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    BsaI
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    BsaI 5 000 units
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    Restriction Enzymes
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    New England Biolabs bsa i
    BsaI
    BsaI 5 000 units
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    Average 99 stars, based on 50 article reviews
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    bsa i - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Clarithromycin-Based Standard Triple Therapy Can Still Be Effective for Helicobacter pylori Eradication in Some Parts of the Korea"

    Article Title: Clarithromycin-Based Standard Triple Therapy Can Still Be Effective for Helicobacter pylori Eradication in Some Parts of the Korea

    Journal: Journal of Korean Medical Science

    doi: 10.3346/jkms.2014.29.9.1240

    Restriction fragment length polymorphism analysis of 23S rRNA amplicons: ( A ) digestion with Bsa I and ( B ) digestion with Bbs I. The A2144G mutations are observed in lanes 1 to 2, but not in lanes 3 to 5. Note that the A2143G mutation detected by digestion with Bbs I was not detected in any of the strains studied. Lanes 3 to 5 reveal the T2183C mutation, as assessed by DNA sequencing. Lane M, 100 bp DNA size markers (indicated to the left of the gels in base pairs); lane C, H. pylori ATCC 43504; lane 1 to 5, clarithromycin-resistant H. pylori strains.
    Figure Legend Snippet: Restriction fragment length polymorphism analysis of 23S rRNA amplicons: ( A ) digestion with Bsa I and ( B ) digestion with Bbs I. The A2144G mutations are observed in lanes 1 to 2, but not in lanes 3 to 5. Note that the A2143G mutation detected by digestion with Bbs I was not detected in any of the strains studied. Lanes 3 to 5 reveal the T2183C mutation, as assessed by DNA sequencing. Lane M, 100 bp DNA size markers (indicated to the left of the gels in base pairs); lane C, H. pylori ATCC 43504; lane 1 to 5, clarithromycin-resistant H. pylori strains.

    Techniques Used: Mutagenesis, DNA Sequencing

    2) Product Images from "FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science"

    Article Title: FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science

    Journal: Human Gene Therapy

    doi: 10.1089/hum.2015.172

    Construction of the FusX1–4 libraries. (A) Component plasmids used to construct the pFusX1–4 libraries. pXX-1 and pXX-10 are single-RVD (repeat-variable diresidue) encoding plasmids from the original Golden Gate system (2.0) 16 ; pXX-M and -MM are new, single RVD modules with designated sequence and Bsa I overhangs for ligation in between pXX-1 and pXX-10 to form 3-mer intermediates in pFusX1–4 libraries. pXX-MM includes extra silent mutations and is used only to construct the pFusX3 library, providing a specific primer-binding site for sequencing of long TALE (transcription activator-like effector) domain. “XX” represents any of the four RVD modules: HD, NG, NI, NN. (B) Schematic diagram showing sequential ligation of single RVD component plasmids into the four intermediate vectors: pFusX1, pFusX2, pFusX3, pFusX4. Dotted arrows indicate ligation at compatible overhangs generated by Bsa I.
    Figure Legend Snippet: Construction of the FusX1–4 libraries. (A) Component plasmids used to construct the pFusX1–4 libraries. pXX-1 and pXX-10 are single-RVD (repeat-variable diresidue) encoding plasmids from the original Golden Gate system (2.0) 16 ; pXX-M and -MM are new, single RVD modules with designated sequence and Bsa I overhangs for ligation in between pXX-1 and pXX-10 to form 3-mer intermediates in pFusX1–4 libraries. pXX-MM includes extra silent mutations and is used only to construct the pFusX3 library, providing a specific primer-binding site for sequencing of long TALE (transcription activator-like effector) domain. “XX” represents any of the four RVD modules: HD, NG, NI, NN. (B) Schematic diagram showing sequential ligation of single RVD component plasmids into the four intermediate vectors: pFusX1, pFusX2, pFusX3, pFusX4. Dotted arrows indicate ligation at compatible overhangs generated by Bsa I.

    Techniques Used: Construct, Sequencing, Ligation, Binding Assay, Generated

    3) Product Images from "PCR-Restriction Fragment Length Polymorphism Can Also Detect Point Mutation A2142C in the 23S rRNA Gene, Associated with Helicobacter pylori Resistance to Clarithromycin"

    Article Title: PCR-Restriction Fragment Length Polymorphism Can Also Detect Point Mutation A2142C in the 23S rRNA Gene, Associated with Helicobacter pylori Resistance to Clarithromycin

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.46.4.1156-1157.2002

    Detection of mutation A2142C by Bce AI-mediated restriction digestion. The restriction fragments of the 267-bp PCR products were analyzed by electrophoresis on a 5% agarose Resophor gel (A) or on a 12% polyacrylamide gel (B) stained with ethidium bromide. (A) PCR-RFLP analysis of mutations A2142G, A2143G, and A2142C occurring in domain V of the 23S rRNA gene of H. pylori . Lanes 1 and 8, 25-bp DNA Step Ladder molecular size markers (Promega). Lanes 2 and 3, PCR products of the wild-type and A2142G H. pylori strains digested with Bbs I, respectively. Lanes 4 and 5, PCR products of the wild-type and A2143G H. pylori strains digested with Bsa I, respectively. Lanes 6 and 7, PCR products of the wild-type and A2142C H. pylori strains digested with Bce AI, respectively. (B) PCR product of the H. pylori strain with mutation A2142C digested with Bce AI. Lanes 2 and 3, amplified wild-type PCR product and amplified PCR product presenting the A2142C mutation, respectively. Lane 1, 25-bp DNA Step Ladder (Promega). The wild-type H. pylori ).
    Figure Legend Snippet: Detection of mutation A2142C by Bce AI-mediated restriction digestion. The restriction fragments of the 267-bp PCR products were analyzed by electrophoresis on a 5% agarose Resophor gel (A) or on a 12% polyacrylamide gel (B) stained with ethidium bromide. (A) PCR-RFLP analysis of mutations A2142G, A2143G, and A2142C occurring in domain V of the 23S rRNA gene of H. pylori . Lanes 1 and 8, 25-bp DNA Step Ladder molecular size markers (Promega). Lanes 2 and 3, PCR products of the wild-type and A2142G H. pylori strains digested with Bbs I, respectively. Lanes 4 and 5, PCR products of the wild-type and A2143G H. pylori strains digested with Bsa I, respectively. Lanes 6 and 7, PCR products of the wild-type and A2142C H. pylori strains digested with Bce AI, respectively. (B) PCR product of the H. pylori strain with mutation A2142C digested with Bce AI. Lanes 2 and 3, amplified wild-type PCR product and amplified PCR product presenting the A2142C mutation, respectively. Lane 1, 25-bp DNA Step Ladder (Promega). The wild-type H. pylori ).

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Electrophoresis, Staining, Amplification

    4) Product Images from "Targeted mutagenesis in soybean using the CRISPR-Cas9 system"

    Article Title: Targeted mutagenesis in soybean using the CRISPR-Cas9 system

    Journal: Scientific Reports

    doi: 10.1038/srep10342

    Construction of binary vectors for genome editing in soybean. Cas9 fused with a single nuclear localization signal (NLS) is expressed with a Cauliflower mosaic virus 35s (CaMV 35s) promoter. Synthetic guide RNA (sgRNA) is derived using U6 promoters. ( a ) Arabidopsis thaliana U6-26 promoter ( b ) Glycine max U6-10 promoter. Sequences containing two Bsa I sites are located between the U6 promoter and the sgRNA scaffold. These sequences can be easily replaced with a gene-specific sgRNA seed. LB: left border; RB: right border.
    Figure Legend Snippet: Construction of binary vectors for genome editing in soybean. Cas9 fused with a single nuclear localization signal (NLS) is expressed with a Cauliflower mosaic virus 35s (CaMV 35s) promoter. Synthetic guide RNA (sgRNA) is derived using U6 promoters. ( a ) Arabidopsis thaliana U6-26 promoter ( b ) Glycine max U6-10 promoter. Sequences containing two Bsa I sites are located between the U6 promoter and the sgRNA scaffold. These sequences can be easily replaced with a gene-specific sgRNA seed. LB: left border; RB: right border.

    Techniques Used: Derivative Assay

    5) Product Images from "Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence"

    Article Title: Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

    Journal: BMC Biochemistry

    doi: 10.1186/1471-2091-12-47

    Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 466 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked). Enzyme purity and reaction steps controls: lane 1, uncut 437 bp PCR fragment amplified from pGCN1 plasmid; lane 2, uncut 480 bp PCR fragment amplified from pGCN2 plasmid; lane 3, BsaI-cut 437 bp fragment; lane 4, BsaI-cut 480 bp fragment; lane 5, BsaI restriction fragment I (191 bp) filled in with BrdUTP isolated from agarose gel; lane 6, BsaI restriction fragment III (270 bp) filled in with BrdUTP isolated from agarose gel; lane 7, BsaI-cut 437 bp fragment, purified and back-ligated; lane 8, BsaI-cut 437 bp fragment, purified, incubated with Bst exo- DNA Pol without dNTPs and back-ligated. Incorporation reaction: lane 9, fragment I (191 bp) filled in with dTTP, ligated to BrdU-labeled fragment III (270 bp); lane 10, fragment I (191 bp) filled in with BrdUTP, ligated to BrdU-labeled fragment III (270 bp). I, III BsaI restriction fragments numbered as in Figure 1.
    Figure Legend Snippet: Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 466 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked). Enzyme purity and reaction steps controls: lane 1, uncut 437 bp PCR fragment amplified from pGCN1 plasmid; lane 2, uncut 480 bp PCR fragment amplified from pGCN2 plasmid; lane 3, BsaI-cut 437 bp fragment; lane 4, BsaI-cut 480 bp fragment; lane 5, BsaI restriction fragment I (191 bp) filled in with BrdUTP isolated from agarose gel; lane 6, BsaI restriction fragment III (270 bp) filled in with BrdUTP isolated from agarose gel; lane 7, BsaI-cut 437 bp fragment, purified and back-ligated; lane 8, BsaI-cut 437 bp fragment, purified, incubated with Bst exo- DNA Pol without dNTPs and back-ligated. Incorporation reaction: lane 9, fragment I (191 bp) filled in with dTTP, ligated to BrdU-labeled fragment III (270 bp); lane 10, fragment I (191 bp) filled in with BrdUTP, ligated to BrdU-labeled fragment III (270 bp). I, III BsaI restriction fragments numbered as in Figure 1.

    Techniques Used: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Isolation, Agarose Gel Electrophoresis, Purification, Incubation, Labeling

    Assessment of various DNA polymerases for their ability to incorporate BrdU . Complete and incomplete specific incorporation reactions (Figure 1) were carried out with 5 DNA Polymerases: Bst exo - (thermophilic), T4 (mesophilic), Taq (thermophilic), OptiTaq (thermophilic blend) and Pfu (hyperthermophilic) in the presence of BrdUTP. Lanes M, Perfect 100 bp Ladder; lane 1, PCR 1 fragment (379 bp); lane 2, BsaI-cleaved PCR 1 fragment; lane 3, PCR 2 fragment (625 bp); lane 4, BsaI-cleaved PCR 2 fragment; lane 5, BsaI restriction fragments: I (363 bp) and III (609 bp). Lanes 6-18 reactions with specified DNA Polymerases: lane 6, restriction fragments: I and III, T4; lane 7, restriction fragments: I and III, Bst exo - ; lane 8, restriction fragments: I and III, Bst exo - , T4 DNA Ligase; lane 9, restriction fragments: I and III, T4; lane 10, restriction fragments: I and III, T4, T4 DNA Ligase; lane 11, restriction fragments: I and III, Taq; lane 12, restriction fragments: I and III, Taq, T4 DNA Ligase; lane 13, restriction fragments: I and III, OptiTaq; lane 14, restriction fragments: I and III, OptiTaq, T4 DNA Ligase; lane 15, restriction fragments: I and III, Tfl; lane 16, restriction fragments: I and III, Tfl, T4 DNA Ligase; lane 17, restriction fragments: I and III, Pfu; lane 18, restriction fragments: I and III, Pfu, T4 DNA Ligase. I, III BsaI restriction fragments numbered as in Figure 1.
    Figure Legend Snippet: Assessment of various DNA polymerases for their ability to incorporate BrdU . Complete and incomplete specific incorporation reactions (Figure 1) were carried out with 5 DNA Polymerases: Bst exo - (thermophilic), T4 (mesophilic), Taq (thermophilic), OptiTaq (thermophilic blend) and Pfu (hyperthermophilic) in the presence of BrdUTP. Lanes M, Perfect 100 bp Ladder; lane 1, PCR 1 fragment (379 bp); lane 2, BsaI-cleaved PCR 1 fragment; lane 3, PCR 2 fragment (625 bp); lane 4, BsaI-cleaved PCR 2 fragment; lane 5, BsaI restriction fragments: I (363 bp) and III (609 bp). Lanes 6-18 reactions with specified DNA Polymerases: lane 6, restriction fragments: I and III, T4; lane 7, restriction fragments: I and III, Bst exo - ; lane 8, restriction fragments: I and III, Bst exo - , T4 DNA Ligase; lane 9, restriction fragments: I and III, T4; lane 10, restriction fragments: I and III, T4, T4 DNA Ligase; lane 11, restriction fragments: I and III, Taq; lane 12, restriction fragments: I and III, Taq, T4 DNA Ligase; lane 13, restriction fragments: I and III, OptiTaq; lane 14, restriction fragments: I and III, OptiTaq, T4 DNA Ligase; lane 15, restriction fragments: I and III, Tfl; lane 16, restriction fragments: I and III, Tfl, T4 DNA Ligase; lane 17, restriction fragments: I and III, Pfu; lane 18, restriction fragments: I and III, Pfu, T4 DNA Ligase. I, III BsaI restriction fragments numbered as in Figure 1.

    Techniques Used: Polymerase Chain Reaction

    Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 441 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked); lane 1, 260 bp BsaI-cleaved PCR (restriction fragment I); lane 2, 208 bp BsaI-cleaved PCR (restriction fragment III); lane 3, BrdUTP-filled restriction fragments I and III, T4 DNA ligase; lane 4, BrdUTP-filled restriction fragments I and III; lane 5, dTTP-filled restriction fragment I and BrdUTP-filled restriction fragment III, T4 DNA ligase; lane 6, dTTP-filled restriction fragment I and BrdU-filled restriction fragment III. Lanes 7-9, controls of enzymes functional purity: lane 7, control PCR fragment with internal BsaI site; lane 8, BsaI-cleaved control PCR fragment; lane 9, BsaI-cleaved control PCR fragment after addition of T4 DNA Ligase; lane M, Perfect 100 bp Ladder. I, III BsaI restriction fragments numbered as in Figure 1.
    Figure Legend Snippet: Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 441 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked); lane 1, 260 bp BsaI-cleaved PCR (restriction fragment I); lane 2, 208 bp BsaI-cleaved PCR (restriction fragment III); lane 3, BrdUTP-filled restriction fragments I and III, T4 DNA ligase; lane 4, BrdUTP-filled restriction fragments I and III; lane 5, dTTP-filled restriction fragment I and BrdUTP-filled restriction fragment III, T4 DNA ligase; lane 6, dTTP-filled restriction fragment I and BrdU-filled restriction fragment III. Lanes 7-9, controls of enzymes functional purity: lane 7, control PCR fragment with internal BsaI site; lane 8, BsaI-cleaved control PCR fragment; lane 9, BsaI-cleaved control PCR fragment after addition of T4 DNA Ligase; lane M, Perfect 100 bp Ladder. I, III BsaI restriction fragments numbered as in Figure 1.

