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    Structured Review

    New England Biolabs bsmbi
    Schematic overview of the basic <t>pWUR_Cas9nt</t> 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 <t>BsmBI</t> 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.
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    Images

    1) 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, Expressing, Synthesized, Clone Assay, Isolation, Transformation Assay, Produced, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Marker, Positive Control

    2) Product Images from "Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining"

    Article Title: Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

    Journal: bioRxiv

    doi: 10.1101/754242

    Golden Transformation method for ADP1 genome engineering. Two PCR reactions are performed to create upstream (U) and downstream (D) genomic target flanks with added terminal BsaI and BsmBI type IIS restriction sites as depicted. The two PCR products can then be combined via BsaI Golden Gate assembly (GGA) with the selection cassette to form a replacement DNA or combined with one another and optionally with additional genetic parts (not shown) via BsmBI GGA to form a rescue cassette. The positive-negative selection cassette ( tdk - kanR ) is maintained on the high-copy pBTK622 plasmid that has an origin that does not replicate in A. baylyi . The first GGA reaction is added to an A. baylyi culture and then plated on LB-Kan to select for transformants with the replacement cassette integrated into the genome. Then, transformation of the second assembly reaction with counterselection on LB-AZT is used to move the unmarked deletions/additions encoded on the rescue cassette into the genome.
    Figure Legend Snippet: Golden Transformation method for ADP1 genome engineering. Two PCR reactions are performed to create upstream (U) and downstream (D) genomic target flanks with added terminal BsaI and BsmBI type IIS restriction sites as depicted. The two PCR products can then be combined via BsaI Golden Gate assembly (GGA) with the selection cassette to form a replacement DNA or combined with one another and optionally with additional genetic parts (not shown) via BsmBI GGA to form a rescue cassette. The positive-negative selection cassette ( tdk - kanR ) is maintained on the high-copy pBTK622 plasmid that has an origin that does not replicate in A. baylyi . The first GGA reaction is added to an A. baylyi culture and then plated on LB-Kan to select for transformants with the replacement cassette integrated into the genome. Then, transformation of the second assembly reaction with counterselection on LB-AZT is used to move the unmarked deletions/additions encoded on the rescue cassette into the genome.

    Techniques Used: Transformation Assay, Polymerase Chain Reaction, Selection, Plasmid Preparation

    3) 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

    4) Product Images from "Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair"

    Article Title: Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair

    Journal: bioRxiv

    doi: 10.1101/481069

    An assay for massively parallel profiling of the outcomes of CRISPR/Cas9-mediated double-stranded DNA break repair. (A) Schematic of library of 200 bp oligonucleotides encoding sgRNAs targeting a large number of designed 20 bp spacers, with their matched target sequence encoded in cis . In our primary experiment, 70,000 targets of random sequence were designed and cloned. (B) After array-based synthesis and PCR amplification of the library, BsmBI restriction sites at either end were used for cloning into a modified lentiviral construct. The library was bottlenecked to 12,286 targets to facilitate greater coverage of independent NHEJ-mediated events corresponding to each target. Monoclonal HEK293T cells expressing Cas9 were transduced with packaged lentivirus. Cells were harvested at 5 days after transduction, and a region including both the spacer and the target was PCR amplified from genomic DNA for high-throughput sequencing. The sequences of mutated targets were aligned to their corresponding unmutated reference (assigned based on the spacer sequence).
    Figure Legend Snippet: An assay for massively parallel profiling of the outcomes of CRISPR/Cas9-mediated double-stranded DNA break repair. (A) Schematic of library of 200 bp oligonucleotides encoding sgRNAs targeting a large number of designed 20 bp spacers, with their matched target sequence encoded in cis . In our primary experiment, 70,000 targets of random sequence were designed and cloned. (B) After array-based synthesis and PCR amplification of the library, BsmBI restriction sites at either end were used for cloning into a modified lentiviral construct. The library was bottlenecked to 12,286 targets to facilitate greater coverage of independent NHEJ-mediated events corresponding to each target. Monoclonal HEK293T cells expressing Cas9 were transduced with packaged lentivirus. Cells were harvested at 5 days after transduction, and a region including both the spacer and the target was PCR amplified from genomic DNA for high-throughput sequencing. The sequences of mutated targets were aligned to their corresponding unmutated reference (assigned based on the spacer sequence).

    Techniques Used: CRISPR, Sequencing, Polymerase Chain Reaction, Amplification, Clone Assay, Modification, Construct, Non-Homologous End Joining, Expressing, Transduction, Next-Generation Sequencing

    5) Product Images from "Joint Universal Modular Plasmids (JUMP): A flexible and comprehensive platform for synthetic biology"

    Article Title: Joint Universal Modular Plasmids (JUMP): A flexible and comprehensive platform for synthetic biology

    Journal: bioRxiv

    doi: 10.1101/799585

    JUMP design and secondary sites. 2A) In SEVA (Standard European Vector Architecture) plasmids, three common short DNA sequences (black) flank three variable regions (coloured). Variable regions are the OriV (origin of replication), AbR (antibiotic selection marker) and “cargo” (any expression cassette). The invariable regions are two transcription terminators flanking the cargo (T1 and T0,) and origin of conjugation (oriT). Invariable regions also contain rare cutting sites, forbidden in the sequence of variable regions. 2B) JUMP is designed as special cargo of SEVA vectors to allow compatibility with future OriV’s and AbR’s of the collection. The cargo contains Upstream Module (outwards AarI); BioBricks prefix (XbaI, EcoRI); Main Module (a screening reporter gene flanked by outwards BsaI and inwards BsmBI for level 1, and vice-versa for level 2); BioBrick suffix (SpeI, PstI); and Downstream Module (outwards BbsI). SEVA’s canonical SpeI site was removed to allow BioBricks compatibility. 2C) Building constructs to test similar genes (G 1 to G n ) as sequences of interest (SOI) that depend on common auxiliary factors (AF) with conventional MoClo might require multiple assembly steps per SOI. D) Introduction of the auxiliary factors in vector chassis using orthogonal use of secondary modules.
    Figure Legend Snippet: JUMP design and secondary sites. 2A) In SEVA (Standard European Vector Architecture) plasmids, three common short DNA sequences (black) flank three variable regions (coloured). Variable regions are the OriV (origin of replication), AbR (antibiotic selection marker) and “cargo” (any expression cassette). The invariable regions are two transcription terminators flanking the cargo (T1 and T0,) and origin of conjugation (oriT). Invariable regions also contain rare cutting sites, forbidden in the sequence of variable regions. 2B) JUMP is designed as special cargo of SEVA vectors to allow compatibility with future OriV’s and AbR’s of the collection. The cargo contains Upstream Module (outwards AarI); BioBricks prefix (XbaI, EcoRI); Main Module (a screening reporter gene flanked by outwards BsaI and inwards BsmBI for level 1, and vice-versa for level 2); BioBrick suffix (SpeI, PstI); and Downstream Module (outwards BbsI). SEVA’s canonical SpeI site was removed to allow BioBricks compatibility. 2C) Building constructs to test similar genes (G 1 to G n ) as sequences of interest (SOI) that depend on common auxiliary factors (AF) with conventional MoClo might require multiple assembly steps per SOI. D) Introduction of the auxiliary factors in vector chassis using orthogonal use of secondary modules.

