, Polymerase Chain Reaction, Amplification, Sequencing, Purification, Next-Generation Sequencing, Expressing, Selection

    Assembly process of the secretory immunoglobulin A (sIgA). ( a ) Collection of basic parts necessary to construct a secretory IgA. Each basic part is cloned in a pGem-T vector. 35S, SP, VH-CH, VL-CL, SC, JC, Tnos, correspond, respectively, to the 35s CMV promoter, pectate lyase signal peptide, variable and constant regions of the heavy chain, variable and constant regions of the light chain, secretory component, J-chain and nopaline synthase terminator; ( b ) Example of domestication of a basic part. The 35s promoter is flanked by fixed BsmbI recognition-cleavage sites. The overhangs left by the BsmbI restriction enzyme converge with GB pDGB vectors on 5′ and on 3′, with the next basic part to assemble; ( c ) Multipartite assembly of the basic parts to form the four different transcriptional units: heavy chain (HC), light chain (LC), secretory component (SC) and J-chain (JC), into level Ω-GB destiny vectors (pDGB_1AB3 and pDGB_3AB2); ( d ) Binary assembly of transcriptional units in level α-GB destination vectors (pDGB_C12B and pDGB_A12C), in order to construct two different composite parts—IgA and JC-SC; ( e ) Last construct of sIgA by binary assembly of two composite parts in a final pDGB; ( f ) Example of restriction analysis of four colonies of each construct: left, BglII (expected bands of 2825, 1886 and 1197) and BglI (expected bands of 2345, 1790, 1498 and 275) restriction analysis of the HC transcriptional unit; middle, BglII (expected bands of 4183, 2495 and 1228 kDa) restriction analysis of IgA; right, BamHI (expected bands of 6815, 5857 and 913 kDa) and BsaI (expected bands of 10,664 + 2921 kDa) restriction analysis of sIgA.

    Journal: International Journal of Molecular Sciences

    Article Title: Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants

    doi: 10.3390/ijms14036205

    Figure Lengend Snippet: Assembly process of the secretory immunoglobulin A (sIgA). ( a ) Collection of basic parts necessary to construct a secretory IgA. Each basic part is cloned in a pGem-T vector. 35S, SP, VH-CH, VL-CL, SC, JC, Tnos, correspond, respectively, to the 35s CMV promoter, pectate lyase signal peptide, variable and constant regions of the heavy chain, variable and constant regions of the light chain, secretory component, J-chain and nopaline synthase terminator; ( b ) Example of domestication of a basic part. The 35s promoter is flanked by fixed BsmbI recognition-cleavage sites. The overhangs left by the BsmbI restriction enzyme converge with GB pDGB vectors on 5′ and on 3′, with the next basic part to assemble; ( c ) Multipartite assembly of the basic parts to form the four different transcriptional units: heavy chain (HC), light chain (LC), secretory component (SC) and J-chain (JC), into level Ω-GB destiny vectors (pDGB_1AB3 and pDGB_3AB2); ( d ) Binary assembly of transcriptional units in level α-GB destination vectors (pDGB_C12B and pDGB_A12C), in order to construct two different composite parts—IgA and JC-SC; ( e ) Last construct of sIgA by binary assembly of two composite parts in a final pDGB; ( f ) Example of restriction analysis of four colonies of each construct: left, BglII (expected bands of 2825, 1886 and 1197) and BglI (expected bands of 2345, 1790, 1498 and 275) restriction analysis of the HC transcriptional unit; middle, BglII (expected bands of 4183, 2495 and 1228 kDa) restriction analysis of IgA; right, BamHI (expected bands of 6815, 5857 and 913 kDa) and BsaI (expected bands of 10,664 + 2921 kDa) restriction analysis of sIgA.

    Article Snippet: Assembly reactions were performed basically as described by [ ] using BsaI and BsmBI (New England Biolabs, Ipswich, MA, USA) as restriction enzymes in 25-cycle digestion/ligation reactions.

    Techniques: Construct, Clone Assay, Plasmid Preparation, Liquid Chromatography