    Techniques Used: Polymerase Chain Reaction, Functional Assay

    6) Product Images from "GoldenBac: a simple, highly efficient, and widely applicable system for construction of multi-gene expression vectors for use with the baculovirus expression vector system"

    Article Title: GoldenBac: a simple, highly efficient, and widely applicable system for construction of multi-gene expression vectors for use with the baculovirus expression vector system

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-020-00616-z

    Efficiency of different BsaI enzymes. To test the efficiency of the BsaI-HFv2 enzyme, the same 15 entry vectors were used in a Golden Gate reaction with either the standard BsaI enzyme or BsaI-HFv2. Ten clones resulting from each assembly reaction were picked, digested with EcoRV, and analyzed via agarose gel electrophoresis. A schematic of the DNA sizing ladder and the predicted band pattern for the assembly reaction is shown to the left of the respective agarose gel. Clones demonstrating correct assembly based on the pattern of bands are marked with a green asterisk. While the reaction performed with the standard BsaI enzyme resulted in no correct clones, the reaction with the BsaI-HFv2 enzyme showed 9/10 correct clones
    Figure Legend Snippet: Efficiency of different BsaI enzymes. To test the efficiency of the BsaI-HFv2 enzyme, the same 15 entry vectors were used in a Golden Gate reaction with either the standard BsaI enzyme or BsaI-HFv2. Ten clones resulting from each assembly reaction were picked, digested with EcoRV, and analyzed via agarose gel electrophoresis. A schematic of the DNA sizing ladder and the predicted band pattern for the assembly reaction is shown to the left of the respective agarose gel. Clones demonstrating correct assembly based on the pattern of bands are marked with a green asterisk. While the reaction performed with the standard BsaI enzyme resulted in no correct clones, the reaction with the BsaI-HFv2 enzyme showed 9/10 correct clones

    Techniques Used: Clone Assay, Agarose Gel Electrophoresis

    7) Product Images from "A novel one-step approach for the construction of yeast surface display Fab antibody libraries"

    Article Title: A novel one-step approach for the construction of yeast surface display Fab antibody libraries

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-017-0853-z

    One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping
    Figure Legend Snippet: One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Expressing, Sequencing, Generated, Plasmid Preparation

    8) Product Images from "Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana"

    Article Title: Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana

    Journal: Plant Methods

    doi: 10.1186/s13007-016-0148-0

    Overview of level 1 (L1) and level 2 (L2) Golden Gate cloning for assembly of the CRISPR-Cas construct pAGM4723:TpCC_Urease. L0 modules are created by PCR amplifying the relevant insert, BsaI sites are added through the primers. Products are then TOPO TA cloned into pCR8/GW/TOPO vectors. Construction of the L1 and L2 modules is based on the ability of BsaI and BpiI restriction enzymes to cut outside of their restriction recognition sites leaving 4 nt overhangs specific to each module. Overhangs between adjacent modules correspond allowing multiple modules to be ligated in a particular order within the same reaction. BsaI is used to assemble L0 modules into L1 destination vectors and BpiI is used to assemble L1 modules into the final L2 destination vector. L1 assemblies of pICH47742:FCP:Cas9YFP and pICH47751:U6:sgRNA_Urease 1 are shown. L2 assembly of the final construct is shown. L1 modules containing the FCP:NAT cassette, U6:sgRNA Urease 2 cassette, the L4E linker and L2 destination vector are shown in a simplified format. Corresponding colours denote a shared 4 nt overhang
    Figure Legend Snippet: Overview of level 1 (L1) and level 2 (L2) Golden Gate cloning for assembly of the CRISPR-Cas construct pAGM4723:TpCC_Urease. L0 modules are created by PCR amplifying the relevant insert, BsaI sites are added through the primers. Products are then TOPO TA cloned into pCR8/GW/TOPO vectors. Construction of the L1 and L2 modules is based on the ability of BsaI and BpiI restriction enzymes to cut outside of their restriction recognition sites leaving 4 nt overhangs specific to each module. Overhangs between adjacent modules correspond allowing multiple modules to be ligated in a particular order within the same reaction. BsaI is used to assemble L0 modules into L1 destination vectors and BpiI is used to assemble L1 modules into the final L2 destination vector. L1 assemblies of pICH47742:FCP:Cas9YFP and pICH47751:U6:sgRNA_Urease 1 are shown. L2 assembly of the final construct is shown. L1 modules containing the FCP:NAT cassette, U6:sgRNA Urease 2 cassette, the L4E linker and L2 destination vector are shown in a simplified format. Corresponding colours denote a shared 4 nt overhang

    Techniques Used: Clone Assay, CRISPR, Construct, Polymerase Chain Reaction, Plasmid Preparation

    9) Product Images from "Reverse Genetic System for the Analysis of Parvovirus Telomeres Reveals Interactions between Transcription Factor Binding Sites in the Hairpin Stem"

    Article Title: Reverse Genetic System for the Analysis of Parvovirus Telomeres Reveals Interactions between Transcription Factor Binding Sites in the Hairpin Stem

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.16.8650-8660.2003

    Relative DNA replication under one-step growth conditions. (A and C) DNA extracted from single-cycle infections using wild-type and no-cre viruses in A9 (A) and 324K (C) cells, with autoradiographs positioned above the gels from which they were created by Southern blotting. Individual lanes are aligned and contain total DNA extracted at 12, 24, and 36 h postinfection and then digested with Bsa I. DNA extracted from equivalent amounts of both cells and medium are shown for each time point. The gels were stained with ethidium bromide, and the position of the double-stranded (ds) 5-kb band is shown. (B and D) On the Southern blots, the positions of 10-kb and 5-kb viral double-stranded bands, as well as those of 5-kb single-stranded (ss) progeny genomes, are indicated. Results for all five viruses, tested in A9 (B) and 324K (D) cells, are expressed in graphic form as the quantity of viral DNA (in nanograms), normalized to the amount of total DNA (in micrograms) in each sample. Each bar represents three independent experiments, with the standard deviations indicated. The numbers under each bar of the graph indicate hours postinfection.
    Figure Legend Snippet: Relative DNA replication under one-step growth conditions. (A and C) DNA extracted from single-cycle infections using wild-type and no-cre viruses in A9 (A) and 324K (C) cells, with autoradiographs positioned above the gels from which they were created by Southern blotting. Individual lanes are aligned and contain total DNA extracted at 12, 24, and 36 h postinfection and then digested with Bsa I. DNA extracted from equivalent amounts of both cells and medium are shown for each time point. The gels were stained with ethidium bromide, and the position of the double-stranded (ds) 5-kb band is shown. (B and D) On the Southern blots, the positions of 10-kb and 5-kb viral double-stranded bands, as well as those of 5-kb single-stranded (ss) progeny genomes, are indicated. Results for all five viruses, tested in A9 (B) and 324K (D) cells, are expressed in graphic form as the quantity of viral DNA (in nanograms), normalized to the amount of total DNA (in micrograms) in each sample. Each bar represents three independent experiments, with the standard deviations indicated. The numbers under each bar of the graph indicate hours postinfection.

    Techniques Used: Southern Blot, Staining

    The MVM-Chop system for generating mutations in the left-hand terminal hairpin. (A) A map of pChopII, indicating the two Bsa I restriction sites, is shown in the upper left, where gray indicates MVM sequence and black indicates bacterial vector. Digestion with Bsa I and subsequent gel purification produced a 6-kb linear DNA with noncohesive 5′ overhangs at each end. Synthetic oligonucleotides that regenerate the terminal hairpin were ligated to the linearized plasmid. This construct was transfected into permissive cells from which mutant virus could be plaque purified for future analysis. (B) The sequence of the wild-type oligonucleotide is shown in hairpin form, with the two PIF half-sites and the CRE indicated by open and shaded boxes, respectively. The arrow with an asterisk indicates the identification of an A residue which differs from the previously published MVMp sequence, as discussed in the text. The sequences of the central regions of mutant hairpin oligonucleotides used in this study are shown below the wild-type hairpin. Altered and deleted sequences are indicated by gray boxes and arrows, respectively. Ellipses (…) indicate continuation of the wild-type sequence.
    Figure Legend Snippet: The MVM-Chop system for generating mutations in the left-hand terminal hairpin. (A) A map of pChopII, indicating the two Bsa I restriction sites, is shown in the upper left, where gray indicates MVM sequence and black indicates bacterial vector. Digestion with Bsa I and subsequent gel purification produced a 6-kb linear DNA with noncohesive 5′ overhangs at each end. Synthetic oligonucleotides that regenerate the terminal hairpin were ligated to the linearized plasmid. This construct was transfected into permissive cells from which mutant virus could be plaque purified for future analysis. (B) The sequence of the wild-type oligonucleotide is shown in hairpin form, with the two PIF half-sites and the CRE indicated by open and shaded boxes, respectively. The arrow with an asterisk indicates the identification of an A residue which differs from the previously published MVMp sequence, as discussed in the text. The sequences of the central regions of mutant hairpin oligonucleotides used in this study are shown below the wild-type hairpin. Altered and deleted sequences are indicated by gray boxes and arrows, respectively. Ellipses (…) indicate continuation of the wild-type sequence.

    Techniques Used: Sequencing, Plasmid Preparation, Gel Purification, Produced, Construct, Transfection, Mutagenesis, Purification

    10) Product Images from "Editing of an Alpha-Kafirin Gene Family Increases, Digestibility and Protein Quality in Sorghum 1Editing of an Alpha-Kafirin Gene Family Increases, Digestibility and Protein Quality in Sorghum 1 [OPEN]"

    Article Title: Editing of an Alpha-Kafirin Gene Family Increases, Digestibility and Protein Quality in Sorghum 1Editing of an Alpha-Kafirin Gene Family Increases, Digestibility and Protein Quality in Sorghum 1 [OPEN]

    Journal: Plant Physiology

    doi: 10.1104/pp.18.00200

    Construction of the sgRNA/Cas9 system. A, Partial sequence alignment of the k1C gene family from the reference and sgRNA design. The gray line at the bottom indicates a PAM; the black line shows a 19-nucleotide consensus DNA target region. sgRNA is complementary to the DNA target region. B, Schematic diagram of the construction of the sgRNA/Cas9 system. Gray arrows indicate the cohesive end produced by Bsa I digestion in the vector. The synthetic double-stranded sgRNA with four-nucleotide 5′ overhangs at both ends was inserted into the sgRNA/Cas9 expression system. The black triangle represents a G base in the TaU3 promoter followed by the 5′ end of the 19-nucleotide sgRNA that resulted in a final 20-bp sgRNA with G (N19-nt) in the guide RNA/Cas9 system and introduced an artificial mismatch in the target site of the k1C members. RB/LB represent the left and right borders of the vector; TaU3P is the wheat U3 gene promoter; the gRNA site refers to the guide RNA clone site; gRNA SC is the gRNA scaffold; Ubi P is the Ubiquitin gene promoter; Zm Cas9 is the maize codon-optimized Cas9 ; Nos t is the Nos terminator; 35S P is the 35S promoter; Npt II is the neomycin phosphotransferase II gene.
    Figure Legend Snippet: Construction of the sgRNA/Cas9 system. A, Partial sequence alignment of the k1C gene family from the reference and sgRNA design. The gray line at the bottom indicates a PAM; the black line shows a 19-nucleotide consensus DNA target region. sgRNA is complementary to the DNA target region. B, Schematic diagram of the construction of the sgRNA/Cas9 system. Gray arrows indicate the cohesive end produced by Bsa I digestion in the vector. The synthetic double-stranded sgRNA with four-nucleotide 5′ overhangs at both ends was inserted into the sgRNA/Cas9 expression system. The black triangle represents a G base in the TaU3 promoter followed by the 5′ end of the 19-nucleotide sgRNA that resulted in a final 20-bp sgRNA with G (N19-nt) in the guide RNA/Cas9 system and introduced an artificial mismatch in the target site of the k1C members. RB/LB represent the left and right borders of the vector; TaU3P is the wheat U3 gene promoter; the gRNA site refers to the guide RNA clone site; gRNA SC is the gRNA scaffold; Ubi P is the Ubiquitin gene promoter; Zm Cas9 is the maize codon-optimized Cas9 ; Nos t is the Nos terminator; 35S P is the 35S promoter; Npt II is the neomycin phosphotransferase II gene.

    Techniques Used: Sequencing, Produced, Plasmid Preparation, Expressing

    11) Product Images from "Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis"

    Article Title: Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14329-5

    Architecture of the MCS-IIS C2. This DNA-sequence is located on the cargo vector between the E. coli ori and the Bacillus antibiotic marker. The recognition sites for five type IIS restriction enzymes (AarI, BtgZI, BbsI, BsaI, BsmBI), each designed to create a 5′ GCGA-overhang are encoded on the DNA stretch. Architecture of all MCS-IIS can be found in Fig. S1 .
    Figure Legend Snippet: Architecture of the MCS-IIS C2. This DNA-sequence is located on the cargo vector between the E. coli ori and the Bacillus antibiotic marker. The recognition sites for five type IIS restriction enzymes (AarI, BtgZI, BbsI, BsaI, BsmBI), each designed to create a 5′ GCGA-overhang are encoded on the DNA stretch. Architecture of all MCS-IIS can be found in Fig. S1 .

    Techniques Used: Sequencing, Plasmid Preparation, Marker

    12) Product Images from "A simple and efficient cloning system for CRISPR/Cas9-mediated genome editing in rice"

    Article Title: A simple and efficient cloning system for CRISPR/Cas9-mediated genome editing in rice

    Journal: PeerJ

    doi: 10.7717/peerj.8491

    Workflow for constructing expression clone containing two target-sgRNA expression cassettes with Golden Gate clone. Primers containing adaptors for Golden Gate cloning (OJH307 and OJH308) were used in the amplification with PJG090 as the template. The reagents were recommended as following: one ul of PCR product, 50 ng of PJG112, one ul of Cutsmart Buffer (NEB), 0.4 ul of T4 ligase buffer (NEB), 5 U of Bsa I (NEB), 20 U of T4 DNA ligase (NEB) and add ddH 2 O to 10 ul. The reaction was incubated for 20–25 cycles (37 °C 2 min, 20 °C 5 min), followed by incubation at 50 °C and 80 °C for 5 min, respectively. Subsequently, one ul of the product was introduced into Trans T1 competent cells. Positive clones were identified by clone PCR and sequenced.
    Figure Legend Snippet: Workflow for constructing expression clone containing two target-sgRNA expression cassettes with Golden Gate clone. Primers containing adaptors for Golden Gate cloning (OJH307 and OJH308) were used in the amplification with PJG090 as the template. The reagents were recommended as following: one ul of PCR product, 50 ng of PJG112, one ul of Cutsmart Buffer (NEB), 0.4 ul of T4 ligase buffer (NEB), 5 U of Bsa I (NEB), 20 U of T4 DNA ligase (NEB) and add ddH 2 O to 10 ul. The reaction was incubated for 20–25 cycles (37 °C 2 min, 20 °C 5 min), followed by incubation at 50 °C and 80 °C for 5 min, respectively. Subsequently, one ul of the product was introduced into Trans T1 competent cells. Positive clones were identified by clone PCR and sequenced.

    Techniques Used: Expressing, Clone Assay, Amplification, Polymerase Chain Reaction, Incubation

    Construction of the destination vectors PJG097 without Bas I sites. There are two BsaI sites in the backbone of pH-Ubi-cas9-7. To remove the BsaI sites, primers containing site mutation in its recognition site was designed (referred to as OJK121 and OJK122). The Bsa I sites and matched adaptors were added to the 5′ stream of primers. An amplification with OJK121 and OJK122 was conducted with pH-Ubi-cas9-7 as the template. Gel-purified PCR product and pH-Ubi-cas9-7 was then digested with BsaI and then submitted to a ligation reaction. The product was then transferred into DB3.1 competent cells (ZOMANBIO Co., Shanghai, China). Positive clones were then verified by DNA sequencing and digestion with BsaI. The new vector removing BsaI sites was renamed as PJG097. An LR reaction was performed with 30 ng pOs-sgRNA and PJG097, producing the destination clone named PJG112. PJG112 could serve as destination vector to construct expression clones containing two spacers.
    Figure Legend Snippet: Construction of the destination vectors PJG097 without Bas I sites. There are two BsaI sites in the backbone of pH-Ubi-cas9-7. To remove the BsaI sites, primers containing site mutation in its recognition site was designed (referred to as OJK121 and OJK122). The Bsa I sites and matched adaptors were added to the 5′ stream of primers. An amplification with OJK121 and OJK122 was conducted with pH-Ubi-cas9-7 as the template. Gel-purified PCR product and pH-Ubi-cas9-7 was then digested with BsaI and then submitted to a ligation reaction. The product was then transferred into DB3.1 competent cells (ZOMANBIO Co., Shanghai, China). Positive clones were then verified by DNA sequencing and digestion with BsaI. The new vector removing BsaI sites was renamed as PJG097. An LR reaction was performed with 30 ng pOs-sgRNA and PJG097, producing the destination clone named PJG112. PJG112 could serve as destination vector to construct expression clones containing two spacers.