    Techniques Used: Plasmid Preparation, Selection, Marker, Expressing, Conjugation Assay, Sequencing, Construct

    6) 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

    7) Product Images from "Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology"

    Article Title: Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology

    Journal: Synthetic Biology

    doi: 10.1093/synbio/ysab003

    JUMP design and secondary sites. ( A ) In Standard European Vector Architecture (SEVA) plasmids, three common short DNA sequences (black) flank three variable regions (colored). Variable regions are the OriV (origin of replication), AbR (antibiotic selection marker) and ‘cargo’ (any expression cassette). The invariable regions are two transcription terminators flanking the cargo (T1 and T0) and origin of conjugation (oriT). Invariable regions also contain rare cutting sites, forbidden in the sequence of variable regions. ( B ) JUMP is designed as a special cargo of SEVA vectors to allow compatibility with future OriV's and AbR's of the collection. The cargo contains the upstream modular site (with outward-facing AarI sites); BioBrick prefix (XbaI, EcoRI); main modular site (a screening reporter gene flanked by outwards-facing BsaI and inwards-facing BsmBI sites for level 1, and vice versa for level 2); BioBrick suffix (SpeI, PstI); and downstream modular site (with outwards-facing BbsI sites). SEVA's canonical SpeI site was removed to allow BioBrick compatibility. ( C ) Building constructs to test similar genes (G 1 to G n ) as sequences of interest (SOI) that depend on common auxiliary factors (Aux.) with conventional modular cloning might require multiple assembly steps per SOI: the SOI is first assembled by itself and then combined with the auxiliary elements. ( D ) Introduction of the auxiliary factors in vector chassis using orthogonal use of secondary sites reduces number of assembly steps to combine the SOI with the auxiliary factor. Squares indicate restriction sites for BsaI (blue), BsmBI (red), AarI (yellow) and BbsI (green).
    Figure Legend Snippet: JUMP design and secondary sites. ( A ) In Standard European Vector Architecture (SEVA) plasmids, three common short DNA sequences (black) flank three variable regions (colored). Variable regions are the OriV (origin of replication), AbR (antibiotic selection marker) and ‘cargo’ (any expression cassette). The invariable regions are two transcription terminators flanking the cargo (T1 and T0) and origin of conjugation (oriT). Invariable regions also contain rare cutting sites, forbidden in the sequence of variable regions. ( B ) JUMP is designed as a special cargo of SEVA vectors to allow compatibility with future OriV's and AbR's of the collection. The cargo contains the upstream modular site (with outward-facing AarI sites); BioBrick prefix (XbaI, EcoRI); main modular site (a screening reporter gene flanked by outwards-facing BsaI and inwards-facing BsmBI sites for level 1, and vice versa for level 2); BioBrick suffix (SpeI, PstI); and downstream modular site (with outwards-facing BbsI sites). SEVA's canonical SpeI site was removed to allow BioBrick compatibility. ( C ) Building constructs to test similar genes (G 1 to G n ) as sequences of interest (SOI) that depend on common auxiliary factors (Aux.) with conventional modular cloning might require multiple assembly steps per SOI: the SOI is first assembled by itself and then combined with the auxiliary elements. ( D ) Introduction of the auxiliary factors in vector chassis using orthogonal use of secondary sites reduces number of assembly steps to combine the SOI with the auxiliary factor. Squares indicate restriction sites for BsaI (blue), BsmBI (red), AarI (yellow) and BbsI (green).

    Techniques Used: Plasmid Preparation, Selection, Marker, Expressing, Conjugation Assay, Sequencing, Construct, Clone Assay

    Use of secondary sites with two-step assembly. ( A ) Two-step assembly works by assembling inserts first and then ligating the assembly reaction with the destination vector. Squares indicate restriction sites for BsaI (blue), BsmBI (red), AarI (yellow) and BbsI (green). ( B ) Domestication and characterization of promoters using level-0 promoter acceptor. ( C ) Domestication and characterization of terminators using level-0 terminator acceptor. T and CT terminators differ in the 5' end of the part ( Supplementary Figure S2 ), with CT terminators including a stop codon. In B and C, fluorescence was normalized to OD (600 nm) and is shown as % of that of the J23100 promoter. Error bars indicate standard deviation, n = 9 (biological and technical triplicate).
    Figure Legend Snippet: Use of secondary sites with two-step assembly. ( A ) Two-step assembly works by assembling inserts first and then ligating the assembly reaction with the destination vector. Squares indicate restriction sites for BsaI (blue), BsmBI (red), AarI (yellow) and BbsI (green). ( B ) Domestication and characterization of promoters using level-0 promoter acceptor. ( C ) Domestication and characterization of terminators using level-0 terminator acceptor. T and CT terminators differ in the 5' end of the part ( Supplementary Figure S2 ), with CT terminators including a stop codon. In B and C, fluorescence was normalized to OD (600 nm) and is shown as % of that of the J23100 promoter. Error bars indicate standard deviation, n = 9 (biological and technical triplicate).

    Techniques Used: Plasmid Preparation, Fluorescence, Standard Deviation

    8) 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

    9) Product Images from "Rapid and efficient synthetic assembly of multiplex luciferase reporter plasmids for the simultaneous monitoring of up to six cellular signaling pathways"

    Article Title: Rapid and efficient synthetic assembly of multiplex luciferase reporter plasmids for the simultaneous monitoring of up to six cellular signaling pathways

    Journal: Current protocols in molecular biology

    doi: 10.1002/cpmb.121

    Synthetic assembly of the final multiplex hextuple luciferase vector (Basic protocol 3). ( A ) Overview of the in silico scarless assembly of a multiplex luciferase reporter in the destination vector Omega Destination-CMV:ELuc:bGH. Five “transcriptional reporter” plasmids, AlphaA 5xNF-KB:RedF:bGHpA (Addgene #124530) reporting on NF-κβ pathway signaling using red firefly luciferase (RF), AlphaB 7xSMAD:FLuc:bGHpA (Addgene #124531) reporting on TGF-β signaling using firefly luciferase (FL), AlphaC 3xDBE:Renilla:bGHpA (Addgene #124535) reporting on FoxO pathway signaling using renilla luciferase (Re), AlphaD 2xp53:NLuc:bGHpA (Addgene #124533) reporting on p53 pathway signaling using nano luciferase (NL), and AlphaE 6xAP1_RE:GrRenilla:bGHpA (Addgene #124534) reporting on MAPK/JNK pathway signaling using green renilla luciferase (GR), as well as the destination vector Omega Destination-CMV:ELuc:bGH containing the control enhanced beetle luciferase (EL) for normalization purposes are incubated together with BsmBI and T4 DNA ligase. Correct multipartitie stitching of all five BsmBI-released transcriptional luciferase reporter units into BsmBI-opended Omega Destination-CMV:ELuc:bGH, results in the final multiplex luciferase vector MLRV2:NF-kb-SMAD-DBE-P53-AP1 (Addgene #124536), consisting of five transcriptional luciferase reporter units stitched together in a specified order, and one control luciferase reporter unit (constitutively expressed ELuc luciferase). Assembled plasmids are identified as white colored colonies that are characterized further (see C ), while religated Omega Destination-CMV:ELuc:bGH plasmids are pink to purple colored due to the presence of the colorimetric marker, tinsel purple. ( B ) Overview of the cloning reaction. Prepare a reaction mix containing 75 ng of the final pink/white destination vector (Omega Destination-CMV:ELuc:bGH), 75 ng of each of the luciferase entry vectors, the type IIs restriction enzyme BsmBI, T4 DNA ligase and 10x T4 DNA ligase buffer. Start the assembly protocol that cycles 25 times between 37°C and 16°C. ( C ) Extended assembly cycling reaction conditions, with 50 cycles of 37°C and 16°C, followed by 1h at 37°C to favor digestion of uncut plasmids, 20 minutes at 85°C to denature the enzymes and a prolonged incubation at 16°C. ( D ) Restriction enzyme digestion of 3 white colored colonies using ScaI (6510, 2219, 1790, 1543, 1088 and 486 bps) and XhoI (6500, 2263, 1508, 1098, 979, 794 and 494 bps), and uncut DNA. All colonies show the correct digestion pattern.
    Figure Legend Snippet: Synthetic assembly of the final multiplex hextuple luciferase vector (Basic protocol 3). ( A ) Overview of the in silico scarless assembly of a multiplex luciferase reporter in the destination vector Omega Destination-CMV:ELuc:bGH. Five “transcriptional reporter” plasmids, AlphaA 5xNF-KB:RedF:bGHpA (Addgene #124530) reporting on NF-κβ pathway signaling using red firefly luciferase (RF), AlphaB 7xSMAD:FLuc:bGHpA (Addgene #124531) reporting on TGF-β signaling using firefly luciferase (FL), AlphaC 3xDBE:Renilla:bGHpA (Addgene #124535) reporting on FoxO pathway signaling using renilla luciferase (Re), AlphaD 2xp53:NLuc:bGHpA (Addgene #124533) reporting on p53 pathway signaling using nano luciferase (NL), and AlphaE 6xAP1_RE:GrRenilla:bGHpA (Addgene #124534) reporting on MAPK/JNK pathway signaling using green renilla luciferase (GR), as well as the destination vector Omega Destination-CMV:ELuc:bGH containing the control enhanced beetle luciferase (EL) for normalization purposes are incubated together with BsmBI and T4 DNA ligase. Correct multipartitie stitching of all five BsmBI-released transcriptional luciferase reporter units into BsmBI-opended Omega Destination-CMV:ELuc:bGH, results in the final multiplex luciferase vector MLRV2:NF-kb-SMAD-DBE-P53-AP1 (Addgene #124536), consisting of five transcriptional luciferase reporter units stitched together in a specified order, and one control luciferase reporter unit (constitutively expressed ELuc luciferase). Assembled plasmids are identified as white colored colonies that are characterized further (see C ), while religated Omega Destination-CMV:ELuc:bGH plasmids are pink to purple colored due to the presence of the colorimetric marker, tinsel purple. ( B ) Overview of the cloning reaction. Prepare a reaction mix containing 75 ng of the final pink/white destination vector (Omega Destination-CMV:ELuc:bGH), 75 ng of each of the luciferase entry vectors, the type IIs restriction enzyme BsmBI, T4 DNA ligase and 10x T4 DNA ligase buffer. Start the assembly protocol that cycles 25 times between 37°C and 16°C. ( C ) Extended assembly cycling reaction conditions, with 50 cycles of 37°C and 16°C, followed by 1h at 37°C to favor digestion of uncut plasmids, 20 minutes at 85°C to denature the enzymes and a prolonged incubation at 16°C. ( D ) Restriction enzyme digestion of 3 white colored colonies using ScaI (6510, 2219, 1790, 1543, 1088 and 486 bps) and XhoI (6500, 2263, 1508, 1098, 979, 794 and 494 bps), and uncut DNA. All colonies show the correct digestion pattern.