    Techniques Used: Mutagenesis, Amplification, Purification, Polymerase Chain Reaction, Ligation, Clone Assay, DNA Sequencing, Plasmid Preparation, Construct, Expressing

    13) 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

    14) 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

    15) Product Images from "Transcriptional Activators of Human Genes with Programmable DNA-Specificity"

    Article Title: Transcriptional Activators of Human Genes with Programmable DNA-Specificity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019509

    Golden TAL Technology for assembly of TAL proteins with programmed repeat composition. (A) Single TAL repeats were cloned with flanking Bpi I sites that generate specific four base pair-overhangs (a–g). Matching sites are indicated by identical letters. A library was constructed with four different repeat types (RVD, repeat variable di-residue: NI, HD, NN, NG) for each repeat position. The repeat types have different DNA-specificities (Spec., only upper DNA-strand is shown). (B) One to six repeats are assembled in specific order into an assembly vector to generate a repeat assembly with flanking Bsa I sites. (C) TALs were directly assembled with N-terminal GFP-tag into an expression vector using fragments with matching Bsa I-generated overhangs (capital letters). Insertion of one to four repeat assemblies generated TALs with 1 to 24 repeats. The last repeat is only a half repeat as typical for TAL proteins. Please see the Text S1 and Figure S4 for details.
    Figure Legend Snippet: Golden TAL Technology for assembly of TAL proteins with programmed repeat composition. (A) Single TAL repeats were cloned with flanking Bpi I sites that generate specific four base pair-overhangs (a–g). Matching sites are indicated by identical letters. A library was constructed with four different repeat types (RVD, repeat variable di-residue: NI, HD, NN, NG) for each repeat position. The repeat types have different DNA-specificities (Spec., only upper DNA-strand is shown). (B) One to six repeats are assembled in specific order into an assembly vector to generate a repeat assembly with flanking Bsa I sites. (C) TALs were directly assembled with N-terminal GFP-tag into an expression vector using fragments with matching Bsa I-generated overhangs (capital letters). Insertion of one to four repeat assemblies generated TALs with 1 to 24 repeats. The last repeat is only a half repeat as typical for TAL proteins. Please see the Text S1 and Figure S4 for details.

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

    16) Product Images from "Highly Efficient One-Step Scarless Protein Tagging by Type IIS Restriction Endonuclease-Mediated Precision Cloning"

    Article Title: Highly Efficient One-Step Scarless Protein Tagging by Type IIS Restriction Endonuclease-Mediated Precision Cloning

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2017.05.153

    Comparison of type II and IIS restriction enzyme-mediated protein tagging strategy ( a ) Type II restriction enzymes (TII-es) recognize palindromic DNA sequences. For example, EcoRI recognizes 5′-GAATTC-3′ (marked by top curly bracket) and creates 4 base pairs overhangs highlighted in red. ( b ) Single or double type II restriction enzymes cassette (highlighted in blue box) for traditional protein tagging. Note that in all destination clones, varying junction sequences exist adjacent to both sides of the tag. ( c ) Type IIS restriction enzymes (TIIS-es) recognize non-palindromic, asymmetrical DNA sequences. For example, BsaI recognizes 5′-GGTCTC-3′ (marked by top curly bracket) and cleaves DNA one bp away (indicated by the two arrows), producing N 2 ′N 3 ′N 4 ′N 5 ′ custom sticky end (highlighted in red). N indicates four bases of DNA, including A, T, G and C. Apostrophe (’) indicates the complementary base of the DNA. ( d ) Type IIS restriction enzyme DNA cassette (TIIS DNA cassette highlighted in blue box) for precision tagging. Note that on both ends of a tag, the flanking sequences (such as BsaI-released 5′-N 2 ′N 3 ′N 4 ′N 5 ′ and 5′-N 2 N 3 N 4 N 5 belong to gene-specific sequences including SP or gene of interest indicated by two closely dotted lines. After Tag replaces type IIS DNA cassette, a scarless tagging clone can be generated. Comparison between traditional and precision tagging were summarized in the bottom table. * Gibson assembly sometimes fails due to certain DNA sequences such as repetitive region or creating one or two nucleotides deletion.
    Figure Legend Snippet: Comparison of type II and IIS restriction enzyme-mediated protein tagging strategy ( a ) Type II restriction enzymes (TII-es) recognize palindromic DNA sequences. For example, EcoRI recognizes 5′-GAATTC-3′ (marked by top curly bracket) and creates 4 base pairs overhangs highlighted in red. ( b ) Single or double type II restriction enzymes cassette (highlighted in blue box) for traditional protein tagging. Note that in all destination clones, varying junction sequences exist adjacent to both sides of the tag. ( c ) Type IIS restriction enzymes (TIIS-es) recognize non-palindromic, asymmetrical DNA sequences. For example, BsaI recognizes 5′-GGTCTC-3′ (marked by top curly bracket) and cleaves DNA one bp away (indicated by the two arrows), producing N 2 ′N 3 ′N 4 ′N 5 ′ custom sticky end (highlighted in red). N indicates four bases of DNA, including A, T, G and C. Apostrophe (’) indicates the complementary base of the DNA. ( d ) Type IIS restriction enzyme DNA cassette (TIIS DNA cassette highlighted in blue box) for precision tagging. Note that on both ends of a tag, the flanking sequences (such as BsaI-released 5′-N 2 ′N 3 ′N 4 ′N 5 ′ and 5′-N 2 N 3 N 4 N 5 belong to gene-specific sequences including SP or gene of interest indicated by two closely dotted lines. After Tag replaces type IIS DNA cassette, a scarless tagging clone can be generated. Comparison between traditional and precision tagging were summarized in the bottom table. * Gibson assembly sometimes fails due to certain DNA sequences such as repetitive region or creating one or two nucleotides deletion.

    Techniques Used: Clone Assay, Generated

    17) Product Images from "YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae"

    Article Title: YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv464

    Overall scheme to construct standard biological parts, transcription units and pathways. ( A ) Overall strategy to construct the standard biological parts and to profile their functions. All parts generated in this study are derived from native sequences, amplified from S. cerevisiae genome by PCR. Each part is verified by sequencing. ( B ) Use of the part libraries to assemble transcription units (TUs) and pathways. Each part within a library is compatible with the parts from other libraries, allowing compositional assemblies. The TUs can be used for a second round of assembly, leading to the construction of multiple-gene pathways. The assembled pathways can be integrated into either a designated genomic locus or a plasmid. ( C ) Schematic representation of the acceptor vectors for parts. Each vector contains two different type IIs restriction enzyme recognition sites. BsaI was used to release the RFP marker, allowing quick identification of the correctly assembled parts. BsmBI was used to put different parts together to construct the transcription units.
    Figure Legend Snippet: Overall scheme to construct standard biological parts, transcription units and pathways. ( A ) Overall strategy to construct the standard biological parts and to profile their functions. All parts generated in this study are derived from native sequences, amplified from S. cerevisiae genome by PCR. Each part is verified by sequencing. ( B ) Use of the part libraries to assemble transcription units (TUs) and pathways. Each part within a library is compatible with the parts from other libraries, allowing compositional assemblies. The TUs can be used for a second round of assembly, leading to the construction of multiple-gene pathways. The assembled pathways can be integrated into either a designated genomic locus or a plasmid. ( C ) Schematic representation of the acceptor vectors for parts. Each vector contains two different type IIs restriction enzyme recognition sites. BsaI was used to release the RFP marker, allowing quick identification of the correctly assembled parts. BsmBI was used to put different parts together to construct the transcription units.

    Techniques Used: Construct, Generated, Derivative Assay, Amplification, Polymerase Chain Reaction, Sequencing, Plasmid Preparation, Marker

    18) Product Images from "MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme"

    Article Title: MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky596

    Design of a standard MetClo vector set. Three types of adaptor sequences were designed for the standard MetClo vector set. The vectors contain two head-to-head BsaI sites (boxed) flanking the negative selection marker LacZα. The pair of unmethylated BsaI sites closest to the negative selection marker can be used to release LacZα and generates adhesive ends ‘p’ and ‘q’. This allows any fragments that start with adhesive end ‘p’ and end with adhesive end ‘q’ to be cloned into any of the three types of adaptor sequences (types ‘Start’, ‘Middle’, ‘End’). The outer pair of BsaI sites closest to the vector backbone overlap with the M.Osp807II recognition sequence and are both methylated at the adenine bases highlighted in bold when the vector is prepared in M.Osp807II-expressing E. coli strain. A DNA fragment assembled into these vectors, following transformation into a normal E. coli strain which lacks this switch methylase activity, can be released from the assembled plasmid using BsaI, which can now recognize this unmethylated BsaI site. The released fragment will carry different adhesive ends depending on the type of the vector used. Assembly into a type ‘Start’ vector will generate a fragment flanked by adaptors ‘p-a’; assembly into a type ‘Middle’ vector will generate a fragment flanked by ‘a-b’, ‘b-c’, ‘c-d’ or ‘d-e’; assembly into a type ‘End’ vector will generate a fragment flanked by ‘a-q’, ‘b-q’, ‘c-q’, ‘d-q’ or ‘e-q’. The design of these LacZα selection cassettes flanked by unique adaptors are represented in the figure by the letter codes for the outside adaptor sequences.
    Figure Legend Snippet: Design of a standard MetClo vector set. Three types of adaptor sequences were designed for the standard MetClo vector set. The vectors contain two head-to-head BsaI sites (boxed) flanking the negative selection marker LacZα. The pair of unmethylated BsaI sites closest to the negative selection marker can be used to release LacZα and generates adhesive ends ‘p’ and ‘q’. This allows any fragments that start with adhesive end ‘p’ and end with adhesive end ‘q’ to be cloned into any of the three types of adaptor sequences (types ‘Start’, ‘Middle’, ‘End’). The outer pair of BsaI sites closest to the vector backbone overlap with the M.Osp807II recognition sequence and are both methylated at the adenine bases highlighted in bold when the vector is prepared in M.Osp807II-expressing E. coli strain. A DNA fragment assembled into these vectors, following transformation into a normal E. coli strain which lacks this switch methylase activity, can be released from the assembled plasmid using BsaI, which can now recognize this unmethylated BsaI site. The released fragment will carry different adhesive ends depending on the type of the vector used. Assembly into a type ‘Start’ vector will generate a fragment flanked by adaptors ‘p-a’; assembly into a type ‘Middle’ vector will generate a fragment flanked by ‘a-b’, ‘b-c’, ‘c-d’ or ‘d-e’; assembly into a type ‘End’ vector will generate a fragment flanked by ‘a-q’, ‘b-q’, ‘c-q’, ‘d-q’ or ‘e-q’. The design of these LacZα selection cassettes flanked by unique adaptors are represented in the figure by the letter codes for the outside adaptor sequences.

    Techniques Used: Plasmid Preparation, Selection, Marker, Clone Assay, Sequencing, Methylation, Expressing, Transformation Assay, Activity Assay

    Identification of suitable methylases for methylation-switching. ( A ) Initial screening of functional methylases for selective blocking of overlapping methylation/restriction sites. The diagrams show the design of overlapping sites for screening of methylase activity. The table shows the screening result using methylases expressed in vivo from an F-ori based low copy number vector. Restriction enzyme recognition sites are boxed in solid lines. The adhesive ends generated by the restriction enzyme are shown by solid lines. Methylase recognition sites are boxed in dashed lines, and methylated bases are in bold font. All the listed methylases modify N6-adenine, except M.SacI and M.AspJHL3I, which modify C5-cytosine and N4-cytosine respectively. ( B ) Experimental designs to test blocking of methylation-switchable type IIS restriction enzyme sites by in vivo methylation. For each methylase/restriction enzyme combination tested, the test plasmid contains a head-to-head potentially methylation-switchable restriction site and a non-methylatable restriction site. Restriction digestion of test plasmid prepared from a normal E. coli strain would result in cutting at both sites and the release of a 600 bp fragment from the 4.3 kb vector backbone. Restriction digestion of test plasmid prepared from a strain expressing the switch methylase would result in a single 4.9 kb band, due to blocking of the methylation-switchable restriction sites by in vivo methylation. The restriction sites of the test plasmids for each restriction enzyme are shown, with the restriction site boxed in solid line, the methylase recognition site boxed in dashed line, and the methylated bases in bold. The head-to-head arrangement of overlapping methylation/restriction site allows the same assay to be used to detect any residual single strand nicking activity of the restriction enzyme towards the methylated restriction site. ( C ) Agarose gel electrophoresis analysis of the test plasmids for each methylation-switchable restriction site after preparation of the plasmids in a normal strain (–) or in a strain expressing the appropriate DNA methylase (+) and digested with the corresponding type IIS restriction enzymes. The combinations tested were BsaI with M.Osp807II methylase using test plasmid pMOP_BsaINC, BpiI with M2.NmeMC58II methylase using test plasmid pMOP_BpiINC, and LguI with M.XmnI methylase using test plasmid pMOP_LguINC. Test conditions were 60 fmol test plasmid digested using 5 U BsaI or BpiI, or 2.5 U LguI in 10 μl reactions at 37°C for 1 h. The results show that in vivo methylation by each of the methylases successfully blocked the restriction site for the corresponding type IIS restriction enzyme when the methylase recognition site overlapped the restriction enzyme site. The data shown represents results from three independent experiments.
    Figure Legend Snippet: Identification of suitable methylases for methylation-switching. ( A ) Initial screening of functional methylases for selective blocking of overlapping methylation/restriction sites. The diagrams show the design of overlapping sites for screening of methylase activity. The table shows the screening result using methylases expressed in vivo from an F-ori based low copy number vector. Restriction enzyme recognition sites are boxed in solid lines. The adhesive ends generated by the restriction enzyme are shown by solid lines. Methylase recognition sites are boxed in dashed lines, and methylated bases are in bold font. All the listed methylases modify N6-adenine, except M.SacI and M.AspJHL3I, which modify C5-cytosine and N4-cytosine respectively. ( B ) Experimental designs to test blocking of methylation-switchable type IIS restriction enzyme sites by in vivo methylation. For each methylase/restriction enzyme combination tested, the test plasmid contains a head-to-head potentially methylation-switchable restriction site and a non-methylatable restriction site. Restriction digestion of test plasmid prepared from a normal E. coli strain would result in cutting at both sites and the release of a 600 bp fragment from the 4.3 kb vector backbone. Restriction digestion of test plasmid prepared from a strain expressing the switch methylase would result in a single 4.9 kb band, due to blocking of the methylation-switchable restriction sites by in vivo methylation. The restriction sites of the test plasmids for each restriction enzyme are shown, with the restriction site boxed in solid line, the methylase recognition site boxed in dashed line, and the methylated bases in bold. The head-to-head arrangement of overlapping methylation/restriction site allows the same assay to be used to detect any residual single strand nicking activity of the restriction enzyme towards the methylated restriction site. ( C ) Agarose gel electrophoresis analysis of the test plasmids for each methylation-switchable restriction site after preparation of the plasmids in a normal strain (–) or in a strain expressing the appropriate DNA methylase (+) and digested with the corresponding type IIS restriction enzymes. The combinations tested were BsaI with M.Osp807II methylase using test plasmid pMOP_BsaINC, BpiI with M2.NmeMC58II methylase using test plasmid pMOP_BpiINC, and LguI with M.XmnI methylase using test plasmid pMOP_LguINC. Test conditions were 60 fmol test plasmid digested using 5 U BsaI or BpiI, or 2.5 U LguI in 10 μl reactions at 37°C for 1 h. The results show that in vivo methylation by each of the methylases successfully blocked the restriction site for the corresponding type IIS restriction enzyme when the methylase recognition site overlapped the restriction enzyme site. The data shown represents results from three independent experiments.

    Techniques Used: Methylation, Functional Assay, Blocking Assay, Activity Assay, In Vivo, Low Copy Number, Plasmid Preparation, Generated, Expressing, Agarose Gel Electrophoresis

    19) Product Images from "GoldenBac: a simple, highly efficient, and widely applicable system for construction of multi-gene expression vectors for use with the baculovirus expression vector system"

    Article Title: GoldenBac: a simple, highly efficient, and widely applicable system for construction of multi-gene expression vectors for use with the baculovirus expression vector system

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-020-00616-z

    Efficiency of different BsaI enzymes. To test the efficiency of the BsaI-HFv2 enzyme, the same 15 entry vectors were used in a Golden Gate reaction with either the standard BsaI enzyme or BsaI-HFv2. Ten clones resulting from each assembly reaction were picked, digested with EcoRV, and analyzed via agarose gel electrophoresis. A schematic of the DNA sizing ladder and the predicted band pattern for the assembly reaction is shown to the left of the respective agarose gel. Clones demonstrating correct assembly based on the pattern of bands are marked with a green asterisk. While the reaction performed with the standard BsaI enzyme resulted in no correct clones, the reaction with the BsaI-HFv2 enzyme showed 9/10 correct clones
    Figure Legend Snippet: Efficiency of different BsaI enzymes. To test the efficiency of the BsaI-HFv2 enzyme, the same 15 entry vectors were used in a Golden Gate reaction with either the standard BsaI enzyme or BsaI-HFv2. Ten clones resulting from each assembly reaction were picked, digested with EcoRV, and analyzed via agarose gel electrophoresis. A schematic of the DNA sizing ladder and the predicted band pattern for the assembly reaction is shown to the left of the respective agarose gel. Clones demonstrating correct assembly based on the pattern of bands are marked with a green asterisk. While the reaction performed with the standard BsaI enzyme resulted in no correct clones, the reaction with the BsaI-HFv2 enzyme showed 9/10 correct clones

    Techniques Used: Clone Assay, Agarose Gel Electrophoresis

    20) Product Images from "A CRISPR/Cas9 toolkit for multiplex genome editing in plants"

    Article Title: A CRISPR/Cas9 toolkit for multiplex genome editing in plants

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-014-0327-y

    Premade gRNA modules used for the assembly of two to four gRNA expression cassettes. (A) gRNA-expressing modules for both dicots and monocots. U6-29p, U6-26p, and U6-1p are three Arabidopsis U6 gene promoters; U6-29t, U6-26t, and U6-1t, corresponding Arabidopsis U6 gene terminators with downstream sequences; OsU3p and TaU3p, rice and wheat U3 promoters, respectively; OsU3t and TaU3t, rice and wheat U3 terminators with downstream sequences, respectively; gRNA-Sc, gRNA scaffold; DT1/2/3/4, dicot target-1/2/3/4; MT1/2/3/4, monocot target-1/2/3/4. The vector pCBC is the cloning vector into which the gRNA modules were inserted separately. (B) Examples of the assembly of two-gRNA expression cassettes for dicots and monocots using the gRNA modules. Note: Each PCR fragment is flanked by two Bsa I sites (not shown).
    Figure Legend Snippet: Premade gRNA modules used for the assembly of two to four gRNA expression cassettes. (A) gRNA-expressing modules for both dicots and monocots. U6-29p, U6-26p, and U6-1p are three Arabidopsis U6 gene promoters; U6-29t, U6-26t, and U6-1t, corresponding Arabidopsis U6 gene terminators with downstream sequences; OsU3p and TaU3p, rice and wheat U3 promoters, respectively; OsU3t and TaU3t, rice and wheat U3 terminators with downstream sequences, respectively; gRNA-Sc, gRNA scaffold; DT1/2/3/4, dicot target-1/2/3/4; MT1/2/3/4, monocot target-1/2/3/4. The vector pCBC is the cloning vector into which the gRNA modules were inserted separately. (B) Examples of the assembly of two-gRNA expression cassettes for dicots and monocots using the gRNA modules. Note: Each PCR fragment is flanked by two Bsa I sites (not shown).