    Techniques Used: Multiplex Assay, Luciferase, Plasmid Preparation, In Silico, Incubation, Marker, Clone Assay

    10) 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

    11) Product Images from "Systematic Assembly and Genetic Manipulation of the Mouse Hepatitis Virus A59 Genome"

    Article Title: Systematic Assembly and Genetic Manipulation of the Mouse Hepatitis Virus A59 Genome

    Journal: SARS- and Other Coronaviruses

    doi: 10.1007/978-1-59745-181-9_21

    Engineering mutations with the No See’m approach: ( A ) The position of interest, a glutamate (CAG) to alanine (GTC) mutation at position 13354-56, is targeted and mapped to the MHV-E fragment. The vector sequence, the wild-type fragment, and the mutated fragment are analyzed to determine which type IIs restriction enzymes do not cut any of them, and one of these is selected as the restriction site to add. In this case BbsI is selected. (B ) Primers are designed that add the mutation and the restriction site in proper orientation so that upon digestion the restriction site is eliminated and complementing sticky ends are produced. The BbsI cut sites are highlighted in gray. The mutated codon is bold and underlined. (C ) PCR is conducted using vector specific primers along with the newly designed primers that incorporate the mutation of interest to produce two amplicons. These are cloned and purified, and then digested with BbsI and BsmBI to produce a mutated MHV-E fragment that can then be incorporated into the full-length clone.
    Figure Legend Snippet: Engineering mutations with the No See’m approach: ( A ) The position of interest, a glutamate (CAG) to alanine (GTC) mutation at position 13354-56, is targeted and mapped to the MHV-E fragment. The vector sequence, the wild-type fragment, and the mutated fragment are analyzed to determine which type IIs restriction enzymes do not cut any of them, and one of these is selected as the restriction site to add. In this case BbsI is selected. (B ) Primers are designed that add the mutation and the restriction site in proper orientation so that upon digestion the restriction site is eliminated and complementing sticky ends are produced. The BbsI cut sites are highlighted in gray. The mutated codon is bold and underlined. (C ) PCR is conducted using vector specific primers along with the newly designed primers that incorporate the mutation of interest to produce two amplicons. These are cloned and purified, and then digested with BbsI and BsmBI to produce a mutated MHV-E fragment that can then be incorporated into the full-length clone.

    Techniques Used: Mutagenesis, Plasmid Preparation, Sequencing, Produced, Polymerase Chain Reaction, Clone Assay, Purification

    12) Product Images from "Targeted insertional mutagenesis libraries for deep domain insertion profiling"

    Article Title: Targeted insertional mutagenesis libraries for deep domain insertion profiling

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz1110

    ( A ) SPINE workflow. A target gene sequence is divided into shorter fragments. For each fragment, an oligo pool is generated with a genetic handle (purple) at each amino acid position. Flanking barcodes (different hues of yellow) mediate specific amplification of each subpool, which is then joined with the PCR-amplified target gene backbone in BsmBI-mediated Golden Gate cloning. This process is repeated for each fragment, and the resulting intermediate libraries are pooled. The genetic handle is replaced by a domain of interest (orange) through BsaI-mediated Golden Gate cloning, resulting in the final domain insertion library. ( B ) Barcode Design. Each OLS subpool is designed with a bio-orthogonal barcode followed by a BsmBI recognition site that cuts within the sequence of a gene. Every barcode and BsmBI cut site are unique to a given subpool minimizing the chance for undesired assembly. The genetic handle is designed with outward-facing BsaI recognition sites that enable cutting within the beginning and ends of short flexible serine–glycine linkers. These linkers are the only scars that result from assembly and can be programmed to be any sequence at least 4 bp long.
    Figure Legend Snippet: ( A ) SPINE workflow. A target gene sequence is divided into shorter fragments. For each fragment, an oligo pool is generated with a genetic handle (purple) at each amino acid position. Flanking barcodes (different hues of yellow) mediate specific amplification of each subpool, which is then joined with the PCR-amplified target gene backbone in BsmBI-mediated Golden Gate cloning. This process is repeated for each fragment, and the resulting intermediate libraries are pooled. The genetic handle is replaced by a domain of interest (orange) through BsaI-mediated Golden Gate cloning, resulting in the final domain insertion library. ( B ) Barcode Design. Each OLS subpool is designed with a bio-orthogonal barcode followed by a BsmBI recognition site that cuts within the sequence of a gene. Every barcode and BsmBI cut site are unique to a given subpool minimizing the chance for undesired assembly. The genetic handle is designed with outward-facing BsaI recognition sites that enable cutting within the beginning and ends of short flexible serine–glycine linkers. These linkers are the only scars that result from assembly and can be programmed to be any sequence at least 4 bp long.

    Techniques Used: Sequencing, Generated, Amplification, Polymerase Chain Reaction, Clone Assay

    13) 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

    14) Product Images from "Multiplexed sgRNA Expression Allows Versatile Single Non-repetitive DNA Labeling and Endogenous Gene Regulation"

    Article Title: Multiplexed sgRNA Expression Allows Versatile Single Non-repetitive DNA Labeling and Endogenous Gene Regulation

    Journal: bioRxiv

    doi: 10.1101/121905

    Hierarchical assembly of multiplexed sgRNA expression plasmid. ( A ). Workflow for the hierarchical assembly of 20 sgRNAs. U6 promoter and sgRNA scaffold are PCR amplified and then ligated with each annealed oligo in 20 separated Golden Gate reaction with a type IIs restriction endonuclease BsmBI (Step 1). Each Golden Gate reaction mix was then used as template for amplification without purification by 20 pair of primers, which can be grouped into 4 categories, each member in a group with different adaptors (Step 2). The purified 20 sgRNAs expression cassettes were then separated into 4 group and implemented with the second round Golden Gate reaction using BsaI (Step 3). The four fragments each carried 5 sgRNAs expression cassettes were purified and then ligated together into a third round Golden Gate reaction with a receptor plasmid using BpiI (Step 4). Due to the repetitive sequence of each sgRNA expression cassette, each step should be verified by agar gel electrophoresis to guarantee that each reaction was carried out successfully. ( B ). A typical gel of the first round PCR result for the successful ligation of U6, annealed oligoes, sgRNA scaffold. Trans2K plus II DNA ladder (TransGen Biotech) was shown in the left. The 20 individual sgRNA expression cascade all show the expected length on the gel, indicating that this cloning method is quite robust. A detail to note is that the first and fifth sgRNA in each group are slightly longer than the remaining one. This is because two handles are added to the 5’ of the first sgRNA and 3’ of the fifth sgRNA to facilitate the PCR of the five sgRNAs in the following step. ( C ). A typical gel of the second round PCR result for the successful ligation of five sgRNA expression cassettes into one fragment. Trans2K plus II DNA ladder (TransGen Biotech) was shown in the left. Due to the repetitive nature of the five sgRNAs fragment, laddering effect was observed. The indicated repeat number was shown in the right. ( D ). A typical gel of the verification of the final all-in-one plasmid. Trans15K DNA ladder (TransGen Biotech) was shown in the left. All the plasmids were linearized by endonuclease enzyme. The empty vector was also shown as a control. All the tested six plasmids have the right insertion size, suggesting the high efficiency of the assembly process.
    Figure Legend Snippet: Hierarchical assembly of multiplexed sgRNA expression plasmid. ( A ). Workflow for the hierarchical assembly of 20 sgRNAs. U6 promoter and sgRNA scaffold are PCR amplified and then ligated with each annealed oligo in 20 separated Golden Gate reaction with a type IIs restriction endonuclease BsmBI (Step 1). Each Golden Gate reaction mix was then used as template for amplification without purification by 20 pair of primers, which can be grouped into 4 categories, each member in a group with different adaptors (Step 2). The purified 20 sgRNAs expression cassettes were then separated into 4 group and implemented with the second round Golden Gate reaction using BsaI (Step 3). The four fragments each carried 5 sgRNAs expression cassettes were purified and then ligated together into a third round Golden Gate reaction with a receptor plasmid using BpiI (Step 4). Due to the repetitive sequence of each sgRNA expression cassette, each step should be verified by agar gel electrophoresis to guarantee that each reaction was carried out successfully. ( B ). A typical gel of the first round PCR result for the successful ligation of U6, annealed oligoes, sgRNA scaffold. Trans2K plus II DNA ladder (TransGen Biotech) was shown in the left. The 20 individual sgRNA expression cascade all show the expected length on the gel, indicating that this cloning method is quite robust. A detail to note is that the first and fifth sgRNA in each group are slightly longer than the remaining one. This is because two handles are added to the 5’ of the first sgRNA and 3’ of the fifth sgRNA to facilitate the PCR of the five sgRNAs in the following step. ( C ). A typical gel of the second round PCR result for the successful ligation of five sgRNA expression cassettes into one fragment. Trans2K plus II DNA ladder (TransGen Biotech) was shown in the left. Due to the repetitive nature of the five sgRNAs fragment, laddering effect was observed. The indicated repeat number was shown in the right. ( D ). A typical gel of the verification of the final all-in-one plasmid. Trans15K DNA ladder (TransGen Biotech) was shown in the left. All the plasmids were linearized by endonuclease enzyme. The empty vector was also shown as a control. All the tested six plasmids have the right insertion size, suggesting the high efficiency of the assembly process.

    Techniques Used: Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Purification, Sequencing, Nucleic Acid Electrophoresis, Ligation, Clone Assay

    15) Product Images from "Systematic Assembly and Genetic Manipulation of the Mouse Hepatitis Virus A59 Genome"

    Article Title: Systematic Assembly and Genetic Manipulation of the Mouse Hepatitis Virus A59 Genome

    Journal: SARS- and Other Coronaviruses

    doi: 10.1007/978-1-59745-181-9_21

    Engineering mutations with the No See’m approach: ( A ) The position of interest, a glutamate (CAG) to alanine (GTC) mutation at position 13354-56, is targeted and mapped to the MHV-E fragment. The vector sequence, the wild-type fragment, and the mutated fragment are analyzed to determine which type IIs restriction enzymes do not cut any of them, and one of these is selected as the restriction site to add. In this case BbsI is selected. (B ) Primers are designed that add the mutation and the restriction site in proper orientation so that upon digestion the restriction site is eliminated and complementing sticky ends are produced. The BbsI cut sites are highlighted in gray. The mutated codon is bold and underlined. (C ) PCR is conducted using vector specific primers along with the newly designed primers that incorporate the mutation of interest to produce two amplicons. These are cloned and purified, and then digested with BbsI and BsmBI to produce a mutated MHV-E fragment that can then be incorporated into the full-length clone.
    Figure Legend Snippet: Engineering mutations with the No See’m approach: ( A ) The position of interest, a glutamate (CAG) to alanine (GTC) mutation at position 13354-56, is targeted and mapped to the MHV-E fragment. The vector sequence, the wild-type fragment, and the mutated fragment are analyzed to determine which type IIs restriction enzymes do not cut any of them, and one of these is selected as the restriction site to add. In this case BbsI is selected. (B ) Primers are designed that add the mutation and the restriction site in proper orientation so that upon digestion the restriction site is eliminated and complementing sticky ends are produced. The BbsI cut sites are highlighted in gray. The mutated codon is bold and underlined. (C ) PCR is conducted using vector specific primers along with the newly designed primers that incorporate the mutation of interest to produce two amplicons. These are cloned and purified, and then digested with BbsI and BsmBI to produce a mutated MHV-E fragment that can then be incorporated into the full-length clone.