    Techniques Used: Expressing, Plasmid Preparation, Clone Assay, Polymerase Chain Reaction

    Physical maps and structures of CRISPR/Cas9 binary vectors. (A) Physical maps of the backbones of pGreen and pCAMBIA from which CRISPR/Cas9 binary vectors were derived. The map of the helper plasmid required for propagation of pGreen in Agrobacterium and the mutated Bsa I site on the pCAMBIA backbone are indicated. LB/RB, left/right border of T-DNA; pSa-ori, required for replication in Agrobacterium engineered with the corresponding replication protein (pSa-repA); KmR, kanamycin resistance gene; pUC-ori, replication origin required for replication in E. coli ; pVS1-staA, pVS1-ori and pVS1-rep are the DNA elements required for replication in Agrobacterium . Only the 225-bp fragment between the LB and RB was left for comparison of the sizes of the pGreen and pCAMBIA backbones. (B, C) Physical maps of the regions between the RB and LB. The sizes of T-DNA regions and the structures of SpR-gRNA-Sc and final working gRNA are indicated. zCas9 , Zea mays codon-optimized Cas9 ; U6-26p, Arabidopsis U6 gene promoter; U6-26t, U6-26 terminator with downstream sequence; OsU3p, rice U3 promoter; OsU3t, rice U3 terminator with downstream sequence; SpR, spectinomycin resistance gene; gRNA-Sc, gRNA scaffold.
    Figure Legend Snippet: Physical maps and structures of CRISPR/Cas9 binary vectors. (A) Physical maps of the backbones of pGreen and pCAMBIA from which CRISPR/Cas9 binary vectors were derived. The map of the helper plasmid required for propagation of pGreen in Agrobacterium and the mutated Bsa I site on the pCAMBIA backbone are indicated. LB/RB, left/right border of T-DNA; pSa-ori, required for replication in Agrobacterium engineered with the corresponding replication protein (pSa-repA); KmR, kanamycin resistance gene; pUC-ori, replication origin required for replication in E. coli ; pVS1-staA, pVS1-ori and pVS1-rep are the DNA elements required for replication in Agrobacterium . Only the 225-bp fragment between the LB and RB was left for comparison of the sizes of the pGreen and pCAMBIA backbones. (B, C) Physical maps of the regions between the RB and LB. The sizes of T-DNA regions and the structures of SpR-gRNA-Sc and final working gRNA are indicated. zCas9 , Zea mays codon-optimized Cas9 ; U6-26p, Arabidopsis U6 gene promoter; U6-26t, U6-26 terminator with downstream sequence; OsU3p, rice U3 promoter; OsU3t, rice U3 terminator with downstream sequence; SpR, spectinomycin resistance gene; gRNA-Sc, gRNA scaffold.

    Techniques Used: CRISPR, Derivative Assay, Plasmid Preparation, SPR Assay, Sequencing

    21) Product Images from "TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE-Mediated Generation and Metabolic Analysis of Camalexin-Deficient cyp71a12 cyp71a13 Double Knockout Lines 1"

    Article Title: TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE-Mediated Generation and Metabolic Analysis of Camalexin-Deficient cyp71a12 cyp71a13 Double Knockout Lines 1

    Journal: Plant Physiology

    doi: 10.1104/pp.15.00481

    Somatic mutagenesis and inheritance of TALEN-mediated mutations in CYP71A12 . A, Schematic representation of the analysis of targeted mutagenesis; the TALEN-binding site targeting one of two Bsa analysis for a wild-type plant and a homozygous mutant plant. C and D, Analysis of TALEN-induced mutations in CYP71A12 . The sequence of the CYP71A12 wild-type allele with TALEN-binding sites is in italic letters, and the targeted Bsa I restriction site is in boldface; insertions are in lowercase letters, and deletions are indicated as dashes. C, Somatic events detected. D, Stable lines generated. prim. transf., Primary transformant; *, of the 82-bp deletion event, only 56 deleted bp are indicated.
    Figure Legend Snippet: Somatic mutagenesis and inheritance of TALEN-mediated mutations in CYP71A12 . A, Schematic representation of the analysis of targeted mutagenesis; the TALEN-binding site targeting one of two Bsa analysis for a wild-type plant and a homozygous mutant plant. C and D, Analysis of TALEN-induced mutations in CYP71A12 . The sequence of the CYP71A12 wild-type allele with TALEN-binding sites is in italic letters, and the targeted Bsa I restriction site is in boldface; insertions are in lowercase letters, and deletions are indicated as dashes. C, Somatic events detected. D, Stable lines generated. prim. transf., Primary transformant; *, of the 82-bp deletion event, only 56 deleted bp are indicated.

    Techniques Used: Mutagenesis, Binding Assay, Sequencing, Generated

    22) Product Images from "Identification of a Mutation Associated with Erythromycin Resistance in Bordetella pertussis: Implications for Surveillance of Antimicrobial Resistance"

    Article Title: Identification of a Mutation Associated with Erythromycin Resistance in Bordetella pertussis: Implications for Surveillance of Antimicrobial Resistance

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.41.3.1167-1172.2003

    Screening for A2058G and A2059G mutations in B. pertussis by PCR-RFLP analysis. (A). Bsa I (lanes 1 to 5) or Bbs I (lanes 6 to 10) digestion of a 521-bp fragment of the 23S rDNA gene of erythromycin-resistant B. pertussis clinical isolates (A228, C353, and MN2531), heterogeneous strain C352, and erythromycin-susceptible strain MN2726. The 521-bp fragment was generated by PCR amplification using primers 1907U and 2408L as described in Materials and Methods. Lanes: M, 100-bp ladder (Life Technologies); 1 and 6, B. pertussis A228; 2 and 7, B. pertussis C352; 3 and 8, B. pertussis C353; 4 and 9, B. pertussis MN2531; 5 and 10, B. pertussis MN2726. (B). Bbs I digestion of the 521-bp fragment of additional isolates of B. pertussis . Lanes: M, 100-bp ladder (Life Technologies); 1 to 7, erythromycin-susceptible clinical isolates B. pertussis MN277, MN973, MN1286, MN1699, MN1773, MN1893, and MN2726; 8, erythromycin-resistant B. pertussis isolate MN253.
    Figure Legend Snippet: Screening for A2058G and A2059G mutations in B. pertussis by PCR-RFLP analysis. (A). Bsa I (lanes 1 to 5) or Bbs I (lanes 6 to 10) digestion of a 521-bp fragment of the 23S rDNA gene of erythromycin-resistant B. pertussis clinical isolates (A228, C353, and MN2531), heterogeneous strain C352, and erythromycin-susceptible strain MN2726. The 521-bp fragment was generated by PCR amplification using primers 1907U and 2408L as described in Materials and Methods. Lanes: M, 100-bp ladder (Life Technologies); 1 and 6, B. pertussis A228; 2 and 7, B. pertussis C352; 3 and 8, B. pertussis C353; 4 and 9, B. pertussis MN2531; 5 and 10, B. pertussis MN2726. (B). Bbs I digestion of the 521-bp fragment of additional isolates of B. pertussis . Lanes: M, 100-bp ladder (Life Technologies); 1 to 7, erythromycin-susceptible clinical isolates B. pertussis MN277, MN973, MN1286, MN1699, MN1773, MN1893, and MN2726; 8, erythromycin-resistant B. pertussis isolate MN253.

    Techniques Used: Polymerase Chain Reaction, Generated, Amplification

    23) Product Images from "Programmed Evolution for Optimization of Orthogonal Metabolic Output in Bacteria"

    Article Title: Programmed Evolution for Optimization of Orthogonal Metabolic Output in Bacteria

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0118322

    Combinatorics Module. (A) Junction-Golden Gate Assembly (J-GGA) introduces genetic variation into a single gene expression cassette as shown, or multiple gene expression cassettes arranged in tandem. PCR amplifies the vector and adds BsaI restriction sites and sticky ends complementary to the elements to be inserted. J-GGA inserts element(s) using standardized PCR primers regardless of the insert sequences. (B) The online Golden Gate Assembly Junction Evaluative Tool (GGAJET) enables users to design junctions with compatible sticky ends and specific primers with similar melting temperatures. GGAJET is available at gcat.davidson.edu/SynBio13/GGAJET/ .
    Figure Legend Snippet: Combinatorics Module. (A) Junction-Golden Gate Assembly (J-GGA) introduces genetic variation into a single gene expression cassette as shown, or multiple gene expression cassettes arranged in tandem. PCR amplifies the vector and adds BsaI restriction sites and sticky ends complementary to the elements to be inserted. J-GGA inserts element(s) using standardized PCR primers regardless of the insert sequences. (B) The online Golden Gate Assembly Junction Evaluative Tool (GGAJET) enables users to design junctions with compatible sticky ends and specific primers with similar melting temperatures. GGAJET is available at gcat.davidson.edu/SynBio13/GGAJET/ .

    Techniques Used: Expressing, Polymerase Chain Reaction, Plasmid Preparation

    24) Product Images from "Sequence-Specific DNA Detection at 10 fM by Electromechanical Signal Transduction"

    Article Title: Sequence-Specific DNA Detection at 10 fM by Electromechanical Signal Transduction

    Journal: Analytical Chemistry

    doi: 10.1021/ac5021408

    Schematic of DNA oligomer preparation. (a) Purified pET-21b plasmids were enzymatically digested by selected pairs of ScaI, PvuI, Pst I, BsaI, and EcoNI restriction enzymes, producing fragments of different lengths. The target DNA sequence complementary to the PNA probe is located beginning at plasmid position 4427 (orange band). Plasmid digestion by ScaI and PvuI produced a 110-base, target-containing fragment, T1. Plasmid digestion by PvuI and PstI produced a 125-base, target-free control fragment, C1. Other fragments were produced similarly: T2 (235 bases) using ScaI and PstI, T3 (419 bases) using ScaI and BsaI, T4 (1613 bases) using by PvuI and EcoNI), C2 (184 bases) using PstI and BsaI, C3 (309 bases) using PvuI and BsaI, and C4 (1503 bases) using ScaI and EcoNI. (b) Following digestion, the DNA was isolated by gel electrophoresis, extracted, and purified. (c) Purified double-stranded DNA was denatured and hybridized with bead–PNA probe conjugates. (d) DNA–PNA–bead mixture was injected into the micropipette for electrical detection.
    Figure Legend Snippet: Schematic of DNA oligomer preparation. (a) Purified pET-21b plasmids were enzymatically digested by selected pairs of ScaI, PvuI, Pst I, BsaI, and EcoNI restriction enzymes, producing fragments of different lengths. The target DNA sequence complementary to the PNA probe is located beginning at plasmid position 4427 (orange band). Plasmid digestion by ScaI and PvuI produced a 110-base, target-containing fragment, T1. Plasmid digestion by PvuI and PstI produced a 125-base, target-free control fragment, C1. Other fragments were produced similarly: T2 (235 bases) using ScaI and PstI, T3 (419 bases) using ScaI and BsaI, T4 (1613 bases) using by PvuI and EcoNI), C2 (184 bases) using PstI and BsaI, C3 (309 bases) using PvuI and BsaI, and C4 (1503 bases) using ScaI and EcoNI. (b) Following digestion, the DNA was isolated by gel electrophoresis, extracted, and purified. (c) Purified double-stranded DNA was denatured and hybridized with bead–PNA probe conjugates. (d) DNA–PNA–bead mixture was injected into the micropipette for electrical detection.

    Techniques Used: Purification, Positron Emission Tomography, Sequencing, Plasmid Preparation, Produced, Isolation, Nucleic Acid Electrophoresis, Injection

    25) Product Images from "GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris"

    Article Title: GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris

    Journal: BMC Systems Biology

    doi: 10.1186/s12918-017-0492-3

    Assembly strategy and hierarchical backbone levels of the cloning systems GoldenMOCS and Golden Pi CS. In the microorganism-independent general platform GoldenMOCS, DNA products (synthetic DNA, PCR products or oligonucleotides) are integrated into BB1 by a Bsa I Golden Gate Assembly and fusion sites Fs1, Fs2, Fs3 and Fs4. Fusion sites are indicated as colored boxes with corresponding fusion site number or letter. Basic genetic elements contained in backbone 1 (BB1) can be assembled in recipient BB2 by performing a Bpi I GGA reaction. The transcription units in BB2 are further used for Bsa I assembly into multigene BB3 constructs. Single transcription units can be obtained by direct Bpi I assembly into recipient BB3 with fusion sites Fs1-Fs4. Fusion sites determine module and transcription unit positions in assembled constructs. Thereby, fusion sites Fs1 to Fs4 are used to construct single expression cassettes in BB2 and are required between promoter (Fs1-Fs2), CDS (Fs2-Fs3) and terminator (Fs3-Fs4). Fusion sites FsA to FsI are designed to construct BB3 plasmids and separate the different expression cassettes from each other. The FSs are almost randomly chosen sequences and only FS2 has a special function, because it includes the start codon ATG. Golden Pi CS additionally includes module-containing BB1s specific for P. pastoris : 20 promoters, 1 reporter gene (eGFP) and 10 transcription terminators, and recipient BB3 vectors containing different integration loci for stable genome integration in P. pastoris and suitable resistance cassettes (Additional file 2 )
    Figure Legend Snippet: Assembly strategy and hierarchical backbone levels of the cloning systems GoldenMOCS and Golden Pi CS. In the microorganism-independent general platform GoldenMOCS, DNA products (synthetic DNA, PCR products or oligonucleotides) are integrated into BB1 by a Bsa I Golden Gate Assembly and fusion sites Fs1, Fs2, Fs3 and Fs4. Fusion sites are indicated as colored boxes with corresponding fusion site number or letter. Basic genetic elements contained in backbone 1 (BB1) can be assembled in recipient BB2 by performing a Bpi I GGA reaction. The transcription units in BB2 are further used for Bsa I assembly into multigene BB3 constructs. Single transcription units can be obtained by direct Bpi I assembly into recipient BB3 with fusion sites Fs1-Fs4. Fusion sites determine module and transcription unit positions in assembled constructs. Thereby, fusion sites Fs1 to Fs4 are used to construct single expression cassettes in BB2 and are required between promoter (Fs1-Fs2), CDS (Fs2-Fs3) and terminator (Fs3-Fs4). Fusion sites FsA to FsI are designed to construct BB3 plasmids and separate the different expression cassettes from each other. The FSs are almost randomly chosen sequences and only FS2 has a special function, because it includes the start codon ATG. Golden Pi CS additionally includes module-containing BB1s specific for P. pastoris : 20 promoters, 1 reporter gene (eGFP) and 10 transcription terminators, and recipient BB3 vectors containing different integration loci for stable genome integration in P. pastoris and suitable resistance cassettes (Additional file 2 )

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Construct, Expressing

    26) Product Images from "Prevalence of A2143G mutation of H. pylori-23S rRNA in Chinese subjects with and without clarithromycin use history"

    Article Title: Prevalence of A2143G mutation of H. pylori-23S rRNA in Chinese subjects with and without clarithromycin use history

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-8-81

    Chromatograms of PCR-RFLP assays and sequencing for detection of nucleotide alterations of 23S rRNA . H. Pylori 26695 and CLR r -1 were used as negative and positive control of A2143G mutation. BsaI digestion of the PCR products of representative samples was displayed on 8% PAGE gel. The 289 bp A2143G-positive PCR products were cleaved into a 199 bp and a 90 bp fragments ( A ). The A2143G mutation was also confirmed by sequencing of the PCR products of 23S rRNA ( B , displayed 2140–2154 fragment). H. Pylori 26695 and a 2142G clone were used as negative and positive control of A2142G mutation. MboII digestion of the PCR products of representative samples was displayed on 2% agarose gel. The 289 bp A2142G-positive PCR products of 2142G were cleaved into an 182 bp and a 107 bp fragments. The PCR product of GJ2040 was cleaved into a 164 bp and a 125 bp fragments; and the product of GJ2111 was cleaved into 245 bp and 44 bp fragment(s) ( C ). The A2142G and other mutations were confirmed by sequencing ( D ). Two new MboII -sensitive sequences were characterized as CTTCA (2222–2226) for GJ2040 and GAAG (2081–2084) for GJ2111.
    Figure Legend Snippet: Chromatograms of PCR-RFLP assays and sequencing for detection of nucleotide alterations of 23S rRNA . H. Pylori 26695 and CLR r -1 were used as negative and positive control of A2143G mutation. BsaI digestion of the PCR products of representative samples was displayed on 8% PAGE gel. The 289 bp A2143G-positive PCR products were cleaved into a 199 bp and a 90 bp fragments ( A ). The A2143G mutation was also confirmed by sequencing of the PCR products of 23S rRNA ( B , displayed 2140–2154 fragment). H. Pylori 26695 and a 2142G clone were used as negative and positive control of A2142G mutation. MboII digestion of the PCR products of representative samples was displayed on 2% agarose gel. The 289 bp A2142G-positive PCR products of 2142G were cleaved into an 182 bp and a 107 bp fragments. The PCR product of GJ2040 was cleaved into a 164 bp and a 125 bp fragments; and the product of GJ2111 was cleaved into 245 bp and 44 bp fragment(s) ( C ). The A2142G and other mutations were confirmed by sequencing ( D ). Two new MboII -sensitive sequences were characterized as CTTCA (2222–2226) for GJ2040 and GAAG (2081–2084) for GJ2111.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Positive Control, Mutagenesis, Polyacrylamide Gel Electrophoresis, Agarose Gel Electrophoresis

    27) Product Images from "MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme"

    Article Title: MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky596

    Identification of suitable methylases for methylation-switching. ( A ) Initial screening of functional methylases for selective blocking of overlapping methylation/restriction sites. The diagrams show the design of overlapping sites for screening of methylase activity. The table shows the screening result using methylases expressed in vivo from an F-ori based low copy number vector. Restriction enzyme recognition sites are boxed in solid lines. The adhesive ends generated by the restriction enzyme are shown by solid lines. Methylase recognition sites are boxed in dashed lines, and methylated bases are in bold font. All the listed methylases modify N6-adenine, except M.SacI and M.AspJHL3I, which modify C5-cytosine and N4-cytosine respectively. ( B ) Experimental designs to test blocking of methylation-switchable type IIS restriction enzyme sites by in vivo methylation. For each methylase/restriction enzyme combination tested, the test plasmid contains a head-to-head potentially methylation-switchable restriction site and a non-methylatable restriction site. Restriction digestion of test plasmid prepared from a normal E. coli strain would result in cutting at both sites and the release of a 600 bp fragment from the 4.3 kb vector backbone. Restriction digestion of test plasmid prepared from a strain expressing the switch methylase would result in a single 4.9 kb band, due to blocking of the methylation-switchable restriction sites by in vivo methylation. The restriction sites of the test plasmids for each restriction enzyme are shown, with the restriction site boxed in solid line, the methylase recognition site boxed in dashed line, and the methylated bases in bold. The head-to-head arrangement of overlapping methylation/restriction site allows the same assay to be used to detect any residual single strand nicking activity of the restriction enzyme towards the methylated restriction site. ( C ) Agarose gel electrophoresis analysis of the test plasmids for each methylation-switchable restriction site after preparation of the plasmids in a normal strain (–) or in a strain expressing the appropriate DNA methylase (+) and digested with the corresponding type IIS restriction enzymes. The combinations tested were BsaI with M.Osp807II methylase using test plasmid pMOP_BsaINC, BpiI with M2.NmeMC58II methylase using test plasmid pMOP_BpiINC, and LguI with M.XmnI methylase using test plasmid pMOP_LguINC. Test conditions were 60 fmol test plasmid digested using 5 U BsaI or BpiI, or 2.5 U LguI in 10 μl reactions at 37°C for 1 h. The results show that in vivo methylation by each of the methylases successfully blocked the restriction site for the corresponding type IIS restriction enzyme when the methylase recognition site overlapped the restriction enzyme site. The data shown represents results from three independent experiments.
    Figure Legend Snippet: Identification of suitable methylases for methylation-switching. ( A ) Initial screening of functional methylases for selective blocking of overlapping methylation/restriction sites. The diagrams show the design of overlapping sites for screening of methylase activity. The table shows the screening result using methylases expressed in vivo from an F-ori based low copy number vector. Restriction enzyme recognition sites are boxed in solid lines. The adhesive ends generated by the restriction enzyme are shown by solid lines. Methylase recognition sites are boxed in dashed lines, and methylated bases are in bold font. All the listed methylases modify N6-adenine, except M.SacI and M.AspJHL3I, which modify C5-cytosine and N4-cytosine respectively. ( B ) Experimental designs to test blocking of methylation-switchable type IIS restriction enzyme sites by in vivo methylation. For each methylase/restriction enzyme combination tested, the test plasmid contains a head-to-head potentially methylation-switchable restriction site and a non-methylatable restriction site. Restriction digestion of test plasmid prepared from a normal E. coli strain would result in cutting at both sites and the release of a 600 bp fragment from the 4.3 kb vector backbone. Restriction digestion of test plasmid prepared from a strain expressing the switch methylase would result in a single 4.9 kb band, due to blocking of the methylation-switchable restriction sites by in vivo methylation. The restriction sites of the test plasmids for each restriction enzyme are shown, with the restriction site boxed in solid line, the methylase recognition site boxed in dashed line, and the methylated bases in bold. The head-to-head arrangement of overlapping methylation/restriction site allows the same assay to be used to detect any residual single strand nicking activity of the restriction enzyme towards the methylated restriction site. ( C ) Agarose gel electrophoresis analysis of the test plasmids for each methylation-switchable restriction site after preparation of the plasmids in a normal strain (–) or in a strain expressing the appropriate DNA methylase (+) and digested with the corresponding type IIS restriction enzymes. The combinations tested were BsaI with M.Osp807II methylase using test plasmid pMOP_BsaINC, BpiI with M2.NmeMC58II methylase using test plasmid pMOP_BpiINC, and LguI with M.XmnI methylase using test plasmid pMOP_LguINC. Test conditions were 60 fmol test plasmid digested using 5 U BsaI or BpiI, or 2.5 U LguI in 10 μl reactions at 37°C for 1 h. The results show that in vivo methylation by each of the methylases successfully blocked the restriction site for the corresponding type IIS restriction enzyme when the methylase recognition site overlapped the restriction enzyme site. The data shown represents results from three independent experiments.

    Techniques Used: Methylation, Functional Assay, Blocking Assay, Activity Assay, In Vivo, Low Copy Number, Plasmid Preparation, Generated, Expressing, Agarose Gel Electrophoresis

    BsaI-M.Osp807II based MetClo system. The donor plasmids contain DNA fragments to be assembled (Fragments A–D) flanked by BsaI sites that generate compatible adhesive ends (schematically labelled aaaa-eeee). The BsaI sites overlap with the M.Osp807II methylase recognition sequence. Donor plasmids were prepared from a normal strain that does not express the M.Osp807II switch methylase. As a result, the BsaI sites are not methylated and so the insert DNA fragments can be released by BsaI digestion. The recipient assembly vector contains a LacZα selection marker flanked by head-to-head BsaI sites. The outer pair of BsaI sites closer to the vector backbone overlap with an M.Osp807II methylation sequence and so are methylation-switchable, whereas the inner pair of BsaI sites are not. Preparation of the assembly vector in the M.Osp807II switch methylase-expressing DH10B strain results in selective blocking of the outer pair of BsaI sites. The LacZα fragment can be released by BsaI through cutting at inner pair of BsaI sites. Following a one-pot reaction using BsaI and T4 DNA ligase, ligation among compatible adhesive ends results in ordered assembly of DNA fragments into the assembly vector backbone. The assembled fragment in the assembled plasmid is flanked by methylated BsaI sites, which are not cut by BsaI. Following transformation into a normal strain that does not express the M.Osp807II switch methylase, methylation of the flanking restriction sites is lost, and the assembled fragment can be released by BsaI for the next stage assembly.
    Figure Legend Snippet: BsaI-M.Osp807II based MetClo system. The donor plasmids contain DNA fragments to be assembled (Fragments A–D) flanked by BsaI sites that generate compatible adhesive ends (schematically labelled aaaa-eeee). The BsaI sites overlap with the M.Osp807II methylase recognition sequence. Donor plasmids were prepared from a normal strain that does not express the M.Osp807II switch methylase. As a result, the BsaI sites are not methylated and so the insert DNA fragments can be released by BsaI digestion. The recipient assembly vector contains a LacZα selection marker flanked by head-to-head BsaI sites. The outer pair of BsaI sites closer to the vector backbone overlap with an M.Osp807II methylation sequence and so are methylation-switchable, whereas the inner pair of BsaI sites are not. Preparation of the assembly vector in the M.Osp807II switch methylase-expressing DH10B strain results in selective blocking of the outer pair of BsaI sites. The LacZα fragment can be released by BsaI through cutting at inner pair of BsaI sites. Following a one-pot reaction using BsaI and T4 DNA ligase, ligation among compatible adhesive ends results in ordered assembly of DNA fragments into the assembly vector backbone. The assembled fragment in the assembled plasmid is flanked by methylated BsaI sites, which are not cut by BsaI. Following transformation into a normal strain that does not express the M.Osp807II switch methylase, methylation of the flanking restriction sites is lost, and the assembled fragment can be released by BsaI for the next stage assembly.

    Techniques Used: Sequencing, Methylation, Plasmid Preparation, Selection, Marker, Expressing, Blocking Assay, Ligation, Transformation Assay

    Design of a standard MetClo vector set. Three types of adaptor sequences were designed for the standard MetClo vector set. The vectors contain two head-to-head BsaI sites (boxed) flanking the negative selection marker LacZα. The pair of unmethylated BsaI sites closest to the negative selection marker can be used to release LacZα and generates adhesive ends ‘p’ and ‘q’. This allows any fragments that start with adhesive end ‘p’ and end with adhesive end ‘q’ to be cloned into any of the three types of adaptor sequences (types ‘Start’, ‘Middle’, ‘End’). The outer pair of BsaI sites closest to the vector backbone overlap with the M.Osp807II recognition sequence and are both methylated at the adenine bases highlighted in bold when the vector is prepared in M.Osp807II-expressing E. coli strain. A DNA fragment assembled into these vectors, following transformation into a normal E. coli strain which lacks this switch methylase activity, can be released from the assembled plasmid using BsaI, which can now recognize this unmethylated BsaI site. The released fragment will carry different adhesive ends depending on the type of the vector used. Assembly into a type ‘Start’ vector will generate a fragment flanked by adaptors ‘p-a’; assembly into a type ‘Middle’ vector will generate a fragment flanked by ‘a-b’, ‘b-c’, ‘c-d’ or ‘d-e’; assembly into a type ‘End’ vector will generate a fragment flanked by ‘a-q’, ‘b-q’, ‘c-q’, ‘d-q’ or ‘e-q’. The design of these LacZα selection cassettes flanked by unique adaptors are represented in the figure by the letter codes for the outside adaptor sequences.
    Figure Legend Snippet: Design of a standard MetClo vector set. Three types of adaptor sequences were designed for the standard MetClo vector set. The vectors contain two head-to-head BsaI sites (boxed) flanking the negative selection marker LacZα. The pair of unmethylated BsaI sites closest to the negative selection marker can be used to release LacZα and generates adhesive ends ‘p’ and ‘q’. This allows any fragments that start with adhesive end ‘p’ and end with adhesive end ‘q’ to be cloned into any of the three types of adaptor sequences (types ‘Start’, ‘Middle’, ‘End’). The outer pair of BsaI sites closest to the vector backbone overlap with the M.Osp807II recognition sequence and are both methylated at the adenine bases highlighted in bold when the vector is prepared in M.Osp807II-expressing E. coli strain. A DNA fragment assembled into these vectors, following transformation into a normal E. coli strain which lacks this switch methylase activity, can be released from the assembled plasmid using BsaI, which can now recognize this unmethylated BsaI site. The released fragment will carry different adhesive ends depending on the type of the vector used. Assembly into a type ‘Start’ vector will generate a fragment flanked by adaptors ‘p-a’; assembly into a type ‘Middle’ vector will generate a fragment flanked by ‘a-b’, ‘b-c’, ‘c-d’ or ‘d-e’; assembly into a type ‘End’ vector will generate a fragment flanked by ‘a-q’, ‘b-q’, ‘c-q’, ‘d-q’ or ‘e-q’. The design of these LacZα selection cassettes flanked by unique adaptors are represented in the figure by the letter codes for the outside adaptor sequences.

    Techniques Used: Plasmid Preparation, Selection, Marker, Clone Assay, Sequencing, Methylation, Expressing, Transformation Assay, Activity Assay

    28) Product Images from "A comprehensive assay for targeted multiplex amplification of human DNA sequences"

    Article Title: A comprehensive assay for targeted multiplex amplification of human DNA sequences

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0803240105

    Construction of single-strand probes. ( A ) The bacteriophage lambda DNA (gray) was used as a template, and the primers containing the common amplification primers as adaptors on the 5′ end were used to PCR amplify the spacer that is common to all of the probes. This common spacer with the amplification sequences was the further template for building the double-stranded probe precursor. The left primer with the target sequence (red) had an adaptor with a BsaI site (blue) The right primer with the target sequences (red) has an adaptor (green) with the MlyI site. ( B ) The construct was digested with BsaI (red triangle) cutting 5 bases inwards from the recognition sequence. The digestion creates a phosphorylated 5′end and a recessed 3′ end. The phosphate group is removed by shrimp alkaline phosphatase digestion. A subsequent digestion with MlyI (black triangle) cuts 5 bases inwards from the right-hand recognition sequence to create a blunt end with a 5′ phosphate. The lambda exonuclease digests the lower strand from the 5′ phosphorylated end created by the Mly I digestion leaving the upper strand intact. ( C ) The dHPLC profile of the double-stranded template has two peaks (blue) at ≈4.5 min. After exonuclease digestion (red) there is only one peak at 4.5 min, and one large early peak that represent the digested products. ( D ) The LPP is hybridized to genomic DNA (gray) and gap-filled (orange) using the genomic DNA as template. Ligation occurs when the polymerase reaches the 5′ end of the probe (yellow circle) to form a circular molecule. The amplification sequences (yellow) in the circle are then used to amplify the targeted exons. Digestion with AscI and ClaI (sites are included in the amplification primers) separate the primers from the genomic target sequences.
    Figure Legend Snippet: Construction of single-strand probes. ( A ) The bacteriophage lambda DNA (gray) was used as a template, and the primers containing the common amplification primers as adaptors on the 5′ end were used to PCR amplify the spacer that is common to all of the probes. This common spacer with the amplification sequences was the further template for building the double-stranded probe precursor. The left primer with the target sequence (red) had an adaptor with a BsaI site (blue) The right primer with the target sequences (red) has an adaptor (green) with the MlyI site. ( B ) The construct was digested with BsaI (red triangle) cutting 5 bases inwards from the recognition sequence. The digestion creates a phosphorylated 5′end and a recessed 3′ end. The phosphate group is removed by shrimp alkaline phosphatase digestion. A subsequent digestion with MlyI (black triangle) cuts 5 bases inwards from the right-hand recognition sequence to create a blunt end with a 5′ phosphate. The lambda exonuclease digests the lower strand from the 5′ phosphorylated end created by the Mly I digestion leaving the upper strand intact. ( C ) The dHPLC profile of the double-stranded template has two peaks (blue) at ≈4.5 min. After exonuclease digestion (red) there is only one peak at 4.5 min, and one large early peak that represent the digested products. ( D ) The LPP is hybridized to genomic DNA (gray) and gap-filled (orange) using the genomic DNA as template. Ligation occurs when the polymerase reaches the 5′ end of the probe (yellow circle) to form a circular molecule. The amplification sequences (yellow) in the circle are then used to amplify the targeted exons. Digestion with AscI and ClaI (sites are included in the amplification primers) separate the primers from the genomic target sequences.