    Techniques Used: Mutagenesis, Plasmid Preparation, Sequencing, Produced, Polymerase Chain Reaction, Clone Assay, Purification

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    New England Biolabs piggybac plasmid pb tre3g bsmbi ef1α hygror p2a rtta home made
    Single non-repetitive sequence labeling by multiplexed sgRNAs expression. ( A ). Schematic diagram of a non-repetitive sequence labeling of the genome. With the cell line that expressed proper level of SunTag system and 20 different sgRNAs tiling a short range sequence, arbitrary target loci can be labeled. The all-in-one plasmid carries 20 sgRNAs expression cassette each with a U6 promoter and poly U terminator so that individual sgRNA can be expressed independently. Six sgRNAs are shown as a demo here. The background fluorescent signal arises from the unbound dCas9 with multiple sfGFP molecules and the free sfGFP single molecules. Minimal level and proper stoichiometry (1:24) of SunTag system is the key point for the successful labeling. In principle, the 20 sgRNAs can direct 20 dCas9-sunTag molecule, that is 480 sfGFP molecules, to the target loci to gather the fluorescent signal. ( B ). Workflow for the establishment of live imaging system with proper expression level. <t>Piggybac</t> plasmids dCas9-(GCN4) X24 <t>-P2A-BFP</t> and scFV-sfGFP were used to construct the stable cell line. The MDA-MB-231 cells were co-transfected with SunTag expression plasmids in PiggyBac backbone and transposase expression plasmid, followed by puromycin and hygromycin selection for about 2 weeks. sfGFP and BFP double positive single clones with minimal background expression and high labeling efficiency were selected and transient transfected with multiple sgRNA expression plasmid to test the performance of each clone in subsequent live imaging experiment. ( C ). The labeling result of non-repetitive sequence in human cells. By transfection of 20 sgRNAs from an all-in-one plasmid targeting the first intron of human MUC4 gene, bright fluorescent puncta (indicated by arrow) can be visualized. Fluorescence in situ hybridization against the third exon of MUC4 was used to verified the labeling result of CRISPR. The nucleus was stained with DAPI. The merged image showed the colonialization of CRISPR signal and FISH signal. All scale bars are 5 μm. ( D ). Single non-repetitive sequence can be labeled in different cell cycle stages. In G1 of interphase, two loci can be visualized. In G2 of interphase, four loci appeared as closely located pairs. During mitosis, the duplicated loci were allocated into two cells. In anaphase and telophase, the position of MUC4 loci nearly mirrored each other All scale bars are 10 μm.
    Piggybac Plasmid Pb Tre3g Bsmbi Ef1α Hygror P2a Rtta Home Made, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/piggybac plasmid pb tre3g bsmbi ef1α hygror p2a rtta home made/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    piggybac plasmid pb tre3g bsmbi ef1α hygror p2a rtta home made - by Bioz Stars, 2022-07
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    86
    New England Biolabs piggybac plasmid pb tre3g bsmbi ef1α puror p2a rtta home made
    Single non-repetitive sequence labeling by multiplexed sgRNAs expression. ( A ). Schematic diagram of a non-repetitive sequence labeling of the genome. With the cell line that expressed proper level of SunTag system and 20 different sgRNAs tiling a short range sequence, arbitrary target loci can be labeled. The all-in-one plasmid carries 20 sgRNAs expression cassette each with a U6 promoter and poly U terminator so that individual sgRNA can be expressed independently. Six sgRNAs are shown as a demo here. The background fluorescent signal arises from the unbound dCas9 with multiple sfGFP molecules and the free sfGFP single molecules. Minimal level and proper stoichiometry (1:24) of SunTag system is the key point for the successful labeling. In principle, the 20 sgRNAs can direct 20 dCas9-sunTag molecule, that is 480 sfGFP molecules, to the target loci to gather the fluorescent signal. ( B ). Workflow for the establishment of live imaging system with proper expression level. <t>Piggybac</t> plasmids dCas9-(GCN4) X24 <t>-P2A-BFP</t> and scFV-sfGFP were used to construct the stable cell line. The MDA-MB-231 cells were co-transfected with SunTag expression plasmids in PiggyBac backbone and transposase expression plasmid, followed by puromycin and hygromycin selection for about 2 weeks. sfGFP and BFP double positive single clones with minimal background expression and high labeling efficiency were selected and transient transfected with multiple sgRNA expression plasmid to test the performance of each clone in subsequent live imaging experiment. ( C ). The labeling result of non-repetitive sequence in human cells. By transfection of 20 sgRNAs from an all-in-one plasmid targeting the first intron of human MUC4 gene, bright fluorescent puncta (indicated by arrow) can be visualized. Fluorescence in situ hybridization against the third exon of MUC4 was used to verified the labeling result of CRISPR. The nucleus was stained with DAPI. The merged image showed the colonialization of CRISPR signal and FISH signal. All scale bars are 5 μm. ( D ). Single non-repetitive sequence can be labeled in different cell cycle stages. In G1 of interphase, two loci can be visualized. In G2 of interphase, four loci appeared as closely located pairs. During mitosis, the duplicated loci were allocated into two cells. In anaphase and telophase, the position of MUC4 loci nearly mirrored each other All scale bars are 10 μm.
    Piggybac Plasmid Pb Tre3g Bsmbi Ef1α Puror P2a Rtta Home Made, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/piggybac plasmid pb tre3g bsmbi ef1α puror p2a rtta home made/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    piggybac plasmid pb tre3g bsmbi ef1α puror p2a rtta home made - by Bioz Stars, 2022-07
    86/100 stars
      Buy from Supplier

    95
    New England Biolabs bsmbi
    Schematic overview of the basic <t>pWUR_Cas9nt</t> 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 <t>BsmBI</t> 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.
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    Single non-repetitive sequence labeling by multiplexed sgRNAs expression. ( A ). Schematic diagram of a non-repetitive sequence labeling of the genome. With the cell line that expressed proper level of SunTag system and 20 different sgRNAs tiling a short range sequence, arbitrary target loci can be labeled. The all-in-one plasmid carries 20 sgRNAs expression cassette each with a U6 promoter and poly U terminator so that individual sgRNA can be expressed independently. Six sgRNAs are shown as a demo here. The background fluorescent signal arises from the unbound dCas9 with multiple sfGFP molecules and the free sfGFP single molecules. Minimal level and proper stoichiometry (1:24) of SunTag system is the key point for the successful labeling. In principle, the 20 sgRNAs can direct 20 dCas9-sunTag molecule, that is 480 sfGFP molecules, to the target loci to gather the fluorescent signal. ( B ). Workflow for the establishment of live imaging system with proper expression level. Piggybac plasmids dCas9-(GCN4) X24 -P2A-BFP and scFV-sfGFP were used to construct the stable cell line. The MDA-MB-231 cells were co-transfected with SunTag expression plasmids in PiggyBac backbone and transposase expression plasmid, followed by puromycin and hygromycin selection for about 2 weeks. sfGFP and BFP double positive single clones with minimal background expression and high labeling efficiency were selected and transient transfected with multiple sgRNA expression plasmid to test the performance of each clone in subsequent live imaging experiment. ( C ). The labeling result of non-repetitive sequence in human cells. By transfection of 20 sgRNAs from an all-in-one plasmid targeting the first intron of human MUC4 gene, bright fluorescent puncta (indicated by arrow) can be visualized. Fluorescence in situ hybridization against the third exon of MUC4 was used to verified the labeling result of CRISPR. The nucleus was stained with DAPI. The merged image showed the colonialization of CRISPR signal and FISH signal. All scale bars are 5 μm. ( D ). Single non-repetitive sequence can be labeled in different cell cycle stages. In G1 of interphase, two loci can be visualized. In G2 of interphase, four loci appeared as closely located pairs. During mitosis, the duplicated loci were allocated into two cells. In anaphase and telophase, the position of MUC4 loci nearly mirrored each other All scale bars are 10 μm.