    Techniques Used: Lambda DNA Preparation, Amplification, Polymerase Chain Reaction, Sequencing, Construct, Ligation

    29) Product Images from "A robust family of Golden Gate Agrobacterium vectors for plant synthetic biology"

    Article Title: A robust family of Golden Gate Agrobacterium vectors for plant synthetic biology

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2013.00339

    Detail schematic of the two-component assembly using pGoldenGate-SE7 as the destination vector . (A) Synthetic promoter (SP), reporter (GFP), and the pGoldenGate-SE7 destination plasmid containing the lacZ α gene are put in the same reaction tube along with BsaI and the T4 DNA ligase. The recognition site for BsaI (5′-GGTCTC-3′) is shown in bold. The underlined segments are XhoI and HindIII sites flanking the lacZ α gene in the destination vector. (B) Shows the partially single-stranded DNA fragment generated after the digestion with BsaI (C) Ligase is used to assemble the desired product. Note that 5′-ATGA-3′ is the first four bps of the GFP coding DNA sequence and therefore this assembly is scar-less.
    Figure Legend Snippet: Detail schematic of the two-component assembly using pGoldenGate-SE7 as the destination vector . (A) Synthetic promoter (SP), reporter (GFP), and the pGoldenGate-SE7 destination plasmid containing the lacZ α gene are put in the same reaction tube along with BsaI and the T4 DNA ligase. The recognition site for BsaI (5′-GGTCTC-3′) is shown in bold. The underlined segments are XhoI and HindIII sites flanking the lacZ α gene in the destination vector. (B) Shows the partially single-stranded DNA fragment generated after the digestion with BsaI (C) Ligase is used to assemble the desired product. Note that 5′-ATGA-3′ is the first four bps of the GFP coding DNA sequence and therefore this assembly is scar-less.

    Techniques Used: Plasmid Preparation, Generated, Sequencing

    Golden Gate assembly of an insert into the destination vector. (A) Recognition sequences for the Type IIS restriction endonucleases BsaI and SapI . 5′ overhang sequences used for annealing fragments are shown in bold magenta lettering. (B) Example of a Golden Gate-compatible destination vector. Note the orientation of the BsaI sites cause excision of the lacZ α gene. (C) Example of a Golden Gate compatible vector containing a gene of interest (GOI) that will be released after digestion with BsaI . (D) A typical Golden Gate Cloning reaction would involve mixing the destination vector and insert storage vector together into one tube at equal molar ratio with BsaI and T4 DNA ligase. The final vector produced would lack BsaI recognition sequences and be resistant to digestion.
    Figure Legend Snippet: Golden Gate assembly of an insert into the destination vector. (A) Recognition sequences for the Type IIS restriction endonucleases BsaI and SapI . 5′ overhang sequences used for annealing fragments are shown in bold magenta lettering. (B) Example of a Golden Gate-compatible destination vector. Note the orientation of the BsaI sites cause excision of the lacZ α gene. (C) Example of a Golden Gate compatible vector containing a gene of interest (GOI) that will be released after digestion with BsaI . (D) A typical Golden Gate Cloning reaction would involve mixing the destination vector and insert storage vector together into one tube at equal molar ratio with BsaI and T4 DNA ligase. The final vector produced would lack BsaI recognition sequences and be resistant to digestion.

    Techniques Used: Plasmid Preparation, Clone Assay, Produced

    30) Product Images from "Seven novel mutations in the long isoform of the USH2A gene in Chinese families with nonsyndromic retinitis pigmentosa and Usher syndrome Type II"

    Article Title: Seven novel mutations in the long isoform of the USH2A gene in Chinese families with nonsyndromic retinitis pigmentosa and Usher syndrome Type II

    Journal: Molecular Vision

    doi:

    A restriction fragment length analysis of the four mutations detected in this study. A : c.2802T > G abolished a HincII restriction site that co-segregated with the affected individuals and the carriers (42 bp, 57 bp, 99 bp, 717 bp, and 774 bp), but not with unaffected individuals and normal controls (42 bp, 57 bp, and 717 bp). B : c.8232G > C created a new HpyCH4V restriction site that co-segregated with the affected individuals and the carriers (88 bp, 186 bp, 218 bp, and 274 bp), but not with unaffected individuals and normal controls (218 bp, 274 bp). C : c.3788G > A abolished a BsaI restriction site that co-segregated with the affected individuals and the carriers (70 bp, 132 bp, 422 bp, and 492 bp), but not with unaffected individuals and normal controls (70 bp, 132 bp, and 422 bp). D : c.14403C > G created a SpeI restriction site that co-segregated with the affected individuals and the carriers (145 bp, 300 bp, and 445 bp), but not with unaffected individuals and normal controls (445 bp). A participant identification number is given above each lane. N represents normal controls.
    Figure Legend Snippet: A restriction fragment length analysis of the four mutations detected in this study. A : c.2802T > G abolished a HincII restriction site that co-segregated with the affected individuals and the carriers (42 bp, 57 bp, 99 bp, 717 bp, and 774 bp), but not with unaffected individuals and normal controls (42 bp, 57 bp, and 717 bp). B : c.8232G > C created a new HpyCH4V restriction site that co-segregated with the affected individuals and the carriers (88 bp, 186 bp, 218 bp, and 274 bp), but not with unaffected individuals and normal controls (218 bp, 274 bp). C : c.3788G > A abolished a BsaI restriction site that co-segregated with the affected individuals and the carriers (70 bp, 132 bp, 422 bp, and 492 bp), but not with unaffected individuals and normal controls (70 bp, 132 bp, and 422 bp). D : c.14403C > G created a SpeI restriction site that co-segregated with the affected individuals and the carriers (145 bp, 300 bp, and 445 bp), but not with unaffected individuals and normal controls (445 bp). A participant identification number is given above each lane. N represents normal controls.

    Techniques Used:

    31) Product Images from "Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis"

    Article Title: Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14329-5

    Architecture of the MCS-IIS C2. This DNA-sequence is located on the cargo vector between the E. coli ori and the Bacillus antibiotic marker. The recognition sites for five type IIS restriction enzymes (AarI, BtgZI, BbsI, BsaI, BsmBI), each designed to create a 5′ GCGA-overhang are encoded on the DNA stretch. Architecture of all MCS-IIS can be found in Fig. S1 .
    Figure Legend Snippet: Architecture of the MCS-IIS C2. This DNA-sequence is located on the cargo vector between the E. coli ori and the Bacillus antibiotic marker. The recognition sites for five type IIS restriction enzymes (AarI, BtgZI, BbsI, BsaI, BsmBI), each designed to create a 5′ GCGA-overhang are encoded on the DNA stretch. Architecture of all MCS-IIS can be found in Fig. S1 .

    Techniques Used: Sequencing, Plasmid Preparation, Marker

    32) Product Images from "A novel one-step approach for the construction of yeast surface display Fab antibody libraries"

    Article Title: A novel one-step approach for the construction of yeast surface display Fab antibody libraries

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-017-0853-z

    One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping
    Figure Legend Snippet: One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Expressing, Sequencing, Generated, Plasmid Preparation

    33) Product Images from "PCR Using 3?-Mismatched Primers To Detect A2142C Mutation in 23S rRNA Conferring Resistance to Clarithromycin in Helicobacter pylori Clinical Isolates"

    Article Title: PCR Using 3?-Mismatched Primers To Detect A2142C Mutation in 23S rRNA Conferring Resistance to Clarithromycin in Helicobacter pylori Clinical Isolates

    Journal: Journal of Clinical Microbiology

    doi:

    PCR-RFLP patterns obtained after digestion with Bsa I or Mbo II. Bsa I digested the 1.4-kbp fragment, producing 1,000- and 400-bp fragments in either susceptible or resistant strains. However, if the A2143G mutation was present, the 1,000-bp fragment was converted to 700- and 300-bp fragments. Mbo II digested the fragment, producing two fragments of 700 bp only when the A2142G mutation was present. Lanes: 1 and 2, strain 1 digested with Bsa I and Mbo II, respectively; 3 and 4, strain 2 digested with Bsa I and Mbo II, respectively; 5 and 6, strain 3 digested with Bsa I and Mbo II, respectively; 7 and 8, strain 4 digested with Bsa I and Mbo II, respectively; 9, DNA markers. Bsa I digested the fragment in strains 1 and 2. Bsa I or Mbo II did not digest strains 3 and 4.
    Figure Legend Snippet: PCR-RFLP patterns obtained after digestion with Bsa I or Mbo II. Bsa I digested the 1.4-kbp fragment, producing 1,000- and 400-bp fragments in either susceptible or resistant strains. However, if the A2143G mutation was present, the 1,000-bp fragment was converted to 700- and 300-bp fragments. Mbo II digested the fragment, producing two fragments of 700 bp only when the A2142G mutation was present. Lanes: 1 and 2, strain 1 digested with Bsa I and Mbo II, respectively; 3 and 4, strain 2 digested with Bsa I and Mbo II, respectively; 5 and 6, strain 3 digested with Bsa I and Mbo II, respectively; 7 and 8, strain 4 digested with Bsa I and Mbo II, respectively; 9, DNA markers. Bsa I digested the fragment in strains 1 and 2. Bsa I or Mbo II did not digest strains 3 and 4.

    Techniques Used: Polymerase Chain Reaction, Mutagenesis

    34) Product Images from "Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha"

    Article Title: Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0205117

    All-in-one vector systems for genome editing in M . polymorpha . (A) Designs of Gateway-based all-in-one binary vectors and entry plasmids for gRNA cloning. pMpGE010 and pMpGE011 contain a cassette for the expression of Atco-Cas9 fused with an NLS under the control of Mp EF pro , a Gateway cassette, and a cassette for the expression of hygromycin phosphotransferase ( HPT ) in pMpGE010 and mutated acetolactate synthase ( mALS ) in pMpGE011. pMpGE_En01 contains recognition sites for two restriction enzymes, SacI and PstI, upstream of a gRNA backbone for the insertion of a guide sequence by In-Fusion/Gibson cloning, which automatically places a G nucleotide for transcription initiation by RNA polymerase III (extra initial G). Expression of single guide RNAs is controlled by a 2 kbp fragment of Mp U6-1 pro . pMpGE_En02 and pMpGE_En03 contain two BsaI recognition sites upstream of the gRNA backbone for the insertion of a guide sequence by ligation without or with an “extra initial G,” whose expression is under the control of a 500 bp Mp U6-1 pro fragment. For all the entry vectors, the gRNA cassette is flanked by the attL1 and attL2 sequences and is thus transferrable to the Gateway cassette in pMpGE010 or pMpGWB011 by the LR reaction. (B) Designs of all-in-one binary vectors for direct gRNA cloning. pMpGE013 ( HPT marker) and pMpGE014 ( mALS marker) contain the Atco-Cas9-NLS expression cassette, a unique AarI site in the upstream of the gRNA backbone for insertion of a guide sequence by ligation with an “extra initial G,” whose expression is under the control of a 500 bp Mp U6-1 pro fragment.
    Figure Legend Snippet: All-in-one vector systems for genome editing in M . polymorpha . (A) Designs of Gateway-based all-in-one binary vectors and entry plasmids for gRNA cloning. pMpGE010 and pMpGE011 contain a cassette for the expression of Atco-Cas9 fused with an NLS under the control of Mp EF pro , a Gateway cassette, and a cassette for the expression of hygromycin phosphotransferase ( HPT ) in pMpGE010 and mutated acetolactate synthase ( mALS ) in pMpGE011. pMpGE_En01 contains recognition sites for two restriction enzymes, SacI and PstI, upstream of a gRNA backbone for the insertion of a guide sequence by In-Fusion/Gibson cloning, which automatically places a G nucleotide for transcription initiation by RNA polymerase III (extra initial G). Expression of single guide RNAs is controlled by a 2 kbp fragment of Mp U6-1 pro . pMpGE_En02 and pMpGE_En03 contain two BsaI recognition sites upstream of the gRNA backbone for the insertion of a guide sequence by ligation without or with an “extra initial G,” whose expression is under the control of a 500 bp Mp U6-1 pro fragment. For all the entry vectors, the gRNA cassette is flanked by the attL1 and attL2 sequences and is thus transferrable to the Gateway cassette in pMpGE010 or pMpGWB011 by the LR reaction. (B) Designs of all-in-one binary vectors for direct gRNA cloning. pMpGE013 ( HPT marker) and pMpGE014 ( mALS marker) contain the Atco-Cas9-NLS expression cassette, a unique AarI site in the upstream of the gRNA backbone for insertion of a guide sequence by ligation with an “extra initial G,” whose expression is under the control of a 500 bp Mp U6-1 pro fragment.

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

    35) Product Images from "Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction"

    Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111538

    Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).
    Figure Legend Snippet: Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).

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

    36) Product Images from "pEVL: A Linear Plasmid for Generating mRNA IVT Templates With Extended Encoded Poly(A) Sequences"

    Article Title: pEVL: A Linear Plasmid for Generating mRNA IVT Templates With Extended Encoded Poly(A) Sequences

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1038/mtna.2016.21

    Characterization of poly(A) tract stability in pEVL. ( a ) Stability of the encoded poly(A) tracts following overnight induction of pEVL for template preparation. BFP-pEVL-200 through 500 were grown overnight with induction at 30 °C and then maxiprepped. Each maxiprepped sample was digested with BsiWI and BsaI to release the poly(A) tail fragment from the rest of the plasmid. The tail length was determined by gel electrophoresis with comparison to a known molecular weight s tandard. ( b ) Shortening of poly(A) tracts upon cloning into standard circular or linear plasmid cloning vectors at 30 °C. BFP followed by poly(A) tract inserts of 70, 172, and 325 base pairs bounded by restriction enzyme sites DraIII and SwaI were generated via restriction enzyme digest from the linear plasmid cloning vectors pEVL-100, pEVL-200, and pEVL-300. The inserts were ligated into the circular cloning vector pWNY or subcloned into pEVL and transformed via electroporation. Transformed bacteria were grown with ampicillin (pWNY) or kanamycin (pEVL) selection at 30 °C. Individual colonies were amplified by PCR using primers flanking the poly(A) tract, and the length of the poly(A) tract was determined based on the resulting band size as in Figure 1 . Typically, a band was obtained at the expected size, or a smaller size, reflecting shortening of the poly(A) tract during transformation. Colonies were scored for whether the poly(A) tract fragment was approximately of the expected size (open circle), or was substantially shortened (closed circle). ( c ) Stability of encoded poly(A) tracts under extended propagation conditions. To test the stability of the poly(A) tail under stringent propagation conditions, pEVL 100 through 500 were grown for 2 weeks at 30 °C and 37 °C with reseeding into fresh media at a 1:1000 dilution every 24 hours. At days 0, 6, and 13, each sample was similarly reseeded into induction media and grown overnight before being miniprepped. Parallel analysis was performed with the circular vectors described in ( b ), in which the poly(A) tract fragment was sub-cloned into a circular vector (pWNY). As these are already high-copy plasmids, no inducing agent was added to the cultures. For the circular vectors, samples were miniprepped daily for 7 days. For both pEVL and the circular vectors, the tail length of the induced minipreps was determined by gel elctrophoresis as described above. The expected tail band size for each construct is indicated with an arrow.
    Figure Legend Snippet: Characterization of poly(A) tract stability in pEVL. ( a ) Stability of the encoded poly(A) tracts following overnight induction of pEVL for template preparation. BFP-pEVL-200 through 500 were grown overnight with induction at 30 °C and then maxiprepped. Each maxiprepped sample was digested with BsiWI and BsaI to release the poly(A) tail fragment from the rest of the plasmid. The tail length was determined by gel electrophoresis with comparison to a known molecular weight s tandard. ( b ) Shortening of poly(A) tracts upon cloning into standard circular or linear plasmid cloning vectors at 30 °C. BFP followed by poly(A) tract inserts of 70, 172, and 325 base pairs bounded by restriction enzyme sites DraIII and SwaI were generated via restriction enzyme digest from the linear plasmid cloning vectors pEVL-100, pEVL-200, and pEVL-300. The inserts were ligated into the circular cloning vector pWNY or subcloned into pEVL and transformed via electroporation. Transformed bacteria were grown with ampicillin (pWNY) or kanamycin (pEVL) selection at 30 °C. Individual colonies were amplified by PCR using primers flanking the poly(A) tract, and the length of the poly(A) tract was determined based on the resulting band size as in Figure 1 . Typically, a band was obtained at the expected size, or a smaller size, reflecting shortening of the poly(A) tract during transformation. Colonies were scored for whether the poly(A) tract fragment was approximately of the expected size (open circle), or was substantially shortened (closed circle). ( c ) Stability of encoded poly(A) tracts under extended propagation conditions. To test the stability of the poly(A) tail under stringent propagation conditions, pEVL 100 through 500 were grown for 2 weeks at 30 °C and 37 °C with reseeding into fresh media at a 1:1000 dilution every 24 hours. At days 0, 6, and 13, each sample was similarly reseeded into induction media and grown overnight before being miniprepped. Parallel analysis was performed with the circular vectors described in ( b ), in which the poly(A) tract fragment was sub-cloned into a circular vector (pWNY). As these are already high-copy plasmids, no inducing agent was added to the cultures. For the circular vectors, samples were miniprepped daily for 7 days. For both pEVL and the circular vectors, the tail length of the induced minipreps was determined by gel elctrophoresis as described above. The expected tail band size for each construct is indicated with an arrow.

    Techniques Used: Plasmid Preparation, Nucleic Acid Electrophoresis, Molecular Weight, Clone Assay, Generated, Transformation Assay, Electroporation, Selection, Amplification, Polymerase Chain Reaction, Construct

    Generation and characterization of mRNA from pEVL-encoded templates . ( a ) IVT mRNA encoding blue fluorescent protein (mTagBFP2) generated from pWNY with enzymatic tailing and pEVL-100 through pEVL-500. BFP-pEVL-100 to 500 were digested with XbaI and BsaI, and pWNY with ScaI and BsiWI, to generate template for IVT. IVT was carried out with antireverse cap analog capping, and for pWNY, enzymatic tailing with EPAP. After purification, 200 ng of each transcript was imaged via gel electrophoresis on the FlashGel system. Typically, pEVL produces a single band of defined length, whereas pWNY with enzymatic tailing produces transcripts of a more heterogenous length. ( b ) Relative potency of mRNA encoding BFP generated from a circular plasmid vector with enzymatic polyadenylation or from pEVL-300 and representative flow plots. 1 μg of IVT mRNA from the indicated template was electroporated into prestimulated primary human T cells. After a 24-hour cold shock at 30 ° C, the cells were analyzed each day for 5 days by flow cytometry for the percentage of cells expressing BFP as well as the mean fluorescence intensity (MFI) of the BFP in BFP+ cells. Flow plots are shown as side scatter (SSC) versus BFP. ( c ) Relative potency of mRNA encoding BFP generated from pEVL-100 through pEVL-500 and representative flow plots. Equimolar amounts of IVT mRNA from BFP-pEVL-100 to 500 were electroporated into prestimulated primary human T cells. After an initial 24-hour cold shock at 30 ° C, the cells were grown at 37 °C for 6 more days. Every 24 hours after electroporation, the percentage of cells expressing BFP and the BFI MFI of the BFP+ cells was analyzed by flow cytometry. Flow plots are shown as side scatter (SSC) versus BFP. BFP, blue fluorescent protein; IVT, in vitro transcribed; pEVL, p(Extended Variable Length).
    Figure Legend Snippet: Generation and characterization of mRNA from pEVL-encoded templates . ( a ) IVT mRNA encoding blue fluorescent protein (mTagBFP2) generated from pWNY with enzymatic tailing and pEVL-100 through pEVL-500. BFP-pEVL-100 to 500 were digested with XbaI and BsaI, and pWNY with ScaI and BsiWI, to generate template for IVT. IVT was carried out with antireverse cap analog capping, and for pWNY, enzymatic tailing with EPAP. After purification, 200 ng of each transcript was imaged via gel electrophoresis on the FlashGel system. Typically, pEVL produces a single band of defined length, whereas pWNY with enzymatic tailing produces transcripts of a more heterogenous length. ( b ) Relative potency of mRNA encoding BFP generated from a circular plasmid vector with enzymatic polyadenylation or from pEVL-300 and representative flow plots. 1 μg of IVT mRNA from the indicated template was electroporated into prestimulated primary human T cells. After a 24-hour cold shock at 30 ° C, the cells were analyzed each day for 5 days by flow cytometry for the percentage of cells expressing BFP as well as the mean fluorescence intensity (MFI) of the BFP in BFP+ cells. Flow plots are shown as side scatter (SSC) versus BFP. ( c ) Relative potency of mRNA encoding BFP generated from pEVL-100 through pEVL-500 and representative flow plots. Equimolar amounts of IVT mRNA from BFP-pEVL-100 to 500 were electroporated into prestimulated primary human T cells. After an initial 24-hour cold shock at 30 ° C, the cells were grown at 37 °C for 6 more days. Every 24 hours after electroporation, the percentage of cells expressing BFP and the BFI MFI of the BFP+ cells was analyzed by flow cytometry. Flow plots are shown as side scatter (SSC) versus BFP. BFP, blue fluorescent protein; IVT, in vitro transcribed; pEVL, p(Extended Variable Length).

    Techniques Used: Generated, Purification, Nucleic Acid Electrophoresis, Plasmid Preparation, Flow Cytometry, Cytometry, Expressing, Fluorescence, Electroporation, In Vitro

    Generation of pEVL: a linear plasmid vector for generation of mRNA with extended encoded poly(A) tracts. ( a ) Schematic of pJazz and conversion to pEVL. The plasmids are shown with orange arrows denoting genes, red circles with T's denoting transcriptional terminators, open circles denoting terminal hairpin loops, yellow blocks denoting BsaI sites, and green blocks denoting the poly(A) tail. ( b ) Schematic of pEVL and method used for generation of extended poly(A) tracts in pEVL.
    Figure Legend Snippet: Generation of pEVL: a linear plasmid vector for generation of mRNA with extended encoded poly(A) tracts. ( a ) Schematic of pJazz and conversion to pEVL. The plasmids are shown with orange arrows denoting genes, red circles with T's denoting transcriptional terminators, open circles denoting terminal hairpin loops, yellow blocks denoting BsaI sites, and green blocks denoting the poly(A) tail. ( b ) Schematic of pEVL and method used for generation of extended poly(A) tracts in pEVL.

    Techniques Used: Plasmid Preparation

    37) Product Images from "MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme"

    Article Title: MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky596

    Design of a standard MetClo vector set. Three types of adaptor sequences were designed for the standard MetClo vector set. The vectors contain two head-to-head BsaI sites (boxed) flanking the negative selection marker LacZα. The pair of unmethylated BsaI sites closest to the negative selection marker can be used to release LacZα and generates adhesive ends ‘p’ and ‘q’. This allows any fragments that start with adhesive end ‘p’ and end with adhesive end ‘q’ to be cloned into any of the three types of adaptor sequences (types ‘Start’, ‘Middle’, ‘End’). The outer pair of BsaI sites closest to the vector backbone overlap with the M.Osp807II recognition sequence and are both methylated at the adenine bases highlighted in bold when the vector is prepared in M.Osp807II-expressing E. coli strain. A DNA fragment assembled into these vectors, following transformation into a normal E. coli strain which lacks this switch methylase activity, can be released from the assembled plasmid using BsaI, which can now recognize this unmethylated BsaI site. The released fragment will carry different adhesive ends depending on the type of the vector used. Assembly into a type ‘Start’ vector will generate a fragment flanked by adaptors ‘p-a’; assembly into a type ‘Middle’ vector will generate a fragment flanked by ‘a-b’, ‘b-c’, ‘c-d’ or ‘d-e’; assembly into a type ‘End’ vector will generate a fragment flanked by ‘a-q’, ‘b-q’, ‘c-q’, ‘d-q’ or ‘e-q’. The design of these LacZα selection cassettes flanked by unique adaptors are represented in the figure by the letter codes for the outside adaptor sequences.
    Figure Legend Snippet: Design of a standard MetClo vector set. Three types of adaptor sequences were designed for the standard MetClo vector set. The vectors contain two head-to-head BsaI sites (boxed) flanking the negative selection marker LacZα. The pair of unmethylated BsaI sites closest to the negative selection marker can be used to release LacZα and generates adhesive ends ‘p’ and ‘q’. This allows any fragments that start with adhesive end ‘p’ and end with adhesive end ‘q’ to be cloned into any of the three types of adaptor sequences (types ‘Start’, ‘Middle’, ‘End’). The outer pair of BsaI sites closest to the vector backbone overlap with the M.Osp807II recognition sequence and are both methylated at the adenine bases highlighted in bold when the vector is prepared in M.Osp807II-expressing E. coli strain. A DNA fragment assembled into these vectors, following transformation into a normal E. coli strain which lacks this switch methylase activity, can be released from the assembled plasmid using BsaI, which can now recognize this unmethylated BsaI site. The released fragment will carry different adhesive ends depending on the type of the vector used. Assembly into a type ‘Start’ vector will generate a fragment flanked by adaptors ‘p-a’; assembly into a type ‘Middle’ vector will generate a fragment flanked by ‘a-b’, ‘b-c’, ‘c-d’ or ‘d-e’; assembly into a type ‘End’ vector will generate a fragment flanked by ‘a-q’, ‘b-q’, ‘c-q’, ‘d-q’ or ‘e-q’. The design of these LacZα selection cassettes flanked by unique adaptors are represented in the figure by the letter codes for the outside adaptor sequences.

    Techniques Used: Plasmid Preparation, Selection, Marker, Clone Assay, Sequencing, Methylation, Expressing, Transformation Assay, Activity Assay

    Identification of suitable methylases for methylation-switching. ( A ) Initial screening of functional methylases for selective blocking of overlapping methylation/restriction sites. The diagrams show the design of overlapping sites for screening of methylase activity. The table shows the screening result using methylases expressed in vivo from an F-ori based low copy number vector. Restriction enzyme recognition sites are boxed in solid lines. The adhesive ends generated by the restriction enzyme are shown by solid lines. Methylase recognition sites are boxed in dashed lines, and methylated bases are in bold font. All the listed methylases modify N6-adenine, except M.SacI and M.AspJHL3I, which modify C5-cytosine and N4-cytosine respectively. ( B ) Experimental designs to test blocking of methylation-switchable type IIS restriction enzyme sites by in vivo methylation. For each methylase/restriction enzyme combination tested, the test plasmid contains a head-to-head potentially methylation-switchable restriction site and a non-methylatable restriction site. Restriction digestion of test plasmid prepared from a normal E. coli strain would result in cutting at both sites and the release of a 600 bp fragment from the 4.3 kb vector backbone. Restriction digestion of test plasmid prepared from a strain expressing the switch methylase would result in a single 4.9 kb band, due to blocking of the methylation-switchable restriction sites by in vivo methylation. The restriction sites of the test plasmids for each restriction enzyme are shown, with the restriction site boxed in solid line, the methylase recognition site boxed in dashed line, and the methylated bases in bold. The head-to-head arrangement of overlapping methylation/restriction site allows the same assay to be used to detect any residual single strand nicking activity of the restriction enzyme towards the methylated restriction site. ( C ) Agarose gel electrophoresis analysis of the test plasmids for each methylation-switchable restriction site after preparation of the plasmids in a normal strain (–) or in a strain expressing the appropriate DNA methylase (+) and digested with the corresponding type IIS restriction enzymes. The combinations tested were BsaI with M.Osp807II methylase using test plasmid pMOP_BsaINC, BpiI with M2.NmeMC58II methylase using test plasmid pMOP_BpiINC, and LguI with M.XmnI methylase using test plasmid pMOP_LguINC. Test conditions were 60 fmol test plasmid digested using 5 U BsaI or BpiI, or 2.5 U LguI in 10 μl reactions at 37°C for 1 h. The results show that in vivo methylation by each of the methylases successfully blocked the restriction site for the corresponding type IIS restriction enzyme when the methylase recognition site overlapped the restriction enzyme site. The data shown represents results from three independent experiments.
    Figure Legend Snippet: Identification of suitable methylases for methylation-switching. ( A ) Initial screening of functional methylases for selective blocking of overlapping methylation/restriction sites. The diagrams show the design of overlapping sites for screening of methylase activity. The table shows the screening result using methylases expressed in vivo from an F-ori based low copy number vector. Restriction enzyme recognition sites are boxed in solid lines. The adhesive ends generated by the restriction enzyme are shown by solid lines. Methylase recognition sites are boxed in dashed lines, and methylated bases are in bold font. All the listed methylases modify N6-adenine, except M.SacI and M.AspJHL3I, which modify C5-cytosine and N4-cytosine respectively. ( B ) Experimental designs to test blocking of methylation-switchable type IIS restriction enzyme sites by in vivo methylation. For each methylase/restriction enzyme combination tested, the test plasmid contains a head-to-head potentially methylation-switchable restriction site and a non-methylatable restriction site. Restriction digestion of test plasmid prepared from a normal E. coli strain would result in cutting at both sites and the release of a 600 bp fragment from the 4.3 kb vector backbone. Restriction digestion of test plasmid prepared from a strain expressing the switch methylase would result in a single 4.9 kb band, due to blocking of the methylation-switchable restriction sites by in vivo methylation. The restriction sites of the test plasmids for each restriction enzyme are shown, with the restriction site boxed in solid line, the methylase recognition site boxed in dashed line, and the methylated bases in bold. The head-to-head arrangement of overlapping methylation/restriction site allows the same assay to be used to detect any residual single strand nicking activity of the restriction enzyme towards the methylated restriction site. ( C ) Agarose gel electrophoresis analysis of the test plasmids for each methylation-switchable restriction site after preparation of the plasmids in a normal strain (–) or in a strain expressing the appropriate DNA methylase (+) and digested with the corresponding type IIS restriction enzymes. The combinations tested were BsaI with M.Osp807II methylase using test plasmid pMOP_BsaINC, BpiI with M2.NmeMC58II methylase using test plasmid pMOP_BpiINC, and LguI with M.XmnI methylase using test plasmid pMOP_LguINC. Test conditions were 60 fmol test plasmid digested using 5 U BsaI or BpiI, or 2.5 U LguI in 10 μl reactions at 37°C for 1 h. The results show that in vivo methylation by each of the methylases successfully blocked the restriction site for the corresponding type IIS restriction enzyme when the methylase recognition site overlapped the restriction enzyme site. The data shown represents results from three independent experiments.

    Techniques Used: Methylation, Functional Assay, Blocking Assay, Activity Assay, In Vivo, Low Copy Number, Plasmid Preparation, Generated, Expressing, Agarose Gel Electrophoresis

    38) Product Images from "Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe"

    Article Title: Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

    Journal: Open Biology

    doi: 10.1098/rsob.150054

    The Golden Gate method to create integration plasmids. ( a ) A schematic of an integration plasmid. (left) An example of an integration plasmid to express a GOI in fusion with a fluorescent protein (FP) tag under a promoter. A target module comprises tar.F and tar.R regions separated by an Fse I restriction site. (right) An integration plasmid can be linearized with Fse I, and tar.F and tar.R sequences are targeted to the homologous sequences on the S. pombe chromosome, to induce homologous recombination. ( b ) A schematic for the Golden Gate reaction. (left) Examples of module elements. Modules are given either as plasmids (1, 3–6) or as PCR products (2). Modules for a promoter module (1), GOI (2), an FPtag (3), a selection marker (4), a target region (5), the vector backbone (6). a–f: cohesive ends to connect modules 1–6 in this order. (right) A reaction protocol for the Golden Gate reaction by the mixture of 1–6 and the resulting circular integration plasmid (7). Each module plasmid (1, 3–6) contains the kanamycin resistance gene (KanR), whereas the final product (integration plasmid, 7) is ampicillin resistant. ( c ) Unique property of Bsa I. (left) Eco RI, a standard restriction enzyme, cleaves its recognition site, therefore digestion and religation can be repeated. (right) By contrast, Bsa I has separate sites for recognition (GGTCTC) and digestion (NNNN; any four bases).
    Figure Legend Snippet: The Golden Gate method to create integration plasmids. ( a ) A schematic of an integration plasmid. (left) An example of an integration plasmid to express a GOI in fusion with a fluorescent protein (FP) tag under a promoter. A target module comprises tar.F and tar.R regions separated by an Fse I restriction site. (right) An integration plasmid can be linearized with Fse I, and tar.F and tar.R sequences are targeted to the homologous sequences on the S. pombe chromosome, to induce homologous recombination. ( b ) A schematic for the Golden Gate reaction. (left) Examples of module elements. Modules are given either as plasmids (1, 3–6) or as PCR products (2). Modules for a promoter module (1), GOI (2), an FPtag (3), a selection marker (4), a target region (5), the vector backbone (6). a–f: cohesive ends to connect modules 1–6 in this order. (right) A reaction protocol for the Golden Gate reaction by the mixture of 1–6 and the resulting circular integration plasmid (7). Each module plasmid (1, 3–6) contains the kanamycin resistance gene (KanR), whereas the final product (integration plasmid, 7) is ampicillin resistant. ( c ) Unique property of Bsa I. (left) Eco RI, a standard restriction enzyme, cleaves its recognition site, therefore digestion and religation can be repeated. (right) By contrast, Bsa I has separate sites for recognition (GGTCTC) and digestion (NNNN; any four bases).