    Journal: bioRxiv

    Article Title: Multiplexed sgRNA Expression Allows Versatile Single Non-repetitive DNA Labeling and Endogenous Gene Regulation

    doi: 10.1101/121905

    Figure Lengend Snippet: Single non-repetitive sequence labeling by multiplexed sgRNAs expression. ( A ). Schematic diagram of a non-repetitive sequence labeling of the genome. With the cell line that expressed proper level of SunTag system and 20 different sgRNAs tiling a short range sequence, arbitrary target loci can be labeled. The all-in-one plasmid carries 20 sgRNAs expression cassette each with a U6 promoter and poly U terminator so that individual sgRNA can be expressed independently. Six sgRNAs are shown as a demo here. The background fluorescent signal arises from the unbound dCas9 with multiple sfGFP molecules and the free sfGFP single molecules. Minimal level and proper stoichiometry (1:24) of SunTag system is the key point for the successful labeling. In principle, the 20 sgRNAs can direct 20 dCas9-sunTag molecule, that is 480 sfGFP molecules, to the target loci to gather the fluorescent signal. ( B ). Workflow for the establishment of live imaging system with proper expression level. Piggybac plasmids dCas9-(GCN4) X24 -P2A-BFP and scFV-sfGFP were used to construct the stable cell line. The MDA-MB-231 cells were co-transfected with SunTag expression plasmids in PiggyBac backbone and transposase expression plasmid, followed by puromycin and hygromycin selection for about 2 weeks. sfGFP and BFP double positive single clones with minimal background expression and high labeling efficiency were selected and transient transfected with multiple sgRNA expression plasmid to test the performance of each clone in subsequent live imaging experiment. ( C ). The labeling result of non-repetitive sequence in human cells. By transfection of 20 sgRNAs from an all-in-one plasmid targeting the first intron of human MUC4 gene, bright fluorescent puncta (indicated by arrow) can be visualized. Fluorescence in situ hybridization against the third exon of MUC4 was used to verified the labeling result of CRISPR. The nucleus was stained with DAPI. The merged image showed the colonialization of CRISPR signal and FISH signal. All scale bars are 5 μm. ( D ). Single non-repetitive sequence can be labeled in different cell cycle stages. In G1 of interphase, two loci can be visualized. In G2 of interphase, four loci appeared as closely located pairs. During mitosis, the duplicated loci were allocated into two cells. In anaphase and telophase, the position of MUC4 loci nearly mirrored each other All scale bars are 10 μm.

    Article Snippet: The scFV-sfGFP-GB1-NLSSV40 fragment was amplified from plasmid pHR-scFv-GCN4-sfGFP-GB1-NLS-dWPRE (Addgene Plasmid #60906) and then ligated into PiggyBac plasmid pB-TRE3G-BsmBI-EF1α-HygroR-P2A-rtTA (home-made) by Golden Gate assembly with BsmBI and T4 ligase (NEB).

    Techniques: Sequencing, Labeling, Expressing, Plasmid Preparation, Imaging, Construct, Stable Transfection, Multiple Displacement Amplification, Transfection, Selection, Clone Assay, Fluorescence, In Situ Hybridization, CRISPR, Staining, Fluorescence In Situ Hybridization

    Single non-repetitive sequence labeling by multiplexed sgRNAs expression. ( A ). Schematic diagram of a non-repetitive sequence labeling of the genome. With the cell line that expressed proper level of SunTag system and 20 different sgRNAs tiling a short range sequence, arbitrary target loci can be labeled. The all-in-one plasmid carries 20 sgRNAs expression cassette each with a U6 promoter and poly U terminator so that individual sgRNA can be expressed independently. Six sgRNAs are shown as a demo here. The background fluorescent signal arises from the unbound dCas9 with multiple sfGFP molecules and the free sfGFP single molecules. Minimal level and proper stoichiometry (1:24) of SunTag system is the key point for the successful labeling. In principle, the 20 sgRNAs can direct 20 dCas9-sunTag molecule, that is 480 sfGFP molecules, to the target loci to gather the fluorescent signal. ( B ). Workflow for the establishment of live imaging system with proper expression level. Piggybac plasmids dCas9-(GCN4) X24 -P2A-BFP and scFV-sfGFP were used to construct the stable cell line. The MDA-MB-231 cells were co-transfected with SunTag expression plasmids in PiggyBac backbone and transposase expression plasmid, followed by puromycin and hygromycin selection for about 2 weeks. sfGFP and BFP double positive single clones with minimal background expression and high labeling efficiency were selected and transient transfected with multiple sgRNA expression plasmid to test the performance of each clone in subsequent live imaging experiment. ( C ). The labeling result of non-repetitive sequence in human cells. By transfection of 20 sgRNAs from an all-in-one plasmid targeting the first intron of human MUC4 gene, bright fluorescent puncta (indicated by arrow) can be visualized. Fluorescence in situ hybridization against the third exon of MUC4 was used to verified the labeling result of CRISPR. The nucleus was stained with DAPI. The merged image showed the colonialization of CRISPR signal and FISH signal. All scale bars are 5 μm. ( D ). Single non-repetitive sequence can be labeled in different cell cycle stages. In G1 of interphase, two loci can be visualized. In G2 of interphase, four loci appeared as closely located pairs. During mitosis, the duplicated loci were allocated into two cells. In anaphase and telophase, the position of MUC4 loci nearly mirrored each other All scale bars are 10 μm.