    Techniques Used: Plasmid Preparation, Homologous Recombination, Polymerase Chain Reaction, Selection, Marker

    Choice of module plasmids for expression of a C-terminal tagged GOI. ( a ) Detailed illustration of an integration plasmid for expression of the GOI–GFP fusion (C-terminal GFP tag). Modules I–V are connected in the pFA6a-based vector (module VI) in that order. In this example, the adh1 promoter (selected from group I modules) drives expression of the fusion gene of the GOI (GOI (bc), II) with GFP (FPtag-C (cd), III). T adh serves as a terminator. kan (P TEF , promoter; T TEF , terminator) is a selection marker used after S. pombe transformation (module IV). Target module (V) is the sequence that is targeted to a homologous sequence in S. pombe chromosomes. Useful restriction sites are also indicated. Digestion with Not I separates the vector and other modules. JB19F and JB20R correspond to sequences commonly used in PCR-based gene targeting [ 12 ]. a–f in module names indicate the names of Bsa I cohesive ends used therein ( c ). AmpR, the ampicillin resistance gene. ( b , c ) List of module plasmids created in this study. ( b ) Modules are categorized as groups I–VI in boxes. Choose one module from each group to mix. II. The GOI (bc) is made through PCR to add cohesive ends (‘b’ and ‘c’). Group IIIa, instead of II and III, can be used to make control strains. Modules for the adh terminator and a selection marker gene can be supplied together (group IV). Alternatively, each module can be chosen separately: a terminator (group IVa) and a selection marker (group IVb). ( c ) Sequences of cohesive ends named a–g. Note that the module vector pBMod contains a Bsa I site with the cohesive end GTTA.
    Figure Legend Snippet: Choice of module plasmids for expression of a C-terminal tagged GOI. ( a ) Detailed illustration of an integration plasmid for expression of the GOI–GFP fusion (C-terminal GFP tag). Modules I–V are connected in the pFA6a-based vector (module VI) in that order. In this example, the adh1 promoter (selected from group I modules) drives expression of the fusion gene of the GOI (GOI (bc), II) with GFP (FPtag-C (cd), III). T adh serves as a terminator. kan (P TEF , promoter; T TEF , terminator) is a selection marker used after S. pombe transformation (module IV). Target module (V) is the sequence that is targeted to a homologous sequence in S. pombe chromosomes. Useful restriction sites are also indicated. Digestion with Not I separates the vector and other modules. JB19F and JB20R correspond to sequences commonly used in PCR-based gene targeting [ 12 ]. a–f in module names indicate the names of Bsa I cohesive ends used therein ( c ). AmpR, the ampicillin resistance gene. ( b , c ) List of module plasmids created in this study. ( b ) Modules are categorized as groups I–VI in boxes. Choose one module from each group to mix. II. The GOI (bc) is made through PCR to add cohesive ends (‘b’ and ‘c’). Group IIIa, instead of II and III, can be used to make control strains. Modules for the adh terminator and a selection marker gene can be supplied together (group IV). Alternatively, each module can be chosen separately: a terminator (group IVa) and a selection marker (group IVb). ( c ) Sequences of cohesive ends named a–g. Note that the module vector pBMod contains a Bsa I site with the cohesive end GTTA.

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

    39) Product Images from "A plug-and-play pathway refactoring workflow for natural product research in Escherichia coli and Saccharomyces cerevisiae"

    Article Title: A plug-and-play pathway refactoring workflow for natural product research in Escherichia coli and Saccharomyces cerevisiae

    Journal: Biotechnology and bioengineering

    doi: 10.1002/bit.26309

    Scheme of the plug-and-play pathway refactoring workflow. (A) The 1 st tier Golden Gate reaction. The gene is either synthesized or PCR amplified with Bbs I cleavage sites at both ends and cloned into a helper plasmid through Bbs I catalyzed Golden Gate reaction. (B) The 2 nd tier Golden Gate reaction. Helper plasmids harboring the corresponding genes are mixed with the appropriate spacer plasmids and the receiver plasmid and assembled into the final construct through Bsa I catalyzed Golden Gate reaction. All helper plasmids and spacer plasmids share the pUC19 backbone, while the receiver plasmid has either a pET28a backbone (for expression in E. coli ) or pRS416 backbone with the ampicillin resistance gene replaced by the kanamycin resistance gene (for expression in S. cerevisiae ).
    Figure Legend Snippet: Scheme of the plug-and-play pathway refactoring workflow. (A) The 1 st tier Golden Gate reaction. The gene is either synthesized or PCR amplified with Bbs I cleavage sites at both ends and cloned into a helper plasmid through Bbs I catalyzed Golden Gate reaction. (B) The 2 nd tier Golden Gate reaction. Helper plasmids harboring the corresponding genes are mixed with the appropriate spacer plasmids and the receiver plasmid and assembled into the final construct through Bsa I catalyzed Golden Gate reaction. All helper plasmids and spacer plasmids share the pUC19 backbone, while the receiver plasmid has either a pET28a backbone (for expression in E. coli ) or pRS416 backbone with the ampicillin resistance gene replaced by the kanamycin resistance gene (for expression in S. cerevisiae ).

    Techniques Used: Synthesized, Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Construct, Expressing

    40) Product Images from "A novel one-step approach for the construction of yeast surface display Fab antibody libraries"

    Article Title: A novel one-step approach for the construction of yeast surface display Fab antibody libraries

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-017-0853-z

    One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping
    Figure Legend Snippet: One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Expressing, Sequencing, Generated, Plasmid Preparation

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    Article Snippet: .. BsaI and BpiI sites were removed in a so-called “domestication” procedure using a Q5 site-directed mutagenesis (SDM) kit (NEB). ..

    Polymerase Chain Reaction:

    Article Title: A novel one-step approach for the construction of yeast surface display Fab antibody libraries
    Article Snippet: .. 160 ng of pooled VH PCR product as well as 200 U Bsa I (New England Biolabs), 800 U T4 DNA ligase (New England Biolabs) and 10 µL 10× T4 Ligase buffer (New England Biolabs). .. After cloning, six reactions were pooled, purified using Wizard® SV Gel and PCR Clean-up System (Promega) and eluted in a final volume of 30 µL which were subsequently used for one electroporation reaction into EBY100 as previously described by Benatuil et al. [ ].

    Article Title: PCR-Restriction Fragment Length Polymorphism Can Also Detect Point Mutation A2142C in the 23S rRNA Gene, Associated with Helicobacter pylori Resistance to Clarithromycin
    Article Snippet: .. The 267-bp PCR products were precipitated and suspended in 15 μl of H2 O, and 5 μl was digested overnight in a final volume of 15 μl with the restriction enzymes Bbs I (5 U), Bsa I (5 U) , and Bce AI (0.5 U) (New England Biolabs). .. PCR-RFLP allowed the identification of mutations A2142G and A2143G using the Bbs I and Bsa I restriction enzymes, respectively (Fig. , lanes 3 and 5), as previously described ( , , ).

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    Article Title: GoldenBac: a simple, highly efficient, and widely applicable system for construction of multi-gene expression vectors for use with the baculovirus expression vector system
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    Article Snippet: .. Briefly, for each reaction, 25 ng of each plasmid (for trimer libraries, 13 plasmids; for dimer libraries, 9 plasmids) ( ) was combined with 10 U of Bsa I (New England BioLabs, Ipswich, MA) and 400 U of T4 DNA ligase (New England BioLabs) in 1× T4 DNA ligase buffer (reaction volume, 20 μl). .. Three digest–ligation cycles were performed (10-min digest at 37°C; 15-min ligation at 16°C), followed by two 5-min inactivation steps at 50°C and 80°C.

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    Restriction fragment length polymorphism analysis of <t>23S</t> rRNA amplicons: ( A ) digestion with <t>Bsa</t> I and ( B ) digestion with Bbs I. The A2144G mutations are observed in lanes 1 to 2, but not in lanes 3 to 5. Note that the A2143G mutation detected by digestion with Bbs I was not detected in any of the strains studied. Lanes 3 to 5 reveal the T2183C mutation, as assessed by DNA sequencing. Lane M, 100 bp DNA size markers (indicated to the left of the gels in base pairs); lane C, H. pylori ATCC 43504; lane 1 to 5, clarithromycin-resistant H. pylori strains.
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    Restriction fragment length polymorphism analysis of 23S rRNA amplicons: ( A ) digestion with Bsa I and ( B ) digestion with Bbs I. The A2144G mutations are observed in lanes 1 to 2, but not in lanes 3 to 5. Note that the A2143G mutation detected by digestion with Bbs I was not detected in any of the strains studied. Lanes 3 to 5 reveal the T2183C mutation, as assessed by DNA sequencing. Lane M, 100 bp DNA size markers (indicated to the left of the gels in base pairs); lane C, H. pylori ATCC 43504; lane 1 to 5, clarithromycin-resistant H. pylori strains.

    Journal: Journal of Korean Medical Science

    Article Title: Clarithromycin-Based Standard Triple Therapy Can Still Be Effective for Helicobacter pylori Eradication in Some Parts of the Korea

    doi: 10.3346/jkms.2014.29.9.1240

    Figure Lengend Snippet: Restriction fragment length polymorphism analysis of 23S rRNA amplicons: ( A ) digestion with Bsa I and ( B ) digestion with Bbs I. The A2144G mutations are observed in lanes 1 to 2, but not in lanes 3 to 5. Note that the A2143G mutation detected by digestion with Bbs I was not detected in any of the strains studied. Lanes 3 to 5 reveal the T2183C mutation, as assessed by DNA sequencing. Lane M, 100 bp DNA size markers (indicated to the left of the gels in base pairs); lane C, H. pylori ATCC 43504; lane 1 to 5, clarithromycin-resistant H. pylori strains.

    Article Snippet: Amplicons (424 bp each) of the 23S rRNA gene were digested with either Bsa I (New England BioLabs, Beverly, MA, USA) for 14 hr at 50℃ or Bbs I (New England BioLabs) for 14 hr at 37℃ to detect the A2144G and A2143G mutations, respectively ( ).

    Techniques: Mutagenesis, DNA Sequencing

    Construction of the FusX1–4 libraries. (A) Component plasmids used to construct the pFusX1–4 libraries. pXX-1 and pXX-10 are single-RVD (repeat-variable diresidue) encoding plasmids from the original Golden Gate system (2.0) 16 ; pXX-M and -MM are new, single RVD modules with designated sequence and Bsa I overhangs for ligation in between pXX-1 and pXX-10 to form 3-mer intermediates in pFusX1–4 libraries. pXX-MM includes extra silent mutations and is used only to construct the pFusX3 library, providing a specific primer-binding site for sequencing of long TALE (transcription activator-like effector) domain. “XX” represents any of the four RVD modules: HD, NG, NI, NN. (B) Schematic diagram showing sequential ligation of single RVD component plasmids into the four intermediate vectors: pFusX1, pFusX2, pFusX3, pFusX4. Dotted arrows indicate ligation at compatible overhangs generated by Bsa I.

    Journal: Human Gene Therapy

    Article Title: FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science

    doi: 10.1089/hum.2015.172

    Figure Lengend Snippet: Construction of the FusX1–4 libraries. (A) Component plasmids used to construct the pFusX1–4 libraries. pXX-1 and pXX-10 are single-RVD (repeat-variable diresidue) encoding plasmids from the original Golden Gate system (2.0) 16 ; pXX-M and -MM are new, single RVD modules with designated sequence and Bsa I overhangs for ligation in between pXX-1 and pXX-10 to form 3-mer intermediates in pFusX1–4 libraries. pXX-MM includes extra silent mutations and is used only to construct the pFusX3 library, providing a specific primer-binding site for sequencing of long TALE (transcription activator-like effector) domain. “XX” represents any of the four RVD modules: HD, NG, NI, NN. (B) Schematic diagram showing sequential ligation of single RVD component plasmids into the four intermediate vectors: pFusX1, pFusX2, pFusX3, pFusX4. Dotted arrows indicate ligation at compatible overhangs generated by Bsa I.

    Article Snippet: Briefly, for each reaction, 25 ng of each plasmid (for trimer libraries, 13 plasmids; for dimer libraries, 9 plasmids) ( ) was combined with 10 U of Bsa I (New England BioLabs, Ipswich, MA) and 400 U of T4 DNA ligase (New England BioLabs) in 1× T4 DNA ligase buffer (reaction volume, 20 μl).

    Techniques: Construct, Sequencing, Ligation, Binding Assay, Generated

    Detection of mutation A2142C by Bce AI-mediated restriction digestion. The restriction fragments of the 267-bp PCR products were analyzed by electrophoresis on a 5% agarose Resophor gel (A) or on a 12% polyacrylamide gel (B) stained with ethidium bromide. (A) PCR-RFLP analysis of mutations A2142G, A2143G, and A2142C occurring in domain V of the 23S rRNA gene of H. pylori . Lanes 1 and 8, 25-bp DNA Step Ladder molecular size markers (Promega). Lanes 2 and 3, PCR products of the wild-type and A2142G H. pylori strains digested with Bbs I, respectively. Lanes 4 and 5, PCR products of the wild-type and A2143G H. pylori strains digested with Bsa I, respectively. Lanes 6 and 7, PCR products of the wild-type and A2142C H. pylori strains digested with Bce AI, respectively. (B) PCR product of the H. pylori strain with mutation A2142C digested with Bce AI. Lanes 2 and 3, amplified wild-type PCR product and amplified PCR product presenting the A2142C mutation, respectively. Lane 1, 25-bp DNA Step Ladder (Promega). The wild-type H. pylori ).

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: PCR-Restriction Fragment Length Polymorphism Can Also Detect Point Mutation A2142C in the 23S rRNA Gene, Associated with Helicobacter pylori Resistance to Clarithromycin

    doi: 10.1128/AAC.46.4.1156-1157.2002

    Figure Lengend Snippet: Detection of mutation A2142C by Bce AI-mediated restriction digestion. The restriction fragments of the 267-bp PCR products were analyzed by electrophoresis on a 5% agarose Resophor gel (A) or on a 12% polyacrylamide gel (B) stained with ethidium bromide. (A) PCR-RFLP analysis of mutations A2142G, A2143G, and A2142C occurring in domain V of the 23S rRNA gene of H. pylori . Lanes 1 and 8, 25-bp DNA Step Ladder molecular size markers (Promega). Lanes 2 and 3, PCR products of the wild-type and A2142G H. pylori strains digested with Bbs I, respectively. Lanes 4 and 5, PCR products of the wild-type and A2143G H. pylori strains digested with Bsa I, respectively. Lanes 6 and 7, PCR products of the wild-type and A2142C H. pylori strains digested with Bce AI, respectively. (B) PCR product of the H. pylori strain with mutation A2142C digested with Bce AI. Lanes 2 and 3, amplified wild-type PCR product and amplified PCR product presenting the A2142C mutation, respectively. Lane 1, 25-bp DNA Step Ladder (Promega). The wild-type H. pylori ).

    Article Snippet: The 267-bp PCR products were precipitated and suspended in 15 μl of H2 O, and 5 μl was digested overnight in a final volume of 15 μl with the restriction enzymes Bbs I (5 U), Bsa I (5 U) , and Bce AI (0.5 U) (New England Biolabs).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Electrophoresis, Staining, Amplification

    Construction of binary vectors for genome editing in soybean. Cas9 fused with a single nuclear localization signal (NLS) is expressed with a Cauliflower mosaic virus 35s (CaMV 35s) promoter. Synthetic guide RNA (sgRNA) is derived using U6 promoters. ( a ) Arabidopsis thaliana U6-26 promoter ( b ) Glycine max U6-10 promoter. Sequences containing two Bsa I sites are located between the U6 promoter and the sgRNA scaffold. These sequences can be easily replaced with a gene-specific sgRNA seed. LB: left border; RB: right border.

    Journal: Scientific Reports

    Article Title: Targeted mutagenesis in soybean using the CRISPR-Cas9 system

    doi: 10.1038/srep10342

    Figure Lengend Snippet: Construction of binary vectors for genome editing in soybean. Cas9 fused with a single nuclear localization signal (NLS) is expressed with a Cauliflower mosaic virus 35s (CaMV 35s) promoter. Synthetic guide RNA (sgRNA) is derived using U6 promoters. ( a ) Arabidopsis thaliana U6-26 promoter ( b ) Glycine max U6-10 promoter. Sequences containing two Bsa I sites are located between the U6 promoter and the sgRNA scaffold. These sequences can be easily replaced with a gene-specific sgRNA seed. LB: left border; RB: right border.

    Article Snippet: These two plasmids were digested completely using Bsa I (NEB, Massachusetts, USA) and purified with a TIANquick Midi purification kit (Tiangen, Beijing, China).

    Techniques: Derivative Assay