    Journal: bioRxiv

    Article Title: Multiplexed sgRNA Expression Allows Versatile Single Non-repetitive DNA Labeling and Endogenous Gene Regulation

    doi: 10.1101/121905

    Figure Lengend Snippet: Single non-repetitive sequence labeling by multiplexed sgRNAs expression. ( A ). Schematic diagram of a non-repetitive sequence labeling of the genome. With the cell line that expressed proper level of SunTag system and 20 different sgRNAs tiling a short range sequence, arbitrary target loci can be labeled. The all-in-one plasmid carries 20 sgRNAs expression cassette each with a U6 promoter and poly U terminator so that individual sgRNA can be expressed independently. Six sgRNAs are shown as a demo here. The background fluorescent signal arises from the unbound dCas9 with multiple sfGFP molecules and the free sfGFP single molecules. Minimal level and proper stoichiometry (1:24) of SunTag system is the key point for the successful labeling. In principle, the 20 sgRNAs can direct 20 dCas9-sunTag molecule, that is 480 sfGFP molecules, to the target loci to gather the fluorescent signal. ( B ). Workflow for the establishment of live imaging system with proper expression level. Piggybac plasmids dCas9-(GCN4) X24 -P2A-BFP and scFV-sfGFP were used to construct the stable cell line. The MDA-MB-231 cells were co-transfected with SunTag expression plasmids in PiggyBac backbone and transposase expression plasmid, followed by puromycin and hygromycin selection for about 2 weeks. sfGFP and BFP double positive single clones with minimal background expression and high labeling efficiency were selected and transient transfected with multiple sgRNA expression plasmid to test the performance of each clone in subsequent live imaging experiment. ( C ). The labeling result of non-repetitive sequence in human cells. By transfection of 20 sgRNAs from an all-in-one plasmid targeting the first intron of human MUC4 gene, bright fluorescent puncta (indicated by arrow) can be visualized. Fluorescence in situ hybridization against the third exon of MUC4 was used to verified the labeling result of CRISPR. The nucleus was stained with DAPI. The merged image showed the colonialization of CRISPR signal and FISH signal. All scale bars are 5 μm. ( D ). Single non-repetitive sequence can be labeled in different cell cycle stages. In G1 of interphase, two loci can be visualized. In G2 of interphase, four loci appeared as closely located pairs. During mitosis, the duplicated loci were allocated into two cells. In anaphase and telophase, the position of MUC4 loci nearly mirrored each other All scale bars are 10 μm.

    Article Snippet: Construction of other plasmids used in this work The NLSSv40 -dCas9-(NLSSv40 )x3 -(GCN4-v4 )x24 -NLSSv40 -P2A-BFP fragment was amplified by PCR from plasmid pHRdSV40-NLS-dCas9-(GCN4-V4 )X24 -NLS-P2A-BFP-dWPRE (Addgene Plasmid #60910) and then ligated into PiggyBac plasmid pB-TRE3G-BsmBI-EF1α-PuroR-P2A-rtTA (home-made) by Golden Gate assembly with BsmBI and T4 ligase (NEB).

    Techniques: Sequencing, Labeling, Expressing, Plasmid Preparation, Imaging, Construct, Stable Transfection, Multiple Displacement Amplification, Transfection, Selection, Clone Assay, Fluorescence, In Situ Hybridization, CRISPR, Staining, Fluorescence In Situ Hybridization

    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.

    Journal: ACS Synthetic Biology

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

    doi: 10.1021/acssynbio.6b00339

    Figure Lengend 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.

    Article Snippet: Annealed oligos and plasmid pWUR_Cas9nt were digested with BspEI and BsmBI (NEB).

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

    Golden Transformation method for ADP1 genome engineering. Two PCR reactions are performed to create upstream (U) and downstream (D) genomic target flanks with added terminal BsaI and BsmBI type IIS restriction sites as depicted. The two PCR products can then be combined via BsaI Golden Gate assembly (GGA) with the selection cassette to form a replacement DNA or combined with one another and optionally with additional genetic parts (not shown) via BsmBI GGA to form a rescue cassette. The positive-negative selection cassette ( tdk - kanR ) is maintained on the high-copy pBTK622 plasmid that has an origin that does not replicate in A. baylyi . The first GGA reaction is added to an A. baylyi culture and then plated on LB-Kan to select for transformants with the replacement cassette integrated into the genome. Then, transformation of the second assembly reaction with counterselection on LB-AZT is used to move the unmarked deletions/additions encoded on the rescue cassette into the genome.

    Journal: bioRxiv

    Article Title: Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

    doi: 10.1101/754242

    Figure Lengend Snippet: Golden Transformation method for ADP1 genome engineering. Two PCR reactions are performed to create upstream (U) and downstream (D) genomic target flanks with added terminal BsaI and BsmBI type IIS restriction sites as depicted. The two PCR products can then be combined via BsaI Golden Gate assembly (GGA) with the selection cassette to form a replacement DNA or combined with one another and optionally with additional genetic parts (not shown) via BsmBI GGA to form a rescue cassette. The positive-negative selection cassette ( tdk - kanR ) is maintained on the high-copy pBTK622 plasmid that has an origin that does not replicate in A. baylyi . The first GGA reaction is added to an A. baylyi culture and then plated on LB-Kan to select for transformants with the replacement cassette integrated into the genome. Then, transformation of the second assembly reaction with counterselection on LB-AZT is used to move the unmarked deletions/additions encoded on the rescue cassette into the genome.

    Article Snippet: Golden Gate assembly DNA fragments for transformation were constructed using Golden Gate Assembly (GGA) reactions containing 1 U/µl BsaI-HF or 0.5 U/µl BsmBI and 150 U/µl of either T7 or T4 DNA ligase in T4 DNA ligase buffer (New England Biolabs) ( , ).

    Techniques: Transformation Assay, Polymerase Chain Reaction, Selection, Plasmid Preparation